Car polypeptides with slamf7 costimulatory domains

Abstract

Disclosed herein are chimeric antigen receptors (CARs) with SLAMF7 co-stimulatory domains. For example, disclosed herein is a chimeric antigen receptor (CAR) polypeptide having a signaling peptide, a ligand binding domain, a hinge domain, a transmembrane domain, and an endodomain that comprises a CD3ζ domain and a SLAMF7 co-stimulatory domain. In some embodiments the endodomain further comprises a CD28 co-stimulatory domain, 4-1BB co-stimulatory domain, or a combination thereof.

Description

CAR POLYPEPTIDES WITH SLAMF7 CO STIMULATORY DOMAINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 643,667, filed May 7, 2024, which is hereby incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled “320805-2230 Sequence Listing” created on May 6, 2025, and having 54,329 bytes. The content of the sequence listing is incorporated herein in its entirety. BACKGROUND OF THE INVENTION

[0003] Tumor immunotherapy is a promising therapeutic strategy for patients with advanced cancers. T cells are key mediators of antitumor function that specifically recognize and react to tumor-expressing antigens and have proven critical for cancer immunotherapy. However, T cells are not as effective against cancer as expected. This is partly because T cells enter a dysfunctional or exhausted state, which is characterized by sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells.SUMMARY OF THE INVENTION

[0004] Disclosed herein is a chimeric antigen receptor (CAR) polypeptide having a signaling peptide, a ligand binding domain, a hinge domain, a transmembrane domain, and an endodomain that comprises a CD3 domain and a SLAMF7 co-stimulatory domain. In some embodiments the endodomain further comprises a CD28 co-stimulatory domain, 4- 1BB co-stimulatory domain, or a combination thereof.

[0005] In some embodiments the CAR polypeptide is defined by the formula: SP-LBD-HG-TM-SLAMF7-CD3 ;SP-LBD-HG-TM-CD28-SLAMF7-CD3 ; SP-LBD-HG-TM-41 BB-SLAMF7-CD3 ;SP-LBD-HG-TM-CD28-41 BB-SLAMF7-CD3 ; or SP-LBD-HG-TM-41 BB-CD28-SLAMF7-CD3 wherein “SP” represents an optional signal peptide, wherein “LBD” represents the ligand binding domain, wherein “HG” represents an optional hinge domain, wherein “TM” represents a transmembrane domain, wherein “CD3 ’ represents a CD3 domain, wherein “CD28” represents a CD28 co-stimulatory domain, wherein “41 BB” represents a 41 BB co-stimulatory domain, wherein “SLAMF7” represents an SLAMF7 co-stimulatory domain, andwherein represents a peptide bond or linker.

[0006] In some embodiments the SLAMF7 domain has a mutated ITSM domain to prefer binding by signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) over a tyrosine phosphatase.

[0007] In some embodiments the ITSM domain is replaced with an ITAM domain. For example, the ITAM domain can have the amino acid sequence Yxxl / L(x)6-8Yxxl / L. In some embodiments the ITAM domain comprises the amino acid sequence YNELNLGRREEYDVL (SEQ ID NO:7), YNELQKDKMAEAYSEI (SEQ ID NO:8), or YQGLSTATKDTYDAL (SEQ ID NO:55). In some embodiments the ITSM domain is replaced with a mutant ITSM domain selected from the group consisting of SEQ ID NO:23-54. In some embodiments the ITSM domain is truncated or deleted. In some embodiments the wherein one or more of the S305, T306, and V307 amino acids of the ITSM domain are deleted. In some embodiments the Y304 of the ITSM domain is substituted. In some embodiments the Y304 of the ITSM domain is substituted with phosphotyrosine or 3’- phosphotyrosine. In some embodiments charged residues within the ITSM domain or within 10 amino acids of the ITSM domain are replaced with polar or nonpolar amino acids. In some embodiments proline residues are substituted into or inserted into the ITSM domain or within 10 amino acids of the ITSM domain. In some embodiments residues within 10 amino acids of Y304 are mutated to tryptophan or phenylalanine. In some embodiments residues within 10 amino acids of the ITSM motif are mutated to create a steric hindrance that prevents binding of the tyrosine phosphatases but allow binding of SAP.

[0008] In some embodiments the CAR further contains a signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) linked to the endodomain by a cleavable peptide. In some embodiments the CAR further contains an EAT2 protein linked to the endodomain by a cleavable peptide.

[0009] Also disclosed is a chimeric receptor having a signaling peptide, an ectodomain, and transmembrane domain, wherein the ectodomain comprises a SLAMF7 protein and / or a signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) protein. CAR further contains SLAMF7 protein has a mutated ITSM domain to prefer binding by signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) over a tyrosine phosphatase.

[0010] Also disclosed is a fusion protein having a chimeric antigen receptor (CAR) polypeptide that comprises a ligand binding domain, a hinge domain, a transmembrane domain, and an endodomain, wherein the chimeric receptor of claim 19 or 20 is linked to the endodomain of the CAR polypeptide by a cleavable linker.

[0011] Also disclosed is an isolated nucleic acid sequence encoding a CAR polypeptide disclosed herein, a chimeric receptor disclosed herein, or the fusion protein disclosed herein.

[0012] Also disclosed is a vector comprising an isolated nucleic acid disclosed herein operably linked to an expression control sequence.

[0013] Also disclosed is an immune effector cell engineered to express a CAR polypeptide disclosed herein, a chimeric receptor disclosed herein, or the fusion protein disclosed herein. In some embodiments, the immune effector cell is selected from the group consisting of an apT cell, y<5T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T (Treg) cell, or any combination thereof.

[0014] Also disclosed is a method of providing an anti-tumor immunity in a subject with a cancer, the method comprising administering to the subject an effective amount of an immune effector cell disclosed herein, thereby providing an anti-tumor immunity in the mammal. In some embodiments, the method further involves administering to the subject a checkpoint inhibitor. For example, the checkpoint inhibitor can be an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.

[0015] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0016] FIG. 1 illustrates a proposed SLAMF7 signaling model in T cells in which SLAMF7 can either provide a positive or negative signal during T-cell activation depending on the presence and amounts of adaptor proteins signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) and tyrosine phosphatases.

[0017] FIG. 2 illustrates a proposed mechanism by which the presence and amounts of SAP and tyrosine phosphatases affect T cell activation or inhibition.

[0018] FIGs. 3A to 3C show proposed methods for modulating the inhibitory function of SLAMF7 in T cells. FIG. 3A shows the use of alternatively spliced SLAMF7 resulting in loss of binding to self-ligand (a1), mutated ITSM motif (a2), truncated ectodomain (a3), or mutated endodomain (a4). FIG. 3B shows overexpression of SLAMF7. FIG. 3C shows SAP deficiency or overexpression of tyrosine phosphatases in T cells.

[0019] FIG. 4 shows tumor types with significant expression of SLAMF7. SKCM - Skin Cutaneous Melanoma; UCEC - Uterine Corpus Endometrial Carcinoma; TGCT - Testicular Germ Cell Tumor; OVSC - Ovarian Serous Cystadenocarcinoma; CESC - Cervical Squamous Cell Carcinoma; LUAD - Lung Adenocarcinoma; LAML - Acute MyeloidLeukemia; KIRC - Kidney Renal Clear Carcinoma; DBLC - Diffuse Large B-cell Lymphoma;PAAD - Pancreatic Adenocarcinoma.

[0020] FIGs. 5A to 5D show SLAMF7 lymphocyte infiltration (L) score (FIG. 5A), cytotoxicity (C) score (FIG. 5B), CD8+ cells (FIG. 5C), and CD4+ cells (FIG. 5D) in primary melanoma (TP), metastatic melanoma (TM), and skin cutaneous melanoma (SKCM).

[0021] FIG. 6 shows the correlation between SLAMF7 expression and checkpoint or co-stimulatory molecules in melanoma patients (TCGA).

[0022] FIG. 7 shows differential expression of SLAMF7 in CD8+ T cell clusters between Responders vs Non-Responders.

[0023] FIG. 8 shows the differential expression of SHIP1 in CD8+ T cell clusters between Responders vs Non-Responders.

[0024] FIG. 9 shows the differential expression of SHP1 in CD8+ T cell clusters between Responders vs Non-Responders.

[0025] FIGs. 10A and 10B show embodiments of SLAMF7 as a co-stimulatory domain of a chimeric antigen receptor (CAR) polypeptide.

[0026] FIGs. 11 A to 11 D show embodiments of SLAMF7 as a co-stimulatory domain and SAP as the Fusion protein of a CAR.

[0027] FIGs. 12A and 12B show embodiments of SLAMF7 and SAP as a fusion protein with a CAR.

[0028] FIG. 13 illustrates the binding of potential effector proteins including ZAP-70, She, PI3K(p85), Grb2, Fyn, and Ras-GAP to the TCR ITAMs was analyzed using synthetic phosphorylated peptides containing individual ITAM sequences. Both ZAP-70 and Syk contain tandem SH2 domains that direct their specific and selective interaction with doubly phosphorylated ITAMs. The combinatorial action of Src kinases and ZAP-70 is sufficient for the full activation of downstream signaling pathways and for eliciting T-cell effector responses.

[0029] FIG. 14 shows potential methods of modulating SLAMF7 in CAR T cells.DETAILED DESCRIPTION

[0030] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0032] Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0033] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0034] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0035] Embodiments of the present disclosure will employ unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0036] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, the temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0037] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for thepurposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequences where this is logically possible.Definitions

[0038] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0039] The term “activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0040] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0041] The term “heterologous” as used herein is defined as DNA or RNA sequences or proteins that are derived from different species.

[0042] The term “operably linked” refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0043] The term “overexpressed” tumor antigen or “overexpression” of the tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.

[0044] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subjectcan be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., a physician.

[0045] The term “therapeutically effective” refers to the amount of the composition used being of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0046] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0047] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, a treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0048] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha-amino group of another.

[0049] The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on the amino acid composition of a portion of a protein, portions of a protein, or the entire protein.

[0050] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0051] A “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by thechemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single-chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in a frame into a single nucleic acid, and then express the nucleic acid in an appropriate host cell under the conditions in which the fusion protein is produced.

[0052] A “ligand”, as used herein, refers generally to all molecules capable of reacting with or otherwise recognizing or binding to a receptor on a target cell.

[0053] The term “vector” refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors can be expression vectors.

[0054] The term “expression vector” refers to a vector that includes one or more expression control sequences

[0055] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.CAR Polypeptides

[0056] CARs generally incorporate an antigen recognition domain from the singlechain variable fragments (scFv) of a monoclonal antibody (mAb) with transmembrane signaling motifs involved in lymphocyte activation (Sadelain M, et al. Nat Rev Cancer 2003 3:35-45). Disclosed herein is a chimeric antigen receptor (CAR) that can be expressed in immune effector cells to enhance antitumor activity against cancers.

[0057] The disclosed CAR is generally made up of three domains: an ectodomain, a transmembrane domain, and an endodomain. The ectodomain comprises the ligand binding domain and is responsible for antigen recognition. It also optionally contains a signal peptide (SP) so that the CAR can be glycosylated and anchored in the cell membrane of the immune effector cell. The transmembrane (TM) domain, as its name suggests, connects the ectodomain to the endodomain and resides within the cell membrane when expressed by a cell. The endodomain is the business end of the CAR that transmits an activation signal to the immune effector cell after antigen recognition. The disclosed CARs contain in the endodomain a SLAMF7 co-stimulatory domain.

[0058] In some embodiments, the disclosed CAR is defined by the formula:SP-LBD-HG-TM-SLAMF7-CD3 ;SP-LBD-HG-TM- CD28-SLAMF7-CD3 ;SP-LBD-HG-TM-41 BB-SLAMF7-CD3 ;SP-LBD-HG-TM-CD28-41 BB-SLAMF7-CD3 ;SP-LBD-HG-TM-41 BB-CD28-S LAM F7-CD3 ; wherein “SP” represents an optional signal peptide, wherein “LBD” represents the ligand binding domain, wherein “HG” represents an optional hinge domain, wherein “TM” represents a transmembrane domain, wherein “CD3 ’ represents a CD3 domain, wherein “CD28” represents a CD28 co-stimulatory domain, wherein “41 BB” represents a 41 BB co-stimulatory domain, wherein “SLAMF7” represents an SLAMF7 co-stimulatory domain, and wherein represents a peptide bond or linker.

[0059] Additional CAR constructs are described, for example, in Fresnak AD, et al. Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer. 2016 Aug 23;16(9):566-81, which is incorporated by reference in its entirety for the teaching of these CAR models.

[0060] For example, the CAR can be a TRUCK, Universal CAR, Self-driving CAR, Armored CAR, Self-destruct CAR, Conditional CAR, Marked CAR, TenCAR, Dual CAR, or sCAR.

[0061] TRUCKS (T cells redirected for universal cytokine killing) co-express a chimeric antigen receptor (CAR) and an antitumor cytokine. Cytokine expression may be constitutive or induced by T-cell activation. Targeted by CAR specificity, localized production of pro-inflammatory cytokines recruits endogenous immune cells to tumor sites and may potentiate an antitumor response.

[0062] Universal, allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0063] Self-driving CARs co-express a CAR and a chemokine receptor, which binds to a tumor ligand, thereby enhancing tumor homing.

[0064] CAR T cells engineered to be resistant to immunosuppression (Armored CARs) may be genetically modified to no longer express various immune checkpoint molecules (for example, cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1 )), with an immune checkpoint switch receptor, or may be administered with a monoclonal antibody that blocks immune checkpoint signaling.

[0065] A self-destruct CAR may be designed using RNA delivered by electroporation to encode the CAR. Alternatively, inducible apoptosis of the T cell may be achieved based on ganciclovir binding to thymidine kinase in gene-modified lymphocytes or the more recently described system of activation of human caspase 9 by a small-molecule dimerizer.

[0066] A conditional CAR T cell is by default unresponsive or switched ‘off’, until the addition of a small molecule to complete the circuit, enabling full transduction of both signal 1 and signal 2, thereby activating the CAR T cell. Alternatively, T cells may be engineered to express an adaptor-specific receptor with an affinity for subsequently administered secondary antibodies directed at the target antigen.

[0067] Marked CAR T cells express a CAR plus a tumor epitope to which an existing monoclonal antibody agent binds. In the setting of intolerable adverse effects, administration of the monoclonal antibody clears the CAR T cells and alleviates symptoms with no additional off-tumor effects.

[0068] A tandem CAR (TanCAR) T cell expresses a single CAR consisting of two linked single-chain variable fragments (scFvs) that have different affinities fused to the intracellular co-stimulatory domain(s) and a CD3 domain. TanCAR T cell activation is achieved only when target cells co-express both targets.

[0069] A safety CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR become activated only when encountering target cells that possess the standard CAR target but lack the sCAR target.

[0070] The binding domain of the disclosed CAR is usually an scFv. There are however many alternatives. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g. CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact, almost anything that binds a given target with high affinity can be used as an antigen recognition region.

[0071] The endodomain is the business end of the CAR that after antigen recognition transmits a signal to the immune effector cell, activating at least one of the normal effector functions of the immune effector cell. The effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. The disclosed CARs contain in the endodomain the intracellular domain of NKG2D. However, in some embodiments, the endodomain may further comprise the “intracellular signaling domain” of a T cell receptor (TCR) and optional co-receptors. For example, the endodomain of the CAR can be designed to further comprise the CD3 signaling domain by itself or combined withany other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the cytoplasmic domain of the CAR can further comprise a CD3 chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that are required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1 BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. Thus, while the CAR is exemplified primarily with CD28 as the co-stimulatory signaling element, other costimulatory elements can be used alone or in combination with other co-stimulatory signaling elements.

[0072] In some embodiments, the CAR comprises a hinge sequence. A hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence may be positioned between the antigen recognition moiety and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule. In some embodiments, the hinge sequence is derived from an NK receptor, preferably the same NK receptor from which the intracellular domain is derived.

[0073] The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD11a, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1 , ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1 , CRTAM, Ly9 (CD229) , CD160 (BY55) , PSGL1, CD100 (SEMA4D) , SLAMF6 (NTB-A, Ly108) , SLAM (SLAMF1 , CD150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD162) , LTBR, and PAG / Cbp. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan, and valine will be found at each end of a synthetic transmembrane domain. Ashort oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR.

[0074] In some embodiments, the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain or can be different transmembrane domains.

[0075] In some embodiments, the CAR is a multi-chain CAR, as described in WO20 15 / 039523, which is incorporated by reference for this teaching. A multi-chain CAR can comprise separate extracellular ligand binding and signaling domains in different transmembrane polypeptides. The signaling domains can be designed to assemble in the juxtamembrane position, which forms flexible architecture closer to natural receptors, that confers optimal signal transduction.

[0076] In some embodiments, the binding domain is a single chain variable fragment (scFv) antibody. The affinity / specificity of an scFv is driven in large part by specific sequences within complementarity-determining regions (CDRs) in the heavy (VH) and light (VL) chains. Each Vn and L sequence will have three CDRs (CDR1, CDR2, CDR3).

[0077] In some cases, the binding domain is an affinity-maturated scFv. In some cases, the binding domain has a dissociation constant (KD) for the TAA that is less than 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, or 10 nM.

[0078] In some embodiments, the binding domain is derived from natural antibodies, such as monoclonal antibodies. In some cases, the antibody is human. In some cases, the antibody has undergone an alteration to render it less immunogenic when administered to humans. For example, the alteration comprises one or more techniques selected from the group consisting of chimerization, humanization, CDR-grafting, deimmunization, and mutation of framework amino acids to correspond to the closest human germline sequence.

[0079] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell-mediated immune responses. The binding domain can be an antibody or a natural ligand of the tumor antigen. The selection of the antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, p-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, CD123, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1 , RUI, RU2, SSX2, AKAP-4, LCK, OY-TESI, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1, RUI, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostatespecific antigen (PSA), PAX3, PAP, NY-ESO-1 , LAGE-la, LMP2, NCAM, p53, p53 mutant,Ras mutant, gp100, prostein, OR51 E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1 , VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1 , prostate-carcinoma tumor antigen- 1 (PCTA-1), ML-IAP, MAGE, MAGE-A1.MAD-CT-1, MAD-CT-2, MelanA / MART 1 , XAGE1 , ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1 , Flt3, TAG72, TN Ag, Tie 2, TEM1 , TEM7R, CLDN6, TSHR, LIPK2, and mesothelin. In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvlll, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1 , CA-IX, MUC1 , HER2, and any combination thereof.

[0080] Non-limiting examples of tumor antigens include the following: Differentiation antigens such as tyrosinase, TRP-1 , TRP-2 and tumor-specific multilineage antigens such as MAGE-1 , MAGE-3, BAGE, GAGE-1 , GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY- ESO, pl85erbB2, pl80erbB-3, c-met, nm- 23H1 , PSA, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7- Ag, MOV18, NB / 70K, NY-CO-1, RCASI, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MLIC16, LMP1, EBMA-1 , BARF-1, CS1 , CD319, HER1 , B7H6, L1CAM, IL6, and MET.SLAMF7

[0081] In some embodiments, human SLAMF7 has the amino acid sequence: MAGSPTCLTLIYILWQLTGSAASGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTI QPEGGTIIVTQNRNRERVDFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVY EHLSKPKVTMGLQSNKNGTCVTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWR WGESDMTFICVARNPVSRNFSSPILARKLCEGAADDPDSSMVLLCLLLVPLLLSLFVLGLFL WFLKRERQEEYIEEKKRVDICRETPNICPHSGENTEYDTIPHTNRTILKEDPANTVYSTVEIP KKMENPHSLLTMPDTPRLFAYENVI (SEQ ID NO:1).

[0082] SLAMF7 protein consists of a single extracellular domain (amino acids 1- 223), a transmembrane domain (amino acids 224-246), and a cytoplasmic tail (amino acids 247-327). The extracellular domain contains the IgV and lgC2 domains, which are involved in ligand binding and receptor signaling.

[0083] In some embodiments, the extracellular domain (amino acids 1-223) has the amino acid sequence: MAGSPTCLTLIYILWQLTGSAASGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTI QPEGGTIIVTQNRNRERVDFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVY EHLSKPKVTMGLQSNKNGTCVTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWR WGESDMTFICVARNPVSRNFSSPILARKLCEGAADDPDSSMVLLCLLLVPLLLSLFVLGLFL WFL (SEQ ID NO:2).

[0084] In some embodiments, the transmembrane domain (amino acids 224-246) has the amino acid sequence: KRERQEEYIEEKKRVDICRETPNI (SEQ ID NO:3).

[0085] In some embodiments, the Cytoplasmic tail (amino acids 247-327) has the amino acid sequence: CPHSGENTEYDTIPHTNRTILKEDPANTVYSTVEIPKKMENPHSLLTMPDTPRLFAYENVI (SEQ ID NO:4).

[0086] In some embodiments, the FYN Binding Region has the amino acid sequence: SGENTEYDTIPHTNRTILK (SEQ ID NO:5).

[0087] In some embodiments, the ITSM Motif has the amino acid sequence: TVYSTV (SEQ ID NO:6).

[0088] Therefore, in some embodiments, the disclosed engineered T cell comprises a transgene with the nucleic acid sequence: ATGGCTGGTTCCCCAACATGCCTCACCCTCATCTATATCCTTTGGCAGCTCACAGGGTC AGCAGCCTCTGGACCCGTGAAAGAGCTGGTCGGTTCCGTTGGTGGGGCCGTGACTTT CCCCCTGAAGTCCAAAGTAAAGCAAGTTGACTCTATTGTCTGGACCTTCAACACAACCC CTCTTGTCACCATACAGCCAGAAGGGGGCACTATCATAGTGACCCAAAATCGTAATAGG GAGAGAGTAGACTTCCCAGATGGAGGCTACTCCCTGAAGCTCAGCAAACTGAAGAAGA ATGACTCAGGGATCTACTATGTGGGGATATACAGCTCATCACTCCAGCAGCCCTCCACC CAGGAGTACGTGCTGCATGTCTACGAGCACCTGTCAAAGCCTAAAGTCACCATGGGTC TGCAGAGCAATAAGAATGGCACCTGTGTGACCAATCTGACATGCTGCATGGAACATGG GGAAGAGGATGTGATTTATACCTGGAAGGCCCTGGGGCAAGCAGCCAATGAGTCCCAT AATGGGTCCATCCTCCCCATCTCCTGGAGATGGGGAGAAAGTGATATGACCTTCATCTG CGTTGCCAGGAACCCTGTCAGCAGAAACTTCTCAAGCCCCATCCTTGCCAGGAAGCTC TGTGAAGGTGCTGCTGATGACCCAGATTCCTCCATGGTCCTCCTGTGTCTCCTGTTGGT GCCCCTCCTGCTCAGTCTCTTTGTACTGGGGCTATTTCTTTGGTTTCTGAAGAGAGAGA GACAAGAAGAGTACATTGAAGAGAAGAAGAGAGTGGACATTTGTCGGGAAACTCCTAACATATGCCCCCATTCTGGAGAGAACACAGAGTACGACACAATCCCTCACACTAATAGAA CAATCCTAAAGGAAGATCCAGCAAATACGGTTTACTCCACTGTGGAAATACCGAAAAAG ATGGAAAATCCCCACTCACTGCTCACGATGCCAGACACACCAAGGCTATTTGCCTATGA GAATGTTATCTAG encoding (SEQ ID NO:58) operably linked to an expression control sequence. In some embodiments, the expression control sequence is selected to provide elevated SLAMF7 expression in the T cell. Therefore, in some embodiments, the expression control sequence is a constitutive promoter (Ubiquitin C (UBC) promoter, Human elongation factor 1 alpha (hEF1a) promoter, Phosphoglycerate kinase 1 (PGK1) promoter, Chicken p- actin (CAG) promoter, and Spleen focus-forming virus (SFFV) promoter), an inducible promoter (Tet-On and Tet-Off systems, Ecdysone-inducible system, and Cre-loxP system), or a tissue-specific promoter (CD2 promoter, Lek promoter, and CD4 promoter).Mutated ITSM Domain

[0089] In some cases, the engineered T cell comprises a recombinant nucleic acid encoding a transgenic CD319 (SLAMF7) protein, wherein the transgenic SLAMF7 protein has a mutated or truncated ectodomain with reduced binding to self-ligand; has a mutated ITSM domain to prefer binding by signaling lymphocyte activation molecule (SLAM)- associated protein (SAP) over a tyrosine phosphatase; or any combination thereof.

[0090] For example, in some embodiments, the ITSM domain is replaced with an ITAM domain, which binds SAP but does not bind tyrosine phosphatases. In some embodiments, the ITAM domain comprises the amino acid sequence Yxxl / L(x)6-sYxxl / L. For example, the ITAM domain can have the amino acid sequence YNELNLGRREEYDVL (SEQ ID NO:7), YNELQKDKMAEAYSEI (SEQ ID NO:8), or YQGLSTATKDTYDAL (SEQ ID NO:55).

[0091] In other embodiments, the ITSM domain is mutated by substituting, inserting, or deleting one or more amino acids within the ITSM domain.

[0092] Therefore, in some embodiments, the ITSM domain can be truncated or deleted. For example, one or more of the S305, T306, and V307 amino acids of the ITSM domain can be deleted.

[0093] In other embodiments, the ITSM domain is replaced with a mutant ITSM domain selected from the group consisting of SEQ ID NO:23-54.

[0094] In some embodiments, Y304 of the ITSM domain is substituted. For example, Y304 of the ITSM domain can be substituted with a phosphotyrosine or 3’-phosphotyrosine.

[0095] In some embodiments, charged residues within the ITSM domain or within 10 amino acids of the ITSM domain are replaced with polar or nonpolar amino acids.

[0096] In some embodiments, proline residues are substituted into or inserted into the ITSM domain or within 10 amino acids of the ITSM domain.

[0097] In some embodiments, residues within 10 amino acids of Y304 are mutated to tryptophan or phenylalanine.

[0098] In some embodiments, residues within 10 amino acids of the ITSM motif are mutated to create a steric hindrance that prevents binding of the tyrosine phosphatases but allows binding of SAP.SAP

[0099] In some embodiments, human SAP has the amino acid sequence: MDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYHGYIYTYRVSQTETG SWSAETAPGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEKKSSARSTQGTTGIREDPDVC LKAP (SEQ ID NO:56).EAT2

[0100] In some embodiments, human EAT2 has the amino acid sequence: MDLPYYHGRLTKQDCETLLLKEGVDGNFLLRDSESIPGVLCLCVSFKNIVYTYRIFREKHGY YRIQTAEGSPKQVFPSLKELISKFEKPNQGMVVHLLKPIKRTSPSLRWRGLKLELETFVNSN SDYVDVLP (SEQ ID NO:57).Expression Vectors

[0101] Also disclosed herein are expression vectors and methods for the introduction of exogenous DNA into T cells with concomitant expression of the exogenous DNA in the T cells such as those described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0102] Also disclosed herein are vectors in which a DNA disclosed herein is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve longterm gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0103] In one embodiment, the expression of natural or synthetic nucleic acids encoding the inhibitor is typically achieved by operably linking a nucleic acid to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for the replication and integration of eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for the regulation of the expression of the desired nucleic acid sequence.

[0104] The desired nucleic can be cloned into a number of types of vectors. However, the present invention should not be construed to be limited to any particular vector. Instead, the present invention should be construed to encompass a wide plethora ofvectors that are readily available and / or well-known in the art. For example, a desired polynucleotide of the invention can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0105] In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector. Numerous expression vector systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and / or eukaryote-vector-based systems can be employed for use with the present invention to produce polynucleotides or their cognate polypeptides. Many such systems are commercially and widely available.

[0106] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193.

[0107] 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. A selected gene 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 cells of the subject either in vivo 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 one embodiment, lentivirus vectors are used.

[0108] For expression of the desired polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0109] Additional promoter elements, i.e. , enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that the promoter’s function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacingbetween promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0110] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Pat. Nos. 4,683,202, 5,928,906). Furthermore, it is contemplated that the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0111] Naturally, it will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2001). The promoters employed may be constitutive, tissuespecific, inducible, and / or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0112] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mousemammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the muscle creatine promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on the expression of the polynucleotide sequence which it is operatively linked to when such expression is desired, or turning off the expression when the expression is not desired. Examples of inducible promoters include but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. Further, the invention includes the use of a tissue-specific promoter, which promoter is active only in a desired tissue. Tissue-specific promoters are well known in the art and include, but are not limited to, the HER-2 promoter and the PSA-associated promoter sequences.

[0113] In order to assess the expression of a transgene disclosed herein, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neo and the like.

[0114] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. Reporter genes that encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

[0115] Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (see, e.g., Ui-Tei et al., 2000 FEBS Lett. 479:79-82). Suitable expression systems are well-known and may be prepared using well-known techniques or obtained commercially. Internal deletion constructs may be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. Constructs may then betransfected into cells that display high levels of the siRNA polynucleotide and / or polypeptide expression. In general, the construct with the minimal 5' flanking region showing the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0116] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means. It is readily understood that the introduction of the expression vector comprising the polynucleotide of the invention yields a silenced cell with respect to a protein.

[0117] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0118] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0119] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e. , an artificial membrane vesicle). The preparation and use of such systems are well-known in the art.

[0120] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into a host cell (in vitro, ex vivo, or in vivo). In another aspect, nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, containedas a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DN, or lipid / expression vector-associated compositions are not limited to any particular structure in the solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0121] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR, and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.Immune effector cells

[0122] Also disclosed are immune effector cells that are engineered to express the disclosed CARs (also referred to herein as “CAR-T cells”). These cells are preferably obtained from the subject to be treated (i.e. are autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for the positive selection of the desired immune effector cells. Alternatively, enrichment of the immune effector cells population can be accomplished bynegative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0123] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, the immune effector cells can comprise T lymphocytes.

[0124] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.

[0125] T helper cells (TH cells) assist other white blood cells in immunologic processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1 , TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate a different type of immune response.

[0126] Cytotoxic T cells (Tccells, or CTLs) destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0127] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4+or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0128] Regulatory T cells (Tregcells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactiveT cells that escaped the process of negative selection in the thymus. Two major classes of CD4+Tregcells have been described — naturally occurring Tregcells and adaptive Tregcells.

[0129] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0130] In some embodiments, the T cells comprise a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T cells comprise cytotoxic CD8+T lymphocytes. In some embodiments, the T cells comprise y<5 T cells, which possess a distinct T-cell receptor (TCR) having one y chain and one 5 chain instead of a and p chains.

[0131] Natural-killer (NK) cells are CD56+CD3~ large granular lymphocytes that can kill virally infected and transformed cells, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8+T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization, and can also eradicate MHC-l-negative cells (Narni- Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells, as they may avoid the potentially lethal complications of cytokine storms (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725- 733), and on-target, off-tumor effects. Although NK cells have a well-known role as killers of cancer cells, NK cell impairment has been extensively documented as crucial for the progression of MM (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676; Fauriat C, et al. Leukemia 2006 20:732-733), the means by which one might enhance NK cell-mediated anti- MM activity has been largely unexplored prior to the disclosed CARs.Sources of T cells

[0132] Prior to the expansion and genetic modification of the T cells disclosed herein, a source of T cells can be obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T-cell lines available in the art may be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove theplasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate- buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Again, surprisingly, initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or theHaemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as for example, Ca2+- free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer.Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0133] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer, and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For the isolation of T cells from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, the use of longer incubation times can increase the efficiency of the capture of CD8+T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisanwould recognize that multiple rounds of selection can also be used in the context of this invention. In certain embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

[0134] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11 b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar methods of selection.

[0135] For the isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e. , increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using a high concentration of cells allows a more efficient selection of CD8+T cells that normally have weaker CD28 expression.

[0136] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example,CD4+T cells express higher levels of CD28 and are more efficiently captured than CD8+T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5x106 / ml. In other embodiments, the concentration used can be from about 1 x105 / ml to 1xio6 / ml, and any integer value in between.

[0137] Whether prior to or after genetic modification of the T cells to express a desirable protein (e.g., a CAR), the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 ; and U.S. Patent Application Publication No. 20060121005.

[0138] In some embodiments, the T cells are expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex-associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For the co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating the proliferation of the T cells. To stimulate the proliferation of either CD4+T cells or CD8+T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, and XR-CD28 (Diaclone, Besangon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0139] In certain embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e. , in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in a solution. In one embodiment, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see, for example, U.S. Patent Application Publication Nos.20040101519 and 20060034810 for artificial antigen-presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

[0140] In some embodiments, the two agents are immobilized on beads, either on the same bead, i.e. , “cis,” or to separate beads, i.e. , “trans.” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 :1 ratio of each antibody bound to the beads for CD4+T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti-CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 :1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the invention, the ratio of anti-CD28 antibody to anti- CD3 antibody bound to the beads is greater than 2:1. In one particular embodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1 :50 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :30 CD3:CD28 ratio of antibody bound to beads is used. In one preferred embodiment, a 1 :10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :3 CD3:CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

[0141] Ratios of particles to cells from 1:500 to 500:1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small-sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments, the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between, and in further embodiments the ratio comprises 1:9 to 9:1 and any integer values in between can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however, certain preferred values include 1 :100, 1:50, 1:40, 1:30, 1 :20, 1:10, 1:9, 1:8, 1:7, 1:6, 1 :5, 1 :4, 1 :3, 1:2, 1:1 , 2:1 , 3:1 , 4:1, 5:1, 6:1, 7:1, 8:1 , 9:1 , 10:1, and 15:1 with one preferred ratio being at least 1 :1 particles per T cell. In one embodiment, a ratio of particles to cells of 1 : 1 or less is used. In oneparticular embodiment, a preferred particle: cell ratio is 1:5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1:1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1:1 to 1 :10 (based on cell counts on the day of addition). In one particular embodiment, the ratio of particles to cells is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1 :5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation. One of the skills in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, ratios will vary depending on particle size as well as cell size and type.Therapeutic Application

[0142] Disclosed herein are engineered T cells having heightened cytolytic activity. Therefore, disclosed herein are methods of cellular therapy in a subject where the engineered T cells are infused into a recipient in need thereof. In some embodiments, the engineered T cells are tumor-infiltrating lymphocytes (TILs). In some embodiments, the engineered T cells are further modified to express a chimeric antigen receptor (CAR).

[0143] In some embodiments, the method involves i) expansion of donor T cells, ii) engineering the donor T cells as described herein to enhance T cell activation, and optionally iii) cryopreservation of the cells prior to adoptive cell transfer.

[0144] Ex vivo procedures are well known in the art. Briefly, cells are isolated from a mammal (preferably a human) and modified to enhance its cytolytic activity according to the methods described herein. For example, the cell is modified to have any one or more DGK isoforms inhibited. The heightened immunogenic cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the cell so modified can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

[0145] In some embodiment, the subject has a type of cancer that expresses a tumor-specific antigen.

[0146] In some embodiments, the engineered T cells disclosed herein may be used in combination with a therapeutic agent such as an anti-tumor agent including but not limited to a chemotherapeutic agent, an anti-cell proliferation agent, or any combination thereof. For example, any conventional chemotherapeutic agents of the following non-limiting exemplaryclasses are included in the invention: alkylating agents; nitrosoureas; antimetabolites; antitumor antibiotics; plant alkyloids; taxanes; hormonal agents; and miscellaneous agents.

[0147] Alkylating agents are so named because of their ability to add alkyl groups to many electronegative groups under conditions present in cells, thereby interfering with DNA replication to prevent cancer cells from reproducing. Most alkylating agents are cell cycle non-specific. In specific aspects, they stop tumor growth by cross-linking guanine bases in DNA double-helix strands. Non-limiting examples include busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, mechlorethamine hydrochloride, melphalan, procarbazine, thiotepa, and uracil mustard.

[0148] Anti-metabolites prevent the incorporation of bases into DNA during the synthesis (S) phase of the cell cycle, prohibiting normal development and division. Nonlimiting examples of antimetabolites include drugs such as 5-fluorouracil, 6-mercaptopurine, capecitabine, cytosine arabinoside, floxuridine, fludarabine, gemcitabine, methotrexate, and thioguanine.

[0149] There are a variety of antitumor antibiotics that generally prevent cell division by interfering with enzymes needed for cell division or by altering the membranes that surround cells. Included in this class are the anthracyclines, such as doxorubicin, which act to prevent cell division by disrupting the structure of the DNA and terminating its function. These agents are cell cycle non-specific. Non-limiting examples of antitumor antibiotics include dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin-C, and mitoxantrone.

[0150] Plant alkaloids inhibit or stop mitosis or inhibit enzymes that prevent cells from making proteins needed for cell growth. Frequently used plant alkaloids include vinblastine, vincristine, vindesine, and vinorelbine. However, the invention should not be construed as being limited solely to these plant alkaloids.

[0151] The taxanes affect cell structures called microtubules that are important in cellular functions. In normal cell growth, microtubules are formed when a cell starts dividing, but once the cell stops dividing, the microtubules are disassembled or destroyed. Taxanes prohibit the microtubules from breaking down such that the cancer cells become so clogged with microtubules that they cannot grow and divide. Non-limiting exemplary taxanes include paclitaxel and docetaxel.

[0152] Hormonal agents and hormone-like drugs are utilized for certain types of cancer, including, for example, leukemia, lymphoma, and multiple myeloma. They are often employed with other types of chemotherapy drugs to enhance their effectiveness. Sex hormones are used to alter the action or production of female or male hormones and are used to slow the growth of breast, prostate, and endometrial cancers. Inhibiting the production (aromatase inhibitors) or action (tamoxifen) of these hormones can often be used as an adjunct to therapy. Some other tumors are also hormone dependent. Tamoxifen is anon-limiting example of a hormonal agent that interferes with the activity of estrogen, which promotes the growth of breast cancer cells.

[0153] Miscellaneous agents include chemotherapeutics such as bleomycin, hydroxyurea, L-asparaginase, and procarbazine which are also useful in the invention.

[0154] An anti-cell proliferation agent can further be defined as an apoptosisinducing agent or a cytotoxic agent. The apoptosis-inducing agent may be a granzyme, a Bcl-2 family member, cytochrome C, a caspase, or a combination thereof. Exemplary granzymes include granzyme A, granzyme B, granzyme C, granzyme D, granzyme E, granzyme F, granzyme G, granzyme H, granzyme I, granzyme J, granzyme K, granzyme L, granzyme M, granzyme N, or a combination thereof. In other specific aspects, the Bcl-2 family member is, for example, Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Hrk, Bok, or a combination thereof.

[0155] In additional aspects, the caspase is caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase- 11, caspase-12, caspase-13, caspase-14, or a combination thereof. In specific aspects, the cytotoxic agent is TNF-a, gelonin, Prodigiosin, a ribosome-inhibiting protein (RIP), Pseudomonas exotoxin, Clostridium difficile Toxin B, Helicobacter pylori VacA, Yersinia enterocolitica opT, Violacein, diethylenetriaminepentaacetic acid, irofulven, Diptheria Toxin, mitogillin, ricin, botulinum toxin, cholera toxin, saporin 6, or a combination thereof.

[0156] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1 :

[0157] SLAMFs are CD2-related surface receptors expressed by many innate and adaptive immune cells, including NK cells, Plasma cells, and activated B and T cells. They exhibit heterotypic (Trans) and homotypic (Cis) interaction with one another on immune cells (self-binding receptors) to transmit co-stimulatory signals through the recruiting adaptor proteins either SLAM-associated protein (SH2D1A|SAP) or Ewing’s sarcoma-associated transcript 2 (SH2D1B|EAT-2), which target immunoreceptor tyrosine-based switch motifs (ITSM) in the cytoplasmic tail of the receptors. SLAMs have a variety of functions, including enhancing T cellular proliferation by stimulating IL-4 and IFN-y.

[0158] SLAMF7 was found to be highly expressed on the surface of suppressive CD8+ T cells and its expression correlates with an exhausted phenotype and exhaustion- related transcription factor signatures in T cells. T cells from patients with a high frequency ofSLAMF7+CD8+ T cells exhibited decreased immunoreactivity towards the MART-1 aa26- 35*A27L antigen (Melanoma antigen peptide). SLAMF7+ CD8+ Treg cells could be eliminated using the humanized anti-SLAMF7 antibody Elotuzumab via antibody-dependent cellular phagocytosis in vitro and in vivo. Activation of SLAMF7 on T cells has been reported to induce STAT1 and STAT3 phosphorylation, expression of multiple inhibitory receptors, and transcription factors associated with T cell exhaustion.

[0159] SLAMF7 is expressed on NK cells, and increased antibody-dependent cellular cytotoxicity (ADCC) by NK cells after incubation with Elotuzumab in vitro has been reported. PD-1 blockade was shown to increase the antitumor efficiency of Elotuzumab in mice, a finding that hinted at a possible role of SLAM7 in the mode of action of PD1 in T cells, and vice versa. SLAMF7-chimeric antigen receptor (CAR) T cells eliminate not only myeloma cells but also SLAMF7-high NK and T cells. CARAMBA, a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma. Despite the promising effects of Elotuzumab in preclinical studies, monotherapy with this immunotherapeutic agent failed to elicit objective responses in patients with relapsed / refractory MM due to NK-cell exhaustion in the tumor microenvironment. (This may be due in part to the contribution of negative regulation by cytoplasmic tyrosine phosphatases).

[0160] Based on the expression patterns of SLAMF7 and its signaling intermediates in various immune cells, it is possible that SLAMF7 may have distinct effects in T cell subsets.

[0161] SLAMF7 has been identified as one of the genes enriched in the CD8+ T cell subset of melanoma patients who fail to respond to checkpoint blockade. Our current understanding of the signaling consequences and downstream molecular mechanisms of SLAMF7 in tumor-infiltrating T cells, particularly in the context of solid tumor TME, is limited. Furthermore, it remains unclear how modulation of SLAMF7 and its adaptors could potentially enhance the efficacy of adoptive TILs and CAR T cell therapies in treating solid tumors. In this study, melanoma served as the primary cancer model.

[0162] The signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a small cytoplasmic protein encoded by the gene sh2d1a. Mutations or deletions of this gene have recently been shown to be directly responsible for the X-linked lymphoproliferative syndrome-1 (XLP1). SLAM receptors have been suggested to provide co-stimulatory signals to T cells, and this function is partly dependent on the presence of SAP. A full T cell activation requires a signal that is delivered by the TCR-CD3 complex and additional co-stimulatory signal from other receptors such as CD28, 4-1 BB, CD2, and the SLAM family receptors, among others. In mature CD8 T cells, the absence of SAP has beensuggested to decrease TCR phosphorylation and signaling strength toward Akt and MAPK pathways, impairing restimulation-induced cell death and preventing target lysis.

[0163] FIG. 1 illustrates a proposed SLAMF7 signaling model in T cells in which SLAMF7 can either provide a positive or negative signal during T-cell activation depending on the presence and amounts of adaptor proteins signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) and tyrosine phosphatases. IRF8 is an essential transcription factor in developing intratumoral DCs.

[0164] SAP / SH2D1A is a 128 amino-acid long protein and, along with EAT-2 and ERT, belongs to the SAP family of small adaptor proteins. These small proteins are composed of a single SH2 domain that is followed, in the case of SAP, by a short C-terminal tail. This SH2 domain has been shown to bind to a specific consensus sequence named an Immunoreceptor Tyrosine-based Switch Motif (ITSM), with an amino sequence: TxYxxV / l / L. The ITSM may interact with either SAP / SH2D1A (T cells) or its homolog EAT-2 / SH2D1 B (Myeloid cells) as well as with SH2-containing inhibitory phosphatases, such as the cytoplasmic tyrosine phosphatases: SHP-1 and SHP-2, as well as the lipid phosphatase SHIP-1. Upon activation of SLAMF7, the ITSM motif recruits SAP which modulates signaling through two mechanisms: SAP promotes activating signaling by recruiting and binding to Src tyrosine kinases, such as Fyn and Lek, while also competing with negative regulators like SHP-1 , SHP-2, and SHIP-1. SAP inhibits negative signaling by sterically hindering the docking of SH2-containing phosphatases, including SHP-1, SHP-2, and SHIP-1. Therefore, the precise balance of these adaptors in the cytosol of TILs or activated T cells plays a crucial role in determining whether SLAM receptor engagement induces a positive or negative signal, thereby shaping TCR signaling, T cell differentiation, and effector function.

[0165] FIG. 2 illustrates a proposed mechanism by which the presence and amounts of SAP and tyrosine phosphatases affect T cell activation or inhibition.

[0166] FIGs. 3A to 3C show proposed methods for modulating the inhibitory function of SLAMF7 in T cells. FIG. 3A shows the use of alternatively spliced SLAMF7 resulting in loss of binding to self-ligand (a1), mutated ITSM motif (a2), truncated ectodomain (a3), or mutated endodomain (a4). FIG. 3B shows overexpression of SLAMF7. FIG. 3C shows SAP deficiency or overexpression of tyrosine phosphatases in T cells.

[0167] Potential solutions, therefore, include KO defective SLAMF7; overexpression of SLAMF7 & / or SAP simultaneously to competitively displace cytoplasmic tyrosine phosphatases from SLAMF7’s ITSM; KO / downregulation of tyrosine phosphatases; mutation or truncation of SLAMF7’s ITSM to prefer SAP binding; or replacement of ITSM of SLAMF7 with ITAM motif.

[0168] While the canonical 8-exon receptor inhibits T cell activation through SAP recruitment, the short isoform SLAMF6A17-65 has a strong agonistic effect. Thecostimulatory action depends on protein phosphatase SHP-1 and leads to a cytotoxic molecular profile governed by transcription factors Tbet, Runx3, and Tcf7. In T cells from individual patients treated with immune checkpoint blockade, a shift was noted towards SLAMF6A17-65. Splice-switching antisense oligonucleotides designed to target the SLAMF6 splice junction, enhanced SLAMF6A17-65 in human tumor-infiltrating lymphocytes and improved their capacity to inhibit human melanoma in mice.

[0169] Therefore, in some embodiments, the strategy involves knocking out SLAMF7 in TILs that express a defective SLAMF7 or simultaneously overexpressing SLAMF7 and SAP.

[0170] In some embodiments, the strategy involves knocking out / down tyrosine phosphatases (SHP1 , SHP2, or SHIP1) in adoptively transferred TILs.

[0171] In some embodiments, the strategy involves recessive or dominant-negative mutation (or truncation) of the N-SH2 or C-SH2 domains of the tyrosine phosphatases (SHP1 or SHP2) to induce loss-of-binding to SLAMF7's ITSM or to checkpoint molecules containing the ITSM motif while preserving their other beneficial signaling roles in adoptively transferred TILs.

[0172] In some embodiments, the strategy involves overexpressing SAP in adoptively transferred TILs.

[0173] In some embodiments, the strategy involves overexpressing SAP in adoptively transferred CAR T cells.

[0174] In some embodiments, the strategy involves mutating the SLAMF7’s ITSM motif to inhibit the binding of tyrosine phosphatases (SHP1 , SHIP1, and SHP2) while enhancing the binding of SAP.

[0175] In some embodiments, the strategy involves creating recessive or dominantnegative mutations of tyrosine phosphatases (SHP1 or SHP2) in CAR T cells.

[0176] In some embodiments, the strategy involves incorporating the cytoplasmic domain of ITSM-mutated / wildtype SLAMF7 as the co-stimulatory domain of CAR construct (with / without CD28 / 4-1 BB).

[0177] In some embodiments, the strategy involves incorporating the cytoplasmic domain of ITSM-mutated / wildtype SLAMF7 as the signaling domain of the CAR T construct (with / without CD3^).

[0178] In some embodiments, the strategy involves replacing the ITSM motif of SLAMF7 with CD3 ITAM (#1) as the signaling domain of 1st - 3rd generation CAR T.

[0179] In some embodiments, the strategy involves eliminating / Mutating CD3 s ITAM #1 in the CAR containing the SAP fusion protein or ITSM-mutated SLAMF7 costimulatory domain, to prevent non-specific / tonic signaling of CAR T cells.

[0180] In some embodiments, the strategy involves incorporating I TSM- mutated SLAMF7 as a fusion protein in CAR T constructs w / o cytokine signaling receptors (IL2R, IL15R, IL17R, IL7R, and IL-18R).

[0181] In some embodiments, the strategy involves incorporating a suicide gene / safety switch (iCasp9 or iCD20) in the ITSM-mutated SLAMF7 / SAP CAR to prevent autoimmune reaction or excessive tonic signaling.

[0182] The CD3 chain is a component of the T cell receptor (TCR) complex that contains three immunoreceptor tyrosine-based activation motifs (ITAMs) in its cytoplasmic tail. Upon TCR engagement with antigen, the ITAMs on CD3 are phosphorylated, leading to downstream signaling cascades that activate T cells.

[0183] In CAR T cell therapy, the CD3 chain is often used as the signaling domain in the CAR construct to activate T cells upon recognition of the CAR target antigen. However, the use of CD3 ITAMs in CARs can also lead to non-specific or tonic signaling of CAR T cells, which can result in undesirable toxicities.

[0184] To prevent this non-specific signaling, one strategy is to eliminate one or more of the ITAMs on the CD3 chain in the CAR construct. In the case of the ITSM-mutant SLAMF7 CAR, the ITSM motif has already been introduced to enhance the binding of SH2D1A and SH2D1B while preventing the binding of phosphatases like SHP1, SHP2, and SHIP1. To further prevent non-specific signaling, one of the ITAMs on the CD3 chain can be eliminated, typically ITAM #1, which is closest to the transmembrane domain.

[0185] By eliminating CD3 ITAM #1 in the ITSM-mutant SLAMF7 CAR, the CAR T cells can still specifically recognize and kill target cells expressing SAP, but with reduced risk of non-specific activation and toxicities.

[0186] FIG. 4 shows tumor types with significant expression of SLAMF7. SKCM - Skin Cutaneous Melanoma; UCEC - Uterine Corpus Endometrial Carcinoma; TGCT - Testicular Germ Cell Tumor; OVSC - Ovarian Serous Cystadenocarcinoma; CESC - Cervical Squamous Cell Carcinoma; LUAD - Lung Adenocarcinoma; LAML - Acute Myeloid Leukemia; KIRC - Kidney Renal Clear Carcinoma; DBLC - Diffuse Large B-cell Lymphoma; PAAD - Pancreatic Adenocarcinoma.

[0187] FIGs. 5A to 5D show SLAMF7 lymphocyte infiltration (L) score (FIG. 5A), cytotoxicity (C) score (FIG. 5B), CD8+ cells (FIG. 5C), and CD4+ cells (FIG. 5D) in primary melanoma (TP), metastatic melanoma (TM), and skin cutaneous melanoma (SKCM).

[0188] FIG. 6 shows the correlation between SLAMF7 expression and checkpoint or co-stimulatory molecules in melanoma patients (TCGA).

[0189] FIG. 7 shows differential expression of SLAMF7 in CD8+ T cell clusters between Responder vs Non-Responders. FIG. 8 shows the differential expression of SHIP1 in CD8+ T cell clusters between Responders vs Non-Responders. FIG. 9 shows thedifferential expression of SHP1 in CD8+ T cell clusters between Responders vs NonResponders.

[0190] RT-qPCR and western blot / flow cytometry were used to determine baseline expression of SLAMF7 in naive, activated, and exhausted T cells from HD-PBMC, TIL- Tumor pair melanoma patient samples, and melanoma cell lines. CRISPR was used to KO SLAMF7 in T cells (Confirmed with Flow cytometry and Western blot). SLAMF7 was overexpressed in T cells using lentiviral or adenoviral-mediated approaches. T-cell killing capacity (RTCA), Cytokine release assay, Exhaustion assay, Flow cytometry staining for inhibitory molecules, and 3D co-culture real-time imaging were performed. Cell pellets were collected for scRNA and proteomics was used to profile differential gene expression between SLAMF7KO, and SLAMF7-overexpressing T cells. In vivo studies were conducted using Adoptive transfer of KO vs WT pmel CD8+ T cells into B16F10 tumor-bearing B6 mice.

[0191] CRACC / SLAMF7 / CD319 (also known as CS1) is a member of the signaling lymphocytic activation molecule (SLAM) family. It is a single-pass type I transmembrane glycoprotein expressed on NK cells, subsets of mature dendritic cells, and activated B and T lymphocytes, but not in promyelocytic B or T cell lines. Expression of this protein has been detected in the spleen, lymph node, peripheral blood leukocytes, bone marrow, small intestine, stomach, appendix, lung, and trachea. Homophilic interactions of CRACC / SLAMF7 / CD319 modulate the activity and differentiation of immune cells. CRACC / SLAMF7 / CD319 may function as an inhibitory or activating receptor in immune cells depending on the cellular context and availability of adapter proteins, SH2D1A / SAP and / or SH2D1 B / EAT-2. In the presence of SH2D1 B / EAT-2, CRACC / SLAMF7 / CD319 activates NK cells and B cells. T cells lack SH2D1 B / EAT-2 expression, and therefore CRACC / SLAMF7 / CD319 acts as an inhibitory receptor. In LPS-activated monocytes, CRACC / SLAMF7 / CD319 negatively regulates the production of proinflammatory cytokines. CRACC / SLAMF7 / CD319 is upregulated in multiple myeloma and is implicated in the uncontrolled proliferation of these cells, and thus has become the target for therapeutic intervention. Seven isoforms of CRACC / SLAMF7 / CD319 produced by alternative splicing have been identified.

[0192] FIG. 13 illustrates the binding of potential effector proteins including ZAP-70, She, PI3K(p85), Grb2, Fyn, and Ras-GAP to the TCR ITAMs was analyzed using synthetic phosphorylated peptides containing individual ITAM sequences. Both ZAP-70 and Syk contain tandem SH2 domains that direct their specific and selective interaction with doubly phosphorylated ITAMs. The combinatorial action of Src kinases and ZAP-70 is sufficient for the full activation of downstream signaling pathways and for eliciting T-cell effector responses.

[0193] Proteins that use ITAM-based signaling with the help of associated proteins that contain the motif include CD3y, CD3S, CD3s, TYROBP (DAP12), FcaRI, FcyRI, FcyRII, FcyRIII, Dectin-1 , CLEC-1 , CD28, and CD72. In other studies, the binding of potential effector proteins including ZAP-70, She, PI3K( p85), Grb2, Fyn, and Ras-GAP to the TCR ITAMs was analyzed using synthetic phosphorylated peptides containing individual ITAM sequences.

[0194] ITIM & ITSM binding Phosphatases include : SHP1 , SHIP1 , and SHP2.

[0195] Common to SHP1 / SHP2-recruiting immunoreceptors is the presence of one or both types of pY-based motifs in their intracellular domains (ICD): immunoreceptor tyrosinebased inhibitory motif (ITIM, consensus sequence S / l / V / LxYxxl / V / L) (Burshtyn et al., 1996; Daeron et al., 1995) and immunoreceptor tyrosine-based switch motif (ITSM, consensus sequence TxYxxV / l / L) (Cannons et al., 2011). Once phosphorylated, ITIM and ITSM act as docking sites for the SH2 domains of SHP1 / SHP2. Moreover, ITSM in some receptors interactswith SH2-containing adaptor proteins SH2D1A and SH2D1 B (Cannons et al., 2011). Despite the general presumption that ITIM / ITSM-containing receptors recruit both SHP1 and SHP2, increasing evidence suggests that these receptors exhibit differential phosphatase-binding specificities. For example, PD-1 strongly recruits SHP2, but not SHPI , in both T cells and B cells.

[0196] Disclosed are mutations of the ITSM motif of SLAM F7.

[0197] In some embodiments, the tyrosine residues are mutated. The ITSM motif of SLAMF7 contains one tyrosine residue (Y304) which is critical for the interaction with SHP1 , SHP2, and SHIP1 . By mutating this tyrosine residue to another amino acid, it is possible to disrupt their interaction with SHP1 , SHP2, and SHIP1 while promoting selective binding of SAP. For example, mutating residues in the vicinity of Y304 to bulkier amino acids such as tryptophan or phenylalanine could achieve this effect.

[0198] In some embodiments, residues adjacent to the ITSM motif are mutated to create a steric hindrance. By mutating residues adjacent to the ITSM motif, it may be possible to create a steric hindrance that prevents the binding of SHP1 , SHP2, or SHIP1 while still allowing for the binding of SAP.

[0199] In some embodiments, tyrosine residues are substituted with tyrosine analogs. Replacing tyrosine in SLAMF7's ITSM motif with phosphotyrosine or 3’-phosphotyrosine could potentially enhance the binding of SAP to the ITSM while preventing the binding of tyrosine phosphatases.

[0200] In some embodiments, the electrostatic properties of the ITSM motif are altered. Mutating charged residues within or adjacent to the ITSM motif may alter its electrostatic properties and affect its binding specificity for different signaling molecules. For example, replacing charged residues with polar or nonpolar amino acids may change the orientation of the ITSM motif and affect its binding properties.

[0201] In some embodiments, proline residues are introduced to disrupt SHP1 / SHP2 binding. The introduction of proline residues within the ITSM motif or adjacent to it may disrupt the binding of SHP1 and SHP2, as these proteins have a proline-rich region that mediates their interaction with the ITSM motif. This could potentially enhance the binding of SAP by reducing competition for binding sites.

[0202] In some embodiments, the ITSM motif is truncated to remove the tyrosine residue or any amino acids flanking the tyrosine residue in the ITSM motif. Shortening the ITSM motif by deleting amino acids may disrupt the binding of SHP1 , SHP2, or SHIP1 whilemaintaining or enhancing the binding of SAP. For example, truncation of the last three amino acids (TVY) of the ITSM motif in human SLAMF7 has been shown to enhance SAP binding.

[0203] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0204] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A chimeric antigen receptor (CAR) polypeptide, comprising a signaling peptide, a ligand binding domain, a hinge domain, a transmembrane domain, and an endodomain that comprises a CD3 domain and a SLAMF7 co-stimulatory domain.

2. The CAR polypeptide of claim 1 , wherein the endodomain further comprises a CD28 costimulatory domain, 4-1 BB co-stimulatory domain, or a combination thereof.

3. The polypeptide of claim 1 or 2, wherein the CAR polypeptide is defined by the formula:wherein “SP” represents an optional signal peptide, wherein “LBD” represents the ligand binding domain, wherein “HG” represents an optional hinge domain, wherein “TM” represents a transmembrane domain, wherein “CD3 ” represents a CD3 domain, wherein “CD28” represents a CD28 co-stimulatory domain, wherein “41 BB” represents a 41 BB co-stimulatory domain, wherein “SLAMF7” represents an SLAMF7 co-stimulatory domain, and wherein represents a peptide bond or linker.

4. The CAR polypeptide of any one of claims 1 to 3, wherein the SLAMF7 domain has a mutated ITSM domain to prefer binding by signaling lymphocyte activation molecule (SLAM)- associated protein (SAP) over a tyrosine phosphatase.

5. The CAR polypeptide of claim 4, wherein the ITSM domain is replaced with an ITAM domain.

6. The CAR polypeptide of claim 4, wherein the ITAM domain comprises the amino acid sequence Yxxl / L(x)6-8Yxxl / L.

7. The CAR polypeptide of claim 6, wherein the ITAM domain comprises the amino acid sequence YNELNLGRREEYDVL (SEQ ID NO:7), YNELQKDKMAEAYSEI (SEQ ID NO:8), or YQGLSTATKDTYDAL (SEQ ID NO:55).

8. The CAR polypeptide of claim 4, wherein the ITSM domain is replaced with a mutant ITSM domain selected from the group consisting of SEQ ID NO:23-54.

9. The CAR polypeptide of claim 4, wherein the ITSM domain is truncated or deleted.

10. The CAR polypeptide of claim 9, wherein the wherein one or more of the S305, T306, and V307 amino acids of the ITSM domain are deleted.

11. The CAR polypeptide of claim 4, wherein Y304 of the ITSM domain is substituted.

12. The CAR polypeptide of claim 11 , wherein Y304 of the ITSM domain is substituted with phosphotyrosine or 3’-phosphotyrosine.

13. The CAR polypeptide of claim 4, wherein charged residues within the ITSM domain or within 10 amino acids of the ITSM domain are replaced with polar or nonpolar amino acids.

14. The CAR polypeptide of claim 4, wherein proline residues are substituted into or inserted into the ITSM domain or within 10 amino acids of the ITSM domain.

15. The CAR polypeptide of claim 4, wherein residues within 10 amino acids of Y304 are mutated to tryptophan or phenylalanine.

16. The CAR polypeptide of claim 4, wherein residues within 10 amino acids of the ITSM motif are mutated to create a steric hindrance that prevents binding of the tyrosine phosphatases but allow binding of SAP.

17. The CAR polypeptide any one of claims 1 to 16, further comprising a signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) linked to the endodomain by a cleavable peptide.

18. The CAR polypeptide any one of claims 1 to 17, further comprising an EAT2 protein linked to the endodomain by a cleavable peptide.

19. A chimeric receptor comprising a signaling peptide, an ectodomain, and transmembrane domain, wherein the ectodomain comprises a SLAMF7 protein and / or a signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) protein.

20. The chimeric receptor of claim 19, wherein the SLAMF7 protein has a mutated ITSM domain to prefer binding by signaling lymphocyte activation molecule (SI-AM)-associated protein (SAP) over a tyrosine phosphatase.

21. A fusion protein comprising a chimeric antigen receptor (CAR) polypeptide that comprises a ligand binding domain, a hinge domain, a transmembrane domain, and an endodomain, wherein the chimeric receptor of claim 19 or 20 is linked to the endodomain of the CAR polypeptide by a cleavable linker.

22. An isolated nucleic acid sequence encoding the CAR polypeptide of any one of claims 1 to 18, the chimeric receptor of claim 19 or 20, or the fusion protein of claim 21.

23. A vector comprising the isolated nucleic acid sequence of claim 22 operably linked to an expression control sequence.

24. An immune effector cell engineered to express the CAR polypeptide of any one of claims 1 to 18, the chimeric receptor of claim 19 or 20, or the fusion protein of claim 21.

25. The immune effector cell of claim 24, wherein the cell is selected from the group consisting of an a[3T cell, y5T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T (Treg) cell, or any combination thereof.

26. A method of providing an anti-tumor immunity in a subject with a cancer, the method comprising administering to the subject an effective amount of the immune effector cell of claim 24 or 25, thereby providing an anti-tumor immunity in the mammal.

27. The method of claim 26, further comprising administering to the subject a checkpoint inhibitor.

28. The method of claim 27, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.

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