WO2023020471A1

WO2023020471A1 - Cd123-targetting antibodies and uses thereof in cancer therapies

Info

Publication number: WO2023020471A1
Application number: PCT/CN2022/112724
Authority: WO
Inventors: Yangbing Zhao; Gengzhen ZHU; Xiaojun Liu
Original assignee: Utc Therapeutics (Shanghai) Co., Ltd.
Priority date: 2021-08-16
Filing date: 2022-08-16
Publication date: 2023-02-23

Abstract

Provided herein are anti-CD123 antibodies and antigen-binding fragments thereof, chimeric antigen receptors (CARs) having the antibodies or the antigen-binding fragments (CD123 CARs), and genetically modified immune effector cells having such CARs. Polynucleotides encoding the antibodies, the antigen-binding fragments or the CARs are also provided. Compositions comprising the antibodies, the antigen-binding fragments or the CARs are also provided. The use of the antibodies, the antigen-binding fragments and the genetically modified immune effector cells having such CARs in cancer treatment are also provided.

Description

CD123-TARGETTING ANTIBODIES AND USES THEREOF IN CANCER THERAPIES

1. Field

The present invention relates to molecular biology, cell biology, and immuno-oncology. In particular, provided herein include anti-CD123-antibodies, chimeric antigen receptors (CARs) comprising such anti-CD123-antibodies ( “CD123 CARs” ) , genetically engineered immune effector cells expressing such CD123 CARs, and uses thereof in treating tumors or cancers.

2. Background

CD123 is widely expressed in various hematological malignancies, including acute myeloid leukemia (AML) , blastic plasmacytoid dendritic cell neoplasm (BPDCN) , B cell precursor acute lymphoblastic leukemia (BCP‐ALL) . Current therapies targeting CD123, however, have only had limited success. Thus, additional CD123-targeting therapeutic options represent unmet needs. The compositions and methods provided herein meet these needs and provide other relative advantages.

3. Summary

Provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123, comprising: (a) a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-29; (2) a light chain CDR2 (VL CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-56; and (3) a light chain CDR3 (VL CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57-88; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VH CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-109; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-133; and (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134-163; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments of the antibodies or antigen-binding fragments provided herein, (a) the VL CDR1, CDR2 and CDR3 have (1) the amino acid sequences of SEQ ID NOs: 3, 33, and 61, respectively; (2) the amino acid sequences of SEQ ID NOs: 17, 45, and 75, respectively; (3) the amino acid sequences of SEQ ID NOs: 7, 37, and 66, respectively; (4) the amino acid sequences of SEQ ID NOs: 18, 32, and 76, respectively; (5) the amino acid sequences of SEQ ID NOs: 23, 50 and 82, respectively; (6) the amino acid sequences of SEQ ID NOs: 24, 51, and 83, respectively; (7) the amino acid sequences of SEQ ID NOs: 8, 38, and 67, respectively; (8) the amino acid sequences of SEQ ID NOs: 4, 34, and 62, respectively; (9) the amino acid sequences of SEQ ID NOs: 25, 52, and 84, respectively; (10) the amino acid sequences of SEQ ID NOs: 26, 53, and 85, respectively; (11) the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; (12) the amino acid sequences of SEQ ID NOs: 17, 46, and 77, respectively; (13) the amino acid sequences of SEQ ID NOs: 27, 54 and 86, respectively; (14) the amino acid sequences of SEQ ID NOs: 9, 39, and 68, respectively; (15) the amino acid sequences of SEQ ID NOs: 10, 40, and 69, respectively; (16) the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively; (17) the amino acid sequences of SEQ ID NOs: 5, 35, and 63, respectively; (18) the amino acid sequences of SEQ ID NOs: 1, 30 and 64, respectively; (19) the amino acid sequences of SEQ ID NOs: 11, 41, and 70, respectively; (20) the amino acid sequences of SEQ ID NOs: 1, 31, and 59, respectively; (21) the amino acid sequences of SEQ ID NOs: 19, 47, and 78, respectively; (22) the amino acid sequences of SEQ ID NOs: 28, 55, and 87, respectively; (23) the amino acid sequences of SEQ ID NOs: 12, 39, and 71, respectively; (24) the amino acid sequences of SEQ ID NOs: 13, 42, and 72, respectively; (25) the amino acid sequences of SEQ ID NOs: 1, 30, and 57, respectively; (26) the amino acid sequences of SEQ ID NOs: 20, 48 and 79, respectively; (27) the amino acid sequences of SEQ ID NOs: 14, 43, and 67, respectively; (28) the amino acid sequences of SEQ ID NOs: 1, 30, and 58, respectively; (29) the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; (30) the amino acid sequences of SEQ ID NOs: 15, 44, and 73, respectively; (31) the amino acid sequences of SEQ ID NOs: 29, 56 and 88, respectively; (32) the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively; (33) the amino acid sequences of SEQ ID NOs: 16, 42, and 74, respectively; (34) the amino acid sequences of SEQ ID NOs: 6, 36, and 65, respectively; or (35) the amino acid sequences of SEQ ID NOs: 21, 45, and 80, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) the VH CDR1, CDR2 and CDR3 have (1) the amino acid sequences of SEQ ID NOs: 91, 112, and 136, respectively; (2) the amino acid sequences of SEQ ID NOs: 89, 122, and 149, respectively; (3) the amino acid sequences of SEQ ID NOs: 95, 116, and 140, respectively; (4) the amino acid sequences of SEQ ID NOs: 100, 123, and 150, respectively; (5) the amino acid sequences of SEQ ID NOs: 99, 129 and 157, respectively; (6) the amino acid sequences of SEQ ID NOs: 106, 128, and 158, respectively; (7) the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively; (8) the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively; (9) the amino acid sequences of SEQ ID NOs: 107, 130, and 159, respectively; (10) the amino acid sequences of SEQ ID NOs: 95, 116, and 160, respectively; (11) the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively; (12) the amino acid sequences of SEQ ID NOs: 101, 124, and 151, respectively; (13) the amino acid sequences of SEQ ID NOs: 105, 131, and 161, respectively; (14) the amino acid sequences of SEQ ID NOs: 97, 118, and 142, respectively; (15) the amino acid sequences of SEQ ID NOs: 98, 119, and 143, respectively; (16) the amino acid sequences of SEQ ID NOs: 105, 128, and 156, respectively; (17) the amino acid sequences of SEQ ID NOs: 92, 113, and 137, respectively; (18) the amino acid sequences of SEQ ID NOs: 93, 114 and 138, respectively; (19) the amino acid sequences of SEQ ID NOs: 91, 120, and 144, respectively; (20) the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively; (21) the amino acid sequences of SEQ ID NOs: 102, 125, and 152, respectively; (22) the amino acid sequences of SEQ ID NOs: 108, 132, and 162, respectively; (23) the amino acid sequences of SEQ ID NOs: 96, 117, and 145, respectively; (24) the amino acid sequences of SEQ ID NOs: 95, 116, and 146, respectively; (25) the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively; (26) the amino acid sequences of SEQ ID NOs: 103, 126 and 153, respectively; (27) the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively; (28) the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively; (29) the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively; (30) the amino acid sequences of SEQ ID NOs: 99, 121, and 147, respectively; (31) the amino acid sequences of SEQ ID NOs: 109, 133 and 163, respectively; (32) the amino acid sequences of SEQ ID NOs: 104, 127, and 155, respectively; (33) the amino acid sequences of SEQ ID NOs: 95, 116, and 148, respectively; (34) the amino acid sequences of SEQ ID NOs: 94, 115, and 139, respectively; or (35) the amino acid sequences of SEQ ID NOs: 89, 122, and 154, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments of the antibodies or antigen-binding fragments provided herein, (1) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 3, 33, and 61, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 91, 112, and 136, respectively; (2) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 17, 45, and 75, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 122, and 149, respectively; (3) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 7, 37, and 66, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 95, 116, and 140, respectively; (4) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 18, 32, and 76, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 100, 123, and 150, respectively; (5) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 23, 50 and 82, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 99, 129 and 157, respectively; (6) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 24, 51, and 83, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 106, 128, and 158, respectively; (7) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 8, 38, and 67, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively; (8) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 4, 34, and 62, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively; (9) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 25, 52, and 84, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 107, 130, and 159, respectively; (10) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 26, 53, and 85, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 95, 116, and 160, respectively; (11) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively; (12) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 17, 46, and 77, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 101, 124, and 151, respectively; (13) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 27, 54 and 86, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 105, 131, and 161, respectively; (14) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 9, 39, and 68, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 97, 118, and 142, respectively; (15) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 10, 40, and 69, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 98, 119, and 143, respectively; (16) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 105, 128, and 156, respectively; (17) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 5, 35, and 63, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 92, 113, and 137, respectively; (18) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 30 and 64, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 93, 114 and 138, respectively; (19) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 11, 41, and 70, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 91, 120, and 144, respectively; (20) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 31, and 59, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively; (21) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 19, 47, and 78, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 102, 125, and 152, respectively; (22) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 28, 55, and 87, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 108, 132, and 162, respectively; (23) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 12, 39, and 71, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 96, 117, and 145, respectively; (24) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 13, 42, and 72, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 95, 116, and 146, respectively; (25) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 30, and 57, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively; (26) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 20, 48 and 79, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 103, 126 and 153, respectively; (27) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 14, 43, and 67, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively; (28) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 30, and 58, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively; (29) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively; (30) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 15, 44, and 73, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 99, 121, and 147, respectively; (31) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 29, 56 and 88, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 109, 133 and 163, respectively; (32) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 104, 127, and 155, respectively; (33) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 16, 42, and 74, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 95, 116, and 148, respectively; (34) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 6, 36, and 65, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 94, 115, and 139, respectively; or (35) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 21, 45, and 80, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 122, and 154, respectively.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3, wherein (a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 23, 50 and 82, respectively; and (b) the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 99, 129 and 157, respectively.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3, wherein (a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 8, 38, and 67, respectively; and (b) the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3, wherein (a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; and (b) the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123, comprising: (a) a VL comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 348-352; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 353-356; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 357-359; and/or (b) a VH comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 360-361; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 362-367; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 368.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123, comprising: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198; and/or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL and a VH, wherein the VL and VH have (1) the amino acid sequences of SEQ ID NOs: 164 and 199, respectively; (2) the amino acid sequences of SEQ ID NOs: 165 and 200, respectively; (3) the amino acid sequences of SEQ ID NOs: 166 and 201, respectively; (4) the amino acid sequences of SEQ ID NOs: 167 and 202, respectively; (5) the amino acid sequences of SEQ ID NOs: 168 and 203, respectively; (6) the amino acid sequences of SEQ ID NOs: 169 and 204, respectively; (7) the amino acid sequences of SEQ ID NOs: 170 and 205, respectively; (8) the amino acid sequences of SEQ ID NOs: 171 and 206, respectively; (9) the amino acid sequences of SEQ ID NOs: 172 and 207, respectively; (10) the amino acid sequences of SEQ ID NOs: 173 and 208, respectively; (11) the amino acid sequences of SEQ ID NOs: 174 and 209, respectively; (12) the amino acid sequences of SEQ ID NOs: 175 and 210, respectively; (13) the amino acid sequences of SEQ ID NOs: 176 and 211, respectively; (14) the amino acid sequences of SEQ ID NOs: 177 and 212, respectively; (15) the amino acid sequences of SEQ ID NOs: 178 and 213, respectively; (16) the amino acid sequences of SEQ ID NOs: 179 and 214, respectively; (17) the amino acid sequences of SEQ ID NOs: 180 and 215, respectively; (18) the amino acid sequences of SEQ ID NOs: 181 and 216, respectively; (19) the amino acid sequences of SEQ ID NOs: 182 and 217, respectively; (20) the amino acid sequences of SEQ ID NOs: 183 and 218, respectively; (21) the amino acid sequences of SEQ ID NOs: 184 and 219, respectively; (22) the amino acid sequences of SEQ ID NOs: 185 and 220, respectively; (23) the amino acid sequences of SEQ ID NOs: 186 and 221, respectively; (24) the amino acid sequences of SEQ ID NOs: 187 and 222, respectively; (25) the amino acid sequences of SEQ ID NOs: 188 and 223, respectively; (26) the amino acid sequences of SEQ ID NOs: 189 and 224, respectively; (27) the amino acid sequences of SEQ ID NOs: 190 and 225, respectively; (28) the amino acid sequences of SEQ ID NOs: 191 and 226, respectively; (29) the amino acid sequences of SEQ ID NOs: 192 and 227, respectively; (30) the amino acid sequences of SEQ ID NOs: 193 and 228, respectively; (31) the amino acid sequences of SEQ ID NOs: 194 and 229, respectively; (32) the amino acid sequences of SEQ ID NOs: 195 and 230, respectively; (33) the amino acid sequences of SEQ ID NOs: 196 and 231, respectively; (34) the amino acid sequences of SEQ ID NOs: 197 and 232, respectively; or (35) the amino acid sequences of SEQ ID NOs: 198 and 233, respectively.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NOs: 168 and 203, respectively. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NOs: 170 and 205, respectively. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NOs: 174 and 209, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123, comprising (a) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198; and/or (b) a VH comprising VH CDR1, CDR2, and CDR3 from a VH having an amino acid sequence selected from group consisting of SEQ ID NOs: 199-233.

In some embodiments, the antibodies or antigen-binding fragments provided herein comprise (1) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 164, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 199; (2) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 165, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 200; (3) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 166, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 201; (4) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 167, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 202; (5) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 168, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 203; (6) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 169, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 204; (7) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 170, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 205; (8) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 171, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 206; (9) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 172, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 207; (10) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 173, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 208; (11) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 174, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 209; (12) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 175, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 210; (13) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 176, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 211; (14) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 177, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 212; (15) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 178, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 213; (16) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 179, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 214; (17) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 180, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 215; (18) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 181, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 216; (19) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 182, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 217; (20) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 183, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 218; (21) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 184, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 219; (22) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 185, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 220; (23) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 186, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 221; (24) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 187, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 222; (25) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 188, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 223; (26) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 189, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 224; (27) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 190, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 225; (28) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 191, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 226; (29) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 192, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 227; (30) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 193, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 228; (31) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 194, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 229; (32) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 195, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 230; (33) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 196, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 231; (34) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 197, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 232; or (35) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 198, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 233.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123, comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 168, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 203. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123, comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 170, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 205. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123, comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 174, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 209.

Provided herein are also antibodies or antigen-binding fragments thereof that compete with any antibody or antigen-binding fragment disclosed herein for binding to CD123.

In some embodiments, the antibody or antigen-binding fragment provided herein is a monoclonal antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment provided herein is a bispecific or multispecific antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment provided herein is a Bi-specific T-cell engagers (BiTE) .

In some embodiments, the antibody or antigen-binding fragment provided herein is selected from the group consisting of IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody. In some embodiments, the antibody or antigen-binding fragment provided herein is selected from the group consisting of a Fab, a Fab’, a F (ab’) ₂, a Fv, a scFv, a (scFv) ₂, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) . In some embodiments, the antibody or antigen-binding fragment is a scFv.

In some embodiments, the antibody or antigen-binding fragment provided herein is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment.

Provided herein are also polynucleotides encoding the antibodies or antigen-binding fragments provided herein. In some embodiments, the polynucleotide is a messenger RNA (mRNA) .

Provided herein are also vectors comprising any polynucleotides disclosed herein.

Provided herein are also host cells comprising any polynucleotide disclosed herein or any vector disclosed herein.

Provided herein are also Chimeric Antigen Receptors (CAR) that specifically bind CD123, comprising, from N-terminus to C-terminus: (a) a CD123-binding domain that comprises an anti-CD123 antibody or antigen-binding fragment disclosed herein; (b) a transmembrane domain; and (c) a cytoplasmic domain.

In some embodiments of the CARs disclosed herein, the transmembrane domain is derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.

In some embodiments of the CARs disclosed herein, the transmembrane domain comprises CD8 transmembrane region or CD28 transmembrane region.

In some embodiments of the CARs disclosed herein, the cytoplasmic domain comprises a signaling domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

In some embodiments of the CARs disclosed herein, the cytoplasmic domain further comprises a co-stimulatory domain derived from CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.

In some embodiments of the CARs disclosed herein, the cytoplasmic domain comprises a CD3 ζ signaling domain and a 4-1BB co-stimulatory domain. In some embodiments of the CARs disclosed herein, the cytoplasmic domain comprises a CD3 ζ signaling domain and a CD28 co-stimulatory domain.

In some embodiments, the CARs disclosed herein further comprise a CD8 hinge between the antibody or antigen-binding fragment and the transmembrane domain.

In some embodiments, provided herein are CARs that specifically bind CD123 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 369-371.

Provided herein are also polynucleotides encoding a CAR that specifically bind CD123 as disclosed herein. In some embodiments, the polynucleotide is a mRNA. Provided herein are also vectors comprising any polynucleotide disclosed herein.

Provided herein are cells comprising any polynucleotide disclosed herein, or any vector disclosed herein. In some embodiments, the cells provided herein comprise a polynucleotide comprising a first fragment encoding a CAR described herein and a second fragment encoding a fusion protein. In some embodiments, the cells provided herein comprise a first polynucleotide encoding a CAR described herein and a second polynucleotide encoding a fusion protein. The fusion protein comprise a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein (i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and (ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

In some embodiments, the N-terminus of the first domain of the fusion protein is linked to the C-terminus of the second domain of the fusion protein. In some embodiments, the N-terminus of the second domain of the fusion protein is linked to the C-terminus of the first domain of the fusion protein. In some embodiments, the first domain and the second domain of the fusion protein are linked via a linker.

The fusion proteins described herein comprise a first domain that activates an APC and a second domain that activates an immune effector cell. In some embodiments, the APC is selected from the group consisting of a dendritic cell, a macrophage, a myeloid derived suppressor cell, a monocyte, a B cell, a T cell, and a Langerhans cell. In some embodiments, the activation receptor of the APC is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN. In some embodiments, the first domain comprises a ligand that binds CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN, or a receptor-binding fragment thereof. In some embodiments, the first domain comprises a receptor-binding domain of CD40 Ligand (CD40L) . In some embodiments, the first domain comprises an antibody that binds the activation receptor of the APC, or an antigen- binding fragment thereof. In some embodiments, the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof.

The fusion proteins described herein comprise a first domain that activates an APC and a second domain that activates an immune effector cell. In some embodiments, the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte. In some embodiments, the second domain comprises a cytoplasmic domain of the co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the second domain comprises a cytoplasmic domain of CD28. In some embodiments, the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof. In some embodiments, the co-stimulatory ligand is selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44. In some embodiments, the second domain comprises an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. The antibody can be an scFv. In some embodiments, the second domain comprises an antibody that binds CD28.

In some embodiments of the fusion proteins described herein, the first domain comprises CD40L or a receptor-binding thereof and the second domain comprises a CD28 cytoplasmic domain. In some embodiments, the first domain comprises CD40L or a receptor-binding thereof and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises a CD28 cytoplasmic domain.

In some embodiments, the fusion protein described herein is at least 85%, 90%, 95%, 98%, or 99%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 418-433 and 490-492.

In some embodiments, the cell provided herein is an immune effector cell. In some embodiments, the cell is derived from a cell isolated from peripheral blood or bone marrow. In some embodiments, the cell is derived from a cell differentiated in vitro from a stem or progenitor cell selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell. In some embodiments, the cell is a T cell or a NK cell.

In some embodiments, the cell is a cytotoxic T cell, a helper T cell, a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, or an effector memory TEMRA cell. In some embodiments, the cell is a cytotoxic T cell.

Provided herein are also populations of the cells comprising any cell disclosed herein, wherein the population of cells are derived from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) .

Provided herein are pharmaceutical compositions comprising a therapeutically effective amount of any antibody or antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier.

Provided herein are also pharmaceutical compositions comprising a therapeutically effective amount of any cell or cell population disclosed herein, and a pharmaceutically acceptable carrier.

Provided herein are uses of any antibody or antigen-binding fragment provided herein in cancer treatment. Provided herein are also uses of any antibody or antigen-binding fragment provided herein for the preparation of a medicament for the treatment of cancer.

Provided herein are uses of any cell or population of cells provided herein in cancer treatment. Provided herein are also uses of any cell or population of cells provided herein for the preparation of a medicament for the treatment of cancer.

Provided herein are uses of any pharmaceutical composition provided herein in cancer treatment. Provided herein are also uses of any pharmaceutical composition disclosed herein for the preparation of a medicament for the treatment of cancer.

In some embodiments of the uses disclosed herein, the cell, population of cells, or pharmaceutical composition is used in combination with an additional therapy.

Provided herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any antibody or antigen-binding fragment disclosed herein. Provided herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any cell or cell population disclosed herein. Provided herein are also method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any pharmaceutical composition disclosed herein.

In some embodiments of the methods disclosed herein, the cell or population of cells is autologous to the subject. In some embodiments, the methods disclosed herein further comprise obtaining cells from the subject. In some embodiments, the methods disclosed herein comprise administering an additional therapy to the subject.

In some embodiments of the methods disclosed herein, the subject is a human.

In some embodiments of the uses or methods disclosed herein, the cancer is a solid tumor. In some embodiments of the uses or methods disclosed herein, the cancer is a hematological cancer. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is acute myeloid leukemia (AML) , B-acute lymphoid leukemia (B-ALL) , T-acute lymphoid leukemia (T-ALL) , B cell precursor acute lymphoblastic leukemia (BCP‐ALL) or blastic plasmacytoid dendritic cell neoplasm (BPDCN) . In some embodiments, the cancer is CD123-expressing cancer. In some embodiments, the cancer is CD123-expressing AML.

Provided herein are methods of preparing a cell capable of expressing a CAR that specifically binds CD123, comprising transferring a polynucleotide disclosed herein into the cell, wherein the polynucleotide encodes a CAR that specifically binds CD123. In some embodiments, the polynucleotide is transferred via electroporation. In some embodiments, the polynucleotide is transferred via viral transduction. In some embodiments, a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus is used for the viral transduction. In some embodiments, the polynucleotide is transferred using a transposon system. In some embodiments, the transposon system is Sleeping Beauty or PiggyBac. In some embodiments, the polynucleotide is transferred using gene-editing. In some embodiments, the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system. In some embodiments, the cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell.

4. Brief Description of Drawings

FIG. 1 provides reads of two 96-well plates of anti-human CD123-Fc monoclonal phage ELISA.

FIG. 2 provides schematic representation of pDA-CAR vector used for CAR mRNA generation.

FIG. 3 provides FACS staining results showing the binding of anti-CD123 scFv that expressed in CART cells to CD123-Fc protein.

FIG. 4 provides FACS staining of the A549 cells that were electroporated with different amount of CD123 mRNA with isotype and anti-CD123 antibodies.

FIG. 5 provides the killing curves of different mRNA-based CD123 CART cells against A549-GFP tumor cells at E/T ratio=10: 1.

FIG. 6 provides the killing curves of different mRNA-based anti-CD123 CART cells against A549-GFP tumor cells at E/T ratio=3: 1.

FIG. 7 provides the killing curves of different mRNA-based anti-CD123 CART cells against A549-GFP tumor cells that were electroporated with 10 μg CD123 mRNA at E/T ratio=10: 1.

FIG. 8 provides the killing curves of different mRNA-based anti-CD123 CART cells against A549-GFP tumor cells that were electroporated with 10 μg CD123 mRNA at E/T ratio=3: 1.

FIG. 9 shows FACS staining of A549, SK-OV3, Jeko-1, Molm-14, SupT-1, 293T, Nalm-6 and PC-3 cells with PE-isotype control and PE-anti-CD123 mAb.

FIG. 10 shows CD107a staining of anti-CD123-C5, anti-CD123-C7, anti-CD123-C11 CART cells in the coculture and killing assay with different tumor cell lines.

FIG. 11 provides the killing curves of different mRNA-based anti-CD123 CART cells with or without LACO (A40C. CD28) against MOLM-14, NALM6 or JEKO-1 tumor cells at E/T ratio=10: 1

FIG. 12 provides the killing curves of different mRNA-based anti-CD123 CART cells with or without LACO (A40C. CD28) against A549 tumor cells that were electroporated with 10 μg, 0.1 μg or 0 CD123 mRNA at E/T ratio=30: 1.

FIG. 13 provides ELISA results showing the IFN-gamma secretion of the T cells electroporated with different CD123 CAR with or without LACO.

5. Detailed Description

Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting.

The present disclosure provides novel antibodies, including antigen-binding fragments that specifically bind CD123 (e.g., human CD123) . Further, the present disclosure also provides chimeric antigen receptors (CARs) that comprise such antibodies or antigen-binding fragments that specifically bind CD123 (e.g., human CD123) , as well as engineered immune effector cells (e.g., T cells) and populations of cells that recombinantly express a CAR (e.g., CARTs) that specifically binds CD123 (e.g., human CD123) . Pharmaceutical compositions comprising a therapeutically effective amount of such antibodies or antigen-binding fragments, and pharmaceutical compositions comprising a therapeutically effective amount of cells or population of cells are also disclosed herein. Also disclosed herein are uses of such pharmaceutical compositions for treating cancer (e.g., CD123-expressing cancer) and methods of cancer treatment.

Cell division cycle protein 123 homolog, or CD123 (other alias: Interleukin 3 Receptor Subunit Alpha, IL3RA, HT-1080, PZ32) is type I transmembrane glycoprotein, with a molecular weight (MW) of approximately 70 kDa. Exemplary amino acid sequences of human CD123 are described (see, e.g., NCBI Reference Sequence: NP_002174.1, XP_005274488.1) . Exemplary polynucleotides that encode human CD123 and equivalents are described (see, e.g., NCBI Reference Sequence: NM_002183.3, XM_005274431.4) . An exemplary amino acid sequence for human CD123 is provided below (SEQ ID NO: 321) . The extracellular region of CD123 comprises three fibronectin-like (FnIII) domains, that can be bound by IL-3. The extracellular region of CD123 includes amino acid residues 19-305 of SEQ ID NO: 321. The transmembrane domain of CD123 includes amino acid residues 306-325 of SEQ ID NO: 321. The cytoplasmic domain of CD123 includes amino acid residues 326-378 of SEQ ID NO: 321.

CD123 alone binds IL-3 with low affinity, when CD123 form complex with common β chain, it binds IL-3 with high affinity. Functionally, CD123 alone does not transduce signals upon binding IL-3 and requires the common β chain for the downstream signaling transduction (Kitamura et al. (1991) Cell 66 (6) : 1165-1174) .

In normal tissues and cells, CD123 is reported to constitutively expressed in monocytes, neutrophils, basophils, eosinophils, megakaryocytes, erythroid precursors, mast cells, macrophages, hematopoietic stem/progenitor cells, and by some CD19+ cells (Testa et al. (2014) . Biomark Res 2 (1) : 4) . Outside the hematopoietic system CD123 is reported to be expressed in Leydig cells of the testis, some endothelial cells, and cells of the placenta and brain.

Testa and colleagues systematically investigated the expression of CD123 in 79 AML patients, 25 patients with B-acute lymphoid leukemia (B-ALL) , and 7 patients with T-acute lymphoid leukemia (T-ALL) . Results demonstrated that CD123 was overexpressed in 40%and 45%of patients with B-ALL and AML, respectively, while CD123 was scarcely expressed in most patients with T-ALL. They also reported the expression of CD123 is closely related with enhanced blast proliferation, increased cycling activity, and poor prognosis (Testa et al (2002) Blood 100 (8) : 2980-2988. ) In another study, CD123 expression levels in 846 acute leukemia patients, including 139 pediatric AML, 316 adult AML, 193 pediatric BCP‐ALL, 69 adult BCP‐ALL, 101 pediatric T‐ALL, and 28 adult T‐ALL patients, were evaluated by detailed flow cytometric analysis. The results showed that CD123 was expressed in the majority of AML and BCP‐ALL patients, but absent in most T‐ALL patients (Bras et al (2019) Cytometry B Clin Cytom 96 (2) : 134-142. ) .

Rollins-Raval and colleagues reported CD123 was aberrantly expressed in 40%of AML patients based on the CD123 immunohistochemistry (IHC) staining of 157 AML bone marrow biopsies. They also found the overexpression of CD123 in AML patients is associated with FLT3-ITD and NPM1 mutations (Rollins-Raval et al (2013) Appl Immunohistochem Mol Morphol 21 (3) : 212-217) . Besides in AML and BCP‐ALL, other studies have also shown the overexpression of CD123 in hairy cell leukemia and neoplastic cells of Hodgkin lymphoma (Munoz et al (2001) Haematologica 86 (12) : 1261-1269, Fromm (2011) . Cytometry B Clin Cytom 80 (2) : 91-99. ) .

In summary, CD123 is widely expressed in various hematological malignancies, including acute myeloid leukemia (AML) , blastic plasmacytoid dendritic cell neoplasm (BPDCN) , B cell precursor acute lymphoblastic leukemia (BCP‐ALL) (Liu et al (2015) . Life Sci 122: 59-64., Testa et al (2019) . Cancers (Basel) 11 (9) ) . Various therapies targeting CD123 have entered clinical trials but met with limited success (Frankel et al (2014) Blood 124 (3) : 385-392; Jabbour et al (2017) Blood 130 (13) : 1514-1522; Frankel et al (2008) Leuk Lymphoma 49 (3) : 543-553; Xie et al (2017) Blood Cancer J. 7, (e567) ; Daver et al (2018) Blood 132 (abst. 27) ; Uy et al (2018) Blood 132 (abst. 764) ; Hofmann et al (2019) . J Clin Med 8 (2) ; Gill et al (2014) Blood 123: 2343–2354; Pemmaraju et al (2019) N Engl J Med 380 (17) : 1628-1637) . To address the unmet need, included in present disclosure are novel cancer therapies targeting CD123 with improved efficacy and safety.

5.1 Definitions

Unless otherwise defined herein, scientific and technical terms used in the present disclosures shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

The term “antibody, ” and its grammatical equivalents as used herein refer to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , single-chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecule comprising an antigen-binding site (e.g., dual variable domain immunoglobulin molecules) as long as the antibodies exhibit the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) , based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. Unless expressly indicated otherwise, the term “antibody” as used herein include “antigen-binding fragment” of intact antibodies. The term “antigen-binding fragment” as used herein refers to a portion or fragment of an intact antibody that is the antigenic determining variable region of an intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F (ab’) 2, Fv, linear antibodies, single chain antibody molecules (e.g., scFv) , heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , disulfide-linked scFv (dsscFv) , diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD) , single variable domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies) , and single variable domain of heavy chain antibodies (VHH) , and bispecific or multispecific antibodies formed from antibody fragments.

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region. The constant region can be one of five distinct types, referred to as alpha (a) , delta (δ) , epsilon (ε) , gamma (γ) and mu (μ) , based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (κ) of lambda (λ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

The term “variable domain” or “variable region” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR) . The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. Numbering of amino acid positions used herein is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, D. C. ) 5thed. A variable region can be a human variable region.

A CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by a variety of methods/systems. These systems and/or definitions have been developed and refined over years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines the regions of most hypervariability within the antibody variable (V) domains (Kabat et al, J. Biol. Chem. 252: 6609-6616 (1977) ; Kabat, Adv. Prot. Chem. 32: 1-75 (1978) ) . The Chothia definition is based on the location of the structural loop regions, which defines CDR region sequences as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) ) . Both terminologies are well recognized in the art. Additionally, the IMGT system is based on sequence variability and location within the structure of the variable regions. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. Software programs (e.g., abYsis) are available and known to those of skill in the art for analysis of antibody sequence and determination of CDRs. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al, J. Mol. Biol. 273: 927-948 (1997) ; Morea et al, Methods 20: 267-279 (2000) ) . Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997) ) . Such nomenclature is similarly well known to those skilled in the art.

For example, CDRs defined according to either the Kabat (hypervariable) or Chothia (structural) designations, are set forth in the table below.

	Kabat ¹	Chothia ²	Loop Location
VHCDRl	31-35	26-32	linking B and C strands
VHCDR2	50-65	53-55	linking C’ and C” strands
VHCDR3	95-102	96-101	linking F and G strands
VLCDRl	24-34	26-32	linking B and C strands
VLCDR2	50-56	50-52	linking C’ and C” strands
VLCDR3	89-97	91-96	linking F and G strands

¹Residue numbering follows the nomenclature of Kabat et al., supra

²Residue numbering follows the nomenclature of Chothia et al., supra

One or more CDRs also can be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin can incorporate the CDR (s) as part of a larger polypeptide chain, can covalently link the CDR (s) to another polypeptide chain, or can incorporate the CDR (s) noncovalently. The CDRs permit the immunoadhesin to bind to a particular antigen of interest. The CDR regions can be analyzed by, for example, abysis website (http: //abysis. org/) .

The term “humanized antibody” as used herein refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulin. In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. In some instances, residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and/or binding capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability. The term “human antibody” as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.

The terms “epitope” and “antigenic determinant” are used interchangeably herein an refer to the site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, such as a localized region on the surface of an antigen. The target molecule can comprise, a protein, a peptide, a nucleic acid, a carbohydrate, or a lipid. An epitope having immunogenic activity is a portion of a target molecule that elicits an immune response in an animal. An epitope of a target molecule having antigenic activity is a portion of the target molecule to which an antibody binds, as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. The term, “epitope” includes linear epitopes and conformational epitopes. A region of a target molecule (e.g., a polypeptide) contributing to an epitope can be contiguous amino acids of the polypeptide or the epitope can come together from two or more non-contiguous regions of the target molecule. The epitope may or may not be a three-dimensional surface feature of the target molecule. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

The term “specifically binds, ” as used herein, means that a polypeptide or molecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. A binding moiety (e.g., antibody) that specifically binds a target molecule (e.g., antigen) can be identified, for example, by immunoassays, ELISAs, SPR (e.g., Biacore) , or other techniques known to those of skill in the art. Typically, a specific reaction will be at least twice background signal or noise and can be more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity. A binding moiety that specifically binds a target molecule can bind the target molecule at a higher affinity than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a target molecule can bind the target molecule with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds a particular target molecule binds a different molecule at such a low affinity that binding cannot be detected using an assay described herein or otherwise known in the art. In some embodiments, “specifically binds” means, for instance, that a binding moiety binds a molecule target with a K _D of about 0.1 mM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a K _D of at about 10 μM or less or about 1 μM or less. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a K _D of at about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It is understood that, in some embodiments, a binding moiety (e.g., antibody) that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, i.e., binding to a single target. Thus, a binding moiety (e.g., antibody) can, in some embodiments, specifically bind more than one target. For example, an antibody can, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities.

The term “binding affinity” as used herein generally refers to the strength of the sum total of noncovalent interactions between a binding moiety (e.g., antibody) and a target molecule (e.g., antigen) . The binding of a binding moiety and a target molecule is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (K _D) . K _D is the ratio of a dissociation rate (k _off or k _d) to the association rate (k _on or k _a) . The lower the K _D of a binding pair, the higher the affinity. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following. In some embodiments, the “K _D” or “K _D value” can be measured by assays known in the art, for example by a binding assay. The K _D may be measured in a radiolabeled antigen binding assay (RIA) (Chen, et al., (1999) J. Mol Biol 293: 865-881) . The K _D or K _D value may also be measured by using surface plasmon resonance assays by Biacore, using, for example, a BIAcoreTM-2000 or a BIAcoreTM-3000 BIAcore, Inc., Piscataway, NJ) , or by biolayer interferometry using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA) .

The term “variant” as used herein in relation to a protein or a polypeptide with particular sequence features (the “reference protein” or “reference polypeptide” ) refers to a different protein or polypeptide having one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and/or additions as compared to the reference protein or reference polypeptide. The changes to an amino acid sequence can be amino acid substitutions. The changes to an amino acid sequence can be conservative amino acid substitutions. A functional fragment or a functional variant of a protein or polypeptide maintains the basic structural and functional properties of the reference protein or polypeptide.

The terms “polypeptide, ” “peptide, ” “protein, ” and their grammatical equivalents as used interchangeably herein refer to polymers of amino acids of any length, which can be linear or branched. It can include unnatural or modified amino acids or be interrupted by non-amino acids. A polypeptide, peptide, or protein can also be modified with, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

The terms “polynucleotide, ” “nucleic acid, ” and their grammatical equivalents as used interchangeably herein mean polymers of nucleotides of any length and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

The terms “identical, ” percent “identity, ” and their grammatical equivalents as used herein in the context of two or more polynucleotides or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

The term “vector, ” and its grammatical equivalents as used herein refer to a vehicle that is used to carry genetic material (e.g., a polynucleotide sequence) , which can be introduced into a host cell, where it can be replicated and/or expressed. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more polynucleotides are to be co-expressed, both polynucleotides can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding polynucleotides can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of polynucleotides into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the polynucleotides are expressed in a sufficient amount to produce a desired product (e.g., an anti-CD123 antibody or antigen-binding fragment as described herein) , and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

The term “chimeric antigen receptor” or “CAR” as used herein refers to an artificially constructed hybrid protein or polypeptide containing a binding moiety (e.g., an antibody) linked to immune cell (e.g., T cell) signaling or activation domains. In some embodiments, CARs are synthetic receptors that retarget T cells to tumor surface antigens (Sadelain et al., Nat. Rev. Cancer 3 (l) : 35-45 (2003) ; Sadelain et al., Cancer Discovery 3 (4) : 388-398 (2013) ) . CARs can provide both antigen binding and immune cell activation functions onto an immune cell such as a T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition can give T-cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a mechanism of tumor escape.

The term “genetic engineering” or its grammatical equivalents when used in reference to a cell is intended to mean alteration of the genetic materials of the cell that is not normally found in a naturally occurring cell. Genetic alterations include, for example, modifications introducing expressible polynucleotides, other additions, mutations/alterations, deletions and/or other functional disruption of the cell’s genes. Such modifications can be done in, for example, coding regions and functional fragments thereof of a gene. Additional modifications can be done in, for example, non-coding regulatory regions in which the modifications alter expression of a gene.

The term “transfer, ” “transduce, ” “transfect, ” and their grammatical equivalents as used herein refer to a process by which an exogenous polynucleotide is introduced into the host cell. A “transferred, ” “transfected, ” or “transduced” cell is one which has been transferred, transduced, or transfected with an exogenous polynucleotide. The cell includes the primary subject cell and its progeny. A polynucleotide can be “transferred” into a host cell using any type of approaches known in the art, including, e.g., a chemical method, a physical method, or a biological method. A polynucleotide is commonly “transduced” into a host cell using a virus. By contrast, a polynucleotide is commonly “transfected” into a host cell using a non-viral approach. These terms are used interchangeable at times, and a person of ordinary skill in the art would readily understand their meanings when used in different contexts.

As used herein, the term “encode” and its grammatical equivalents refer to the inherent property of specific sequences of nucleotides in a polynucleotide or a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

A polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

The term “immune effector cell” and its grammatical equivalents as used herein and understood in the art refer to cells that are of hematopoietic origin and play a direct role in the immune response against a target, such as a pathogen, a cancer cell, or a foreign substance. Immune effector cells include T cells, B cell, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils.

The term “treat” and its grammatical equivalents as used herein in connection with a disease or a condition, or a subject having a disease or a condition refer to an action that suppresses, eliminates, reduces, and/or ameliorates a symptom, the severity of the symptom, and/or the frequency of the symptom associated with the disease or disorder being treated. For example, when used in reference to a cancer or tumor, the term “treat” and its grammatical equivalents refer to an action that reduces the severity of the cancer or tumor, or retards or slows the progression of the cancer or tumor, including (a) inhibiting the growth, or arresting development of the cancer or tumor, (b) causing regression of the cancer or tumor, or (c) delaying, ameliorating or minimizing one or more symptoms associated with the presence of the cancer or tumor.

The term “administer” and its grammatical equivalents as used herein refer to the act of delivering, or causing to be delivered, a therapeutic or a pharmaceutical composition to the body of a subject by a method described herein or otherwise known in the art. The therapeutic can be a compound, a polypeptide, antibody, a cell, or a population of cells. Administering a therapeutic or a pharmaceutical composition includes prescribing a therapeutic or a pharmaceutical composition to be delivered into the body of a subject. Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV) , intramuscular (IM) , or intraperitoneal (IP) ; transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

The terms “effective amount, ” “therapeutically effective amount, ” and their grammatical equivalents as used herein refer to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to the subject. The therapeutically effective amount can be ascertained by measuring relevant physiological effects. The exact amount required vary from subject to subject, depending on the age, weight, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. An appropriate “effective amount” in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to a material that is suitable for drug administration to an individual along with an active agent without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.

The term “subject” as used herein refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. A subject can be a human. A subject can have a particular disease or condition.

The term “autologous” as used herein refers to any material derived from the same individual to which it is later to be re-introduced into the individual.

The term “allogeneic” as used herein refers to a graft derived from a different animal of the same species.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and/or SEQ ID NOS. It is understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to GenBank (ncbi. nlm. nih. gov/genbank/) and EMBL (embl. org/) .

5.2 Anti-CD123 antibodies and antigen-binding fragments

Provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) . In some embodiments, provided herein are anti-CD123 antibodies. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibodies provided herein can be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

In some embodiments, provided herein are antigen-binding fragments of an anti-CD123 antibody. In some embodiments, antigen-binding fragments provided herein can be a single domain antibody (sdAb) , a heavy chain antibody (HCAb) , a Fab, a Fab’, a F (ab’) ₂, a Fv, a single-chain variable fragment (scFv) , or a (scFv) ₂. In some embodiments, the antigen-binding fragment of an anti-CD123 antibody is a single domain antibody (sdAb) . In some embodiments, the antigen-binding fragment of an anti-CD123 antibody is a heavy chain antibody (HCAb) . In some embodiments, the antigen-binding fragment of an anti-CD123 antibody is a Fab. In some embodiments, the antigen-binding fragment of an anti-CD123 antibody is a Fab’. In some embodiments, the antigen-binding fragment of an anti-CD123 antibody is a F (ab’) ₂. In some embodiments, the antigen-binding fragment of an anti-CD123 antibody is a Fv. In some embodiments, the antigen-binding fragment of an anti-CD123 antibody is a scFv. In some embodiments, the antigen-binding fragment of an anti-CD123 antibody is a disulfide-linked scFv [ (scFv) ₂] . In some embodiments, the antigen-binding fragment of an anti-CD123 antibody is a diabody (dAb) .

In some embodiments, the anti-CD123 antibodies or antigen-binding fragments provided herein comprise recombinant antibodies or antigen-binding fragments. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments provided herein comprise monoclonal antibodies or antigen-binding fragments. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments provided herein comprise polyclonal antibodies or antigen-binding fragments. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments provided herein comprise camelid (e.g., camels, dromedary and llamas) antibodies or antigen-binding fragments. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments provided herein comprise chimeric antibodies or antigen-binding fragments. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments provided herein comprise humanized antibodies or antigen-binding fragments. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments provided herein comprise human antibodies or antigen-binding fragments. In some embodiments, provided herein are anti-CD123 human scFvs.

In some embodiments, the anti-CD123 antibodies or antigen-binding fragments provided herein are isolated. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments provided herein are substantially pure.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment provided herein comprises a multispecific antibody or antigen-binding fragment. In some embodiments, the anti-CD123 antibody or antigen-binding fragment provided herein comprises a bispecific antibody or antigen-binding fragment. In some embodiments, provided herein is a Bi-specific T-cell engager (BiTE) . BiTEs are bispecific antibodies that bind to a T cell antigen (e.g., CD3) and a tumor antigen. BiTEs have been shown to induce directed lysis of target tumor cells and thus provide great potential therapies for cancers and other disorders. In some embodiments, provided herein are BiTEs that specifically bind CD3 and CD123. In some embodiments, the BiTEs comprises an anti-CD123 antibody or antigen-binding fragment provided herein. In some embodiments, the BiTEs comprises an anti-CD123 scFv provided herein.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment provided herein comprises a monovalent antigen-binding site. In some embodiments, an anti-CD123 antibody or antigen-binding fragment comprises a monospecific binding site. In some embodiments, an anti-CD123 antibody or antigen-binding fragment comprises a bivalent binding site.

In some embodiments, an anti-CD123 antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment. Monoclonal antibodies can be prepared by any method known to those of skill in the art. One exemplary approach is screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228: 1315-1317; and WO 92/18619. In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries expressing variable regions or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art.

In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to one of skill in the art. For example, using a hybridoma method, a mouse, rat, rabbit, hamster, or other appropriate host animal, is immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a human protein or a fragment thereof.

Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. The hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas that produce monoclonal antibodies directed to a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore) , and radioimmunoassay) . Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution or other techniques. The hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal. The monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.

In some embodiments, monoclonal antibodies are made using recombinant DNA techniques as known to one skilled in the art. For example, the polynucleotides encoding an antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

In some embodiments, a monoclonal antibody is modified by using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light chain and heavy chain of a mouse monoclonal antibody are replaced with the constant regions of a human antibody to generate a chimeric antibody. In some embodiments, the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region (s) is used to optimize specificity and/or affinity of a monoclonal antibody.

In some embodiments, an anti-CD123 antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment. Various methods for generating humanized antibodies are known in the art. Methods are known in the art for achieving high affinity binding with humanized antibodies. A non-limiting example of such a method is hypermutation of the variable region and selection of the cells expressing such high affinity antibodies (affinity maturation) . In addition to the use of display libraries, the specified antigen (e.g., recombinant CD123 or an epitope thereof) can be used to immunize a non-human animal, e.g., a rodent. In certain embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be generated and isolated using methods known in the art and/or disclosed herein. In some embodiments, a mouse can be immunized with an antigen (e.g., recombinant CD123 or an epitope thereof) .

In some embodiments, an anti-CD123 antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from an immunized individual. In any case, cells that produce an antibody directed against a target antigen can be generated and isolated. In some embodiments, a human antibody is selected from a phage library, where that phage library expresses human antibodies. Alternatively, phage display technology may be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable region gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are well-known in the art. Once antibodies are identified, affinity maturation strategies known in the art, including but not limited to, chain shuffling and site-directed mutagenesis, may be employed to generate higher affinity human antibodies. In some embodiments, human antibodies are produced in transgenic mice that contain human immunoglobulin loci. Upon immunization these mice are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

The specific CDR sequences defined herein are generally based on a combination of Kabat and Chothia definitions. However, it is understood that reference to a heavy chain CDR or CDRs and/or a light chain CDR or CDRs of a specific antibody will encompass all CDR definitions as known to those of skill in the art.

Anti-CD123 antibodies or antigen-binding fragments provided herein include the followings clones: C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34 and C35. The sequence features are described below.

In some embodiments, anti-CD123 antibodies or antigen-binding fragments provided herein comprise one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein. In some embodiments, anti-CD123 antibodies or antigen-binding fragments provided herein comprise a VL comprising one, two, and/or three, VL CDRs from Table 1. In some embodiments, anti-CD123 antibodies or antigen-binding fragments provided herein comprise a VH comprising one, two, and/or three VH CDRs from Table 2. In some embodiments, anti-CD123 antibodies or antigen-binding fragments provided herein comprise one, two, and/or three VL CDRs from Table 1 and one, two, and/or three VH CDRs from Table 2.

Table 1 Amino acid sequences of light chain variable region CDRs (VL CDRs) of anti-CD123 Abs

Table 2 Amino acid sequences of heavy chain variable region CDRs (VH CDRs) of anti-CD123 Abs

Table 3 Consensus Amino acid sequences of VL CDRs and VH CDRs of anti-CD123 Abs

In some embodiments, an anti-CD123 antibody or antigen-binding fragment thereof comprises a humanized antibody or antigen-binding fragment. In some embodiments, an anti-CD123 antibody or antigen-binding fragment thereof comprises a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3 from an antibody or antigen-binding fragment described herein. In some embodiments, an anti-CD123 antibody or antigen-binding fragment thereof comprises a variant of an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises one to 30 amino acid substitutions, additions, and/or deletions in the anti-CD123 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises one to 25 amino acid substitutions, additions, and/or deletions in the anti-CD123 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises one to 20 substitutions, additions, and/or deletions in the anti-CD123 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises one to 15 substitutions, additions, and/or deletions in the anti-CD123 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises one to 10 substitutions, additions, and/or deletions in the anti-CD123 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises one to five conservative amino acid substitutions, additions, and/or deletions in the anti-CD123 antibody or antigen-binding fragment. In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises one to three amino acid substitutions, additions, and/or deletions in the anti-CD123 antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions, additions, and/or deletions are conservative amino acid substitutions. In some embodiments, the conservative amino acid substitution (s) is in a CDR of the antibody or antigen-binding fragment. In some embodiments, the conservative amino acid substitution (s) is not in a CDR of the antibody or antigen-binding fragment. In some embodiments, the conservative amino acid substitution (s) is in a framework region of the antibody or antigen-binding fragment.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) , comprising a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-29; (2) a light chain CDR2 (VL CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-56; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57-88; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, the variant has about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) , comprising a VL comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-29; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-56; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57-88; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VH CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-109; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-133; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134-163; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-109; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-133; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134-163; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) , comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-29; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-56; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57-88; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-109; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-133; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134-163; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, wherein the VL comprises VL CDR1, CDR2 and CDR3 having the amino acid sequences of (1) SEQ ID NOs: 3, 33, and 61, respectively; (2) SEQ ID NOs: 17, 45, and 75, respectively; (3) SEQ ID NOs: 7, 37, and 66, respectively; (4) SEQ ID NOs: 18, 32, and 76, respectively; (5) SEQ ID NOs: 23, 50 and 82, respectively; (6) SEQ ID NOs: 24, 51, and 83, respectively; (7) SEQ ID NOs: 8, 38, and 67, respectively; (8) SEQ ID NOs: 4, 34, and 62, respectively; (9) SEQ ID NOs: 25, 52, and 84, respectively; (10) SEQ ID NOs: 26, 53, and 85, respectively; (11) SEQ ID NOs: 2, 32, and 60, respectively; (12) SEQ ID NOs: 17, 46, and 77, respectively; (13) SEQ ID NOs: 27, 54 and 86, respectively; (14) SEQ ID NOs: 9, 39, and 68, respectively; (15) SEQ ID NOs: 10, 40, and 69, respectively; (16) SEQ ID NOs: 22, 49, and 81, respectively; (17) SEQ ID NOs: 5, 35, and 63, respectively; (18) SEQ ID NOs: 1, 30 and 64, respectively; (19) SEQ ID NOs: 11, 41, and 70, respectively; (20) SEQ ID NOs: 1, 31, and 59, respectively; (21) SEQ ID NOs: 19, 47, and 78, respectively; (22) SEQ ID NOs: 28, 55, and 87, respectively; (23) SEQ ID NOs: 12, 39, and 71, respectively; (24) SEQ ID NOs: 13, 42, and 72, respectively; (25) SEQ ID NOs: 1, 30, and 57, respectively; (26) SEQ ID NOs: 20, 48 and 79, respectively; (27) SEQ ID NOs: 14, 43, and 67, respectively; (28) SEQ ID NOs: 1, 30, and 58, respectively; (29) SEQ ID NOs: 2, 32, and 60, respectively; (30) SEQ ID NOs: 15, 44, and 73, respectively; (31) SEQ ID NOs: 29, 56 and 88, respectively; (32) SEQ ID NOs: 22, 49, and 81, respectively; (33) SEQ ID NOs: 16, 42, and 74, respectively; (34) SEQ ID NOs: 6, 36, and 65, respectively; or (35) SEQ ID NOs: 21, 45, and 80, respectively; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VL CDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, wherein the VH comprises VH CDR1, CDR2 and CDR3 having the amino acid sequences of (1) SEQ ID NOs: 91, 112, and 136, respectively; (2) SEQ ID NOs: 89, 122, and 149, respectively; (3) SEQ ID NOs: 95, 116, and 140, respectively; (4) SEQ ID NOs: 100, 123, and 150, respectively; (5) SEQ ID NOs: 99, 129 and 157, respectively; (6) SEQ ID NOs: 106, 128, and 158, respectively; (7) SEQ ID NOs: 96, 117, and 141, respectively; (8) SEQ ID NOs: 89, 110, and 134, respectively; (9) SEQ ID NOs: 107, 130, and 159, respectively; (10) SEQ ID NOs: 95, 116, and 160, respectively; (11) SEQ ID NOs: 90, 111, and 135, respectively; (12) SEQ ID NOs: 101, 124, and 151, respectively; (13) SEQ ID NOs: 105, 131, and 161, respectively; (14) SEQ ID NOs: 97, 118, and 142, respectively; (15) SEQ ID NOs: 98, 119, and 143, respectively; (16) SEQ ID NOs: 105, 128, and 156, respectively; (17) SEQ ID NOs: 92, 113, and 137, respectively; (18) SEQ ID NOs: 93, 114 and 138, respectively; (19) SEQ ID NOs: 91, 120, and 144, respectively; (20) SEQ ID NOs: 89, 110, and 134, respectively; (21) SEQ ID NOs: 102, 125, and 152, respectively; (22) SEQ ID NOs: 108, 132, and 162, respectively; (23) SEQ ID NOs: 96, 117, and 145, respectively; (24) SEQ ID NOs: 95, 116, and 146, respectively; (25) SEQ ID NOs: 89, 110, and 134, respectively; (26) SEQ ID NOs: 103, 126 and 153, respectively; (27) SEQ ID NOs: 96, 117, and 141, respectively; (28) SEQ ID NOs: 89, 110, and 134, respectively; (29) SEQ ID NOs: 90, 111, and 135, respectively; (30) SEQ ID NOs: 99, 121, and 147, respectively; (31) SEQ ID NOs: 109, 133 and 163, respectively; (32) SEQ ID NOs: 104, 127, and 155, respectively; (33) SEQ ID NOs: 95, 116, and 148, respectively; (34) SEQ ID NOs: 94, 115, and 139, respectively; or (35) SEQ ID NOs: 89, 122, and 154, respectively; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the variant has up about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL and a VH. In some embodiments, the VL and VH are connected by a linker. In some embodiments, the VL and VH are connected by a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 484) . In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 485) . In some embodiments, the linker has the amino acid sequence of (PA) nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 486) . In some embodiments, the linker has the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 320) .

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL and a VH, wherein (a) the VL comprises VL CDR1, CDR2 and CDR3 having the amino acid sequences of (1) SEQ ID NOs: 3, 33, and 61, respectively; (2) SEQ ID NOs: 17, 45, and 75, respectively; (3) SEQ ID NOs: 7, 37, and 66, respectively; (4) SEQ ID NOs: 18, 32, and 76, respectively; (5) SEQ ID NOs: 23, 50 and 82, respectively; (6) SEQ ID NOs: 24, 51, and 83, respectively; (7) SEQ ID NOs: 8, 38, and 67, respectively; (8) SEQ ID NOs: 4, 34, and 62, respectively; (9) SEQ ID NOs: 25, 52, and 84, respectively; (10) SEQ ID NOs: 26, 53, and 85, respectively; (11) SEQ ID NOs: 2, 32, and 60, respectively; (12) SEQ ID NOs: 17, 46, and 77, respectively; (13) SEQ ID NOs: 27, 54 and 86, respectively; (14) SEQ ID NOs: 9, 39, and 68, respectively; (15) SEQ ID NOs: 10, 40, and 69, respectively; (16) SEQ ID NOs: 22, 49, and 81, respectively; (17) SEQ ID NOs: 5, 35, and 63, respectively; (18) SEQ ID NOs: 1, 30 and 64, respectively; (19) SEQ ID NOs: 11, 41, and 70, respectively; (20) SEQ ID NOs: 1, 31, and 59, respectively; (21) SEQ ID NOs: 19, 47, and 78, respectively; (22) SEQ ID NOs: 28, 55, and 87, respectively; (23) SEQ ID NOs: 12, 39, and 71, respectively; (24) SEQ ID NOs: 13, 42, and 72, respectively; (25) SEQ ID NOs: 1, 30, and 57, respectively; (26) SEQ ID NOs: 20, 48 and 79, respectively; (27) SEQ ID NOs: 14, 43, and 67, respectively; (28) SEQ ID NOs: 1, 30, and 58, respectively; (29) SEQ ID NOs: 2, 32, and 60, respectively; (30) SEQ ID NOs: 15, 44, and 73, respectively; (31) SEQ ID NOs: 29, 56 and 88, respectively; (32) SEQ ID NOs: 22, 49, and 81, respectively; (33) SEQ ID NOs: 16, 42, and 74, respectively; (34) SEQ ID NOs: 6, 36, and 65, respectively; or (35) SEQ ID NOs: 21, 45, and 80, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and (b) the VH comprises VH CDR1, CDR2 and CDR3 having the amino acid sequences of (1) SEQ ID NOs: 91, 112, and 136, respectively; (2) SEQ ID NOs: 89, 122, and 149, respectively; (3) SEQ ID NOs: 95, 116, and 140, respectively; (4) SEQ ID NOs: 100, 123, and 150, respectively; (5) SEQ ID NOs: 99, 129 and 157, respectively; (6) SEQ ID NOs: 106, 128, and 158, respectively; (7) SEQ ID NOs: 96, 117, and 141, respectively; (8) SEQ ID NOs: 89, 110, and 134, respectively; (9) SEQ ID NOs: 107, 130, and 159, respectively; (10) SEQ ID NOs: 95, 116, and 160, respectively; (11) SEQ ID NOs: 90, 111, and 135, respectively; (12) SEQ ID NOs: 101, 124, and 151, respectively; (13) SEQ ID NOs: 105, 131, and 161, respectively; (14) SEQ ID NOs: 97, 118, and 142, respectively; (15) SEQ ID NOs: 98, 119, and 143, respectively; (16) SEQ ID NOs: 105, 128, and 156, respectively; (17) SEQ ID NOs: 92, 113, and 137, respectively; (18) SEQ ID NOs: 93, 114 and 138, respectively; (19) SEQ ID NOs: 91, 120, and 144, respectively; (20) SEQ ID NOs: 89, 110, and 134, respectively; (21) SEQ ID NOs: 102, 125, and 152, respectively; (22) SEQ ID NOs: 108, 132, and 162, respectively; (23) SEQ ID NOs: 96, 117, and 145, respectively; (24) SEQ ID NOs: 95, 116, and 146, respectively; (25) SEQ ID NOs: 89, 110, and 134, respectively; (26) SEQ ID NOs: 103, 126 and 153, respectively; (27) SEQ ID NOs: 96, 117, and 141, respectively; (28) SEQ ID NOs: 89, 110, and 134, respectively; (29) SEQ ID NOs: 90, 111, and 135, respectively; (30) SEQ ID NOs: 99, 121, and 147, respectively; (31) SEQ ID NOs: 109, 133 and 163, respectively; (32) SEQ ID NOs: 104, 127, and 155, respectively; (33) SEQ ID NOs: 95, 116, and 148, respectively; (34) SEQ ID NOs: 94, 115, and 139, respectively; or (35) SEQ ID NOs: 89, 122, and 154, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences of (1) SEQ ID NOs: 3, 33, 61, 91, 112, and 136, respectively; (2) SEQ ID NOs: 17, 45, 75, 89, 122, and 149, respectively; (3) SEQ ID NOs: 7, 37, 66, 95, 116, and 140, respectively; (4) SEQ ID NOs: 18, 32, 76, 100, 123, and 150, respectively; (5) SEQ ID NOs: 23, 50, 82, 99, 129 and 157, respectively; (6) SEQ ID NOs: 24, 51, 83, 106, 128, and 158, respectively; (7) SEQ ID NOs: 8, 38, 67, 96, 117, and 141, respectively; (8) SEQ ID NOs: 4, 34, 62, 89, 110, and 134, respectively; (9) SEQ ID NOs: 25, 52, 84, 107, 130, and 159, respectively; (10) SEQ ID NOs: 26, 53, 85, 95, 116, and 160, respectively; (11) SEQ ID NOs: 2, 32, 60, 90, 111, and 135, respectively; (12) SEQ ID NOs: 17, 46, 77, 101, 124, and 151, respectively; (13) SEQ ID NOs: 27, 54, 86, 105, 131, and 161, respectively; (14) SEQ ID NOs: 9, 39, 68, 97, 118, and 142, respectively; (15) SEQ ID NOs: 10, 40, 69, 98, 119, and 143, respectively; (16) SEQ ID NOs: 22, 49, 81, 105, 128, and 156, respectively; (17) SEQ ID NOs: 5, 35, 63, 92, 113, and 137, respectively; (18) SEQ ID NOs: 1, 30, 64, 93, 114 and 138, respectively; (19) SEQ ID NOs: 11, 41, 70, 91, 120, and 144, respectively; (20) SEQ ID NOs: 1, 31, 59, 89, 110, and 134, respectively; (21) SEQ ID NOs: 19, 47, 78, 102, 125, and 152 respectively; (22) SEQ ID NOs: 28, 55, 87, 108, 132, and 162, respectively; (23) SEQ ID NOs: 12, 39, 71, 96, 117, and 145, respectively; (24) SEQ ID NOs: 13, 42, 72, 95, 116, and 146, respectively; (25) SEQ ID NOs: 1, 30, 57, 89, 110, and 134, respectively; (26) SEQ ID NOs: 20, 48, 79, 103, 126 and 153, respectively; (27) SEQ ID NOs: 14, 43, 67, 96, 117, and 141, respectively; (28) SEQ ID NOs: 1, 30, 58, 89, 110, and 134, respectively; (29) SEQ ID NOs: 2, 32, 60, 90, 111, and 135, respectively; (30) SEQ ID NOs: 15, 44, 73, 99, 121, and 147, respectively; (31) SEQ ID NOs: 29, 56, 88, 109, 133 and 163, respectively; (32) SEQ ID NOs: 22, 49, 81, 104, 127, and 155, respectively; (33) SEQ ID NOs: 16, 42, 74, 95, 116, and 148, respectively; (34) SEQ ID NOs: 6, 36, 65, 94, 115, and 139, respectively; or (35) SEQ ID NOs: 21, 45, 80, 89, 122, and 154, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs.

Consensus CDR sequences of the antibodies provided herein are analyzed and provided in Table 3. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) , comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 348-352; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 353-356; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 357-359; and/or (b) a VH comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 360-361; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 362-367; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 368.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences of (1) SEQ ID NOs: 349, 353, 357, 89, 110, and 134, respectively; (2) SEQ ID NOs: 348, 354, 358, (89 or 360) , 365 and (135, 149, 154, or 159) , respectively; (3) SEQ ID NOs: 348, 354, 358, 360, 365 and (135 or 159) , respectively; (4) SEQ ID NOs: 348, 45, 358, 89, 122 and (149 or 154) , respectively; (5) SEQ ID NOs: 350, 355, 359, 95, 116, and (140 or 148) , respectively; (6) SEQ ID NOs: 22, 49, 81, 360, 362, and 367, respectively; (7) SEQ ID NOs: 352, 353, 358, 361, 367, and (160 or 162) , respectively; (8) SEQ ID NOs: 350, 355, 359, (97 or 99) , (118 or 121) and (142 or 147) , respectively; (8) SEQ ID NOs: 350, 355, 359, (97 or 99) , (118 or 121) and (142 or 147) , respectively, (9) SEQ ID NOs: 351, 356, 67, 96, 117, and 141, respectively; (10) SEQ ID NOs: 350, 355, 359, (91, 95 or 96) , (116, 117 or 210) and 368, respectively; 11) SEQ ID NOs: 348, 354, (76, 77 or 358) , (101, 103 or 360) , (123, 124, 125, or 126) and (150, 151, 152, or 153) , respectively; or 12) SEQ ID NOs: 349, 353, (61, 62, 63, 64, or 65) , (89, 91, 92, 93, or 94) , (110, 112, 113, 114 or 115) and (134, 136, 137, 138, or 139) , respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 17, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 45, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 75. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 17, 45, and 75, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 89, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 122, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 149. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 89, 122, and 149, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 17, 45, and 75, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 89, 122, and 149, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 18, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 32, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 76. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 18, 32, and 76, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 100, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 123, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 150. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 100, 123, and 150, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 18, 32, and 76, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 100, 123, and 150, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 23, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 50, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 82. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 23, 50 and 82, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 99, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 129, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 157. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 99, 129 and 157, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 23, 50 and 82, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 99, 129 and 157, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 24, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 51, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 83. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 24, 51, and 83, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 106, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 128, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 158. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 106, 128, and 158, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 24, 51, and 83, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 106, 128, and 158, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 8, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 38, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 67. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 8, 38, and 67, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 96, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 117, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 141. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 8, 38, and 67, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 25, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 52, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 84. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 25, 52, and 84, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 107, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 130, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 159. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 107, 130, and 159, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 25, 52, and 84, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 107, 130, and 159, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 2, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 32, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 60. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 90, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 111, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 135. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 9, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 39, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 68. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 9, 39, and 68, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 97, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 118, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 142. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 97, 118, and 142, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 9, 39, and 68, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 97, 118, and 142, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 40, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 69. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 10, 40, and 69, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 98, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 119, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 143. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 98, 119, and 143, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 10, 40, and 69, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 98, 119, and 143, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 22, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 49, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 81. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 105, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 128, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 156. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 105, 128, and 156, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 105, 128, and 156, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 5, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 35, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 63. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 5, 35, and 63, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 92, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 113, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 137. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 92, 113, and 137, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 5, 35, and 63, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 92, 113, and 137, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 30, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 64. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 30 and 64, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 93, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 114, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 138. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 93, 114 and 138, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 30 and 64, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 93, 114 and 138, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 30, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 57. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 30, and 57, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 89, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 110, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 134. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 30, and 57, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 20, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 48, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 79. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 20, 48 and 79, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 103, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 126, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 153. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 103, 126 and 153, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 20, 48 and 79, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 103, 126 and 153, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 1, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 30, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 58. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 30, and 58, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 89, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 110, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 134. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 1, 30, and 58, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 15, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 44, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 73. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 15, 44, and 73, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 99, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 121, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 147. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 99, 121, and 147, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 15, 44, and 73, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 99, 121, and 147, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VL, comprising (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 45, or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 80. The VL can have VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 21, 45, and 80, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) having a VH, comprising (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 89, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 122, or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 154. The VH can have VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 89, 122, and 154, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL that comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 21, 45, and 80, respectively; and (b) a VH that comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 89, 122, and 154, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233.

Table 4 Amino acid sequences of light chain variable regions (VLs) and heavy chain variable region (VHs) of anti-CD123 antibodies

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198; and (b) a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 165. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 165. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 165. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 165. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 165. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 165.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 200. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 200. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 200. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 200. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 200. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 200.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 167. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 167. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 167. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 167. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 167. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 167.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 202. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 202. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 202. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 202. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 202. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 202.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 168. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 168. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 168. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 168. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 168. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 168.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 203. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 203. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 203. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 203. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 203. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 203.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 169. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 169. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 169. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 169. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 169. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 169.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 204. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 204. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 204. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 204. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 204. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 204.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 170. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 170. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 170. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 170. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 170. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 170.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 205. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 205. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 205. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 205. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 205. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 205.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 172. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 172. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 172. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 172. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 172. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 172.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 207. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 207. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 207. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 207. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 207. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 207.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 174. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 174. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 174. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 174. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 174. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 174.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 209. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 209. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 209. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 209. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 209. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 209.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 177. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 177. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 177. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 177. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 177. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 177.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 212. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 212. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 212. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 212. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 212. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 212.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 178. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 178. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 178. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 178. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 178. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 178.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 213. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 213. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 213. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 213. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 213. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 213.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 179. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 179. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 179. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 179. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 179. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 179.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 214. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 214. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 214. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 214. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 214. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 214.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 180. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 180. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 180. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 180. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 180. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 180.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 215. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 215. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 215. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 215. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 215. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 215.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 181. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 181. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 181. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 181. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 181. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 181.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 216. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 216. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 216. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 216. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 216. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 216.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 188. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 188. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 188. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 188. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 188. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 188.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 223. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 223. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 223. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 223. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 223. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 223.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 189. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 189. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 189. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 189. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 189. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 189.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 224. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 224. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 224. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 224. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 224. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 224.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 191. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 191. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 191. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 191. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 191. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 191.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 226. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 226. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 226. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 226. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 226. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 226.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 192. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 192. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 192. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 192. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 192. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 192.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 227. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 227. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 227. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 227. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 227. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 227.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 193. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 193. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 193. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 193. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 193. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 193.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 228. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 228. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 228. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 228. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 228. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 228.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 198. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 85%sequence identity to SEQ ID NO: 198. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 90%sequence identity to SEQ ID NO: 198. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 95%sequence identity to SEQ ID NO: 198. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VL having at least 98%sequence identity to SEQ ID NO: 198. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL having the amino acid sequence of SEQ ID NO: 198.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 233. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 85%sequence identity to SEQ ID NO: 233. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 90%sequence identity to SEQ ID NO: 233. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 95%sequence identity to SEQ ID NO: 233. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof has a VH having at least 98%sequence identity to SEQ ID NO: 233. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH having the amino acid sequence of SEQ ID NO: 233.

The anti-CD123 antibodies or antigen-binding fragments thereof can comprise a combination of any VL disclosed herein and any VH disclosed herein. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL and a VH, wherein the VL has the amino acid sequence of SEQ ID NO: 164, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 165, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 166, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 167, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 168, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 169, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 170, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 171, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 172, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 173, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 174, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 175, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 176, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 177, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 178, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 179, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 180, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 181, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 182, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 183, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 184, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 185, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 186, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 187, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 188, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 189, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 190, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 191, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 192, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 193, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 194, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 195, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 196, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 197, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the VL has the amino acid sequence of SEQ ID NO: 198, and the VH has an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL and a VH, wherein the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 199. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 200. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 201. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 202. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 203. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 204. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 205. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 206. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 207. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 208. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 209. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 210. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 211. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 212. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 213. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 214. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 215. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 216. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 217. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 218. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 219. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 220. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 221. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 222. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 223. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 224. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 225. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 226. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 227. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 229. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 230. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 231. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 232. In some embodiments, the VL has an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198, and the VH has the amino acid sequence of SEQ ID NO: 233.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NOs: 164 and 199, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 165 and 200, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 166 and 201, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 167 and 202, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 168 and 203, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 169 and 204, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 170 and 205, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 171 and 206, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 172 and 207, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 173 and 208, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 174 and 209, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 175 and 210, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 176 and 211, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 177 and 212, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 178 and 213, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 179 and 214, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 180 and 215, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 181 and 216, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 182 and 217, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 183 and 218, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 184 and 219, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 185 and 220, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 186 and 221, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 187 and 222, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 188 and 223, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 189 and 224, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 190 and 225, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 191 and 226, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 192 and 227, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 193 and 228, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 194 and 229, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 195 and 230, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 196 and 231, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 197 and 232, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 198 and 233, respectively.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising (a) a VL comprising

VL CDRs

1, 2, and 3 from a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198; and/or (b) a VH comprising

VH CDRs

1, 2, and 3 from a VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL, wherein the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 164. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 165. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 166. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 167. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 168. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 169. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 170. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 171. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 172. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 173. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 174. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 175. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 176. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 177. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 178. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 179. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 180. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 181. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 182. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 183. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 184. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 185. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 186. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 187. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 188. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 189. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 190. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 191. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 192. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 193. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 194. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 195. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 196. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 197. In some embodiments, the VL comprises

VL CDRs

1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 198.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VH, wherein the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 199. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 200. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 201. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 202. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 203. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 204. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 205. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 206. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 207. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 208. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 209. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 210. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 211. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 212. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 213. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 214. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 215. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 216. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 217. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 218. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 219. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 220. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 221. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 222. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 223. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 224. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 225. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 226. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 227. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 229. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 230. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 231. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 232. In some embodiments, the VH comprises

VH CDRs

1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 233.

In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind CD123 (e.g., human CD123) comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 164, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 199. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 165, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 200. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 166, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 201. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 167, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 202. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 168, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 203. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 169, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 204. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 170, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 205. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 171, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 206. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 172, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 207. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 173, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 208. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 174, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 209. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 175, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 210. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 176, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 211. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 177, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 212. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 178, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 213. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 179, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 214. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 180, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 215. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 181, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 216. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 182, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 217. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 183, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 218. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 184, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 219. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 185, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 220. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 186, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 221. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 187, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 222. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 188, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 223. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 189, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 224. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 190, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 225. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 191, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 226. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 192, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 227. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 193, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 194, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 229. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 195, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 230. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 196, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 231. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 197, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 232. In some embodiments, the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 198, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 233.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C2 (SEQ ID NO: 446) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 446. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C2 (SEQ ID NO: 165) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C2 (SEQ ID NO: 200) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C2. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C2 (SEQ ID NO: 165) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C2 (SEQ ID NO: 200) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C2, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C2. The C2 variant can have a VL that is a variant of the VL of C2 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 165. The C2 variant can have a VH that is a variant of the VH of C2 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 200. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C2 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C2 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C4 (SEQ ID NO: 448) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 448. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C4 (SEQ ID NO: 167) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C4 (SEQ ID NO: 202) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C4. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C4 (SEQ ID NO: 167) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C4 (SEQ ID NO: 202) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C4, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C4. The C4 variant can have a VL that is a variant of the VL of C4 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 167. The C4 variant can have a VH that is a variant of the VH of C4 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 202. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C4 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C4 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C5 (SEQ ID NO: 449) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 449. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C5 (SEQ ID NO: 168) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C5 (SEQ ID NO: 203) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C5. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C5 (SEQ ID NO: 168) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C5 (SEQ ID NO: 203) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C5, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C5. The C5 variant can have a VL that is a variant of the VL of C5 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 168. The C5 variant can have a VH that is a variant of the VH of C5 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 203. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C5 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C5 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C6 (SEQ ID NO: 450) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 450. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C6 (SEQ ID NO: 169) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C6 (SEQ ID NO: 204) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C6. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C6 (SEQ ID NO: 169) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C6 (SEQ ID NO: 204) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C6, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C6. The C6 variant can have a VL that is a variant of the VL of C6 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 169. The C6 variant can have a VH that is a variant of the VH of C6 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 204. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C6 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C6 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C7 (SEQ ID NO: 451) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: . 451. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C7 (SEQ ID NO: 170) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C7 (SEQ ID NO: 205) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C7. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C7 (SEQ ID NO: 170) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C7 (SEQ ID NO: 205) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C7, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C7. The C7 variant can have a VL that is a variant of the VL of C7 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 170. The C7 variant can have a VH that is a variant of the VH of C7 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 205. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C7 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C7 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C9 (SEQ ID NO: 453) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: . 453. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C9 (SEQ ID NO: 172) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C9 (SEQ ID NO: 207) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C9. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C9 (SEQ ID NO: 172) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C9 (SEQ ID NO: 207) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C9, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C9. The C9 variant can have a VL that is a variant of the VL of C9 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 172. The C9 variant can have a VH that is a variant of the VH of C9 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 207. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C9 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C9 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C11 (SEQ ID NO: 455) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 455. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C11 (SEQ ID NO: 174) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C11 (SEQ ID NO: 209) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C11. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C11 (SEQ ID NO: 174) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C11 (SEQ ID NO: 209) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C11, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C11. The C11 variant can have a VL that is a variant of the VL of C11 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 174. The C11 variant can have a VH that is a variant of the VH of C11 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 209. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C11 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C11 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C14 (SEQ ID NO: 458) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 458. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C14 (SEQ ID NO: 177) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C14 (SEQ ID NO: 212) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C14. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C14 (SEQ ID NO: 177) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C14 (SEQ ID NO: 212) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C14, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C14. The C14 variant can have a VL that is a variant of the VL of C14 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 177. The C14 variant can have a VH that is a variant of the VH of C14 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 212. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C14 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C14 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C15 (SEQ ID NO: 459) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 459. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C15 (SEQ ID NO: 178) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C15 (SEQ ID NO: 213) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C15. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C15 (SEQ ID NO: 178) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C15 (SEQ ID NO: 213) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C15, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C15. The C15 variant can have a VL that is a variant of the VL of C15 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 178. The C15 variant can have a VH that is a variant of the VH of C15 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 213. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C15 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C15 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C16 (SEQ ID NO: 460) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 460. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C16 (SEQ ID NO: 179) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C16 (SEQ ID NO: 214) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C16. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C16 (SEQ ID NO: 179) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C16 (SEQ ID NO: 214) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C16, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C16. The C16 variant can have a VL that is a variant of the VL of C16 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 179. The C16 variant can have a VH that is a variant of the VH of C16 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 214. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C16 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C16 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C17 (SEQ ID NO: 461) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 461. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C17 (SEQ ID NO: 180) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C17 (SEQ ID NO: 215) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C17. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C17 (SEQ ID NO: 180) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C17 (SEQ ID NO: 215) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C17, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C17. The C17 variant can have a VL that is a variant of the VL of C17 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 180. The C17 variant can have a VH that is a variant of the VH of C17 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 215. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C17 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C17 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C18 (SEQ ID NO: 462) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 462. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C18 (SEQ ID NO: 181) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C18 (SEQ ID NO: 216) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C18. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C18 (SEQ ID NO: 181) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C18 (SEQ ID NO: 216) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C18, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C18. The C18 variant can have a VL that is a variant of the VL of C18 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 181. The C18 variant can have a VH that is a variant of the VH of C18 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 216. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C18 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C18 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C25 (SEQ ID NO: 469) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 469. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C25 (SEQ ID NO: 188) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C25 (SEQ ID NO: 223) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C25. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C25 (SEQ ID NO: 188) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C25 (SEQ ID NO: 223) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C25, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C25. The C25 variant can have a VL that is a variant of the VL of C25 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 188. The C25 variant can have a VH that is a variant of the VH of C25 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 223. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C25 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C25 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C26 (SEQ ID NO: 470) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 470. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C26 (SEQ ID NO: 189) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C26 (SEQ ID NO: 224) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C26. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C26 (SEQ ID NO: 189) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C26 (SEQ ID NO: 224) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C26, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C26. The C26 variant can have a VL that is a variant of the VL of C26 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 189. The C26 variant can have a VH that is a variant of the VH of C26 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 224. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C26 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C26 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C28 (SEQ ID NO: 472) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 472. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C28 (SEQ ID NO: 191) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C28 (SEQ ID NO: 226) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C28. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C28 (SEQ ID NO: 191) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C28 (SEQ ID NO: 226) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C28, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C28. The C28 variant can have a VL that is a variant of the VL of C28 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 191. The C28 variant can have a VH that is a variant of the VH of C28 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 226. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C28 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C28 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C29 (SEQ ID NO: 473) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 473. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C29 (SEQ ID NO: 192) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C29 (SEQ ID NO: 227) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C29. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C29 (SEQ ID NO: 192) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C29 (SEQ ID NO: 227) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C29, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C29. The C29 variant can have a VL that is a variant of the VL of C29 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 192. The C29 variant can have a VH that is a variant of the VH of C29 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 227. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C29 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C29 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C30 (SEQ ID NO: 474) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 474. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C30 (SEQ ID NO: 193) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C30 (SEQ ID NO: 228) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C30. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C30 (SEQ ID NO: 193) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C30 (SEQ ID NO: 228) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C30, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C30. The C30 variant can have a VL that is a variant of the VL of C30 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 193. The C30 variant can have a VH that is a variant of the VH of C30 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 228. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C30 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C30 has up to 3 conservative amino acid substitutions.

In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is the scFv designated as C35 (SEQ ID NO: 475) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%identical to SEQ ID NO: 475. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL from C35 (SEQ ID NO: 198) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH from C35 (SEQ ID NO: 233) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have both a VL and a VH from C35. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VL that comprises

VL CDRs

1, 2, and 3 from the VL from C35 (SEQ ID NO: 198) . In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein has a VH that comprises

VH CDRs

1, 2, and 3 from the VH from C35 (SEQ ID NO: 233) . The anti-CD123 antibody or antigen-binding fragment thereof provided herein can have a VL comprising

VL CDRs

1, 2, and 3 and a VH comprising

VH CDRs

1, 2, and 3 from the VL and VH of C35, respectively. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof provided herein is a variant of C35. The C35 variant can have a VL that is a variant of the VL of C35 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 198. The C35 variant can have a VH that is a variant of the VH of C35 having up to about 5 amino acid substitutions, additions, and/or deletions in SEQ ID NO: 233. The amino acid substitutions, additions, and/or deletions can be in the VH CDRs or VL CDRs. In some embodiments, the amino acid substitutions, additions, and/or deletions are not in the CDRs. In some embodiments, the variant of C35 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of C35 has up to 3 conservative amino acid substitutions.

In some embodiments, provided herein are also antibodies or antigen-binding fragments that compete with the antibody or antigen-binding fragment provided above for binding to CD123 (e.g., human CD123) . Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, can be determined using known competition experiments, e.g.,

surface plasmon resonance (SPR) analysis. In some embodiments, an anti-CD123 antibody or antigen-binding fragment competes with, and inhibits binding of another antibody or antigen-binding fragment to CD123 by at least 50%, 60%, 70%, 80%, 90%or 100%. Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: l0. H0l/pdb. prot4277 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C1 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C2 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C3 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C4 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C5 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C6 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C7 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C8 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C9 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C10 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C11 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C12 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C12 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C13 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C14 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C15 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C16 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C17 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C18 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C19 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C20 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C21 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C22 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C23 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C24 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C25 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C26 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C27 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C28 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C29 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C30 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C31 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C32 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C33 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C34 for binding to CD123. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with C35 for binding to CD123.

The present disclosure further contemplates additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate biological properties of the antibody, including but not limited to, specificity, thermostability, expression level, effector function (s) , glycosylation, immunogenicity, and/or solubility. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of an antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

Variations may be a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native antibody or polypeptide sequence. In some embodiments, amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Insertions or deletions can be in the range of about 1 to 5 amino acids. In some embodiments, the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule. In some embodiments, variations in the amino acid sequence that are biologically useful and/or relevant can be determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parent protein.

It is known in the art that the constant region (s) of an antibody mediates several effector functions and these effector functions can vary depending on the isotype of the antibody. For example, binding of the C1 component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind a cell expressing a Fc receptor (FcR) . There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors) , IgE (epsilon receptors) , IgA (alpha receptors) and IgM (mu receptors) . Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell cytotoxicity or ADCC) , release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgA antibody. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgD antibody. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgE antibody. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG antibody. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgM antibody. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG1 antibody. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG2 antibody. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG3 antibody. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG4 antibody.

In some embodiments, at least one or more of the constant regions has been modified or deleted in the anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, the antibodies comprise modifications to one or more of the three heavy chain constant regions (CH1, CH2 or CH3) and/or to the light chain constant region (CL) . In some embodiments, the heavy chain constant region of the modified antibodies comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibodies comprises more than one human constant region. In some embodiments, modifications to the constant region comprise additions, deletions, or substitutions of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or entirely deleted from the constant regions of the modified antibodies. In some embodiments, the entire CH2 domain has been removed from an antibody (ΔCH2 constructs) . In some embodiments, a deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically imparted by the absent constant region. In some embodiments, a modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In some embodiments, a modified antibody comprises a peptide spacer inserted between the hinge region and modified CH2 and/or CH3 domains.

In some embodiments, an anti-CD123 antibody or antigen-binding fragment comprises a Fc region. In some embodiments, the Fc region is fused via a hinge. The hinge can be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequences of the Fc region of human IgG1, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art. In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, the modified antibodies (e.g., modified Fc region) provide for altered effector functions that, in turn, affect the biological profile of the antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region reduces Fc receptor binding of the modified antibody as it circulates. In some embodiments, the constant region modifications reduce the immunogenicity of the antibody. In some embodiments, the constant region modifications increase the serum half-life of the antibody. In some embodiments, the constant region modifications reduce the serum half-life of the antibody. In some embodiments, the constant region modifications decrease or remove ADCC and/or complement dependent cytotoxicity (CDC) of the antibody. In some embodiments, specific amino acid substitutions in a human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduce effector functions (e.g., ADCC and CDC) in the modified antibody. In some embodiments, an antibody does not have one or more effector functions (e.g., “effectorless” antibodies) . In some embodiments, the antibody has no ADCC activity and/or no CDC activity. In some embodiments, the antibody does not bind an Fc receptor and/or complement factors. In some embodiments, the antibody has no effector function (s) . In some embodiments, the constant region modifications increase or enhance ADCC and/or CDC of the antibody. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. In some embodiments, the constant region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites. In some embodiments, an anti-CD123 antibody or antigen-binding fragment comprises a variant Fc region that is engineered with substitutions at specific amino acid positions as compared to a native Fc region. In some embodiments, an anti-CD123 antibody or antigen-binding fragment described herein comprises an IgG1 heavy chain constant region that comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E, and L358M, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, D356E, and L358M, per EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S, per EU numbering.

In some embodiments, variants can include addition of amino acid residues at the amino-and/or carboxyl-terminal end of the antibody or polypeptide. The length of additional amino acids residues can range from one residue to a hundred or more residues. In some embodiments, a variant comprises an N-terminal methionyl residue. In some embodiments, the variant comprises an additional polypeptide/protein (e.g., Fc region) to create a fusion protein. In some embodiments, a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g., a fluorescent tag or an enzyme) .

The variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment disclosed herein can retain the ability to bind CD123 to a similar extent, the same extent, or to a higher extent, as the parent antibody or antigen-binding fragment. In some embodiments, the variant can be at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%or more identical in amino acid sequence to the parent antibody or antigen-binding fragment. In certain embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises the amino acid sequence of the parent anti-CD123 antibody or antigen-binding fragment with one or more conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties.

In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises the amino acid sequence of the parent antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions. In some embodiments, a variant of an anti-CD123 antibody or antigen-binding fragment comprises the amino acid sequence of the parent binding antibody or antigen-binding fragment with one or more non-conservative amino acid substitution, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant (e.g., CD123 binding) . In certain embodiments, the one or more conservative amino acid substitutions and/or the one or more non-conservative amino acid substitutions can enhance a biological activity of the variant, such that the biological activity of the functional variant is increased as compared to the parent binding moiety.

In some embodiments, the variant can have 1, 2, 3, 4, or 5 amino acid substitutions in the CDRs (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3) of the binding moiety.

In some embodiments, anti-CD123 antibodies or antigen-binding fragments described herein are chemically modified naturally or by intervention. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications can be carried out by known techniques. The anti-CD123 antibodies or antigen-binding fragments can comprise one or more analogs of an amino acid (including, for example, unnatural amino acids) , as well as other modifications known in the art.

In some embodiments, an anti-CD123 antibody or antigen-binding fragment (e.g., an antibody) binds CD123 (e.g., human CD123) with a dissociation constant (K _D) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. In some embodiments, an anti-CD123 antibody or antigen-binding fragment binds CD123 (e.g., human CD123) with a K _D of about 20 nM or less. In some embodiments, an anti-CD123 antibody or antigen-binding fragment binds CD123 (e.g., human CD123) with a K _D of about 10 nM or less. In some embodiments, an anti-CD123 antibody or antigen-binding fragment binds CD123 (e.g., human CD123) with a K _D of about 1 nM or less. In some embodiments, an anti-CD123 antibody or antigen-binding fragment binds CD123 (e.g., human CD123) with a K _D of about 0.5 nM or less. In some embodiments, an anti-CD123 antibody or antigen-binding fragment binds CD123 (e.g., human CD123) with a K _D of about 0.1 nM or less. In some embodiments, an anti-CD123 antibody or antigen-binding fragment binds CD123 (e.g., human CD123) with a K _D of about 50 pM or less. In some embodiments, an anti-CD123 antibody or antigen-binding fragment binds CD123 (e.g., human CD123) with a K _D of about 25 pM or less. In some embodiments, an anti-CD123 antibody or antigen-binding fragment binds CD123 (e.g., human CD123) with a K _D of about 10 pM or less. In some embodiments, an anti-CD123 antibody or antigen-binding fragment binds CD123 (e.g., human CD123) with a K _D of about 1 pM or less. In some embodiments, the dissociation constant of the binding agent (e.g., an antibody) for CD123 is the dissociation constant determined using a CD123 protein immobilized on a Biacore chip and the binding agent flowed over the chip. In some embodiments, the dissociation constant of the binding agent (e.g., an antibody) for CD123 is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and soluble CD123 flowed over the chip.

The anti-CD123 antibodies or antigen-binding fragments of the present disclosure can be analyzed for their physical, chemical and/or biological properties by various methods known in the art. In some embodiments, an anti-CD123 antibody is tested for its ability to bind CD123 (e.g., human CD123) . Binding assays include, but are not limited to, SPR (e.g., Biacore) , ELISA, and FACS. In addition, antibodies can be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.

Epitope mapping is a method of identifying the binding site, region, or epitope on a target protein where an antibody binds. A variety of methods are known in the art for mapping epitopes on target proteins. These methods include mutagenesis, including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology) ; display methods (e.g., phage display, microbial display, and ribosome/mRNA display) ; methods involving proteolysis and mass spectroscopy; and structural determination (e.g., X-ray crystallography and NMR) . In some embodiments, anti-CD123 antibodies or antigen-binding fragments described herein are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography, and papain digestion.

In some embodiments, an anti-CD123 antibody or antigen-binding fragment is conjugated to a cytotoxic agent or moiety. In some embodiments, an anti-CD123 antibody or antigen-binding fragment is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate) . In some embodiments, the cytotoxic moiety is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs) , or other intercalating agents. In some embodiments, the cytotoxic moiety is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4) , and tubulysins. In some embodiments, the cytotoxic moiety is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In some embodiments, an antibody is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065.

In some embodiments, an anti-CD123 antibody or antigen-binding fragment described herein is conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and/or detection. A detectable substance can include, but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine (s) ; fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC) , rhodamine, tetramethylrhodamine isothiocyanate (TRITC) , dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3) , and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials, such as ²¹²Bi, ¹⁴C, ⁵⁷Co, ⁵¹Cr, ⁶⁷Cu, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ¹⁵³Gd, ¹⁵⁹Gd, ⁶⁸Ge, ³H, ¹⁶⁶Ho, ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I, ¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In, ¹⁴⁰La, ¹⁷⁷Lu, ⁵⁴Mn, ⁹⁹Mo, ³²P, ¹⁰³Pd, ¹⁴⁹Pm, ¹⁴²Pr, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁰⁵Rh, ⁹⁷Ru, ³⁵S, ⁴⁷Sc, ⁷⁵Se, ¹⁵³Sm, ¹¹³Sn, ¹¹⁷Sn, ⁸⁵Sr, ^99mTc, ²⁰¹Ti, ¹³³Xe, ⁹⁰Y, ⁶⁹Yb, ¹⁷⁵Yb, ⁶⁵Zn; positron emitting metals; and magnetic metal ions positron emitting metals; and magnetic metal ions.

An anti-CD123 antibody or antigen-binding fragment described herein can be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, an immobilized anti-CD123 antibody or antigen-binding fragment is used in an immunoassay. In some embodiments, an immobilized anti-CD123 antibody or antigen-binding fragment is used in purification of the target antigen (e.g., human CD123) .

5.3 CARs, TCRs, and Genetically Engineered Immune Effector Cells

The anti-CD123 antibodies or antigen-binding fragments described herein can be used as part of a chimeric antigen receptor (CAR) or a T-Cell Receptor (TCR) that can be expressed in an immune effector cell for cancer treatment. As such, provided herein are also CARs or TCRs that specifically bind CD123, immune effector cells that express such CARs or TCRs, and the uses of such cells.

5.3.1 TCRs

Provided herein are T cell receptors (TCRs) that specifically bind CD123 ( “CD123 TCR” ) . TCRs are antigen-specific molecules that are responsible for recognizing antigenic peptides presented in the context of a product of the MHC on the surface of APCs or any nucleated cells. This system endows T cells, via their TCRs, with the potential ability to recognize the entire array of intracellular antigens expressed by a cell (including virus proteins) that are processed into short peptides, bound to an intracellular MHC molecule, and delivered to the surface as a peptide-MHC complex. This system allows foreign protein (e.g., mutated cancer antigen or virus protein) or aberrantly expressed protein to serve a target for T cells (e.g., Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544) .

The interaction of a TCR and a peptide-MHC complex can drive the T cell into various states of activation, depending on the affinity (or dissociation rate) of binding. The TCR recognition process allows a T cell to discriminate between a normal, healthy cell and, for example, one that has become transformed via a virus or malignancy, by providing a diverse repertoire of TCRs, wherein there is a high probability that one or more TCRs will be present with a binding affinity for the foreign peptide bound to an MHC molecule that is above the threshold for stimulating T cell activity (Manning and Kranz (1999) Immunology Today, 20, 417-422) .

Wild type TCRs isolated from either human or mouse T cell clones that were identified by in vitro culturing have been shown to have relatively low binding affinities (K _D = 1 -300 μΜ) (Davis et al. (1998) Annu Rev Immunol, 16, 523-544) . This is partly because that T cells that develop in the thymus are negatively selected (tolerance induction) on self-peptide-MHC ligands, such that T cells with too high of an affinity are deleted (Starr et al. (2003) Annu Rev Immunol, 21, 139-76) . To compensate for these relatively low affinities, T cells have evolved a co-receptor system in which the cell surface molecules CD4 and CD8 bind to the MHC molecules (class II and class I, respectively) and synergize with the TCR in mediating signaling activity. CD8 is particularly effective in this process, allowing TCRs with very low affinity (e.g., K _D =300 μΜ) to mediate potent antigen-specific activity.

Directed evolution can be used to generate TCRs with higher affinity for a specific peptide-MHC complex. Methods that can be used include yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci U S A, 97, 5387-92) , phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54) , and T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84) . All three approaches involve engineering, or modifying, a TCR that exhibits the normal, low affinity of the wild-type TCR, to increase the affinity for the cognate peptide-MHC complex (the original antigen that the T cells were specific for) .

As such, in some embodiments, provided herein are TCRs comprising an anti-CD123 antibody or antigen-binding fragment described herein. The anti-CD123 antibody or antigen-binding fragment can be any anti-CD123 antibody or antigen-binding fragment described herein. For illustrative purposes, in some embodiments, the TCRs provided herein can comprise an anti-CD123 antibody or antigen-binding fragment having a VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences of (1) SEQ ID NOs: 3, 33, 61, 91, 112, and 136, respectively; (2) SEQ ID NOs: 17, 45, 75, 89, 122, and 149, respectively; (3) SEQ ID NOs: 7, 37, 66, 95, 116, and 140, respectively; (4) SEQ ID NOs: 18, 32, 76, 100, 123, and 150, respectively; (5) SEQ ID NOs: 23, 50, 82, 99, 129 and 157, respectively; (6) SEQ ID NOs: 24, 51, 83, 106, 128, and 158, respectively; (7) SEQ ID NOs: 8, 38, 67, 96, 117, and 141, respectively; (8) SEQ ID NOs: 4, 34, 62, 89, 110, and 134, respectively; (9) SEQ ID NOs: 25, 52, 84, 107, 130, and 159, respectively; (10) SEQ ID NOs: 26, 53, 85, 95, 116, and 160, respectively; (11) SEQ ID NOs: 2, 32, 60, 90, 111, and 135, respectively; (12) SEQ ID NOs: 17, 46, 77, 101, 124, and 151, respectively; (13) SEQ ID NOs: 27, 54, 86, 105, 131, and 161, respectively; (14) SEQ ID NOs: 9, 39, 68, 97, 118, and 142, respectively; (15) SEQ ID NOs: 10, 40, 69, 98, 119, and 143, respectively; (16) SEQ ID NOs: 22, 49, 81, 105, 128, and 156, respectively; (17) SEQ ID NOs: 5, 35, 63, 92, 113, and 137, respectively; (18) SEQ ID NOs: 1, 30, 64, 93, 114 and 138, respectively; (19) SEQ ID NOs: 11, 41, 70, 91, 120, and 144, respectively; (20) SEQ ID NOs: 1, 31, 59, 89, 110, and 134, respectively; (21) SEQ ID NOs: 19, 47, 78, 102, 125, and 152 respectively; (22) SEQ ID NOs: 28, 55, 87, 108, 132, and 162, respectively; (23) SEQ ID NOs: 12, 39, 71, 96, 117, and 145, respectively; (24) SEQ ID NOs: 13, 42, 72, 95, 116, and 146, respectively; (25) SEQ ID NOs: 1, 30, 57, 89, 110, and 134, respectively; (26) SEQ ID NOs: 20, 48, 79, 103, 126 and 153, respectively; (27) SEQ ID NOs: 14, 43, 67, 96, 117, and 141, respectively; (28) SEQ ID NOs: 1, 30, 58, 89, 110, and 134, respectively; (29) SEQ ID NOs: 2, 32, 60, 90, 111, and 135, respectively; (30) SEQ ID NOs: 15, 44, 73, 99, 121, and 147, respectively; (31) SEQ ID NOs: 29, 56, 88, 109, 133 and 163, respectively; (32) SEQ ID NOs: 22, 49, 81, 104, 127, and 155, respectively; (33) SEQ ID NOs: 16, 42, 74, 95, 116, and 148, respectively; (34) SEQ ID NOs: 6, 36, 65, 94, 115, and 139, respectively; or (35) SEQ ID NOs: 21, 45, 80, 89, 122, and 154, respectively. In some embodiments, the TCRs provided herein can comprise an anti-CD123 antibody or antigen-binding fragment that are the human scFv designated as C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, or C35. In some embodiments, the CARs provided herein can comprise C5. In some embodiments, the CARs provided herein can comprise C7. In some embodiments, the CARs provided herein can comprise C11.

In some embodiments, the TCRs provided herein comprise an alpha (α) chain and a beta (β) chain. The constant region of TCR α chain and β chain are encoded by TRAC and TRBC, respectively. A human TRAC can have an amino acid sequence corresponding to UniProtKB/Swiss-Prot No.: P01848.2 (Accession: P01848.2 GI: 1431906459) . A human TRBC can have an amino acid sequence corresponding to the GenBank sequence ALC78509.1 (Accession: ALC78509.1 GI: 924924895) . In some embodiments, the TCRs provided herein comprise a TCR α chain comprising an anti-CD123 antibody or antigen-binding fragment provided herein. In some embodiments, the TCRs provided herein comprise a TCR β chain comprising an anti-CD123 antibody or antigen-binding fragment provided herein. In some embodiments, the TCR comprises a gamma chain (γ) and a delta (δ) chain. The constant region of TCR γ chain and δ chain are encoded by encoded by TRGC and TRDC, respectively. A human TRGC can have an amino acid sequence corresponding to UniProtKB/Swiss-Prot: P0CF51.1 (Accession: P0CF51.1 GI: 294863156) , or an amino acid sequence corresponding to UniProtKB/Swiss-Prot: P03986.2 (Accession: P03986.2 GI: 1531253869) . A human TRDC can have an amino acid sequence corresponding to the UniProtKB/Swiss-Prot: B7Z8K6.2 (Accession: B7Z8K6.2 GI: 294863191) . In some embodiments, the TCRs provided herein comprise a TCR γ chain comprising an anti-CD123 antibody or antigen-binding fragment provided herein. In some embodiments, the TCRs provided herein comprise a TCR δ chain comprising an anti-CD123 antibody or antigen-binding fragment provided herein.

5.3.2 CARs

CARs are engineered receptors that provide both antigen binding and immune effector cell activation functions. CARs can be used to graft the specificity of an antibody, such as a monoclonal antibody, onto an immune effector cell such as a T cell, a NK cell, or a macrophage. CARs can retarget immune effector cells (e.g., T cells) to tumor surface antigens in HLA-independent manner (Sadelain et al., Nat. Rev. Cancer. 3 (1) : 35-45 (2003) ; Sadelain et al., Cancer Discovery 3 (4) : 388-398 (2013) ; Rafiq and Brentjens (2016) . Nat Rev Clin Oncol 13 (6) : 370-383) . The typical structure of a CAR molecule includes an extracellular antigen-binding domain (e.g., scFv) , a spacer, a transmembrane domain (TM) and an intracellular signaling domain. CAR-expressing T cells ( “CART” s) can be classified into three generations according to the presence of intracellular co-stimulatory signals. The extracellular antigen-binding domain of a CAR is usually derived from a monoclonal antibody (mAb) or from receptors or their ligands. Antigen binding by the CARs triggers phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the intracellular domain, initiating a signaling cascade required for cytolysis induction, cytokine secretion, and proliferation.

In some embodiments, provided herein are CARs that specifically binds CD123 ( “CD123 CAR” ) . In some embodiments, the CAR can be a “first generation, ” “second generation” or “third generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3 (4) : 388-398 (2013) ; Jensen et al., Immunol. Rev. 257: 127-133 (2014) ; Sharpe et al., Dis. Model Mech. 8 (4) : 337-350 (2015) ; June et al (2018) , Science 359 (6382) : 1361-1365) .

“First generation” CARs are typically composed of an extracellular antigen binding domain, for example, a single-chain variable fragment (scFv) , fused to a transmembrane domain, which is fused to a cytoplasmic/intracellular domain of the T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3 -chain, which is the primary transmitter of signals from endogenous T cell receptors (TCRs) . “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4 ⁺ and CD8 ⁺ T cells through their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. “Second-generation” CARs comprise a cancer antigen-binding domain fused to an intracellular signaling domain capable of activating immune effector cells such as T cells and a co-stimulatory domain designed to augment immune effector cell, such as T cell, potency and persistence (Sadelain et al., Cancer Discov. 3: 388-398 (2013) ) . CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically borne by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co-stimulatory receptors, for example, CD28, 4-1BB, ICOS, OX40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell. “Second generation” CARs provide both co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3ζ signaling domain. Studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of T cells. In 2017, FDA approved two anti-CD19 CAR T cell products for the treatment of relapsed B-cell precursor acute lymphoblastic leukemia (B-ALL) and B-cell Non-Hodgkin Lymphoma. “Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4-1BB domains, and activation, for example, by comprising a CD3ζ activation domain.

As such, provided herein are CARs that specifically binds CD123, comprising, from N-terminus to C-terminus: (a) a CD123-binding domain comprising an anti-CD123 antibody or antigen-binding fragment provided herein, (b) a transmembrane domain, and (c) a cytoplasmic domain.

The anti-CD123 antibody or antigen-binding fragment can be any anti-CD123 antibody or antigen-binding fragment described herein. For illustrative purposes, in some embodiments, the CARs provided herein can comprise an anti-CD123 antibody or antigen-binding fragment having a VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences of (1) SEQ ID NOs: 3, 33, 61, 91, 112, and 136, respectively; (2) SEQ ID NOs: 17, 45, 75, 89, 122, and 149, respectively; (3) SEQ ID NOs: 7, 37, 66, 95, 116, and 140, respectively; (4) SEQ ID NOs: 18, 32, 76, 100, 123, and 150, respectively; (5) SEQ ID NOs: 23, 50, 82, 99, 129 and 157, respectively; (6) SEQ ID NOs: 24, 51, 83, 106, 128, and 158, respectively; (7) SEQ ID NOs: 8, 38, 67, 96, 117, and 141, respectively; (8) SEQ ID NOs: 4, 34, 62, 89, 110, and 134, respectively; (9) SEQ ID NOs: 25, 52, 84, 107, 130, and 159, respectively; (10) SEQ ID NOs: 26, 53, 85, 95, 116, and 160, respectively; (11) SEQ ID NOs: 2, 32, 60, 90, 111, and 135, respectively; (12) SEQ ID NOs: 17, 46, 77, 101, 124, and 151, respectively; (13) SEQ ID NOs: 27, 54, 86, 105, 131, and 161, respectively; (14) SEQ ID NOs: 9, 39, 68, 97, 118, and 142, respectively; (15) SEQ ID NOs: 10, 40, 69, 98, 119, and 143, respectively; (16) SEQ ID NOs: 22, 49, 81, 105, 128, and 156, respectively; (17) SEQ ID NOs: 5, 35, 63, 92, 113, and 137, respectively; (18) SEQ ID NOs: 1, 30, 64, 93, 114 and 138, respectively; (19) SEQ ID NOs: 11, 41, 70, 91, 120, and 144, respectively; (20) SEQ ID NOs: 1, 31, 59, 89, 110, and 134, respectively; (21) SEQ ID NOs: 19, 47, 78, 102, 125, and 152 respectively; (22) SEQ ID NOs: 28, 55, 87, 108, 132, and 162, respectively; (23) SEQ ID NOs: 12, 39, 71, 96, 117, and 145, respectively; (24) SEQ ID NOs: 13, 42, 72, 95, 116, and 146, respectively; (25) SEQ ID NOs: 1, 30, 57, 89, 110, and 134, respectively; (26) SEQ ID NOs: 20, 48, 79, 103, 126 and 153, respectively; (27) SEQ ID NOs: 14, 43, 67, 96, 117, and 141, respectively; (28) SEQ ID NOs: 1, 30, 58, 89, 110, and 134, respectively; (29) SEQ ID NOs: 2, 32, 60, 90, 111, and 135, respectively; (30) SEQ ID NOs: 15, 44, 73, 99, 121, and 147, respectively; (31) SEQ ID NOs: 29, 56, 88, 109, 133 and 163, respectively; (32) SEQ ID NOs: 22, 49, 81, 104, 127, and 155, respectively; (33) SEQ ID NOs: 16, 42, 74, 95, 116, and 148, respectively; (34) SEQ ID NOs: 6, 36, 65, 94, 115, and 139, respectively; or (35) SEQ ID NOs: 21, 45, 80, 89, 122, and 154, respectively. In some embodiments, the CARs provided herein can comprise an anti-CD123 antibody or antigen-binding fragment that are the human scFv designated as C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, or C35. In some embodiments, the CARs provided herein can comprise C5. In some embodiments, the CARs provided herein can comprise C7. In some embodiments, the CARs provided herein can comprise C11.

In some embodiments, the transmembrane domain of the CARs provided herein comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In some embodiments, the transmembrane domain of the CAR provided herein can be derived from a protein or polypeptide that is naturally expressed in an immune effector cell. A transmembrane domain derived from a protein or polypeptide means that the transmembrane domain comprises the entire transmembrane region of the protein or polypeptide, or a fragment thereof. In some embodiments, the CAR provided herein can have a transmembrane domain derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T-cell receptor (TCR) α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, or other polypeptides expressed in the immune effector cell. In some embodiments, the transmembrane domain of CARs provided herein comprises the transmembrane region of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T-cell receptor (TCR) α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, or other polypeptides expressed in the immune effector cell.

In some embodiments, the transmembrane domain of CARs provided herein is derived from CD8. In some embodiments, the transmembrane domain comprises the transmembrane region of CD8. In some embodiments, the transmembrane domain is derived from CD28. In some embodiments, the transmembrane domain comprises the transmembrane region of CD28. In some embodiments, the transmembrane domain is derived from CD3ζ. In some embodiments, the transmembrane domain comprises the transmembrane region of CD3ζ. In some embodiments, the transmembrane domain is derived from CD4. In some embodiments, the transmembrane domain comprises the transmembrane region of CD4. In some embodiments, the transmembrane domain is derived from 4-1BB. In some embodiments, the transmembrane domain comprises the transmembrane region of 4-1BB. In some embodiments, the transmembrane domain is derived from OX40. In some embodiments, the transmembrane domain comprises the transmembrane region of OX40. In some embodiments, the transmembrane domain is derived from ICOS. In some embodiments, the transmembrane domain comprises the transmembrane region of ICOS. In some embodiments, the transmembrane domain is derived from CTLA-4. In some embodiments, the transmembrane domain comprises the transmembrane region of CTLA-4. In some embodiments, the transmembrane domain is derived from PD-1. In some embodiments, the transmembrane domain comprises the transmembrane region of PD-1. In some embodiments, the transmembrane domain is derived from LAG-3. In some embodiments, the transmembrane domain comprises the transmembrane region of LAG-3. In some embodiments, the transmembrane domain is derived from 2B4. In some embodiments, the transmembrane domain comprises the transmembrane region of 2B4. In some embodiments, the transmembrane domain is derived from BTLA. In some embodiments, the transmembrane domain comprises the transmembrane region of BTLA. In some embodiments, the transmembrane domain is derived from TCR α chain. In some embodiments, the transmembrane domain comprises the transmembrane region of TCR α chain. In some embodiments, the transmembrane domain is derived from TCR β chain. In some embodiments, the transmembrane domain comprises the transmembrane region of TCR β chain. In some embodiments, the transmembrane domain is derived from TCR ζ chain. In some embodiments, the transmembrane domain comprises the transmembrane region of TCR ζ chain. In some embodiments, the transmembrane domain is derived from CD3ε. In some embodiments, the transmembrane domain comprises the transmembrane region of CD3ε. In some embodiments, the transmembrane domain is derived from CD45. In some embodiments, the transmembrane domain comprises the transmembrane region of CD45. In some embodiments, the transmembrane domain is derived from CD5. In some embodiments, the transmembrane domain comprises the transmembrane region of CD5. In some embodiments, the transmembrane domain is derived from CD8. In some embodiments, the transmembrane domain comprises the transmembrane region of CD8. In some embodiments, the transmembrane domain is derived from CD9. In some embodiments, the transmembrane domain comprises the transmembrane region of CD9. In some embodiments, the transmembrane domain is derived from CD16. In some embodiments, the transmembrane domain comprises the transmembrane region of CD16. In some embodiments, the transmembrane domain is derived from CD22. In some embodiments, the transmembrane domain comprises the transmembrane region of CD22. In some embodiments, the transmembrane domain is derived from CD33. In some embodiments, the transmembrane domain comprises the transmembrane region of CD33. In some embodiments, the transmembrane domain is derived from CD37. In some embodiments, the transmembrane domain comprises the transmembrane region of CD37. In some embodiments, the transmembrane domain is derived from CD64. In some embodiments, the transmembrane domain comprises the transmembrane region of CD64. In some embodiments, the transmembrane domain is derived from CD80. In some embodiments, the transmembrane domain comprises the transmembrane region of CD80. In some embodiments, the transmembrane domain is derived from CD86. In some embodiments, the transmembrane domain comprises the transmembrane region of CD86. In some embodiments, the transmembrane domain is derived from CD134. In some embodiments, the transmembrane domain comprises the transmembrane region of CD134. In some embodiments, the transmembrane domain is derived from CD154. In some embodiments, the transmembrane domain comprises the transmembrane region of CD154. Exemplary transmembrane domains are described below in more detail.

Alternatively, the transmembrane domain can be synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine. Optionally, the transmembrane domain can be derived from a polypeptide that is not naturally expressed in the immune effector cell, so long as the transmembrane domain can function in transducing signal from antigen bound to the CAR to the intracellular signaling and/or co-stimulatory domains. In some embodiments, the transmembrane domain can comprise a triplet of phenylalanine, tryptophan and valine at each end. Optionally, a short oligo-or polypeptide linker, preferably between 2 and 10 amino acids in length can form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

Cytoplasmic domains of CARs provided herein can contain a signaling domain that functions in the immune effector cell expressing the CAR. Such a signaling domain can be, for example, derived from CD3, Fc receptor, FcγRIIa, FcRβ (FcεR1b) , CD3, CD3δ, CD3ε, CD79a, CD79b, DAP10, or DAP12. A signaling domain can also be a combination of signaling domains derived from molecules selected from CD3, Fc receptor, FcγRIIa, FcRβ (FcεR1b) , CD3, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. A signaling domain derived from a protein or polypeptide refers to the domain of the protein or polypeptide that is responsible for activating the immune effector cell (such as a T cell) , or a fragment thereof that retains its activation function. In general, the signaling domain induces persistence, trafficking and/or effector functions in the transduced immune effector cells such as T cells (Sharpe et al., Dis. Model Mech. 8: 337-350 (2015) ; Finney et al., J. Immunol. 161: 2791-2797 (1998) ; Krause et al., J. Exp. Med. 188: 619-626 (1998) ) . The signaling domain of a protein or polypeptide can be the intracellular domain of the protein or polypeptide. In some embodiments, the signaling domain comprises the intracellular domain of CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or a combination thereof.

In some embodiments, the cytoplasmic domain of CARs provided herein comprises a signaling domain derived from CD3ζ. In some embodiments, the signaling domain comprises the intracellular domain of CD3ζ. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from FcRγ. In some embodiments, the signaling domain comprises the intracellular domain of FcRγ. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from FcγRIIa. In some embodiments, the signaling domain comprises the intracellular domain of FcγRIIa. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from FcRβ. In some embodiments, the signaling domain comprises the intracellular domain of FcRβ. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from CD3γ. In some embodiments, the signaling domain comprises the intracellular domain of CD3γ. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from CD3δ. In some embodiments, the signaling domain comprises the intracellular domain of CD3δ. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from CD3ε. In some embodiments, the signaling domain comprises the intracellular domain of CD3ε. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from CD5. In some embodiments, the signaling domain comprises the intracellular domain of CD5. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from CD22. In some embodiments, the signaling domain comprises the intracellular domain of CD22. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from CD79a. In some embodiments, the signaling domain comprises the intracellular domain of CD79a. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from CD79b. In some embodiments, the signaling domain comprises the intracellular domain of CD79b. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from DAP10. In some embodiments, the signaling domain comprises the intracellular domain of DAP10. In some embodiments, the cytoplasmic domain comprises a signaling domain derived from DAP12. In some embodiments, the signaling domain comprises the intracellular domain of DAP12. Exemplary signaling domains are described below in more detail.

In some embodiments, the cytoplasmic domain of CARs provided herein further comprises a co-stimulatory domain. In some embodiments, the cytoplasmic domain of CARs provided herein further comprises two co-stimulatory domains. Such a co-stimulatory domain can provide increased activation of an immune effector cell (e.g., T cell) . A co-stimulatory signaling domain can be derived from, for example, CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, or CD43. A co-stimulatory domain derived from a protein or polypeptide refers to the domain of the protein or polypeptide that is responsible for providing increased activation of an immune effector cell (e.g., T cell) , or a fragment thereof that retains its activation function. In some embodiments, the co-stimulatory domain of CARs provided herein comprises the intracellular domain of CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, TIGIT, GITR, TLR, DR3, or CD43. In some embodiments, the cytoplasmic domain of CARs provided herein comprises a co-stimulatory domain derived from CD28. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD28. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from 4-1BB. In some embodiments, the co-stimulatory domain comprises the intracellular domain of 4-1BB. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from OX40. In some embodiments, the co-stimulatory domain comprises the intracellular domain of OX40. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from ICOS. In some embodiments, the co-stimulatory domain comprises the intracellular domain of ICOS. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from DAP10. In some embodiments, the co-stimulatory domain comprises the intracellular domain of DAP10. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from 2B4. In some embodiments, the co-stimulatory domain comprises the intracellular domain of 2B4. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from CD27. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD27. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from CD30. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD30. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from CD40. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD40. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from CD2. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD2. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from CD7. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD7. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from LIGHT. In some embodiments, the co-stimulatory domain comprises the intracellular domain of LIGHT. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from TIGIT. In some embodiments, the co-stimulatory domain comprises the intracellular domain of TIGIT. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from GITR. In some embodiments, the co-stimulatory domain comprises the intracellular domain of GITR. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from TLR. In some embodiments, the co-stimulatory domain comprises the intracellular domain of TLR. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from DR3. In some embodiments, the co-stimulatory domain comprises the intracellular domain of DR3. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from CD43. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD43. Exemplary co-stimulatory domains are described below in more detail.

CARs comprising an intracellular domain that comprises a co-stimulatory domain derived from 4-1BB, ICOS or DAP-10 have been described previously (see U.S. 7,446,190, which is incorporated herein by reference, which also describes representative sequences for 4-1BB, ICOS and DAP-10) . In some embodiments, the cytoplasmic domain of a CAR can comprise two co-stimulatory domains derived from two co-stimulatory receptors, such as CD28 and 4-1BB (see Sadelain et al., Cancer Discov. 3 (4) : 388-398 (2013) ) , or CD28 and OX40, or other combinations of co-stimulatory ligands, as disclosed herein.

The extracellular domain of a CAR can be fused to a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum and subsequent translocation to the cell surface. It is understood that, once a polypeptide containing a signal peptide is expressed at the cell surface, the signal peptide has generally been proteolytically removed during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, a polypeptide such as a CAR is generally expressed at the cell surface as a mature protein lacking the signal peptide, whereas the precursor form of the polypeptide includes the signal peptide. A signal peptide or leader can be essential if a CAR is to be glycosylated and/or anchored in the cell membrane. The signal sequence or leader is a peptide sequence generally present at the N-terminus of newly synthesized proteins that directs their entry into the secretory pathway. The signal peptide is covalently joined to the N-terminus of the extracellular antigen-binding domain of a CAR as a fusion protein. Any suitable signal peptide, as are well known in the art, can be applied to a CAR to provide cell surface expression in an immune cell (see Gierasch Biochem. 28: 923-930 (1989) ; von Heijne, J. Mol. Biol. 184 (1) : 99–105 (1985) ) . Particularly useful signal peptides can be derived from cell surface proteins naturally expressed in the immune cell provided herein, including any of the signal peptides of the polypeptides disclosed herein. Thus, any suitable signal peptide can be utilized to direct a CAR to be expressed at the cell surface of an immune effector cell provided herein.

In some embodiments, a CAR can also comprise a spacer region or sequence that links the domains of the CAR to each other. For example, a spacer can be included between a signal peptide and an antigen binding domain, between the antigen binding domain and the transmembrane domain, between the transmembrane domain and the intracellular domain, and/or between domains within the intracellular domain, for example, between a stimulatory domain and a co-stimulatory domain. The spacer region can be flexible enough to allow interactions of various domains with other polypeptides, for example, to allow the antigen binding domain to have flexibility in orientation in order to facilitate antigen recognition. The spacer region can be, for example, the hinge region from an IgG, the CH ₂CH ₃ (constant) region of an immunoglobulin, and/or portions of CD3 (cluster of differentiation 3) or some other sequence suitable as a spacer. In some embodiments, a CAR disclosed herein comprises a hinge domain that connects the CD123 binding domain and the transmembrane domain. In some embodiments, the hinge domain comprises human CD8 hinge domain. In some embodiments, the hinge domain comprises human CD28 hinge domain.

Provided below are some exemplary molecules from which domains of the CARs provided herein can be derived.

CD3ζ. CD3ζ comprises 3 Immune-receptor-Tyrosine-based-Activation-Motifs (ITAMs) , and transmits an activation signal to the cell, for example, a cell of the lymphoid lineage such as a T cell, after antigen is bound. A CD3ζ polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_932170 (NP_932170.1, GI: 37595565; see below) , or fragments thereof. In some embodiments, a CD3ζ signaling domain has an amino acid sequence of amino acids 52 to 164 of the CD3ζ polypeptide sequence provided below, or a fragment thereof that is sufficient for signaling activity. See GenBank NP_932170 for reference to domains within CD3ζ, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 30; transmembrane region, amino acids 31 to 51; intracellular domain, amino acids 52 to 164. In some embodiments, a CAR can have a transmembrane domain derived from CD3ζ. The transmembrane domain can comprise the transmembrane region of CD3ζ (e.g., amino acids 31 to 51 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from CD3ζ. In some embodiments, a signaling domain of CD3ζ can comprise the intracellular domain of CD3ζ (e.g., amino acids 52 to 164 of the sequence below) , or a fragment thereof. It is understood that sequences of CD3ζ that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

FcRγ Activating types of IgG receptor FcγRs form multimeric complexes including the Fc receptor common γ chain (FcRγ) that contains an intracellular tyrosine-based activating motif (ITAM) , whose activation triggers oxidative bursts, cytokine release, phagocytosis, antibody-dependent cell-mediated cytotoxicity, and degranulation. An FcRγ polypeptide can have an amino acid sequence corresponding to the sequence having NCBI Reference Sequence: NP_004097.1 (GI: 4758344) , provided below, or fragments thereof. See GenBank NP_004097 for reference to domains within FcRγ, for example, signal peptide, amino acids 1 to 18; extracellular domain, amino acids 19 to 23; transmembrane region, amino acids 24 to 44; intracellular domain, amino acids 45 to 86. In some embodiments, a CAR can comprise a transmembrane domain derived from FcRγ. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of FcRγ(e.g., amino acids 24 to 44 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from FcRγ. In some embodiments, the signaling domain comprises the intracellular domain of FcRγ (e.g., amino acids 45 to 86 of the sequence below) , or a fragment thereof. It is understood that sequences of FcRγ that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

FcγRIIa is a cell surface receptor found on phagocytic cells such as macrophages and neutrophils, and is involved in the process of phagocytosis and clearing of immune complexes. By binding to IgG it initiates cellular responses against pathogens and soluble antigens. FcγRIIa also promotes phagocytosis of opsonized antigens. An FcγRIIa polypeptide can have an amino acid sequence corresponding to the sequence having NCBI Reference Sequence: NP_001129691.1, provided below, or fragments thereof. See NCBI Reference Sequence NP_001129691.1 for reference to domains within FcγRIIa, for example, signal peptide, amino acids 1 to 33; extracellular domain, amino acids 34 to 217; transmembrane region, amino acids 218 to 240; intracellular domain, amino acids 241 to 317. In some embodiments, a CAR can comprise a transmembrane domain derived from FcγRIIa. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of FcγRIIa (e.g., amino acids 218 to 240 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from FcγRIIa. In some embodiments, the signaling domain comprises the intracellular domain of FcγRIIa (e.g., amino acids 241 to 317 of the sequence below) , or a fragment thereof. It is understood that sequences of FcγRIIa that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

FcRβ (FcεR1b) is a high affinity receptor that binds to the Fc region of immunoglobulins epsilon. Aggregation of FcRβ by multivalent antigens is required for the full mast cell response, including the release of preformed mediators (such as histamine) by degranulation and de novo production of lipid mediators and cytokines. FcRβ also mediates the secretion of important lymphokines. Binding of allergen to receptor-bound IgE leads to cell activation and the release of mediators responsible for the manifestations of allergy. An FcRβ polypeptide can have an amino acid sequence corresponding to the sequence having NCBI Reference Sequence: NP_000130.1, provided below, or fragments thereof. See NCBI Reference Sequence: NP_000130.1 for reference to domains within FcRβ, for example, intracellular domain, amino acids 1 to 59, 118 to 130, and 201 to 244; transmembrane region, amino acids 60 to 79, 98 to 117, 131 to 150, and 181 to 200; extracellular domain, amino acids 80 to 97, and 151 to 180. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from FcRβ. In some embodiments, the signaling domain comprises an intracellular domain of FcRβ (e.g., amino acids 1 to 59, 118 to 130, or 201 to 244 of the sequence below, or a combination thereof) , or a fragment thereof. It is understood that sequences of FcRβ that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD3γ (T-cell surface glycoprotein CD3 gamma chain) , is part of the TCR-CD3 complex present on T-lymphocyte cell surface that plays an essential role in adaptive immune response. CD3γ contains immunoreceptor tyrosine-based activation motifs (ITAMs) in its cytoplasmic domain. In addition to this role of signal transduction in T-cell activation, CD3γ plays an essential role in the dynamic regulation of TCR expression at the cell surface. A CD3γ polypeptide can have an amino acid sequence corresponding to the sequence having NCBI Reference Sequence: NP_004097.1 (GI: 4758344) , provided below, or fragments thereof. See GenBank NP_004097 for reference to domains within CD3γ, for example, signal peptide, amino acids 1 to 22; extracellular domain, amino acids 23 to 116; transmembrane region, amino acids 117 to 137; intracellular domain, amino acids 138 to 182. In some embodiments, a CAR can comprise a transmembrane domain derived from CD3γ. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD3γ (e.g., amino acids 117 to 137 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from CD3γ. In some embodiments, the signaling domain comprises the intracellular domain of CD3γ (e.g., amino acids 138 to 182 of the sequence below) , or a fragment thereof. It is understood that sequences of CD3γ that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD3δ (T-cell surface glycoprotein CD3 delta chain) , is part of the TCR-CD3 complex present on T-lymphocyte cell surface that plays an essential role in adaptive immune response. CD3δ contains immunoreceptor tyrosine-based activation motifs (ITAMs) in its cytoplasmic domain. In addition of this role of signal transduction in T-cell activation, CD3δ plays an essential role in thymocyte differentiation and participates in correct intracellular TCR-CD3 complex assembly and surface expression. CD3δ interacts with CD4 and CD8 and thus serves to establish a functional link between the TCR and coreceptors CD4 and CD8, which is needed for activation and positive selection of CD4 or CD8 T-cells. A CD3δ polypeptide can have an amino acid sequence corresponding to the sequence having NCBI Reference Sequence: NP_000723.1, provided below, or fragments thereof. See NCBI Reference Sequence: NP_000723.1 for reference to domains within CD3δ, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 105; transmembrane region, amino acids 106 to 126; intracellular domain, amino acids 127 to 171. In some embodiments, a CAR can comprise a transmembrane domain derived from CD3δ. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD3δ (e.g., amino acids 106 to 126 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from CD3δ. In some embodiments, the signaling domain comprises the intracellular domain of CD3δ (e.g., amino acids 127 to 171 of the sequence below) , or a fragment thereof. It is understood that sequences of CD3δ that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD3ε (T-cell surface glycoprotein CD3 epsilon chain) , is part of the TCR-CD3 complex present on T-lymphocyte cell surface that plays an essential role in adaptive immune response. CD3ε contains immunoreceptor tyrosine-based activation motifs (ITAMs) in its cytoplasmic domain. In addition of this role of signal transduction in T-cell activation, CD3ε plays an essential role in correct T-cell development. CD3ε initiates the TCR-CD3 complex assembly by forming the two heterodimers CD3δ/CD3γ and CD3γ/CD3γ. A CD3ε polypeptide can have an amino acid sequence corresponding to the sequence having NCBI Reference Sequence: NP_000724.1, provided below, or fragments thereof. See NCBI Reference Sequence: NP_000724.1 for reference to domains within CD3ε, for example, signal peptide, amino acids 1 to 22; extracellular domain, amino acids 23 to 126; transmembrane region, amino acids 127 to 152; intracellular domain, amino acids 153 to 207. In some embodiments, a CAR can comprise a transmembrane domain derived from CD3ε. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD3ε (e.g., amino acids 127 to 152 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from CD3ε. In some embodiments, the signaling domain comprises the intracellular domain of CD3ε (e.g., amino acids 153 to 207 of the sequence below) , or a fragment thereof. It is understood that sequences of CD3ε that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD79a (B-cell antigen receptor complex-associated protein alpha chain) is required in cooperation with CD79b for initiation of the signal transduction cascade activated by binding of antigen to the B-cell antigen receptor complex (BCR) which leads to internalization of the complex, trafficking to late endosomes and antigen presentation. CD79a stimulates SYK autophosphorylation and activation. CD79a also binds to BLNK, bringing BLNK into proximity with SYK and allowing SYK to phosphorylate BLNK, and interacts with and increases activity of some Src-family tyrosine kinases. A CD79a polypeptide can have an amino acid sequence corresponding to the sequence having NCBI Reference Sequence: NP_001774.1, provided below, or fragments thereof. See NCBI Reference Sequence: NP_001774.1 for reference to domains within CD79a, for example, signal peptide, amino acids 1 to 32; extracellular domain, amino acids 33 to 143; transmembrane region, amino acids 144 to 165; intracellular domain, amino acids 166 to 226. In some embodiments, a CAR can comprise a transmembrane domain derived from CD79a. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD79a (e.g., amino acids 144 to 165 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from CD79a. In some embodiments, the signaling domain comprises the intracellular domain of CD79a (e.g., amino acids 166 to 226 of the sequence below) , or a fragment thereof. It is understood that sequences of CD79a that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD79b (B-cell antigen receptor complex-associated protein beta chain) is required in cooperation with CD79a for initiation of the signal transduction cascade activated by the B-cell antigen receptor complex (BCR) which leads to internalization of the complex, trafficking to late endosomes and antigen presentation. CD79b enhances phosphorylation of CD79a. A CD79b polypeptide can have an amino acid sequence corresponding to the sequence having NCBI Reference Sequence: NP_000617.1, provided below, or fragments thereof. See NCBI Reference Sequence: NP_000617.1 for reference to domains within CD79b, for example, signal peptide, amino acids 1 to 28;extracellular domain, amino acids 29 to 159; transmembrane region, amino acids 160 to 180; intracellular domain, amino acids 181 to 229. In some embodiments, a CAR can comprise a transmembrane domain derived from CD79b. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD79b (e.g., amino acids 160 to 180 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from CD79b. In some embodiments, the signaling domain comprises the intracellular domain of CD79b (e.g., amino acids 181 to 229 of the sequence below) , or a fragment thereof. It is understood that sequences of CD79b that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

DAP10. DAP10, also referred to as hematopoietic cell signal transducer, is a signaling subunit that associates with a large family of receptors in hematopoietic cells. A DAP10 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_055081.1 (GI: 15826850) , provided below, or fragments thereof. See GenBank NP_055081 for reference to domains within DAP10, for example, signal peptide, amino acids 1 to 18; extracellular domain, amino acids 19 to 48; transmembrane region, amino acids 49 to 69; intracellular domain, amino acids 70 to 93. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from DAP10. In some embodiments, the signaling domain comprises the intracellular domain of DAP10 (e.g., amino acids 70 to 93 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from DAP10. In some embodiments, the co-stimulatory domain comprises the intracellular domain of DAP10 (e.g., amino acids 70 to 93 of the sequence below) , or a fragment thereof. It is understood that sequences of DAP10 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

DAP12. DAP12 is found in cells of the myeloid lineage, such as macrophages and granulocytes, where it associates, for instance, with the triggering receptor expressed on myeloid cell members (TREM) and MDL1 (myeloid DAP12-associating lectin 1/CLEC5A) , both involved in inflammatory responses against pathogens like viruses and bacteria. In the lymphoid lineage, DAP12 is expressed in NK cells and associates with activating receptors such as the C-type lectin receptor NKG2C, the natural cytotoxicity receptor NKp44, and the short-tailed KIR3DS1 and KIR2DS1/2/5, respectively. In particular, NGK2C is the dominant activating NK cell receptor for controlling CMV infection in both humans and mice. It was found that a DAP12-containing CAR generated sufficient activating signals in NK cells upon cross-linking with its Ag.

et al., J Immunol 194: 3201-12 (2015) . A DAP12 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. AAD09437.1 (GI: 2905996) , provided below, or fragments thereof. See GenBank No. AAD09437.1 for reference to domains within DAP12, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 40; transmembrane region, amino acids 41 to 61; intracellular domain, amino acids 62 to 113. In some embodiments, the cytoplasmic domain of a CAR can comprise a signaling domain derived from DAP12. In some embodiments, the signaling domain comprises the intracellular domain of DAP12 (e.g., amino acids 62 to 113 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain comprises a co-stimulatory domain derived from DAP12. In some embodiments, the co-stimulatory domain comprises the intracellular domain of DAP12 (e.g., amino acids 62 to 113 of the sequence below) , or a fragment thereof. It is understood that sequences of DAP12 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD28. Cluster of Differentiation 28 (CD28) is a protein expressed on T cells that provides co-stimulatory signals for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. A CD28 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P10747 (P10747.1, GI: 115973) or NP_006130 (NP_006130.1, GI: 5453611) , as provided below, or fragments thereof. See GenBank NP_006130 for reference to domains within CD28, for example, signal peptide, amino acids 1 to 18; extracellular domain, amino acids 19 to 152; transmembrane domain, amino acids 153 to 179; intracellular domain, amino acids 180 to 220. In some embodiments, a CAR can comprise a hinge domain derived from CD28 (e.g., amino acids 114 to 152 of the sequence below, or a fragment thereof) . In some embodiments, a CAR can comprise a transmembrane domain derived from CD28. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD28 (e.g., amino acids 153 to 179 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from CD28. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD28 (e.g., amino acids 180 to 220 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from CD28, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of CD28 and comprises amino acids 153 to 220 of CD28. In some embodiments, a CAR can comprise three domains derived from CD28, a transmembrane domain, a hinge domain and a co-stimulatory signaling domain. In another embodiment, a CAR comprises amino acids 114 to 220 of CD28. It is understood that sequences of CD28 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

4-1BB. 4-1BB, also referred to as tumor necrosis factor receptor superfamily member 9, can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. A 4-1BB polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P41273 (P41273.1, GI: 728739) or NP_001552 (NP_001552.2, GI: 5730095) or fragments thereof. See GenBank NP_001552 for reference to domains within 4-1BB, for example, signal peptide, amino acids 1 to 17; extracellular domain, amino acids 18 to 186; transmembrane domain, amino acids 187 to 213; intracellular domain, amino acids 214 to 255. In some embodiments, a CAR can comprise a transmembrane domain derived from 4-1BB. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of 4-1BB (e.g., amino acids 187 to 213 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from 4-1BB. In some embodiments, the co-stimulatory domain comprises the intracellular domain of 4-1BB (e.g., amino acids 214 to 255 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from 4-1BB, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of 4-1BB and comprises amino acids 187 to 255 of 4-1BB. It is understood that sequences of 4-1BB that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

OX40. OX40, also referred to as tumor necrosis factor receptor superfamily member 4 precursor or CD134, is a member of the TNFR-superfamily of receptors. An OX40 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. P43489 (P43489.1, GI: 1171933) or NP_003318 (NP_003318.1, GI: 4507579) , provided below, or fragments thereof. See GenBank NP_003318 for reference to domains within OX40, for example, signal peptide, amino acids 1 to 28; extracellular domain, amino acids 29 to 214; transmembrane domain, amino acids 215 to 235; intracellular domain, amino acids 236 to 277. It is understood that sequences of OX40 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired. In some embodiments, a CAR can comprise a transmembrane domain derived from OX40. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of OX40 (e.g., amino acids 215 to 235 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from OX40. In some embodiments, the co-stimulatory domain comprises the intracellular domain of OX40 (e.g., amino acids 236 to 277 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from OX40, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of OX40 and comprises amino acids 215 to 277 of OX40.

ICOS. Inducible T-cell co-stimulator precursor (ICOS) , also referred to as CD278, is a CD28-superfamily co-stimulatory receptor that is expressed on activated T cells. An ICOS polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_036224 (NP_036224.1, GI: 15029518) , provided below, or fragments thereof. See GenBank NP_036224 for reference to domains within ICOS, for example, signal peptide, amino acids 1 to 20; extracellular domain, amino acids 21 to 140; transmembrane domain, amino acids 141 to 161; intracellular domain, amino acids 162 to 199. In some embodiments, a CAR can comprise a transmembrane domain derived from ICOS. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of ICOS (e.g., amino acids 141 to 161 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from ICOS. In some embodiments, the co-stimulatory domain comprises the intracellular domain of ICOS (e.g., amino acids 162 to 199 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from ICOS, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of ICOS and comprises amino acids 141 to 199 of ICOS. It is understood that sequences of ICOS that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

2B4 2B4 (CD244) is a co-stimulatory receptor expressed on both NK cells and CD8+ T cells. It targets a non-MHC like molecule (CD48) expressed on hematopoietic cells, including B and T cells, as well as on activated monocytes and granulocytes. Activation of 2B4 by binding of its ligand on target cells leads to NK (or T cell) activation, and target killing. 2B4 polypeptide can have an amino acid sequence corresponding to the sequence having Accession No: Q9BZW8.2 (NP_001160135.1; GI: 47605541) , provided below, or fragments thereof. See GenBank NP_001160135.1 for reference to domains within 2B4, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 229; transmembrane domain, amino acids 230 to 250; intracellular domain, amino acids 251 to 370. In some embodiments, a CAR can comprise a transmembrane domain derived from 2B4. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of 2B4 (e.g., amino acids 230 to 250 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from 2B4. In some embodiments, the co-stimulatory domain comprises the intracellular domain of 2B4 (e.g., amino acids 251 to 370 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from 2B4, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of 2B4 and comprises amino acids 230 to 370 of 2B4. It is understood that sequences of 2B4 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD27: CD27 (TNFRSF7) is a transmembrane receptor expressed on subsets of human CD8+and CD4+ T-cells, NKT cells, NK cell subsets and hematopoietic progenitors and induced in FOXP3+ CD4 T-cells and B cell subsets. Previously studies have found that CD27 can either actively provide costimulatory signals that improve human T-cell survival and anti-tumor activity in vivo. (See Song and Powell; Oncoimmunology 1, no. 4 (2012) : 547-549) . A CD27 polypeptide can have an amino acid sequence corresponding to the sequence having UniProtKB/Swiss-Prot No.: P26842.2 (GenBank NP_001233.1; GI: 269849546) , provided below, or fragments thereof. See GenBank NP_001233 for reference to domains within CD27, for example, signal peptide, amino acids 1 to 19; extracellular domain, amino acids 20 to 191; transmembrane domain, amino acids 192 to 212; intracellular domain, amino acids 213 to 260. In some embodiments, a CAR can comprise a transmembrane domain derived from CD27. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD27 (e.g., amino acids 192 to 212 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from CD27. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD27 (e.g., amino acids 213 to 260 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from CD27, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of CD27 and comprises amino acids 192 to 260 of CD27. It is understood that sequences of CD27 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD30: CD30 and its ligand (CD30L) are members of the tumor necrosis factor receptor (TNFR) and tumor necrosis factor (TNF) superfamilies, respectively. CD30, in many respects, behaves similarly to Ox40 and enhances proliferation and cytokine production induced by TCR stimulation. (Goronzy and Weyand, Arthritis research &therapy 10, no. S1 (2008) : S3. ) A CD30 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No.: AAA51947.1 (GenBank NP_001234.3; GI: 180096) , provided below, or fragments thereof. See GenBank NP_001234.3 for reference to domains within CD30, for example, signal peptide, amino acids 1 to 18; extracellular domain, amino acids 19 to 385; transmembrane domain, amino acids 386 to 406; intracellular domain, amino acids 407 to 595. In some embodiments, a CAR can comprise a transmembrane domain derived from CD30. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD30 (e.g., amino acids 386 to 406 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from CD30. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD30 (e.g., amino acids 407 to 595 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from CD30, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of CD30 and comprises amino acids 386 to 595 of CD30. It is understood that sequences of CD30 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD40: CD40 is a 48 kD transmembrane glycoprotein surface receptor that is a member of the Tumor Necrosis Factor Receptor superfamily (TNFRSF) . Exemplary amino acid sequences of human CD40 are described (see, e.g., Accession: ALQ33424.1, GenBank NP_001241.1, GI: 957949089) , CD40 was initially characterized as a co-stimulatory receptor expressed on APCs that played a central role in B and T cell activation. The ligand for CD40, CD154 (also known as TRAP, T-BAM, CD40 Ligand or CD40L) is a type II integral membrane protein. See GenBank NP_001241.1 for reference to domains within CD40, for example, signal peptide, amino acids 1 to 20; extracellular domain, amino acids 21 to 193; transmembrane domain, amino acids 194 to 215; intracellular domain, amino acids 216 to 277. In some embodiments, a CAR can comprise a transmembrane domain derived from CD40. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD40 (e.g., amino acids 194 to 215 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from CD40. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD40 (e.g., amino acids 216 to 277 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from CD40, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of CD40 and comprises amino acids 194 to 277 of CD40. It is understood that sequences of CD40 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD2 The engagement of the CD2 molecule by its ligand CD58 co-stimulates proliferation, cytokine production, and effector function in this T cells, especially the CD28-deficient T cells subset. CD58 is broadly expressed on APCs including dendritic cells. Engagement of CD2 amplifies TCR signals in CD28 ^-CD8 ⁺ T cells, demonstrating that the CD2–CD58 interaction has a genuine costimulatory effect. CD2 signals could promote the control of viral infection by CD28 ^-CD8 ⁺ T cells, but they could also contribute to the continuous expansion of CD28 ^-CD8 ⁺ T cells during chronic stimulation by persistent Ag. (Judith Leitner J et al., Immunol, 2015, 195 (2) 477-487) . A CD2 polypeptide can have an amino acid sequence corresponding to the sequence having Accession: NP_001758.2 GI: 156071472, provided below, or fragments thereof. See GenBank NP_001758.2 for reference to domains within CD2, for example, signal peptide, amino acids 1 to 24; extracellular domain, amino acids 25 to 209; transmembrane domain, amino acids 210 to 235; intracellular domain, amino acids 236 to 351. In some embodiments, a CAR can comprise a transmembrane domain derived from CD2. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD2 (e.g., amino acids 210 to 235 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from CD2. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD2 (e.g., amino acids 236 to 351 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from CD2, a co-stimulatory domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of CD2 and comprises amino acids 210 to 351 of CD2. It is understood that sequences of CD2 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

LIGHT TNF superfamily member 14 (also known as LTg, CD258, HVEML, LIGHT) is a co-stimulatory receptor involved in cellular immune responses. LIGHT can function as a costimulatory factor for the activation of lymphoid cells and as a deterrent to infection by herpesvirus. LIGHT has been shown to stimulate the proliferation of T cells, and trigger apoptosis of various tumor cells. LIGHT is found in T cells and stromal cells. LIGHT is expressed on immature dendritic cells (DCs) generated from human PBMCs. Engagement of LIGHT co-stimulates human T cell proliferation, amplifies the NF-κB signaling pathway, and preferentially induces the production of IFN-γ, but not IL-4, in the presence of an antigenic signal. (Tamada K et al., J Immunol, 2000, 164 (8) 4105-4110) . A LIGHT polypeptide can have an amino acid sequence corresponding to the sequence provided below (Accession: NP_001363816.1 GI: 1777376047) , or fragments thereof. See GenBank NP_001363816.1 for reference to domains within LIGHT, for example, intracellular domain, amino acids 1 to 37; transmembrane domain, amino acids 38 to 58; extracellular domain, amino acids 59 to 240. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from LIGHT. In some embodiments, the co-stimulatory domain comprises the intracellular domain of LIGHT (e.g., amino acids 1 to 37 of the sequence below) , or a fragment thereof. It is understood that sequences of LIGHT that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

GITR TNF receptor superfamily member 18 (also known as TNFRSF18, AITR, GITR; CD357; GITR-D; ENERGEN) has been shown to have increased expression upon T-cell activation. Stimulation of T cells through GITR has been shown to enhance immunity to tumors and viral pathogens, and to exacerbate autoimmune disease. The effects of stimulation through GITR are generally thought to be caused by attenuation of the effector activity of immunosuppressive CD4+CD25+ regulatory T (TReg) cells. (Shevach, E. and Stephens, G. Nat Rev Immunol 6, 613–618 (2006) ) . A GITR polypeptide can have an amino acid sequence corresponding to the sequence provided below (Accession: AAI52382.1, GenBank NP_004186.1, GI: 158931986) , or fragments thereof. See GenBank NP_004186.1 for reference to domains within GITR, for example, signal peptide, amino acids 1 to 25; extracellular domain, amino acids 26 to 162; transmembrane domain, amino acids 163 to 183; intracellular domain, amino acids 184 to 241. In some embodiments, a CAR can comprise a transmembrane domain derived from GITR. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of GITR (e.g., amino acids 163 to 183 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from GITR. In some embodiments, the co-stimulatory domain comprises the intracellular domain of GITR (e.g., amino acids 184 to 241 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from GITR, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of GITR and comprises amino acids 163 to 241 of GITR. It is understood that sequences of GITR that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

DR3 TNF receptor superfamily member 25 (also known as DR3, TR3, DDR3, LARD, APO-3, TRAMP, WSL-1, GEF720, WSL-LR, PLEKHG5, or TNFRSF12) is expressed preferentially in the tissues enriched in lymphocytes, and it plays a role in regulating lymphocyte homeostasis. This receptor has been shown to stimulate NF-kappa B activity and regulate cell apoptosis. The signal transduction of this receptor is mediated by various death domain containing adaptor proteins. Multiple alternatively spliced transcript variants of this gene encoding distinct isoforms have been reported, most of which are potentially secreted molecules. The alternative splicing of this gene in B and T cells encounters a programmed change upon T-cell activation, which predominantly produces full-length, membrane bound isoforms, and is involved in controlling lymphocyte proliferation induced by T-cell activation. A DR3 polypeptide can have an amino acid sequence corresponding to the sequence provided below (Accession: Accession: Accession: AAI17190.1, GenBank NP_003781.1 GI: 109658976) , or fragments thereof. See GenBank NP_003781.1 for reference to domains within DR3, for example, signal peptide, amino acids 1 to 24; extracellular domain, amino acids 25 to 199; transmembrane domain, amino acids 200 to 220; intracellular domain, amino acids 221 to 417. In some embodiments, a CAR can comprise a transmembrane domain derived from DR3. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of DR3 (e.g., amino acids 200 to 220 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from DR3. In some embodiments, the co-stimulatory domain comprises the intracellular domain of DR3 (e.g., amino acids 221 to 417 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from DR3, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of DR3 and comprises amino acids 200 to 417 of DR3. It is understood that sequences of DR3 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD43 CD43 (also known as SPN sialophorin, LSN, GALGP, GPL115) is a highly sialylated glycoprotein that functions in antigen-specific activation of T cells, and is found on the surface of thymocytes, T lymphocytes, monocytes, granulocytes, and some B lymphocytes. It contains a mucin-like extracellular domain, a transmembrane region and a carboxy-terminal intracellular region. In stimulated immune effector cells, proteolytic cleavage of the extracellular domain occurs in some cell types, releasing a soluble extracellular fragment. A CD43 polypeptide can have an amino acid sequence corresponding to the sequence provided below (GenBank NP_003114.1, Accession: EAW80016.1 GI: 119600422) , or fragments thereof. See GenBank NP_003114.1 for reference to domains within CD43, for example, signal peptide, amino acids 1 to 19; extracellular domain, amino acids 20 to 253; transmembrane domain, amino acids 254 to 276; intracellular domain, amino acids 277 to 400. In some embodiments, a CAR can comprise a transmembrane domain derived from CD43. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD43 (e.g., amino acids 254 to 276 of the sequence below) , or a fragment thereof. In some embodiments, the cytoplasmic domain of a CAR can comprise a co-stimulatory domain derived from CD43. In some embodiments, the co-stimulatory domain comprises the intracellular domain of CD43 (e.g., amino acids 277 to 400 of the sequence below) , or a fragment thereof. In some embodiments, a CAR can comprise two domains derived from CD43, a co-stimulatory signaling domain and a transmembrane domain. In some embodiments, a CAR has an amino acid sequence comprising the transmembrane domain and the intracellular domain of CD43 and comprises amino acids 254 to 400 of CD43. It is understood that sequences of CD43 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD4. Cluster of differentiation 4 (CD4) , also referred to as T-cell surface glycoprotein CD4, is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells. In some embodiments, a CAR can comprise a transmembrane domain derived from CD4. CD4 exists in various isoforms. It is understood that any isoform can be selected to achieve a desired function. Exemplary isoforms include isoform 1 (NP_000607.1, GI: 10835167) , isoform 2 (NP_001181943.1, GI: 303522479) , isoform 3 (NP_001181944.1, GI: 303522485; or NP_001181945.1, GI: 303522491; or NP_001181946.1, GI: 303522569) , and the like. One exemplary isoform sequence, isoform 1, is provided below. See GenBank NP_000607.1 for reference to domains within CD4, for example, signal peptide, amino acids 1 to 25; extracellular domain, amino acids 26 to 396; transmembrane domain amino acids, 397 to 418; intracellular domain, amino acids 419 to 458. In some embodiments, a CAR can comprise a transmembrane domain derived from CD4. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD4 (e.g., amino acids 397 to 418 of the sequence below) , or a fragment thereof. It is understood that additional sequence of CD4 beyond the transmembrane domain of amino acids 397 to 418 can be included in a CAR, if desired. It is further understood that sequences of CD4 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

CD8. Cluster of differentiation 8 (CD8) is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR) . CD8 binds to a major histocompatibility complex (MHC) molecule and is specific for the class I MHC protein. In some embodiments, a CAR can comprise a transmembrane domain derived from CD8. A CD8 polypeptide can have an amino acid sequence corresponding to the sequence having GenBank No. NP_001139345.1 (GI: 225007536) , as provided below, or fragments thereof. See GenBank NP_001139345.1 for reference to domains within CD8, for example, signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 182; transmembrane domain amino acids, 183 to 203; intracellular domain, amino acids 204 to 235. In some embodiments, a CAR can comprise a hinge domain derived from CD8. In some embodiments, the hinge domain can comprise amino acids 137-182 of the CD8 polypeptide provided below. In some embodiments, a CAR can comprise a signal peptide derived from CD8 (e.g., amino acids 1 to 21 of the sequence below) . In some embodiments, a CAR can comprise a transmembrane domain derived from CD8. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane region of CD8 (e.g., amino acids 183 to 203 of the sequence below) , or a fragment thereof. In another embodiment, a CAR can comprise amino acids 137-203 of the CD8 polypeptide provided below. In yet another embodiment, a CAR can comprise amino acids 137 to 209 of the CD8 polypeptide provided below. It is understood that additional sequence of CD8 beyond the hinge domain of amino acids 137-182 and the transmembrane domain of amino acids 183 to 203 can be included in a CAR, if desired. It is further understood that sequences of CD8 that are shorter or longer than a specific delineated domain can be included in a CAR, if desired.

As such, for exemplary purposes, a CAR disclosed herein can comprise, from N-terminus to the C-terminus, an anti-CD123 antibody or antigen-binding fragment (e.g., scFvs disclosed herein) , a hinge (e.g., CD8 hinge or CD28 hinge) , a transmembrane region (e.g., CD8 transmembrane region or CD28 transmembrane region) , a costimulatory domain (e.g., the intracellular signaling domain of 4-1BB, CD28, or both) , and a signaling domain (e.g., the T cell signaling domain of CD3ζ) .

In some embodiments, the anti-CD123 CAR provided herein has the amino acid sequence of SEQ ID NO: 369, which can be encoded by, for example, the nucleotide sequence of SEQ ID NO: 372. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 85%identical to SEQ ID NO: 369. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 88%identical to SEQ ID NO: 369. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 90%identical to SEQ ID NO: 369. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 95%identical to SEQ ID NO: 369. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 97%identical to SEQ ID NO: 369. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 99%identical to SEQ ID NO: 369.

In some embodiments, the CAR provided herein has the amino acid sequence of SEQ ID NO: 370, which can be encoded by, for example, the nucleotide sequence of SEQ ID NO: 373. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 85%identical to SEQ ID NO: 370. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 88%identical to SEQ ID NO: 370. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 90%identical to SEQ ID NO: 370. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 95%identical to SEQ ID NO: 370. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 97%identical to SEQ ID NO: 370. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 99%identical to SEQ ID NO: 370.

In some embodiments, the CAR provided herein has the amino acid sequence of SEQ ID NO: 371, which can be encoded by, for example, the nucleotide sequence of SEQ ID NO: 374. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 85%identical to SEQ ID NO: 371. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 88%identical to SEQ ID NO: 371. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 90%identical to SEQ ID NO: 371. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 95%identical to SEQ ID NO: 371. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 97%identical to SEQ ID NO: 371. In some embodiments, the CAR provided herein has an amino acid sequence that is at least 99%identical to SEQ ID NO: 371.

5.4 LACO-Stim fusion proteins

In some embodiments, the anti-CD123 antibodies and TCRs/CARs provided herein can be used in combination with fusion proteins that are referred to as Lymphocytes- Antigen presenting cells Co- stimulators ( “LACO-Stims” or “LACOs” ) . LACOs are fusion proteins comprising a first domain that activates an antigen-presenting cell ( “APC” ; e.g., a dendritic cell) and a second domain that activates an immune effector cell (e.g., a T cell) , wherein the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the antigen-presenting cell, or an antigen-binding fragment thereof; and wherein the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

In some embodiments, the fusion protein is a membrane protein. In some embodiments, the fusion protein is a soluble protein. In some embodiments, the fusion protein is a bispecific antibody. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain.

5.4.1 APC Activators

Fusion proteins (LACOs) provided herein comprise a first domain that activates an APC, wherein the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof. An APC refers to any cell that displays one or more antigens on its surface, for example, in combination with one or more major histocompatibility complex (MHC) proteins. The MHC/antigen complex can be recognized by T-cells using their T-cell receptors (TCRs) and illicit an immune response.

5.4.1.1 Antigen-presenting cells (APCs)

APCs include, for example, dendritic cells (DCs) , macrophages, monocytes, myeloid derived suppressor cells, certain B cells, T cells and Langerhans cells.

Dendritic Cells: Dendritic cells (DCs) are bone marrow-derived cells that function as professional antigen presenting cells. Immature DCs are characterized by a high capacity for antigen capture and processing, but low T cell stimulatory capability. Inflammatory mediators promote DC maturation. Once DCs have reached the mature stage, they have undergone a dramatic change in their properties. Specifically, they have substantially lost the ability to capture antigen and have acquired an increased capacity to stimulate T cells. Typically, mature DCs present antigen that has been captured at the level of peripheral tissues to naive T cells.

Macrophages: Macrophages are immune cells that are specialized for detection, phagocytosis, and destruction of target cells including pathogens and tumor cells. As such, macrophages are potent effectors of the innate immune system and are capable of at least three distinct anti-tumor functions: phagocytosis of dead and dying cells, cytotoxicity against tumor cells themselves, and presentation of tumor antigens to orchestrate an adaptive anti-tumor immune response. In adult humans, unpolarized, uncommitted, or resting macrophages (M0) differentiate from bone marrow-derived monocyte precursors and express the common markers of the lineage, including CD 14, CD 16, CD64, CD68, CD71, and CCR5. Exposure to various stimuli can induce M0 macrophages to polarize into several distinct populations identified by surface marker and cytokine/chemokine secretion.

Monocytes: Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen, and generally do not proliferate when in a steady state. Typically, they comprise chemokine receptors and pathogen recognition receptors that mediate migration from blood to tissues, for example, during an infection. Monocytes can produce inflammatory cytokines and/or take up cells and toxic molecules and can also differentiate into inflammatory DCs or macrophages.

Myeloid derived suppressor cells: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that expand in cancer, inflammation, infection and transplantation. MDSCs have a remarkable ability to regulate adaptive and innate immune responses. Despite the widely accepted immunosuppressive capacities of MDSC, a new function has emerged which is immune stimulation and antitumor activity. MDSCs consist of myeloid progenitor cells and immature myeloid cells, which can propagate continuously in pathological conditions, and are the main source of APCs. (Li A et al, Int J Clin Exp Med 2017; 10 (8) : 12217-12222)

B Cells: B cells account for up to 25%of all cells in some tumors and that 40%of tumor-infiltrating lymphocytes in some breast cancer subjects are B cells (Yuen et al. Trends Cancer, 2016, 2 (12) : 747-757) . Additionally, therapeutic immune checkpoint blockade may also target activated B cells, in additional to activated T cells, since PD-l, PD-L1, CTLA-4, and the B7 molecules are expressed on B cells. In addition to the immune-regulatory function of producing antibodies and antibody-antigen complexes, B cells can affect the functions of other immune cells by presenting antigens, providing co-stimulation and secreting cytokines. Membrane-bound immunoglobulin on the B cell surface serves as the cell’s receptor for antigen and is known as a B cell receptor (BCR) . Activation of BCRs on the surface of a B cell leads to clonal expansion of that B cell and specific antibody production. Additionally, B cells can internalize an antigen that binds to a BCR and present it to helper (CD4+) T cells. Unlike T cells, B cells can recognize soluble antigen for which their BCR is specific.

T cells: T cells are immune effector cells that play important roles in the induction and maintenance of an effective immune response, such as an antiviral response or antitumor response. It has been recognized in the art that T cells can also present peptide epitopes from both viral antigens and tumor antigens. See e.g., Atanackovic et al., Journal of immunological methods 278.1-2 (2003) : 57-66.

Langerhans cells: Langerhans cells constitute the first line of immunologic defense in the skin. These cells are derived from the bone marrow and can normally be found scattered among the keratinocytes of the stratum spinosum. Langerhans cells are APCs derived from the monocyte lineage and function in the afferent limb of the immune response. They take up foreign invaders and process them to present to T cells. Once they present antigens, they migrate to lymph nodes to activate T cells. These cells are essential for the induction of delayed-type hypersensitivity reactions.

5.4.1.2 Activation Receptor of APCs

As understood in the art, a molecule can activate an APC by promoting its maturation, pro-inflammatory status, cytotoxicity, antigen-presentation, epitope-spreading, cytokine production, co-stimulation of immune effector cells (e.g., T cells) , or any combination thereof. In some embodiments, the first domains of fusion proteins provided herein activate an APC by promoting the maturation and activation of the APC (e.g., a DC) . In some embodiments, the first domains of fusion proteins provided herein activate an APC by promoting epitope spreading among the APCs and other immune effector cells (e.g., T cells) . In some embodiments, the first domains of fusion proteins provided herein activate an APC by promoting antigen-presentation of the APC. In some embodiments, the first domains of fusion proteins provided herein activate an APC by promoting its cytotoxicity against the foreign substance (e.g., the cancer cell) .

In some embodiments, fusion proteins provided herein comprise a first domain that activates an APC, which comprises a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof. An “activation receptor” refers to a membrane protein expressed on the APC that can elicit signaling to promote the mobilization, differentiation, proliferation, and/or activation of the APC upon binding with a ligand or an antibody. APC activation receptors include, for example, CD40, CD80, CD86, CD91, DEC-205, and DC-SIGN.

A “ligand” of a receptor refers to a molecule that can selectively bind the receptor. In some embodiments, the ligand is a polypeptide. A “receptor-binding fragment” of a ligand refers to a fragment of the ligand that retains its capacity to bind its receptor. Various ligands can stimulate the growth, differentiation, migration, and/or activation of dendritic cells or other APCs by binding to an activation receptor on the APCs. (See, e.g., Banchereau J et al., Nature (1998) 392: 245-52; Young J W et al., Stem Cells (1996) 14: 376-387; Cella M et al., Curr Opin Immunol. (1997) 9: 10-16; Curti A et al., J. Biol. Regul. Homeost. Agents (2001) 15: 49-52) . Examples of ligands that can modulate differentiation, maturation, expansion and/or activation of dendritic cells or other APCs include, for example, CD40 ligand (CD40L) , CD80 ligand, CD86 ligand, CD91 ligand (RAP1) , DEC-205 ligand, and DC-SIGN ligand. In some embodiments, fusion proteins provided herein include a first domain that comprises a ligand disclosed herein that binds an activation receptor of APCs, or a receptor-binding fragment thereof.

In some embodiments, the first domain of the fusion proteins provided herein comprises an antibody that binds an activation receptor of an APC, or an antigen-binding fragment thereof. In some embodiments, the first domain of the fusion proteins provided herein comprises an antibody or an antigen-binding fragment that binds CD40, CD80, CD86, CD91, DEC-205, or DC-SIGN.

CD40/CD40L: CD40 is a 48 kD transmembrane glycoprotein surface receptor that is a member of the Tumor Necrosis Factor Receptor superfamily (TNFRSF) . Exemplary amino acid sequences of human CD40 are described (see, e.g., Accession: ALQ33424.1 GI: 957949089; SEQ ID NO: 340) , CD40 was initially characterized as a co-stimulatory receptor expressed on APCs that played a central role in B and T cell activation. The ligand for CD40, CD154 (also known as TRAP, T-BAM, CD40 Ligand or CD40L) is a type II integral membrane protein. CD40L has been reported to promote induction of dendritic cells and facilitate development of immunogenic responses. See, e.g., Elgueta R et al., Immunol Rev. (2009) 229 (1) : 10.1111; Ma D &Clark EA, Semin Immunol. 2009 21 (5) : 265-72; Borges L et al., J Immunol. (1999) 163: 1289-1297; Grewal I, Immunol Res. (1997) 16: 59-70. Exemplary polynucleotides that encode CD40 ligand and equivalents are described (see, e.g., Genbank Accession Nos. X65453 and L07414) , as are preparations, compositions, and methods of use (U.S. Pat. No. 6,290,972) . An exemplary amino acid sequence for human CD40L is provided below. The extracellular domain of CD40L includes amino acids 47-261 of SEQ ID NO: 375.

In some embodiments, the first domain of the fusion proteins provided herein comprises CD40L or a receptor-binding fragment of CD40L. In some embodiments, the receptor-binding fragment of CD40L comprises amino acids 119-261 of CD40L (SEQ ID NO: 375) . In some embodiments, the receptor-binding fragment of CD40L comprises the extracellular domain of CD40L. In some embodiments, the first domain of the fusion proteins provided herein comprises an antibody that binds CD40, or an antigen-binding fragment thereof.

CD80 and CD86: CD80 (B7.1) and CD86 (B7.2) expressed on APC play important roles of costimulatory molecules necessary for a sustained immune response. Exemplary amino acid sequences of human CD80 are described (see, e.g., Accession: EAW79565.1 GI: 119599971; SEQ ID NO: 387) . Exemplary amino acid sequences of human CD86 are described (see, e.g., Accession: NP_787058.5 GI: 1519311816; SEQ ID NO: 388) . CD80 and CD86 can bind to either CD28 or CTLA-4 (ligands for CD80/CD86, also referred to as their counter receptors on T cells) . CD80 is expressed on activated B cells and interferon induced monocytes, but not resting B cells. CD86 is constitutively expressed at very low levels on resting monocytes, dendritic cells and B cells, and its expression is enhanced on activated T cells, NK cells and B lymphocytes. CD80 is a 44-54kD glycoprotein comprised of a 223 amino acid extracellular domain, a 23 amino acid transmembrane domain, and a 61 amino acid cytoplasmic tail. CD80 contains 3 potential protein kinase phosphorylation sites. CD86 is a 306 amino acid membrane glycoprotein. It consists of a 220 amino acid extracellular region, a 23 amino acid hydrophobic transmembrane domain and a 60 amino acid cytoplasmic tail.

In some embodiments, the first domain of the fusion proteins provided herein comprises a CD80 ligand or a receptor-binding fragment of the CD80 ligand. In some embodiments, the first domain of the fusion proteins provided herein comprises a CD86 ligand or a receptor-binding fragment of the CD86 ligand. In some embodiments, the CD80/CD86 ligand is CD28. In some embodiments, the receptor-binding fragment of the CD80/CD86 ligand comprises the extracellular domain of CD28. In some embodiments, the CD80/CD86 ligand is CTLA-4. In some embodiments, the receptor-binding fragment of the CD80/CD86 ligand comprises the extracellular domain of CTLA-4. In some embodiments, the CD80 ligand is PD-L1. In some embodiments, the receptor-binding fragment of the CD80 ligand comprises the extracellular domain of PD-L1. In some embodiments, the first domain of the fusion proteins provided herein comprises an antibody that binds CD80, or an antigen-binding fragment thereof. In some embodiments, the first domain of the fusion proteins provided herein comprises an antibody that binds CD86, or an antigen-binding fragment thereof.

CD91/RAP1: CD91 is a receptor on APCs that influences response to nascent tumors. (Sedlacek AL et al., JCI Insight. 2019; 4 (7) : e127239) . Exemplary amino acid sequences of human CD91 are described (see, e.g., Accession: NP_002323.2 GI: 126012562; SEQ ID NO: 376) . CD91 provides an essential and highly efficient conduit for cross-presentation of tumor antigens to T cells, and this pathway is necessary for mounting successful immune responses for surveillance of tumors. CD91 is also involved in activating NK cell responses, activating DCs to produce costimulation, and priming T cells. Receptor-associated protein (RAP1) with a molecular weight of 39 kDa is an ER resident protein and molecular chaperone for LDL receptor-related protein that has a high binding affinity to CD91 (Kd: about 3 nM) and capable of activating the CD91 signaling in APCs. Exemplary polynucleotides that encode RAP1 and equivalents are described (see, e.g., Genbank Accession Nos. AAI12068.1, AAI05075.1, and P30533.1) . An exemplary amino acid sequence for human RAP1 is provided below. Domain 3 of RAP1 (amino acid resides 219-323 of RAP1, SEQ ID NO: 377) is known to bind CD91.

In some embodiments, the first domain of the fusion proteins provided herein comprises RAP1 or a receptor-binding fragment of RAP1. In some embodiments, the receptor-binding fragment of RAP1 comprises domain 3 of RAP1. In some embodiments, the first domain of the fusion proteins provided herein comprises an antibody that binds CD91, or an antigen-binding fragment thereof.

DEC-205: The function of an APC has been connected to high levels in the expression of the DEC-205 receptor, also called CD205 or lymphocytic antigen 75, especially in dendritic cells located in areas of T cells of peripheral or secondary lymph organs. Exemplary amino acid sequences of human DEC-205 are described (see, e.g., Accession: NP_002340.2 GI: 144446030; SEQ ID NO: 378) . The DEC-205 receptor is an endocytic receptor with a broad extracellular domain that contains various subdomains: a cysteine-rich (CR) domain, a fibronectin type II (FN) domain and 10 contiguous carbohydrate recognition domains (CRDs) . These multi-lectin domains affect the efficiency of the processing and presentation of antigens in vivo. The pioneering experiments that described the cellular processes of directing an antigen were carried out using the DEC-205 human receptor, where the T-cell-mediated response changes dramatically when the maturation stimulus of the dendritic cells is added at the same time as the directing of the antigen using an antibody directed against the DEC-205 receptor. The proliferation of T cells increases by various orders of magnitude when compared to a classic immunization protocol. When the antigens are directed at the dendritic cells via DEC-205, there is an increase in the stimulation of the cooperating T cells (Th) ; this triggers or promotes the humoral immune response or antibody production.

In some embodiments, the first domain of the fusion proteins provided herein comprises a DEC-205 ligand or a receptor-binding fragment of a DEC-205 ligand. Keratins are natural ligands for DEC-205. In some embodiments, the first domain of the fusion proteins provided herein comprises a keratin or a receptor-binding fragment of a keratin. In some embodiments, the first domain of the fusion proteins provided herein comprises an antibody that binds DEC-205, or an antigen-binding fragment thereof.

DC-SIGN: DC specific ICAM-3 grabbing non-integrin (DC-SIGN) receptor is a C-type lectin containing an external calcium-dependent mannose binding lectin domain. DC-SIGN interacts with a variety of compounds such as the envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) , HIV-2 and simian immunodeficiency virus (SIV) as well as other pathogens such as hepatitis C, Ebola, cytomegalovirus, Dengue virus, Mycobacterium, Leishmania, Candida albicans and Helicobacter pylori. DC-SIGN plays an important role in pathogen transmission and the establishment of infection. Exemplary amino acid sequences of human DC-SIGN are described (see, e.g., Accession: AAK20997.1 GI: 13383468; SEQ ID NO: 379) .

The DC-SIGN receptor is also capable of binding ICAM2 and ICAM3. ICAM2 is expressed on endothelial cells and ICAM3 is expressed on T cells. DC-SIGN furthermore interacts with β2-integrin Mac-1 (CD11b/CD18) , which is expressed on neutrophils and promotes the interaction with DC cells, therefore controlling the immune responses mounted. As another example, CEACAM1, which is expressed on neutrophils is also capable of interacting with DC-SIGN.

In some embodiments, the first domain of the fusion proteins provided herein comprises a DC-SIGN ligand or a receptor-binding fragment of the DC-SIGN ligand. In some embodiments, the DC-SIGN ligand is ICAM2, ICAM3, CD18, or CEACAM1, or a receptor-binding fragment thereof. In some embodiments, the DC-SIGN ligand is ICAM2 or a receptor-binding fragment thereof. Exemplary amino acid sequences of human ICAM2 are described (see, e.g., Accession: CAG46611.1 GI: 49456581; SEQ ID NO: 380) . In some embodiments, the DC-SIGN ligand is ICAM3 or a receptor-binding fragment thereof. Exemplary amino acid sequences of human ICAM3 are described (see, e.g., Accession: P32942.2 GI: 206729872; SEQ ID NO: 381) . In some embodiments, the DC-SIGN ligand is CD18 or a receptor-binding fragment thereof. Exemplary amino acid sequences of human CD18 are described (see, e.g., Accession: P05107.2 GI: 124056465; SEQ ID NO: 382) . In some embodiments, the DC-SIGN ligand is CEACAM1 or a receptor-binding fragment thereof. Exemplary amino acid sequences of human CEACAM1 are described (see, e.g., Accession: AAH14473.1 GI: 15680237; SEQ ID NO: 383) . In some embodiments, the first domain of the fusion proteins provided herein comprises an antibody that binds DC-SIGN, or an antigen-binding fragment thereof.

5.4.2 Co-stimulatory receptors of immune effector cells

In some embodiments, provided herein are fusion proteins comprising a first domain that activates an antigen-presenting cell (e.g., a dendritic cell) and a second domain that activates an immune effector cell (e.g., a T cell) , wherein the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

“Immune effector cells” as used herein and understood in the art refer to cells that are of hematopoietic origin and play a direct role in the immune response against a target, such as a pathogen, a cancer cell, or a foreign substance. Immune effector cells include T cells, B cell, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils. In some embodiments, the second domain of the fusion proteins provided herein that activates an immune effector cell comprises a co-stimulatory receptor of the immune effector cell. In some embodiments, the immune effector cell is a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, or a granulocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a macrophage.

“Stimulation” of an immune effector cell means a primary response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event in the immune effector cell which can alter expression of certain genes and/or reorganization of cytoskeletal structures, and the like. A “stimulatory molecule” of an immune effector cell refers to a molecule on the immune effector cell that, upon binding with its cognate ligand, which is commonly present on an APC, can mediate signal transduction to promote the maturation, differentiation, proliferation, and/or activation of the immune effector cell. For example, a stimulatory molecule of the T cells, the TCR/CD3 complex triggers the activation of the T cells. The ligand for a stimulatory molecule, or “stimulatory ligand, ” means a ligand that is commonly present on an APC and can bind with a stimulatory molecule on the immune effector cell to mediate a primary response by the immune effector cell, including, but not limited to, maturation, differentiation, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, for example, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

A “co-stimulatory signal, ” as used herein and understood in the art, refers to a signal from a co-stimulatory receptor (e.g., CD28 or 4-1BB) , which in combination with a primary signal (e.g., TCR/CD3) promotes optimal clonal expansion, differentiation and effector functions of immune effector cells (e.g., T cells) . A “co-stimulatory receptor” of an immune effector cell, s used herein and understood in the art, refers to a molecule on the immune effector cell that specifically binds with a “co-stimulatory ligand” to mediate a co-stimulatory response by the immune effector cell, such as heightened activation or proliferation of the immune effector cell. Co-stimulatory receptors for immune effector cells include, but are not limited to, CD28, 4-1BB, ICOS, CD27, OX40, DAP10, CD30, 2B4, CD2, LIGHT, GITR, TLR, DR3, and CD43. A “functional fragment” of a co-stimulatory receptor is a fragment of the co-stimulatory receptor that retains its function to mediate a co-stimulatory signal and stimulate the immune effector cell. In some embodiments, a functional fragment of a co-stimulatory receptor retains the co-stimulatory domain of the co-stimulatory receptor. In some embodiments, the co-stimulatory domain is the cytoplasmic domain of the co-stimulatory receptor. In some embodiments, signals from co-stimulatory receptors of immune effector cells (e.g., T cells) lower the activation threshold for the immune effector cells. In some embodiments, signals from co-stimulatory receptors of T cells lead to the augmentation of TCR signaling events necessary for efficient cytokine production (via augmented transcriptional activity and messenger RNA stabilization) , cell cycle progression, survival, regulation of metabolism and T cell responses.

A “co-stimulatory ligand, ” as used herein and understood in the art, refers to a molecule that specifically binds a cognate co-stimulatory receptor on an immune effector cell, thereby providing a signal which, in addition to the primary signal provided by the stimulatory molecule, mediates a response in the immune effector cell, including, but not limited to, proliferation, activation, differentiation, and the like. The co-stimulatory ligand can be present on an APC (e.g., a dendritic cell) . Co-stimulatory ligands include, but are not limited to, CD58, CD70, CD83, CD80, CD86, CD137L (4-1BBL) , CD252 (OX40L) , CD275 (ICOS-L) , CD54 (ICAM-1) , CD49a, CD112 (PVRL2) , CD150 (SLAM) , CD155 (PVR) , CD265 (RANK) , CD270 (HVEM) , TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153 (CD30L) , CD48, CD160, CD200R (OX2R) , and CD44. A “receptor-binding fragment” of a co-stimulatory ligand refers to a fragment of the ligand that retains its capacity to bind its receptor.

Some co-stimulatory receptors and co-stimulatory ligands are exemplified below. It is understood that any co-stimulatory receptors and/or co-stimulatory ligands provided herein or otherwise known in the art can be used as part of the fusion proteins provided herein.

CD28: Cluster of Differentiation 28 (CD28) is a protein expressed on T cells that provides co-stimulatory signals for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins. CD28 is a co-stimulatory receptor for optimal T cell clonal expansion, differentiation and effector functions. CD28 engagement lowers the T cell activation threshold and leads to the augmentation of TCR signaling events necessary for efficient cytokine production (via augmented transcriptional activity and messenger RNA stabilization) , cell cycle progression, survival, regulation of metabolism and T cell responses. CD28 is a crucial player for immunological synapse (IS) organization, where it enhances close contact between T cells and APCs.

Provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises a CD28 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of CD28. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD28. In some embodiments, the ligand of CD28 is CD80. In some embodiments, the second domain comprises an antibody that binds CD28, or an antigen-binding fragment thereof. In some embodiments, the second domain of fusion proteins provided herein comprises a functional fragment of CD28, which comprises a portion of an intracellular/cytoplasmic domain of CD28 that can function as a co-stimulatory signaling domain. A CD28 can have the amino acid sequence of GenBank No. P10747 (P10747.1, GI: 115973) or NP_006130 (NP_006130.1, GI: 5453611) , as provided below, or functional fragments thereof. In some embodiments, a fusion protein disclosed herein can have an amino acid sequence comprising the cytoplasmic domain of CD28 corresponding to amino acids 180 to 220 of the sequence below, or a fragment thereof. In another embodiment, a fusion protein disclosed herein can have an amino acid sequence further comprising the transmembrane domain of CD28 corresponding to amino acids 153 to 179, or a functional fragment thereof. It is understood that sequences of CD28 that are shorter or longer than a specific delineated domain can be included in a fusion protein disclosed herein, if desired.

4-1BB. 4-1BB, also referred to as tumor necrosis factor receptor superfamily member 9, can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity (Stephan MT et al., Nat Med (2007) 13 (12) : 1440-1449) . Provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises a 4-1BB polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of 4-1BB. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds 4-1BB. In some embodiments, the ligand of 4-1BB is 4-1BBL. In some embodiments, the second domain comprises an antibody that binds 4-1BB, or an antigen-binding fragment thereof. In some embodiments, the second domain of fusion proteins provided herein can comprise a co-stimulatory signaling domain derived from 4-1BB. A 4-1BB polypeptide can have the amino acid sequence of GenBank No. P41273 (P41273.1, GI: 728739) or NP_001552 (NP_001552.2, GI: 5730095) or fragments thereof. In some embodiments, the second domain of fusion proteins provided herein can have a co-stimulatory domain comprising the cytoplasmic domain of 4-1BB corresponding to amino acids 214 to 255 of the sequence below, or a functional fragment thereof. It is understood that sequences of 4-1BB that are shorter or longer than a specific delineated domain can be included in a fusion protein disclosed herein, if desired.

ICOS. Inducible T-cell co-stimulator precursor (ICOS) , also referred to as CD278, is a CD28-superfamily co-stimulatory receptor that is expressed on activated T cells. Provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises an ICOS polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of ICOS. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds ICOS. In some embodiments, the ligand of ICOS is CD275 (ICOS-L) . In some embodiments, the second domain comprises an antibody that binds ICOS, or an antigen-binding fragment thereof. In some embodiments, the second domain of fusion proteins provided herein can comprise a co-stimulatory signaling domain derived from ICOS. An ICOS polypeptide can have the amino acid sequence of GenBank No. NP_036224 (NP_036224.1, GI: 15029518) , provided below, or fragments thereof. In some embodiments, the second domain of fusion proteins provided herein can have a co-stimulatory domain comprising the cytoplasmic domain of ICOS corresponding to amino acids 162 to 199 of the sequence below, or a functional fragment thereof. It is understood that sequences of ICOS that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

CD27: CD27 (TNFRSF7) is a transmembrane receptor expressed on subsets of human CD8+and CD4+ T-cells, NKT cells, NK cell subsets and hematopoietic progenitors and induced in FOXP3+ CD4 T-cells and B cell subsets. Previous studies have found that CD27 can provide costimulatory signals that improve human T-cell survival and anti-tumor activity in vivo. (See Song and Powell; Oncoimmunology 1 (4) : 547-549 (2012) ) . Provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell. In some embodiments, the second domain comprises a CD27 polypeptide, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of CD27. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD27. In some embodiments, the ligand of CD27 is CD70. In some embodiments, the second domain comprises an antibody that binds CD27, or an antigen-binding fragment thereof. In some embodiments, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from CD27. A CD27 polypeptide can have the amino acid sequence of UniProtKB/Swiss-Prot No.: P26842.2 (GI: 269849546) , provided below, or fragments thereof. In some embodiments, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain comprising the cytoplasmic domain of CD27 corresponding to amino acids 213 to 260 of the sequence below, or a functional fragment thereof. It is understood that sequences of CD27 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

Co-stimulatory receptors for immune effector cells include, but are not limited to, CD28, 4-1BB, ICOS, CD27, OX40, DAP10, CD30, 2B4, CD2, LIGHT, GITR, TLR, DR3, and CD43. In addition to the exemplified co-stimulatory receptors above, the following embodiments are explicitly contemplated herein.

Provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell. In some embodiments, the second domain of fusion proteins provided herein comprises an OX40 polypeptide, or a functional fragment thereof. An OX40 polypeptide can have an amino acid sequence corresponding to GenBank No. P43489 (P43489.1, GI: 1171933) or NP_003318 (NP_003318.1, GI: 4507579) . An OX40 polypeptide can have an amino acid sequence of SEQ ID NO: 335, or fragments thereof. In some embodiments, the second domain comprises the cytoplasmic domain of OX40. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds OX40. In some embodiments, the ligand of OX40 is CD252. In some embodiments, the second domain comprises an antibody that binds OX40, or an antigen-binding fragment thereof. In some embodiments, the second domain can comprise a co-stimulatory signaling domain derived from OX40. An OX40 co-stimulatory signaling domain can comprise the cytoplasmic domain of OX40 corresponding to amino acids 236 to 277 of SEQ ID NO: 335, or a functional fragment thereof. It is understood that sequences of OX40 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

In some embodiments, the second domain of fusion proteins provided herein comprises a DAP10 polypeptide, or a functional fragment thereof. A DAP10 polypeptide can have the amino acid sequence of GenBank No. NP_055081.1 (GI: 15826850) , or fragments thereof. A DAP10 polypeptide can have the amino acid sequence of SEQ ID NO: 331. In some embodiments, the second domain comprises the cytoplasmic domain of DAP10. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds DAP10. In some embodiments, the second domain comprises an antibody that binds DAP10, or an antigen-binding fragment thereof. In some embodiments, the second domain can comprise a co-stimulatory signaling domain derived from DAP10. A DAP10 co-stimulatory signaling domain can have the cytoplasmic domain of DAP10 corresponding to amino acids 70 to 93 of SEQ ID NO: 331, or a functional fragment thereof. It is understood that sequences of DAP10 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

In some embodiments, the second domain of fusion proteins provided herein comprises a CD30 polypeptide, or a functional fragment thereof. A CD30 polypeptide can have the amino acid sequence of GenBank No.: AAA51947.1 (GI: 180096) or fragments thereof. A CD30 polypeptide can have the amino acid sequence of SEQ ID NO: 339. In some embodiments, the second domain comprises the cytoplasmic domain of CD30. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD30. In some embodiments, the ligand of CD30 is CD153. In some embodiments, the second domain comprises an antibody that binds CD30, or an antigen-binding fragment thereof. In some embodiments, the second domain can comprise a co-stimulatory domain derived from CD30. A CD30 co-stimulatory signaling domain can have the cytoplasmic domain corresponding to amino acids 407 to 595 of SEQ ID NO: 339, or a functional fragment thereof. It is understood that sequences of CD30 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

In some embodiments, the second domain of fusion proteins provided herein comprises a 2B4 polypeptide, or a functional fragment thereof. A 2B4 polypeptide can have the amino acid sequence of Accession No: Q9BZW8.2 (GI: 47605541) or fragments thereof. A 2B4 polypeptide can have the amino acid sequence of SEQ ID NO: 337. In some embodiments, the second domain comprises the cytoplasmic domain of 2B4. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds 2B4. In some embodiments, the second domain comprises an antibody that binds 2B4, or an antigen-binding fragment thereof. In some embodiments, the second domain of fusion proteins provided herein can comprise a co-stimulatory domain derived from 2B4. In some embodiments, the second domain can comprise a co-stimulatory domain comprising the cytoplasmic domain of 2B4 corresponding to amino acids 251 to 370 of SEQ ID NO: 337, or a functional fragment thereof. It is understood that sequences of 2B4 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

In some embodiments, the second domain of fusion proteins provided herein comprises a CD2 polypeptide, or a functional fragment thereof. A CD2 polypeptide can have the amino acid sequence of Accession: NP_001758.2 GI: 156071472 or fragments thereof. A CD2 polypeptide can have the amino acid sequence of SEQ ID NO: 341. In some embodiments, the second domain comprises the cytoplasmic domain of CD2. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD2. In some embodiments, the ligand of CD2 is CD58. In some embodiments, the ligand of CD2 is CD48. In some embodiments, the second domain comprises an antibody that binds CD2, or an antigen-binding fragment thereof. In some embodiments, the second domain can comprise a co-stimulatory domain derived from CD2. A CD2 co-stimulatory signaling domain can have the cytoplasmic domain of CD2 corresponding to amino acids 236 to 351 of SEQ ID NO: 341, or a functional fragment thereof. It is understood that sequences of CD2 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

In some embodiments, the second domain of fusion proteins provided herein comprises a LIGHT polypeptide, or a functional fragment thereof. A LIGHT polypeptide can have an amino acid sequence corresponding to the sequence provided below (Accession: NP_001363816.1 GI: 1777376047) , or fragments thereof. A LIGHT polypeptide can have the amino acid sequence of SEQ ID NO: 342. In some embodiments, the second domain comprises the cytoplasmic domain of LIGHT. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds LIGHT. In some embodiments, the second domain comprises an antibody that binds LIGHT, or an antigen-binding fragment thereof. In some embodiments, the second domain can comprise a co-stimulatory domain derived from LIGHT. A LIGHT co-stimulatory signaling domain can have the cytoplasmic domain of LIGHT corresponding to amino acids 1 to 37 of SEQ ID NO: 342, or a functional fragment thereof. It is understood that sequences of LIGHT that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

In some embodiments, the second domain of fusion proteins provided herein comprises a GITR polypeptide, or a functional fragment thereof. A GITR polypeptide can have an amino acid sequence corresponding to the sequence of GENBANK Accession: AAI52382.1 (GI: 158931986) , or fragments thereof. A GITR polypeptide can have the amino acid sequence of SEQ ID NO: 343. In some embodiments, the second domain comprises the cytoplasmic domain of GITR. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds GITR. In some embodiments, the ligand of GITR is GITR-L. In some embodiments, the second domain comprises an antibody that binds GITR, or an antigen-binding fragment thereof. In some embodiments, the second domain can comprise a co-stimulatory domain derived from GITR. A GITR co-stimulatory signaling domain can have the cytoplasmic domain of GITR corresponding to amino acids 184 to 241 of SEQ ID NO: 343, or a functional fragment thereof. It is understood that sequences of GITR that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

In some embodiments, the second domain of fusion proteins provided herein comprises a DR3 polypeptide, or a functional fragment thereof. A DR3 polypeptide can have an amino acid sequence corresponding to the sequence of GENBANK Accession No.: AAI17190.1 GI: 109658976) , or fragments thereof. A DR3 polypeptide can have the amino acid sequence of SEQ ID NO: 344. In some embodiments, the second domain comprises the cytoplasmic domain of DR3. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds DR3. In some embodiments, the second domain comprises an antibody that binds DR3, or an antigen-binding fragment thereof. In some embodiments, the second domain can comprise a co-stimulatory domain derived from DR3. A DR3 co-stimulatory signaling domain can have the cytoplasmic domain of DR3 corresponding to amino acids 221 to 417 of SEQ ID NO: 344, or a functional fragment thereof. It is understood that sequences of DR3 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

In some embodiments of fusion proteins provided herein, the second domain comprises a CD43 polypeptide, or a functional fragment thereof. A CD43 polypeptide can have an amino acid sequence corresponding to the sequence provided of GENBANK Accession: EAW80016.1 (GI: 119600422) or Accession: EAW80015.1 (GI: 119600421) , or fragments thereof. A CD43 polypeptide can have the amino acid sequence of SEQ ID NO: 345. In some embodiments, the second domain comprises the cytoplasmic domain of CD43. In some embodiments, the second domain comprises a ligand or a receptor-binding fragment thereof that binds CD43. In some embodiments, the second domain comprises an antibody that binds CD43, or an antigen-binding fragment thereof. In some embodiments, the second domain can comprise a co-stimulatory domain derived from CD43. A CD43 co-stimulatory signaling domain can have the cytoplasmic domain of CD43 corresponding to amino acids 277 to 400 of SEQ ID NO: 345, or a functional fragment thereof. It is understood that sequences of CD43 that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

In some embodiments, provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the second domain comprises a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof. In some embodiments, the second domain comprises a co-stimulatory signaling domain of a co-stimulatory receptor. The co-stimulatory ligand can CD58, CD70, CD83, CD80, CD86, CD137L (4-1BBL) , CD252 (OX40L) , CD275 (ICOS-L) , CD54 (ICAM-1) , CD49a, CD112 (PVRL2) , CD150 (SLAM) , CD155 (PVR) , CD265 (RANK) , CD270 (HVEM) , TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153 (CD30L) , CD48, CD160, CD200R (OX2R) , or CD44.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD58 polypeptide, or a receptor-binding fragment thereof. A CD58 polypeptide can have the amino acid sequence of GENBANK Accession No. NP_001770 or NP_001138294. A CD58 polypeptide can have the amino acid sequence of SEQ ID NO: 384. In some embodiments, the second domain comprises the extracellular domain of CD58 corresponding to amino acids 29-215 of SEQ ID NO: 384.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD70 polypeptide, or a receptor-binding fragment thereof. A CD70 polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_001243; NP_001317261; or XP_016883012. A CD70 polypeptide can have the amino acid sequence of SEQ ID NO: 385. In some embodiments, the second domain comprises the extracellular domain of CD70 corresponding to amino acids 39-193 of SEQ ID NO: 385.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD83 polypeptide, or a receptor-binding fragment thereof. A CD83 polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_001035370, NP_001238830, or NP_004224. A CD83 polypeptide can have the amino acid sequence of SEQ ID NO: 386. In some embodiments, the second domain comprises the extracellular domain of CD83 corresponding to amino acids 20-144 of SEQ ID NO: 386.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD80 polypeptide, or a receptor-binding fragment thereof. A CD80 polypeptide can have the amino acid sequence of GENBANK Accession NO: EAW79565.1; or NP_005182. A CD80 polypeptide can have the amino acid sequence of SEQ ID NO: 387. In some embodiments, the second domain comprises the extracellular domain of CD80 corresponding to amino acids 35-242 of SEQ ID NO: 387.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD86 polypeptide, or a receptor-binding fragment thereof. A CD86 polypeptide can have the amino acid sequence of GENBANK Accession No.: NP_787058.5, or NP_001193853. A CD86 polypeptide can have the amino acid sequence of SEQ ID NO: 388. In some embodiments, the second domain comprises the extracellular domain of CD86 corresponding to amino acids 24-247 of SEQ ID NO: 388.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD137L polypeptide, or a receptor-binding fragment thereof. A CD137L polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_003802.1. A CD137L polypeptide can have the amino acid sequence of SEQ ID NO: 389. In some embodiments, the second domain comprises the extracellular domain of CD137L corresponding to amino acids 50-254 of SEQ ID NO: 389.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD252 polypeptide, or a receptor-binding fragment thereof. A CD252 polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_001284491, XP_005245532, or NP_003317. A CD252 polypeptide can have the amino acid sequence of SEQ ID NO: 390. In some embodiments, the second domain comprises the extracellular domain of CD252 corresponding to amino acids 51-183 of SEQ ID NO: 390.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD275 polypeptide, or a receptor-binding fragment thereof. A CD275polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_001269979, NP_001269980, NP_001269981, NP_056074, NP_001352688, or XP_016883799. A CD275 polypeptide can have the amino acid sequence of SEQ ID NO: 391. In some embodiments, the second domain comprises the extracellular domain of CD275 corresponding to amino acids 19-256 of SEQ ID NO: 391.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD54 polypeptide, or a receptor-binding fragment thereof. A CD54 polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_000192. A CD54 polypeptide can have the amino acid sequence of SEQ ID NO: 392. In some embodiments, the second domain comprises the extracellular domain of CD54 corresponding to amino acids 28-480 of SEQ ID NO: 392.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD49a polypeptide, or a receptor-binding fragment thereof. A CD49a polypeptide can have the amino acid sequence of GENBANK NP_852478. A CD49a polypeptide can have the amino acid sequence of SEQ ID NO: 393. In some embodiments, the second domain comprises the extracellular domain of CD49a corresponding to amino acids 29-1141 of SEQ ID NO: 393.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD112 polypeptide, or a receptor-binding fragment thereof. A CD112 polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_001036189, NP_002847. A CD112 polypeptide can have the amino acid sequence of SEQ ID NO: 394. In some embodiments, the second domain comprises the extracellular domain of CD112 corresponding to amino acids 32-360 of SEQ ID NO: 394.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD150 polypeptide, or a receptor-binding fragment thereof. A CD150 polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_001317683, XP_016857618, or NP_003028. A CD150 polypeptide can have the amino acid sequence of SEQ ID NO: 395. In some embodiments, the second domain comprises the extracellular domain of CD150 corresponding to amino acids 21-237 of SEQ ID NO: 395.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD155 polypeptide, or a receptor-binding fragment thereof. A CD155 polypeptide can have the amino acid sequence of GENBANK Accession No: NP_001129240; NP_001129241; NP_001129242; or NP_006496. A CD155 polypeptide can have the amino acid sequence of SEQ ID NO: 396. In some embodiments, the second domain comprises the extracellular domain of CD155 corresponding to amino acids 21-343 of SEQ ID NO: 396.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD265 polypeptide, or a receptor-binding fragment thereof. A CD265 polypeptide can have the amino acid sequence of GENBANK Accession No: NP_001257878, NP_001257879, or NP_003830. A CD265 polypeptide can have the amino acid sequence of SEQ ID NO: 397. In some embodiments, the second domain comprises the extracellular domain of CD265 corresponding to amino acids 30-212 of SEQ ID NO: 397.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD270 polypeptide, or a receptor-binding fragment thereof. A CD270 polypeptide can have the amino acid sequence of GENBANK Accession No: NP_001284534 or NP_003811. A CD270 polypeptide can have the amino acid sequence of SEQ ID NO: 398. In some embodiments, the second domain comprises the extracellular domain of CD270 corresponding to amino acids 39-202 corresponding to amino acids of SEQ ID NO: 398.

In some embodiments, the second domain of the fusion proteins provided herein comprises a TL1A polypeptide, or a receptor-binding fragment thereof. A TL1A polypeptide can have the amino acid sequence of GENBANK Accession No. NP_005109; or NP_001191273. A TL1A polypeptide can have the amino acid sequence of SEQ ID NO: 399. In some embodiments, the second domain comprises the extracellular domain of TL1A corresponding to amino acids 57-251 of SEQ ID NO: 399.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD127 polypeptide, or a receptor-binding fragment thereof. A CD127 polypeptide can have the amino acid sequence of GENBANK Accession No: NP_002176, or XP_942460. A CD127 polypeptide can have the amino acid sequence of SEQ ID NO: 400. In some embodiments, the second domain comprises the extracellular domain of CD127 corresponding to amino acids 21-239 SEQ ID NO: 400.

In some embodiments, the second domain of the fusion proteins provided herein comprises an IL-4R polypeptide, or a receptor-binding fragment thereof. An IL-4R polypeptide can have the amino acid sequence of GENBANK Accession No.: NP_000409, NP_001244335, NP_001244336, or NP_001244926. An IL-4R polypeptide can have the amino acid sequence of SEQ ID NO: 401. In some embodiments, the second domain comprises the extracellular domain of IL-4R corresponding to amino acids 26-232 of SEQ ID NO: 401.

In some embodiments, the second domain of the fusion proteins provided herein comprises a GITR-L polypeptide, or a receptor-binding fragment thereof. A GITR-L polypeptide can have the amino acid sequence of GENBANK Accession No: NP_005083. A GITR-L polypeptide can have the amino acid sequence of SEQ ID NO: 402. In some embodiments, the second domain comprises the extracellular domain of GITR-L corresponding to amino acids 72-199 of SEQ ID NO: 402.

In some embodiments, the second domain of the fusion proteins provided herein comprises a TIM-4 polypeptide, or a receptor-binding fragment thereof. A TIM-4 polypeptide can have the amino acid sequence of GENBANK Accession: NP_001140198.1; NP_612388.2; or Q96H15.2. A TIM-4 polypeptide can have the amino acid sequence of SEQ ID NO: 403. In some embodiments, the second domain comprises the extracellular domain of TIM-4 corresponding to amino acids 25-314 of SEQ ID NO: 403.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD153 polypeptide, or a receptor-binding fragment thereof. A CD153 polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_001235, or NP_001239219. A CD153 polypeptide can have the amino acid sequence of SEQ ID NO: 404. In some embodiments, the second domain comprises the extracellular domain of CD153 corresponding to amino acids 63-234 of SEQ ID NO: 404.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD48 polypeptide, or a receptor-binding fragment thereof. A CD48 polypeptide can have the amino acid sequence of GENBANK Accession: EAW52705.1 (GI: 119573090) ; or Accession: CAG33293.1 (GI: 48146141) . A CD48 polypeptide can have the amino acid sequence of SEQ ID NO: 405. In some embodiments, the second domain comprises the extracellular domain of CD48 corresponding to amino acids 27-220 of SEQ ID NO: 405.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD160 polypeptide, or a receptor-binding fragment thereof. A CD160 polypeptide can have the amino acid sequence of GENBANK. A CD160 polypeptide can have the amino acid sequence of SEQ ID NO: 406. In some embodiments, the second domain comprises the mature form of CD160 corresponding to amino acids 25-159 of SEQ ID NO: 406.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD200R polypeptide, or a receptor-binding fragment thereof. A CD200R polypeptide can have the amino acid sequence of GENBANK Accession NO: NP_620161; or NP_620385. A CD200R polypeptide can have the amino acid sequence of SEQ ID NO: 407. In some embodiments, the second domain comprises the extracellular domain of CD200R corresponding to amino acids 29-243 of SEQ ID NO: 407.

In some embodiments, the second domain of the fusion proteins provided herein comprises a CD44 polypeptide, or a receptor-binding fragment thereof. A CD44 polypeptide can have the amino acid sequence of GENBANK Accession No. NP_000601, or NP_001001389. A CD44 polypeptide can have the amino acid sequence of SEQ ID NO: 408. In some embodiments, the second domain comprises the extracellular domain of CD44 corresponding to amino acids 21-649 of SEQ ID NO: 408.

It is understood that sequences of the co-stimulatory ligand described herein that are shorter or longer than a specific delineated domain can be included in a fusion protein, if desired.

5.4.3 Exemplary LACO-Stim Fusion Proteins

In some embodiments of the fusion proteins provided herein, the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof, wherein the activation receptor of the APC is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN. In some embodiments, the first domain comprises a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof. In some embodiments, the first domain comprises a ligand that binds CD40, or a receptor-binding fragment thereof. In some embodiments, the first domain comprises the extracellular domain of CD40L. In some embodiments, the receptor-binding fragment of CD40L comprises a receptor-binding fragment (e.g., amino acids 119-261) of CD40L. In some embodiments, the first domain comprises CD40L. In some embodiments, the first domain of the fusion proteins provided herein comprises three copies of CD40L or a receptor-binding fragment of CD40L. In some embodiments, the first domain of the fusion proteins provided herein comprises three copies of amino acids 119-261 of CD40L. In some embodiments, the first domain comprises a ligand that binds CD80, or a receptor-binding fragment thereof. In some embodiments, the first domain comprises a ligand that binds CD86, or a receptor- binding fragment thereof. In some embodiments, the first domain comprises the extracellular domain of CD28. In some embodiments, the first domain comprises CD28. In some embodiments, the first domain comprises the extracellular domain of CTLA-4. In some embodiments, the first domain comprises CTLA-4. In some embodiments, the first domain comprises a ligand that binds CD91, or a receptor-binding fragment thereof. In some embodiments, the first domain comprises domain 3 of RAP1. In some embodiments, the first domain comprises RAP1. In some embodiments, the first domain comprises a ligand that binds DEC-205, or a receptor-binding fragment thereof. In some embodiments, the first domain comprises a ligand that binds DC-SIGN, or a receptor-binding fragment thereof. In some embodiments, the first domain comprises ICAM2, ICAM3, CD18, or CEACAM1, or a receptor-binding fragment of. In some embodiments, the first domain comprises ICAM2, or a receptor-binding fragment of. In some embodiments, the first domain comprises ICAM3, or a receptor-binding fragment of. In some embodiments, the first domain comprises CD18, or a receptor-binding fragment of. In some embodiments, the first domain comprises CEACAM1, or a receptor-binding fragment of.

In some embodiments, the first domain comprises an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof. In some embodiments, the activation receptor of the APC is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN. In some embodiments, the first domain comprises an antibody that binds CD40, or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an antibody that binds CD80, or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an antibody that binds CD86, or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an antibody that binds CD91, or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an antibody that binds DEC-205, or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an antibody that binds DC-SIGN, or an antigen-binding fragment thereof. In some embodiments, the first domain comprises a monoclonal antibody. In some embodiments the first domain comprises a chimeric antibody. In some embodiments the first domain comprises a humanized antibody. In some embodiments the first domain comprises a human antibody. In some embodiments, the first domain comprises a Fab, Fab’, F (ab’) 2, Fv, scFv, (scFv) 2, single chain antibody, dual variable region antibody, diabody, nanobody, or single variable region antibody. In some embodiments the first domain comprises a human antibody. In some embodiments, the first domain comprises a scFv.

In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 antibody or antigen-binding fragment thereof. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises the antibody designated as F2.103, F5.157, F5.77, 4D11, A40C, 119, 40-45, or 40-52 as provided below in Table 5.

Table 5: Anti-CD40 Antibodies

In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv. In some embodiments, the first domain of the fusion proteins provided herein comprise an anti-CD40 scFv having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 409-416. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 409. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 410. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 411. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 412. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 413. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 414. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 415. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence of SEQ ID NO: 416.

In some embodiments, the second domain of fusion proteins provided herein comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, or (b) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. The immune effector cell can be selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte. In some embodiments, the second domain of fusion proteins provided herein comprises a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, wherein the immune cell is a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, or a granulocyte. In some embodiments, the co-stimulatory receptor of the immune effector cell is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, DR3, and CD43. In some embodiments, the second domain of fusion proteins provided herein comprises a functional fragment of a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, DR3, and CD43. In some embodiments, the functional fragment comprises the cytoplasmic domain of the co-stimulatory receptor. In some embodiments, the second domain of fusion proteins provided herein further comprises the transmembrane domain of the co-stimulatory receptor. In some embodiments, the second domain comprises a functional fragment of CD28. In some embodiments, the second domain comprises the cytoplasmic domain of CD28. In some embodiments, the second domain comprises a functional fragment of 4-1BB. In some embodiments, the second domain comprises the cytoplasmic domain of 4-1BB. In some embodiments, the second domain comprises a functional fragment of ICOS. In some embodiments, the second domain comprises the cytoplasmic domain of ICOS. In some embodiments, the second domain comprises a functional fragment of CD27. In some embodiments, the second domain comprises the cytoplasmic domain of CD27. In some embodiments, the second domain comprises a functional fragment of OX40. In some embodiments, the second domain comprises the cytoplasmic domain of OX40. In some embodiments, the second domain comprises a functional fragment of DAP10. In some embodiments, the second domain comprises the cytoplasmic domain of DAP10. In some embodiments, the second domain comprises a functional fragment of 2B4. In some embodiments, the second domain comprises the cytoplasmic domain of 2B4. In some embodiments, the second domain comprises a functional fragment of CD30. In some embodiments, the second domain comprises the cytoplasmic domain of CD30. In some embodiments, the second domain comprises a functional fragment of CD2. In some embodiments, the second domain the cytoplasmic domain of CD2. In some embodiments, the second domain comprises a functional fragment of LIGHT. In some embodiments, the second domain comprises the cytoplasmic domain of LIGHT. In some embodiments, the second domain comprises a functional fragment of GITR. In some embodiments, the second domain comprises the cytoplasmic domain of GITR. In some embodiments, the second domain comprises a functional fragment of DR3. In some embodiments, the second domain comprises the cytoplasmic domain of DR3. In some embodiments, the second domain comprises a functional fragment of CD43. In some embodiments, the second domain comprises the cytoplasmic domain of CD43.

In some embodiments, the second domain of fusion proteins provided herein comprises an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. The immune effector cell can be selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte. In some embodiments, the co-stimulatory receptor of the immune effector cell is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, DR3, and CD43. In some embodiments, the second domain comprises an antibody that binds CD28, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds 4- 1BB, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds ICOS, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds CD27, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds OX40, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds DAP10, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds 2B4, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds CD30, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds CD2, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds LIGHT, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds GITR, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds DR3, or an antigen-binding fragment thereof. In some embodiments, the second domain comprises an antibody that binds CD43, or an antigen-binding fragment thereof.

In some embodiments, the second domain comprises a monoclonal antibody. In some embodiments the second domain comprises a chimeric antibody. In some embodiments the second domain comprises a humanized antibody. In some embodiments the second domain comprises a human antibody. In some embodiments, the second domain comprises a Fab, Fab’, F (ab’) 2, Fv, scFv, (scFv) 2, single chain antibody, dual variable region antibody, diabody, nanobody, or single variable region antibody. In some embodiments the second domain comprises a human antibody. In some embodiments, the second domain comprises a scFv.

In some embodiments, the second domain of the fusion proteins provided herein comprise an anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the second domain of the fusion proteins provided herein comprise an anti-CD28 scFv. In some embodiments, the anti-CD28 antibody or antigen-binding fragment thereof comprises the antibody that is designated 1412.

Table 6: Exemplary Anti-CD28 Antibody

In some embodiments, the second domain of the fusion proteins provided herein comprise an anti-CD28 scFv having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 417. In some embodiments, the second domain comprises an anti-CD28 scFv having the amino acid sequence of SEQ ID NO: 417.

The fusion proteins described herein (i.e., the LACO-Stim molecules or LACOs) can include any combinations of APC activators (ligands or antibodies that bind activation receptors) and immune effector cell activators (co-stimulatory receptors or antibodies that bind co-stimulatory receptors) disclosed herein or otherwise known in the art. For illustration purposes, provided below are various forms of the CD40-C28 LACO-Stim fusion proteins that activates APCs (e.g., the dendritic cells) via the CD40/CD40L signaling and activates immune effector cells (e.g., the T cells) via the CD28 signaling.

5.4.3.1 Exemplary LACO-Stim (1) : ligand for APC activation receptor + co-stimulatory receptor (e.g., CD40L-CD28)

In some embodiments, fusion proteins provided herein comprise a first domain that activates APC and a second domain that activates an immune effector cell, wherein the first domain comprises a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, and the second domain comprises a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of a co-stimulatory receptor of the immune effector cell. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain. In some embodiments, provided herein are fusion proteins that are membrane fusion proteins. In some embodiments, the first domain and the second domain are linked via a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 484) . In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 485) . In some embodiments, the linker has the amino acid sequence of (PA) nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 486) . In some embodiments, the linker has the amino acid sequence of GSGGGGSGGGGSGGGGS (SEQ ID NO: 489) . In some embodiments, the linker has the amino acid sequence of GGGGS (SEQ ID NO: 488) .

In some embodiments, the first domain comprises a ligand that binds an APC activation receptor selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205, and DC-SIGN, or a functional-fragment thereof. In some embodiments, the first domain of the fusion proteins provided herein comprises the extracellular domain of CD40L (e.g., amino acids 47-261 of SEQ ID NO: 375) . In some embodiments, the first domain is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to amino acids 47-261 of SEQ ID NO: 375. In some embodiments, the first domain has amino acids 47-261 of SEQ ID NO: 375. In some embodiments, the first domain has amino acids 119-261 of SEQ ID NO: 375. In some embodiments, the first domain comprises full length CD40L. In some embodiments, the first domain can have an amino acid sequence that is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to SEQ ID NO: 375. In some embodiments, the first domain has the amino acid sequence of SEQ ID NO: 375. In some embodiments, the first domain of the fusion proteins provided herein comprises three copies of the CD40L, or a functional fragment thereof. In some embodiments, the first domain comprises three copies of the extracellular domain of the CD40L (e.g., amino acids 47-261 of SEQ ID NO: 375) . In some embodiments, the first domain of the fusion proteins provided herein comprises three copies of amino acids 119-261 of CD40L (SEQ ID NO: 375) .

In some embodiments, the second domain comprises a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43, or a functional fragment thereof. In some embodiments, the second domain comprises the cytoplasmic domain of a co-stimulatory receptor selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the second domain comprises a CD28 cytoplasmic domain (e.g., amino acids 180 to 220 of SEQ ID NO: 333) . In some embodiments, the second domain can is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to amino acids 180 to 220 of SEQ ID NO: 333. In some embodiments, the second domain has amino acids 180 to 220 of SEQ ID NO: 333. In some embodiments, the second domain further comprises a CD28 transmembrane domain (e.g., amino acids 153 to 179 of SEQ ID NO: 333) . In some embodiments, the second domain comprises a 4-1BB cytoplasmic domain (e.g., amino acids 214 to 255 of SEQ ID NO: 334) . In some embodiments, the second domain is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to amino acids 214 to 255 of SEQ ID NO: 334. In some embodiments, the second domain has amino acids 214 to 255 of SEQ ID NO: 334. In some embodiments, the second domain further comprises a 4-1BB transmembrane domain (e.g., amino acids 187 to 213 of SEQ ID NO: 334) .

In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a CD28 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a 4-1BB cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises an ICOS cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a CD27 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises an OX40 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a DAP10 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a 2B4 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a CD30 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a CD2 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a LIGHT cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a GITR cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a TLR cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a DR3 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises CD40L or a receptor-binding domain, and a second domain that comprises a CD43 cytoplasmic domain. The receptor-binding fragment of CD40L can be amino acids 119-261 of CD40L (SEQ ID NO: 375) . In some embodiments, the first domain comprises full length CD40L.

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 40L. 28.40L. 40L (SEQ ID NO: 418) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 418. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as TriCD40L_8-28 (SEQ ID NO: 490) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 490. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as TriCD40L_28-28 (SEQ ID NO: 491) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 491.

As a person of ordinary skill in the art would understand, the extracellular domain of CD40L or full length CD40L in the fusion proteins exemplified herein can be replaced with another ligand for an activation receptor for APC that is disclosed herein or otherwise known in the art, including, for example, the extracellular domain or the full length of a CD80 ligand (e.g., CD28 or CTLA-4) , a CD86 ligand (e.g., CD28 or CTLA-4) , a CD91 ligand (e.g., RAP1) , a DEC-205 ligand or a DC-SIGN ligand (e.g., ICAM2, ICAM3, CD18, or CEACAM1) . As a person of ordinary skill in the art would understand, the CD28 cytoplasmic domain in the fusion proteins exemplified herein can be replaced with the cytoplasmic domain of another co-stimulator for immune effector cells that is disclosed herein or otherwise known in the art, including, for example, the cytoplasmic domain of 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43; or a different functional fragment of 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43, that retains the function of the full-length protein to activate the immune effector cell.

5.4.3.2 Exemplary LACO-Stim (2) : ligand for APC activation receptor +antibody binding co-stimulatory receptor (e.g., aCD28-CD40L)

In some embodiments, fusion proteins provided herein comprise a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the first domain comprises an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof, and wherein the second domain comprises an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain. In some embodiments, fusion proteins provided herein are antibody-based soluble proteins.

In some embodiments, the two domains of the fusion proteins disclosed herein are linked via a trimerization motif. In some embodiments, the linker is a trimerization motif selected from the group consisting of a T4 fibritin trimerization motif, an isoleucine zipper, a GCN4II motif, a Matrilin-1 motif, and a collagen XV trimerization motif. In some embodiments, the linker is a T4 fibritin trimerization motif (e.g., SEQ ID NO: 434) . In some embodiments, the linker is an isoleucine zipper (e.g., SEQ ID NO: 435 or 436. In some embodiments, the linker is a GCN4II motif (e.g., SEQ ID NO: 437 or 438) . In some embodiments, the linker is a Matrilin-1 motif (e.g., SEQ ID NO: 439 or 440) . In some embodiments, the linker is a collagen XV trimerization motif (e.g., SEQ ID NO: 441) .

In some embodiments, the first domain comprises a ligand that binds an APC activation receptor selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205, and DC-SIGN, or a functional-fragment thereof. In some embodiments, the first domain of the fusion proteins provided herein comprises the extracellular domain of CD40L (e.g., amino acids 47-261 of SEQ ID NO: 375) . In some embodiments, the first domain is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to amino acids 47-261 of SEQ ID NO: 375. In some embodiments, the first domain has amino acids 47-261 of SEQ ID NO: 375. In some embodiments, the first domain has amino acids 119-261 of SEQ ID NO: 375. In some embodiments, the first domain comprises full length CD40L. In some embodiments, the first domain can have an amino acid sequence that is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to SEQ ID NO: 375. In some embodiments, the first domain has the amino acid sequence of SEQ ID NO: 375. In some embodiments, the first domain of the fusion proteins provided herein comprises three copies of the CD40L, or a functional fragment thereof. In some embodiments, the first domain comprises three copies of the extracellular domain of the CD40L (e.g., amino acids 47-261 of SEQ ID NO: 375) . In some embodiments, the first domain comprises three copies of amino acids 119-261 of CD40L (SEQ ID NO: 375) .

In some embodiments, the second domain comprises an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-4-1BB antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-ICOS antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-CD27 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-OX40 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-DAP10 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-2B4 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-CD30 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-CD2 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-LIGHT antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-GITR antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-TLR antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-DR3 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are fusion proteins having a first domain that comprises CD40L or a receptor-binding domain and a second domain that comprises an anti-CD43 antibody or an antigen-binding fragment thereof.

In some embodiments, provided herein are fusion proteins having a first domain that comprises a CD40L ECD and a second domain that comprises an anti-CD28 antibody or an antigen-binding fragment thereof. The anti-CD28 antibody or antigen-binding fragment can be any anti-CD28 antibody or antigen-binding fragment disclosed herein or otherwise known in the art that can activate CD28 signaling. In some embodiments, the anti-CD28 antibody or antigen-binding fragment is the antibody designated 1412 (SEQ ID NO: 417) .

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 1412-T4-CD40L (SEQ ID NO: 419) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 419.

As a person of ordinary skill in the art would understand, the extracellular domain of CD40L in the fusion proteins exemplified herein can be replaced with the extracellular domain or a receptor-binding fragment of another ligand for an activation receptor for APC that is disclosed herein or otherwise known in the art, including, for example, the extracellular domain or the receptor-binding domain of a CD80 ligand (e.g., CD28 or CTLA-4) , a CD86 ligand (e.g., CD28 or CTLA-4) , a CD91 ligand (e.g., RAP1) , a DEC-205 ligand or a DC-SIGN ligand (e.g., ICAM2, ICAM3, CD18, or CEACAM1) . As a person of ordinary skill in the art would understand, the anti-CD28 antibody or antigen-binding fragment in the fusion proteins exemplified herein can be replaced with an antibody or antigen-binding fragment that binds and activates another co-stimulator for immune effector cells that is disclosed herein or otherwise known in the art, including, for example, an antibody or antigen-binding fragment that binds 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43.

5.4.3.3 Exemplary LACO-Stim (3) : antibody for APC activation receptor +antibody for co-stimulatory receptor (e.g., aCD40/aCD28 bispecific Ab)

In some embodiments, provided herein are bispecific antibodies. A “bispecific antibody, ” as used herein and understood in the art, refers to an antibody having binding specificities for at least two different antigenic epitopes. The epitopes can be from the same antigen or two different antigens. In some embodiments, provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the first domain comprises an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof, and wherein the second domain comprises an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. Accordingly, bispecific antibodies disclosed herein have binding specificities for (1) an activation receptor for an APC (e.g., a dendritic cell) and (2) a co-stimulatory receptor for an immune effector cell (e.g., a T cell) . In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain.

In some embodiments, the first domain and the second domain are linked via a linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 484) . In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 485) . In some embodiments, the linker has the amino acid sequence of (PA) nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 486) . In some embodiments, the linker has the amino acid sequence of GSGGGGSGGGGSGGGGS (SEQ ID NO: 489) . In some embodiments, the linker has the amino acid sequence of GGGGS (SEQ ID NO: 488) .

In some embodiments, provided herein are fusion proteins are bispecific antibodies comprising a first domain that binds an activation receptor of the APC, or an antigen-binding fragment thereof, and a second domain comprises an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN. In some embodiments, the second domain comprise an antibody or antigen-binding fragment thereof that binds CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43.

In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-4-1BB antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-ICOS antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-CD27 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-OX40 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-DAP10 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-2B4 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-CD30 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-CD2 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-LIGHT antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-GITR antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-TLR antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-DR3 antibody or an antigen-binding fragment thereof. In some embodiments, provided herein are bispecific antibodies comprising a first domain that is an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain comprises an anti-CD43 antibody or an antigen-binding fragment thereof.

Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs. See, e.g., Milstein et al. (1983) Nature 305: 537-39. Alternatively, bispecific antibodies can be prepared using chemical linkage. See, e.g., Brennan et al. (1985) Science 229: 81. Bispecific antibodies include bispecific antigen-binding fragments. See, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 6444-48; Gruber et al. (1994) J. Immunol. 152: 5368. Techniques for making bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983) , WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991) ) , and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168) . Multi-specific antibodies can also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1) ; cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science 229: 81 (1985) ) ; using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. 148 (5) : 1547-1553 (1992) ) ; using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) ) ; and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152: 5368 (1994) ) ; and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991) . Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies, ” are also included herein (see, e.g., US 2006/0025576A1) . Bispecific antibodies can be constructed by linking two different antibodies, or portions thereof. For example, a bispecific antibody can comprise Fab, F (ab′) ₂, Fab′, scFv, and sdAb from two different antibodies.

In some embodiments, the first domain of the fusion proteins provided herein comprises an anti-CD40 antibody or an antigen-binding fragment thereof. The anti-CD40 antibody or antigen-binding fragment can be any anti-CD40 antibody or antigen-binding fragment disclosed herein or otherwise known in the art. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises the scFv designated as F2.103, F5.157, F5.77, 4D11, A40C, 119, 40-45, or 40-52 as provided above in Table 5. In some embodiments, the anti-CD28 antibody or antigen-binding fragment can be any anti-CD28 antibody or antigen-binding fragment disclosed herein or otherwise known in the art that activate CD28 signaling. In some embodiments, the anti-CD28 antibody or antigen-binding fragment is the antibody designated 1412 (SEQ ID: 417) .

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 1412-F2.103 (SEQ ID NO: 420) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 420. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 1412-F5.157 (SEQ ID NO: 421) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 421. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 1412-F5.77 (SEQ ID NO: 422) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 422. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 1412-4D11 (SEQ ID NO: 492) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 492.

As a person of ordinary skill in the art would understand, the anti-CD40 antibody or antigen-binding fragment thereof in the fusion proteins exemplified herein can be replaced with an antibody or antigen-binding fragment that binds another activation receptor for APC that is disclosed herein or otherwise known in the art, including, for example, CD80, CD86, CD91, DEC-205 or DC-SIGN. As a person of ordinary skill in the art would understand, the anti-CD28 antibody or antigen-binding fragment in the fusion proteins exemplified herein can be replaced with an antibody or antigen-binding fragment that binds another co-stimulator for immune effector cells that is disclosed herein or otherwise known in the art, including, for example, an antibody or antigen-binding fragment that binds 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43.

5.4.3.4 Exemplary LACO-Stim (4) : antibody for activation receptor + co-stimulatory receptor (e.g., aCD40-CD28; aCD40-4-1BB)

In some embodiments, provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the first domain comprises an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof, and wherein the second domain comprises a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain. In some embodiments, provided herein are antibody-based membrane fusion protein. In some embodiments, the first and second domains are linked via a CD8 hinge (e.g., SEQ ID NO: 442) , a CD28 hinge (e.g., SEQ ID NO: 443) , or an IgG Fc region (e.g., SEQ ID NO: 444) .

In some embodiments, provided herein are fusion proteins comprising a first domain comprising an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof, and a second domain comprising an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN. In some embodiments, the first domain of the fusion proteins provided herein comprises an anti-CD40 antibody or an antigen-binding fragment thereof. The anti-CD40 antibody or antigen-binding fragment can be any anti-CD40 antibody or antigen-binding fragment disclosed herein or otherwise known in the art that activate CD40 signaling. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises the antibody designated as F2.103, F5.157, F5.77, 4D11, A40C, 119, 40-45, or 40-52 as provided above in Table 5.

In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a CD28 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a 4-1BB cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises an ICOS cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a CD27 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises an OX40 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a DAP10 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a 2B4 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a CD30 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a CD2 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a LIGHT cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a GITR cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a TLR cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a DR3 cytoplasmic domain. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a CD43 cytoplasmic domain. In some embodiments, the first domain comprises full length CD40L.

In some embodiments, fusion proteins provided herein further comprise a transmembrane region. In some embodiments, the transmembrane region is derived from the same co-stimulatory receptor. In some embodiments, the transmembrane region is derived from a different co-stimulatory receptor. In some embodiments, fusion proteins provided herein have a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a CD28 transmembrane region and a CD28 cytoplasmic domain. In some embodiments, the second domain further comprises. In some embodiments, provided herein are fusion proteins having a first domain that comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and a second domain that comprises a 4-1BB transmembrane region and a 4-1BB cytoplasmic domain.

In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as F2.103. CD28 (SEQ ID NO: 423) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 423. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as F5.157. CD28 (SEQ ID NO: 424) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 424. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as F5.77. CD28 (SEQ ID NO: 425) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 425. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as F2.103. BB (SEQ ID NO: 426) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 426. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as F5.157. BB (SEQ ID NO: 427) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 427. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as F5.77. BB (SEQ ID NO: 428) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 428. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 4D11. CD28 (SEQ ID NO: 429) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 429. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as A40C. CD28 (SEQ ID NO: 430) . In some embodiments, fusion proteins provided herein have an amino acid sequence that is identical to SEQ ID NO: 430. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 119. CD28 (SEQ ID NO: 431) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 431. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 40-45.28 (SEQ ID NO: 432) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 432. In some embodiments, fusion proteins provided herein have an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the sequence of the fusion protein designated as 40-45.28 (SEQ ID NO: 433) . In some embodiments, fusion proteins provided herein have the amino acid sequence of SEQ ID NO: 433.

As a person of ordinary skill in the art would understand, the anti-CD40 antibody or antigen-binding fragment thereof in the fusion proteins exemplified herein can be replaced with an antibody or antigen-binding fragment that binds and activates another activation receptor for APC that is disclosed herein or otherwise known in the art, including, for example, CD80, CD86, CD91, DEC-205 or DC-SIGN. As a person of ordinary skill in the art would understand, the CD28 cytoplasmic domain or 4-1BB cytoplasmic domain in the fusion proteins exemplified herein can be replaced with the cytoplasmic domain of another co-stimulator for immune effector cells that is disclosed herein or otherwise known in the art, including, for example, the cytoplasmic domain of ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43; or a different functional fragment of 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, or CD43 that retains the function of the full-length protein to activate the immune effector cell.

5.4.3.5 Exemplary LACO-Stim (5) : antibody for APC activation receptor +ligand for co-stimulatory receptor (e.g., aCD40-CD80; aCD40-CD86)

In some embodiments, provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the first domain comprises an antibody that binds an activation receptor of the antigen-presenting cell, or an antigen-binding fragment thereof; and wherein the second domain comprises a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain. In some embodiments, provided herein are antibody-based soluble fusion protein.

In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN. In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds CD40. In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds CD80. In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds CD86. In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds CD91. In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds DEC-205. In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds DC-SIGN. The antibodies and antigen-binding fragments can be any antibody or antigen-binding fragment disclosed herein or otherwise known in the art.

In some embodiments, provided herein are fusion proteins comprising a first domain comprising an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof, and a second domain comprising a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof. In some embodiments, the first domain comprises an antibody or antigen-binding fragment thereof that binds CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN. In some embodiments, the second domain comprises a ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44, or a receptor-binding fragments thereof.

In some embodiments, the first domain of the fusion proteins provided herein comprises an anti-CD40 antibody or an antigen-binding fragment thereof. The anti-CD40 antibody or antigen-binding fragment can be any anti-CD40 antibody or antigen-binding fragment disclosed herein or otherwise known in the art that activate CD40 signaling. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof comprises the antibody designated as F2.103, F5.157, F5.77, 4D11, A40C, 119, 40-45, or 40-52 as provided above in Table 5.

In some embodiments, the fusion protein comprises a first domain that comprises an antibody or antigen-binding fragment thereof that binds CD40, and a second domain comprises the ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, CD44, and receptor-binding fragments thereof. In some embodiments, the second domain comprises CD58 (e.g., SEQ ID NO: 384) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD70 (e.g., SEQ ID NO: 385) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD83 (e.g., SEQ ID NO: 386) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD80 (e.g., SEQ ID NO: 387) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD86 (e.g., SEQ ID NO: 388) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD137L (e.g., SEQ ID NO: 389) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD252 (e.g., SEQ ID NO: 390) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD275 (e.g., SEQ ID NO: 391) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD54 (e.g., SEQ ID NO: 392) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD49a (e.g., SEQ ID NO: 393) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD112 (e.g., SEQ ID NO: 394) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD150 (e.g., SEQ ID NO: 395) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD155 (e.g., SEQ ID NO: 396) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD265 (e.g., SEQ ID NO: 397) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD270 (e.g., SEQ ID NO: 398) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises TL1A (e.g., SEQ ID NO: 399) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD127 (e.g., SEQ ID NO: 400) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises IL-4R (e.g., SEQ ID NO: 401) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises GITR-L (e.g., SEQ ID NO: 402) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises TIM-4 (e.g., SEQ ID NO: 403) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD153 (e.g., SEQ ID NO: 404) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD48 (e.g., SEQ ID NO: 405) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD160 (e.g., SEQ ID NO: 406) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD200R (e.g., SEQ ID NO: 407) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD44 (e.g., SEQ ID NO: 408) or a receptor-binding fragment thereof. A person of ordinary skill in the art can readily determine a proper receptor-binding fragment of a ligand that retains its binding affinity toward its receptor and function to activate the receptor.

As a person of ordinary skill in the art would understand, the anti-CD40 antibody or antigen-binding fragment thereof in the fusion proteins exemplified herein can be replaced with an antibody or antigen-binding fragment that binds another activation receptor for APC that is disclosed herein or otherwise known in the art, including, for example, CD80, CD86, CD91, DEC-205 or DC-SIGN.

5.4.3.6 Exemplary LACO-Stim (6) : ligand for APC activation receptor + co-stimulatory ligand (e.g., CD40L ECD-CD86; CD40L ECD-CD80)

In some embodiments, provided herein are fusion proteins comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein the first domain comprises a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, and wherein the second domain comprises a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof. In some embodiments, the C-terminus of the first domain is linked to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is linked to the C-terminus of the second domain.

In some embodiments, fusion proteins provided herein comprise a first domain comprising a ligand that binds an activation receptor selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205, and DC-SIGN, or a functional-fragment thereof, and a second domain comprising a co-stimulatory ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, CD44 or a receptor-binding fragment thereof.

In some embodiments, the first domain of the fusion proteins provided herein comprises the extracellular domain of CD40L (e.g., amino acids 47-261 of SEQ ID NO: 375) . In some embodiments, the first domain is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to amino acids 47-261 of SEQ ID NO: 375. In some embodiments, the first domain has the amino acids 47-261 of SEQ ID NO: 375. In some embodiments, the first domain has amino acids 119-261 of SEQ ID NO: 375. In some embodiments, the first domain comprises full length CD40L. In some embodiments, the first domain can have an amino acid sequence that is at least 85%, at least 88%, at least 90%, at least 95%, at least 98%, or 100%identical to SEQ ID NO: 375. In some embodiments, the first domain has the amino acid sequence of SEQ ID NO: 375. In some embodiments, the first domain of the fusion proteins provided herein comprises three copies of the CD40L, or a functional fragment thereof. In some embodiments, the first domain comprises three copies of the extracellular domain of the CD40L (e.g., amino acids 47-261 of SEQ ID NO: 375) . In some embodiments, the first domain of the fusion proteins provided herein comprises three copies of amino acids 119-261 of CD40L (SEQ ID NO: 375) .

In some embodiments, the fusion protein comprises a first domain that comprises CD40L or a receptor-binding domain, and a second domain comprises a ligand selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, CD44, or receptor-binding fragments thereof. In some embodiments, the second domain comprises CD58 (e.g., SEQ ID NO: 384) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD70 (e.g., SEQ ID NO: 385) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD83 (e.g., SEQ ID NO: 386) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD80 (e.g., SEQ ID NO: 387) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD86 (e.g., SEQ ID NO: 388) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD137L (e.g., SEQ ID NO: 389) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD252 (e.g., SEQ ID NO: 390) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD275 (e.g., SEQ ID NO: 391) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD54 (e.g., SEQ ID NO: 392) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD49a (e.g., SEQ ID NO: 393) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD112 (e.g., SEQ ID NO: 394) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD150 (e.g., SEQ ID NO: 395) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD155 (e.g., SEQ ID NO: 396) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD265 (e.g., SEQ ID NO: 397) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD270 (e.g., SEQ ID NO: 398) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises TL1A (e.g., SEQ ID NO: 399) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD127 (e.g., SEQ ID NO: 400) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises IL-4R (e.g., SEQ ID NO: 401) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises GITR-L (e.g., SEQ ID NO: 402) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises TIM-4 (e.g., SEQ ID NO: 403) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD153 (e.g., SEQ ID NO: 404) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD48 (e.g., SEQ ID NO: 405) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD160 (e.g., SEQ ID NO: 406) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD200R (e.g., SEQ ID NO: 407) or a receptor-binding fragment thereof. In some embodiments, the second domain comprises CD44 (e.g., SEQ ID NO: 408) or a receptor-binding fragment thereof. A person of ordinary skill in the art can readily determine a proper receptor-binding fragment of a ligand that retains its binding affinity toward its receptor and function to activate the receptor.

As a person of ordinary skill in the art would understand, the extracellular domain of CD40L or full length CD40L in the fusion proteins exemplified herein can be replaced with another ligand for an activation receptor for APC that is disclosed herein or otherwise known in the art, including, for example, the extracellular domain or the full length of a CD80 ligand (e.g., CD28 or CTLA-4) , a CD86 ligand (e.g., CD28 or CTLA-4) , a CD91 ligand (e.g., RAP1) , a DEC-205 ligand or a DC-SIGN ligand (e.g., ICAM2, ICAM3, CD18, or CEACAM1) .

5.5 Polynucleotides and Vectors

Also provided herein are polynucleotides that encode a polypeptide (e.g., an anti-CD123 antibody or antigen-binding fragment or a CAR that specifically binds CD123, or a fusion protein) described herein. The term “polynucleotide that encode a polypeptide” encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA can be cDNA, genomic DNA, or synthetic DNA, and can be double-stranded or single-stranded. Single stranded DNA can be the coding strand or non-coding (anti-sense) strand. The polynucleotides of the disclosure can be mRNA.

Expressly contemplated herein are polynucleotides encode any anti-CD123 antibody or antigen-binding fragment disclosed herein. In some embodiments, the polynucleotides provided herein encode an anti-CD123 antibody or antigen-binding fragment disclosed herein comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-29; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-56; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57-88; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or (b) a VH comprising (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-109; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-133; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134-163; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs. In some embodiments, the polynucleotides provided herein encode an anti-CD123 antibody or antigen-binding fragment disclosed herein comprising (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198; and/or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode an anti-CD123 antibody or antigen-binding fragment disclosed herein comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2 and CDR3 and the VH comprises VH CDR1, CDR2 and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 have the amino acid sequences of (1) SEQ ID NOs: 3, 33, 61, 91, 112, and 136, respectively; (2) SEQ ID NOs: 17, 45, 75, 89, 122, and 149, respectively; (3) SEQ ID NOs: 7, 37, 66, 95, 116, and 140, respectively; (4) SEQ ID NOs: 18, 32, 76, 100, 123, and 150, respectively; (5) SEQ ID NOs: 23, 50, 82, 99, 129 and 157, respectively; (6) SEQ ID NOs: 24, 51, 83, 106, 128, and 158, respectively; (7) SEQ ID NOs: 8, 38, 67, 96, 117, and 141, respectively; (8) SEQ ID NOs: 4, 34, 62, 89, 110, and 134, respectively; (9) SEQ ID NOs: 25, 52, 84, 107, 130, and 159, respectively; (10) SEQ ID NOs: 26, 53, 85, 95, 116, and 160, respectively; (11) SEQ ID NOs: 2, 32, 60, 90, 111, and 135, respectively; (12) SEQ ID NOs: 17, 46, 77, 101, 124, and 151, respectively; (13) SEQ ID NOs: 27, 54, 86, 105, 131, and 161, respectively; (14) SEQ ID NOs: 9, 39, 68, 97, 118, and 142, respectively; (15) SEQ ID NOs: 10, 40, 69, 98, 119, and 143, respectively; (16) SEQ ID NOs: 22, 49, 81, 105, 128, and 156 respectively; (17) SEQ ID NOs: 5, 35, 63, 92, 113, and 137 respectively; (18) SEQ ID NOs: 1, 30 64, 93, 114 and 138 respectively; (19) SEQ ID NOs: 11, 41, 70, 91, 120, and 144 respectively; (20) SEQ ID NOs: 1, 31, 59, 89, 110, and 134 respectively; (21) SEQ ID NOs: 19, 47, 78, 102, 125, and 152 respectively; (22) SEQ ID NOs: 28, 55, 87, 108, 132, and 162, respectively; (23) SEQ ID NOs: 12, 39, 71, 96, 117, and 145, respectively; (24) SEQ ID NOs: 13, 42, 72, 95, 116, and 146, respectively; (25) SEQ ID NOs: 1, 30, 57, 89, 110, and 134, respectively; (26) SEQ ID NOs: 20, 48, 79, 103, 126 and 153, respectively; (27) SEQ ID NOs: 14, 43, 67, 96, 117, and 141, respectively; (28) SEQ ID NOs: 1, 30, 58, 89, 110, and 134, respectively; (29) SEQ ID NOs: 2, 32, 60, 90, 111, and 135, respectively; (30) SEQ ID NOs: 15, 44, 73, 99, 121, and 147, respectively; (31) SEQ ID NOs: 29, 56, 88, 109, 133 and 163, respectively; (32) SEQ ID NOs: 22, 49, 81, 104, 127, and 155, respectively; (33) SEQ ID NOs: 16, 42, 74, 95, 116, and 148, respectively; (34) SEQ ID NOs: 6, 36, 65, 94, 115, and 139, respectively; or (35) SEQ ID NOs: 21, 45, 80, 89, 122, and 154, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the CDRs. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode an anti-CD123 antibody or antigen-binding fragment disclosed herein comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of (1) SEQ ID NOs: 164 and 199, respectively; (2) SEQ ID NOs: 165 and 200, respectively; (3) SEQ ID NOs: 166 and 201, respectively; (4) SEQ ID NOs: 167 and 202, respectively; (5) SEQ ID NOs: 168 and 203, respectively; (6) SEQ ID NOs: 169 and 204, respectively; (7) SEQ ID NOs: 170 and 205, respectively; (8) SEQ ID NOs: 171 and 206, respectively; (9) SEQ ID NOs: 172 and 207, respectively; (10) SEQ ID NOs: 173 and 208, respectively; (11) SEQ ID NOs: 174 and 209, respectively; (12) SEQ ID NOs: 175 and 210, respectively; (13) SEQ ID NOs: 176 and 211, respectively; (14) SEQ ID NOs: 177 and 212, respectively; (15) SEQ ID NOs: 178 and 213, respectively; (16) SEQ ID NOs: 179 and 214, respectively; (17) SEQ ID NOs: 180 and 215, respectively; (18) SEQ ID NOs: 181 and 216, respectively; (19) SEQ ID NOs: 182 and 217, respectively; (20) SEQ ID NOs: 183 and 218, respectively; (21) SEQ ID NOs: 184 and 219, respectively; (22) SEQ ID NOs: 185 and 220, respectively; (23) SEQ ID NOs: 186 and 221, respectively; (24) SEQ ID NOs: 187 and 222, respectively; (25) SEQ ID NOs: 188 and 223, respectively; (26) SEQ ID NOs: 189 and 224, respectively; (27) SEQ ID NOs: 190 and 225, respectively; (28) SEQ ID NOs: 191 and 226, respectively; (29) SEQ ID NOs: 192 and 227, respectively; (30) SEQ ID NOs: 193 and 228, respectively; (31) SEQ ID NOs: 194 and 229, respectively; (32) SEQ ID NOs: 195 and 230, respectively; (33) SEQ ID NOs: 196 and 231, respectively; (34) SEQ ID NOs: 197 and 232, respectively; or (35) SEQ ID NOs: 198 and 233, respectively. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode an anti-CD123 antibody or antigen-binding fragment disclosed herein comprising a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 234-268. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 234. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 235. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 236. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 237. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 238. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 239. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 240. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 241. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 242. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 243. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 244. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 245. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 246. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 247. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 248. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 249. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 250. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 251. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 252. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 253. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 254. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 255. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 256. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 257. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 258. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 259. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 260. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 261. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 262. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 263. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 264. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 265. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 266. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 267. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 268. Also provided is a polynucleotide that hybridizes to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 234-268. In some embodiments, the hybridization is under conditions of high stringency as is known to those skilled in the art. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the polynucleotides provided herein encode an anti-CD123 antibody or antigen-binding fragment disclosed herein comprising a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 269-303. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 269. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 270. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 271. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 272. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 273. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 274. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 275. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 276. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 277. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 278. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 279. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 280. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 281. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 282. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 283. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 284. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 285. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 286. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 287. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 288. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 289. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 290. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 291. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 292. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 293. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 294. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 295. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 296. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 297. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 298. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 299. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 300. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 301. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 302. In some embodiments, the polynucleotides provided herein have a nucleotide sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 303. Also provided is a polynucleotide that hybridizes to a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 269-303. In some embodiments, the hybridization is under conditions of high stringency as is known to those skilled in the art. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

In some embodiments, the VL and VH are connected by a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has the amino acid sequence of (GGGGS) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 484) . In some embodiments, the linker has the amino acid sequence of (EAAAK) n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 485) . In some embodiments, the linker has the amino acid sequence of (PA) nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 486) . In some embodiments, the linker has the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 320) .

The present disclosure also provides variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and/or derivatives of an anti-CD123 antibody or antigen-binding fragment disclosed herein. In some embodiments, the present disclosure provides a polynucleotide having a nucleotide sequence at least about 80%identical, at least about 85%identical, at least about 90%identical, at least about 95%identical, at least about 96%identical, at least about 97%identical, at least about 98%identical, or at least about 99%identical to a polynucleotide sequence encoding an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, the present disclosure provides a polynucleotide having a nucleotide sequence at least about 80%identical, at least about 85%identical, at least about 90%identical, at least about 95%identical, at least about 96%identical, at least about 97%identical, at least about 98%identical, or at least about 99%identical to a polynucleotide sequence encoding an anti-CD123 antibody or antigen-binding fragment described herein.

In some embodiments, the polynucleotides provided herein encode an anti-CD123 antibody or antigen-binding fragment that is the scFv designated as C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34 or C35. In some embodiments, the polynucleotides provided herein encode an anti-CD123 antibody or antigen-binding fragment having the amino acid sequence selected from the group consisting of SEQ ID NOs: 445-479.

Provided herein are also polynucleotides that encode the TCRs disclosed herein. In some embodiments, provided herein are polynucleotides that encode a TCR α chain that comprises an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, provided herein are polynucleotides that encode a TCR β chain that comprises an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, provided herein are polynucleotides that encode a TCR γ chain that comprises an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, provided herein are polynucleotides that encode a TCR δ chain that comprises an anti-CD123 antibody or antigen-binding fragment described herein. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

Provided herein are also polynucleotides that encode the CARs disclosed herein. In some embodiments, provided herein are polynucleotides encoding CARs that specifically binds CD123, comprising, from N-terminus to C-terminus: (a) a CD123-binding domain comprising an anti-CD123 antibody or antigen-binding fragment provided herein, (b) a transmembrane domain, and (c) a cytoplasmic domain. The transmembrane and cytoplasmic domains can be any transmembrane and cytoplasmic domains disclosed herein. For illustrative purposes, provided herein are, for example, CARs that specifically binds CD123, comprising, from N-terminus to C-terminus: (a) a CD123-binding domain comprising an anti-CD123 scFv provided herein, (b) a transmembrane domain comprising the CD28 transmembrane region, and (c) a cytoplasmic domain comprising a CD3 signaling domain and a 4-1BB co-stimulatory domain. The polynucleotides can be in the form of DNA. The polynucleotides can be in the form of mRNA.

As used herein, the phrase “a polynucleotide having a nucleotide sequence at least about 95%identical to a polynucleotide sequence” means that the nucleotide sequence of the polynucleotide is identical to a reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5%of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code) . Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. coli) . In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

In some embodiments, a polynucleotide has a nucleotide sequence at least about 80%identical, at least 85%identical, at least 90%identical, at least 95%identical, at least 96%identical, at least 97%identical, at least 98%identical, or at least 99%identical to a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 164-233. Also provided is a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 164-233. In some embodiments, the hybridization is under conditions of high stringency as is known to those skilled in the art.

In some embodiments, a polynucleotide comprises the coding sequence for a polypeptide (e.g., a CAR or an antibody) fused in the same reading frame to a polynucleotide which aids in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide) . The polypeptide can have the leader sequence cleaved by the host cell to form a “mature” form of the polypeptide.

In some embodiments, a polynucleotide comprises the coding sequence for a polypeptide (e.g., a CAR or an antibody) fused in the same reading frame to a marker or tag sequence. For example, in some embodiments, a marker sequence is a hexa-histidine tag (HIS-tag) that allows for efficient purification of the polypeptide fused to the marker. In some embodiments, a marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. In some embodiments, the marker sequence is a FLAG ^TM tag. In some embodiments, a marker may be used in conjunction with other markers or tags.

In some embodiments, a polynucleotide is isolated. In some embodiments, a polynucleotide is substantially pure.

Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments, provided herein are vectors comprising a polynucleotide provided herein. The vectors can be expression vectors. In some embodiments, vectors provided herein comprise a polynucleotide encoding an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a polypeptide that is part of an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a CAR or TCR described herein. In some embodiments, vectors provided herein comprise a polynucleotide encoding a polypeptide that is part of a CAR or TCR described herein.

In some embodiments, provided herein are recombinant expression vectors, which can be used to amplify and express a polynucleotide encoding a CAR or TCR described herein that specifically binds CD123 or an anti-CD123 antibody or antigen-binding fragment described herein. For example, a recombinant expression vector can be a replicable DNA construct that includes synthetic or cDNA-derived DNA fragments encoding a CAR or TCR or a polypeptide chain of an anti-CD123 antibody, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. In some embodiments, a viral vector is used. DNA regions are “operatively linked” when they are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in certain expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In some embodiments, in situations where recombinant protein is expressed without a leader or transport sequence, a polypeptide can include an N-terminal methionine residue.

A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

In some embodiments, a CAR or TCR described herein or an anti-CD123 antibody or antigen-binding fragment described herein is expressed from one or more vectors. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well-known in the art.

Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived) , L-929 (murine fibroblast-derived) , C127 (murine mammary tumor-derived) , 3T3 (murine fibroblast-derived) , CHO (Chinese hamster ovary-derived) , HeLa (human cervical cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

The present disclosure also provides host cells comprising the polypeptides described herein, polynucleotides encoding polypeptides described herein, or vectors comprising such polynucleotides. In some embodiments, provided herein are host cells comprising a vector comprising a polynucleotide disclosed herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding a polypeptide that is part of an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, host cells provided herein comprise a polynucleotide encoding an anti-CD123 antibody or antigen-binding fragment described herein. In some embodiments, the cells produce the anti-CD123 antibodies or antigen-binding fragments described herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide encoding a CAR or TCR described herein. In some embodiments, host cells provided herein comprise a vector comprising a polynucleotide molecule encoding a polypeptide that is part of a CAR or TCR described herein. In some embodiments, host cells provided herein comprise a polynucleotide encoding a CAR or TCR described herein. In some embodiments, the host cells produce the CD123 CARs or TCRs described herein.

5.6 Cells

Provided herein are cells comprising the polynucleotides disclosed herein. In some embodiments, provided herein are cells comprising a polynucleotide that encodes a polypeptide disclosed herein. In some embodiments, provided herein are cells comprising a vector having a polynucleotide disclosed herein. In some embodiments, provided herein are cells recombinantly expressing a polypeptide disclosed herein. The polypeptide can be an anti-CD123 antibody or antigen-binding fragment. The polypeptide can be CD123 CAR or TCR. In some embodiments, the cells provided herein can further express the fusion proteins disclosed herein or comprise a polynucleotide encoding a fusion protein disclosed herein. The fusion protein can be any fusion protein disclosed herein.

In some embodiments, the cells provided herein express a CAR or TCR disclosed herein and a fusion protein disclosed herein. In some embodiments, the cells provided herein comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a CAR or TCR. In some embodiments, the cells provided herein comprise a polynucleotide that comprises a first fragment encoding a CAR or TCR and a second fragment encoding a fusion protein. In some embodiments, the polynucleotide has the first fragment and the second fragment from N-terminus to the C-terminus. In some embodiments, the polynucleotide has the second fragment and the first fragment from N-terminus to the C-terminus.

In some embodiments, the cells provided herein comprise a polynucleotide having a first fragment encoding a CAR or a TCR provided herein and a second fragment that encodes a fusion protein disclosed herein. The first fragment and the second fragment can be linked by a nucleotide sequence encoding a linker. The linker can be a self-cleaving linker. In some embodiments, the first and second fragment are linked by a nucleotide sequence encoding a 2A peptide. In some embodiments, the 2A linker is a P2A peptide (SEQ ID NO: 480) . In some embodiments, the linker is a T2A peptide (SEQ ID NO: 481) . In some embodiments, the linker is an E2A peptide (SEQ ID NO: 482) . In some embodiments, the linker is an F2A peptide (SEQ ID NO: 483) . In some embodiments, provided herein are polynucleotides comprising a first fragment encoding a CAR or TCR provided herein and a second fragment encoding a fusion protein provided herein, wherein the first and second fragments are linked by a nucleotide sequence encoding a F2A peptide (SEQ ID NO: 483) . In some embodiments, the first fragment (CAR/TCR-encoding) is located at the 5’ end of the second fragment (fusion protein-encoding) . In some embodiments, the first fragment (CAR/TCR-encoding) is located at the 3’ end of the second fragment (fusion protein encoding) .

In some embodiments, cells provided herein are immune effector cells. In some embodiments, the immune effector cells are selected from the group consisting of T cells, B cell, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils. In some embodiments, the immune effector cell provided herein is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte. In some embodiments, the immune effector cell provided herein is a T cell. In some embodiments, the immune effector cell provided herein is an NK cell. In some embodiments, the immune effector cell provided herein is an NKT cell. In some embodiments, the immune effector cell provided herein is a macrophage. In some embodiments, the immune effector cell provided herein is a neutrophil. In some embodiments, the immune effector cell provided herein is a granulocyte.

In some embodiments, the immune effector cells provided herein can be genetically engineered. In some embodiments, the genetically engineered immune effector cells provided herein are isolated. In some embodiments, the genetically engineered immune effector cells provided herein are substantially pure.

As such, in some embodiments, provided herein are immune effector cells recombinantly expressing a polypeptide (e.g., an antibody or a CAR) disclosed herein. Provided herein are also immune effector cells (e.g., T cells) comprising a polynucleotide encoding a polypeptide (e.g., an antibody or a CAR) disclosed herein, or a vector having a polynucleotide disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) comprising a polynucleotide that encodes an anti-CD123 antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) recombinantly expressing an anti-CD123 antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are immune effector cells comprising a polynucleotide that encodes a CD123 CAR disclosed herein. In some embodiments, provided herein are immune effector cells (e.g., T cells) recombinantly expressing a CD123 CAR disclosed herein (e.g., CD123 CART cell) .

In some embodiments, the immune effector cell provided herein is a T cell. The T cell can be a cytotoxic T cell, a helper T cell, or a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a cytotoxic T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, an effector memory TEMRA cell, or a gamma delta T cell. In some embodiments, the T cell is a cytotoxic T cell. In some embodiments, the T cell is genetically engineered. In some embodiments, the T cells provided herein are isolated. In some embodiments, the T cells provided herein are substantially pure.

In some embodiments, genetically engineered cells provided herein are derived from cells isolated from a subject. As used herein, a genetically engineered cell that is “derived from” a source cell means that the genetically engineered cell is obtained by taking the source cell and genetically manipulating the source cell. The source cell can be from a natural source. For example, the source cell can be a primary cell isolated from a subject. The subject can be an animal or a human. The source cell can also be a cell that has undergone passages or genetically manipulation in vitro.

In some embodiments, genetically engineered cells provided herein are derived from cells isolated from a human. Immune effector cells (e.g., T cells) can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cell lines available in the art can be used. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from peripheral blood. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from bone marrow. In some embodiments, genetically engineered cells provided herein are derived from cells isolated from peripheral blood mononuclear cells (PBMC) .

In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a stem or progenitor cell. In some embodiments, the stem or progenitor cell is selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a T cell progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a hematopoietic stem and progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from a hematopoietic multipotent progenitor cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from an embryonic stem cell. In some embodiments, genetically engineered cells provided herein are derived from cells differentiated in vitro from an induced pluripotent cell.

In some embodiments, provided herein are a population of cells comprising a cell disclosed herein. The cells disclosed herein can comprise a polynucleotide that encodes a polypeptide disclosed herein or recombinantly express a polypeptide disclosed herein. The polypeptide can be an anti- CD123 antibody or antigen-binding fragment, or a CD123 CAR/TCR. In some embodiments, cells disclosed herein can further express the fusion proteins disclosed herein or comprise a polynucleotide encoding a fusion protein disclosed herein. The fusion protein can be any fusion protein disclosed herein. In some embodiments, the population of cells provided herein express a CAR/TCR disclosed herein and a fusion protein disclosed herein. In some embodiments, the population of cells provided herein comprise a first polynucleotide encoding a fusion protein provided herein, and a second polynucleotide encoding a CAR/TCR. In some embodiments, the population of cells provided herein comprise a polynucleotide that comprises a first fragment encoding a CAR/TCR and a second fragment encoding a fusion protein. In some embodiments, the polynucleotide has the first fragment and the second fragment from N-terminus to the C-terminus. In some embodiments, the polynucleotide has the second fragment and the first fragment, from N-terminus to the C-terminus.

The population of cells can be a homogenous population of cells. The population of cells can be a heterogeneous population of cells. In some embodiments, the population of cells can be a heterogeneous population of cells comprising any combination of the cells disclosed herein. In some embodiments, the population of cells are derived from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) . In some embodiments, the population of cells provided herein are derived from PBMC. In some embodiments, the population of cells provided herein are derived from PBL. In some embodiments, the population of cells provided herein are derived from TIL. In some embodiments, the population of cells provided herein are derived from CIK. In some embodiments, the population of cells provided herein are derived from LAK. In some embodiments, the population of cells provided herein are derived from MILs. The population of cells can be genetically engineered to recombinantly expressing a polypeptide (e.g., an antibody or a CAR) disclosed herein. In some embodiments, provided herein are population of cells comprising a polynucleotide encoding a polypeptide (e.g., an antibody or a CAR) disclosed herein, or a vector having a polynucleotide disclosed herein. In some embodiments, provided herein are population of cells comprising a polynucleotide that encodes an anti-CD123 antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are population of cells recombinantly expressing an anti-CD123 antibody or antigen-binding fragment disclosed herein. In some embodiments, provided herein are population of cells comprising a polynucleotide that encodes a CD123 CAR/TCR disclosed herein. In some embodiments, provided herein are population of cells recombinantly expressing a CD123 CAR disclosed herein (e.g., CD123 CART cell) .

5.7 Pharmaceutical Compositions

Provided herein are also pharmaceutical compositions comprising the anti-CD123 antibodies or antigen-binding fragments disclosed herein. Provided herein are also pharmaceutical compositions comprising the genetically engineered immune effector cells disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the anti-CD123 antibodies or antigen-binding fragments disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of genetically engineered cells disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are useful in immunotherapy. In some embodiments, the pharmaceutical compositions are useful in immuno-oncology. In some embodiments, the pharmaceutical compositions are useful in inhibiting tumor growth in a subject (e.g., a human patient) . In some embodiments, the pharmaceutical compositions are useful in treating cancer in a subject (e.g., a human patient) .

In some embodiments, the pharmaceutical compositions provided herein comprise anti-CD123 antibodies or antigen-binding fragments provided herein. The anti-CD123 antibodies or antigen-binding fragments can be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise soluble anti-CD123 antibodies or antigen-binding fragments provided herein at 1-1000 mg/ml. In some embodiments, the pharmaceutical compositions comprise soluble anti-CD123 antibodies or antigen-binding fragments provided herein at 10-500 mg/ml, 10-400 mg/ml, 10-300 mg/ml, 10-200 mg/ml, 10-100 mg/ml, 20-100 mg/ml, or 50-100 mg/ml. In some embodiments, the pharmaceutical compositions provided herein comprise anti-CD123 antibodies or antigen-binding fragments provided herein at about 10 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 120 mg/ml, about 150 mg/ml, about 180 mg/ml, about 200 mg/ml, about 300 mg/ml, about 500 mg/ml, about 800 mg/ml, or about 1000 mg/ml.

The pharmaceutical compositions comprising genetically engineered immune effector cells (e.g., T cells) disclosed herein can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of cells in a cell population using various well-known methods, as described herein. The ranges of purity in cell populations comprising genetically engineered cells provided herein can be from about 20%to about 25%, from about 25%to about 30%, from about 30%to about 35%, from about 35%to about 40%, from about 40%to about 45%, from about 45%to about 50%, from about 55%to about 60%, from about 65%to about 70%, from about 70%to about 75%, from about 75%to about 80%, from about 80%to about 85%; from about 85%to about 90%, from about 90%to about 95%, or from about 95 to about 100%. In some embodiments, the ranges of purity in cell populations comprising immune effector cells provided herein can be from about 20%to about 30%, from about 20%to about 50%, from about 20%to about 80%, from about 20%to about 100%, from about 50%to about 80%, or from about 50%to about 100%. Dosages can be readily adjusted by those skilled in the art; for example, a decrease in purity may require an increase in dosage.

Provided herein are also kits for preparation of pharmaceutical compositions having the anti-CD123 antibodies or antigen-binding fragments disclosed herein. In some embodiments, the kit comprises the anti-CD123 antibodies or antigen-binding fragments disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kits can comprise anti-CD123 antibodies or antigen-binding fragments disclosed herein for administration to a subject. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the anti-CD123 antibodies or antigen-binding fragments.

Provided herein are also kits for preparation of cells disclosed herein. In some embodiments, the kits comprise one or more vectors for generating a genetically engineered cell, such as a T cell, that expresses the anti-CD123 antibodies or antigen-binding fragments disclosed herein. The kits can be used to generate genetically engineered cells from autologous or non-autologous cells to be administered to a compatible subject. In another embodiment, the kits can comprise cells disclosed herein for administration to a subject. In specific embodiments, the kits comprise the cells disclosed herein in one or more containers. In specific embodiments, the kits comprise instructions regarding the preparation and/or administration of the genetically engineered cells.

In some embodiments, provided herein is a pharmaceutical composition comprising anti-CD123 antibodies or antigen-binding fragments or cells provided herein wherein the composition is suitable for local administration. In some embodiments, local administration comprises intratumoral injection, peritumoral injection, juxtatumoral injection, intralesional injection and/or injection into a tumor draining lymph node, or essentially any tumor-targeted injection where the antitumor agent is expected to leak into primary lymph nodes adjacent to targeted solid tumor.

Pharmaceutically acceptable carriers that can be used in compositions provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) . Depending on the route of administration, the active ingredient (i.e., anti-CD123 antibodies or antigen-binding fragments or immune effector cells provided herein) , can be coated in a material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.

Provided herein are also pharmaceutical compositions or formulations that improve the stability of the anti-CD123 antibodies or antigen-binding fragments to allow for their long-term storage. In some embodiments, the pharmaceutical composition or formulation disclosed herein comprises: (a) anti-CD123 antibodies or antigen-binding fragments disclosed herein; (b) a buffering agent; (c) a stabilizing agent; (d) a salt; (e) a bulking agent; and/or (f) a surfactant. In some embodiments, the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years or more. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4℃, 25℃, or 40℃.

The pharmaceutical compositions disclosed herein can further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer and/or a surfactant, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well-known to the skilled person. Reference may be made to Remington: The Science and Practice of Pharmacy, 19 ^th edition, 1995. Pharmaceutical compositions disclosed herein can also include a pharmaceutically acceptable antioxidant. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

In some embodiments, the pharmaceutical composition is an aqueous formulation. Such a formulation is typically a solution or a suspension, but can also include colloids, dispersions, emulsions, and multi-phase materials. The term “aqueous formulation” is defined as a formulation comprising at least 50%w/w water. Likewise, the term “aqueous solution” is defined as a solution comprising at least 50 %w/w water, and the term “aqueous suspension” is defined as a suspension comprising at least 50 %w/w water.

In some embodiments, the pharmaceutical compositions disclosed herein are freeze-dried, to which the physician or the patient adds solvents and/or diluents prior to use.

Examples of suitable aqueous and nonaqueous carriers that can be employed in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) , and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions described herein is contemplated. A pharmaceutical composition or formulation can comprise a preservative or can be devoid of a preservative. Supplementary active compounds can be incorporated into the compositions.

Pharmaceutical compositions or formulations typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, the compositions can include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material in the pharmaceutical compositions or formulations disclosed herein can vary. In some embodiments, the amount of active ingredient which can be combined with a carrier material is the amount that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

The pharmaceutical compositions disclosed herein can be prepared with carriers that protect the active ingredient against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and poly lactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See. e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In some embodiments, the anti-CD123 antibodies or antigen-binding fragments or cells described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the activate ingredient described herein cross the BBB (if desired, e.g., for brain cancers) , they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes can comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29: 685) . Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al) mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153: 1038) ; antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180) ; surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134) ; pl20 (Schreier et al. (1994) J. Biol. Chem. 269: 9090) ; see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346: 123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4: 273.

5.8 Methods and Uses

The present disclosure also provides methods of uses of the anti-CD123 antibodies or antigen-binding fragments, CD123 CARs, polynucleotides encoding such anti-CD123 antibodies or antigen-binding fragments and CD123 CARs, vectors comprising such polynucleotides, CD123 CAR -expressing cells or pharmaceutical compositions having such cells disclosed herein in treating cancer. Without being bound by theory, anti-CD123 antibodies or antigen-binding fragments and the CD123 CAR -expressing cells disclosed herein can specifically target CD123-expressing cancer cells in vivo, thereby delivering their therapeutic effect of eliminating, lysing and/or killing cancer cells. In some embodiments, the methods include administering a therapeutically effective amount of the anti-CD123 antibodies or antigen-binding fragments disclosed herein to a subject in need thereof. In some embodiments, the methods include administering a therapeutically effective amount of CD123 CAR-expressing immune effector cells disclosed herein to a subject in need thereof. In one embodiment, the methods can include administering a therapeutically effective amount of CD123 CARTs disclosed herein to a subject in need thereof.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-CD123 antibodies or antigen-binding fragments disclosed herein. In some embodiments, provided herein are uses of the anti-CD123 antibodies or antigen-binding fragments disclosed herein in the treatment of tumor or cancer. In some embodiments, provided herein are uses of the anti-CD123 antibodies or antigen-binding fragments provided herein for the preparation of a medicament for the treatment of tumor or cancer.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immune effector cells (e.g., CD123 CARTs) disclosed herein. In some embodiments, provided herein are uses of the immune effector cells disclosed herein (e.g., CD123 CARTs) in treatment of tumor or cancer. In some embodiments, provided herein are uses of the immune effector cells (e.g., CD123 CARTs) provided herein for the preparation of a medicament for the treatment of tumor or cancer. In some embodiments, a population of cells comprising the immune effector cell disclosed herein is used in the treatment. The population of cells can be homogenous. The population of cells can be heterogenous.

In some embodiments, provided herein are methods of treating tumor or cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, provided herein are uses of the pharmaceutical composition disclosed herein in treatment of tumor or cancer. In some embodiments, provided herein are uses of the pharmaceutical composition provided herein for the preparation of a medicament for the treatment of tumor or cancer.

Actual dosage levels of the active ingredients (i.e., the anti-CD123 antibodies or antigen-binding fragments or the immune effector cells provided herein) in the pharmaceutical compositions described herein can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The anti-CD123 antibodies or antigen-binding fragments can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the anti-CD123 antibodies or antigen-binding fragments in the patient. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease.

In some embodiments, immune effector cells provided herein that recombinantly express the CD123 CARs disclosed herein can be used in the therapeutic methods disclosed herein. As described above, the immune effector cells can express a fusion protein (LACO) disclosed herein. When a cell therapy is adopted, the cells provided herein can be administered as a dose based on cells per kilogram (cells/kg) of body weight of the subject to which the cells are administered. The cell doses can be in the range of about 10 ⁴ to about 10 ¹⁰ cells/kg of body weight, for example, about 10 ⁵ to about 10 ⁹, about 10 ⁵ to about 10 ⁸, about 10 ⁵ to about 10 ⁷, or about 10 ⁵ to 10 ⁶ cells/kg of body weight, depending on the mode and location of administration. In general, in the case of systemic administration, a higher dose is used than in regional administration, where the immune effector cells are administered in the region of a tumor. The precise determination of what would be considered an effective dose can be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject, as described above. Dosages can be readily determined by those skilled in the art based on the disclosure herein and knowledge in the art.

The anti-CD123 antibodies or antigen-binding fragments, immune effector cells, and pharmaceutical compositions provided herein can be administered to a subject by any methods known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal or other parenteral routes of administration, for example by injection or infusion, or direct administration to the thymus. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. In some embodiments, subcutaneous administration is adopted. In some embodiments, intravenous administration is adopted. In some embodiments, oral administration is adopted. In one embodiment, the cells provided herein can be delivered regionally to a tumor using well known methods, including but not limited to, hepatic or aortic pump; limb, lung or liver perfusion; in the portal vein; through a venous shunt; in a cavity or in a vein that is nearby a tumor, and the like. In another embodiment, the cells provided herein can be administered systemically. In a preferred embodiment, the cells are administered regionally at the site of a tumor. The cells can also be administered intratumorally, for example, by direct injection of the cells at the site of a tumor and/or into the tumor vasculature. For example, in the case of malignant pleural disease, mesothelioma or lung cancer, administration is preferably by intrapleural administration (see Adusumilli et al., Science Translational Medicine 6 (261) : 261ra151 (2014) ) . One skilled in the art can select a suitable mode of administration based on the type of cancer and/or location of a tumor to be treated. The cells can be introduced by injection or catheter. In one embodiment, the cells are pleurally administered to the subject in need, for example, using an intrapleural catheter. Optionally, expansion and/or differentiation agents can be administered to the subject prior to, during or after administration of cells to increase production of the cells provided herein in vivo.

Proliferation of the cells provided herein is generally done ex vivo, prior to administration to a subject, and can be desirable in vivo after administration to a subject (see Kaiser et al., Cancer Gene Therapy 22: 72-78 (2015) ) . Cell proliferation should be accompanied by cell survival to permit cell expansion and persistence, such as with T cells.

In some embodiments, cancers or tumors that can be treated with the anti-CD123 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions disclosed herein are hematological cancers. The hematological cancers can be CD123-expressing hematological cancers. In some embodiments, the hematological cancer can be leukemia, lymphoma, multiple myeloma (MM) , or myelodysplastic syndrome (MDS) . In some embodiments, the hematological cancer can be acute leukemia, acute myeloid leukemia (AML) , B-acute lymphoid leukemia (B-ALL) , T-acute lymphoid leukemia (T-ALL) , B cell precursor acute lymphoblastic leukemia (BCP-ALL) , blastic plasmacytoid dendritic cell neoplasm (BPDCN) , acute lymphocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia (CML) , chronic myelocytic leukemia, chronic lymphocytic leukemia, chronic myelomonocytic leukemia (CMML) , natural killer cell leukemia (NK leukemia) , Hodgkin’s disease, non-Hodgkin’s disease, Waldenstrom’s macroglobulinemia, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma, natural killer cell lymphoma (NK lymphoma) , cutaneous T-Cell lymphoma (CTCL) , or peripheral T-cell lymphoma (PTCL) . In some embodiments, cancers or tumors that can be treated with the anti-CD123 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions disclosed herein are leukemias. In some embodiments, the leukemia is AML. In some embodiments, the leukemia is B-ALL. In some embodiments, the leukemia is T-ALL. In some embodiments, the leukemia is BCP-ALL. In some embodiments, the leukemia is BPDCN.

In some embodiments, provided herein are methods of treating CD123-expressing leukemia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-CD123 antibodies or antigen-binding fragments, the CD123 CARTs, or the pharmaceutical compositions disclosed herein.

In some embodiments, cancers or tumors that can be treated with the anti-CD123 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions disclosed herein are solid tumors. In some embodiments, the cancer or tumor can be carcinomas, sarcoma, melanoma (e.g., cutaneous or intraocular malignant melanoma) , glioma, glioblastoma, brain and spinal cord tumors, germ cell tumors, neuroendocrine tumors, carcinoid tumors, gastric cancer, esophageal cancer, liver cancer, lung cancer (e.g., small cell lung cancer, or non-small cell lung cancer) , head and neck cancer, skin cancer, nasopharyngeal cancer, kidney cancer, colorectal cancer, breast cancer, pancreatic cancer, testicular cancer, cervical cancer, ovarian cancer, uterine cancer, prostate cancer (for example, hormone refractory prostate adenocarcinoma) , bladder cancer, colon cancer, endocrine cancer, basal cell cancer, squamous cell cancer, dermatofibrosarcoma protuberans, mesothelioma, Merkel cell carcinoma, bone cancer, intestinal cancer, renal cancer (for example, clear cell carcinoma) , throat cancer, rectal cancer, cancer of the anal region, brain cancer, stomach cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the small intestine, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, synovial sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, neuroblastoma, or retinoblastoma.

In cancer treatment, eliminating cancer or tumor cells in a subject can occur, but any clinical improvement constitutes a benefit. An anti-tumor effect can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An anti-tumor effect can also be manifested by the ability of the cells or pharmaceutical compositions provided herein in prevention of the occurrence of tumor in the first place. In some embodiments, an “anti-tumor effect” can be manifested by the reduction in cancer-induced immunosuppression. Clinical improvement comprises decreased risk or rate of progression or reduction in pathological consequences of the cancer or tumor. It is also understood that a method of treating cancer can include any effect that ameliorates a sign or symptom associated with cancer. Such signs or symptoms include, but are not limited to, reducing tumor burden, including inhibiting growth of a tumor, slowing the growth rate of a tumor, reducing the size of a tumor, reducing the number of tumors, eliminating a tumor, all of which can be measured using routine tumor imaging techniques well known in the art. Other signs or symptoms associated with cancer include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers.

In some embodiments, the methods or uses provided herein can reduce tumor burden. Thus, administration of the anti-CD123 antibodies or antigen-binding fragments, cells or pharmaceutical compositions disclosed herein can reduce the number of tumor cells, reduce tumor size, and/or eradicate the tumor in the subject. Methods for monitoring patient response to administration of a pharmaceutical composition disclosed herein are known in the art and can be employed in accordance with methods disclosed herein.

In the methods disclosed herein, a therapeutically effective amount of the anti-CD123 antibodies or antigen-binding fragments, cells or pharmaceutical compositions disclosed herein is administered to a subject in need of cancer treatment. The subject can be a mammal. In some embodiments, the subject is a human. In some embodiments, these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts and are suitably defined for different types of cancers. Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.

Anti-CD123 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein can be administered with medical devices known in the art. For example, in some embodiments, a needleless hypodermic injection device can be used, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use described herein include: U.S. Patent No.4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

Combination therapy using agents with different mechanisms of action can result in additive or synergetic effects. Combination therapy can allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent disclosed herein. Combination therapy can decrease the likelihood that resistant cancer cells will develop. In some embodiments, the additional therapy results in an increase in the therapeutic index of the cells or pharmaceutical compositions described herein. In some embodiments, the additional therapy results in a decrease in the toxicity and/or side effects of cells or pharmaceutical compositions described herein. In some embodiments, the anti-CD123 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions described herein can be administered in combination with an additional therapy. In some embodiments, the additional therapy can be surgical resection, radiotherapy, or chemotherapy.

The additional therapy can be administered prior to, concurrently with, or subsequent to administration of the anti-CD123 antibodies or antigen-binding fragments, cells, or pharmaceutical compositions described herein. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. A person skilled in the art can readily determine appropriate regimens for administering a pharmaceutical composition described herein and an additional therapy in combination, including the timing and dosing of an additional agent to be used in a combination therapy, based on the needs of the subject being treated.

5.9 Methods of production

5.9.1 Polynucleotides, polypeptides, and antibodies

Polynucleotides provided herein can be prepared, manipulated, and/or expressed using any of a variety of well-established techniques known and available in the art. Many vectors can be used. Examples of vectors are plasmid, autonomously replicating sequences, and transposable elements. Exemplary transposon systems such as Sleeping Beauty and PiggyBac can be used, which can be stably integrated into the genome (e.g., Ivics et al., Cell, 91 (4) : 501–510 (1997) ;

et al., (2007) Nucleic Acids Research. 35 (12) : e87) . Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST ^TM, pLenti6/V5-DEST ^TM, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.

In some embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term “episomal” refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally. The vector is engineered to harbor the sequence coding for the origin of DNA replication or “ori” from a lymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically a replication origin of a lymphotrophic herpes virus or a gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotrophic herpes virus may be Epstein Barr virus (EBV) , Kaposi's sarcoma herpes virus (KSHV) , Herpes virus saimiri (HS) , or Marek's disease virus (MDV) . Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examples of a gamma herpesvirus. Typically, the host cell comprises the viral replication transactivator protein that activates the replication.

“Expression control sequences, ” “control elements, ” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector-origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions-which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters can be used.

Illustrative ubiquitous expression control sequences that can be used in present disclosure include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) promoter (e.g., early or late) , a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1) , ferritin H (FerH) , ferritin L (FerL) , Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , eukaryotic translation initiation factor 4A1 (EIF4A1) , heat shock 70kDa protein 5 (HSPA5) , heat shock protein 90kDa beta, member 1 (HSP90B1) , heat shock protein 70kDa (HSP70) , β-kinesin (β-KIN) , the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477 -1482 (2007) ) , a Ubiquitin C promoter (UBC) , a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer/chicken β-actin (CAG) promoter, and a β-actin promoter.

Illustrative examples of inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone) , metallothionine promoter (inducible by treatment with various heavy metals) , MX-1 promoter (inducible by interferon) , the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323: 67) , the cumate inducible gene switch (WO 2002/088346) , tetracycline-dependent regulatory systems, etc.

The anti-CD123 antibodies or antigen-binding fragments described herein can be produced by any method known in the art, including chemical synthesis, recombinant expression, conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique (See e.g., Kohler and Milstein, Nature 256: 495 (1975) ) , viral or oncogenic transformation of B lymphocytes. The practice of the invention employs, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature. See, e.g., Maniatis et al. (1982) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989) , MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons (1987 and annual updates) ; CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley &Sons (1987 and annual updates) Gait (ed. ) (1984) OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press; Eckstein (ed. ) (1991) OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, IRL Press; Birren et al. (eds. ) (1999) GENOME ANALYSIS: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Borrebaeck (ed. ) (1995) ANTIBODY ENGINEERING, Second Edition, Oxford University Press; Lo (ed. ) (2006) ANTIBODY ENGINEERING: METHODS AND PROTOCOLS (METHODS IN MOLECULAR BIOLOGY) ; Vol. 248, Humana Press, Inc; each of which is incorporated herein by reference in its entirety.

The polypeptides described herein (e.g., the anti-CD123 antibodies or antigen-binding fragments) can be produced and isolated using methods known in the art. Peptides can be synthesized, in whole or in part, using chemical methods (see, e.g., Caruthers (1980) . Nucleic Acids Res. Symp. Ser. 215; Horn (1980) ; and Banga, A.K., THERAPEUTIC PEPTIDES AND PROTEINS, FORMULATION, PROCESSING AND DELIVERY SYSTEMS (1995) Technomic Publishing Co., Lancaster, PA) . Peptide synthesis can be performed using various solid phase techniques (see, e.g., Roberge Science 269: 202 (1995) ; Merrifield, Methods. Enzymol. 289: 3 (1997) ) and automated synthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the manufacturer’s instructions. Peptides can also be synthesized using combinatorial methodologies. Synthetic residues and polypeptides can be synthesized using a variety of procedures and methodologies known in the art (see, e.g., ORGANIC SYNTHESES Collective Volumes, Gilman, et al. (Eds) John Wiley &Sons, Inc., NY) . Modified peptides can be produced by chemical modification methods (see, for example, Belousov, Nucleic Acids Res. 25: 3440 (1997) ; Frenkel, Free Radic. Biol. Med. 19: 373 (1995) ; and Blommers, Biochemistry 33: 7886 (1994) ) . Peptide sequence variations, derivatives, substitutions and modifications can also be made using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR based mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13: 4331 (1986) ; Zoller et al., Nucl. Acids Res. 10: 6487 (1987) ) , cassette mutagenesis (Wells et al., Gene 34: 315 (1985) ) , restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 317: 415 (1986) ) and other techniques can be performed on cloned DNA to produce invention peptide sequences, variants, fusions and chimeras, and variations, derivatives, substitutions and modifications thereof.

The polypeptides described herein can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant technologies, or a combination thereof. In some embodiments, a recombinant expression vector is used to express a polynucleotide encoding a polypeptide described herein. For example, a recombinant expression vector can be a replicable DNA construct that includes synthetic or cDNA-derived DNA fragments encoding a polypeptide operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. In some embodiments, coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for their expression in mammalian cells. In some embodiments, a viral vector is used. DNA regions are “operatively linked” when they are functionally related to each other. For example, a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In some embodiments, in situations where recombinant protein is expressed without a leader or transport sequence, a polypeptide can include an N-terminal methionine residue.

A wide variety of expression host/vector combinations can be employed. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well-known in the art. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney-derived) , L-929 (murine fibroblast-derived) , C127 (murine mammary tumor-derived) , 3T3 (murine fibroblast-derived) , CHO (Chinese hamster ovary-derived) , HeLa (human cervical cancer-derived) , BHK (hamster kidney fibroblast-derived) , HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5’ or 3’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

Provided herein are anti-CD123 antibodies and antigen-binding fragments thereof that include but are not limited to monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, and antigen-binding fragments thereof.

Methods of antibody production are well-known in the art. See for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) ; Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981) , each of which is incorporated herein by reference in its entirety. For in vivo use of antibodies in humans, it may be preferable to use human antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements to these techniques. See, also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. A human antibody can also be an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human DNA.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. For example, anti-CD123 antibodies directed against the human CD123 antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies, including, but not limited to, IgG1 (gamma 1) and IgG3. For an overview of this technology for producing human antibodies, see, Lonberg and Huszar (Int. Rev. Immunol., 13: 65-93 (1995) ) . For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif. ) and Genpharm (San Jose, Calif. ) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. For a specific discussion of transfer of a human germ-line immunoglobulin gene array in germ-line mutant mice that will result in the production of human antibodies upon antigen challenge see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993) ; Jakobovits et al., Nature, 362: 255-258 (1993) ; Bruggermann et al., Year in Immunol., 7: 33 (1993) ; and Duchosal et al., Nature, 355: 258 (1992) .

Human antibodies can also be derived from phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581-597 (1991) ; Vaughan et al., Nature Biotech., 14: 309 (1996) ) . Phage display technology (McCafferty et al., Nature, 348: 552-553 (1990) ) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3: 564-571 (1993) . Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of unimmunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol., 222: 581-597 (1991) , or Griffith et al., EMBO J., 12: 725-734 (1993) . See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905, each of which is incorporated herein by reference in its entirety.

Human antibodies can also be generated by in vitro activated B cells (see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which is incorporated herein by reference in its entirety) . Human antibodies can also be generated in vitro using hybridoma techniques such as, but not limited to, that described by Roder et al. (Methods Enzymol., 121: 140-167 (1986) ) .

Alternatively, in some embodiments, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human. In some embodiment, the antigen binding domain portion is humanized.

A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No.

EP

239, 400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference) , veneering or resurfacing (see, e.g., European Patent Nos. EP 592, 106 and EP 519, 596; Padlan, 1991, Molecular Immunology, 28 (4/5) : 489-498; Studnicka et al., 1994, Protein Engineering, 7 (6) : 805-814; and Roguska et al., 1994, PNAS, 91: 969-973, each of which is incorporated herein by its entirety by reference) , chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference) , and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002) , Caldas et al., Protein Eng., 13 (5) : 353-60 (2000) , Morea et al., Methods, 20 (3) : 267-79 (2000) , Baca et al., J. Biol. Chem., 272 (16) : 10678-84 (1997) , Roguska et al., Protein Eng., 9 (10) : 895-904 (1996) , Couto et al., Cancer Res., 55 (23 Supp) : 5973s-5977s (1995) , Couto et al., Cancer Res., 55 (8) : 1717-22 (1995) , Sandhu J S, Gene, 150 (2) : 409-10 (1994) , and Pedersen et al., J. Mol. Biol., 235 (3) : 959-73 (1994) , each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332: 323, which are incorporated herein by reference in their entireties. )

A humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Thus, humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human. Humanization of antibodies is well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ) , by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (

EP

239, 400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference herein in their entirety) . In such humanized chimeric antibodies, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies can also be achieved by veneering or resurfacing (EP 592, 106; EP 519, 596; Padlan, 1991, Molecular Immunology, 28 (4/5) : 489-498; Studnicka et al., Protein Engineering, 7 (6) : 805-814 (1994) ; and Roguska et al., PNAS, 91: 969-973 (1994) ) or chain shuffling (U.S. Pat. No. 5,565,332) , the contents of which are incorporated herein by reference herein in their entirety.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993) ; Chothia et al., J. Mol. Biol., 196: 901 (1987) , the contents of which are incorporated herein by reference herein in their entirety) . Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992) ; Presta et al., J. Immunol., 151: 2623 (1993) , the contents of which are incorporated herein by reference herein in their entirety) .

Antibodies can be humanized with retention of high affinity for the target antigen and other favorable biological properties. For example, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

A “humanized” antibody retains a similar antigenic specificity as the original antibody, for example, the ability to bind human CD123 antigen. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody for a particular antigen can be increased using methods of “directed evolution, ” as described by Wu et al., J. Mol. Biol., 294: 151 (1999) , the contents of which are incorporated herein by reference herein in their entirety.

5.9.2 Genetically engineered immune effector cells

In some embodiments, provided herein is a genetically engineered immune effector cell that recombinantly expresses a CD123 CAR disclosed herein. In some embodiments, provided herein is a genetically engineered immune effector cell that comprises a polynucleotide encoding a CD123 CAR disclosed herein. In some embodiments, provided herein is a genetically engineered immune effector cell that comprises a vector comprising a polynucleotide encoding a CD123 CAR disclosed herein. In some embodiments, the immune effector cells are T cells.

5.9.2.1 Methods of genetic engineering

With respect to generating cells recombinantly expressing a CD123 CAR disclosed herein, one or more polynucleotides encoding the CD123 CAR is introduced into the target cell using a suitable expression vector. The target immune effector cells (e.g., T cells) are transferred with one or more polynucleotides encoding a CD123 CAR. The genetically engineered cells can also express the anti-CD123 antibodies or antigen-binding fragments disclosed herein.

In some embodiments, provided herein are methods of genetically engineering an immune effector cell by transferring a polynucleotide provided herein into the cell using a non-viral delivery system. The CD123 CAR encoding polynucleotide can be mRNA, which allows transient expression and the self-elimination of the immune effector cells expressing such CD123 CAR. Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. In some embodiments, RNA electroporation can be used (Van Driessche et al. Folia histochemica et cytobiologica 43: 4 213-216 (2005) ) . The methods can further include preparing the mRNA by in vitro transcribing the polynucleotides described herein. In some embodiments, provided herein are methods of genetically engineering an immune effector cell by transferring a polynucleotide encoding anti-CD123 antibodies or antigen-binding fragments disclosed herein into the cell using electroporation. In some embodiments, provided herein are methods of genetically engineering an immune effector cell by transferring a polynucleotide encoding CD123 CARs disclosed herein into the cell using electroporation.

In some embodiments, DNA transfection and transposon can be used. In some embodiments, the Sleeping Beauty system or PiggyBac system is used (e.g., Ivics et al., Cell, 91 (4) : 501-510 (1997) ;

et al. (2007) Nucleic Acids Research. 35 (12) : e87) . Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle) .

For example, a polynucleotide encoding a CD123 CAR disclosed herein can be cloned into a suitable vector, and introduced into the target cell using well known molecular biology techniques (see Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999) ) . Any vector suitable for expression in a cell, particularly a human cell, can be used. The vectors contain suitable expression elements such as promoters that provide for expression of the encoded nucleic acids in the target cell.

Particularly useful vectors for expressing a CAR, or antibody disclosed herein include vectors that have been used in human gene therapy. In some embodiments, a vector is a retroviral vector. The use of retroviral vectors for expression in T cells or other immune effector cells, including engineered T cells, has been described (see Scholler et al., Sci. Transl. Med. 4: 132-153 (2012; Parente-Pereira et al., J. Biol. Methods 1 (2) : e7 (1-9) (2014) ; Lamers et al., Blood 117 (1) : 72-82 (2011) ; Reviere et al., Proc. Natl. Acad. Sci. USA 92: 6733-6737 (1995) ) . In one embodiment, the vector is a gamma retroviral. In one embodiment, the vector is an SGF retroviral vector such as an SGF γ-retroviral vector, which is Moloney murine leukemia-based retroviral vector. SGF vectors have been described previously (see, for example, Wang et al., Gene Therapy 15: 1454-1459 (2008) ) . Cells can optionally be activated to increase transduction efficiency (see Parente-Pereira et al., J. Biol. Methods 1 (2) e7 (doi 10.14440/jbm. 2014.30) (2014) ; Movassagh et al., Hum. Gene Ther. 11: 1189-1200 (2000) ; Rettig et al., Mol. Ther. 8: 29-41 (2003) ; Agarwal et al., J. Virol. 72: 3720-3728 (1998) ; Pollok et al., Hum. Gene Ther. 10: 2221-2236 (1998) ; Quinn et al., Hum. Gene Ther. 9: 1457-1467 (1998) ; see also commercially available methods such as Dynabeads ^TM human T cell activator products, Thermo Fisher Scientific, Waltham, MA) .

It is understood that any suitable viral vector or non-viral delivery system can be used. Combinations of a retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., Mol. Cell. Biol. 5: 431-437 (1985) ) ; PA317 (Miller et al., Mol. Cell. Biol. 6: 2895-2902 (1986) ) ; and CRIP (Danos et al., Proc. Natl. Acad. Sci. USA 85: 6460-6464 (1988) ) . Non-amphotropic particles are suitable too, for example, particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art (Relander et al., Mol. Therap. 11: 452-459 (2005) ) . Possible methods of transduction also include direct co-culture of the cells with producer cells (for example, Bregni et al., Blood 80: 1418-1422 (1992) ) , or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations (see, for example, Xu et al., Exp. Hemat. 22: 223-230 (1994) ; Hughes, et al. J. Clin. Invest. 89: 1817-1824 (1992) ) .

Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus derived vector, or a herpes virus, such as Epstein-Barr Virus (see, for example, Miller, Hum. Gene Ther. 1 (1) : 5-14 (1990) ; Friedman, Science 244: 1275-1281 (1989) ; Eglitis et al., BioTechniques 6: 608-614 (1988) ; Tolstoshev et al., Current Opin. Biotechnol. 1: 55-61 (1990) ; Sharp, Lancet 337: 1277-1278 (1991) ; Cornetta et al., Prog. Nucleic Acid Res. Mol. Biol. 36: 311-322 (1989) ; Anderson, Science 226: 401-409 (1984) ; Moen, Blood Cells 17: 407-416 (1991) ; Miller et al., Biotechnology 7: 980-990 (1989) ; Le Gal La Salle et al., Science 259: 988-990 (1993) ; and Johnson, Chest 107: 77S-83S (1995) ) . Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med. 323: 370 (1990) ; Anderson et al., U.S. Pat. No. 5,399,346) . Generally, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8: 423-430 (1997) ; Kido et al., Current Eye Research 15: 833-844 (1996) ; Bloomer et al., J. Virol. 71: 6641-6649 (1997) ; Naldini et al., Science 272: 263-267 (1996) ; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319-10323 (1997) ) .

The vectors used herein employ suitable promoters for expression in a particular host cell. The promoter can be an inducible promoter or a constitutive promoter. In some embodiments, the promoter of an expression vector provides expression in a stem cell, such as a hematopoietic stem cell. In some embodiments, the promoter of an expression vector provides expression in an immune effector cell, such as a T cell. Non-viral vectors can be used as well, so long as the vector contains suitable expression elements for expression in the target cell. Some vectors, such as retroviral vectors, can integrate into the host genome.

In some embodiments, provided herein are methods of genetically engineering an immune effector cell by transferring a polynucleotide provided herein into the cell using gene-editing. If desired, targeted integration can be implemented using technologies such as a nuclease, transcription activator-like effector nucleases (TALENs) , Zinc-finger nucleases (ZFNs) , clustered regularly interspaced short palindromic repeats (CRISPRs) , homologous recombination, non-homologous end joining, microhomology-mediated end joining, homology-mediated end joining and the like (Gersbach et al., Nucl. Acids Res. 39: 7868-7878 (2011) ; Vasileva, et al. Cell Death Dis. 6: e1831. (Jul 23 2015) ; Sontheimer, Hum. Gene Ther. 26 (7) : 413-424 (2015) ; Yao et al. Cell Research volume 27, 801-814 (2017) ) . In some embodiments, methods provided herein use a ZFN system. A zinc-finger nuclease consists of a DNA recognition domain and a non-specific endonuclease. The DNA recognition domain consists of a series of Cys2-His2 zinc-finger proteins linked in series, and each zinc-finger unit includes about 30 amino acids for specifically binding to DNA. The non-specific endonuclease is a FokI endonuclease which forms a dimer to cleave the DNA. In some embodiments, methods provided herein use a TALEN system. TALEN is a transcription activator-like effector nuclease. The TALE protein is a core component of a DNA binding domain, and generally consists of a plurality of basic repeat units linked in series. The designed and combined series of units can specifically recognize a DNA sequence and cleave a specific DNA sequence by coupling the FokI endonuclease.

In some embodiments, methods provided herein use a CRISPR-Cas system. The CRISPR-Cas system can be a CRISPR-Cas9 system. CRISPR/Cas system is a nuclease system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR binding proteins (i.e., Cas proteins) , which can cleave nearly all genomic sequences adjacent to protospacer-adjacent motifs (PAM) in eukaryocytes (Cong et al. Science 2013.339: 819-823) . The “CRISPR/Cas system” is used to refer collectively to transcripts involving CRISPR-related ( “Cas” ) genes, as well as other elements involving the expression thereof or directing the activity thereof, including sequences encoding a Cas gene, tracr (trans-activated CRISPR) sequences (for example, tracrRNA or active partial tracrRNA) , tracr pairing sequences (in the background of an endogenous CRISPR system, cover “direct repeats” and processed partial direct repeats) , guide sequences, or other sequences from the CRISPR locus and transcripts. In general, the CRISPR system is characterized as an element that facilitates the formation of a CRISPR complex at a site of a target sequence (also called a protospacer in the endogenous CRISPR system) . Unrestricted examples of the Cas protein include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12) , Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 homologues, or modified forms thereof. In some embodiments, the Cas protein is a Cas9 protein (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Deltcheva, Chylinski et al. 2011; Makarova, Grishin et al. (2006) ) . Amino acid sequences of the Cas9 protein are known in the art. Exemplary sequences can be found, for example, in the SwissProt database under the accession number Q99ZW2, in the UniProt database under the number A1IQ68, Q03LF7, or J7RUA5.

The vectors and constructs can optionally be designed to include a reporter. For example, the vector can be designed to express a reporter protein, which can be useful to identify cells comprising the vector or polynucleotides provided on the vector, such as polynucleotides that have integrated into the host chromosome. In one embodiment, the reporter can be expressed as a bicistronic or multicistronic expression construct with the anti-CD123 antibody or antigen-binding fragment or the CD123 CAR. Exemplary reporter proteins include, but are not limited to, fluorescent proteins, such as mCherry, green fluorescent protein (GFP) , blue fluorescent protein, for example, EBFP, EBFP2, Azurite, and mKalama1, cyan fluorescent protein, for example, ECFP, Cerulean, and CyPet, and yellow fluorescent protein, for example, YFP, Citrine, Venus, and YPet.

Assays can be used to determine the transduction efficiency using routine molecular biology techniques. If a marker has been included in the construct, such as a fluorescent protein, gene transfer efficiency can be monitored by FACS analysis to quantify the fraction of transduced (for example, GFP ⁺) immune effector cells, such as T cells, and/or by quantitative PCR. Using a well-established cocultivation system (Gade et al., Cancer Res. 65: 9080-9088 (2005) ; Gong et al., Neoplasia 1: 123-127 (1999) ; Latouche et al., Nat. Biotechnol. 18: 405-409 (2000) ) it can be determined whether fibroblast AAPCs expressing cancer antigen (vs. controls) direct cytokine release from transduced immune effector cells, such as T cells, expressing a CAR (cell supernatant LUMINEX (Austin TX) assay for IL-2, IL-4, IL-10, IFN-γ, TNF-α, and GM-CSF) , T cell proliferation (by carboxyfluorescein succinimidyl ester (CFSE) labeling) , and T cell survival (by Annexin V staining) . The influence of CD80 and/or 4-1BBL on T cell survival, proliferation, and efficacy can be evaluated. T cells can be exposed to repeated stimulation by cancer antigen positive target cells, and it can be determined whether T cell proliferation and cytokine response remain similar or diminished with repeated stimulation. The cancer antigen CAR constructs can be compared side by side under equivalent assay conditions. Cytotoxicity assays with multiple E: T ratios can be conducted using chromium-release assays.

Combinations and permutations of various methods described herein or otherwise known in the art are expressly contemplated to prepare the genetically engineered cells disclosed herein.

5.9.2.2 Manipulation of immune effector cells

Immune effector cells provided herein can be obtained from a subject. Sources for the immune effector cells provided herein include, but are not limited to, peripheral blood, umbilical cord blood, bone marrow, or other sources of hematopoietic cells. Immune effector cells (e.g., T cells) can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, cell lines available in the art can be used. Immune effector cells provided herein can be isolated by methods well known in the art, including commercially available isolation methods (see, for example, Rowland-Jones et al., LYMPHOCYTES: A PRACTICAL APPROACH, Oxford University Press, New York (1999) ) . Various methods for isolating immune effector cells have been described previously, and can be used, including but not limited to, using peripheral donor lymphocytes (Sadelain et al., Nat. Rev. Cancer 3 : 35-45 (2003) ; Morgan et al., Science 314: 126-129 (2006) , and using selectively in v/Yro-expanded antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or dendritic cells (Dupont et al., Cancer Res. 65: 5417-5427 (2005) ; Papanicolaou et al., Blood 102: 2498-2505 (2003) ) .

In certain embodiments, immune effector cells (e.g., T cells) disclosed herein can be obtained from a unit of blood collected from a subject using any techniques known to the skilled artisan, such as Ficoll ^TM separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS) . In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step can be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca ²⁺-free, Mg ²⁺-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample can be removed, and the cells directly resuspended in culture media.

In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL ^TM gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3 ⁺, CD28 ⁺, CD4 ⁺, CD8 ⁺, CD45RA ⁺, and CD45RO ⁺T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3×28) -conjugated beads, such as

M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune-compromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein) , subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention.

Various techniques can be employed to separate the cells to enrich for desired immune effector cells. For instance, negative selection methods can be used to remove cells that are not the desired immune effector cells. Additionally, positive selection methods can be used to isolate or enrich for desired immune effector cells or precursor cells thereof, or a combination of positive and negative selection methods can be employed. Monoclonal antibodies (MAbs) are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation for both positive and negative selections. If a particular type of cell is to be isolated, for example, a particular type of T cell, various cell surface markers or combinations of markers, including but not limited to, CD3, CD4, CD8, CD34 (for hematopoietic stem and progenitor cells) and the like, can be used to separate the cells, as is well known in the art (see Kearse, T CELL PROTOCOLS: DEVELOPMENT AND ACTIVATION, Humana Press, Totowa NJ (2000) ; De Libero, T CELL PROTOCOLS, Vol. 514 of Methods in Molecular Biology, Humana Press, Totowa NJ (2009) ) . In some embodiments, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4 ⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4 ⁺, CD25 ⁺, CD62L ^hi, GITR ⁺, and FoxP3 ⁺. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.

Procedures for separation of immune effector cells include, but are not limited to, density gradient centrifugation, coupling to particles that modify cell density, magnetic separation with antibody-coated magnetic beads, affinity chromatography; cytotoxic agents joined to or used in conjunction with a monoclonal antibody (mAb) , including, but not limited to, complement and cytotoxins, and panning with an antibody attached to a solid matrix, for example, a plate or chip, elutriation, flow cytometry, or any other convenient technique (see, for example, Recktenwald et al., CELL SEPARATION METHODS AND APPLICATIONS, Marcel Dekker, Inc., New York (1998) ) . It is understood that the immune effector cells used in methods provided herein can be substantially pure cells or can be a polyclonal population. In some embodiments, a polyclonal population can be enriched for a desired immune effector cell. Such an enrichment can take place prior to or after genetically engineering the cells to express a CD123 CAR provided herein, as desired.

The immune effector cells can be autologous or non-autologous to the subject to which they are administered in the methods of treatment disclosed herein. Autologous cells are isolated from the subject to which the engineered cells are to be administered. Optionally, the cells can be obtained by leukapheresis, where leukocytes are selectively removed from withdrawn blood, made recombinant, and then retransfused into the donor. Alternatively, allogeneic cells from a non-autologous donor that is not the subject can be used. In the case of a non-autologous donor, the cells are typed and matched for human leukocyte antigen (HLA) to determine an appropriate level of compatibility, as is well known in the art. The cells can optionally be cryopreserved after isolation and/or genetic engineering, and/or expansion of genetically engineered cells (see Kaiser et al., supra, 2015) ) . Methods for cyropreserving cells are well known in the art (see, for example, Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, 4th ed., Wiley-Liss, New York (2000) ; Harrison and Rae, GENERAL TECHNIQUES OF CELL CULTURE, Cambridge University Press (1997) ) .

In some embodiments, isolated immune effector cells are genetically engineered ex vivo for recombinant expression of a polypeptide (e.g., CAR) . In some embodiments, isolated immune effector cells are genetically engineered ex vivo for recombinant expression of a CD123 CAR. In some embodiments, immune effector cells provided herein are obtained by in vitro sensitization, wherein the sensitization can occur before or after the immune effector cells are genetically engineered to recombinantly express a polypeptide disclosed herein. In an embodiment where the sensitized immune effector cells, such T cells, are isolated from in vivo sources, it will be self-evident that genetic engineering occurs of the already-sensitized immune effector cells.

Also contemplated in the present disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the genetically engineered cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells can be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun. 5: 763-773, 1993) . In a further embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT) , cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.

In a further embodiment, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained can be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated to collect blood cells, including T cells, NK cells, or other immune effector cells of the hematopoietic lineage, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

The immune effector cells disclosed herein can be subjected to conditions that favor maintenance or expansion of cells as well known in the art. (De Libero, T Cell Protocols, Vol. 514 of Methods in Molecular Biology, Humana Press, Totowa NJ (2009) ; Parente-Pereira et al., J. Biol. Methods 1 (2) e7 (doi 10.14440/jbm. 2014.30) (2014) ; Movassagh et al., Hum. Gene Ther. 11: 1189-1200 (2000) ; Rettig et al., Mol. Ther. 8: 29-41 (2003) ; Agarwal et al., J. Virol. 72: 3720-3728 (1998) ; Pollok et al., Hum. Gene Ther. 10: 2221-2236 (1999) ; Quinn et al., Hum. Gene Ther. 9: 1457-1467 (1998) ; see also commercially available methods such as Dynabeads ^TM human T cell activator products, Thermo Fisher Scientific, Waltham, MA) ) . The immune effector cells disclosed herein (e.g., T cells) can optionally be expanded prior to or after ex vivo genetic engineering. Expansion of the cells is particularly useful to increase the number of cells for administration to a subject. Such methods for expansion of cells are well known in the art (see e.g., Kaiser et al., Cancer Gene Therapy 22: 72-78 (2015) ; Wolfl et al., Nat. Protocols 9: 950-966 (2014) ) . Furthermore, the cells can optionally be cryopreserved after isolation and/or genetic engineering, and/or expansion of genetically engineered cells (see Kaiser et al., supra, 2015) ) . Methods for cyropreserving cells are well known in the art (see, for example, Freshney, Culture of Animal Cells: A Manual of Basic Techniques, 4th ed., Wiley-Liss, New York (2000) ; Harrison and Rae, General Techniques of Cell Culture, Cambridge University Press (1997) ) .

Generally, the T cells provided herein can be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory receptor on the surface of the T cells. In particular, T cell populations can be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4 ⁺ T cells or CD8 ⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30 (8) : 3975-3977, 1998; Haanen et al., J. Exp. Med. 190 (9) : 13191328, 1999; Garland et al., J. Immunol Meth. 227 (1-2) : 53-63, 1999) .

The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless disclosed herein.

Particular embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Upon reading the foregoing description, variations of the disclosed embodiments shall become apparent to individuals working in the art, and it is expected that those skilled artisans can employ such variations as appropriate. Accordingly, it is intended that the invention be practiced otherwise than as specifically described herein, and that the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference in its entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided can be different from the actual publication dates which need to be independently confirmed.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the descriptions in the Experimental are intended to illustrate but not limit the scope of invention described in the claims.

5.10 Experimental

5.10.1 Background

Interleukin 3 (IL-3) , mainly produced by activated T-lymphocytes, is a pluripotent cytokine required for the proliferation and differentiation of a wide range of primitive hematopoietic progenitor cells and lineage-committed cells, including granulocytes, macrophages, megakaryocytes, erythroid cells, and mast cells, resulting in formation of multiple lineages (Ihle 1992, Broughton, Nero et al. 2015) . IL-3 belongs to the beta chain common (βc) cytokine family, it signals through the activation of heterodimeric cell surface receptors, composed by interleukin-3 receptor alpha chain (IL-3Rα, also known as CD123) and the shared βc subunit. The IL-3 receptor heterodimer complex activates JAK2/STAT5 and MAP kinase pathways, and promoted cell proliferation and differentiation (Hercus, Dhagat et al. 2013, Broughton, Nero et al. 2015) .

5.10.2 Preparation of human anti-CD123 antibodies

The following steps were taken to prepare fully human anti-CD123 antibodies:

1) Expression and purification of phage display library: infecting the log phase TG1 library culture with freshly thawed M13K07 helper phage with a multiplicity of infection of 20: 1 (phage-to-cell-ratio) and overnight induction by IPTG; the phage library was purified by PEG/NaCl precipitated method and phage titer was determined.

2) Selection of CD123-specific scFv-phages: in the first round of selection, Maxisorp plate was coated with 20 μg/ml CD123-6His protein overnight at 4℃, washed with PBS, blocked with 5%milk+1%BSA in 1×PBS, incubate in the phage solution for 2 hours, and washed 10 times with PBST. The antigen-bound scFv-phages were eluted using acid elution buffer (pH 2.2) , neutralized, inoculated in 15 ml of log-phase TG1 culture (OD600=0.5) , cultured at 37℃ by 30 min standing and 30 min shaking, plated onto 2xYT-GA agar plate, and cultured overnight at 30℃ for subsequent selection. For the subsequence rounds of selection, more stringent selection conditions were used, including decreased protein concentration for coating (2 μg/ml in the 2nd round and 0.5 ug/ml in the 3rd round bio-panning) and increased wash cycles (20 cycles in the 2 ^nd round, 30 cycles in the 3 ^rd round) .

3) mpELISA screening. After 3 rounds of bio-panning, 292 positive colonies were selected for monoclonal phage ELISA (mpELISA) screening. Phage supernatant generated from individual bacterial clones was incubated with pre-blocked Maxisorp plate that coated with 2 μg/ml CD123-6His protein. After three washes, 100 μl/well of HRP-conjugated anti-M13 antibody diluted 1: 5000 in blocking buffer (5%milk+1%BSA in 1×PBS) was added and incubate for 60 min at RT. After washing plate 5 times with PBST, 100 μl/well TMB substrate solution was added and incubated for 10-30 min until blue color had appeared. Reaction was stopped by adding 50 μl/well of stop solution (2N H ₂SO ₄) . Absorbance was read at 450 nm in a microplate reader. FIG. 1 shows the resultant absorbance read of the mpELISA screening. As shown, a number of positive colonies (450 nm absorbance ≥ 0.5) were identified to produce anti-CD123 antibodies capable of binding CD123-6His protein (FIG. 1) .

4) Cloning and sequence analysis: positive clones were selected according to the ELISA results, and used as templates for PCR cloning of the scFv sequence (Forward primer sequence: tgcagctggcacgacaggtttc, reverse primer sequence: cgtcagactgtagcacgtt) . The PCR products were then sequenced by sanger sequencing method (Forward primer sequence: aacaattgaattcaggagga, reverse primer sequence: cctcctaagaagcgtagtc) . The CDR regions of scFv were analyzed through abysis website ( http: //abysis. org/) , and are provided above in Tables 1 and 2.

5.10.3 Preparation of CD123 CAR

Vectors for generating mRNA of anti-CD123 CAR were constructed. First, the scFv sequences and CAR fragment (from hinge domain to CD3-zeta domain) were amplified by PCR and cloned into pDA vector (Xba1/Sal1) . FIG. 2 provides the schematic representation of pDA-CAR vector used for CAR mRNA generation. Second, CD123 CAR mRNA was prepared by in vitro transcription (IVT) . The pDA-CAR plasmid was linearized by digestion with Spe1 enzyme and purified by PCR Cleanup kit. After the DNA concentration was measured by nanodrop and checked by running agarose DNA gel, IVT was performed following the protocol of manufacturer (Thermofisher, Cat No: AM13455) . The concentration of RNA product was measured by nanodrop and checked by running PAGE gel.

5.10.4 Preparation and characterization of CD123 CARTs

CD123 CAR mRNA was introduced into T cells by electroporation with the following procedures: T cells were collected and washed with Opti-MEM medium, and resuspended with Opti-MEM medium at 1×10e7/ml; 10 μg RNA was aliquoted with 100 μl T cells, mixed well for electroporation at the following parameters (BTX machine) : 500 voltage, 0.7 ms; the cells were then transferred to pre-warmed culture medium and culture at 37℃.

Binding of CD123 CART cells to CD123-Fc recombinant protein was measured by FACS staining. As shown in FIG. 3, T cells expressing CARs having anti-CD123 scFv-C1, -C2, -C3, -C4, -C5, -C6, -C7, -C9, -C10, -C11, -C13, -C14, -C15, -C16, -C17, -C18, -C19, -C21, -C23, -C24, -C25, -C26, -C27, -C28, -C29, -C30, -C32, -C33, -C34 and -C35 were able to bind to CD123-Fc recombinant protein. Mock was control T cell without CAR molecule.

A549 tumor cells were electroporated with different amount of CD123 mRNA. The electroporation procedure was the same as described above for T cells, except that a setting of 300 voltage, 0.5 ms was used. Expression of CD123 in A549 tumor cells was measured by FACS staining of the A549 cells electroporated with different amount of CD123 mRNA with isotype or anti-CD123 antibody. As shown in FIG. 4, A549 cells weakly expressed endogenous CD123, and the ectopic expression level of CD123 correlated with the amount of CD123 mRNA that was electroporated into A549 cells.

The cytotoxicity of the CD123 CART cells against tumor cells was measured in in vitro cytotoxicity assay. EGFP-expressing tumor cells or EGFP-A549 cells that were electroporated with different amount of tumor antigen were seeded on flat-bottomed 96-well plate at 3000 cells/100 ul/well; CART cells were diluted to appropriate concentration and seeded with 100 ul/well tumor cells at different E/T ratios, such as 10: 1, 3: 1, 1: 1; the co-culture plates were then placed in IncuCyte S3 machine, and scanning parameters were set. After 3 days scanning, the Total Green Object Integrated Intensity (GCU x μm ²/Well) was analyzed to calculate the killing efficiency.

FIG. 5 and FIG. 6 show the killing curves of different mRNA-based anti-CD123 CART cells against A549-GFP tumor cells at E/T ratio of 10: 1 (FIG. 5) or 3: 1 (FIG. 6) . As shown, CART cells expressing anti-CD123 scFv-C2, -C3, -C4, -C6, -C9, -C11, -C13, -C14, -C15, -C16, -C17, -C19, -C21, -C23, -C24 and -C32 effectively arrested the growth and even eliminated A549 cells, despite that A549 cells expressed low level endogenous CD123, indicating that these scFv-based CART cells had comparably high cytotoxicity against tumor cells.

FIG. 7 and FIG. 8 show the killing curves of different mRNA-based anti-CD123 CART cells against A549-GFP tumor cells that expressed exogenous CD123 (electroporated with 10 μg CD123 mRNA) at E/T ratio of 10: 1 (FIG. 7) or 3: 1 (FIG. 8) . As shown, CART cells expressing anti-CD123 scFv-C1, -C2, -C3, -C4, -C5, -C6, -C7, -C9, -C11, -C13, -C14, -C15, -C16, -C17, -C18, -C19, -C21, -C23, -C24, -C25, -C26, -C27, -C28, -C29, -C30, -C32, -C33, -C34 and -C35 CART cells effectively arrested the growth and even reduced the number of the CD123 expressing-A549 tumor cells, confirming their abilities to kill CD123 expressing tumor cells.

5.10.5 CD107a staining of CART cells.

The CD123 expression by various tumor cell lines were measured by FACS staining. FIG. 9 shows the FACS staining of A549, SK-OV3, Jeko-1, Molm-14, SupT-1, 293T, Nalm-6 and PC-3 cells with PE-isotype control and PE-anti-CD123 mAb. As shown, most tested tumor cell lines did not express CD123, with only Molm-14 expressing CD123 at a relatively high level.

CD107a is an early phase-activating marker for T cells. Activation of CD123 CARTs by CD123-expressing tumor cells was measured by CD107a staining with the following procedures: 20μl PE-CD107a mAb was added to each well of a 96-well plate; tumor cells were diluted to 2×10e6/ml and seeded in 96-well round plates (100 μl/well) ; CART cells were diluted to 1×10e6/ml and seeded in 96-well round plates (100 μl/well) ; the plates were centrifuged at 500 rpm×5 min to attach cells well and cultured at 37℃ for 1 hour; Golgi stop was diluted by 1500× with medium and added to each well (20 μl/well) ; cells were cultured at 37℃ for another 2.5 hours, stained with anti- CD3-APC and anti-CD8-FITC antibodies at 37℃ for 30 min, washed and analyze by flow cytometry.

In our studies, activation of CARTs (expressing anti-CD123-C5, anti-CD123-C7, anti-CD123-C11) by CD123-expressing tumor cells was measured by CD107a staining. Tested cells including A549 electroporated with 10 μg, 0.1 μg and 0 μg CD123 mRNA, SK-OV3, PC-3, cord blood derived CD34+ hematopoietic stem cells (CD34+ cord) , bone marrow derived hematopoietic stem cells (CD34+ M) , Molm-14, Nalm6, Jeko-1 tumor cell lines and fresh isolated patient AML tumor cells (CD123+) . As shown in FIG. 10, CART cells expressing anti-CD123-C5, -C7 and -C11 were specifically activated by tumor cells having relatively high CD123 expression, especially the CD123+ AML tumor cells, but not tumor cell lines with low CD123 expression. These results indicated that CD123 expression by tumor cells could activate CD123 CARTs.

5.10.6 Tumor killing of LACO-expressing CD123 CART cells

The cytolytic activities of the provided CD123 CARTs cells were measured in the tumor killing assay. LACO molecule A40C28 was used in this study. Various mRNA-based anti-CD123 CART cells, including mock T cells (NO EP) , T cells expressing C5 CAR (SEQ ID NO: 369) , C5 CAR with A40C28 (SEQ ID NO: 430) , C7 CAR (SEQ ID NO: 370) , C7 CAR with A40C28, C11 CAR (SEQ ID NO: 371) , and C11 CAR with A40C28, were co-cultured with tumor cells Molm-14, Nalm6, Jeko-1, at E/T ratio=10: 1. As shown in FIG. 11, the co-expression of LACO (A40C28) improved the killing efficiency of provided CART cells against all tumor cells.

The cytolytic activity of the provided CD123 CARTs cells was further examined in A549 cells electroporated with 0, 0.1 μg or 10 μg CD123 mRNA. As shown in FIG. 12, the ectopic expression levels of CD123 in A549 cells correlated with cytolytic activities of the CD123 CARTs against such tumors. Again, the co-expression of LACO consistently enhanced the anti-tumor effects of the CART cells (FIG. 12) .

IFN-γ release was detected by ELISA in the CART killing assays. As shown in FIG. 13, CD123 expressing cancer cells, such as MOLM14 cells, and especially AML cells (patient-001) , promoted the release of IFN-γ by the CART cells; and the co-expression of LACO (A40C28) further enhanced such release.

5.11 The instant application disclosed the following specific embodiments:

Embodiment 1: An antibody or antigen-binding fragment thereof that specifically binds CD123, comprising:

(a) a light chain variable region (VL) comprising

(1) a light chain CDR1 (VL CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-29;

(2) a light chain CDR2 (VL CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-56; and

(3) a light chain CDR3 (VL CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57-88;

or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or

(b) a heavy chain variable region (VH) comprising

(1) a heavy chain CDR1 (VH CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-109;

(2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 110-133; and

(3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 134-163;

or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

Embodiment 2: The antibody or antigen-binding fragment of embodiment 1, wherein

(a) the VL CDR1, CDR2 and CDR3 have

(1) the amino acid sequences of SEQ ID NOs: 3, 33, and 61, respectively;

(2) the amino acid sequences of SEQ ID NOs: 17, 45, and 75, respectively;

(3) the amino acid sequences of SEQ ID NOs: 7, 37, and 66, respectively;

(4) the amino acid sequences of SEQ ID NOs: 18, 32, and 76, respectively;

(5) the amino acid sequences of SEQ ID NOs: 23, 50 and 82, respectively;

(6) the amino acid sequences of SEQ ID NOs: 24, 51, and 83, respectively;

(7) the amino acid sequences of SEQ ID NOs: 8, 38, and 67, respectively;

(8) the amino acid sequences of SEQ ID NOs: 4, 34, and 62, respectively;

(9) the amino acid sequences of SEQ ID NOs: 25, 52, and 84, respectively;

(10) the amino acid sequences of SEQ ID NOs: 26, 53, and 85, respectively;

(11) the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively;

(12) the amino acid sequences of SEQ ID NOs: 17, 46, and 77, respectively;

(13) the amino acid sequences of SEQ ID NOs: 27, 54 and 86, respectively;

(14) the amino acid sequences of SEQ ID NOs: 9, 39, and 68, respectively;

(15) the amino acid sequences of SEQ ID NOs: 10, 40, and 69, respectively;

(16) the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively;

(17) the amino acid sequences of SEQ ID NOs: 5, 35, and 63, respectively;

(18) the amino acid sequences of SEQ ID NOs: 1, 30 and 64, respectively;

(19) the amino acid sequences of SEQ ID NOs: 11, 41, and 70, respectively;

(20) the amino acid sequences of SEQ ID NOs: 1, 31, and 59, respectively;

(21) the amino acid sequences of SEQ ID NOs: 19, 47, and 78, respectively;

(22) the amino acid sequences of SEQ ID NOs: 28, 55, and 87, respectively;

(23) the amino acid sequences of SEQ ID NOs: 12, 39, and 71, respectively;

(24) the amino acid sequences of SEQ ID NOs: 13, 42, and 72, respectively;

(25) the amino acid sequences of SEQ ID NOs: 1, 30, and 57, respectively;

(26) the amino acid sequences of SEQ ID NOs: 20, 48 and 79, respectively;

(27) the amino acid sequences of SEQ ID NOs: 14, 43, and 67, respectively;

(28) the amino acid sequences of SEQ ID NOs: 1, 30, and 58, respectively;

(29) the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively;

(30) the amino acid sequences of SEQ ID NOs: 15, 44, and 73, respectively;

(31) the amino acid sequences of SEQ ID NOs: 29, 56 and 88, respectively;

(32) the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively;

(33) the amino acid sequences of SEQ ID NOs: 16, 42, and 74, respectively;

(34) the amino acid sequences of SEQ ID NOs: 6, 36, and 65, respectively; or

(35) the amino acid sequences of SEQ ID NOs: 21, 45, and 80, respectively;

(b) the VH CDR1, CDR2 and CDR3 have

(1) the amino acid sequences of SEQ ID NOs: 91, 112, and 136, respectively;

(2) the amino acid sequences of SEQ ID NOs: 89, 122, and 149, respectively;

(3) the amino acid sequences of SEQ ID NOs: 95, 116, and 140, respectively;

(4) the amino acid sequences of SEQ ID NOs: 100, 123, and 150, respectively;

(5) the amino acid sequences of SEQ ID NOs: 99, 129 and 157, respectively;

(6) the amino acid sequences of SEQ ID NOs: 106, 128, and 158, respectively;

(7) the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively;

(8) the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively;

(9) the amino acid sequences of SEQ ID NOs: 107, 130, and 159, respectively;

(10) the amino acid sequences of SEQ ID NOs: 95, 116, and 160, respectively;

(11) the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively;

(12) the amino acid sequences of SEQ ID NOs: 101, 124, and 151, respectively;

(13) the amino acid sequences of SEQ ID NOs: 105, 131, and 161, respectively;

(14) the amino acid sequences of SEQ ID NOs: 97, 118, and 142, respectively;

(15) the amino acid sequences of SEQ ID NOs: 98, 119, and 143, respectively;

(16) the amino acid sequences of SEQ ID NOs: 105, 128, and 156, respectively;

(17) the amino acid sequences of SEQ ID NOs: 92, 113, and 137, respectively;

(18) the amino acid sequences of SEQ ID NOs: 93, 114 and 138, respectively;

(19) the amino acid sequences of SEQ ID NOs: 91, 120, and 144, respectively;

(20) the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively;

(21) the amino acid sequences of SEQ ID NOs: 102, 125, and 152, respectively;

(22) the amino acid sequences of SEQ ID NOs: 108, 132, and 162, respectively;

(23) the amino acid sequences of SEQ ID NOs: 96, 117, and 145, respectively;

(24) the amino acid sequences of SEQ ID NOs: 95, 116, and 146, respectively;

(25) the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively;

(26) the amino acid sequences of SEQ ID NOs: 103, 126 and 153, respectively;

(27) the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively;

(28) the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively;

(29) the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively;

(30) the amino acid sequences of SEQ ID NOs: 99, 121, and 147, respectively;

(31) the amino acid sequences of SEQ ID NOs: 109, 133 and 163, respectively;

(32) the amino acid sequences of SEQ ID NOs: 104, 127, and 155, respectively;

(33) the amino acid sequences of SEQ ID NOs: 95, 116, and 148, respectively;

(34) the amino acid sequences of SEQ ID NOs: 94, 115, and 139, respectively; or

(35) the amino acid sequences of SEQ ID NOs: 89, 122, and 154, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.

Embodiment 3: The antibody or antigen-binding fragment of embodiment 1, wherein

(1) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 3, 33, and 61, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 91, 112, and 136, respectively;

(2) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 17, 45, and 75, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 122, and 149, respectively;

(3) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 7, 37, and 66, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 95, 116, and 140, respectively;

(4) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 18, 32, and 76, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 100, 123, and 150, respectively;

(5) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 23, 50 and 82, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 99, 129 and 157, respectively;

(6) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 24, 51, and 83, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 106, 128, and 158, respectively;

(7) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 8, 38, and 67, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively;

(8) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 4, 34, and 62, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively;

(9) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 25, 52, and 84, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 107, 130, and 159, respectively;

(10) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 26, 53, and 85, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 95, 116, and 160, respectively;

(11) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively;

(12) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 17, 46, and 77, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 101, 124, and 151, respectively;

(13) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 27, 54 and 86, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 105, 131, and 161, respectively;

(14) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 9, 39, and 68, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 97, 118, and 142, respectively;

(15) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 10, 40, and 69, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 98, 119, and 143, respectively;

(16) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 105, 128, and 156, respectively;

(17) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 5, 35, and 63, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 92, 113, and 137, respectively;

(18) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 30 and 64, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 93, 114 and 138, respectively;

(19) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 11, 41, and 70, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 91, 120, and 144, respectively;

(20) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 31, and 59, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively;

(21) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 19, 47, and 78, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 102, 125, and 152, respectively;

(22) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 28, 55, and 87, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 108, 132, and 162, respectively;

(23) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 12, 39, and 71, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 96, 117, and 145, respectively;

(24) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 13, 42, and 72, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 95, 116, and 146, respectively;

(25) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 30, and 57, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively;

(26) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 20, 48 and 79, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 103, 126 and 153, respectively;

(27) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 14, 43, and 67, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively;

(28) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 30, and 58, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 110, and 134, respectively;

(29) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively;

(30) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 15, 44, and 73, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 99, 121, and 147, respectively;

(31) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 29, 56 and 88, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 109, 133 and 163, respectively;

(32) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 22, 49, and 81, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 104, 127, and 155, respectively;

(33) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 16, 42, and 74, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 95, 116, and 148, respectively;

(34) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 6, 36, and 65, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 94, 115, and 139, respectively; or

(35) the VL CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 21, 45, and 80, respectively; and/or the VH CDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 89, 122, and 154, respectively.

Embodiment 4: The antibody or antigen-binding fragment of embodiment 1, comprising a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3, wherein

(a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 23, 50 and 82, respectively; and

(b) the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 99, 129 and 157, respectively.

Embodiment 5: The antibody or antigen-binding fragment of embodiment 1, comprising a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3, wherein

(a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 8, 38, and 67, respectively; and

(b) the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 96, 117, and 141, respectively.

Embodiment 6: The antibody or antigen-binding fragment of embodiment 1, comprising a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3, wherein

(a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; and

(b) the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively.

Embodiment 7: An antibody or antigen-binding fragment thereof that specifically binds CD123, comprising:

(a) a VL comprising

(1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 348-352;

(2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 353-356; and

(3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 357-359; and/or

(b) a VH comprising

(1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 360-361;

(2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 362-367; and

(3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 368.

Embodiment 8: An antibody or antigen-binding fragment thereof that specifically binds CD123, comprising:

(a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198; and/or

(b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 199-233.

Embodiment 9: The antibody or antigen-binding fragment of embodiment 8 comprising a VL and a VH, wherein the VL and VH have

(1) the amino acid sequences of SEQ ID NOs: 164 and 199, respectively;

(2) the amino acid sequences of SEQ ID NOs: 165 and 200, respectively;

(3) the amino acid sequences of SEQ ID NOs: 166 and 201, respectively;

(4) the amino acid sequences of SEQ ID NOs: 167 and 202, respectively;

(5) the amino acid sequences of SEQ ID NOs: 168 and 203, respectively;

(6) the amino acid sequences of SEQ ID NOs: 169 and 204, respectively;

(7) the amino acid sequences of SEQ ID NOs: 170 and 205, respectively;

(8) the amino acid sequences of SEQ ID NOs: 171 and 206, respectively;

(9) the amino acid sequences of SEQ ID NOs: 172 and 207, respectively;

(10) the amino acid sequences of SEQ ID NOs: 173 and 208, respectively;

(11) the amino acid sequences of SEQ ID NOs: 174 and 209, respectively;

(12) the amino acid sequences of SEQ ID NOs: 175 and 210, respectively;

(13) the amino acid sequences of SEQ ID NOs: 176 and 211, respectively;

(14) the amino acid sequences of SEQ ID NOs: 177 and 212, respectively;

(15) the amino acid sequences of SEQ ID NOs: 178 and 213, respectively;

(16) the amino acid sequences of SEQ ID NOs: 179 and 214, respectively;

(17) the amino acid sequences of SEQ ID NOs: 180 and 215, respectively;

(18) the amino acid sequences of SEQ ID NOs: 181 and 216, respectively;

(19) the amino acid sequences of SEQ ID NOs: 182 and 217, respectively;

(20) the amino acid sequences of SEQ ID NOs: 183 and 218, respectively;

(21) the amino acid sequences of SEQ ID NOs: 184 and 219, respectively;

(22) the amino acid sequences of SEQ ID NOs: 185 and 220, respectively;

(23) the amino acid sequences of SEQ ID NOs: 186 and 221, respectively;

(24) the amino acid sequences of SEQ ID NOs: 187 and 222, respectively;

(25) the amino acid sequences of SEQ ID NOs: 188 and 223, respectively;

(26) the amino acid sequences of SEQ ID NOs: 189 and 224, respectively;

(27) the amino acid sequences of SEQ ID NOs: 190 and 225, respectively;

(28) the amino acid sequences of SEQ ID NOs: 191 and 226, respectively;

(29) the amino acid sequences of SEQ ID NOs: 192 and 227, respectively;

(30) the amino acid sequences of SEQ ID NOs: 193 and 228, respectively;

(31) the amino acid sequences of SEQ ID NOs: 194 and 229, respectively;

(32) the amino acid sequences of SEQ ID NOs: 195 and 230, respectively;

(33) the amino acid sequences of SEQ ID NOs: 196 and 231, respectively;

(34) the amino acid sequences of SEQ ID NOs: 197 and 232, respectively; or

(35) the amino acid sequences of SEQ ID NOs: 198 and 233, respectively.

Embodiment 10: The antibody or antigen-binding fragment of embodiment 8 comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NOs: 168 and 203, respectively.

Embodiment 11: The antibody or antigen-binding fragment of embodiment 8 comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NOs: 170 and 205, respectively.

Embodiment 12: The antibody or antigen-binding fragment of embodiment 8 comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NOs: 174 and 209, respectively.

Embodiment 13: An antibody or antigen-binding fragment thereof that specifically binds CD123, comprising

(a) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 164-198; and/or

(b) a VH comprising VH CDR1, CDR2, and CDR3 from a VH having an amino acid sequence selected from group consisting of SEQ ID NOs: 199-233.

Embodiment 14: The antibody or antigen-binding fragment thereof embodiment 13, comprising

(1) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 164, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 199;

(2) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 165, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 200;

(3) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 166, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 201;

(4) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 167, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 202;

(5) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 168, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 203;

(6) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 169, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 204;

(7) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 170, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 205;

(8) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 171, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 206;

(9) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 172, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 207;

(10) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 173, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 208;

(11) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 174, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 209;

(12) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 175, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 210;

(13) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 176, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 211;

(14) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 177, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 212;

(15) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 178, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 213;

(16) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 179, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 214;

(17) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 180, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 215;

(18) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 181, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 216;

(19) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 182, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 217;

(20) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 183, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 218;

(21) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 184, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 219;

(22) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 185, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 220;

(23) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 186, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 221;

(24) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 187, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 222;

(25) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 188, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 223;

(26) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 189, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 224

(27) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 190, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 225;

(28) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 191, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 226;

(29) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 192, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 227;

(30) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 193, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 228;

(31) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 194, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 229;

(32) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 195, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 230;

(33) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 196, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 231;

(34) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 197, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 232; or

(35) a VL comprising VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 198, and/or a VH comprising VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 233.

Embodiment 15: The antibody or antigen-binding fragment thereof embodiment 13 comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 168, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 203.

Embodiment 16: The antibody or antigen-binding fragment thereof embodiment 13 comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 170, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 205.

Embodiment 17: The antibody or antigen-binding fragment thereof embodiment 13 comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 174, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 209.

Embodiment 18: An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of any one of embodiments 1 to 17 for binding to CD123.

Embodiment 19: The antibody or antigen-binding fragment of any one of embodiments 1 to 18 that is a monoclonal antibody or antigen-binding fragment.

Embodiment 20: The antibody or antigen-binding fragment of any one of embodiments 1 to 19 that is a bispecific or multispecific antibody.

Embodiment 21: The antibody or antigen-binding fragment of embodiment 20 that is a Bi-specific T-cell engager (BiTE) .

Embodiment 22: The antibody or antigen-binding fragment of any one of embodiments 1 to 21 that is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

Embodiment 23: The antibody or antigen-binding fragment of any one of embodiments 1 to 21 that is selected from the group consisting of a Fab, a Fab’, a F (ab’) ₂, a Fv, a scFv, a (scFv) ₂, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .

Embodiment 24: The antibody or antigen-binding fragment of embodiment 23 that is a scFv.

Embodiment 25: The antibody or antigen-binding fragment of any one of embodiments 1 to 24 that is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.

Embodiment 26: The antibody or antigen-binding fragment of embodiment 25 that is a human antibody or antigen-binding fragment.

Embodiment 27: A polynucleotide encoding the antibody or antigen-binding fragment of any one of embodiments 1 to 26.

Embodiment 28: The polynucleotide of embodiment 27 that is a messenger RNA (mRNA) .

Embodiment 29: A vector comprising the polynucleotide of embodiment 27.

Embodiment 30: A host cell comprising the polynucleotide of embodiment 27 or 28, or the vector of embodiment 29.

Embodiment 31: A Chimeric Antigen Receptor (CAR) that specifically binds CD123, comprising, from N-terminus to C-terminus:

(a) a CD123-binding domain that comprises the antibody or antigen-binding fragment of any one of embodiments 1 to 26;

(b) a transmembrane domain; and

(c) a cytoplasmic domain.

Embodiment 32: The CAR of embodiment 31, wherein the transmembrane domain is derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.

Embodiment 33: The CAR of embodiment 31, wherein the transmembrane domain comprises CD8 transmembrane region or CD28 transmembrane region.

Embodiment 34: The CAR of any one of embodiments 31 to 33, wherein the cytoplasmic domain comprises a signaling domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

Embodiment 35: The CAR of embodiment 34, wherein the cytoplasmic domain further comprises a co-stimulatory domain derived from CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.

Embodiment 36: The CAR of any one of embodiments 31 to 35, wherein the cytoplasmic domain comprises a CD3 signaling domain and a 4-1BB co-stimulatory domain.

Embodiment 37: The CAR of any one of embodiments 31 to 35, wherein the cytoplasmic domain comprises a CD3 signaling domain and a CD28 co-stimulatory domain.

Embodiment 38: The CAR of any one of embodiments 31 to 37, further comprising a CD8 hinge between the antibody or antigen-binding fragment and the transmembrane domain.

Embodiment 39: A CAR that specifically binds CD123 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 369-371.

Embodiment 40: A polynucleotide encoding CAR of any one of the embodiments 31 to 39.

Embodiment 41: The polynucleotide of embodiment 40 that is a mRNA.

Embodiment 42: A vector comprising the polynucleotide of embodiment 40.

Embodiment 43: A cell comprising the polynucleotide of embodiment 40 or 41, or the vector of embodiment 42.

Embodiment 44: The cell of embodiment 43 wherein the polynucleotide comprising a first fragment encoding the CAR and a second fragment encoding a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein (i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and (ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

Embodiment 45: The cell of embodiment 43 further comprising a second polynucleotide encoding a fusion protein comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein (i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and (ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.

Embodiment 46: The cell of embodiment 44 or 45, wherein the N-terminus of the first domain is linked to the C-terminus of the second domain.

Embodiment 47: The cell of embodiment 44 or 45, wherein the N-terminus of the second domain is linked to the C-terminus of the first domain.

Embodiment 48: The cell of any one of embodiments 44 to 47, wherein the first domain and the second domain are linked via a linker.

Embodiment 49: The cell of any one of embodiments 44 to 48, wherein the APC is selected from the group consisting of a dendritic cell, a macrophage, a myeloid derived suppressor cell, a monocyte, a B cell, a T cell, and a Langerhans cell.

Embodiment 50: The cell of any one of embodiments 44 to 49, wherein the activation receptor of the APC is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN.

Embodiment 51: The cell of embodiment 50, wherein the first domain comprises a ligand that binds CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN, or a receptor-binding fragment thereof.

Embodiment 52: The cell of embodiment 50, wherein the first domain comprises a receptor-binding domain of CD40 Ligand (CD40L) .

Embodiment 53: The cell of any one of embodiments 44 to 49, wherein the first domain comprises an antibody that binds the activation receptor of the APC, or an antigen-binding fragment thereof.

Embodiment 54: The cell of embodiment 53, wherein the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof.

Embodiment 55: The cell of any one of embodiments 44 to 54, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.

Embodiment 56: The cell of any one of embodiments 44 to 55 wherein the second domain comprises a cytoplasmic domain of the co-stimulatory receptor.

Embodiment 57: The cell of embodiment 56, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.

Embodiment 58: The cell of embodiment 56, wherein the co-stimulatory receptor is CD28.

Embodiment 59: The cell of any one of embodiments 44 to 55, wherein the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof.

Embodiment 60: The cell of embodiment 59, wherein the co-stimulatory ligand is selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44.

Embodiment 61: The cell of any one of embodiments 44 to 55, wherein the second domain is an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof.

Embodiment 62: The cell of embodiment 61, wherein the second domain is an scFv.

Embodiment 63: The cell of embodiment 61 or 62, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.

Embodiment 64: The cell of embodiment 61 or 62, wherein the co-stimulatory receptor is CD28.

Embodiment 65: The cell of embodiment 44 or 45, wherein the first domain comprises CD40L or a receptor-binding thereof and the second domain comprises a CD28 cytoplasmic domain.

Embodiment 66: The cell of embodiment 44 or 45, wherein the first domain comprises CD40L or a receptor-binding thereof and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

Embodiment 67: The cell of embodiment 44 or 45, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

Embodiment 68: The cell of embodiment 44 or 45, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises a CD28 cytoplasmic domain.

Embodiment 69: The cell of embodiment 44 or 45, wherein the fusion protein is at least 85%, 90%, 95%, 98%, or 99%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 418-433 and 490-492.

Embodiment 70: The cell of any one of embodiments 43 to 69 that is an immune effector cell.

Embodiment 71: The cell of embodiment 70 that is derived from a cell isolated from peripheral blood or bone marrow.

Embodiment 72: The cell of embodiment 70 that is derived from a cell differentiated in vitro from a stem or progenitor cell selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.

Embodiment 73: The cell of any one of embodiments 43 to 72 that is a T cell or a NK cell.

Embodiment 74: The cell of embodiment 73 that is a cytotoxic T cell, a helper T cell, a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, or an effector memory TEMRA cell.

Embodiment 75: The cell of embodiment 73 that is a cytotoxic T cell.

Embodiment 76: A population of the cells comprising the cell of any one of embodiments 43 to 69, wherein the population of cells are derived from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) .

Embodiment 77: A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of embodiments 1 to 26, and a pharmaceutically acceptable carrier.

Embodiment 78: A pharmaceutical composition comprising a therapeutically effective amount of the cell or cell population of any one of embodiments 70 to 76, and a pharmaceutically acceptable carrier.

Embodiment 79: Use of the antibody or antigen-binding fragment of any one of embodiments 1 to 26, the cell or population of cells of any one of embodiments 70 to 76, or the pharmaceutical composition of embodiment 77 or 78, in cancer treatment.

Embodiment 80: Use of the antibody or antigen-binding fragment of any one of embodiments 1 to 26, the cell or population of cells of any one of embodiments 70 to 76, or the pharmaceutical composition of embodiment 77 or 78, for the preparation of a medicament for the treatment of cancer.

Embodiment 81: The use of embodiment 79 or 80, wherein the cell, population of cells, or pharmaceutical composition is used in combination with an additional therapy.

Embodiment 82: A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of embodiments 1 to 26 or the pharmaceutical composition of embodiment 77 or 78.

Embodiment 83: A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the cell or cell population of any one of embodiments 70 to 76.

Embodiment 84: The method of embodiment 83, wherein the cell or population of cells is autologous to the subject.

Embodiment 85: The method of embodiment 84, further comprising obtaining cells from the subject.

Embodiment 86: The method of any one of embodiments 82 to 85, further comprising administering an additional therapy to the subject.

Embodiment 87: The method of any one of embodiments 82 to 86, wherein the subject is a human.

Embodiment 88: The use or method of any one of embodiments 79 to 87, wherein the cancer is a solid tumor.

Embodiment 89: The use or method of any one of embodiments 79 to 87, wherein the cancer is a hematological cancer.

Embodiment 90: The use or method of embodiment 89, wherein the cancer is leukemia.

Embodiment 91: The use or method of embodiment 89, wherein the cancer is acute myeloid leukemia (AML) , B-acute lymphoid leukemia (B-ALL) , T-acute lymphoid leukemia (T-ALL) , B cell precursor acute lymphoblastic leukemia (BCP‐ALL) or blastic plasmacytoid dendritic cell neoplasm (BPDCN) .

Embodiment 92: The use or method of any one of embodiments 79 to 91, wherein the cancer is CD123-expressing cancer.

Embodiment 93: The use or method of embodiment 92, wherein the cancer is CD123-expressing AML.

Embodiment 94: A method of preparing a cell capable of expressing a CAR that specifically binds CD123, comprising transferring the polynucleotide of embodiment 40 or 41 into the cell.

Embodiment 95: The method of embodiment 94, wherein the polynucleotide is transferred via electroporation.

Embodiment 96: The method of embodiment 94, wherein the polynucleotide is transferred via viral transduction.

Embodiment 97: The method of embodiment 96, comprising using a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus for the viral transduction.

Embodiment 98: The method of embodiment 94, wherein the polynucleotide is transferred using a transposon system.

Embodiment 99: The method of embodiment 98, wherein the transposon system is Sleeping Beauty or PiggyBac.

Embodiment 100: The method of embodiment 94, wherein the polynucleotide is transferred using gene-editing.

Embodiment 101: The method of embodiment 100, wherein the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.

Embodiment 102: The method of any one of embodiments 94 to 101, wherein the cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell.

Claims

An antibody or antigen-binding fragment thereof that specifically binds CD123, comprising:

(a) a light chain variable region (VL) comprising

(1) a light chain CDR1 (VL CDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 2;

(2) a light chain CDR2 (VL CDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 32; and

(3) a light chain CDR3 (VL CDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 60;

or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or

(b) a heavy chain variable region (VH) comprising

(1) a heavy chain CDR1 (VH CDR1) comprising an amino acid sequence as set forth in SEQ ID NO: 90;

(2) a heavy chain CDR2 (VH CDR2) comprising an amino acid sequence as set forth in SEQ ID NO: 111; and

(3) a heavy chain CDR3 (VH CDR3) comprising an amino acid sequence as set forth in SEQ ID NO: 135;

or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.
The antibody or antigen-binding fragment of claim 1, wherein

(a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VL CDRs; and/or

(b) the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions, and/or deletions in the VH CDRs.
The antibody or antigen-binding fragment of claim 1, comprising a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and a VH CDR3, wherein

(a) the VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 2, 32, and 60, respectively; and

(b) the VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 90, 111, and 135, respectively.
An antibody or antigen-binding fragment thereof that specifically binds CD123, comprising:

(a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence as set forth in SEQ ID NO: 174; and/or

(b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to an amino acid sequence as set forth in SEQ ID NO: 209.
The antibody or antigen-binding fragment of claim 4 comprising a VL and a VH, wherein the VL and VH have the amino acid sequences of SEQ ID NOs: 174 and 209, respectively.
An antibody or antigen-binding fragment thereof that specifically binds CD123, comprising a VL and a VH, wherein the VL comprises VL CDR1, CDR2, and CDR3 from a VL having the amino acid sequence of SEQ ID NO: 174, and the VH comprises VH CDR1, CDR2, and CDR3 from a VH having the amino acid sequence of SEQ ID NO: 209.
An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of any one of claims 1 to 6 for binding to CD123.
The antibody or antigen-binding fragment of any one of claims 1 to 7 that is a monoclonal antibody or antigen-binding fragment.
The antibody or antigen-binding fragment of any one of claims 1 to 8 that is a bispecific or multispecific antibody.
The antibody or antigen-binding fragment of claim 9 that is a Bi-specific T-cell engager (BiTE) .
The antibody or antigen-binding fragment of any one of claims 1 to 10 that is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.
The antibody or antigen-binding fragment of any one of claims 1 to 10 that is selected from the group consisting of a Fab, a Fab’, a F (ab’) ₂, a Fv, a scFv, a (scFv) ₂, a single domain antibody (sdAb) , and a heavy chain antibody (HCAb) .
The antibody or antigen-binding fragment of claim 12 that is a scFv.
The antibody or antigen-binding fragment of any one of claims 1 to 13 that is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.
The antibody or antigen-binding fragment of claim 14 that is a human antibody or antigen-binding fragment.
A polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1 to15.
The polynucleotide of claim 16 that is a messenger RNA (mRNA) .
A vector comprising the polynucleotide of claim 16.
A host cell comprising the polynucleotide of claim 16 or 17, or the vector of claim 18.
A Chimeric Antigen Receptor (CAR) that specifically binds CD123, comprising, from N-terminus to C-terminus:

(a) a CD123-binding domain that comprises the antibody or antigen-binding fragment of any one of claims 1 to15;

(b) a transmembrane domain; and

(c) a cytoplasmic domain.
The CAR of claim 20, wherein the transmembrane domain is derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain, or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, or CD154.
The CAR of claim 21, wherein the transmembrane domain comprises CD8 transmembrane region or CD28 transmembrane region.
The CAR of any one of claims 20 to 22, wherein the cytoplasmic domain comprises a signaling domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.
The CAR of claim 23, wherein the cytoplasmic domain further comprises a co-stimulatory domain derived from CD28, 4-1BB (CD137) , OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.
The CAR of any one of claims 20 to 24, wherein the cytoplasmic domain comprises a CD3 signaling domain and a 4-1BB co-stimulatory domain.
The CAR of any one of claims 20 to 24, wherein the cytoplasmic domain comprises a CD3 signaling domain and a CD28 co-stimulatory domain.
The CAR of any one of claims 20 to 26, further comprising a CD8 hinge between the antibody or antigen-binding fragment and the transmembrane domain.
A CAR that specifically binds CD123 comprising an amino acid sequence as set forth in SEQ ID NO: 371.
A polynucleotide encoding CAR of any one of the claims 20 to 28.
The polynucleotide of claim 29 that is a mRNA.
A vector comprising the polynucleotide of claim 29.
A cell comprising the polynucleotide of claim 29 or 30, or the vector of claim 31.
The cell of claim 32 wherein the polynucleotide comprising a first fragment encoding the CAR and a second fragment encoding a fusion protein comprising a first domain that activates an antigen-presenting cell (APC) and a second domain that activates an immune effector cell, wherein (i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and (ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.
The cell of claim 32 further comprising a second polynucleotide encoding a fusion protein comprising a first domain that activates an APC and a second domain that activates an immune effector cell, wherein (i) the first domain comprises (a) a ligand that binds an activation receptor of the APC, or a receptor-binding fragment thereof, or (b) an antibody that binds an activation receptor of the APC, or an antigen-binding fragment thereof; and (ii) the second domain comprises (a) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (b) a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof, or (c) an antibody that binds a co-stimulatory receptor of the immune effector cell, or an antigen-binding fragment thereof.
The cell of claim 33 or 34, wherein the N-terminus of the first domain is linked to the C-terminus of the second domain.
The cell of claim 33 or 34, wherein the N-terminus of the second domain is linked to the C-terminus of the first domain.
The cell of any one of claims 33 to 36, wherein the first domain and the second domain are linked via a linker.
The cell of any one of claims 33 to 37, wherein the APC is selected from the group consisting of a dendritic cell, a macrophage, a myeloid derived suppressor cell, a monocyte, a B cell, a T cell, and a Langerhans cell.
The cell of any one of claims 33 to 38, wherein the activation receptor of the APC is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN.
The cell of claim 39, wherein the first domain comprises a ligand that binds CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN, or a receptor-binding fragment thereof.
The cell of claim 39, wherein the first domain comprises a receptor-binding domain of CD40 Ligand (CD40L) .
The cell of any one of claims 33 to 38, wherein the first domain comprises an antibody that binds the activation receptor of the APC, or an antigen-binding fragment thereof.
The cell of claim 42, wherein the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof.
The cell of any one of claims 33 to 43, wherein the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte.
The cell of any one of claims 33 to 44 wherein the second domain comprises a cytoplasmic domain of the co-stimulatory receptor.
The cell of claim 45, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.
The cell of claim 45, wherein the co-stimulatory receptor is CD28.
The cell of any one of claims 33 to 44, wherein the second domain is a co-stimulatory ligand of the immune effector cell, or a receptor-binding fragment thereof.
The cell of claim 48, wherein the co-stimulatory ligand is selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44.
The cell of any one of claims 33 to 44, wherein the second domain is an antibody that binds the co-stimulatory receptor, or an antigen-binding fragment thereof.
The cell of claim 50, wherein the second domain is an scFv.
The cell of claim 50 or 51, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.
The cell of claim 50 or 51, wherein the co-stimulatory receptor is CD28.
The cell of claim 33 or 34, wherein the first domain comprises CD40L or a receptor-binding thereof and the second domain comprises a CD28 cytoplasmic domain.
The cell of claim 33 or 34, wherein the first domain comprises CD40L or a receptor-binding thereof and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof.
The cell of claim 33 or 34, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof.
The cell of claim 33 or 34, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises a CD28 cytoplasmic domain.
The cell of claim 33 or 34, wherein the fusion protein is at least 85%, 90%, 95%, 98%, or 99%identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 418-433 and 490-492.
The cell of any one of claims 32 to 58 that is an immune effector cell.
The cell of claim 59 that is derived from a cell isolated from peripheral blood or bone marrow.
The cell of claim 59 that is derived from a cell differentiated in vitro from a stem or progenitor cell selected from the group consisting of a T cell progenitor cell, a hematopoietic stem and progenitor cell, a hematopoietic multipotent progenitor cell, an embryonic stem cell, and an induced pluripotent cell.
The cell of any one of claims 32 to 61 that is a T cell or a NK cell.
The cell of claim 62 that is a cytotoxic T cell, a helper T cell, a gamma delta T, a CD4+/CD8+ double positive T cell, a CD4+ T cell, a CD8+ T cell, a CD4/CD8 double negative T cell, a CD3+ T cell, a naive T cell, an effector T cell, a helper T cell, a memory T cell, a regulator T cell, a Th0 cell, a Th1 cell, a Th2 cell, a Th3 (Treg) cell, a Th9 cell, a Th17 cell, a Thαβ helper cell, a Tfh cell, a stem memory TSCM cell, a central memory TCM cell, an effector memory TEM cell, or an effector memory TEMRA cell.
The cell of claim 62 that is a cytotoxic T cell.
A population of the cells comprising the cell of any one of claims 32 to 58, wherein the population of cells are derived from peripheral blood mononuclear cells (PBMC) , peripheral blood leukocytes (PBL) , tumor infiltrating lymphocytes (TIL) , cytokine-induced killer cells (CIK) , lymphokine-activated killer cells (LAK) , or marrow infiltrate lymphocytes (MILs) .
A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 15, and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising a therapeutically effective amount of the cell or cell population of any one of claims 59 to 65, and a pharmaceutically acceptable carrier.
Use of the antibody or antigen-binding fragment of any one of claims 1 to 15, the cell or population of cells of any one of claims 59 to 65, or the pharmaceutical composition of claim 66 or 67, in cancer treatment.
Use of the antibody or antigen-binding fragment of any one of claims 1 to 15, the cell or population of cells of any one of claims 59 to 65, or the pharmaceutical composition of claim 66 or 67, for the preparation of a medicament for the treatment of cancer.
The use of claim 68 or 69, wherein the cell, population of cells, or pharmaceutical composition is used in combination with an additional therapy.
A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 15 or the pharmaceutical composition of claim 66 or 67.
A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the cell or cell population of any one of claims 59 to 65.
The method of claim 72, wherein the cell or population of cells is autologous to the subject.
The method of claim 73, further comprising obtaining cells from the subject.
The method of any one of claims 71 to 74, further comprising administering an additional therapy to the subject.
The method of any one of claims 71 to 75, wherein the subject is a human.
The use or method of any one of claims 68 to 76, wherein the cancer is a solid tumor.
The use or method of any one of claims 68 to 76, wherein the cancer is a hematological cancer.
The use or method of claim 78, wherein the cancer is leukemia.
The use or method of claim 78, wherein the cancer is acute myeloid leukemia (AML) , B-acute lymphoid leukemia (B-ALL) , T-acute lymphoid leukemia (T-ALL) , B cell precursor acute lymphoblastic leukemia (BCP‐ALL) or blastic plasmacytoid dendritic cell neoplasm (BPDCN) .
The use or method of any one of claims 68 to 80, wherein the cancer is CD123-expressing cancer.
The use or method of claim 81, wherein the cancer is CD123-expressing AML.
A method of preparing a cell capable of expressing a CAR that specifically binds CD123, comprising transferring the polynucleotide of claim 29 or 30 into the cell.
The method of claim 83, wherein the polynucleotide is transferred via electroporation.
The method of claim 84, wherein the polynucleotide is transferred via viral transduction.
The method of claim 85, comprising using a lentivirus, a retrovirus, an adenovirus, or an adeno-associated virus for the viral transduction.
The method of claim 83, wherein the polynucleotide is transferred using a transposon system.
The method of claim 87, wherein the transposon system is Sleeping Beauty or PiggyBac.
The method of claim 83, wherein the polynucleotide is transferred using gene-editing.
The method of claim 89, wherein the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.
The method of any one of claims 83 to 90, wherein the cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a neutrophil, and a granulocyte cell.