WO2024153211A1

WO2024153211A1 - Fusion polypeptides for targeted protein degradation and mehtods of use thereof

Info

Publication number: WO2024153211A1
Application number: PCT/CN2024/073146
Authority: WO
Inventors: Yuncheng Zhao; Xu Fang; Jun Wang; Xin Huang; Changmeng XU; Qiuchuan ZHUANG
Original assignee: Nanjing Legend Biotech Co., Ltd.; Legend Biotech Ireland Limited
Priority date: 2023-01-19
Filing date: 2024-01-19
Publication date: 2024-07-25

Abstract

Provided herein are fusion polypeptides for degradation of target proteins and methods of use thereof. In some embodiments, the fusion protein includes (1) a non-antibody protein that binds to a target protein, and (2) an E3 ubiquitin ligase or fragment thereof. Also provided herein are engineered immune cells (e.g., CAR-T cells) expressing the fusion polypeptide.

Description

FUSION POLYPEPTIDES FOR TARGETED PROTEIN DEGRADATION AND MEHTODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority of International Patent Application No. PCT/CN2023/073151 filed on January 19, 2023, the content of which is incorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: IEC232092PCT_Sequence Listing. XML, date recorded: January 17, 2024, size: 66 KB) .

TECHNICAL FIELD

This disclosure relates to fusion polypeptides for degradation of target proteins and methods of use thereof. The disclosure also relates to engineered cells (e.g., CAR-T cells) expressing the fusion polypeptides.

BACKGROUND

Universal allogeneic CAR-T or TCR-T therapy is considered as an ideal model, with T cells derived from healthy donors. However, the key challenge is how to effectively eliminate graft-versus-host disease (GvHD) during treatment due to histoincompatibility. TCR is a cell surface receptor involved in T cell activation in response to antigen presentation. 95%of T cells in human have TCR consisting of an alpha (α) chain and a beta (β) chain. TCRα and TCRβchains combine to form a heterodimer and associate with CD3 subunits to form a TCR complex present on the cell surface. GvHD happens when donor’s T cells recognize non-self MHC molecules via TCR and perceive host (transplant recipient) tissues as antigenically foreign and attack them. In order to eliminate endogenous TCR from donor T cells thereby preventing GvHD, gene editing technologies such as Zinc Finger Nuclease (ZFN) , transcription activator-like effector nucleases (TALEN) , and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR associated (Cas) (CRISPR/Cas) have been used for endogenous TCRα or TCRβ gene knockout (KO) , enriching TCR-negative T cells for allogeneic CAR-T or TCR-T production.

Targeted ubiquitination and degradation of oncoproteins via the ubiquitin-proteasome pathway (UPP) represents an alternative degradation strategy. PROTACs (proteolysis targeting chimeras) are bispecific molecules that induce substrate degradation by simultaneously binding a protein of interest (POI) and an E3 ligase (e.g., ARV-110, a degrader of the androgen receptor and the first-in-class PROTAC to enter clinical trials) . Pharmacologically, small molecule degraders offer several advantages over traditional inhibitor-based therapeutics, while the discovery of corresponding clinical candidates is not without its challenges. Unlike traditional small-molecule PROTAC, bioPROTACs are generated by directly replacing the natural substrate recognition domain of the E3 ligase with a peptide or a miniprotein that binds a POI. Because bioPROTAC is an artificially engineered gene-encoded product, it can fuse POI ligands to any of the more than 600 E3 ligase enzymes, making full use of the cell’s ubiquitin-proteasome system.

Therefore, there exist needs for novel strategies for targeted protein degradation in T cells or other cells via the ubiquitin-proteasome pathway.

SUMMARY

The present application provides a method of producing modified or engineered T cells (such as TCR-T cells (e.g., cTCR-T cells) , TAC-T cells, TAC-like-T cells, or CAR-T cells) that can elicit reduced GvHD response in a histoincompatible individual during treatment, such as cancer immunotherapy. Briefly, a precursor T cell (i.e., the initial T cell to be modified) can be modified to express a fusion polypeptide that includes a first moiety (e.g., a non-antibody protein (e.g., a Nef protein) ) that binds to a target protein and a second moiety (e.g., a E3 ubiquitin ligase or fragment thereof) . In some embodiments, the target protein is CD3ζ and the degradation of CD3ζ triggers irreversible down-regulation of the TCR complex.

In one aspect, the disclosure is related to a fusion polypeptide, comprising: a first moiety comprising a non-antibody protein that binds to a target protein, and a second moiety comprising an E3 ubiquitin ligase or fragment thereof. In some embodiments, the target protein is associated with graft-versus-host disease (GvHD) and/or host-versus-graft disease (HvGD) in a cell therapy. In some embodiments, the target protein is CD3ζ. In some embodiments, the non-antibody protein is a viral protein. In some embodiments, the viral protein is a Negative Regulatory Factor (Nef) protein. In some embodiments, the Nef protein is from simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) . In some embodiments, the Nef protein is a wildtype or a non-naturally occurring mutant. In some embodiments, the Nef protein is selected from the group consisting of SIV Nef M116, HIV1 Nef M094, HIV2 Nef M787, HIV2 Nef M092, SIV Nef M1006, SIV Nef M1034, HIV1 Nef M1069, and SIV Nef M998. In some embodiments, the Nef protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 30-37. In some embodiments, the E3 ubiquitin ligase or fragment thereof is selected from the group consisting of Speckle Type BTB/POZ Protein (SPOP) , Beta-Transducin Repeat Containing E3 Ubiquitin Protein Ligase (βTrCP) , S-Phase Kinase Associated Protein 2 (SKP2) , Von Hippel-Lindau Tumor Suppressor (VHL) , Suppressor Of Cytokine Signaling 2 (SOCS2) , Ankyrin Repeat And SOCS Box Containing 1 (ASB1) , Carboxy Terminus Of Hsp70-interacting Protein CHIP (CHIP) , and Tripartite Motif Containing 21 (TRIM21) . In some embodiments, the E3 ubiquitin ligase or fragment thereof is truncated. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to: (a) . amino acids 167-374 of SPOP; (b) . amino acids 2-264 of βTrCP; (c) . amino acids 2-147 of SKP2; (d) . amino acids 152-213 of VHL; (e) . amino acids 143-198 of SOCS2; (f) . amino acids 266-335 of ASB1; (g) . amino acids 128-303 of CHIP; (h) . amino acids 1-267 of TRIM21; (i) . amino acids 1-127 of TRIM21; (j) . amino acids 1-85 of TRIM21; or (k) . amino acids 1-55 of TRIM21. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 19-29. In some embodiments, the first moiety and the second moiety are connected to each other via a linker peptide. In some embodiments, the linker peptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 46. In some embodiments, the first moiety is N-terminal to the second moiety or C-terminal to the second moiety. In some embodiments, the fusion polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 1-18.

In one aspect, the disclosure is related to a polynucleotide comprising a nucleic acid sequence encoding the fusion polypeptide as described herein. In some embodiments, the polynucleotide further comprises a second nucleic acid sequence encoding a functional exogenous receptor; optionally in some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR) . In some embodiments, the polynucleotide further comprises a third nucleic acid sequence encoding a protein marker; optionally in some embodiments, the protein marker is DHFR L22F/F31S. In some embodiments, the polynucleotide further comprises a fourth nucleic acid sequence encoding an exogenous cytokine, or a fragment thereof; optionally in some embodiments, the exogenous cytokine or fragment thereof is a membrane-bound IL12p40. In some embodiments, each nucleic acid sequence is connected via a linking sequence; optionally in some embodiments, the linking sequence comprises a nucleic acid sequence encoding P2A or T2A.

In one aspect, the disclosure is related to a vector comprising the polynucleotide as described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector comprises a human EF1α promoter.

In one aspect, the disclosure is related to an engineered cell comprising the fusion polypeptide, the polynucleotide, or the vector as described herein. In some embodiments, the engineered cell further comprises a functional exogenous receptor; in some embodiments, the functional exogenous receptor comprises (a) an extracellular ligand binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain (ISD) comprising a chimeric signaling domain (CMSD) , in some embodiments, the CMSD comprises one or a plurality of immune-receptor Tyrosine-based Activation Motifs (CMSD ITAMs) , in some embodiments, the plurality of CMSD ITAMs are optionally connected by one or more linkers (CMSD linkers) . In some embodiments, at least one of the CMSD ITAMs is not derived from CD3ζ. In some embodiments, at least one of the CMSD ITAMs is derived from an ITAM-containing parent molecule selected from the group consisting of CD3ε, CD3δ, CD3γ, Igα (CD79a) , Igβ (CD79b) , FcεRIβ, FcεRIγ, DAP12, CNAIP/NFAM1, STAM-1, STAM-2, and Moesin. In some embodiments, the CMSD comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 42. In some embodiments, the functional exogenous receptor further comprises a signal peptide located at the N-terminus of the extracellular ligand binding domain. In some embodiments, the extracellular ligand binding domain of the functional exogenous receptor binds to an antigen, in some embodiments, the antigen is a tumor antigen selected from the group consisting of BCMA, CLL1, GPC3, GU2CYC, CD19, CD7, CD20, CD22, CD38, CD41, CD123, Claudin 18.2, Claudin 6, NKG2D, DLL-3, GPRC5D, and CD70. In some embodiments, the tumor antigen is GPC3 or CD20. In some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR) , a T cell receptor (TCR) , a chimeric TCR (cTCR) , or a T cell antigen coupler (TAC) -like chimeric receptor. In some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR) . In some embodiments, the intracellular signaling domain of the CAR further comprises a co-stimulatory signaling domain. In some embodiments, the CAR further comprises a hinge domain located between the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain. In some embodiments, the CAR comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 38-41. In some embodiments, the engineered cell further comprises a protein marker; optionally in some embodiments, the protein marker is DHFR L22F/F31S. In some embodiments, the engineered cell further comprises an exogenous cytokine, or a fragment thereof; optionally in some embodiments, the exogenous cytokine or fragment thereof is a membrane-bound IL12p40. In some embodiments, the engineered cell is a T cell or an NK cell. In some embodiments, the engineered cell is a primary T cell obtained from a subject; optionally the subject is a cancer patient or healthy donor. In some embodiments, the engineered cell expresses a decreased level of CD3ζ as compared to that in a precursor T cell and/or a decreased level of endogenous T cell receptor (TCR) as compared to that in a precursor T cell. In some embodiments, the engineered cell elicits no or reduced graft-versus-host disease (GvHD) response in a histoincompatible individual as compared to the GvHD response elicited by a primary T cell isolated from the donor of the precursor T cell from which the engineered cell is derived.

In one aspect, the disclosure is related to a chimeric antigen receptor (CAR) , comprising: (a) an extracellular ligand binding domain that binds to GPC3 antigen, (b) a transmembrane domain, and (c) an intracellular signaling domain (ISD) comprising a chimeric signaling domain (CMSD) , in some embodiments, the CMSD comprises one or a plurality of immune-receptor Tyrosine-based Activation Motifs (CMSD ITAMs) , in some embodiments, the plurality of CMSD ITAMs are optionally connected by one or more linkers (CMSD linkers) ; optionally in some embodiments, at least one of the CMSD ITAMs is not derived from CD3ζ. In some embodiments, the CAR described herein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 41.

In one aspect, the disclosure is related to a method for producing an engineered T cell, comprising introducing the polynucleotide or the vector described herein into a precursor cell. In some embodiments, the method described herein further comprises introducing into the precursor cell a second polynucleotide encoding a functional exogenous receptor that binds to an antigen; optionally in some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR) . In some embodiments, the method described herein further comprises isolating and/or enriching TCR-negative and functional exogenous receptor-positive T cells from the engineered T cells.

In one aspect, the disclosure is related to an engineered T cell obtained by the method described herein.

In one aspect, the disclosure is related to a pharmaceutical composition comprising the engineered cell or the engineered T cell as described herein, and a pharmaceutically acceptable carrier.

In one aspect, the disclosure is related to a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the engineered cell, the engineered T cell, or the pharmaceutical composition as described herein.

In one aspect, the disclosure is related to a method of downregulating and/or degrading a target protein in a T cell from a subject, comprising introducing into the T cell the polynucleotide, or the vector as described herein; optionally in some embodiments, the target protein is CD3ζ and/or TCR.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

FIG. 1 shows Western blot detection results of CD3ζ protein and β-actin (control) expressed by Jurkat cells containing different SIV Nef M116-E3 ubiquitin ligase fusion proteins. M2339-Jurkat, M2342-Jurkat, M2346-Jurkat, M2348-Jurkat, M2352-Jurkat, M2354-Jurkat, M2356-Jurkat, and M2360-Jurkat cells overexpressed SIV Nef M116-SPOP, SIV Nef M116-βTrCP, SIV Nef M116-SKP2, SIV Nef M116-VHL, SIV Nef M116-SOCS2, SIV Nef M116-ASB1, SIV Nef M116-CHIP, and SIV Nef M116-TRIM21 fusion proteins, respectively. "Mock" represents untransduced Jurkat cells, which were used as a control.

FIG. 2 shows flow cytometry detection results of CD3ζ protein expressed by Jurkat cells containing SIV Nef M116-TRIM21 truncated fusion proteins. M2360-Jurkat, M2542-Jurkat, M2543-Jurkat, and M2544-Jurkat cells overexpressed SIV Nef M116-TRIM21, SIV Nef M116-TRIM21 Trun1, SIV Nef M116-TRIM21 Trun2, and SIV Nef M116-TRIM21 Trun3 fusion proteins, respectively. "Mock" represents untransduced Jurkat cells, which were used as a control.

FIG. 3 shows Western blot detection results of CD3ζ protein and β-actin (control) expressed by GPC3 CAR-T cells (M1909-T) containing SIV Nef M116 protein and GPC3 CAR-T cells (M2259-T) containing SIV Nef M116-TRIM21 Trun2 fusion protein. "UnT" represents untransduced T cells that were used as a control.

FIG. 4A shows flow cytometry detection results of BCMA CAR (CAR) expression in Jurkat-BCMA-BBz cells containing different subtypes of Nef. "Mock" represents untransduced Jurkat-BCMA-BBz cells, which were used as a control.

FIG. 4B shows flow cytometry detection results of CD3ζ protein expression in CD20 CAR-Jurkat cells containing different subtypes of Nef and TRIM21 Truncate2 fusion proteins. "Mock" represents untransduced Jurkat cells, which were used as a control.

FIG. 5A and FIG. 5B show flow cytometry detection results of GPC3 CAR (CAR) , TCRα/β, and MB12 (IL12p40) expressed in M2259-T cells that containing SIV Nef M116-TRIM21 Trun2 fusion protein. "UnT" represents untransduced T cells that were used as a control.

FIG. 5C shows Western blot detection results of CD3ζ protein and β-actin (control) expressed in M2259-T and UnT cells.

FIG. 5D shows the killing efficiency of M2259-T cells on GPC3-positive liver cancer cell line HuH-7. Luc at an E: T ratio of 2: 1 or 1: 2.

FIG. 6A and FIG. 6B show flow cytometry detection results of CD20 CAR (CAR) , TCRα/β, and MB12 (IL12p40) expressed in M2426-T cells that containing SIV Nef M116-SPOP fusion protein. "UnT" represents untransduced T cells that were used as a control.

FIG. 6C shows Western blot detection results of CD3ζ protein and β-actin (control) expressed in M2426-T and UnT cells.

FIG. 6D shows the killing efficiency of M2426-T cells on CD20-positive lymphoma cell line Raji. Luc at an E: T ratio of 20: 1, 10: 1, or 5: 1.

FIG. 7 lists amino acid sequences discussed in the disclosure.

DETAILED DESCRIPTION

The present disclosure relates to a fusion polypeptide that includes a first moiety (e.g., antibody protein (e.g., a Nef protein) , and a second moiety (e.g., a E3 ubiquitin ligase or fragment thereof) , for targeted degradation of a target protein.

The Nef (Negative Regulatory Factor) protein can down-modulate endogenous TCR, such as down-regulating cell surface expression of endogenous TCRα or TCRβ, thereby inhibiting endogenous TCR-mediated signal transduction. Further, by fusing Nef with a functional E3 ligase peptide (e.g., either a full-length E3 ligase protein or its functional truncation) , the present disclosure found that a target protein (e.g., CD3ζ) can be specifically degraded. Without being bound by theory, the degradation of CD3ζ triggers irreversible down-regulation of the TCR complex. Vectors encoding different combinations of Nef proteins and E3 ligase peptides were used to transduce T cells, and those with desired properties (e.g., degradation of CD3ζ and decreased expression levels of TCR complex) were obtained.

These T cells can be further engineered to express a functional exogenous receptor, such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) . In some embodiments, the functional exogenous receptor comprises: (a) an extracellular ligand binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain (ISD) comprising a chimeric signaling domain (CMSD) , wherein the CMSD comprises one or a plurality of ITAMs ( “CMSD ITAMs” ) . In some embodiments, at least one of the CMSD ITAMs is not based on CD3ζ sequences. As a result, expression of the fusion polypeptide described herein has little to no effect on the expression of the functional exogenous receptor.

The present application also provides an one-step method of producing GvHD-minimized modified T cells (such as TCR-T cells (e.g., cTCR-T cells) , TAC-T cells, TAC-like-T cells, or CAR-T cells) , either by co-transducing a precursor T cell with a vector encoding the fusion polypeptide and a vector encoding the functional exogenous receptor (such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) , or by transducing a precursor T cell with an “all-in-one” vector encoding both the fusion polypeptide and functional exogenous receptor. Engineered T cells derived from methods described herein can effectively down-regulate cell surface expression of TCR and CD3ζ, while preserves or even improve the expression and function of the functional exogenous receptor. The disclosure effectively minimizes or eliminates the occurrence of GvHD during allogeneic transplantation, and provides a convenient, effective, and low-cost strategy for universal allogeneic CAR-T, TCR-T (e.g., cTCR-T) , TAC-T, or TAC-like-T therapy.

Accordingly, one aspect of the present application provides a method of producing a engineered T cell, comprising introducing into a precursor T cell a nucleic acid encoding the fusion polypeptide and engineered T cells obtained by such methods. In another aspect, there are provided engineered T cells comprising a first nucleic acid encoding a fusion polypeptide described herein, and optionally a second nucleic acid encoding a functional exogenous receptor (such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) . Also provided are vectors (such as viral vectors) comprising a nucleic acid encoding the fusion polypeptide and optionally a nucleic acid encoding the functional exogenous receptor.

Definitions

As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.

“Allogeneic” refers to a graft derived from a different individual of the same species. “Allogeneic T cell” refers to a T cell from a donor having a tissue human leukocyte antigen (HLA) type that matches the recipient. Typically, matching is performed on the basis of variability at three or more loci of the HLA gene, and a perfect match at these loci is preferred. In some instances allogeneic transplant donors may be related (usually a closely HLA matched sibling) , syngeneic (a monozygotic “identical” twin of the patient) or unrelated (donor who is not related and found to have very close degree of HLA matching) . The HLA genes fall in two categories (Type I and Type II) . In general, mismatches of the Type-I genes (i.e., HLA-A, HLA-B, or HLA-C) increase the risk of graft rejection. A mismatch of an HLA Type II gene (i.e., HLA-DR, or HLA-DQB1) increases the risk of graft-versus-host disease (GvHD) .

A "patient" as used herein includes any human who is afflicted with a disease (e.g., cancer, viral infection, GvHD) . The terms "subject, " “individual, ” and "patient" are used interchangeably herein. The term "donor subject" or “donor” refers to herein a subject whose cells are being obtained for further in vitro engineering. The donor subject can be a patient that is to be treated with a population of cells generated by the methods described herein (i.e., an autologous donor) , or can be an individual who donates a blood sample (e.g., lymphocyte sample) that, upon generation of the population of cells generated by the methods described herein, will be used to treat a different individual or patient (i.e., an allogeneic donor) . Those subjects who receive the cells that were prepared by the present methods can be referred to as “recipient” or "recipient subject. "

The term “T cell receptor, ” or “TCR, ” refers to a heterodimeric receptor composed of αβ or γδ chains that pair on the surface of a T cell. Each α, β, γ, and δ chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition through the complementarity determining regions (CDR) , followed by a constant domain (C) that is anchored to cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region associates with the invariant subunits of the CD3 signaling apparatus. Each of the V domains has three CDRs. These CDRs interact with a complex between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy (2012) , xix, 868 p. ) .

The term "moiety" refers to a specific segment or a functional portion of a molecule (e.g., a protein, a fusion polypeptide) . In some embodiments, the moiety is a full length protein or a full length polypeptide (Nef protein) . In some embodiments, the moiety is a protein fragment (e.g., Nef protein fragment or a E3 ubiquitin ligase fragment) . In some embodiments, the moiety comprises or consists of one or more functional domains.

“Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN^TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The term “antibody” includes monoclonal antibodies (including full length 4-chain antibodies or full length heavy-chain only antibodies which have an immunoglobulin Fc region) , antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules) , as well as antibody fragments (e.g., Fab, F (ab′) 2, and Fv) . The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein. Antibodies contemplated herein include single-domain antibodies, such as heavy chain only antibodies.

An “antibody fragment” or “antigen-binding fragment” comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody. Examples of antibody fragments (or antigen-binding fragment) include Fab, Fab′, F (ab′) 2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8 (10) : 1057-1062 [1995] ) ; single-chain antibody molecules; single-domain antibodies (such as VHH) , and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH) , and the first constant domain of one heavy chain (CH1) . Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F (ab′) 2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab′ fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue (s) of the constant domains bear a free thiol group. F (ab′) 2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The term “non-antibody protein” as used herein refers to a protein that is not an antibody or antibody fragment.

The term “down-modulation” when used in the context of a target protein (e.g., endogenous CD3ζ or TCR) in cells refers to down-regulate or reduce expression of the target protein, for example, via degradation through, e.g., ubiquitination.

The terms “polypeptide” and “peptide” and “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. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a “polypeptide” can occur as a single chain or as two or more associated chains.

In connection with the binding molecules described herein terms such as “bind to, ” “that specifically bind to, ” and analogous terms are also used interchangeably herein and refer to binding molecules of antigen binding domains that specifically bind to an antigen, such as a polypeptide. A binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, or other techniques known to those of skill in the art. In certain embodiments, a binding molecule or antigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) . Typically, a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity. In certain embodiments, the extent of binding of a binding molecule or antigen binding domain to a “non-target” protein is less than about 10%of the binding of the binding molecule or antigen binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA. A binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the binding molecule is useful, for example, as a therapeutic and/or diagnostic agent in targeting the antigen. In certain embodiments, a binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (KD) of less than or equal to 1μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain embodiments, a binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species.

It is understood that embodiments of the present application described herein include “consisting” and/or “consisting essentially of” embodiments.

Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X" .

As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

The term “about X-Y” used herein has the same meaning as “about X to about Y. ”

As used herein and in the appended claims, the singular forms "a, " "or, " and "the" include plural referents unless the context clearly dictates otherwise.

Fusion polypeptides

The present disclosure provides fusion polypeptides for degradation of target proteins through ubiquitin-proteasome pathway. In some embodiments, the fusion polypeptides comprise a first moiety comprising a non-antibody protein that binds to a target protein and a second moiety comprising an E3 ubiquitin ligase or fragment thereof. In some embodiments, the non-antibody protein is a Nef protein.

Target Proteins

In some embodiments, the target protein is expressed on a cell. In some embodiments, the target protein is expressed on the surface of a cell. In some embodiments, the target protein is expressed on an immune cell. In some embodiments, the target protein is expressed on a T cell or a NK cell. In some embodiments, the target protein is associated with graft-versus-host disease (GvHD) and/or host-versus-graft disease (HvGD) in a cell therapy. In some embodiments, the target protein is associated with a Nef protein. In some embodiments, the target protein binds to a Nef protein. Non-limiting exemplary target proteins bound by the first moiety of the presently disclosed fusion polypeptides include CD3ζ, MHC I, B2M, CD4, and CD28.

In some embodiments, the target protein is CD3ζ. CD3ζ is a homodimer-forming type 1 transmembrane (TM) protein and is part of the T-cell antigen receptor (TCR-CD3) complex along with TCRαβ, CD3γε, and CD3δε dimers expressed on the surface of T cells. CD3ζpossesses a small extracellular part, a TM region, and a long cytoplasmic part that contains three immunoreceptor tyrosine-based activation motifs (ITAMs) , which correspond to the six tyrosines that get phosphorylated upon antigen binding to the extracellular part of TCRαβ. Phosphorylation subsequently activates several downstream signaling cascades. Without being bound by theory, the degradation of CD3ζ triggers irreversible down-regulation of the TCR complex. More details for CD3ζ can be found e.g., in Deswal, S., Schamel, W. W. A. (2012) . CD3ζ. In: Choi, S. (eds) Encyclopedia of Signaling Molecules. Springer, New York, NY, which is incorporated herein by reference in its entirety.

In one aspect, the present disclosure provides a fusion polypeptide that can specifically degrade CD3ζ, and/or down-regulate endogenous CD3ζ and TCR complex expression in an immune cell. In some embodiments, the CD3ζ is expressed in a T cell (e.g., allogeneic T cell) . In some embodiments, the fusion polypeptide includes a first moiety that binds to CD3ζ (e.g., any of the Nef proteins described herein) , and a second moiety that includes an E3 ubiquitin ligase or fragment thereof.

In some embodiments, the fusion polypeptide described herein can alter endogenous CD3ζ expression or CD3ζ-mediated signal transduction, downregulate endogenous CD3ζexpression, and/or down-modulate CD3ζ-mediated signal transduction to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%as compared to that in a T cell from the same donor source.

In some embodiments, the fusion polypeptide described herein can alter endogenous TCR expression or TCR-mediated signal transduction, downregulate endogenous TCR expression, and/or down-modulate TCR-mediated signal transduction to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%as compared to that in a T cell from the same donor source.

In some embodiments, the expression of a fusion polypeptide (any of the fusion polypeptides described herein) in a T cell (e.g., allogeneic T cell) does not down-modulate (e.g., down-regulate cell surface expression) an exogenous receptor (e.g., CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, ACTR) , engineered TCR (e.g., traditional engineered TCR, chimeric TCR, TAC-like chimeric receptor) , or chimeric receptor comprising a ligand binding domain) in the same T cell. In some embodiments, the exogenous receptor in an engineered T cell expressing the fusion polypeptide described herein is down-modulated (e.g., cell surface expression is down-regulated) by at most about any of 50%, 40%, 30%, 20%, 10%, or 5%, compared to when the exogenous receptor is expressed in a T cell from the same donor source without fusion polypeptide expression. In some embodiments, the cell surface expression and/or the signal transduction of the exogenous receptor is unaffected, or down-regulated by at most about any of 50%, 40%, 30%, 20%, 10%, or 5%, when the engineered T cell expresses the fusion polypeptide described herein.

In some embodiments, the expression of a fusion polypeptide (any of the fusion polypeptides described herein) in a T cell (e.g., allogeneic T cell) down-modulates endogenous MHC I, CD4, B2M and/or CD28, such as downregulating cell surface expression of endogenous MHC I, CD4, B2M and/or CD28 (e.g., via endocytosis and degradation) . In some embodiments, the cell surface expression of endogenous MHC I, CD4, B2M and/or CD28 in a T cell expressing a fusion polypeptide described herein is down-regulated by at least about any of 50%, 60%, 70%, 80%, 90%, or 95%compared to that of a T cell from the same donor source.

Nef protein

The fusion polypeptide described herein comprises a non-antibody protein. In some embodiments, the non-antibody protein is a viral protein. In some embodiments, the viral protein binds to a target protein that is associated with graft-versus-host disease (GvHD) and/or host-versus-graft disease (HvGD) in a cell therapy. In some embodiments, the viral protein is a Nef protein. In some embodiments, the Nef protein is associated with CD3ζ. In some embodiments, the fusion polypeptide described herein comprise any of the Nef proteins, e.g., wildtype Nef, mutant Nef such as non-naturally occurring mutant Nef.

Wildtype Nef (negative regulatory factor) is a small 27-35 kDa myristoylated protein encoded by primate lentiviruses, including Human Immunodeficiency Viruses (HIV-1 and HIV- 2) and Simian Immunodeficiency Virus (SIV) . Nef localizes primarily to the cytoplasm but is also partially recruited to the Plasma Membrane (PM) . It functions as a virulence factor, which can manipulate the host’s cellular machinery and thus allow infection, survival or replication of the pathogen.

Nef is highly conserved in all primate lentiviruses. The HIV-2 and SIV Nef proteins are 10-60 amino acids longer than HIV-1 Nef. From N-terminus to C-terminus, a Nef protein comprises the following domains: myristoylation site (involved in CD4 downregulation, MHC I downregulation, and association with signaling molecules, required for inner plasma membrane targeting of Nef and virion incorporation, and thereby for infectivity) , N-terminal α-helix (involved in MHC I downregulation and protein kinase recruitment) , tyrosine-based AP recruitment (HIV-2 /SIV Nef) , CD4 binding site (WL residue, involved in CD4 downregulation, characterized for HIV-1 Nef) , acidic cluster (involved in MHC I downregulation, interaction with host PACS1 and PACS2) , proline-based repeat (involved in MHC I downregulation and SH3 binding) , PAK (p21 activated kinase) binding domain (involved in association with signaling molecules and CD4 downregulation) , COP I recruitment domain (involved in CD4 downregulation) , di-leucine based AP recruitment domain (involved in CD4 downregulation, HIV-1 Nef) , and V-ATPase and Raf-1 binding domain (involved in CD4 downregulation and association with signaling molecules) .

CD4 is a 55 kDa type I integral cell surface glycoprotein. It is a component of the T cell receptor on MHC class II-restricted cells such as helper/inducer T-lymphocytes and cells of the macrophage/monocyte lineage. It serves as the primary cellular receptor for HIV and SIV.

In some embodiments, the Nef protein is obtained or derived from primary HIV-1 subtype C Indian isolates. In some embodiments, the Nef protein is expressed from F2 allele of the Indian isolate encoding the full-length protein (HIV F2-Nef) . In some embodiments, the Nef protein is expressed from C2 allele the Indian isolate with in-frame deletions of CD4 binding site, acidic cluster, proline-based repeat, and PAK binding domain (HIV C2-Nef) . In some embodiments, the Nef protein is expressed from D2 allele the Indian isolate with in-frame deletions of CD4 binding site (HIV D2-Nef) .

In some embodiments, the Nef protein is a mutant Nef, such as Nef proteins comprising one or more of insertion, deletion, point mutation (s) , and/or rearrangement. In some embodiments, the present application provides non-naturally occurring mutant Nef proteins. The Nef protein may comprise one or more mutations (e.g., non-naturally occurring mutation) in one or more domains or motifs selected from the group consisting of myristoylation site, N-terminal α-helix, tyrosine-based AP recruitment, CD4 binding site, acidic cluster, proline-based repeat, PAK binding domain, COP I recruitment domain, di-leucine based AP recruitment domain, V-ATPase and Raf-1 binding domain, and any combinations thereof.

For example, in some embodiments, the mutant (e.g., non-naturally occurring mutant) Nef comprises one or more mutations in di-leucine based AP recruitment domain. In some embodiments, the mutant (e.g., non-naturally occurring mutant) Nef comprises mutations in di-leucine based AP recruitment domain and PAK binding domain. In some embodiments, the mutant (e.g., non-naturally occurring mutant) Nef comprises mutations in di-leucine based AP recruitment domain, PAK binding domain, COP I recruitment domain, and V-ATPase and Raf-1 binding domain. In some embodiments, the mutant (e.g., non-naturally occurring mutant) Nef comprises one or more mutations in di-leucine based AP recruitment domain, COP I recruitment domain, and V-ATPase and Raf-1 binding domain. In some embodiments, the mutant (e.g., non-naturally occurring mutant) Nef comprises one or more mutations in di-leucine based AP recruitment domain and V-ATPase and Raf-1 binding domain. In some embodiments, the mutant (e.g., non-naturally occurring mutant) Nef comprises a truncation deleting partial or the entire domain. In some embodiments, the Nef protein comprises one or more mutations (e.g., non-naturally occurring mutation) not in any of the aforementioned domains/motifs.

In some embodiments, the mutant (e.g., non-naturally occurring mutant) Nef comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residue mutations (e.g., to Ala) at any of amino acid residues of the Nef proteins described herein.

In some embodiments, the Nef protein is derived from the group consisting of SIV Nef, HIV1 Nef, and HIV2 Nef. In some embodiments, the Nef protein is a wildtype Nef. In some embodiments, the Nef protein is a mutated Nef. In some embodiments, the Nef protein includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 30-37. In some embodiments, the Nef protein is derived from any one of SIV Nef M116, HIV1 Nef M094, HIV2 Nef M787, HIV2 Nef M092, SIV Nef M1006, SIV Nef M1034, HIV1 Nef M1069, and SIV Nef M998. In some embodiments, the Nef protein includes all or a portion of SIV Nef M116 (SEQ ID NO: 30) , HIV1 Nef M094 (SEQ ID NO: 31) , HIV2 Nef M787 (SEQ ID NO: 32) , HIV2 Nef M092 (SEQ ID NO: 33) , SIV Nef M1006 (SEQ ID NO: 34) , SIV Nef M1034 (SEQ ID NO: 35) , HIV1 Nef M1069 (SEQ ID NO: 36) , and SIV Nef M998 (SEQ ID NO: 37) .

Also provided are nucleic acids (e.g., isolated nucleic acid) encoding any of the Nef protein described herein (e.g., wt Nef or mutant Nef) . Further provided are vectors (e.g., viral vectors such as lentiviral vectors, bacteria expression vectors) comprising a nucleic acid encoding any of the Nef proteins described herein. These vectors can be replaced with any of the vectors described herein. More details of Nef protein and its sequences can be found, e.g., in WO2020020359A1, which is incorporated herein by reference in its entirety.

E3 Ubiquitin Ligases

In some embodiments, the presently disclosed fusion polypeptides comprise an E3 ubiquitin ligase or fragment thereof. In some embodiments, the presently disclosed fusion polypeptides comprise a truncated E3 ubiquitin ligase or fragment thereof.

In some embodiments, the E3 ubiquitin ligase or fragment thereof is selected from Speckle Type BTB/POZ Protein (SPOP) , Beta-Transducin Repeat Containing E3 Ubiquitin Protein Ligase (βTrCP) , S-Phase Kinase Associated Protein 2 (SKP2) , Von Hippel-Lindau Tumor Suppressor (VHL) , Suppressor Of Cytokine Signaling 2 (SOCS2) , Ankyrin Repeat And SOCS Box Containing 1 (ASB1) , Carboxy Terminus Of Hsp70-interacting Protein CHIP (CHIP) , and Tripartite Motif Containing 21 (TRIM21) .

In some embodiments, the E3 ubiquitin ligase or fragment thereof is SPOP or a fragment thereof. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 167-374 of SPOP. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 19.

In some embodiments, the E3 ubiquitin ligase or fragment thereof is βTrCP or a fragment thereof. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 2-264 of βTrCP. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 20.

In some embodiments, the E3 ubiquitin ligase or fragment thereof is SKP2 or a fragment thereof. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 2-147 of SKP2. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 21.

In some embodiments, the E3 ubiquitin ligase or fragment thereof is VHL or a fragment thereof. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 152-213 of VHL. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 22.

In some embodiments, the E3 ubiquitin ligase or fragment thereof is SOCS2 or a fragment thereof. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 143-198 of SOCS2. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 23.

In some embodiments, the E3 ubiquitin ligase or fragment thereof is ASB1 or a fragment thereof. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 266-335 of ASB1. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 24.

In some embodiments, the E3 ubiquitin ligase or fragment thereof is CHIP or a fragment thereof. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 128-303 of CHIP. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 25

In some embodiments, the E3 ubiquitin ligase or fragment thereof is TRIM21 or a fragment thereof. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 1-267 of TRIM21. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 1-127 of TRIM21. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 1-85 of TRIM21. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to amino acids 1-55 of TRIM21. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. In some embodiments, the E3 ubiquitin ligase or fragment thereof comprises the C RING domain of TRIM21.

In some embodiments, the E3 ubiquitin ligase or fragment thereof provided herein contains substitutions (e.g., conservative substitutions) , insertions, or deletions relative to the reference sequence, but the E3 ubiquitin ligase or fragment thereof comprising that sequence retains the ability to induce protein degradation through ubiquitin-proteasome pathway. In some embodiments, a total of 1 to 212 amino acids have been deleted in a reference amino acid sequence.

Linkers

In some embodiments, the fusion polypeptide further comprises a linker peptide between the first moiety and the second moiety. Exemplary and non-limiting linker peptides include a GS linker, an α-helical linker, a glycine-alanine polymer linker, an alanine-serine polymer linker, and an IgG4-Fc linker.

In some embodiments, the linker peptide includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 46.

Exemplary Fusion Polypeptides

In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the first moiety, a linker peptide, and the second moiety. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the second moiety, a linker peptide, and the first moiety.

In some embodiments, the first moiety includes any one of the Nef proteins described herein, and the second moiety includes any one of the E3 ubiquitin ligase or fragment thereof described herein. In some embodiments, the fusion polypeptide has the first moiety and the second moiety, optionally from N-terminus to C-terminus, as indicated in Table 1, Table 2, and Table 3. In some embodiments, the fusion polypeptide includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 1-18.

In some embodiments, the first moiety is SIV Nef M116, and the second moiety is SPOP or a fragment thereof (e.g., amino acids 167-374 of SPOP) . In some embodiments, first moiety is SIV Nef M116, and the second moiety is βTrCP or a fragment thereof (e.g., amino acids 2-264 of βTrCP) . In some embodiments, first moiety is SIV Nef M116, and the second moiety is SKP2 or a fragment thereof (e.g., amino acids 2-147 of SKP2) . In some embodiments, first moiety is SIV Nef M116, and the second moiety is VHL or a fragment thereof (e.g., amino acids 152-213 of VHL) . In some embodiments, first moiety is SIV Nef M116, and the second moiety is SOCS2 or a fragment thereof (e.g., amino acids 143-198 of SOCS2) . In some embodiments, first moiety is SIV Nef M116, and the second moiety is ASB1 or a fragment thereof (e.g., amino acids 266-335 of ASB1) . In some embodiments, first moiety is SIV Nef M116, and the second moiety is CHIP or a fragment thereof (e.g., amino acids 128-303 of CHIP) . In some embodiments, first moiety is SIV Nef M116, and the second moiety is TRIM21 or a fragment thereof (e.g., amino acids 1-267, amino acids 1-127, amino acids 1-85 of TRIM21, or amino acids 1-55 of TRIM21) .

In some embodiments, first moiety is HIV1 Nef M094, and the second moiety is TRIM21 or a fragment thereof (e.g., amino acids 1-85 of TRIM21) . In some embodiments, first moiety is HIV2 Nef M787, and the second moiety is TRIM21 or a fragment thereof (e.g., amino acids 1-85 of TRIM21) . In some embodiments, first moiety is HIV2 Nef M092, and the second moiety is TRIM21 or a fragment thereof (e.g., amino acids 1-85 of TRIM21) . In some embodiments, first moiety is SIV Nef M1006, and the second moiety is TRIM21 or a fragment thereof (e.g., amino acids 1-85 of TRIM21) . In some embodiments, first moiety is SIV Nef M1034, and the second moiety is TRIM21 or a fragment thereof (e.g., amino acids 1-85 of TRIM21) . In some embodiments, first moiety is HIV1 Nef M1069, and the second moiety is TRIM21 or a fragment thereof (e.g., amino acids 1-85 of TRIM21) . In some embodiments, first moiety is SIV Nef M998, and the second moiety is TRIM21 or a fragment thereof (e.g., amino acids 1-85 of TRIM21) .

Engineered cells

The present disclosure provides engineered cells expressing the fusion polypeptide (e.g., any of the fusion polypeptides described herein) and methods of producing such engineered cells. In some embodiments, the engineered cells further express a functional exogenous receptor (such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) . The present disclosure thus provides engineered T cells co-expressing any one of the fusion polypeptides described herein and optionally any one of the functional exogenous receptors described herein.

In some embodiments, the engineered cells further comprise a protein marker for screening purposes (e.g., DHFR L22F/F31S) . In some embodiments, DHFR L22F/F31S has an amino acid sequence that is at least 70%, 80%, 90%, or 95%identical to SEQ ID NO: 43. In some embodiments, the engineered cells further comprises a sequence encoding a functional IL12 polypeptide (e.g., a membrane-bound IL12p40 polypeptide) . In some embodiments, the membrane-bound IL12p40 polypeptide has an amino acid sequence that is at least 70%, 80%, 90%, or 95%identical to SEQ ID NO: 44. In some embodiments, the membrane-bound IL12p40 polypeptide includes the hinge region, transmembrane region, and/or intracellular cytoplasmic region derived from CD8α.

In some embodiments, provided herein is an engineered T cell (e.g., allogeneic T cell) comprising a first nucleic acid encoding a fusion polypeptide (e.g., any one of the fusion polypeptide described herein) , wherein the fusion polypeptide upon expression results in down-modulation of the endogenous TCR (e.g., TCRα and/or TCRβ) in the modified T cell. In some embodiments, the down-modulation comprises down-regulating cell surface expression of endogenous TCR. In some embodiments, the cell surface expression of endogenous TCR is down-regulated by at least about any of 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the cell surface expression of endogenous MHC, CD3ε, CD3γ, CD3δ, and/or CD3ζ is down-regulated by the fusion polypeptide by at least about any of 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the fusion polypeptide (e.g., any one of the fusion polypeptide described herein) does not down-regulate cell surface expression of CD4 and/or CD28. In some embodiments, the fusion polypeptide (e.g., any one of the fusion polypeptide described herein) down-regulates cell surface expression of CD4 and/or CD28. In some embodiments, the fusion polypeptide (e.g., any one of the fusion polypeptide described herein) down-regulates cell surface expression of endogenous TCR, but does not down-modulate (e.g., down-regulate cell surface expression) exogenous receptor (such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) . In some embodiments, the functional exogenous receptor (such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) is down-modulated (e.g., down-regulated for cell surface expression) by the fusion polypeptide (e.g., any one of the fusion polypeptide described herein) by at most about any of 50%, 40%, 30%, 20%, 10%, or 5%.

In some embodiments, the engineered T cell described herein comprises unmodified endogenous TCR loci. In some embodiments, the modified T cell expressing fusion polypeptide comprises a modified endogenous TCR locus, such as TCRα or TCRβ. In some embodiments, the endogenous TCR locus is modified by a gene editing system selected from CRISPR-Cas, TALEN, shRNA, and ZFN.

In various embodiments, the cell that is engineered can be obtained from e.g., humans and non-human animals. In various embodiments, the cell that is engineered can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pig or any other species. In some embodiments, the cell is from humans, rats or mice. In some embodiments, the cells are mouse lymphocytes and engineered (e.g., transduced) to express the CAR, or antigen-binding fragment thereof. In some embodiments, the cell is obtained from humans. In various embodiments, the cell that is engineered is a blood cell. In some embodiments, the cell is a leukocyte (e.g., a T cell) , lymphocyte or any other suitable blood cell type. In some embodiments, the cell is a peripheral blood cell. In some embodiments, the cell is a tumor-infiltrating lymphocyte (TIL) . In some embodiments, the cell is a T cell, B cell or NK cell. In some embodiments, the cells are human peripheral blood mononuclear cells (PBMCs) . In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, the human PBMCs are CD8+ cells or CD4+ cells.

In some embodiments, the cell is a T cell. In some embodiments, the T cells can express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell. The cell surface receptor can be a wild type or recombinant T cell receptor (TCR) , a chimeric antigen receptor (CAR) , or any other surface receptor capable of recognizing an antigenic moiety that is associated with the target cell. T cells can be obtained by various methods known in the art, e.g., in vitro culture of T cells (e.g., tumor infiltrating lymphocytes) isolated from patients. Genetically modified T cells can be obtained by transducing T cells (e.g., isolated from the peripheral blood of patients) , with a viral vector. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells. In some embodiments, the T cells are T helper type 1 T cells and T helper type 2 T cells. In some embodiments, the T cell expressing this receptor is an αβ-T cell. In alternate embodiments, the T cell expressing this receptor is a γδ-T cell. In some embodiments, the T cells are central memory T cells. In some embodiments, the T cells are effector memory T cells. In some embodiments, the T cells areT cells.

In some embodiments, the cell is an NK cell. In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the binding molecule, e.g., CAR, can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.

In some embodiments, the cells are stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs) . The cells can be primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the stem cells are cultured with additional differentiation factors to obtain desired cell types (e.g., T cells) .

Different cell types can be obtained from appropriate isolation methods. The isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is affinity-or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

Also provided are methods, nucleic acids, compositions, and kits, for expressing the binding molecules, and for producing the genetically engineered cells expressing such binding molecules. The genetic engineering generally involves introduction of a nucleic acid encoding the therapeutic molecule, e.g. CAR, polypeptides, fusion proteins, into the cell, such as by retroviral transduction, transfection, or transformation. In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical application.

In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40) , adenoviruses, adeno-associated virus (AAV) . In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors. In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR) , e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV) , myeloproliferative sarcoma virus (MPSV) , murine embryonic stem cell virus (MESV) , murine stem cell virus (MSCV) , or spleen focus forming virus (SFFV) . Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In some embodiments, the vector is a lentivirus vector. In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation. In some embodiments, recombinant nucleic acids are transferred into T cells via transposition. Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment and strontium phosphate DNA co-precipitation. Many of these methods are descried e.g., in WO2019195486, which is incorporated herein by reference in its entirety.

In some embodiments, the modified T cell expressing the fusion polypeptide elicits no or a reduced GvHD response in a histoincompatible individual as compared to the GvHD response elicited by a primary T cell isolated from the donor of the precursor T cell from which the modified T cell is derived.

Functional exogenous receptor

The term “functional exogenous receptor” as used herein, refers to an exogenous receptor (such as e.g. CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, ACTR) , engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , T cell antigen coupler (TAC) , or TAC-like chimeric receptor) that retains its biological activity after being introduced into the T cells or the engineered T cell described herein. The biological activity include but are not limited to the ability of the exogenous receptor in specifically binding to a molecule (e.g., cancer antigen, or an antibody for ACTR) , properly transducing downstream signals, such as inducing cellular proliferation, cytokine production and/or performance of regulatory or cytolytic effector functions.

In some embodiments, the functional exogenous receptor is monovalent and monospecific. In some embodiments, the functional exogenous receptor is multivalent and monospecific. In some embodiments, the functional exogenous receptor is multivalent and multispecific.

In some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR) . In some embodiments, the functional exogenous receptor is an ITAM-modified T cell receptor (TCR) , an ITAM-modified chimeric antigen receptor (CAR) , an ITAM-modified chimeric TCR (cTCR) , or an ITAM-modified T cell antigen coupler (TAC) -like chimeric receptor.

In some embodiments, the functional exogenous receptor comprises: (a) an extracellular ligand binding domain, (b) a transmembrane domain (e.g., derived from CD8α) , and (c) an intracellular signaling domain (ISD) comprising a chimeric signaling domain (CMSD) , wherein the CMSD comprises one or a plurality of ITAMs ( “CMSD ITAMs” ) , wherein the plurality of CMSD ITAMs are optionally connected by one or more linkers ( “CMSD linkers” ) . In some embodiments, the CMSD comprises one or more of the characteristics selected from the group consisting of: (a) the plurality (e.g., 2, 3, 4, or more) of CMSD ITAMs are directly linked to each other; (b) the CMSD comprises two or more (e.g., 2, 3, 4, or more) CMSD ITAMs connected by one or more linkers not derived from an ITAM-containing parent molecule (e.g., G/Slinker) ; (c) the CMSD comprises one or more CMSD linkers derived from an ITAM-containing parent molecule that is different from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs are derived from; (d) the CMSD comprises two or more (e.g., 2, 3, 4, or more) identical CMSD ITAMs; (e) at least one of the CMSD ITAMs is not derived from CD3ζ; (f) at least one of the CMSD ITAMs is not ITAM1 or ITAM2 of CD3ζ; (g) the plurality of CMSD ITAMs are each derived from a different ITAM-containing parent molecule; and/or (h) at least one of the CMSD ITAMs is derived from an ITAM-containing parent molecule selected from the group consisting of CD3ε, CD3δ, CD3γ, Igα (CD79a) , Igβ (CD79b) , FcεRIβ, FcεRIγ, DAP12, CNAIP/NFAM1, STAM-1, STAM-2, and Moesin. In some embodiments, the CMSD consists essentially of (e.g., consists of) one CMSD ITAM. In some embodiments, the CMSD consists essentially of (e.g., consists of) one CMSD ITAM and a CMSD N-terminal sequence and/or a CMSD C-terminal sequence that is heterologous to the ITAM-containing parent molecule (e.g., a G/Slinker) . In some embodiments, the plurality (e.g., 2, 3, 4, or more) of CMSD ITAMs are directly linked to each other. In some embodiments, the CMSD comprises two or more (e.g., 2, 3, 4, or more) CMSD ITAMs connected by one or more linkers not derived from an ITAM-containing parent molecule (e.g., G/Slinker) . In some embodiments, the CMSD comprises one or more CMSD linkers derived from an ITAM-containing parent molecule that is different from the ITAM-containing parent molecule from which one or more of the CMSD ITAMs are derived from. In some embodiments, the CMSD comprises two or more (e.g., 2, 3, 4, or more) identical CMSD ITAMs. In some embodiments, at least one of the CMSD ITAMs is not derived from CD3ζ. In some embodiments, at least one of the CMSD ITAMs is not ITAM1 or ITAM2 of CD3ζ. In some embodiments, the plurality of CMSD ITAMs are each derived from a different ITAM-containing parent molecule. In some embodiments, at least one of the CMSD ITAMs is derived from an ITAM-containing parent molecule selected from the group consisting of CD3ε, CD3δ, CD3γ, Igα (CD79a) , Igβ (CD79b) , FcεRIβ, FcεRIγ, DAP12, CNAIP/NFAM1, STAM-1, STAM-2, and Moesin. In some embodiments, the CMSD comprises ITAM010 (SEQ ID NO: 42) . Details of ITAM010 and other ITAMs can be found, e.g., in PCT Application Publication No. WO2021037221, which is incorporated herein by reference in its entirety. In some embodiments, the functional exogenous receptor has an intracellular signaling domain derived from CD3ζ. In some embodiments, the functional exogenous receptor does not have any sequence derived from CD3ζ (e.g., human CD3ζ) .

In some embodiments, the extracellular ligand binding domain of the functional exogenous receptor binds to an antigen, wherein the antigen is a tumor antigen selected from the group consisting of BCMA, CLL1, GPC3, GU2CYC, CD19, CD7, CD20, CD22, CD38, CD41, CD123, Claudin 18.2, Claudin 6, NKG2D, DLL-3, GPRC5D, and CD70. In some embodiments, the extracellular ligand binding domain comprises, optionally from N-terminus to C-terminus: a signal peptide (SP) from CD8α; a scFv that binds to a target antigen (e.g., BCMA, GPC3 or CD20) ; and a hinge region from CD8α. In some embodiments, the transmembrane region comprises the transmembrane region of CD8α. In some embodiments, the intracellular signaling domain (ISD) further comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory domain is N-terminal to the CMSD. In some embodiments, the co-stimulatory domain is C-terminal to the CMSD. In some embodiments, the co-stimulatory signaling domain is derived from 4-1BB or CD28.

In some embodiments, the scFv can bind to GPC3, and the corresponding CAR can be found, e.g., in PCT Application Publication No. WO2021170100, which is incorporated herein by reference in its entirety. In some embodiments, the GPC3 CAR comprises an amino acid sequence that is at least 70%, 80%, 90%, or 95%identical to SEQ ID NO: 38. In some embodiments, the scFv can bind to CD20, and the corresponding CAR can be found, e.g., in PCT Application Publication No. WO2021037221, which is incorporated herein by reference in its entirety. In some embodiments, the scFv can bind to BCMA, and the corresponding CAR can be found, e.g., in PCT Application Publication No. WO2021037221, which is incorporated herein by reference in its entirety. In some embodiments, the BCMA CAR comprises an amino acid sequence that is at least 70%, 80%, 90%, or 95%identical to SEQ ID NO: 39.

In some embodiments, the functional exogenous receptor is an ITAM-modified CAR. In some embodiments, the GPC3 CAR modified by ITAM010 comprises an amino acid sequence that is at least 70%, 80%, 90%, or 95%identical to SEQ ID NO: 41. In some embodiments, the CD20 CAR modified by ITAM010 comprises an amino acid sequence that is at least 70%, 80%, 90%, or 95%identical to SEQ ID NO: 40.

Recombinant vectors

The present application provides vectors for cloning and expressing any one of fusion polypeptides or functional exogenous receptors (such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) described herein. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vector, retroviral vectors, herpes simplex viral vector, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals.

A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the engineered mammalian cell in vitro or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying the fusion polypeptide-coding sequence and/or self-inactivating lentiviral vectors carrying exogenous receptor (e.g. such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) can be packaged with protocols known in the art. The resulting lentiviral vectors can be used to transduce a mammalian cell (such as primary human T cells) using methods known in the art. Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, because they allow long-term, stable integration of a transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity, and can transduce non-proliferating cells.

In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a transposon, such as a Sleeping Beauty (SB) transposon system, or a PiggyBac transposon system. In some embodiments, the vector is a polymer-based non-viral vector, including for example, poly (lactic-co-glycolic acid) (PLGA) and poly lactic acid (PLA) , poly (ethylene imine) (PEI) , and dendrimers. In some embodiments, the vector is a cationic-lipid based non-viral vector, such as cationic liposome, lipid nanoemulsion, and solid lipid nanoparticle (SLN) . In some embodiments, the vector is a peptide-based gene non-viral vector, such as poly-L-lysine. Any of the known non-viral vectors suitable for genome editing can be used for introducing the fusion polypeptide-encoding nucleic acid and/or exogenous receptor (such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) -encoding nucleic acid to the engineered immune effector cells (e.g., T cell) . See, for example, Yin H. et al. Nature Rev. Genetics (2014) 15: 521-555; Aronovich EL et al. “The Sleeping Beauty transposon system: a non-viral vector for gene therapy. ” Hum. Mol. Genet. (2011) R1: R14-20; and Zhao S. et al. “PiggyBac transposon vectors: the tools of the human gene editing. ” Transl. Lung Cancer Res. (2016) 5 (1) : 120-125, which are incorporated herein by reference. In some embodiments, any one or more of the nucleic acids encoding the fusion polypeptide and/or exogenous receptor (e.g. such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) described herein is introduced to the engineered immune effector cells (e.g., T cell) by a physical method, including, but not limited to electroporation, sonoporation, photoporation, magnetofection, hydroporation.

In some embodiments, the vector (e.g., viral vector such as lentiviral vector) comprises any one of the nucleic acids encoding the fusion polypeptide and/or the exogenous receptor (e.g. such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) described herein. The nucleic acid can be cloned into the vector using any known molecular cloning methods in the art, including, for example, using restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter (e.g., human EF1α promoter) . Varieties of promoters have been explored for gene expression in mammalian cells, and any of the promoters known in the art may be used in the present disclosure. Promoters may be roughly categorized as constitutive promoters or regulated promoters, such as inducible promoters. In some embodiments, the first nucleic acid and the second nucleic acid are on separate vectors. In some embodiments, the first nucleic acid and the second nucleic acid are on the same vector. In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to the same promoter. In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to different promoters. some embodiments, the first nucleic acid is upstream of the second nucleic acid. In some embodiments, the first nucleic acid is downstream of the second nucleic acid. In some embodiments, the first nucleic acid and the second nucleic acid are connected via a linking sequence. In some embodiments, the linking sequence comprises any of nucleic acid sequence encoding P2A, T2A, E2A, F2A, BmCPV 2A, BmIFV 2A, (GS) _n, (GSGGS) _n, (GGGS) _n, (GGGGS) _n, or nucleic acid sequence of IRES, SV40, CMV, UBC, EF1α, PGK, CAGG, or any combinations thereof, wherein n is an integer of at least one.

In some embodiments, the vector is a viral vector. In some embodiments, the viral vector selected from the group consisting of adenoviral vector, adeno-associated virus vector, retroviral vector, lentiviral vector, herpes simplex viral vector, and derivatives thereof. In some embodiments, the vector is a non-viral vector, such as episomal expression vector, Enhanced Episomal Vector (EEV) , PiggyBac Transposase Vector, or Sleeping Beauty (SB) transposon system.

In some embodiments, the vector further includes a sequence encoding a protein marker for screening purposes (e.g., DHFR L22F/F31S) . In some embodiments, DHFR L22F/F31S has an amino acid sequence that is at least 70%, 80%, 90%, or 95%identical to SEQ ID NO: 43. In some embodiments, the vector further comprises a sequence encoding a functional IL12 polypeptide (e.g., a membrane-bound IL12p40 polypeptide) . In some embodiments, the membrane-bound IL12p40 polypeptide has an amino acid sequence that is at least 70%, 80%, 90%, or 95%identical to SEQ ID NO: 44. In some embodiments, the membrane-bound IL12p40 polypeptide includes the hinge region, transmembrane region, and/or intracellular cytoplasmic region derived from CD8α. In some embodiments, the membrane-bound IL12p40 polypeptide further includes a signal peptide, optionally wherein the signal peptide has an amino acid sequence of SEQ ID NO: 49. In some embodiments, the sequences encoding the fusion polypeptide described herein, the functional exogenous receptor described herein, the protein marker described herein, and/or the functional IL12 polypeptide described herein, are linked by (i) a nucleic acid sequence encoding any of P2A, T2A, E2A, F2A, BmCPV 2A, BmIFV 2A, (GS) _n, (GGGS) _n, and (GGGGS) _n, wherein n is an integer of at least one; (ii) a nucleic acid sequence of any of IRES, SV40, CMV, UBC, EF1α, PGK, and CAGG; or (iii) any combinations thereof.

In some embodiments, the nucleic acid encoding a fusion polypeptide (e.g., any of the fusion polypeptides described herein) and/or the exogenous receptor (e.g. such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) described herein is operably linked to a constitutive promoter. Constitutive promoters allow heterologous genes (also referred to as transgenes) to be expressed constitutively in the host cells. Exemplary promoters contemplated herein include, but are not limited to, cytomegalovirus immediate-early promoter (CMV) , human elongation factors-1alpha (hEF1α) , ubiquitin C promoter (UbiC) , phosphoglycerokinase promoter (PGK) , simian virus 40 early promoter (SV40) , chicken β-Actin promoter coupled with CMV early enhancer (CAGG) , a Rous Sarcoma Virus (RSV) promoter, a polyoma enhancer/herpes simplex thymidine kinase (MC1) promoter, a beta actin (β-ACT) promoter, a “myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer-binding site substituted (MND) ” promoter. The efficiencies of such constitutive promoters on driving transgene expression have been widely compared in a huge number of studies. For example, Michael C. Milone et al. compared the efficiencies of CMV, hEF1α, UbiC and PGK to drive CAR expression in primary human T cells, and concluded that hEF1αpromoter not only induced the highest level of transgene expression, but was also optimally maintained in the CD4 and CD8 human T cells (Molecular Therapy, 17 (8) : 1453-1464 (2009) ) . In some embodiments, the nucleic acid encoding the fusion polypeptide and/or the exogenous receptor (e.g. such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) described herein is operably linked to a hEF1α promoter or a PGK promoter.

In some embodiments, the promoter is selected from the group consisting of an EF-1 promoter, a CMV IE gene promoter, an EF-la promoter, an ubiquitin C promoter, a phosphoglycerate kinase (PGK) promoter, a Rous Sarcoma Virus (RSV) promoter, an Simian Virus 40 (SV40) promoter a cytomegalovirus immediate early gene promoter (CMV) , an elongation factor 1 alpha promoter (EF1-α) , a phosphoglycerate kinase-1 promoter (PGK) , a ubiquitin-C promoter (UBQ-C) , a cytomegalovirus enhancer/chicken beta-actin promoter (CAG) , polyoma enhancer/herpes simplex thymidine kinase promoter (MC1) , a beta actin promoter (β-ACT) , a simian virus 40 promoter (SV40) , and a myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer-binding site substituted (MND) promoter, an NFAT promoter, apromoter, and an NFκB promoter.

In some embodiments, the nucleic acid encoding a fusion polypeptide (e.g., any of the fusion polypeptides described herein) and/or the exogenous receptor (e.g. such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) described herein is operably linked to an inducible promoter. Inducible promoters belong to the category of regulated promoters. The inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the engineered immune effector cell (e.g., T cell) , or the physiological state of the engineered immune effector cell, an inducer (i.e., an inducing agent) , or a combination thereof. In some embodiments, the inducing condition does not induce the expression of endogenous genes in the engineered mammalian cell, and/or in the subject that receives the pharmaceutical composition. In some embodiments, the inducing condition is selected from the group consisting of: inducer, irradiation (such as ionizing radiation, light) , temperature (such as heat) , redox state, tumor environment, and the activation state of the engineered mammalian cell. In some embodiments, the inducible promoter can be an NFAT promoter, apromoter, or an NFκB promoter.

In some embodiments, the vector also contains a selectable marker gene or a reporter gene to select cells expressing a fusion polypeptide (e.g., any of the fusion polypeptides described herein) and/or the exogenous receptor (e.g. such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) described herein from the population of host cells transfected through vectors (e.g., lentiviral vectors) . Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in the host cells. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid sequences.

The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant polypeptides or fragments thereof by recombinant techniques.

A vector is a construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) . Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

The present disclosure provides a recombinant vector comprising a nucleic acid construct suitable for genetically modifying a cell, which can be used for treatment of pathological disease or condition.

Any vector or vector type can be used to deliver genetic material to the cell. These vectors include but are not limited to plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs) , yeast artificial chromosomes (YACs) , and human artificial chromosomes (HACs) . Viral vectors can include but are not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy virus vectors, recombinant adeno-associated viral (AAV) vectors, hybrid vectors, and plasmid transposons (e.g., sleeping beauty transposon system, and PiggyBac transposon system) or integrase based vector systems. Other vectors that are known in the art can also be used in connection with the methods described herein.

In some embodiments, the vector is a viral vector. The viral vector can be grown in a culture medium specific for viral vector manufacturing. Any suitable growth media and/or supplements for growing viral vectors can be used in accordance with the embodiments described herein. In some embodiments, the viral vector contains an EF1α promoter to facilitate expression.

Various cell lines can be used in connection with the vectors as described herein. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; HEK293 cells, including HEK293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; cells; and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the antibodies or CAR molecule. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in HEK293 cells. In one aspect, the disclosure relates to a cell comprising the vector or the pair of vectors as described herein.

The present disclosure also provides a nucleic acid sequence comprising a nucleotide sequence encoding any of the antibodies, CAR, antigen binding fragments thereof, and/or CAR-derived binding molecules (including e.g., functional portions and functional variants thereof, polypeptides, or proteins described herein) . “Nucleic acid” as used herein can include “polynucleotide, ” “oligonucleotide, ” and “nucleic acid molecule, ” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained from natural sources, which can contain natural, non-natural or altered nucleotides. Furthermore, the nucleic acid comprises complementary DNA (cDNA) .

In some embodiments, the nucleotide sequence encoding the CARs are separated by a peptide sequence that causes ribosome skipping. In some embodiments, the peptide that causes ribosome skipping is a P2A or T2A peptide. In some embodiments, the nucleic acid is synthetic. In some embodiments, the nucleic acid is cDNA.

Pharmaceutical compositions

Further provided by the present application are pharmaceutical compositions comprising any one of the engineered T cells (e.g., allogeneic T cells, endogenous TCR-deficient T cell, GvHD-minimized T cell) expressing a fusion polypeptide (e.g., any of the fusion polypeptides described herein) and/or a functional exogenous receptor (such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing a chimeric antibody immune effector cell engager having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) , in the form of lyophilized formulations or aqueous solutions.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes) ; chelating agents such as EDTA and/or non-ionic surfactants.

Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typically present in a range from 0.2%-1.0% (w/v) . Suitable preservatives for use with the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide) , benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

Tonicity agents, sometimes known as “stabilizers” are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1%to 25%by weight, preferably 1 to 5%, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients include: polyhydric sugar alcohols (enumerated above) ; amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc. ; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol) , polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thio sulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose) ; trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

Non-ionic surfactants or detergents (also known as “wetting agents” ) are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants are present in a range of about 0.05 mg/mL to about 1.0 mg/mL, preferably about 0.07 mg/mL to about 0.2 mg/mL.

Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc. ) , polyoxamers (184, 188, etc. ) , polyols, polyoxyethylene sorbitan monoethers (etc. ) , lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means.

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl-methacrylate) , or poly (vinylalcohol) ) , polylactides (U.S. Pat. No. 3,773,919) , copolymers of L-glutamic acid and. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) , and poly-D- (-) -3-hydroxybutyric acid.

The pharmaceutical compositions described herein may also contain more than one active compound or agent as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

The present disclosure provides compositions (including pharmaceutical and therapeutic compositions) containing the engineered cells and populations thereof, produced by the methods disclosed herein. Also provided are methods, e.g., therapeutic methods for administrating the engineered cells and compositions thereof to subjects, e.g., patients or animal models (e.g., mice) .

Compositions including the engineered cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof are provided. The pharmaceutical compositions and formulations can include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

The cells and compositions can be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous, allogenic, or heterologous. For example, immunoresponsive T cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject after genetically modifying them in accordance with various embodiments described herein. Peripheral blood derived immunoresponsive T cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. Usually, when administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell) , it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion) .

Formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral, ” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose) , pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts can in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtration membranes.

The compositions or pharmaceutical compositions as described herein can be included in a container, pack, or dispenser together with instructions for administration.

Method for preparation of engineered cells

The present disclosure provides a method or process for preparing, manufacturing and/or using the engineered cells for treatment of pathological diseases or conditions.

The cells for introduction of the protein described herein, e.g., CAR, can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.

Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector) , washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs) , leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, or non-human primate. In some embodiments, the cells are isolated from mouse lymph nodes.

In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS) . In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated "flow-through" centrifuge. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) . In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca²⁺/Mg²⁺ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

In some embodiments, the method comprises one or more steps of: e.g., isolating the T cells from a patient’s blood; transducing the population T cells with a viral vector including the nucleic acid construct encoding a genetically engineered antigen receptor; expanding the transduced cells in vitro; and/or infusing the expanded cells into the patient, where the engineered T cells will seek and destroy antigen positive tumor cells. In some embodiments, the method further comprises: transfection of T cells with the viral vector containing the nucleic acid construct.

In some embodiments, the methods involve introducing any vectors described herein into a cell in vitro or ex vivo. In some embodiments, the vector is a viral vector and the introducing is carried out by transduction. In some embodiments, the cell is transduced for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or longer. In some embodiments, the methods further involve introducing into the cell one or more agent, wherein each of the one or more agent is independently capable of inducing a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene. In some embodiments, the one or more agent is an inhibitory nucleic acid (e.g., siRNA) . In some embodiments, the one or more agent is a fusion protein comprising a DNA-targeting protein and a nuclease or an RNA-guided nuclease (e.g., a clustered regularly interspaced short palindromic nucleic acid (CRISPR) -associated nuclease) .

The transfection of T cells can be achieved by using any standard method such as calcium phosphate, electroporation, liposomal mediated transfer, microinjection, biolistic particle delivery system, or any other known methods by skilled artisan. In some embodiments, transfection of T cells is performed using the calcium phosphate method.

The present disclosure provides a method to create a personalized anti-tumor immunotherapy. Genetically engineered T cells can be produced from a patient’s blood cells. These engineered T cells are then reinfused into the patient as a cellular therapy product.

Methods of treatment

The present application further provides methods of treating a disease (such as cancer, infectious disease, GvHD, transplantation rejection, autoimmune disorders, or radiation sickness) in an individual comprising administering to the individual an effective amount of any one of the pharmaceutical compositions or the engineered T cells (e.g., allogeneic T cell, endogenous TCR-deficient T cell, GvHD-minimized T cell) expressing a fusion polypeptide (e.g., any of the fusion polypeptides described herein) and/or a functional exogenous receptor (such as CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, ACTR) , or engineered TCR (e.g., traditional engineered TCR, chimeric TCR, TAC-like chimeric receptor) ) described herein.

The methods described herein are suitable for treating various cancers, including both solid cancer and liquid cancer. The methods are applicable to cancers of all stages, including early stage, advanced stage and metastatic cancer. The methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of cancer therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.

In some embodiments, the methods described herein are suitable for treating a solid cancer selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS) , primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

In some embodiments, the methods described herein are suitable for treating a hematologic cancer chosen from one or more of chronic lymphocytic leukemia (CLL) , acute leukemias, acute lymphoid leukemia (ALL) , B-cell acute lymphoid leukemia (B-ALL) , T-cell acute lymphoid leukemia (T-ALL) , chronic myelogenous leukemia (CML) , B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or pre-leukemia.

In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is stage I, stage II or stage III, and/or stage A or stage B multiple myeloma based on the Durie-Salmon staging system. In some embodiments, the cancer is stage I, stage II or stage III multiple myeloma based on the International staging system published by the International Myeloma Working Group (IMWG) . In some embodiments, the cancer is monoclonal gammopathy of undetermined significance (MGUS) . In some embodiments, the cancer is asymptomatic (smoldering/indolent) myeloma. In some embodiments, the cancer is symptomatic or active myeloma. In some embodiments, the cancer is refractory multiple myeloma. In some embodiments, the cancer is metastatic multiple myeloma. In some embodiments, the individual did not respond to a previous treatment for multiple myeloma. In some embodiments, the individual has progressive disease after a previous treatment of multiple myeloma. In some embodiments, the individual has previously received at least about any one of 2, 3, 4, or more treatment for multiple myeloma. In some embodiments, the cancer is relapsed multiple myeloma.

In some embodiments, the methods described herein are suitable for treating an autoimmune disease. Autoimmune disease, or autoimmunity, is the failure of an organism to recognize its own constituent parts (down to the sub-molecular levels) as “self, ” which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Prominent examples include Coeliac disease, diabetes mellitus type 1 (IDDM) , systemic lupus erythematosus (SLE) , syndrome, multiple sclerosis (MS) , Hashimoto's thyroiditis, Graves'disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis (RA) .

In some embodiments, the methods described herein are suitable for treating an inflammatory diseases, including autoimmune diseases are also a class of diseases associated with B-cell disorders. Examples of autoimmune diseases include, but are not limited to, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcalnephritis, erythema nodosurn, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans. Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosing alveolitis. The most common treatments are corticosteroids and cytotoxic drugs, which can be very toxic. These drugs also suppress the entire immune system, can result in serious infection, and have adverse effects on the bone marrow, liver, and kidneys. Other therapeutics that has been used to treat Class III autoimmune diseases to date have been directed against T cells and macrophages. There is a need for more effective methods of treating autoimmune diseases, particularly Class III autoimmune diseases.

Administration of the pharmaceutical compositions may be carried out in any convenient manner, including by injection, ingestion, transfusion, implantation or transplantation. The compositions may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously, or intraperitoneally. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered to an individual by infusion, such as intravenous infusion. Infusion techniques for immunotherapy are known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988) ) . In some embodiments, the pharmaceutical composition is administered to an individual by intradermal or subcutaneous injection. In some embodiments, the compositions are administered by intravenous injection. In some embodiments, the compositions are injected directly into a tumor, or a lymph node. In some embodiments, the pharmaceutical composition is administered locally to a site of tumor, such as directly into tumor cells, or to a tissue having tumor cells.

Dosages and desired drug concentration of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics, ” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46. It is within the scope of the present application that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue.

In one aspect, the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.

In one aspect, the disclosure features methods that include administering a therapeutically effective amount of antibodies or antigen binding fragments thereof, or engineered cells expressing CAR, to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer) .

In some embodiments, the subject has GPC3-positive cancer. In some embodiments, the subject has liver cancer (e.g., hepatocellular carcinoma) , glioma, lung cancer, colorectal cancer, head and neck cancer, stomach cancer, renal cancer, urothelial cancer, testis cancer, breast cancer, cervical cancer, endometrial cancer, and/or ovarian cancer. In some embodiments, the subject has squamous cell lung carcinoma, or solid tumor. In some embodiments, the subject has a CNS tumor, thyroid cancer, gastrointestinal cancer, skin cancer, sarcoma, urogenital cancer, and/or germ cell tumor. GPC3-related cancers can be found, e.g., in Moek, Kirsten L., et al., The American Journal of Pathology 188.9 (2018) : 1973-1981, which is incorporated herein by reference in its entirety.

In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the therapeutic agent and/or therapeutic compositions is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

An effective amount can be administered in one or more administrations. By way of example, an effective amount of a composition is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro. As is understood in the art, an effective may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of compositions used.

Effective amounts and schedules for administrations may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the treatment, the route of administration, the particular type of therapeutic agents and other drugs being administered to the mammal. Guidance in selecting appropriate doses can be found in the literature. In addition, a treatment does not necessarily result in the 100%or complete treatment or prevention of a disease or a condition. There are multiple treatment/prevention methods available with a varying degree of therapeutic effect which one of ordinary skill in the art recognizes as a potentially advantageous therapeutic mean.

In any of the methods described herein, the engineered cells, optionally with at least one additional therapeutic agent, can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) . In some embodiments, at least two different engineered cells (e.g., cells expressing different CARs) are administered in the same composition (e.g., a liquid composition) . In some embodiments, engineered cells and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) . In some embodiments, engineered cells and at least one additional therapeutic agent are administered in two different compositions. In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation. In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, concurrently with, or after administering the engineered cells to the subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the T cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the T cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type (s) and/or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

In some embodiments, the populations or sub-types of cells, such as CD8+ and CD4+T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some embodiments, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some embodiments, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some embodiments, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4+ to CD8+ ratio) , e.g., within a certain tolerated difference or error of such a ratio.

In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some embodiments, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some embodiments, the desired dose is at or above a minimum number of cells of the population or sub-type, or minimum number of cells of the population or sub-type per unit of body weight.

Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4+ to CD8+ cells, and/or is based on a desired fixed or minimum dose of CD4+ and/or CD8+ cells.

Optimal response to therapy can depend on the ability of the engineered recombinant receptors such as CARs, to be consistently and reliably expressed on the surface of the cells and/or bind the target antigen. For example, in some cases, properties of certain recombinant receptors, e.g., CARs, can affect the expression and/or activity of the recombinant receptor, in some cases when expressed in a cell, such as a human T cell, used in cell therapy. In some contexts, the level of expression of particular recombinant receptors, e.g., CARs, can be low, and activity of the engineered cells, such as human T cells, expressing such recombinant receptors, may be limited due to poor expression or poor signaling activity. In some cases, consistency and/or efficiency of expression of the recombinant receptor, and activity of the receptor is limited in certain cells or certain cell populations of available therapeutic approaches. In some cases, a large number of engineered T cells (a high effector to target (E: T) ratio) is required to exhibit functional activity. In some embodiments, the desired ratio (E: T ratio) is between at or about 1: 10 and at or about 10: 1 (or greater than about 1: 10 and less than about 10: 1) , or between at or about 1: 1 and at or about 10: 1 (or greater than about 1: 1 and less than about 5: 1) , such as between at or about 2: 1 and at or about 10: 1. In some embodiments, the E: T ratio is greater than or about 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1. In some embodiments, the E: T ratio is about 3: 1, about 1: 1, or about 0.3: 1.

For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

The cells described herein can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells.

Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of engineered T cells to the antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., "Construction and pre-clinical evaluation of an anti-CD19 chimeric antigen receptor. " Journal of immunotherapy (Hagerstown, Md. : 1997) 32.7 (2009) : 689 and Hermans et al., "The VITAL assay: a versatile fluorometric technique for assessing CTL-and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo. " Journal of immunological methods 285.1 (2004) : 25-40. In certain embodiments, the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

Kits and articles of manufacture

Further provided are kits, unit dosages, and articles of manufacture comprising any one of the engineered T cells (e.g., allogeneic T cell, endogenous TCR-deficient T cell, GvHD-minimized T cell) expressing a fusion polypeptide (e.g., any of the fusion polypeptides described herein) and/or a functional exogenous receptor (such as engineered TCR (e.g., traditional engineered TCR, chimeric TCR (cTCR) ) , TAC, TAC-like chimeric receptor, or CAR (e.g., antibody-based CAR, ligand/receptor-based CAR, or ACTR) ) described herein. In some embodiments, a kit is provided which contains any one of the pharmaceutical compositions described herein and preferably provides instructions for its use.

The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Kits may optionally provide additional components such as buffers and interpretative information. The present application thus also provides articles of manufacture, which include vials (such as sealed vials) , bottles, jars, flexible packaging, and the like.

The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. Generally, the container holds a composition which is effective for treating a disease or disorder (such as cancer, autoimmune disease, or infectious disease) as described herein, or reducing/preventing GvHD or transplantation rejection when treating a disease or disorder, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . The label or package insert indicates that the composition is used for treating the particular condition in an individual. The label or package insert will further comprise instructions for administering the composition to the individual. The label may indicate directions for reconstitution and/or use. The container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. The kits or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Regulation of CD3ζ protein expression by fusion proteins of SIV Nef M116 and E3 ubiquitin ligase

1) Construction of expression plasmids encoding the SIV Nef M116-E3 ubiquitin ligase fusion protein

The pLVX-Puro plasmid purchased from Clontech was digested with ClaI and EcoRI restriction enzymes to remove the CMV promoter, and the human EF1α promoter (GenBank: J04617.1) was cloned into the digested plasmid, thereby obtaining the pLVX-hEF1α plasmid. Schematic structures and sequences of the fusion proteins formed by SIV Nef M116 and various E3 ubiquitin ligases are shown in the table below.

Table 1. Exemplary SIV Nef M116-E3 ubiquitin ligase fusion proteins

Specifically, a nucleic acid sequence encoding the fusion protein was cloned downstream of the human EF1α promoter on the expression plasmid pLVX-hEF1α. The expression plasmid encodes the fusion protein of SIV Nef M116 and E3 ubiquitin ligase. The fusion protein was connected with the screening marker protein (Puro; SEQ ID NO: 45) by a self-cleaving peptide T2A (SEQ ID NO: 47) . The corresponding recombinant expression plasmids M2339, M2342, M2346, M2348, M2352, M2354, M2356 and M2360 were isolated and mixed with helper plasmids (e.g., psPAX2 and pMD2. G) , to co-transfect HEK 293T cells for lentivirus production. After 60 hours of transfection, cell culture supernatant containing the lentivirus was collected and centrifuged at 4℃, 3000 rpm for 5 minutes. Afterwards, the supernatant was filtered through a 0.45 μm filter, and the 500 KD hollow fiber membrane column tangential flow technology was used to further concentrate the supernatant to prepare the lentivirus concentrate, which was stored at -80℃ for later use.

Details of SIV Nef M116 (SEQ ID NO: 30) can be found. e.g., in PCT Application Publication No. WO2020020359, which is incorporated herein by reference in its entirety. The E3 ubiquitin ligase is selected from the following proteins or truncated forms thereof: SPOP [167-374] (i.e., amino acids 167-374 of E3 ubiquitin ligase SPOP) with amino acid sequence set forth in SEQ ID NO: 19; βTrCP [2-264] (i.e., amino acids 2-264 of E3 ubiquitin ligase βTrCP) with sequence set forth in SEQ ID NO: 20; SKP2 [2-147] (i.e., amino acids 2-147 of E3 ubiquitin ligase SKP2) with sequence set forth in SEQ ID NO: 21; VHL [152-213] (i.e., amino acids 152-213 of E3 ubiquitin ligase VHL) with sequence set forth in SEQ ID NO: 22; SOCS2 [143-198] (i.e., amino acids 143-198 of E3 ubiquitin ligase SOCS2) with sequence set forth in SEQ ID NO: 23; ASB1 [266-335] (i.e., amino acids 266-335 of E3 ubiquitin ligase ASB1) with sequence set forth in SEQ ID NO: 24; CHIP [128-303] (i.e., amino acids 128-303 of E3 ubiquitin ligase CHIP) with sequence set forth in SEQ ID NO: 25; and TRIM21 [1-267] (i.e., amino acids 1-267 of E3 ubiquitin ligase TRIM21) with sequence set forth in SEQ ID NO: 26. The linker sequence connecting the SIV Nef M116 and various E3 ubiquitin ligases has a sequence set forth in SEQ ID NO: 46.

2) Preparation of Jurkat cell line expressing the SIV Nef M116-E3 ubiquitin ligase fusion protein

Jurkat cells (Human T Lymphocyte Cell, clone E6‐1, ATCC, TIB‐152^TM) were cultured in 90%RPMI 1640 medium (Gibco, Cat#: 22400-089) supplemented with 10%fetal bovine serum (FBS, Gibco, Cat#: 10099-141C) . 5 × 10⁶ Jurkat cells were infected with the lentivirus prepared above, and the cell suspension was added to a 10 cm culture dish, which was placed in a 37℃, 5%CO₂ incubator for 3 days. On the third day of viral transduction, the culture medium was replaced to include 1 μg/mL puromycin (Gibco, Cat#: A1113803) . The culture was continued for three days for drug resistance screening. After the screening, the TCRα/β sorting kit was used to enrich and sort the TCRα/β-negative cells to complete the preparation of the Jurkat cell line expressing the SIV Nef M116-E3 ubiquitin ligase fusion protein.

3) Detection of CD3ζ by Western blot

5 ×10⁶ non-transduced Jurkat cells or the transduced Jurkat cells expressing the SIV Nef M116-E3 ubiquitin ligase fusion protein were selected. The cells were washed twice with cold Dulbecco's Phosphate Buffered Saline (DPBS; Cytiva, Cat#: SH30028.02) , and then centrifuged at 2500 g for 5 minutes. After centrifugation, the cells were collected and 1 mL cell lysis buffer (Thermo Scientific, Cat#: 89900) was added. The cell lysate was shaken gently on ice for 15 minutes, and then centrifuged at 14,000 g at 4℃ for 15 minutes. Supernatant was transferred to a new centrifuge tube, and stored in a -80℃ refrigerator for later use. The expression level of CD3ζ protein was detected with a CD3ζ-specific antibody (Santa Cruz, Cat#: sc-1239) by Western blot, and β-actin was detected as a control.

As shown in FIG. 1, CD3ζ expression was significantly down-regulated in Jurkat cells transduced with M2339, M2346, M2352, M2356 and M2360 plasmids, respectively, as compared to the blank mock group (non-transduced Jurkat cells) . The results indicate that SIV Nef M116-SPOP, SIV Nef M116-SKP2, SIV Nef M116-SOCS2, SIV Nef M116-CHIP and SIV Nef M116-TRIM21 fusion proteins can capture and significantly degrade CD3ζ protein on T lymphocytes.

Example 2. Regulation of CD3ζ protein expression by fusion proteins of SIV Nef M116 and truncated TRIM21

1) Construction of expression plasmids encoding the SIV Nef M116-truncated TRIM21 fusion protein

Full-length TRIM21 protein can be truncated to obtain TRIM21 Truncate1 (amino acids 1-127 of TRIM21: SEQ ID NO: 27) , TRIM21 Truncate2 (amino acids 1-85 of TRIM21; SEQ ID NO: 28) , and TRIM21 Truncate3 (amino acids 1-55 of TRIM21; SEQ ID NO: 29) . Expression plasmids encoding the SIV Nef M116-truncated TRIM21 fusion proteins were constructed using the methods described in Example 1. Schematic structures and sequences of the fusion proteins are shown in Table 2. Lentivirus was produced and concentrated as described in Example 1.

Jurkat cell lines were prepared according to the methods described in Example 1, to obtain M2360-Jurkat, M2542-Jurkat, M2543-Jurkat, and M2544-Jurkat cells, respectively.

Table 2. Exemplary SIV Nef M116-truncated TRIM21 fusion proteins

2) Detection of SIV Nef M116 and CD3ζ expression by flow cytometry

Detection of SIV Nef M116 and CD3ζ expression by flow cytometry was performed as follows. 3×10⁶ cell suspension was centrifuged at room temperature at 1000 rpm for 1 minute, and supernatant was discarded. 200 μL of BD Cytofix^TM Fixation Buffer (BD Biosciences, Cat#: 554655) was added to the cell samples above. The cells were suspended and incubated at room temperature for 10 minutes. The cell suspension was centrifuged at 300 g at room temperature for 1 minute, and supernatant was discarded. The cells were suspended with 200 μL BD Perm/Wash^TM Buffer (BD Biosciences, Cat#: 51-2091KZ) , and then washed once. Afterwards, 1 μL APC anti-SIV Nef M116 Antibody (produced by GeneScript, Order No: LGBUADAb-3) and 1 μL (1: 100) PE anti-CD247 (TCRζ, CD3ζ) Antibody (BioLegend, Cat#: 644106) were added, and the cells were incubated at room temperature for 30 minutes. The cells were washed twice, and then subject to flow cytometry detection.

The results are shown in FIG. 2, in SIV Nef M116 positive cells, CD3ζ positive rates of mock (untransduced Jurkat cells) , M2360-Jurkat, M2542-Jurkat, M2543-Jurkat, and M2544-Jurkat are 98.03%, 25.47%, 33.93%, 32.10%, and 92.83%, respectively. CD3ζ expression was significantly down-regulated in Jurkat cells transduced with M2360, M2542, and M2543 plasmids, respectively, as compared to the blank mock group. The results indicate that SIV Nef M116-TRIM21, SIV Nef M116-TRIM21 Trun1, and SIV Nef M116-TRIM21 Trun2 fusion proteins can capture and significantly degrade CD3ζ protein on T lymphocytes.

Example 3. Regulation of CD3ζ protein expression in CAR-T cells by the fusion protein of SIV Nef M116 -TRIM21 Trun2

1) Construction of expression plasmids

Similar to the methods described in Example 1, the DNA fragment encoding SIV Nef M116-T2A-CD8α SP-GPC3 scFv-CD8α Hinge-CD8α TM-4-1BB-ITAM010-P2A-DHFR L22F/F31S-P2A-SP-MB12 was cloned into the expression plasmid pLVX-hEF1α, to obtain the expression plasmid PLSINK-Modified1909 ( “M1909” for short) . The DNA fragment encoding SIV Nef M116-linker-TRIM21 Truncate2-T2A-CD8α SP-GPC3 scFv-CD8α Hinge-CD8α TM-4-1BB-ITAM010-P2A-DHFR L22F/F31S-P2A-SP-MB12 was cloned into the expression plasmid pLVX-hEF1α, to obtain the expression plasmid PLSINK-Modified2259 ( “M2259” for short) . The recombinant expression plasmids were isolated and mixed with helper plasmids (e.g., pMDLg-pRRE, pRSV-Rev, and pMD2. G) , to co-transfect HEK 293T cells for lentivirus production. The amino acid sequence of ITAM010-modified GPC3 CAR (CD8α SP-GPC3 scFv-CD8α Hinge-CD8α TM-4-1BB-ITAM010) is shown in SEQ ID NO: 41. ITAM010 was disclosed in PCT Application Publication No. WO2021037221, and its sequence is shown in SEQ ID NO: 42. The GPC3 scFv and the CAR with CD3ζ intracellular signaling domain thereof were disclosed in PCT Application Publication No. WO2021170100. The amino acid sequence of dihydrofolate reductase (DHFR) L22F/F31S is shown in SEQ ID NO: 43. MB12 is an exemplary membrane-bound IL12p40 polypeptide, with a schematic structure of IL12p40-CD8αHinge-CD8α transmembrane domain-CD8α intracellular cytoplasmic domain, and its amino acid sequence is shown in SEQ ID NO: 44. The amino acid sequences for self-cleaving peptides T2A, P2A, and signal peptide (SP) are shown in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, respectively.

2) T cell isolation, transduction and preparation

50 mL of fresh peripheral blood from volunteers was collected, and peripheral blood mononuclear cells (PBMCs) were isolated by lymphocyte separation medium and density gradient centrifugation. The cells were labeled with magnetic beads using the Pan T Cell Isolation Kit (Miltenyi, Cat#: 130-096-535) , to isolate and purify T lymphocytes. Purified T cells were treated with CD3/CD28 magnetic beads for T lymphocyte activation and proliferation. After culturing in a 37℃, 5%CO₂ incubator for 24 hours, 5 × 10⁶ activated T lymphocytes were infected with the lentivirus prepared above. The cell suspension was added to a 6-well plate, and cultured in a 37℃, 5%CO₂ incubator. On the seventeenth day of viral transduction, the TCRα/βsorting kit was used to enrich and sort TCRα/β negative cells to complete the preparation of M1909-T and M2259-T cells. The protein samples of UnT (untransduced T cells) , M1909-T, and M2259-T cells were prepared using the methods described in Example 1. The expression level of CD3ζ protein was detected with a CD3ζ-specific antibody by Western blot, and β-actin was detected as a control.

As shown in FIG. 3, CD3ζ expression in M2259-T cells was significantly down-regulated, as compared to that in UnT and M1909-T cells. The results indicate that SIV Nef M116-TRIM21 Trun2 fusion protein has the function of capturing and degrading CD3ζ protein on CAR-T cells.

Example 4. Regulation of CD3ζ protein expression by fusion proteins of different subtypes of Nef and TRIM21 Truncate2

The BCMA-BBz CAR (CD8α SP-BCMA scFv-CD8α hinge-CD8α TM-4-lBB-CD3ζ) was disclosed in PCT Application Publication No. WO2021037221, and its amino acid sequence is shown in SEQ ID NO: 39.

Expression plasmids encoding BCMA-BBz CAR was constructed using the methods described in Example 1. Lentivirus was produced and concentrated as described in Example 1.

Expression plasmids encoding fusion proteins containing different subtypes of Nef and TRIM21 Truncate2 (Trun2) were constructed using the methods described in Example 1. Schematic structures and sequences of the fusion proteins are shown in Table 3. Lentivirus was produced and concentrated as described in Example 3.

Table 3. Exemplary fusion proteins having different subtypes of Nef and TRIM21 Truncate2 in corresponding CD20 CAR expression structures

The amino acid sequences of Nef subtypes HIV1 Nef M094, HIV2 Nef M787, HIV2 Nef M092, SIV Nef M1006, SIV Nef M1034, HIV1 Nef M1069, and SIV Nef M998 are shown in SEQ ID NOs: 31, 32, 33, 34, 35, 36, and 37, respectively. The ITAM010-modified CD20 CAR (CD8α SP-CD20 scFv (Leu16) -CD8α Hinge-CD8α TM-4-1BB-ITAM010) was disclosed in PCT Application Publication No. WO2021037221, and its amino acid sequence is shown in SEQ ID NO: 40.

Jurkat cell line expressing BCMA-BBz CAR was prepared according to the methods described in Example 1, to obtain Jurkat-BCMA-BBz (hereinafter referred to as “Jurkat-BCMA-BBz” ) . CAR expression in Jurkat-BCMA-BBz cells was 96.16%after puromycin resistance enrichment.

Detection of BCMA CAR expression by flow cytometry was performed as follows. The cell suspension was centrifuged at room temperature at 1000 rpm for 1 minute, and supernatant was discarded. Cells were suspended in DPBS, and incubated with 1μL biotinylated human BCMA/TNFRSF17 regent (ACROBIOSYSTEM, Cat#: BCA-H522y) at 4℃ for 30 minutes. After the incubation, the cell suspension was centrifuged at room temperature at 1000 rpm for 1 minutes. The supernatant was discarded, and the cells were suspended in 1 mL DPBS. The cells were washed once as described above.

Jurkat-BCMA-BBz cells were infected with the lentiviruses carrying fusion protein of different subtypes of Nef and TRIM21 Truncate2 sequences according to the methods described in Example 1, to obtain M2268-Jurkat-BCMA-BBz (Jurkat-BCMA-BBz cells transduced with M2268 vector) , M2545-Jurkat-BCMA-BBz, M2546-Jurkat-BCMA-BBz, M2547-Jurkat-BCMA-BBz, M2549-Jurkat-BCMA-BBz, M2550-Jurkat-BCMA-BBz, M2551-Jurkat-BCMA-BBz, and M2553-Jurkat-BCMA-BBz cells, respectively. 5 days post-transduction, 1×10⁶ cell suspension was collected for flow cytometry detection of BCMA CAR as described above. Jurkat cell lines were prepared according to the methods described in Example 1, to obtain M2268-Jurkat (Jurkat cells transduced with M2268 vector) , M2545-Jurkat, M2546-Jurkat, M2547-Jurkat, M2549-Jurkat, M2550-Jurkat, M2551-Jurkat, and M2553-Jurkat cells, respectively.

Expressions of CD20 CAR was detected according to the methods as described above. 0.64 μL Alexa Flour^TM 488 HDA-1 antibody (GeneScript, Order No: LGBUADAb-1) was used for cell staining. Expressions of CD3ζ was detected according to the methods described in Example 2.

FIG. 4A shows that fusion proteins of different subtypes of Nef and TRIM21 Truncate2 regulate traditional CAR expression. BCMA CAR positive rates of mock (untransduced Jurkat-BCMA-BBz cells) , M2268-Jurkat-BCMA-BBz, M2545-Jurkat-BCMA-BBz, M2546-Jurkat-BCMA-BBz, M2547-Jurkat-BCMA-BBz, M2549-Jurkat-BCMA-BBz, M2550-Jurkat-BCMA-BBz, M2551-Jurkat-BCMA-BBz, and M2553-Jurkat-BCMA-BBz cells are 96.16%, 60.99%, 97.28%, 75.18%, 97.44%, 77.13%, 87.77%, 97.53%, and 96.15%, respectively.

FIG. 4B shows that fusion protein of different subtypes of Nef and TRIM21 Truncate2 regulate CD3ζ expression. In CD20 CAR positive cells, CD3ζ positive rates of mock (untransduced Jurkat cells) , M2268-Jurkat, M2545-Jurkat, M2546-Jurkat, M2547-Jurkat, M2549-Jurkat, M2550-Jurkat, M2551-Jurkat, and M2553-Jurkat cells are 94.27%, 25.37%, 84.04%, 49.92%, 97.22%, 63.89%, 69.54%, 97.09%, and 94.49%, respectively. CD3ζ expression was significantly down-regulated in Jurkat cells transduced with M2268, M2546, M2549, and M2550 plasmids, respectively, as compared to the blank mock group. The results indicate that SIV Nef M116-TRIM21 Trun2, HIV2 Nef M787-TRIM21 Trun2, SIV Nef M1006-TRIM21 Trun2, and SIV Nef M1034-TRIM21 Trun2 fusion proteins can capture and significantly degrade CD3ζ protein on T lymphocytes.

Example 5. Cell phenotype, CD3ζ protein expression, and specific cytotoxicity of GPC3 CAR-T cells containing SIV Nef M116-TRIM21 Trun2

CAR-T cells were prepared according to the methods described in Example 3, to obtain M2259-T cells.

Expressions of CD5, CAR, TCRα/β and MB12 were detected according to the flow cytometry detection methods of CAR in Example 4. Specifically, 1 μL (1: 100) of PE/Cyanine7 anti-human CD5 antibody, 0.64 μL Alexa Flour^TM 7488 HDA-1 antibody (produced by GeneScript, Order No: LGBUADAb-2) , 1 μL (1: 100) APC anti-human TCRα/β antibody (Miltenyi, Cat#: 130-113-527) , and 1 μL (1: 100) PE anti-human IL-12/IL-23 p40 antibody (BioLegend, Cat#: B307375; used for detecting MB12) were used for cell staining.

The protein samples of UnT (untransduced T cells) and M2259-T cells were prepared using the methods described in Example 1. The expression level of CD3ζ protein was detected with a CD3ζ-specific antibody by Western blot, and β-actin was detected as a control.

Each T cell group was mixed with HuH-7. Luc cells (GPC3-positive liver cancer cells, JCRB, Cat#: JCRB0403) at 2: 1 and 1: 2 effect-to-target (E: T) ratios, respectively, in384-well plates (white) , and incubated for 18-24 hours. The ONE-Glo^TM Luciferase Assay System (TAKARA, Cat#: B6120) was used. Specifically, 25 μL One-Glo^TM reagent was added to each well of the 384-well plates, and the fluorescence signal from Luciferase was detected with a microplate reader (TECAN, 10M) . The killing efficiency of each group of lymphocytes on target cells was calculated as follows: Killing efficiency (%) = (1- (RLUexperimental group-RLUblank control group) /RLUtumor cell group) × 100%.

FIGS. 5A-5D show the M2259-T cell phenotype, CD3ζ protein expression, and specific cytotoxicity results, respectively. FIG. 5A and FIG. 5B show that after TCRα/β sorting and enrichment of M2259-T cells, the ratio of TCRα/β-CAR+ (TCRα/β negative and CAR positive) cells was 95.27%, and the ratio of CAR+MB12+ cells was 72.21%. The Western blot analysis results in FIG. 5C indicate that the SIV Nef M116-TRIM21 Trun2 fusion protein on CAR-T cells had the function of capturing and degrading CD3ζ protein. Moreover, the specific cytotoxicity of CAR-T cells expressing the SIV Nef M116-TRIM21 Trun2 fusion protein was verified in FIG. 5D.

Example 6. Cell phenotype, CD3ζ protein expression, and specific cytotoxicity of CD20 CAR-T cells containing SIV Nef M116-SPOP

Similar to the methods described in Example 1, the DNA fragment encoding SIV Nef M116-linker-SPOP [167-374] -T2A-CD8α SP-CD20 scFv (Leu16) -CD8α Hinge-CD8α TM-4-1BB-ITAM010-P2A-DHFR L22F/F31S-P2A-SP-MB12 was cloned into the expression plasmid pLVX-hEF1α, to obtain the expression plasmid PLSINK-Modified2426 ( “M2426” for short) . CAR-T cells were prepared according to the methods described in Example 2, to obtain M2426-T cells.

Expressions of CD5, CAR, TCRα/β and MB12 were detected according to the flow cytometry detection methods of CAR in Example 4. Specifically, 1 μL (1: 100) of PE/Cyanine7 anti-human CD5 antibody, 0.64 μL Alexa Flour^TM 7488 LUCAR-20S ADA antibody (produced by GeneScript, Order No: LGBUADAb-1) , 1 μL (1: 100) APC anti-human TCRα/β antibody, and 1 μL (1: 100) PE anti-human IL-12/IL-23 p40 antibody were used for cell staining.

The protein samples of UnT (untransduced T cells) and M2426-T cells were prepared using the methods described in Example 1. The expression level of CD3ζ protein was detected with a CD3ζ-specific antibody by Western blot, and β-actin was detected as a control.

Specific cytotoxicity assays were performed as described in Example 5. Each T cell group was mixed with Raji. Luc (CD20-positive lymphoma cells, ATCC, Cat#: CCL-86) cells at 20: 1, 10: 1 and 5: 1 effect-to-target ratios, respectively, and the killing efficiency of each group of lymphocytes on target cells was calculated.

FIGS. 6A-6D show the M2426-T cell phenotype, CD3ζ protein expression, and specific cytotoxicity results, respectively. FIG. 6A and FIG. 6B show that after TCRα/β sorting and enrichment of M2426-T cells, the ratio of TCRα/β-CAR+ cells was 97.49%, and the ratio of CAR+MB12+ cells was 69.54%. The Western blot analysis results in FIG. 6C indicate that the SIV Nef M116-SPOP fusion protein on CAR-T cells had the function of capturing and degrading CD3ζ protein. Moreover, the specific cytotoxicity of CAR-T cells expressing the SIV Nef M116-SPOP fusion protein was verified in FIG. 6D.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

A fusion polypeptide, comprising:

a first moiety comprising a non-antibody protein that binds to a target protein, and a second moiety comprising an E3 ubiquitin ligase or fragment thereof.
The fusion polypeptide of claim 1, wherein the target protein is associated with graft-versus-host disease (GvHD) and/or host-versus-graft disease (HvGD) in a cell therapy; optionally wherein the target protein is CD3ζ.
The fusion polypeptide of claim 1 or 2, wherein the non-antibody protein is a viral protein; optionally wherein the viral protein is a Negative Regulatory Factor (Nef) protein.
The fusion polypeptide of claim 3, wherein the Nef protein is from simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) .
The fusion polypeptide of claim 3 or 4, wherein the Nef protein is a wildtype or a non-naturally occurring mutant.
The fusion polypeptide of any one of claims 3-5, wherein the Nef protein is selected from the group consisting of SIV Nef M116, HIV1 Nef M094, HIV2 Nef M787, HIV2 Nef M092, SIV Nef M1006, SIV Nef M1034, HIV1 Nef M1069, and SIV Nef M998.
The fusion polypeptide of any one of claims 3-6, wherein the Nef protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 30-37.
The fusion polypeptide of any one of claims 1-7, wherein the E3 ubiquitin ligase or fragment thereof is selected from the group consisting of Speckle Type BTB/POZ Protein (SPOP) , Beta-Transducin Repeat Containing E3 Ubiquitin Protein Ligase (βTrCP) , S-Phase Kinase Associated Protein 2 (SKP2) , Von Hippel-Lindau Tumor Suppressor (VHL) , Suppressor Of Cytokine Signaling 2 (SOCS2) , Ankyrin Repeat And SOCS Box Containing 1 (ASB1) , Carboxy Terminus Of Hsp70-interacting Protein (CHIP) , and Tripartite Motif Containing 21 (TRIM21) .
The fusion polypeptide of any one of claims 1-8, wherein the E3 ubiquitin ligase or fragment thereof is truncated.
The fusion polypeptide of any one of claims 1-9, wherein the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to:

(a) . amino acids 167-374 of SPOP;

(b) . amino acids 2-264 of βTrCP;

(c) . amino acids 2-147 of SKP2;

(d) . amino acids 152-213 of VHL;

(e) . amino acids 143-198 of SOCS2;

(f) . amino acids 266-335 of ASB1;

(g) . amino acids 128-303 of CHIP;

(h) . amino acids 1-267 of TRIM21;

(i) . amino acids 1-127 of TRIM21;

(j) . amino acids 1-85 of TRIM21; or

(k) . amino acids 1-55 of TRIM21.
The fusion polypeptide of any one of claims 1-10, wherein the E3 ubiquitin ligase or fragment thereof comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 19-29.
The fusion polypeptide of any one of claims 1-11, wherein the first moiety and the second moiety are connected to each other via a linker peptide.
The fusion polypeptide of claim 12, wherein the linker peptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 46.
The fusion polypeptide of any one of claims 1-13, wherein the first moiety is N-terminal to the second moiety or C-terminal to the second moiety.
The fusion polypeptide of any one of claims 1-14, wherein the fusion polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 1-18.
A polynucleotide comprising a nucleic acid sequence encoding the fusion polypeptide of any one of claims 1-15.
The polynucleotide of claim 16, wherein the polynucleotide further comprises a second nucleic acid sequence encoding a functional exogenous receptor; optionally wherein the functional exogenous receptor is a chimeric antigen receptor (CAR) .
The polynucleotide of claim 16 or 17, wherein the polynucleotide further comprises a third nucleic acid sequence encoding a protein marker; optionally wherein the protein marker is DHFR L22F/F31S.
The polynucleotide of any one of claims 16-18, wherein the polynucleotide further comprises a fourth nucleic acid sequence encoding an exogenous cytokine, or a fragment thereof; optionally wherein the exogenous cytokine or fragment thereof is a membrane-bound IL12p40.
The polynucleotide of any one of claims 17-19, wherein each nucleic acid sequence is connected via a linking sequence; optionally wherein the linking sequence comprises a nucleic acid sequence encoding P2A or T2A.
A vector comprising the polynucleotide of any one of claims 16-20.
The vector of claim 21, wherein the vector is a viral vector.
The vector of claim 21 or 22, wherein the vector comprises a human EF1α promoter.
An engineered cell comprising the fusion polypeptide of any one of claims 1-15, the polynucleotide of any one of claims 16-20, or the vector of any one of claims 21-23.
The engineered cell of claim 24, wherein the engineered cell further comprises a functional exogenous receptor; wherein the functional exogenous receptor comprises

(a) an extracellular ligand binding domain,

(b) a transmembrane domain, and

(c) an intracellular signaling domain (ISD) comprising a chimeric signaling domain (CMSD) , wherein the CMSD comprises one or a plurality of immune-receptor Tyrosine-based Activation Motifs (CMSD ITAMs) , wherein the plurality of CMSD ITAMs are optionally connected by one or more linkers (CMSD linkers) .
The engineered cell of claim 25, wherein at least one of the CMSD ITAMs is not derived from CD3ζ.
The engineered cell of claim 25 or 26, wherein at least one of the CMSD ITAMs is derived from an ITAM-containing parent molecule selected from the group consisting of CD3ε, CD3δ, CD3γ, Igα (CD79a) , Igβ (CD79b) , FcεRIβ, FcεRIγ, DAP12, CNAIP/NFAM1, STAM-1, STAM-2, and Moesin.
The engineered cell of any one of claims 25-27, wherein the CMSD comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 42.
The engineered cell of any one of claims 25-28, wherein the functional exogenous receptor further comprises a signal peptide located at the N-terminus of the extracellular ligand binding domain.
The engineered cell of any one of claims 25-29, wherein the extracellular ligand binding domain of the functional exogenous receptor binds to an antigen, wherein the antigen is a tumor antigen selected from the group consisting of BCMA, CLL1, GPC3, GU2CYC, CD19, CD7, CD20, CD22, CD38, CD41, CD123, Claudin 18.2, Claudin 6, NKG2D, DLL-3, GPRC5D, and CD70.
The engineered cell of claim 30, wherein the tumor antigen is GPC3 or CD20.
The engineered cell of any one of claims 25-31, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR) , a T cell receptor (TCR) , a chimeric TCR (cTCR) , or a T cell antigen coupler (TAC) -like chimeric receptor.
The engineered cell of claim 32, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR) .
The engineered cell of any one of claims 33, wherein the intracellular signaling domain of the CAR further comprises a co-stimulatory signaling domain.
The engineered cell of claim 33 or 34, wherein the CAR further comprises a hinge domain located between the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain.
The engineered cell of any one of claims 33-35, wherein the CAR comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to any one of SEQ ID NOs: 38-41.
The engineered cell of any one of claims 24-36, wherein the engineered cell further comprises a protein marker; optionally wherein the protein marker is DHFR L22F/F31S.
The engineered cell of any one of claims 24-37, wherein the engineered cell further comprises an exogenous cytokine, or a fragment thereof; optionally wherein the exogenous cytokine or fragment thereof is a membrane-bound IL12p40.
The engineered cell of any one of claims 24-38, wherein the engineered cell is a T cell or an NK cell.
The engineered cell of claim 39, wherein the engineered cell is a primary T cell obtained from a subject; optionally the subject is a cancer patient or healthy donor.
The engineered cell of claim 40, wherein the engineered cell expresses a decreased level of CD3ζ as compared to that in a precursor T cell and/or a decreased level of endogenous T cell receptor (TCR) as compared to that in a precursor T cell.
The engineered cell of claim 40 or 41, wherein the engineered cell elicits no or reduced graft-versus-host disease (GvHD) response in a histoincompatible individual as compared to the GvHD response elicited by a primary T cell isolated from the donor of the precursor T cell from which the engineered cell is derived.
A chimeric antigen receptor (CAR) , comprising:

(a) an extracellular ligand binding domain that binds to GPC3 antigen,

(b) a transmembrane domain, and

(c) an intracellular signaling domain (ISD) comprising a chimeric signaling domain (CMSD) , wherein the CMSD comprises one or a plurality of immune-receptor Tyrosine-based Activation Motifs (CMSD ITAMs) , wherein the plurality of CMSD ITAMs are optionally connected by one or more linkers (CMSD linkers) ; optionally wherein at least one of the CMSD ITAMs is not derived from CD3ζ.
The CAR of claim 43, comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 41.
A method for producing an engineered T cell, comprising introducing the polynucleotide of any one of claims 16-20, or the vector of any one of claims 21-23 into a precursor cell.
The method of claim 45, further comprises introducing into the precursor cell a second polynucleotide encoding a functional exogenous receptor that binds to an antigen; optionally wherein the functional exogenous receptor is a chimeric antigen receptor (CAR) .
The method of claim 46, further comprising isolating and/or enriching TCR-negative and functional exogenous receptor-positive T cells from the engineered T cells.
An engineered T cell obtained by the method of any one of claims 45-47.
A pharmaceutical composition comprising the engineered cell of any one of claims 24-42, or the engineered T cell of claim 48, and a pharmaceutically acceptable carrier.
A method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the engineered cell of any one of claims 24-42, the engineered T cell of claim 48, or the pharmaceutical composition of claim 49.
A method of downregulating and/or degrading a target protein in a T cell from a subject, comprising introducing into the T cell the polynucleotide of any one of claims 16-20, or the vector of any one of claims 21-23; optionally wherein the target protein is CD3ζ and/or TCR.