WO2016179319A1

WO2016179319A1 - Chimeric antigen receptors with ctla4 signal transduction domains

Info

Publication number: WO2016179319A1
Application number: PCT/US2016/030834
Authority: WO
Inventors: Bob Liu; Jagath R. Junutula; Timothy Fong; Ying Ping JIANG
Original assignee: Cellerant Therapeutics, Inc.
Priority date: 2015-05-04
Filing date: 2016-05-04
Publication date: 2016-11-10

Abstract

This disclosure provides chimeric antigen receptors (CAR) having (1) a target binding domain, (2) a hinge region, (3) a transmembrane domain (TM), and (4) an intracellular domain comprising cytotoxic T-lymphocyte associated protein 4 (CTLA4) signal transduction domain or a CTLA4-CD28 signal transduction domain. Also provided are nucleic acid molecules encoding CARs, cells expressing CARs, pharmaceutical compositions and methods of use thereof.

Description

CHIMERIC ANTIGEN RECEPTORS WITH CTLA4 SIGNAL

TRANSDUCTION DOMAINS

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present patent application claims benefit of priority to US Provisional Patent Application No.62/156,786, filed May 4, 2015, which is incorporated by reference for all purposes. BACKGROUND OF THE INVENTION

[0002] The most common practice for treating cancer with cellular immunotherapy has been the use of T cells. This approach, which has been evaluated over several decades with many different T cell product compositions, has had variable clinical results. The variability of the clinical responses has been the result of several factors including but not limited to: lack of or weak target specificity of the T cell receptor (TCR) on T cells to tumor antigen or tumor cells, a lack of full activation of T cells to induce an anti-tumor immune response, and a lack of sufficient T cells per dose.

[0003] International patent application WO 2012/079000 refers to administering a genetically modified T cell to express a CAR wherein the CAR comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain.

[0004] Previously T-cell product compositions used in cancer immunotherapy had variable clinical responses. Variability of the clinical response was the consequence of numerous factors such as absence or weak target specificity of the TCR on T cells to tumor antigen or tumor cells. Also, often there was no or inefficient activation of T cells to induce an anti- tumor immune response. Additionally, the titer of T cells per dose was lacking or insufficient. These issues have been partially addressed by using T cells transiently or stably modified to express a genetically engineered CAR or TCR (Jena, Dotti et al.2010, Curran, Pegram et al. 2012, Restifo, Dudley et al.2012, Shirasu and Kuroki 2012, Turtle, Hudecek et al.2012, Essand and Loskog 2013, Han, Li et al.2013, Kershaw, Westwood et al.2013, Zhang, Li et al.2013, Dotti, Gottschalk et al.2014, Fujiwara 2014, Jena, Moyes et al.2014, Maus, Grupp et al.2014).

BRIEF SUMMARY OF THE INVENTION

[0005] In one aspect, provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (1) a target binding domain; (2) optionally a hinge region ; (3) a transmembrane domain (TM); and (4) an intracellular domain comprising a signal transduction domain selected the group consisting of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) signal transduction domain and a CTLA4-CD28 hybrid signal transduction domain. In some embodiments, the CTLA4 signal transduction domain comprises an amino acid sequence having substantial (e.g., at least 80, 85, 90, or 95%) sequence identity to the amino acid sequence from positions 183-223 of SEQ ID NO: 1. In other instances, the CTLA4 signal transduction domain comprises an amino acid sequence having 100% sequence identity to the amino acid sequence from positions 183-223 of SEQ ID NO: 1.

[0006] In some embodiments, the amino acid sequence having substantial (e.g., at least 80, 85, 90, or 95%) sequence identity to the amino acid sequence from positions 182-223 of SEQ ID NO: 1 comprises one or more amino acid substitutions located at one or more positions in SEQ ID NO: 1 selected from the group consisting of position 188, position 191, position 192, position 201, position 205, position 209, position 218 or a combination thereof. In some cases, the one or more amino acid substitutions are selected from the group consisting of: (i) K188A, K191A and K192A; (ii) Y201F; (iii) P205A and P209A; (iv) Y218F; (v) Y201F and Y218F; and (vi) Y201F, P205A and P209A (SEQ ID NO:157).

[0007] In some embodiments, the CTLA4-CD28 hybrid signal transduction domain comprises an amino acid sequence of SEQ ID NO: 1 having fewer than 20 amino acid substitutions, wherein the amino acid substitutions are amino acids at corresponding positions in the amino acid sequence of the signal transduction domain of CD28 as set forth in SEQ ID NO: 2 In some instances, the fewer than 20 amino acid substitutions are selected from the group consisting of V202M, K203N, P205T, T207R, E208R, F219A, I220P, I222R, N223D and a combination thereof (SEQ ID NO:158). In some embodiments, the CTLA4-CD28 hybrid signal transduction domain comprises an amino acid insertion comprising fewer than 6 amino acids after position 209 and/or position 223 of SEQ ID NO:1. In some instances, the amino acid insertion after position 173 is G and/or the amino acid insertion after position 187 is FAAYRS as set forth in SEQ ID NO: 3. In some embodiments, the CTLA4-CD28 hybrid signal transduction domain comprises the amino acid sequence of SEQ ID NO: 4

[0008] In some embodiments the nucleic acid molecule further comprises: (5) a CD3-zeta (ς) signal transduction domain or an Fc receptor signal transduction domain. In some embodiments, the nucleic acid molecule further comprises an Fc receptor signal transduction domain, wherein the Fc receptor is selected from the group consisting of Fc-alpha, Fc- gamma, Fc-epsilon, Fc-mu, and Fc-delta. In some embodiments, the nucleic acid molecule further comprises: (6) at least one co-stimulatory domain selected from a signal transduction domain of CD28, 4-1BB, CD27, OX40, CD30, CD40, PD-1, PD-L1, PD-L2, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3 and B7- H4.

[0009] In some embodiments, the nucleic acid molecule further comprises: a signal peptide (e.g., in addition to, or instead of, independently any one or more of the CD3-zeta signal transduction domain or Fc receptor signal transduction domain, co-stimulatory domain, and/or linker domain). In some embodiments the signal peptide comprises a CD8 signal peptide. In some embodiments the nucleic acid molecule further comprises: a linker positioned between the target binding domain and TM domain (e.g., in addition to, or instead of, independently any one or more of the CD3-zeta signal transduction domain or Fc receptor signal transduction domain, co-stimulatory domain, and/or signal peptide). In some embodiments, at least one of the domains is heterologous to at least one of the other domains (e.g., not found together in this arrangement in nature). In some embodiments, the domains of the CAR are all heterologous to each other.

[0010] In some embodiments the target binding domain comprises an antibody. In some embodiments the antibody is an antibody fragment. In some embodiments the antibody is an antibody single chain fragment variable (“scFV”). In some embodiments the target binding domain comprises a T-cell receptor (TCR) variable alpha (VĮ) and variable beta (Vȕ) domains, or a single chain fragment containing a TCR variable delta (Vį) and variable gamma (VȖ) domains. In some embodiments, the target binding domain binds to a tumor associated antigen, a cancer stem cell associated antigen or a viral antigen. In another embodiment the target binding domain binds a target selected from the group consisting of CLL-1, IL1RAP, TIM-3, GPR-114, CD19, CD20, CD22, ROR1, mesothelin, CD33, CD123/IL3Ra, c-Met, PSMA, Prostatic acid phosphatase (PAP), CEA, CA-125, Muc-1, AFP, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, Tyrosinase, TRPI/gp75, gp100/pmel-17, Melan-A/MART-1, Her2/neu, WT1, EphA3, telomerase, HPV E6, HPV E7, EBNA1, BAGE, GAGE, MAGE A3 TCR, TCRSLITRK6, ENPP3, Nectin-4, CD27, SLC44A4, CAIX, Cripto, CD30, MUC16, GPNMB, BCMA, Trop-2, Tissue Factor (TF), CanAg, EGFR, Įv-integrin, CD37, Folate Receptor, CD138, CEACAM5, CD56, CD70, CD74, GCC, 5T4, CD79b, Steap1, Napi2b, Lewis Y Antigen, LIV1 (ZIP6), Lymphocyte Antigen 6 Complex, c-RET, Locus E (LY6E), LIV, DLL3, EFNA4, Endosalin/CD248 and B7-H4.

[0011] In some embodiments, the target binding domain binds to a biomolecule that binds to a cell surface marker on a target cell. In some embodiments, the biomolecule comprises an antibody, a peptide (e.g., 5-40 amino acids), a polypeptide (e.g., of 40-10,000 amino acids), or an aptamer. In some embodiments, the cell surface marker is selected from the group consisting elected from the group consisting of CLL-1, IL1RAP, TIM-3, GPR-114, CD19, CD20, CD22, ROR1, mesothelin, CD33, CD123/IL3Ra, c-Met, PSMA, Prostatic acid phosphatase (PAP), CEA, CA-125, Muc-1, AFP, Glycolipid F77, EGFRvIII, GD-2, NY- ESO-1 TCR, Tyrosinase, TRPI/gp75, gp100/pmel-17, Melan-A/MART-1, Her2/neu, WT1, EphA3, telomerase, HPV E6, HPV E7, EBNA1, BAGE, GAGE, MAGE A3 TCR,

TCRSLITRK6, ENPP3, Nectin-4, CD27, SLC44A4, CAIX, Cripto, CD30, MUC16, GPNMB, BCMA, Trop-2, Tissue Factor (TF), CanAg, EGFR, Įv-integrin, CD37, Folate Receptor, CD138, CEACAM5, CD56, CD70, CD74, GCC, 5T4, CD79b, Steap1, Napi2b, Lewis Y Antigen, LIV1 (ZIP6), Lymphocyte Antigen 6 Complex, c-RET, Locus E (LY6E), LIV, DLL3, EFNA4, Endosalin/CD248 and B7-H4.

[0012] In some embodiments, the transmembrane domain is selected from a TM domain of CD2, CD3, CD16, CD32, CD64, CD28, 4-1BBL, CD4, and CD8.

[0013] In some embodiments, the hinge region is selected from a hinge region of a CD4 extracellular domain, CD8 extracellular domain and Fc region of IgG1 antibody.

[0014] In some embodiments, the CD3ς signaling domain comprises an amino acid sequence selected a signal transduction domain of CD3ς (e.g., NP_000725.1) and amino acid sequences having at least 70, 80, 85, 90, 95% or higher identity to a signal transduction domain of CD3ς (e.g., NP_000725.1). In other embodiments, the CD3ς signaling domain comprises an amino acid having at least 80, 85, 90, or 95% identity to a signal transduction domain of CD3ς. [0015] In some embodiments the nucleic acid molecule comprises RNA or DNA. In another embodiment the nucleic acid molecule is comprised within a plasmid or a viral vector. In some embodiments the nucleic acid molecule further comprises an expression control sequence operatively linked with the nucleotide sequence encoding the CAR. In some embodiments the nucleic acid molecule further comprises one or more restriction sites for ligating a nucleotide sequence encoding the intracellular domain to a nucleotide sequence encoding CD3ς domain, and/or a nucleotide sequence encoding the hinge domain to a nucleotide sequence encoding the target binding domain.

[0016] In some embodiments, the CAR further comprises: a signal peptide (e.g., in addition to or instead of, independently any one or more of the CD3-zeta signal transduction domain or Fc receptor signal transduction domain, at least one third signal transduction domain, and/or linker). In another embodiment the CAR further comprises: a linker positioned between the target binding domain and TM domain (e.g., in addition to or instead of, independently any one or more of the CD3-zeta signal transduction domain or Fc receptor signal transduction domain, at least one third signal transduction domain, and/or signal peptide). In some embodiments, at least one of the domains is heterologous to at least one of the other domains (e.g., not found together in this arrangement in nature). In some embodiments, the domains of the CAR are all heterologous to each other.

[0017] In another aspect, provided herein is a chimeric antigen receptor (CAR) comprising: (1) a target binding domain; (2) optionally a hinge region; (3) a transmembrane domain (TM); and (4) an intracellular domain comprising a signal transduction domain selected the group consisting of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) signal transduction domain and a CTLA4-CD28 hybrid signal transduction domain, as encoded by the nucleic acid molecule described above. In some embodiments, the CAR also includes (5) a CD3ς signal transduction domain or an Fc receptor signal transduction domain. In other embodiments, the CAR further includes (6) at least one third signal transduction domain selected from a signal transduction domain of an Fc receptor, CD28, 4-1BB, CD27, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 and CD83L.

Alternatively, the CAR includes a signal peptide for localizing the CAR to the surface of a cell and/or a linker positioned between the target binding domain and TM domain.

[0018] Also disclosed herein is a recombinant cell comprising a nucleic acid molecule of a chimeric antigen receptor of this disclosure. In some embodiments, the cell is a T-cell, e.g., a CD4 T-cell, a CD8 T-cell, a memory T cell, or a T memory stem cell (TSCM). In some embodiments, the cell is a myeloid or lymphoid progenitor cell (e.g., a Common Myeloid Progenitor (CMP), a Granulocyte/Monocyte Progenitor (GMP) or a Megakaryocyte

Progenitor (MKP), Common Lymphoid Progenitor (CLP)). In some embodiments, the nucleic acid molecule further comprises an expression control sequence operatively linked with the nucleotide sequence encoding the CAR.

[0019] Also disclosed herein is a pharmaceutical composition comprising a recombinant cell (e.g., a human cell, human T cell, human myeloid or lymphoid cell, etc.), a CAR, or a nucleic acid encoding a CAR of this disclosure, with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for intravenous injection into a human.

[0020] Also disclosed herein is a method for stimulating a T cell-mediated immune response to a target cell in a mammalian subject, the method comprising administering to the mammalian subject an effective amount of the recombinant cell of this disclosure, wherein said cells express the CAR, thereby stimulating an adaptive immune response to the target cell. In some embodiments, the mammalian subject is a human. In some embodiments, the mammalian subject suffers from cancer, and the target binding domain binds a tumor associated antigen. In some embodiments, the mammalian subject suffers from cancer, and the target binding domain binds a viral associated antigen. In some embodiments, the mammalian subject suffers from a viral disease, and the target binding domain binds a viral antigen. In some embodiments, the human is resistant to at least one chemotherapeutic agent.

[0021] In some embodiments, the recombinant cell is a human cell. In some embodiments, the recombinant cell is a T cell. In some embodiments, the T cell is an autologous T cell. In some embodiments, the recombinant cell is a myeloid or lymphoid cell. In another embodiment the myeloid or lymphoid cell is an autologous or allogeneic cell. In some embodiments the myeloid or lymphoid cell is a pool of allogeneic cells from two or more normal healthy donors.

[0022] Also disclosed herein is a method for providing anti-tumor immunity in a mammalian subject, the method comprising administering to the mammalian subject an effective amount of the recombinant cell as described herein, wherein the target binding domain binds a tumor associated antigen, thereby providing direct anti-tumor immunity and initiating or boosting the adaptive immune response to the mammalian subject. In some embodiments, the mammalian subject is a human. In some embodiments, the mammalian subject does not have a detectable tumor. In some embodiments, progeny of the recombinant cell (e.g., T cells) persist in the subject for at least three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years or more than three years after administration. In some embodiments, progeny of the immune cells from the initiated or boosted adapted immune response persist in the subject for at least three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years or more after administration.

[0023] Genetically engineered T cells having a CTLA4 signaling domain or a CTLA4- CD28 hybrid domain confer better T cell target specificity, activation and generation of T cell memory cells (T central memory (T_cm); T effector memory (T_em);T memory stem cell (T_scm)) and other T cell populations that result in long lasting anti-tumor immunity.

[0024] In some embodiments, genetically engineered MPCs having a CTLA4 signaling domain or a CTLA4-CD28 hybrid domain confer an innate immune response to the tumor, activate adaptive immunity, and provide antimicrobial immunity during chemo/radiation therapy.

[0025] Also disclosed herein is a method for treating cancer in a mammalian subject, the method comprising administering to the mammalian subject an effective amount of: (a) a recombinant cell as provided herein; and (b) an antibody conjugate, wherein the antibody (i) specifically binds to a target antigen on the target cell, and (ii) is conjugated to a chemical inducer of dimerization (“CID”) with which the FKBP moiety is able to undergo chemically induced homo-dimerization or heterodimerization; wherein activated recombinant cells kill cancer cells, thereby treating the cancer. In certain embodiments, the mammal is a human.

[0026] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG.1 shows a schematic diagram of an exemplary embodiment of a CAR construct disclosed herein. The CAR construct fuses a CD19 scFv, CD 8 hinge and TM, CTLA4 ICD, and CD3zeta domains. [0028] FIG.2 provides an amino acid sequence encoding the CAR depicted in FIG 1. (SEQ ID NO:145) Signal peptide is in italic-underline. Plain text is anti-CD19. Bold areas are amino acids encoded by restriction sites. Underlined is the CD8 hinge region and transmembrane region. Bold and colored grey is the CTLA4 intracellular domain. Italic is the CD3zeta signaling domain

[0029] FIG.3 provides a nucleotide sequence encoding the CAR depicted in FIG.1. (SEQ ID NO:146) Signal peptide is in italic-underline. Plain text is anti-CD19. Bold areas are restriction sites that facilitate cloning. Underlined is the CD8 hinge region and

transmembrane region. Bold and colored grey is the CTLA4 intracellular domain. Italic is the CD3zeta signaling domain.

[0030] FIG.4 provides a comparison of CTLA4 wild-type ICD sequences and mutations in CTLA4 that may increase cell surface expression and potentially increasing CAR activity (SEQ ID NOS:142-144, SEQ ID NO:4). The YMNM, PRRP, and PYAPP domains in CD28 are swapped into CTLA4 ICD. The mutations and amino acids swaps are shown in grey. The amino acid position information corresponds to the sequence of CTLA4 protein set forth in GenBank Accession No. NP_005205.2.

[0031] FIG.5 shows the expression of the CTLA4 CAR construct in donor T cells. T cells were transfected with the either negative control GFP gene, 4-1BB positive CAR control construct, or with CTLA4construct. Expression of CAR construct on the cell surface is detected by protein L staining and analyzed by flow cytometry. The Y axis detects GFP signal, and the X axis detects CAR construct expression as stained by protein L. Expression of CTLA4 CAR construct is similar to that of positive control 4-1BB CAR construct (28% CTLA4 CAR expressing cells vs.30% 4-1BB expressing cells).

[0032] FIG.6 shows target cell killing by T cells transfected with CTLA4 CAR construct relative to T cells transfected with negative controls (untransfected (UnT) or GFP transfected Tcells) and the positive control (41BB CAR construct). T cells to Daudi cells at a 1:1 ratio is shown by white bars, and T cells to Daudi cells at a 20:1 ratio is shown by black bars. Error bars represent standard deviations of triplicate plated cells.

[0033] FIG.7 illustrates the cytotoxic killing of T cells transfected with CTLA4 mutation CAR constructs relative to T cells transfected with negative control GFP construct and positive control CD28 CAR construct. [0034] FIG.8 shows killing of CLL-1-expressing cells with T cells expressing a CAR having an anit-CLL-1 targeting domain and a CTLA4 co-stimulatory domain.

[0035] FIG.9 shows expression of a variety of CARs having a CTLA4 signalling domain in T cells.

[0036] FIG.10 shows binding properties of CARs containing CLEC12A scFV binding domain arranged either with light chain following heavy chain or heavy chain following light chain.

[0037] FIGS.11A-B show CLEC12A ScFv amino acid sequence that displays binding activity in transfected T cells. FIG.11A: Amino acid sequence of CLEC12A ScFv fused to 4 1BB signaling domain CAR construct is presented (SEQ ID NO:148). FIG 11B: Various amino acid sequence domain is described as indicated in the figure (SEQ ID NOS:149-156). DETAILED DESCRIPTION OF THE INVENTION

I. INTRODUCTION [0038] The inventors have discovered that the CTLA4 signal transduction domain can act as a co-stimulatory domain in a chimeric antigen receptor (CAR). It is surprising that a CTLA4 signal transduction domain can act as a co-stimulatory domain in a CAR in part because CTLA-4 plays a role in the inhibition of T-cell activation. See, e.g., Sansom, Immunology 101(2): 169–177 (2000).

[0039] Disclosed herein are compositions and methods that provide a chimeric antigen receptor (CAR) having a CTLA4 signal transduction domain. In some embodiments, the CAR construct is incorporated within T-cells, myeloid or lymphoid cells, or progenitor cells transiently or stably modified to express a genetically engineered CAR. This disclosure provides novel CAR constructs in which the intracellular domain is derived from a CTLA4 signal transduction domain or a CTLA4-CD28 hybrid signal transduction domain.

II. DEFINITIONS [0040] As used herein, the following terms have the meanings ascribed to them unless specified otherwise. [0041] Unless otherwise specified, terms and symbols of biochemistry, nucleic acid chemistry, molecular biology, developmental biology and molecular genetics follow those of standard treaties and texts in the field, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, 1989); Alberts and Singer, Developmental Biology, Eighth Edition (Sinauer Associates Inc., Sunderland, MA, 2006); Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Gaits, ed., Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Eckstein, ed., Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); and the like.

[0042] As used herein, the term“at least”,“at most” or“between” before a series of measures modifies each of the measures.

[0043] As used herein, a“CTLA4 signal transduction domain” refers to a domain derived from CTLA4 having signal transduction function. In some cases, a CTLA4 signal transduction domain comprises the amino acid sequence of positions 183-223 of SEQ ID NO: 1 or a substantially identical variant thereof having at least 80, 85, 90, or 95% sequence identity to the amino acid sequence from position 183 to position 223 of SEQ ID NO: 1. The amino acid sequence of the wild-type full-length CTLA4 polypeptide is represented in SEQ ID NO: 1. In some embodiments, a CTLA4 signal transduction domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or 100% sequence identity to the wild- type signal transduction domain of CTLA4 (e.g., the amino acid sequence from positions 183-223 of SEQ ID NO: 1).

[0044] The CTLA4 signal transduction domain of the present disclosure can include one or more amino acid substitutions, insertions and/or deletions. Examples include, without limitation, the wild-type signal transduction domain of CTLA4 comprising any of the following amino acid substitutions to the signal transduction domain (i.e., intracellular domain) of CTLA4 : (1) K152A, K155A and K156A (“KLESS”), (2) Y165F, (3) P169A and P173A (“PRO-“), (4) Y182F, and (5) Y165F/Y182F as described in Teft et al., BMC

Immunology, 2009, 10:23. The amino acid substitutions K152A, K155A, K156A, Y165F, P169A, P173A, Y182F, Y165F, Y182F refer to the amino acid positions of the wild-type CTLA4 without that signal peptide that spans from position 1 to 36 of SEQ ID NO: 1. Thus, the amino acid substitutions (i) K188A, K191A and K192A; (ii) Y201F; (iii) P205A and P209A; (iv) Y218F and (v) Y201F and Y218F relative to SEQ IDN NO:1 correspond to the amino acid substitutions (i) K152A, K155A and K156A; (ii) Y165F; (iii) P169A and P173A; (iv) Y182F; (v) Y165F and Y182F, respectively, as described in Teft et al., 2009 (SEQ ID NO:157). Also included are any sequences of CTLA4 signal transduction domains found in the human genome or in other genomes, such as genomes of hominids, primates or mammals.

[0045] As used herein, a“CTLA4-CD28 hybrid signal transduction domain” or“CTLA4- CD28 chimeric signal transduction domain” refers to a signal transduction domain in which an amino acid of the CTLA4 signal transduction domain (e.g., positions 183-223 of SEQ ID NO:1) is substituted with up to 20 amino acids from a corresponding position in CD28. “Corresponding” positions can be identified based on alignment, for example using a BLAST algorithm. A CTLA4-CD28 chimeric signal transduction domain can include an substitution of V202M, K203N, P205T, T207R, E208R, F219A, I220P, I222R, N223D of SEQ ID NO:1 (see SEQ ID NO:158). Additionally after N223 of SEQ ID NO:1, all or a portion of the amino acid sequence FAAYRS (SEQ ID NO: 141) can be inserted or added. A CTLA4- CD28 hybrid signal transduction domain that has having up to 4 amino acid substitutions can have about 90% sequence identity to the wild-type CTLA4 signal transduction domain.

[0046] The term“antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site or antigen-binding site within the variable region of the immunoglobulin molecule. As used herein, the term“antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen recognition site of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. 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. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules including, but not limited to, toxins and radioisotopes. [0047] The term“variable region” or“V region” of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain, (VH), either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions connected by three complementarity determining regions (CDRs) also known as hypervariable regions. (i.e., Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4). The CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two several techniques for determining CDRs:, for example, (1) an approach based on cross-species sequence variability (e.g.,“Kabat determination” disclosed in, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda Md.); and (2) an approach based on crystallographic studies of antigen- antibody complexes (e.g.,“Chothia determination” or“enhanced Chothia determination” disclosed in, e.g., Al-Lazikani et al., 1997, J. Molec. Biol.273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs. Additional methods for determining CDR sequences include AbM and Contact

determinations, disclosed, at the website found at bioinf.org.uk/abs. As used herein, the CDR of a given antibody can refer to the CDR determined according to any method known in the art.

[0048] The term“chimeric” used in reference to molecules refers to molecules having portions that are not normally attached in nature.

[0049] The term“chimeric antibodies” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or capability while the constant regions are homologous to the sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0050] The antibody binds to an“epitope” on the antigen. The epitope is the specific antibody binding interaction site on the antigen, and can include a few amino acids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions of those amino acids. In some cases, the epitope includes non-protein components, e.g., from a carbohydrate, nucleic acid, or lipid. In some cases, the epitope is a three-dimensional moiety. Thus, for example, where the target is a protein, the epitope can be comprised of consecutive amino acids, or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., a discontinuous epitope). The same is true for other types of target molecules that form three-dimensional structures.

[0051] The term“specifically bind” refers to the ability of a molecule (e.g., antibody, TCR, or fragments thereof) to bind to a target with at least 2-fold greater affinity than non-target compounds, e.g., at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25- fold, 50-fold, or 100-fold greater affinity. For example, an antibody that specifically binds a particular cancer target will typically bind to the cancer target with at least a 2-fold greater affinity than a different target (e.g., a different member of the same protein family).

[0052] The term“binds with” or“binds to” respect to an antibody target (e.g., antigen, analyte, immune complex), typically indicates that an antibody binds a majority of the antibody targets in a pure population (assuming appropriate molar ratios). For example, an antibody that binds a given antibody target typically binds to at least 2/3 of the antibody targets in a solution (e.g., at least any of 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). One of skill will recognize that some variability will arise depending on the method and/or threshold of determining binding.

[0053] As used herein, a first target binding molecule (e.g., antibody, TCR, or target binding portion thereof),“competes” for binding to a target with a second target binding molecule, when binding of the second target binding molecule with the target is detectably decreased in the presence of the first target binding molecule compared to the binding of the second target binding molecule in the absence of the first target binding molecule. The alternative, where the binding of the first target binding molecule to the target is also detectably decreased in the presence of the second target binding molecule, can, but need not be the case. For example, a second antibody can inhibit the binding of a first antibody to the target without that first antibody inhibiting the binding of the second antibody to the target. However, where each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to“cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing target binding molecules are encompassed in the present disclosure. The term“competitor” target binding molecule can be applied to the first or second target binding molecule as can be determined by one of skill in the art. In some cases, the presence of the competitor target binding molecule (e.g., a first target binding molecule) reduces binding of the second target binding molecule to the target by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more, e.g., so that binding of the second target binding molecule to target is undetectable in the presence of the first (competitor) target binding molecule.

[0054] The terms“polypeptide” or“peptide” or“protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure are based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.

[0055] The terms“polynucleotide” or“nucleic acid,” are used interchangeably herein and refer to 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. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.

[0056] The phrase“effective amount”, as used herein refers to an amount of a therapeutic agent to effect beneficial or desirable biological and/or clinical results.

[0057] The terms“treat”,“treating” or“treatment”, as used herein refers to the treatment of a disease state in a mammal, particularly in a human, and includes: preventing, slowing the progression of, improving the condition of (e.g., causing remission of), or curing a disease.

[0058] The phrase“effective amount”, as used herein refers to an amount of a therapeutic agent effective to treat a disease or condition or to ameliorate at least one symptom of the disease or condition. [0059] The terms“identical” or percent“identity” in the context of two or more polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of 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 may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that may be used to obtain alignments of amino acid sequences. These include, but are not limited to, BLAST, ALIGN, Megalign, and BestFit. In some embodiments, two polypeptides of the disclosure 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%, 96%, 97%, 98%, 99% 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 sequences that is at least about 10, at least about 20, at least about 40-60 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 90-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared.

[0060] Two amino acid sequences are“substantially homologous” or“substantially identical” when greater than 80%, preferably greater than 85%, preferably greater than 90% of the amino acids are identical, or greater than about 90%, preferably greater than 95%, are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or any of the sequence comparison algorithms such as BLAST, FASTA, etc.

[0061] Unless otherwise specified, the comparison window used to compare two sequences is the length of the shorter sequence. Accordingly, a fragment of a polymer has less than 100% sequence identity with a parent molecule, and can be substantially identical to the parent molecule. In the case of a polymer that is longer than a reference polynucleotide or polypeptide (e.g., a polynucleotide or polypeptide having more monomeric units than the reference polynucleotide or polypeptide), the longer polynucleotide or polypeptide may be used instead as the reference sequence, such that it is considered to have less than 100% sequence identity with the shorter reference polynucleotide or polypeptide. [0062] A“conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution.

Preferably, conservative substitutions in the sequences of the polypeptides and antibodies of the disclosure do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the tumor associated antigen, viral antigen or a viral associated antigen to which the polypeptide or antibody binds. Methods of identifying amino acid conservative substitutions that do not eliminate antigen binding are well-known in the art.

[0063] A polypeptide, antibody, polynucleotide, vector, cell, or composition that is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is the predominant biomolecular species (polynucleotide, polypeptide, polysaccharide, lipid) in a mixture, e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.9%.

[0064] As used herein,“variants” and“function-conservative variants” are those variants in which a given amino acid residue in a protein has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, an amino acid having the same polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A“variant” or“function-conservative variant” also includes a polypeptide which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%, and which has the same or substantially similar polypeptide with the same or substantially similar properties or functions as the native or parent protein to which it is compared. [0065] As used herein, the phrase a nucleotide sequence having a nucleotide sequence at least, for example, 95%“identical” to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the nucleotide 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.

[0066] Two nucleic acid sequences are“substantially homologous” or“substantially identical” when greater than 80%, preferably greater than 85%, preferably greater than 90% of the nucleotides are identical, or greater than about 90%, preferably greater than 95%, are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or any of the sequence comparison algorithms such as BLAST, FASTA, etc.

[0067] The term“vector” means a construct, which is capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

III. DETAILED DESCRIPTIONS OF EMBODIMENTS A. Chimeric Antigen Receptors [0068] Chimeric antigen receptors (“CARs”) can include the following elements: (1) a signal peptide, (2) a target binding domain, (3) a hinge region; (4) a transmembrane region and (5) an intracellular domain comprising a CTLA4 signal transduction domain. The CTLA4 signal transduction domain can have substantial (e.g., at least 80, 85, 90, or 95%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or complete (i.e., 100%) sequence identity to amino acids from position 183-223 of SEQ ID NO:1. In other instances, the CTLA4 signal transduction domain can be a CTLA4-CD28 chimeric signal transduction domain. Optionally, the CAR can further include any of: a signal peptide, a CD3ς signal transduction domain, an Fc receptor signal transduction domain, a co-stimulatory (signal transduction) domain. That is, these optional elements can be included in addition to or instead of, independently any one or more of the other optional elements. At least one of the domains is heterologous to at least one of the other domains. That is, at least two domains are not found together in this arrangement in nature. In some embodiments, the domains of the CAR are all heterologous to each other. The signal transduction domains are involved in the initiation of the activation program in T cells to generate a cytotoxic and/or a pro- inflammatory response against the tumor cell target.

1. Signal Peptide [0069] A signal peptide can be any peptide having the function of allowing a polypeptide to traverse a cell membrane. The signal peptide can be derived from CD4, CD8, CD28, CTLA4 or immunoglobulin family of receptors. In some embodiments, the signal peptide is at or within 10 amino acids of the amino terminus of the CAR protein.

2. Target Binding Domain– Structure [0070] The target binding domain can include any polypeptide comprising a target binding function. The polypeptide can comprise an antibody, a binding domain of a T cell receptor, a polypeptide ligand of a target or a phage display polypeptide or any other peptide (e.g., 5-40 amino acids) or polypeptide that has specific binding affinity for the target.

[0071] In one embodiment, the target binding domain can comprise a single chain antibody (scFV). In one embodiment, the CAR composition uses a scFv containing a T-cell receptor variable alpha and variable beta domain or a single chain fragment containing TCR Vį and VȖ domains.. In some embodiments, the antibody- or TCR-based target binding domain recognizes cells expressing MHCI (e.g., binds to MHCI). In some embodiments, the antibody- or TCR-based target binding domain recognizes cells expressing MHCII (e.g., binds to MHCII). The scFv can be connected to the transmembrane domain via a hinge domain whose length, flexibility and origin provides variability in the CAR’s design (Hudecek, M. et al.2013, Hornbach A., et al.2010, and Hornbach A., et al.2000) and can, along with the transmembrane domain, contribute to the interaction with antigen, building of the immunologic synapse and impact the CAR’s association with additional proteins needed to impart a robust activation signal.

[0072] In one embodiment, the CAR construct’s target binding domain can bind to a tumor associated antigen, a cancer stem cell associated antigen or a viral antigen. The target binding domain can be customized to the antigen(s) of the disease that is afflicting each patient being treated with a CAR. Additionally, CTLA4 can provide potentially greater co- stimulatory signal compared to CD28 and/or 4-1BB signaling on partially activated T cells. Furthermore, co-stimulation via CTLA4 signaling can potentially generate greater or longer- lasting Tcm (T central memory) and/or Tem (T effector memory) cells compared to other co- stimulatory signals. The disclosed CARs in conjunction with antibody target identification that optionally is patient specific provides a unique treatment customized to each individual patients tumor profile resulting in patient specific T cell immunotherapeutic products for all cancers, both known and novel tumor specific antigens.

[0073] In some embodiments, the target binding domain can comprise an antibody (or target binding fragment or variant thereof) that specifically binds to TIM3 (T-cell immunoglobulin domain and mucin domain 3), e.g., human TIM3 (SEQ ID NO:101). For example, the TIM3 antibody (anti-TIM3) can be any one of the TIM3 antibodies described in WO2013/006490. In some embodiments, the TIM3 antibody competes for binding to TIM3 with a TIM3 antibody comprising the heavy chain complementarity determining regions (CDRs) and light chain CDRs of any one of the 1.7E10 (SEQ ID NOS:5-12) , 7.10F6 (SEQ ID NOS:13-20), 8.16C10 (SEQ ID NOS:21-28), 27.2H4 (SEQ ID NOS:29-36), 27.12A6 (SEQ ID NOS:37-44), 6.14B9 (SEQ ID NOS:45-52), 6.16F9 (SEQ ID NOS:53-60), 33.1G12 (SEQ ID NOS:61-68), 33.2A5 (SEQ ID NOS:69-76), 33.14A5 (SEQ ID NOS:77-84), 27.12E12 (SEQ ID NOS:85-92), and 9.1G12 (SEQ ID NOS:93-100) antibody clones disclosed herein. In some embodiments, the TIM3 antibody comprises the heavy chain CDRs and/or light chain CDRs of any one of the 1.7E10, 7.10F6, 8.16C10, 27.2H4, 27.12A6, 6.14B9, 6.16F9, 33.1G12, 33.2A5, and 9.1G12 antibody clones. In some embodiments, the TIM3 antibody comprising the heavy chain variable region and/or light chain variable region of any one of the 1.7E10, 7.10F6, 8.16C10, 27.2H4, 27.12A6, 6.14B9, 6.16F9, 33.1G12, 33.2A5, and 9.1G12 antibody clones. [0074] In some embodiments, the target binding domain can comprise an antibody (or target binding fragment or variant thereof) that specifically binds CLL-1 (C-type lectin like molecule 1), e.g., human CLL-1 (SEQ ID NO:102). For example, the CLL-1 antibody (anti- CLL-1) can be any one of the antibodies described in WO2013/169625. In some embodiments, the CLL-1 antibody competes for binding to CLL-1 with a CLL-1 antibody comprising the heavy chain CDRs and light chain CDRs of any one of the M26 (SEQ ID NOS:103-104), M31 (SEQ ID NOS:105-106), G4 (SEQ ID NOS:107-108), M22 (SEQ ID NOS:109-110), M29 (SEQ ID NOS:111-112), M2 (SEQ ID NOS:113-114), M5 (SEQ ID NOS:115-116), and G12 (SEQ ID NOS:117-118) antibody clones disclosed herein. In some embodiments, the CLL-1 antibody comprises the heavy chain CDRs and/or light chain CDRs of any one of the M26, M31, G4, M22, M29, M2, M5, and G12 antibody clones. In some embodiments, the CLL-1 antibody comprising the heavy chain variable region and/or light chain variable region of any one of the M26, M31, G4, M22, M29, M2, M5, and G12 antibody clones.

[0075] In some embodiments, the target binding domain comprises an antibody (or target binding fragment or variant thereof) that specifically binds IL1RAP (IL1 receptor associated protein), e.g., human IL1RAP (SEQ ID NO:119). For example, the IL1RAP antibody (anti- IL1RAP) can be any one of the antibodies described in WO2014/100772. In some embodiments, the IL1RAP antibody competes for binding to IL1RAP with an IL1RAP antibody comprising the heavy chain CDRs and light chain CDRs of any one of the 1F5 (SEQ ID NOS:120, 121, 126-131), 4G9 (SEQ ID NOS:122-123), and 4B6 (SEQ ID

NOS:124, 125, 132-137) antibody clones disclosed herein. In some embodiments, the IL1RAP antibody comprises the heavy chain CDRs and/or light chain CDRs of any one of the 1F5, 4G9, and 4B6 antibody clones. In some embodiments, the IL1RAP antibody comprising the heavy chain variable region and/or light chain variable region of any one of the 1F5, 4G9, and 4B6 antibody clones.

3. Target Binding Domain– Targets [0076] The target can be any biomolecule on a target cell or that binds to a molecule on a target cell. Target cells can include cancer cells. Therefore, the target can comprise, for example, a polypeptide expressed on a cancer cell, e.g., a tumor associated antigen. The CAR can bind an antigen determinant comprising amino acids within the extracellular domain of a tumor associated antigen, a viral antigen or a viral associated antigen or a fragment of such a polypeptide.

[0077] Cancers that can be targeted include, for example, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML)), breast cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, head and neck cancer, bladder cancer, gynecological cancer, liposarcoma, and multiple myeloma. Additional cancers that can be targeted include lung cancer (e.g., non-small cell lung cancer or NSCLC), ovarian cancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma), thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, pancreatic cancer, bile duct cancer, gastrointestinal carcinoid tumors, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancers of the central and peripheral nervous systems (i.e., schwannomas, neurofibromatosis 1 and 2), skin cancer, choriocarcinoma; blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin's lymphoma, Burkitt’s lymphoma, Small Cell lymphoma, Large Cell lymphoma, monocytic leukemia, and myelogenous leukemia.

[0078] In some embodiments, the target can be a biomolecule on a target cell. In some embodiments, the target binding domain within the CAR of this disclosure is capable of binding any of a broad group of targets, including but not limited to, CLL-1, IL1RAP, TIM- 3, GPR-114, CD19, CD20, CD22, ROR1, mesothelin, CD33, CD123/IL3Ra, c-Met, PSMA, Prostatic acid phosphatase (PAP), CEA, CA-125, Muc-1, AFP, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, Tyrosinase, TRPI/gp75, gp100/pmel-17, Melan-A/MART-1, Her2/neu, WT1, EphA3, telomerase, HPV E6, HPV E7, EBNA1, BAGE, GAGE, MAGE A3 TCR, TCRSLITRK6, ENPP3, Nectin-4, CD27, SLC44A4, CAIX, Cripto, CD30, MUC16, GPNMB, BCMA, Trop-2, Tissue Factor (TF), CanAg, EGFR, Įv-integrin, CD37, Folate Receptor, CD138, CEACAM5, CD56, CD70, CD74, GCC, 5T4, CD79b, Steap1, Napi2b, Lewis Y Antigen, LIV1 (ZIP6), Lymphocyte Antigen 6 Complex, c-RET, Locus E (LY6E), LIV, DLL3, EFNA4, Endosalin/CD248 and B7-H4, and other targets. One of skill in the art will appreciate that the CAR can include a targeting domain that specifically binds to any other known cancer- or virus-associated target.

[0079] In some embodiments, the target can be a biomolecule that binds to a cell surface marker on a target cell. For example, the target can be an antibody or an antibody conjugate or other specific binding molecule (e.g., a peptide, an aptamer, a polypeptide, etc.) that specifically binds to a cell surface marker on a cell (e.g., a cancer cell). The cell surface molecule can be, but is not limited to, those targets listed above or elsewhere herein. In these embodiments, the antibody or specific binding molecule can be administered to an individual, such that the antibody or specific binding molecule binds to the cell surface marker on a cancer or other cell. The target binding domain of the CAR is administered (separately or with the antibody or specific binding molecule) and specifically binds to the antibody or specific binding molecule. The antibody or specific binding molecule can contain a molecular tag that will be a unique target for the target binding domain. Aspects of this configuration are described in, e.g., International Patent Publication WO2012/082841 and Kudo et al, Cancer Res. 74(1): 93-103 (2014).

4. Hinge Region [0080] In some embodiments, the hinge region of the disclosed CARs can be selected from the CD8, CD4, or CD28 extracellular domain, the Fc region of an IgG1 antibody, or the extracellular domain of a CTLA4 or a variant thereof as is known to one of skill in the art and can be found in the GenBank database.

5. Transmembrane Domain [0081] The transmembrane domain can comprise a transmembrane domain of an immunoglobulin family receptor, such as CD8. The intracellular domain can be selected from any membrane-spanning molecule on a T cell. For example, the transmembrane (TM) domain of the disclosed CAR can comprise the TM domain of CTLA4 or the TM domain of CD2, CD3, CD16, CD32, CD64, CD28, CD247, 4-1BBL, CD4, or CD8.

6. Intracellular Signaling Domain Comprising CTLA4 Signal Transduction Domain [0082] As used herein,“signal transduction domain” refers to a moiety capable of transmitting activation, proliferation and/or survival signals to a cell. The intracellular signaling domain includes a CTLA4 signal transduction domain, that is, a domain of a CTLA4 protein having signal transduction function. This can include a complete intracellular domain of a CTLA4 or a portion of an intracellular domain having this function. Also, a portion of a CTLA4 comprising a transmembrane region and an intracellular signaling domain can be used in a CAR of the present disclosure. The intracellular domain of a human CTLA4 polypeptide comprises the amino acid sequence at position 183-223 of SEQ ID NO: 1 or a substantially identical variant thereof. In some cases, the signal transduction domain of CTLA4 of the CAR construct disclosed herein comprises the amino acid sequence at position 183-223 of SEQ ID NO: 1 or a substantially identical variant thereof. Variants of the signal transduction domain of CTLA4 can include one or more amino acid substitution such that the amino acid sequence of the mutant domain has at least 80, 85, 90, or 95% sequence identity to the wild-type (native) CTLA4 signal transduction domain. In other cases, a variant of the signal transduction domain of CTLA4 is a CTLA4-CD28 hybrid signal transduction domain. In yet other cases, a CTLA4-CD28 hydrid domain has fewer than 20, 15, 10, 5, 4, 3, or 2 amino acid substitutions relative to the wild-type CTLA4 signal transduction domain, wherein the substituted amino acids are amino acids from a

corresponding position in the signal transduction domain of CD28.

[0083] CTLA4 belongs to the immunoglobulin (Ig) family of co-receptors. T-cell activation is modulated by antigen specific signals from T-cell receptors and additional signals by coreceptors such as CD28 and CTLA4. CD28 provides a positive signal that promotes and sustains T-cell responses, while CTLA4 provides an inhibitory signal that limits or dampens the response. This balance between stimulatory and inhibitory co-signals calibrates a T-cell response to a foreign pathogen without excess inflammation and autoimmunity. In addition, CTLA4 can generate positive signals that affect cell adhesion, motility, and prosurvival pathways (Rudd et al., Immunol Rev, 2009, 229(1):12-26). CTLA- 4 is primarily located in intracellular compartments such as the trans-Golgi network, endosomes and lysosomes (Leung et al., J Biol Chem, 1995, 270:25107-25114; Valk et al., Immunity, 2006, 25:807-821). A low level of CTLA4 protein can be detected on the cell surface, and the translocation of CTLA4 to the cell surface can be modulated by its intracellular domain (ICD). It has been demonstrated that mutations in the Y165F and Y165F/P169A/P173A can increase the amount of CTLA4 expression on the cell surface (Teft et al., BMC Immunol, 2009, 10:23). The inventors have surprisingly discovered that chimeric antigen receptors containing a mutated CTLA4 intracellular domain (signal transduction domain) can increase cytotoxic activity and may increase cell surface expression, compared to CARs containing either a CD28 or 4-1BB signaling domain.

[0084] The amino acid sequence of CTLA4 is set forth in, for example, GenBank

Accession No. NP_005205 or UniProt Accession No. P16410. The corresponding nucleotide sequence is set forth in, for example, NCBI Ref. Seq. No. NM_005214. There are three isoforms of CTLA4: a full-length isoform, a form lacking the ectodomain, and a soluble form that lacks exon 3 encoding the transmembrane domain (Ueda et al., Nature, 2003, 423:506- 511). In some embodiments, any CTLA4 sequence found in the human genome or in genomes of hominids, primates or other mammals can be used to generate a CTLA4 signal transduction domain for use in a CAR construct.

[0085] In some embodiments, the signal transduction domain of CTLA4 corresponds to position 183-223 of the wild-type CTLA4 amino acid sequence (SEQ ID NO: 1). One or more amino acid substitutions are made in the signal transduction domain of CTLA4.

Suitable CTLA4 signal transduction domains can have an amino acid sequence with substantial (e.g., at least 80, 85, 90, or 95%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence from positions 183- 223 of SEQ ID NO: 1 or fragments thereof of at least 20, 25, 30 or 35 amino acids. In some cases, the CTLA4 domain of the CAR is substantially identical to the wild-type CTLA4 intracellular domain which is represented in SEQ ID NO:1 from amino acid positions 183- 223. In some embodiments, the mutant CTLA4 domain has an amino acid substitution at position 188, 191, 192, 201, 205, 209, 218 or any combination thereof of SEQ ID NO: 1. For instance, the CTLA4 domain can have the following amino acid substitutions: (i) K188A, K191A and K192A; (ii) Y201F; (iii) P205A and P209A; (iv) Y218F; (v) Y201F and Y218F; and(vi) Y201F, P205A and P209A. The amino acid substitutions (i) K188A, K191A and K192A; (ii) Y201F; (iii) P205A and P209A; (iv) Y218F and (v) Y201F and Y218F correspond to the amino acid substitutions (i) K152A, K155A and K156A; (ii) Y165F; (iii) P169A and P173A; (iv) Y182F; (v) Y165F and Y182F, respectively, as described in Teft et al., 2009 (SEQ ID NO:157). Useful CTLA4 signal transduction domains include those set forth in SEQ ID NOS: 142-144. These domains can be used in the CAR constructs described herein.

[0086] The signal transduction domain of CTLA4 can be a chimeric domain comprising sequences from a plurality of CTLA4 from different species such as from humans, hominids, primates and other mammals. It also can be an analog of a CTLA4 in which the native sequence of a CTLA4 has been modified. In some instances, the CTLA4 signal transduction domain is a chimeric domain comprising sequences from the signal transduction domains of CTLA4 and CD28. In other instances, the CTLA4 signal transduction domain is a chimeric domain comprising sequences from the signal transduction domains of CTLA4 and CD28. In other instances, the CTLA4 signal transduction domain is a chimeric domain comprising sequences from the signal transduction domains of CTLA4 and 4-1BB.

[0087] The YMNM (SEQ ID NO:159), PRRP (SEQ ID NO:160) and PYAP (SEQ ID NO:161) domains of CD28 play a role in its co-stimulation function during cytokine production and T cell activation (Isakov et al., Front Immunol, 2012, 3:273). For instance, the YVKM domain (SEQ ID NO:162), PTEP domain (SEQ ID NO:163), and/or PYFI domain (SEQ ID NO:164) of the CTLA4 signal transduction domain can be replaced with the YMNM domain, PRRP domain, and/or PYAP domain of CD28, respectively. In some embodiments, the amino acid sequence APPRDFAAYRS (SEQ ID NO: 141) or a fragment thereof can replace the FIPIN sequence at position 219-223 of SEQ ID NO: 1. Useful CTLA4-CD28 chimeric domain include, without limitation, the amino acid sequence of SEQ ID NO: 4 and variants thereof. In some cases, the CTLA4-CD28 chimeric signal transduction domain can have fewer than 20 amino acid substitutions, e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, relative to the wild-type CTLA4 signal transduction domain (e.g., positions 183-223 of SEQ ID NO: 1).

[0088] Examples of a CTLA4-CD28-hybrid signal transduction domain include, without limitation, any or all of the following substitutions: V202M, K203N, P205T, T207R, E208R, F219A, I220P, I222R, N223D, relative to the wild-type CTLA4 signal transduction domain (see SEQ ID NO:158). In another embodiment of a CTLA4-CD28-hybrid signal transduction domain, amino acids at positions 201-209 of the wild-type CTLA4 sequence (SEQ ID NO:1) are substituted with amino acids at positions 191-199 of the CD28 sequence (SEQ ID NO: 2), and/or amino acids at positions 217-223 of the wild-type CTLA4 sequence (SEQ ID NO:1) are substituted with amino acids at positions 211-220 of the CD28 sequence (SEQ ID NO: 2). In some embodiments, the CTLA4-CD28-hybrid signal transduction domain has the amino acid sequence of SEQ ID NO: 4.

7. CD3ȗ Signal Transduction Domain [0089] In some embodiments, the CAR construct further contains a CD3ς signaling domain. For example, the CD3ς signaling domain can be located at the carboxyl terminus of the CTLA4 signal transduction domain or the CTLA4-CD28 hybrid signal transduction domain. In some embodiments, the CD3ς signaling domain of the disclosed CAR molecule can comprise a CD3ς amino acid sequence, e.g., a signal transduction domain of CD3 zeta (full length sequence shown as SEQ ID NO:138). This element is particularly useful for constructs used in T cells. This sequence can have an amino acid sequence substantially identical to a signal transduction domain of the CD3ς sequence set forth herein. For example the CD3 zeta signal transduction domain can include amino acids 21-163, 31-142, 68-89 and/or 138-158 of the sequence shown in SEQ ID NO:138, or functional variants thereof (e.g., with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10-20 amino acid substitutions, deletions, or additions). In some embodiments, the CD3 zeta signal transduction domain comprises amino acids involved in ZAP70 interaction, e.g., one or more ITAM domains (see, e.g., Steinberg et al. (2004) Blood 104:760). An ITAM domain can be identified by the consensus sequence: D/E-X7-D/E-X2-Y-X-X-L-X7-Y-X-X-L (SEQ ID NO:139).

8. Fc Receptor Signal Transduction Domain [0090] The CAR can have a signal transduction domain of any one of the Fc-alpha, Fc- gamma, Fc-epsilon, Fc-mu, and Fc-delta receptors. For example, the Fc receptor signaling domain can comprise amino acids involved in interaction with Src (e.g., Fgr, Fyn, Hck, Lyn, Yes, and Src) and ZAP-70 family kinases, e.g., one or more ITAM domains (see, e.g., Sanchez-Mejorada et al. (1998) J. Leukocyte Biol.63:531; Garcia-Garcia et al. (2002) J. Leukocyte Biol.72:1092). In some embodiments, the Fc receptor signal transduction domain includes at least one ITAM domain, e.g., from any one of the Fc-alpha, Fc-gamma, Fc- epsilon, Fc-mu, and Fc-delta receptors, or substantially identical thereto.

9. 4-1BB Signal Transduction Domain [0091] In another embodiment the intracellular domain comprises a 4-1BB signal transduction domain.4-1BB transmits a survival signal to a T cell once the CAR is bound to an antigen. A 4-1BB signal transduction domain can have a sequence with substantial identity to KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 147).

10. Co-stimulatory Domain [0092] The CARs of this disclosure can include one or additional co-stimulatory domains in addition to a signal transduction domain of CD3ς. Additional co-stimulatory domains can be derived from for example, CD28, 4-1BB, CD2, CD27, CD30, OX40, CD40, PD-1, PD-L1, PD-L2, ICOS, LFA-1, CD7, LIGHT, NKG2C, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3, B7-H4 and others. It has been shown by others that CARs using an anti-CD19 scFV and CD28 and 4-1BB signaling domains have shown promising clinical results in chronic lymphocytic leukemia (CLL) and acute B lymphocytic leukemia (B-ALL). In some embodiments, the CAR constructs can contain a CTLA4 signal transduction domain or a CTLA4-CD28 hybrid signal transduction domain and also one or more co-stimulatory domains (e.g., a 4-1BB costimulatory domain, and additionally, a CD28 and or CD2 co- stimulatory domain).

[0093] Preliminary data indicates that signaling domains may influence the duration of the T cell mediated anti-tumor response and creation of a long-lasting immune“memory” that is required for anti-tumor immune surveillance. Initial data with CD28 construct suggested that despite an initial burst of activity, long lasting immunity may not be generated or may be suppressed by various suppressor cells or immunomodulatory cytokines. Published data suggests that signaling through 4-1BB may generate more memory T cells than CD28 signaling alone which resulted in the second generation of CARs using two co-stimulatory domains in addition to TCR zeta. Therefore, other signaling domains not yet identified or evaluated can potentially confer better T cell activation and generation of T cell memory cells (T central memory, Tcm; T effector memory, Tem; T memory stem cell (Tscm)) and other T cell populations that may result in long lasting anti-tumor immunity.

[0094] The co-stimulatory domain or domains can be positioned between the CTLA4 signal transduction domain and the CD3ς signal transduction domain.

B. Nucleic Acids [0095] Disclosed herein are nucleic acid molecules (polynucleotides) comprising a nucleotide sequence that encodes a CAR of this disclosure. The nucleic acid of the disclosed CAR can be in the form of DNA or in the form of RNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. RNA includes mRNA, siRNA, sRNA, ssRNA and so on. The nucleic acids can be purified or isolated.

[0096] The present disclosure further relates to variants of the hereinabove described nucleotide sequences encoding, for example, fragments, analogs, and/or derivatives of the disclosed CARs. In some embodiments, the nucleic acid molecule encoding the disclosed CAR can comprise RNA or DNA and form of each as is known to one of skill in the art.

[0097] In some embodiments, the intracellular domain of the disclosed CAR can have a signal transduction domain from a molecule encoding CTLA4 or an isoform thereof. [0098] Polynucleotides encoding CARs of this disclosure can include regulatory elements operatively linked with a nucleotide sequence encoding the CAR. For example, a polynucleotide can include one or more transcription regulatory elements, such as promoters or enhancers, which, when the polynucleotide is present in a cell, cause the sequence encoding the CAR to be expressed within the cell.

[0099] Polynucleotides encoding CARs be isolated molecules, or can be included within a vector, such as a plasmid, a cosmid, an artificial chromosome or a virus. Such vectors can be used to transfect target cells.

[0100] In some embodiments, the present disclosure provides isolated nucleotide sequences comprising nucleotide sequences having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a nucleotide sequences encoding a CAR polypeptide comprising a CAR, or fragment thereof, described herein.

[0101] The polynucleotide variants can contain alterations in the coding regions, non- coding regions, or both. In some embodiments, the polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded CAR polypeptide. In some embodiments, the polynucleotide variants contain alterations that do not produce any changes in the amino acid sequence. In some embodiments, polynucleotide variants contain“silent” substitutions 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.

[0102] In some embodiments, the polynucleotides as described herein are isolated. In certain embodiments, the polynucleotides as described herein are substantially pure.

[0103] An exemplary embodiment of a chimeric antigen receptor includes a CD19 specific CAR having the following domains: a CD19 scFv domain, CD8 hinge-CD8 transmembrane domain, CTLA4 or mutant CTLA4 signal transduction domain, and CD3zeta domain. The amino acid sequence of the exemplary CD19 specific CAR is provided in SEQ ID NO: 145 and the corresponding nucleic acid sequence is provided in SEQ ID NO.146.

[0104] CD19 scFv-CD8 hinge-CD8 TM-CTLA4 ICD-CD3zeta CAR fusion protein (SEQ ID NO: 145)

MALPVTALLL PLALLLHAAR

[0105] An amino acid sequence of an exemplary CAR, e.g., CD19 scFv-CD8 hinge-CD8 TM-CTLA4 ICD-CD3zeta CAR fusion protein. Signal peptide is in italic-underline. Plain text is anti-CD19. Bold areas are amino acids encoded by restriction sites. Underlined is the CD8 hinge region and transmembrane region. Double-underlined is the CTLA4 intracellular domain. Italic is the CD3zeta signaling domain.

[0106] CD19 scFv-CD8 hinge-CD8 TM-CTLA4 ICD-CD3zeta CAR fusion DNA (SEQ ID NO: 146)

[0107] A nucleotide sequence encoding an exemplary CAR, such as a CD19 scFv-CD8 hinge-CD8 TM-CTLA4 ICD-CD3zeta CAR. Signal peptide is in italic-underline. Plain text is anti-CD19. Bold areas are restriction sites that facilitate cloning. Underlined is the CD8 hinge region and transmembrane region. Double-underlined is the CTLA4 intracellular domain. Italic is the CD3zeta signaling domain.

C. Cells [0108] This disclosure also provides cells (e.g., recombinant cells) comprising nucleic acid molecules encoding CARs and/or expressing CARs.

[0109] In some embodiments, the nucleic acid molecule encoding the disclosed CAR can be delivered to a host cell, including but not limited to a T cell, B cell, myeloid or lymphoid progenitor, macrophage, and so on, by a plasmid or a viral vector as is known to one of skill in the art. The resulting recombinant (host) cell can include but is not limited to a T-cell, a CD4 T-cell, a CD8 alpha T-cell, CD8 beta T cell, T helper cell, granulocyte (neutrophils, basophils, eosinophils), megakaryocytes, monocyte, macrophage and a dendritic cell, a T memory stem cell as well as cells expressing MHC class I or class II as is known to one of skill in the art. In some embodiments, the recombinant (host) cell having the nucleic acid molecule encoding the disclosed CAR can be a myeloid or lymphoid progenitor cell selected from the group consisting of a common myeloid or lymphoid progenitor, a granulocyte macrophage progenitor, a megakaryocyte erythrocyte progenitor, a granulocyte progenitor and a monocyte progenitor as is known to one of skill in the art. In some embodiments, the myeloid or lymphoid cell is an autologous or allogeneic cell.

[0110] In some embodiments, the recombinant (host) cell having the nucleic acid molecule encoding the disclosed CAR wherein the nucleic acid molecule can further comprises an expression control sequence operatively linked with the nucleotide sequence encoding the CAR. The assembled CAR (by synthesis, site-directed mutagenesis or another method, as is known to one of skill in the art), the nucleic acid molecule encoding the disclosed CAR can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the disclosed CAR in a desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and/or expression of the CAR polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0111] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding CAR polypeptides or antibodies, or fragments thereof. For example, recombinant expression vectors can be replicable DNA constructs which have a nucleotide sequence encoding a CAR operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A

transcriptional unit generally comprises an assembly of (1) a regulatory element or elements having a role in gene expression, for example, transcriptional promoters and/or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences.

Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are “operatively linked” when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; 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. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.

Alternatively, where recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0112] The proteins produced by a transformed/recombinant host can be purified according to any suitable method. Such methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. In some embodiments, proteins can also be physically characterized using such techniques as proteolysis, high performance liquid chromatography (HPLC), nuclear magnetic resonance and x-ray crystallography.

D. Compositions [0113] In some embodiments, a pharmaceutical composition is provided comprising a recombinant cell having the nucleic acid molecule encoding the disclosed CAR polypeptide and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for intravenous injection into a mammalian subject, including but not limited to a human, mouse, rat, monkey and so on. In some embodiments, the mammalian subject suffers from cancer, and the target binding domain within the CAR within the recombinant cell binds a tumor associated antigen. In some embodiments, the mammalian subject suffers from cancer, and the target binding domain within the CAR within the recombinant cell binds a viral antigen or a viral associated antigen. In some embodiments, the mammal is a human with resistance to at least one chemotherapeutic agent. In some embodiments, the mammal subject does not have a detectable tumor.

E. Methods [0114] Also, disclosed is a method for stimulating a T cell-mediated immune response to a target cell in a mammalian subject. The T cell-mediated immune response can be stimulated by administering to the mammalian subject an effective amount of recombinant cells having the nucleic acid molecule encoding the disclosed CAR polypeptide, wherein said cells express the CAR, thereby stimulating a T cell-mediated immune response to the target cell. In some embodiments, the T cell is an autologous T cell, an allogeneic T cell or a xenogeneic T cell. In some embodiments, the progeny of the T cells persist in the subject for at least three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years or more after administration.

[0115] In some embodiments, a method for stimulating a myeloid or lymphoid progenitor cell response to a target cell in a mammalian subject is provided. The myeloid or lymphoid progenitor cell response can be stimulated by administering to the mammalian subject an effective amount of recombinant cells having the nucleic acid molecule encoding the disclosed CAR polypeptide, wherein said cells express the CAR, thereby stimulating a myeloid or lymphoid progenitor cell response to the target cell. In some embodiments, the myeloid or lymphoid progenitor cell is a common myeloid or lymphoid progenitor cell, a megakaryocyte/erythroid progenitor, or a granulocyte/macrophage progenitor. In some embodiments, the progeny of the myeloid or lymphoid progenitor cells persist in the subject for at least three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years or more after administration.

[0116] In some embodiments, a method for providing anti-tumor immunity in a mammalian subject is provided The anti-tumor immunity can be stimulated by administering to the mammalian subject an effective amount of recombinant cells having the nucleic acid molecule encoding the disclosed CAR polypeptide, wherein the target binding domain binds a tumor associated antigen, thereby providing direct anti-tumor immunity and initiating or boosting the adaptive immune response to the mammalian subject. In some embodiments, the progeny of the immune cells from the initiated or boosted adapted immune response persist in the subject for at least three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years or more than three years after administration.

IV. EXAMPLES [0117] The following examples are offered to illustrate, but not to limit, the claimed invention.

Example 1.

[0118] A CAR construct (CD19 scFv-CD8 hinge-CD8 TM-CTLA4 ICD-CD3zeta) fusing CD19ScFv, CD8 hinge and TM, CTLA4 ICD, and CD3zeta domains are shown in FIG.2. The components include a signal peptide from CD8, CD19 ScFv , CD8 extracellular hinge and transmembrane domain, CTLA4 intracellular domain, and CD3zeta domain . Amino acid length of each component is indicated. The amino acid sequence of the CAR construct depicted in FIG.1 is provided in FIG.2. The signal peptide is in italic-underline. Plain text is anti-CD19. Bold areas are amino acids encoded by restriction sites. Underlined is the CD8 hinge region and transmembrane region. Bold and colored grey is the CTLA4 intracellular domain. Italic is the CD3zeta signaling domain.

[0119] The nucleic acid sequence of the CAR construct (CD19 scFv-CD8 hinge-CD8 TM- CTLA4 ICD-CD3zeta) depicted in FIG.1 is provided in FIG.3. The signal peptide is in italic-underline. Plain text is anti-CD19. Bold areas are restriction sites that facilitate cloning. Underlined is the CD8 hinge region and transmembrane region. Bold and colored grey is the CTLA4 intracellular domain. Italic is the CD3zeta signaling domain.

Example 2. Rationale for designing engineered CTLA4 variants that can provide enhanced co-stimulatory signaling

[0120] The majority of CTLA4 wild-type molecule is localized in intracellular compartments such as trans-golgi network, endosomes, and lysosomes (Rudd et al., Immunol Rev, 2009, 229(1)-:12-26). Only a small amount of CTLA4 can be found on the cell surface, and the translocation of CTLA4 to the cell surface can be modulated by the ICD. It has been demonstrated that mutations in the Y165F and Y165F/P169A/P173A will increase the amount of CTLA4 expression on the cell surface (Teft et al., BMC Immunol, 2009, 10:23). However, use of these mutations in the context of a CAR construct is unknown.

[0121] FIG.4 depicts CTLA4 wild-type ICD sequences and mutations in CTLA4 that may increase cell surface expression and potentially increasing CAR activity. The YMNM, PRRP, and PYAPP domains in CD28 were swapped into the CTLA4 ICD. The mutations and amino acids swaps are shown in grey.

Example 3. Expression of CTLA4 ICD CAR constructs in T cells and cytotoxic T cell activity

[0122] For transient expression of CAR constructs containing CD19 ScFv-CD8 hinge-CD8 TM-CTLA4 ICD(or various mutations in CTLA4 ICD)-CD3zeta domains, DNA sequences (~1.5 kilobase pairs) encoding the amino acid sequence of the CAR constructs were synthesized and cloned into the Pmax vector (Lonza) under the control of the CMV promoter using HindIII and EcoRI restriction enzymes. For lentiviral expression of CAR constructs, the CAR encoding amino acid sequences were cloned into pCD711B-1 vector under the control of the MSCV promoter (System Bioscience Inc.) using the NheI and EcoRI restriction enzymes.

[0123] The cell surface expression of the various CTLA4 CARs on donor T cells was compared to the expression of CARs with either CD28 or 4-1BB signal transduction domains. In particular, the expression of CD19 ScFv-CD8 hinge-CD8 TM-CTLA4-CD3zeta was compared to the expression of CD19 ScFv-CD8 hinge-CD8 TM-CD28-CD3zeta and CD19 ScFv-CD8 hinge-CD8 TM-4-1BB-CD3zeta. CD3 positive T cells from a healthy donor leukoreduction blood sample were isolated using the pan-T cell isolation kit (Miltenyi Biotech). T cells from a single donor were transfected with CAR constructs containing various co-stimulatory domains by using the Amexa transfection system (Lonza), and expression of the CAR constructs was confirmed by staining cells with protein L (FIG.5). The wild-type CTLA4 CAR construct was express on the cell surface of T cells similarly to the control 4-1BB CAR construct (FIG.5). Transfection efficiency of T cells varied from 10- 25%.

[0124] FIG.5 depicts the cell surface expression of the wild-type CTLA4 CAR construct transfected into T cells. The expression was compared to that of either the negative control GFP gene, or the positive control 4-1BB CAR construct. Expression of CAR construct on the cell surface was detected by protein L staining and analyzed by flow cytometry. The Y axis shows GFP signal, and the X axis shows CAR construct expression as stained by protein L. Expression of the wild-type CTLA4 CAR construct is similar to that of positive control 4- 1BB CAR construct (28% CTLA4 CAR expressing cells vs.30% 4-1BB expressing cells).

[0125] The cytotoxic activity of the wild-type CTLA4 CAR-expressing T cells and mutant CTLA4 CAR-expressing T cells was also evaluated compared to 4-1BB CAR-expressing T cells. The transfected T cells were incubated with Daudi cells (B-cell lymphoma cells) at a ratio of 20:1 or 1:1 of T cells to Daudi cells for 20 hours. Live and died Daudi cells were analyzed by flow cytometry. Specific killing of Daudi cells was calculated using the following equation:

{[(# of dead target cells) - (# of target cells spontaneous dead in target cells only) / [(total # of target cells plated)– (# of target cells spontaneous dead in target cells only)]}*100.

[0126] FIG.6 illustrates target cell killing by T cells transfected with the CTLA4 CAR construct relative to T cells transfected with negative controls untransfected (UnT) or GFP transfected T cells and the positive control 4-1BB CAR transfected T cells. T cell to Daudi cells at a 1:1 ratio is shown by white bars, and T cell to Daudi cells at a 20:1 ratio is shown by black bars. Error bars represent standard deviations of triplicate plated cells.

[0127] FIG.7 shows cytotoxic killing of T cells transfected with CTLA4 mutant CAR constructs relative to T cells transfected with negative control GFP construct and positive control CD28 CAR construct.

Example 4. T cells expressing CLEC12A ScFv fused to CTLA4 costimulatory domain can display target dependent cytotoxicity.

[0128] Referring to Fig.8, T cells were transiently transfected with CLEC12A (clone M26) ScFv fused to 41BB, wild-type CTLA4, CTLA4 with Y165F mutation, CTLA4 with Y165/P169A/P173A mutations, and CTLA4-CD28 chimeric signal transduction domains and incubated with target cell at 20:1 or 4:1 T cell to target cell ratio. T cells expressing

CLEC12A ScFv fused to wild-type CTLA4, CTLA4 with Y165/P169A/P173A mutations, and chimeric CTLA4-CD28 domains displayed comparable target cell killing (OCI-AML5) of 12-20% relative to that of the same CLEC12A ScFv fused to positive control 41BB signaling domain. Target cells were labelled with GFP and/or CMFDA (fluorescent marker), and the total target cells alone or when mixed with CAR expressing T cells is determined by flow cytometry. The percentage of target cell killing is determined by the formula: 1- [number of target cells when mixed with T cells expressing CAR constructs / number of target cells without mixing with T cells] X 100. Percent specific killing is determined by subtracting percentage of target cell killing when target cells were mixed with CLEC12A ScFv CAR expressing T cells from percentage of target cell killing when target cells were mixed with T cells expressing none target binding CD19 Scfv CAR construct control.

Example 5. Expression of CLEC12A ScFv CAR constructs fused to various CTLA costimulatory domains in T cells.

[0129] Referring to Fig.9, T cells were transfected with CLEC12A (clone M26) ScFv fused to 41BB, wild-type CTLA4, CTLA4 with Y165F mutation, CTLA4 with

Y165/P169A/P173A mutations, and CTLA4-CD28 chimeric signal transduction domains. Expression of the CAR constructs were determined by incubating the transfected T cells with CLEC12A extracellular domain conjugated to Alexa 647 dye, and analyzed by flow cytometry. T cells expressing CD19 ScFv fused to 41BB signaling domain is used as a control for none relevant binding to CLEC12A extracellular domain.

Example 6. Determination of CLEC12A ScFv with proper binding activity.

[0130] Referring to Fig 10, T cells expressing CLEC12A ScFv with heavy chain followed by light chain and light chain followed by heavy chain were incubated with CLEC12A extracellular domain or protein L conjugated to Alexa 647 dye, and analyzed by flow cytometry. CLEC12A ScFv heavy chain followed by light chain displayed better binding to CLEC12A extracellular domain or protein L.

[0131] We have demonstrated that CAR constructs with the wild-type CTLA4 signal transduction domain surprisingly has activity as a CAR construct. We have also shown that CAR constructs with mutations in the CTLA4 signal transduction domain, such as the Y201F mutant, Y201F/P205A/P209A mutant, and CTLA4-CD28 chimera mutant, all surprising have cytotoxicity activity as CAR constructs.

[0132] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Informal sequence listing

SEQ ID NO: 1– CTLA4 from GenBank Accession No. NP_005205 or UniProt Accession No. P16410

Claims

WHAT IS CLAIMED IS: 1. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises:

(1) a target binding domain;

(2) a hinge region (optional);

(3) a transmembrane domain (TM); and

(4) an intracellular domain comprising a signal transduction domain selected the group consisting of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) signal transduction domain and a CTLA4-CD28 hybrid signal transduction domain.

2. The nucleic acid molecule of claim 1, wherein the CTLA4 signal transduction domain comprises an amino acid sequence having at least 80, 85, 90, or 95% sequence identity to the amino acid sequence from positions 183-223 of SEQ ID NO: 1.

3. The nucleic acid molecule of claim 2, wherein the CTLA4 signal transduction domain comprises an amino acid sequence having 100% sequence identity to the amino acid sequence from positions 183-223 of SEQ ID NO: 1.

4. The nucleic acid molecule of claim 2, wherein the amino acid sequence having at least 80, 85, 90, or 95% sequence identity to the amino acid sequence from positions 182-223 of SEQ ID NO: 1 comprises one or more amino acid substitutions located at one or more positions in SEQ ID NO: 1 selected from the group consisting of position 188, position 191, position 192, position 201, position 205, position 209, position 218 or a combination thereof.

5. The nucleic acid molecule of claim 4, wherein the one or more amino acid substitutions are selected from the group consisting of

(i) K188A, K191A and K192A;

(ii) Y201F;

(iii) P205A and P209A;

(iv) Y218F;

(v) Y201F and Y218F; and

(vi) Y201F, P205A and P209A.

51

6. The nucleic acid molecule of claim 1, wherein the CTLA4-CD28 hybrid signal transduction domain comprises an amino acid sequence of SEQ ID NO: 1 having fewer than 20 amino acid substitutions, wherein the amino acid substitutions are amino acids at corresponding positions in the amino acid sequence of the signal transduction domain of CD28 as set forth in SEQ ID NO: 2.

7. The nucleic acid molecule of claim 6, wherein the fewer than 20 amino acid substitutions are selected from the group consisting of V202M, K203N, P205T, T207R, E208R, F219A, I220P, I222R, N223D and a combination thereof.

8. The nucleic acid molecule of claim 6 or claim 7, wherein the CTLA4- CD28 hybrid signal transduction domain comprises an amino acid insertion comprising fewer than 6 amino acids after position 209 and/or position 223 of SEQ ID NO:1.

9. The nucleic acid molecule of claim 8, wherein the amino acid insertion after position 209 is G and/or the amino acid insertion after position 223 is FAAYRS (SEQ ID NO: 3).

10. The nucleic acid molecule of claim 1, wherein the CTLA4-CD28 hybrid signal transduction domain comprises the amino acid sequence of SEQ ID NO: 4.

11. The nucleic acid molecule of claim 1, further comprising: (5) a CD3ς signal transduction domain or an Fc receptor signal transduction domain.

12. The nucleic acid molecule of claim 11, further comprising an Fc receptor signal transduction domain, wherein the Fc receptor is selected from the group consisting of Fc-alpha, Fc-gamma, Fc-epsilon, Fc-mu, and Fc-delta.

13. The nucleic acid molecule of claim 11, further comprising: (6) at least one co-stimulatory domain selected from a signal transduction domain of CD28, 4-1BB, CD27, OX40, CD30, CD40, PD-1, PD-L1, PD-L2, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3 and B7- H4.

14. The nucleic acid molecule of claim 1, further comprising: (5) a signal peptide for localizing the CAR to the surface of a cell. 52

15. The nucleic acid molecule of claim 14, wherein the signal peptide comprises a CD8 signal peptide.

16. The nucleic acid molecule of claim 1, further comprising: (5) a linker positioned between the target binding domain and TM domain.

17. The nucleic acid molecule of claim 1, wherein the target binding domain comprises an antibody or peptide between 5-40 amino acids long, apolypeptide.

18. The nucleic acid molecule of claim 17, wherein the antibody is an antibody fragment.

19. The nucleic acid molecule of claim 17, wherein the antibody is an antibody single chain fragment variable (scFV).

20. The nucleic acid molecule of claim 1, wherein the target binding domain comprises a T-cell receptor (TCR) variable alpha (VĮ) and variable beta (Vȕ) domains, or a single chain fragment containing a TCR variable delta (Vį) and variable gamma (VȖ) domains.

21. The nucleic acid molecule of claim 1, wherein the target binding domain binds to a tumor associated antigen, a cancer stem cell associated antigen or a viral antigen.

22. The nucleic acid molecule of claim 1, wherein the target binding domain binds a target selected from the group consisting of CLL-1, IL1RAP, TIM-3, GPR- 114, CD19, CD20, CD22, ROR1, mesothelin, CD33, CD123/IL3Ra, c-Met, PSMA, Prostatic acid phosphatase (PAP), CEA, CA-125, Muc-1, AFP, Glycolipid F77, EGFRvIII, GD-2, NY- ESO-1 TCR, Tyrosinase, TRPI/gp75, gp100/pmel-17, Melan-A/MART-1, Her2/neu, WT1, EphA3, telomerase, HPV E6, HPV E7, EBNA1, BAGE, GAGE, MAGE A3 TCR,

53

23. The nucleic acid molecule of claim 1, wherein the target binding domain binds to a biomolecule that binds to a cell surface marker on a target cell.

24. The nucleic acid molecule of claim 23, wherein the biomolecule comprises an antibody, a peptide, or an aptamer.

25. The nucleic acid molecule of claim 23 or 24, wherein the cell surface marker is selected from the group consisting of CLL-1, IL1RAP, TIM-3, GPR-114, CD19, CD20, CD22, ROR1, mesothelin, CD33, CD123/IL3Ra, c-Met, PSMA, Prostatic acid phosphatase (PAP), CEA, CA-125, Muc-1, AFP, Glycolipid F77, EGFRvIII, GD-2, NY- ESO-1 TCR, Tyrosinase, TRPI/gp75, gp100/pmel-17, Melan-A/MART-1, Her2/neu, WT1, EphA3, telomerase, HPV E6, HPV E7, EBNA1, BAGE, GAGE, MAGE A3 TCR,

26. The nucleic acid molecule of claim 1, wherein the transmembrane domain is selected from a TM domain of CD2, CD3, CD16, CD32, CD64, CD28, 4-1BBL, CD4, and CD8.

27. The nucleic acid molecule of claim 1, wherein the hinge region is selected from a hinge region of a CD4 extracellular domain, CD8 extracellular domain, and an Fc region of IgG1 antibody.

28. The nucleic acid molecule of claim 11, wherein the CD3ς signaling domain comprises an amino acid having at least 80, 85, 90, or 95% identity to a signal transduction domain of CD3ς.

29. The nucleic acid molecule of claim 1, comprising RNA or DNA.

30. The nucleic acid molecule of claim 1, comprised within a plasmid or a viral vector.

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31. The nucleic acid molecule of claim 1 or claim 11, further comprising one or more restriction sites for ligating a nucleotide sequence encoding the intracellular domain to a nucleotide sequence encoding CD3ς domain, and/or a nucleotide sequence encoding the hinge domain to a nucleotide sequence encoding the target binding domain.

32. A chimeric antigen receptor (CAR) comprising:

(1) a target binding domain,

(2) a hinge region (optional),

(3) a transmembrane domain (TM), and

(4) an intracellular domain comprising a signal transduction domain selected the group consisting of a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) signal transduction domain and a CTLA4-CD28 hybrid signal transduction domain,

as encoded by the nucleic acid molecule of any one of claim 1-31.

33. The CAR of claim 32, further comprising:

(5) a CD3ς signal transduction domain or an Fc receptor signal transduction domain.

34. The CAR of claim 33, further comprising:

(6) at least one third signal transduction domain selected from a signal transduction domain of an Fc receptor, CD28, 4-1BB, CD27, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3 and CD83L.

35. The CAR of claim 32, further comprising:

(5) a signal peptide for localizing the CAR to the surface of a cell.

36. The CAR of claim 32, further comprising:

(5) a linker positioned between the target binding domain and TM domain.

37. A recombinant cell comprising the nucleic acid molecule of any one of claims 1-28.

38. The recombinant cell of claim 37, which is a T cell.

39. The recombinant cell of claim 37, which is a CD4 T cell.

40. The recombinant cell of claim 37, which is a CD8 T cell. 55

41. The recombinant cell of claim 37, which is a memory T cell.

42. The recombinant cell of claim 37, which is a T memory stem cell.

43. The recombinant cell of claim 37, which is a myeloid or lymphoid progenitor cell (e.g., a Common Myeloid Progenitor (CMP), a Granulocyte/Monocyte Progenitor (GMP), Common Lymphoid Progenitor (CLP),or a Megakaryocyte Progenitor (MKP)).

44. The recombinant cell of claim 37, wherein the nucleic acid molecule further comprises an expression control sequence operatively linked with the nucleotide sequence encoding the CAR.

45. A pharmaceutical composition comprising the recombinant cell of any one of claims 37-41 and a pharmaceutically acceptable carrier.

46. The pharmaceutical composition of claim 45, further formulated for intravenous injection into a human.

47. A method for stimulating a T cell-mediated immune response to a target cell in a mammalian subject, the method comprising administering to the mammalian subject an effective amount of the recombinant cell of any one of claims 37-41, wherein the cell expresses the CAR, thereby stimulating an adaptive immune response to the target cell.

48. The method of claim 47, wherein the mammalian subject is a human.

49. The method of claim 47, wherein the mammalian subject suffers from cancer, and the target binding domain binds a tumor associated antigen.

50. The method of claim 47, wherein the mammalian subject suffers from cancer, and the target binding domain binds a viral associated antigen.

51. The method of claim 47, wherein the mammalian subject suffers from a viral disease, and the target binding domain binds a viral antigen.

52. The method of claim 47, wherein the recombinant cell is a T cell.

53. The method of claim 52, the T cell is an autologous T cell. 56

54. The method of claim 47, wherein the recombinant cell is a myeloid or lymphoid cell.

55. The method of claim 54, wherein the myeloid or lymphoid cell is an autologous or allogeneic cell.

56. The method of claim 54, wherein the myeloid or lymphoid cell is a pool of allogeneic cells from two or more normal healthy donors.

57. The method of claim 48, wherein the human is resistant to at least one chemotherapeutic agent.

58. A method for providing anti-tumor immunity in a mammalian subject, the method comprising administering to the mammalian subject an effective amount of the recombinant cell of any one of claims 37-41, wherein the target binding domain binds a tumor associated antigen, thereby providing direct anti-tumor immunity and initiating or boosting the adaptive immune response to the mammalian subject.

59. The method of claim 58, wherein the mammalian subject is a human.

60. The method of claim 58, wherein the mammalian subject does not have a detectable tumor.

61. The method of claim 58, wherein progeny of the recombinant cell persist in the subject for at least three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years or more than three years after administration.

62. The method of claim 58, wherein progeny of immune cells from the initiated or boosted adapted immune response persist in the subject for at least three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, three years or more after administration.

63. A method for treating cancer in a mammalian subject, the method comprising administering to the mammalian subject an effective amount of:

(a) a recombinant cell of any of claims 37-41; and

57 (b) an antibody conjugate, wherein the antibody (i) specifically binds to a target antigen on the target cell, and (ii) is conjugated to a chemical inducer of dimerization (“CID”) with which the FKBP moiety is able to undergo chemically induced homo- dimerization or heterodimerization;

wherein activated recombinant cells kill cancer cells, thereby treating the cancer.

64. The method of claim 63 wherein the mammal is a human.

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