Chimeric antigen receptors (CARS) targeting met

Abstract

The present invention provides chimeric antigen receptors (CARs) comprising an extracellular domain which specifically binds to Mesenchymal Epithelial Transition factor (MET); a transmembrane (TM) domain; a co-stimulatory signaling domain; and, a primary intracellular signaling domain. The application further relates to polynucleotides and recombinant vectors encoding the CARs, as well as to host cells and methods for producing host cells that express the CARs. The application further relates to pharmaceutical compositions comprising the CAR modified cells. Methods for stimulating elimination of a MET-expressing cell and for treating a MET-positive cancer using the CAR modified cells in a subject are also disclosed.

Description

Attorney Docket No: 251609.000137 CHIMERIC ANTIGEN RECEPTORS (CARS) TARGETING MET CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 651,094, filed May 23, 2024, the disclosure of which is herein incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R21CA263437 and R21CA282629 awarded by National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 18, 2025, is named 251609_000137_SL.xml and is 125,643 bytes in size. FIELD OF THE INVENTION

[0004] The present invention relates to chimeric antigen receptors (CARs) comprising an extracellular domain which specifically binds to Mesenchymal Epithelial Transition factor (MET); a transmembrane (TM) domain; a co-stimulatory signaling domain; and, a primary intracellular signaling domain. The application further relates to polynucleotides and recombinant vectors encoding the CARs, as well as to host cells and methods for producing host cells that express the CARs. The application further relates to pharmaceutical compositions comprising the CAR modified cells. Methods for stimulating elimination of a MET-expressing cell and for treating a MET-positive cancer using the CAR modified cells in a subject are also disclosed. BACKGROUND

[0005] MET overexpression is seen in multiple types of tumors, including approximately 50% of triple negative breast cancers1, 60% of non-small cell lung cancer2, 30-70% of colorectal cancers3, 50-90% of gastric cancers2, as well as those derived from liver, pancreas, kidney, head and neck regions, and brain3. The MET oncogene encodes the MET receptor tyrosine kinase (MET RTK). MET is normally expressed at low levels in tissues such as skin and liver,Attorney Docket No: 251609.000137 and plays important roles during normal organ development and tissue repair4. Activation of MET by its ligand, hepatocyte growth factor (HGF), promotes tumor proliferation, metastasis, and resistance to apoptosis. Overexpression of MET, either through transcriptional dysregulation, MET gene amplification or mutation, is a major mechanism that mediates resistance to primary therapies5. Increased HGF / MET signaling was observed in ~70% melanoma treated with BRAF inhibitors6and 20-30% non-small cell lung cancers treated with EGFR inhibitors7.

[0006] To date, FDA-approved MET inhibitors show only limited efficacy except in tumors harboring specific MET alterations8. However, these alterations, such as MET exon 14 skip, MET amplifications, or MET kinase domain mutations are rare and account for <1% in cBioPortal database3–5.

[0007] Several pilot studies have used chimeric antigen receptor (CAR) expressed in T cells to target MET positive tumors with limited efficacy. In chimeric antigen receptor T-cell (CAR- T) therapy, genetically modified T cells express an artificial, chimeric protein that combines an extracellular target-recognition motif and an intracellular T-cell activation motif. A CAR-T cell therapy therefore combines the tumor-targeting specificity of an antibody and the cytotoxic function of T cells to kill tumors. A recently published Phase I trial showed safety in intravenously injected MET CAR-T cells targeting triple negative breast cancers and melanoma. Nonetheless, all seven patients exhibited either stable or progressive disease, suggesting limited efficacy of the MET CAR-T therapy.

[0008] Many current MET CAR constructs lead to tonic signaling in T cells, a state in which T cells become activated irrespective of the presence of the target antigens. Tonic signaling leads to early T cell exhaustion, which correlates with elevated expression of multiple inhibitory receptors, impaired effector functions, decreased proliferation, and reduced cytokine production. SUMMARY OF THE INVENTION

[0009] As specified in the Background section above, there is a great need in the art for optimized MET CAR construct designs which demonstrate reduced tonic signaling and enhanced cytotoxicity and can target a vast majority of MET-positive tumors. Such MET CAR constructs can be used, e.g., in the development of effective MET CAR-T cell therapies for patients suffering from MET-positive tumors. The present application addresses these and other needs.Attorney Docket No: 251609.000137

[0010] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising: a) an extracellular domain which specifically binds to MET and comprises at least (i) a VHH domain comprising a complementarity determining region (CDR) 1, CDR2, and CDR3, and wherein the CDR1, CDR2, and CDR3 comprise: 1) SEQ ID NOs: 2, 4, and 6, respectively; 2) SEQ ID NOs: 34, 36, and 38, respectively; 3) SEQ ID NOs: 47, 49, and 51, respectively; 4) SEQ ID NOs: 59, 61, and 63, respectively; or 5) SEQ ID NOs: 72, 74, and 76, respectively, or (ii) a scFv comprising a light chain variable domain (VL) comprising a light chain CDR1 (LCDR1), LCDR2, and LCDR3 comprising SEQ ID NOs: 90, 91, and 92, respectively, and a heavy chain variable domain (VH) comprising a heavy chain CDR1 (HCDR1), HCDR2, and HCDR3 comprising SEQ ID NOs: 86, 87, and 88, respectively; b) a transmembrane (TM) domain; c) a co-stimulatory signaling domain; and d) a primary intracellular signaling domain.

[0011] In some embodiments, the extracellular domain comprises at least a VHH domain comprising the amino acid sequence of SEQ ID NO: 9, 41, 53, 65, or 79, or a sequence having at least 80% identity thereto.

[0012] In some embodiments, the nucleotide sequence encoding the at least a VHH domain comprises the nucleotide sequence of SEQ ID NO: 8, 40, 52, 64, or 78, or a sequence having at least 80% identity thereto.

[0013] In some embodiments, the extracellular domain comprises two or more VHH domains.

[0014] In some embodiments, the extracellular domain comprises an scFv comprising a VL comprising the amino acid sequence of SEQ ID NO: 93, or a sequence having at least 80% identity thereto; and a VH comprising the amino acid sequence of SEQ ID NO: 89, or a sequence having at least 80% identity thereto.

[0015] In some embodiments, the extracellular domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 70, or a sequence having at least 80% identity thereto. In some embodiments, the nucleotide sequence encoding the scFv comprises the nucleotide sequence of SEQ ID NO: 32, or a sequence having at least 80% identity thereto.Attorney Docket No: 251609.000137

[0016] In some embodiments, the primary intracellular signaling domain is CD3^ signaling domain.

[0017] In some embodiments, the CD3^ signaling domain comprises the amino acid sequence of SEQ ID NO: 19, or a sequence having at least 80% identity thereto. In some embodiments, the nucleotide sequence encoding the CD3^ signaling domain comprises the nucleotide sequence of SEQ ID NO: 18, or a sequence having at least 80% identity thereto.

[0018] In some embodiments, the TM domain is derived from CD8^ or CD28.

[0019] In some embodiments, the CAR further comprises a hinge domain.

[0020] In some embodiments, the CAR comprises a hinge and TM domain derived from CD8^. In some embodiments, the hinge and TM domain derived from CD8^ comprises the amino acid sequence of SEQ ID NO: 15, or a sequence having at least 80% identity thereto. In some embodiments, the nucleotide sequence encoding the hinge and TM domain derived from CD8^ comprises the nucleotide sequence of SEQ ID NO: 14, or a sequence having at least 80% identity thereto.

[0021] In some embodiments, the CAR comprises a hinge and TM domain derived from CD28. In some embodiments, the hinge and TM domain derived from CD28 comprises the amino acid sequence of SEQ ID NO: 25, or a sequence having at least 80% identity thereto. In some embodiments, the nucleotide sequence encoding the hinge and TM domain derived from CD28 comprises the nucleotide sequence of SEQ ID NO: 24, or a sequence having at least 80% identity thereto.

[0022] In some embodiments, the co-stimulatory signaling domain is derived from 4-1BB (CD137) or CD28. In some embodiments, the co-stimulatory signaling domain derived from 4-1BB (CD137) comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having at least 80% identity thereto. In some embodiments, the nucleotide sequence encoding the co- stimulatory signaling domain derived from 4-1BB (CD137) comprises the nucleotide sequence of SEQ ID NO: 16, or a sequence having at least 80% identity thereto. In some embodiments, the co-stimulatory signaling domain derived from CD28 comprises the amino acid sequence of SEQ ID NO: 27, or a sequence having at least 80% identity thereto. In some embodiments, the nucleotide sequence encoding the co-stimulatory signaling domain derived from 4-1BB (CD137) comprises the nucleotide sequence of SEQ ID NO: 26, or a sequence having at least 80% identity thereto.

[0023] In some embodiments, the CAR comprises a hinge and TM domain derived from CD8^, a co-stimulatory signaling domain derived from 4-1BB (CD137), and a CD3^ signaling domain.Attorney Docket No: 251609.000137

[0024] In some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 21, 31, 43, 45, 55, 57, 67, 69, 81, 83, or 85, or a sequence having at least 80% identity thereto. In some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 21 or 43, or a sequence having at least 80% identity thereto.

[0025] In another aspect, the present disclosure provides a polynucleotide molecule comprising a nucleotide sequence encoding a CAR described herein. In some embodiments, the nucleotide sequence encoding the CAR comprises SEQ ID NO: 20, 30, 42, 44, 54, 56, 66, 68, 90, 82, or 84, or a sequence having at least 80% identity thereto. In some embodiments, the nucleotide sequence encoding the CAR comprises SEQ ID NO: 20 or 42, or a sequence having at least 80% identity thereto.

[0026] In some embodiments, the nucleotide sequence encoding the CAR is operably linked to a promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) or a T7 promoter.

[0027] In some embodiments, the nucleotide sequence encoding the CAR is flanked by 5’ and 3’ homology arms for genomic insertion.

[0028] In some embodiments, the nucleic acid molecule is mRNA.

[0029] In another aspect, provided herein is a recombinant vector comprising a polynucleotide molecule described herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector.

[0030] In another aspect, provided herein is an isolated host cell comprising a CAR described herein.

[0031] In another aspect, provided herein is an isolated host cell comprising a polynucleotide molecule described herein.

[0032] In another aspect, provided herein is an isolated host cell comprising a vector described herein.

[0033] In some embodiments of the isolated host cell described herein, the nucleotide sequence encoding the CAR has been inserted into the T-cell Receptor Alpha Constant (TRAC) locus or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) locus. In some embodiments, the TRAC gene is knocked out.

[0034] In some embodiments, the isolated host cell described herein is a mammalian cell. In some embodiments, the isolated host cell is selected from a cytotoxic cell, a T cell, a stem cell, a progenitor cell, and a cell derived from a stem cell or a progenitor cell. In some embodiments, the T cell is selected from CD8+ T cells, CD4+ T cells, T-helper cells, cytotoxic T cells, T-Attorney Docket No: 251609.000137 regulatory cells (Treg), and gamma-delta T cells. In some embodiments, the cytotoxic cell is a cytotoxic T cell or a cytotoxic NK cell.

[0035] In some embodiments, the host cell has been activated and / or expanded ex vivo.

[0036] In some embodiments, the host cell is an allogeneic cell.

[0037] In some embodiments, the host cell is an autologous cell. In some embodiments, the host cell has been isolated from a subject having a cancer. In some embodiments, the subject is human.

[0038] In another aspect, provided herein is a pharmaceutical composition comprising a host cell described herein and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents comprise an immunomodulatory agent, a chemotherapeutic agent, an anti-angiogenic agent, an anti-inflammatory agent, or a cell death inducer, or a combination thereof. In some embodiments, the cell death inducer is a^BCL-2 family inhibitor. In some embodiments, the cell death inducer is a^BCL-XL inhibitor. In some embodiments, the BCL-2 family inhibitor is selected from Navitoclax (ABT-263), Venetoclax, A-1331852, WEHI-539, A-1155463, and ABT-737, or a combination thereof.

[0039] In another aspect, provided herein is a method for producing a host cell described herein comprising genetically modifying said cell with a polynucleotide molecule or vector described herein. In some embodiments, the genetic modification is conducted ex vivo. In some embodiments, the method further comprises activation and / or expansion of the cell ex vivo.

[0040] In another aspect, provided herein is a method for stimulating elimination of a MET- expressing cell in a subject in need thereof, said method comprising administering to the subject an effective amount of the host cells described herein or the pharmaceutical composition described herein.

[0041] In another aspect, provided herein is a method for treating a MET-positive cancer in a subject in need thereof, said method comprising administering to the subject an effective amount of the host cells or the pharmaceutical composition described herein. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from a breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, prostate cancer, pancreatic cancer, kidney cancer, bladder cancer, testicular cancer, ovarian cancer, endometrial cancer, cervical cancer, neuroendocrine cancer, head and neck cancer, brain cancer, sarcoma, melanoma, and lymphoma. In some embodiments, the breast cancer is a triple negative breastAttorney Docket No: 251609.000137 cancer. In some embodiments, the lung cancer is a non-small cell lung cancer (NSCLC). In some embodiments, the lung cancer is a small cell lung cancer (SCLC).

[0042] In various embodiments of the methods described herein, the method may comprise administering to the subject one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents comprise an immunomodulatory agent, a chemotherapeutic agent, an anti-angiogenic agent, an anti-inflammatory agent, a cell death inducer, a surgery, a radiotherapy, a bone marrow transplantation, or a combination thereof. In some embodiments, the cell death inducer is a^BCL-2 family inhibitor. In some embodiments, the cell death inducer is a^BCL-XL inhibitor. In some embodiments, the BCL-2 family inhibitor is selected from Navitoclax (ABT-263), Venetoclax, A-1331852, WEHI-539, A-1155463, and ABT-737, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figures 1A-1F depict MET expression in solid tumor cell lines and in a human biopsy of a papillary renal cell carcinoma (PRCC). Histograms of MET expression in tumor cell lines triple negative breast cancer (MDA-MB-231) (Figure 1A), diffuse large B-cell lymphoma (Karpas-422) (Figure 1B), lung adenocarcinoma (A549) (Figure 1C), hepatocellular carcinoma (HepG2) (Figure 1D), and renal cell carcinoma (786-O) (Figure 1E) are shown. The x-axis represents the intensity of MET expression and the y-axis represents the cell count. Lined area = IgG negative control. Dotted area = MET expression probed by an anti-MET antibody conjugated with allophycocyanin (APC) fluorophore. MET expression is also shown in a human biopsy of a papillary renal cell carcinoma (PRCC), with negative expression in the neighboring background tissues (Figure 1F).

[0044] Figures 2A-2B demonstrate improved in vitro cytotoxicity of nanobody-based CARs PC15 and PC16. Second generation design of MET CAR (Figure 2A). PC15 and PC16 CAR demonstrated superior cytotoxicity against A549 cells in comparison with 5D5 MET CAR-T (Figure 2B). Right, representative images showing PC16 nanobody-based MET CAR-treated tumors showed decreased proliferation. Quantification of cytotoxicity using flow cytometry showed significantly improved cytotoxicity of PC15 and PC16 CAR compared with earlier versions of MET CARs 5D5 and PC11 (Figure 2C).

[0045] Figure 3 shows expression of T-cell activation marker cluster of differentiation 69 (CD69) as a function of the amount of target. With increasing amount of target (decreasing effector:target [E:T] ratio), a CAR without tonic signaling showed a target-dependent increaseAttorney Docket No: 251609.000137 in CD69, as seen for PC15, PC16, and CD19 CAR. PC11 CAR showed persistent CD69 activation even without target.

[0046] Figure 4 demonstrates that MET positive tumors are specifically sensitive to PC16 MET CAR-T. MDA-MB-231, BoM, and LM are three breast cancer cell lines that express MET but not CD19. While CD19 CAR-T only mediated basal cell death, PC16 MET CAR-T showed up to 70% of tumor lysis.

[0047] Figures 5A-5C show CRISPR / Cas9 targeting of T-cell Receptor Alpha Constant (TRAC) locus in human T cells. Schematic of TRAC targeting site (top) and knock-in DNA template design (bottom) (Figure 5A). Flow cytometry of primary human T cells electroporated with PC16 CAR homology-directed repair template (HDRT) and Cas9 / sgRNA (Figure 5B). Successful knock-in is evidenced by reduced TCR expression (y-axis) and increased CAR expression (x-axis). Cytotoxicity of TRAC-PC16 CAR-T cells versus mock T cells against Karpas-422 cells at multiple E:T ratios at 16 hours (Figure 5C).

[0048] Figure 6 demonstrates in vivo efficacy of PC16 mRNA CAR against triple-negative breast cancer (TNBC) metastatic to lungs. Forkhead box N1 (Foxn1) athymic mice were intravenously (IV) injected with the LM2 human cancer cell line. This is a triple-negative breast cancer (TNBC) cell line selected to metastasize to the lung and engineered to express luciferase. After approximately 10 days, PBS, 5 million mock electroporated T cells (“Mock T”), or 5 million MET CAR-T cells (“PC16”) were IV injected on days 1, 5, and 14 (arrows). Luminescence was measured as Normalized Total Flux (y-axis).

[0049] Figures 7A-7C demonstrate the effectiveness of select BCL-2 family inhibitors against MDA-MB-231. Figure 7A shows the functional mechanism of BCL-2 family inhibitors on cancer cells. Figure 7B displays the protein targets of the tested BCL-2 family inhibitors. Figure 7C shows dose-dependent killing of MDA-MB-231 cells using four different BCL-2 family inhibitors.

[0050] Figures 8A-8C show that BCL-XL inhibition enhances MET CAR-T killing in solid tumors. Figure 8A shows a schematic of the experimental setup. Figure 8B shows tumor killing of MDA-MB-231 or A549 cells using PC16 MET CAR-T cells in combination with Navitoclax or Venetoclax. Figure 8C shows tumor killing of MDA-MB-231 cells using PC16 MET CAR-T cells in combination with ABT-199, S-63845, or A-1331852.

[0051] Figures 9A-9B demonstrate the avidity of nanobody MET CAR-T cells.Attorney Docket No: 251609.000137 DETAILED DESCRIPTION

[0052] Given the lack of effective MET CAR-T therapy to date, described herein are exemplary methods for the systemic screening and identification of MET CAR constructs with improved cytotoxicity (e.g., in vitro cytotoxicity) and avidity, in comparison with published MET CARs. Improvement of MET CAR-T function through CRISPR / Cas9 genome editing and combination therapies with cell death inducers are also described. Compositions and methods of the present disclosure can benefit patients with MET-positive tumors, especially in the population that is refractory to primary therapy. The present disclosure describes, in particular, the use of nanobodies as the target-recognition motif in MET CAR-T designs, which dramatically reduces tonic signaling and improves cytotoxicity of MET CAR-T, as well as CRISPR / Cas9 genome editing to improve MET CAR-T to treat solid tumors.

[0053] In certain aspects, the present disclosure provides novel CAR constructs that, when added to T cells or natural killer (NK) cells, can direct cellular killing against target cells expressing MET. In various embodiments, CARs described herein can contain, for example, without limitation, an extracellular domain which specifically binds to MET, a hinge domain, a transmembrane domain, a co-stimulatory signaling domain and / or a CD3^ signaling domain. In some embodiments, the extracellular domain can include, e.g., at least one VHH or single chain variable fragment (scFv). In some embodiments, two or more VHHs can be added together in combinations. Non-limiting examples of transmembrane domains include CD8 and CD28 domains. Co-stimulatory signaling domains can include, e.g., CD28 and 4-1BB. MET CAR designs described herein can exhibit improved cytotoxicity against MET positive cells, with decreased tonic signaling, and can be differentially activated by different levels of target antigen expression.

[0054] As described in further detail below, various combinations of the above domains were tested in both T cell activation and cytotoxicity experiments to find advantageous CAR-T constructs. An exemplary CAR-T construct based on in vitro tests can include one of the nanobodies described herein, the CD8 hinge and transmembrane domains, and 4-1BB co- stimulatory domains. Such nanobody-based MET CAR-T cells show the highest killing, the least tonic signaling, and the ability to kill cells with the lowest amount of target antigen.

[0055] Given the problem of scFv-induced tonic signaling in CAR-T, some CARs described herein bypass the traditional scFv, and instead use antibodies derived from camelid and llamas called nanobodies. While use of nanobodies in CAR-T has gained attention in recent years, none has been published in the MET CAR-T field.Attorney Docket No: 251609.000137

[0056] Experimentally comparing nanobody-based MET CAR-T to one of the scFv-based MET CAR-T shows superiority of killing and avidity and less tonic signaling. In some embodiments, the 4-1BB co-stimulatory domain can be preferred to the CD28 co-stimulatory domain due to decreased tonic signaling. Finding the optimal combination of domains for the CAR constructs of the disclosure was surprising and could not have been predicted without performing the experiments. Definitions

[0057] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present application. For purposes of the present application, the following terms are defined.

[0058] It is understood that embodiments of the application described terms of “comprising” herein include “consisting of” and / or “consisting essentially of” embodiments.

[0059] The term “chimeric antigen receptor” or “CAR” as used herein refers to a cell- surface receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and a intracellular domain comprising a primary intracellular signaling domain (e.g., a lymphocyte activation domain) and optionally a co-stimulatory signaling domain(s), all in a combination that is not found together in nature on a single protein. This includes, without limitation, receptors wherein the extracellular domain and the intracellular domain are not found together in nature on a single receptor protein. The chimeric antigen receptors of the present disclosure can be used with lymphocytes such as T cells and natural killer (NK) cells.

[0060] The terms “T cell” and “T lymphocyte” are interchangeable and used synonymously herein. Non-limiting examples of T cells include naive T lymphocytes, thymocytes, mature T lymphocytes, immature T lymphocytes, activated T lymphocytes, or resting T lymphocytes. A T cell may be a T helper (Th) cell, e.g., a T helper 1 (Th1) or a T helper 2 (Th2) cell. The T cell may be a cytotoxic T cell (CTL; CD8+ T cell), a helper T cell (HTL; CD4+ T cell) CD4+ T cell, CD4+CD8+ T cell, a tumor infiltrating cytotoxic T cell (TIL; CD8+ T cell), or any other subclass of T cells. Additional exemplary populations of T cells include memory T cells and naive T cells. Further included are “NKT cells”, which comprise a specialized population of T cells that express a semi-invariant ^^ T-cell receptor, as well as various molecular markers that are commonly associated with natural killer cells (NK cells), e.g., NK1.1. NKT cells include and NK1.1- and NK1.1+, as well as, CD4-, CD4+, CD8- and CD8+ cells. The T cellAttorney Docket No: 251609.000137 receptor (TCR) on NKT cells is distinct insofar as it recognizes glycolipid antigens presented by the Major Histocompatibility Complex I (MHC-I)-like molecule CD Id. Also included are “Gamma-delta T cells (^^ T cells)” which may refer to a specialized subset of T cells possessing a distinct TCR on their surface, and unlike most T cells in which the TCR is comprised of two glycoprotein chains called ^- and ^-TCR chains, the TCR in ^^ T cells is comprised of a ^- chain and a ^-chain. ^^ T cells can contribute to immunosurveillance and immunoregulation, and may be a key source of IL-17 and induce a strong CD8+ cytotoxic T cell response. Also included are “regulatory T cells” or “Tregs”, which refer to T cells that suppress an abnormal or excessive immune response and contribute to immune tolerance. Tregs are commonly transcription factor Foxp3-positive CD4+T cells and can also comprise transcription factor Foxp3-negative regulatory T cells that are IL-10-producing CD4+T cells.

[0061] The term “antibody” refers to all isotypes of immunoglobulins (e.g., IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric, polymeric and chimeric forms, unless otherwise specified. Specifically encompassed by the term “antibody” are polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. Immunoglobulin molecules can be of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1 and IgA2) or subclass.

[0062] The term “antigen-binding fragment” refers to any proteinaceous structure that may exhibit binding affinity for a particular antigen. Antigen-binding fragments include those produced by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments are composed of portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, antigen-binding fragments may comprise at least one variable region (either a heavy chain or light chain variable region) or one or more complementarity determining regions (CDRs) of an antibody known to bind a particular antigen. Examples of suitable antigen- binding fragments include, but not limited to, single-chain molecules such as Fab, F(ab’)2, Fc, Fabc, Fv molecules, scFv, and disulfide-linked Fvs (sdFv), intrabodies, diabodies, minibodies, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid nanobodies (VHH domains), multi-specific antibodies formed from antibody fragments, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782 (which is incorporated herein by reference in itsAttorney Docket No: 251609.000137 entirety), bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting essentially of the VH and CH1 domains, a dAb fragment, or an isolated CDR, and the like. All antibody isotypes may be used to produce antigen-binding fragments. Additionally, antigen-binding fragments may include non- antibody proteinaceous frameworks that may successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds. The phrase “an antibody or antigen-binding fragment thereof” may be used to denote that a given antigen-binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.

[0063] The terms “heavy-chain antibody (VHH)” and “nanobody”, used interchangeably herein, refer to the variable region of the heavy chain of an antibody, a heavy-chain antibody (VHH) consisting of only one heavy chain variable region. “Heavy-chain antibody (VHH)” or “nanobody” is one of the smallest antigen-binding fragments with complete functions. Usually, a VHH consisting of only one heavy chain variable region is constructed by cloning the variable region of the heavy chain that naturally lacks light chains and heavy chain constant region 1 (CH1) from the serum of alpaca. Generally, a VHH domain can be defined as an amino acid sequence with the general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively.

[0064] As used herein, the term “antigen” refers to any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) capable of being bound by an antibody or a T-cell receptor. An antigen is also able to provoke an immune response. An example of an immune response may involve, without limitation, antibody production, or the activation of specific immunologically competent cells, or both. A skilled artisan will understand that an antigen need not be encoded by a “gene” at all. An antigen can be generated, synthesized or can be derived from a biological sample (e.g., a tissue sample, a tumor sample, a cell or a body fluid), or might be macromolecule besides a polypeptide.

[0065] The term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. FunctionalAttorney Docket No: 251609.000137 epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0066] The terms “polypeptide” or “peptide” are used herein to encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0067] The terms “percent (%) sequence identity” or “homology” with respect to the polypeptide and nucleotide sequences described herein is defined as the percentage of amino acid or nucleic acid residues in a candidate sequence that are identical with the amino acid or nucleic acid residues in the reference sequence being compared after aligning the sequences. In some cases, conservative substitutions are considered as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004, each of which are incorporated herein by reference in their entirety for all purposes).

[0068] Also, in determining the degree of sequence identity between two polypeptide sequences, the skilled person may take into account “conservative” amino acid substitutions. As used herein, the term “conservative amino acid” when used in the context of amino acid substitution is one which has a similar side chain to the amino acid to be substituted. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic sideAttorney Docket No: 251609.000137 chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in a peptide motif can be replaced with another amino acid residue from the same side chain family.

[0069] As use herein, the terms “specifically binds,” “binds specifically,” “specifically recognizing,” and “is specific for” or derivatives thereof when used in the context of antibodies, or antibody fragments, means the antibody or antibody fragment forms a complex with an antigen (e.g., epiregulin) that is relatively stable under physiologic conditions. In certain embodiments, specific binding is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules (e.g., cell surface receptors). For example, an antibody that specifically recognizes a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and / or with greater duration than its bindings to other molecules. In some embodiments, the extent of binding of an antibody to an unrelated molecule is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds a target has a dissociation constant (KD) of ^10-5M, ^10-6M, ^10-7M, ^10-8M, ^10-9M, ^10-10M, ^10-11M, or ^10-12M. In some embodiments, an antibody specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA, RIA, ECL, IRMA, EIA, BIACORETM bio-layer interferometry assays, and peptide scans.

[0070] As used herein, the term “host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term “host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0071] As used herein, the term “inhibit” means to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, forAttorney Docket No: 251609.000137 example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be, for example, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels.

[0072] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease or disorder, diminishing the extent of the disease or disorder, stabilizing the disease or disorder (e.g., preventing or delaying the worsening of the disease or disorder), preventing or delaying the spread of the disease or disorder, preventing or delaying the recurrence of the disease or disorder, delay or slowing the progression of the disease or disorder, ameliorating the disease or disorder state, providing a remission (partial or total) of the disease or disorder, decreasing the dose of one or more other medications required to treat the disease or disorder, increasing the quality of life, and / or prolonging survival. Also encompassed by “treatment” is a reversal of one or more pathological consequences of the disease or disorder. The methods of the application contemplate any one or more of these aspects of treatment. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0073] The term “effective amount” used herein refers to an amount of an agent or composition sufficient to treat a specified state, disorder, condition, or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms (e.g., clinical or sub- clinical symptoms). For therapeutic use, beneficial or desired results include, e.g., decreasing one or more symptoms resulting from the disease (biochemical, histologic and / or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, delaying the progression of the disease, and / or prolonging survival of patients. An effective amount can be administered in one or more administrations. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.Attorney Docket No: 251609.000137

[0074] The term “simultaneous administration,” as used herein, means that a first therapy and second therapy in a combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition comprising both a first and second therapy) or in separate compositions (e.g., a first therapy in one composition and a second therapy is contained in another composition).

[0075] As used herein, the term “sequential administration” means that the first therapy and second therapy in a combination therapy are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60, or more minutes. Either the first therapy or the second therapy may be administered first. The first and second therapies are contained in separate compositions, which may be contained in the same or different packages or kits.

[0076] As used herein, the term “concurrent administration” means that the administration of the first therapy and that of a second therapy in a combination therapy overlap with each other.

[0077] As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration or other state / federal government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0078] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described inAttorney Docket No: 251609.000137 “Remington’s Pharmaceutical Sciences” by E.W. Martin, incorporated by reference in its entirety for all purposes.

[0079] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the subject is a human. In a preferred embodiment, the subject is a human.

[0080] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. In certain embodiments, a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0081] The term “about X-Y” used herein has the same meaning as “about X to about Y.”

[0082] As used herein and in the appended claims, the singular forms “a,” “an,” “or,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure. As is apparent to one skilled in the art, a subject assessed, selected for, and / or receiving treatment is a subject in need of such activities.

[0083] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rded. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g., in U.S. Patent No.7,912,698 and U.S. Patent Appl.Attorney Docket No: 251609.000137 Pub. Nos.2011 / 0202322 and 2011 / 0307437, each of which is incorporated by reference in their entirety for all purposes.

[0084] The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. Chimeric Antigen Receptors (CARs)

[0085] In some aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising: a) an extracellular domain which specifically binds to MET and comprises at least a VHH domain or a scFv; b) a transmembrane (TM) domain; c) optionally a co-stimulatory signaling domain, and d) a primary intracellular signaling domain.

[0086] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular domain which specifically binds to MET and comprises at least a VHH domain or a scFv; b) a transmembrane (TM) domain; c) optionally a co-stimulatory signaling domain, and d) a primary intracellular signaling domain.

[0087] The intracellular domain of the CARs described herein may comprise a primary intracellular signaling domain (e.g., ^-chain from the T cell receptor CD3 or “CD3^”) and optionally one or more co-stimulatory signaling domain. The chimeric antigen receptors described herein can be used with lymphocytes such as T-cells and natural killer (NK) cells. Upon transduction, the lymphocyte expresses the CAR on its surface, and upon contact and ligation with the target antigen, it signals through the signaling domain (e.g., CD3^ chain), inducing cytotoxicity and cellular activation. Extracellular Target-Binding Domain

[0088] In certain aspects, CARs of the present disclosure comprise an extracellular target- binding domain which specifically binds to MET.

[0089] Non-limiting examples of antigen-binding moieties include antibodies; polypeptides derived from antibodies, such as, for example, single chain variable fragments (scFv), Fab, Fab^, F(ab^)2, and Fv fragments; polypeptides derived from T Cell receptors, such as, for example, TCR variable domains; secreted factors (e.g., cytokines, growth factors) that can be artificially fused to signaling domains; and any ligand or receptor fragment (e.g., CD27, NKG2D) that binds to the antigen of interest. Combinatorial libraries could also be used to identify peptides binding with high affinity to the therapeutic target. In certain embodiments,Attorney Docket No: 251609.000137 the antigen-binding polypeptide is an antibody or an antibody fragment that binds to an antigen. Such antibodies and / or antibody fragments include, but are not limited to, monoclonal antibodies, multispecific antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab^) fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, single domain antibody variable domains, nanobodies (VHHs), diabodies and anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antigen specific TCR), and epitope-binding fragments of any of the above. Antibodies and / or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0090] In some embodiments, the extracellular target-binding domain of the CARs of the present disclosure comprises at least one VHH domain or a scFv.

[0091] In certain embodiments, the extracellular domain comprises a VHH domain. In certain embodiments, the extracellular domain comprises more than one VHH domain (e.g., 2, 3, 4, etc). In certain embodiments, the extracellular domain comprises two VHH domains. In certain embodiments, the extracellular domain comprises three VHH domains. In certain embodiments, the extracellular domain comprises four VHH domains.

[0092] In certain embodiments, the VHH domain comprises a complementarity determining region (CDR) 1, CDR2, and CDR3, and wherein: 1) the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 2, 4, and 6, respectively; 2) the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 34, 36, and 38, respectively; 3) the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 47, 49, and 51, respectively; 4) the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 59, 61, and 63, respectively; or 5) the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 72, 74, and 76, respectively.

[0093] In certain embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 2, 4, and 6, respectively.

[0094] In certain embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 34, 36, and 38, respectively.

[0095] In certain embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 47, 49, and 51, respectively.

[0096] In certain embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 59, 61, and 63, respectively.Attorney Docket No: 251609.000137

[0097] In certain embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 72, 74, and 76, respectively.

[0098] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 2, 4, and 6, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 34, 36, and 38, respectively.

[0099] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 2, 4, and 6, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 47, 49, and 51, respectively.

[0100] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 2, 4, and 6, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 59, 61, and 63, respectively.

[0101] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 2, 4, and 6, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 72, 74, and 76, respectively.

[0102] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 34, 36, and 38, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 47, 49, and 51, respectively.

[0103] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 34, 36, and 38, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 59, 61, and 63, respectively.

[0104] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 34, 36, and 38, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 72, 74, and 76, respectively.

[0105] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 47, 49, and 51, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 59, 61, and 63, respectively.Attorney Docket No: 251609.000137

[0106] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 47, 49, and 51, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 72, 74, and 76, respectively.

[0107] In certain embodiments, the extracellular domain comprises a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 59, 61, and 63, respectively; and a VHH comprising the CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 72, 74, and 76, respectively.

[0108] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 9, 41, 53, 65, or 79, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 9, 41, 53, 65, or 79.

[0109] In certain embodiments, the nucleotide sequence encoding the at least a VHH domain comprising or consisting essentially of the nucleotide sequence of SEQ ID NO: 8, 40, 52, 64, or 78, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 8, 40, 52, 64, or 78.

[0110] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 9. In certain embodiments, the VHH domain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 9.

[0111] In certain embodiments, the nucleotide sequence encoding the at least a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 8, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 8.

[0112] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 41, or aAttorney Docket No: 251609.000137 sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 41. In certain embodiments, the VHH domain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 41.

[0113] In certain embodiments, the nucleotide sequence encoding the at least a VHH domain comprising or consisting essentially of the nucleotide sequence of SEQ ID NO: 40, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 40. In certain embodiments, the nucleotide sequence encoding the VHH domain comprising or consisting essentially of the nucleotide sequence of SEQ ID NO: 40.

[0114] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 53, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 53. In certain embodiments, the VHH domain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 53.

[0115] In certain embodiments, the nucleotide sequence encoding the at least a VHH domain comprising or consisting essentially of the nucleotide sequence of SEQ ID NO: 52, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 52. In certain embodiments, the nucleotide sequence encoding the VHH domain comprising or consisting essentially of the nucleotide sequence of SEQ ID NO: 52.

[0116] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 65. In certain embodiments, the VHH domain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 65.Attorney Docket No: 251609.000137

[0117] In certain embodiments, the nucleotide sequence encoding the at least a VHH domain comprising or consisting essentially of the nucleotide sequence of SEQ ID NO: 64, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 64. In certain embodiments, the nucleotide sequence encoding the VHH domain comprising or consisting essentially of the nucleotide sequence of SEQ ID NO: 64.

[0118] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 79, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 79. In certain embodiments, the VHH domain comprises or consists essentially of the amino acid sequence of SEQ ID NO: 79.

[0119] In certain embodiments, the nucleotide sequence encoding the at least a VHH domain comprising or consisting essentially of the nucleotide sequence of SEQ ID NO: 78, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 78. In certain embodiments, the nucleotide sequence encoding the VHH domain comprising or consisting essentially of the nucleotide sequence of SEQ ID NO: 78.

[0120] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 9; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 41, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 41.

[0121] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96,Attorney Docket No: 251609.000137 at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 9; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 53, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 53.

[0122] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 9; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 65.

[0123] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 9; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 79, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 79.

[0124] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 41, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 41; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 53, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 53.

[0125] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 41, or aAttorney Docket No: 251609.000137 sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 41; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 65.

[0126] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 41, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 41; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 79, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 79.

[0127] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 53, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 53; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 65.

[0128] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 53, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 53; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 79, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 79.Attorney Docket No: 251609.000137

[0129] In certain embodiments, the extracellular domain comprises a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 65, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 65; and a VHH domain comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 79, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 79.

[0130] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising a CDR1, CDR2 and CDR3 comprised within the amino acid sequence of SEQ ID NO: 9, 41, 53, 65, or 79, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 9, 41, 53, 65, or 79.

[0131] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising a CDR1, CDR2 and CDR3 comprised within the amino acid sequence of SEQ ID NO: 9, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 9.

[0132] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising a CDR1, CDR2 and CDR3 comprised within the amino acid sequence of SEQ ID NO: 41, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 41.

[0133] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising a CDR1, CDR2 and CDR3 comprised within the amino acid sequence of SEQ ID NO: 53, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 53.

[0134] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising a CDR1, CDR2 and CDR3 comprised within the amino acid sequence of SEQ ID NO: 65, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at leastAttorney Docket No: 251609.000137 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 65.

[0135] In certain embodiments, the extracellular domain comprises at least a VHH domain comprising a CDR1, CDR2 and CDR3 comprised within the amino acid sequence of SEQ ID NO: 79, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 79.

[0136] Determination of CDR regions in a VHH domain may be accomplished using different methods, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme), each reference cited herein is incorporated by reference in its entirety.

[0137] The boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software.

[0138] VHH domains of the present disclosure may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring VHH sequences (i.e. from a suitable species of Camelid, e.g. llama) or synthetic or semi-synthetic VHs or VLs (e.g.Attorney Docket No: 251609.000137 from human). Such VHH domains may include “humanized” or otherwise “sequence optimized” VHHs, “camelized” immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences, i.e. camelized VHs), as well as human VHs, human VLs, camelid VHHs that have been altered by techniques such as affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, combining fragments derived from different immunoglobulin sequences, veneering, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences.

[0139] Humanization can be achieved based on the homology between camelid VHH and human VH fragments, for example, using resurfacing or CDR grafting. Resurfacing strategies have been described in e.g., Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et al., 2018; Vincke et al., 2009 J Biol Chem, and CDR grafting strategies have been described in e.g., ben Abderrazek et al., 2011; van Faassen et al., 2020 FASEB; Li et al., 2018; Vaneycken et al., 2010; Vincke et al., 2009 J Biol Chem; and Yu et al., 2017, each of which is incorporated herein by reference in its entirety.

[0140] In certain embodiments, the extracellular domain comprises at least a single chain variable fragment (scFv). In certain embodiments, the extracellular domain comprises more than one scFv domain (e.g., 2, 3, 4, etc).

[0141] In certain embodiments, the scFv comprises a light chain variable domain (VL) comprising a CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 90, 91, and 92, respectively, and a heavy chain variable domain (VH) comprising a CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 86, 87, and 88, respectively.

[0142] In certain embodiments, the scFv comprises a VL comprising the amino acid sequence of SEQ ID NO: 93, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any of SEQ ID NO: 93; and a VH comprising the amino acid sequence of SEQ ID NO: 89, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 89. In certain embodiments, the scFv comprises a VL comprising the amino acid sequence of SEQ ID NO: 93, and a VH comprising the amino acid sequence of SEQ ID NO: 89.

[0143] In certain embodiments, the scFv comprises or consists essentially of the amino acid sequence of SEQ ID NO: 70, or a sequence having at least 50, at least 55, at least 60, at leastAttorney Docket No: 251609.000137 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 70. In certain embodiments, the nucleotide sequence that encodes the scFv comprises or consists essentially of the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 32, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 32. In certain embodiments, the scFv comprises or consists essentially of the amino acid sequence of SEQ ID NO: 70. In certain embodiments, the nucleotide sequence encoding the scFv comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 32.

[0144] In certain embodiments, the extracellular domain comprises a leader sequence. The leader sequence may be positioned at the N-terminus of the extracellular target-binding domain. The leader sequence may be optionally cleaved from the antigen-binding moiety during cellular processing and localization of the CAR to the cellular membrane.

[0145] In certain embodiments, the leader sequence comprises a CD8^ signal peptide. In certain embodiments, the leader sequence comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 11. In certain embodiments, the nucleotide sequence encoding the leader sequence comprises or consists essentially of the sequence set forth in SEQ ID NO: 10, , or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 10. In certain embodiments, the leader sequence comprises or consists essentially of the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the nucleotide sequence encoding the leader sequence comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 10. Domain

[0146] In certain embodiments, CARs of the present disclosure comprise a hinge domain between the extracellular target-binding domain and the transmembrane domain. A hinge domain can provide flexibility and accessibility for the extracellular target-binding domain. A hinge domain may be derived from all or part of naturally occurring molecules, or from all orAttorney Docket No: 251609.000137 part of an antibody constant region. For example, the hinge domain may be derived from CD8^ stalk, CD28, or an immunoglobulin, such as IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof. Alternatively, the hinge domain may comprise a synthetic sequence that corresponds to a naturally occurring linker region sequence, or may be an entirely synthetic sequence. In certain embodiments, additional linking amino acids are added to the hinge domain to ensure that the target-binding domain is at an optimal distance from the transmembrane domain.

[0147] In certain embodiments, the hinge domain is derived from IgG1, CD8^ stalk, CD28, IgD, or IgG4.

[0148] In certain embodiments, the hinge domain is derived from CD8^ stalk. In certain embodiments, the hinge domain derived from CD8^ stalk comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 97, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 97. In certain embodiments, the nucleotide sequence that encodes the CD8^ hinge domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 96, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with SEQ ID NO: 98. In certain embodiments, the CD8^ hinge domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 97. In certain embodiments, the nucleotide sequence that encodes the CD8^ hinge domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 96.

[0149] In certain embodiments, the hinge domain is derived from CD28. In certain embodiments, the hinge domain derived from CD28 comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 99, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 99. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 98, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with SEQ ID NO: 98. In certain embodiments, the CD28 hinge domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO:Attorney Docket No: 251609.000137 99. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 98. Transmembrane Domain

[0150] In certain aspects, CARs of the present disclosure comprise a transmembrane domain, fused in frame between the extracellular target-binding domain and the intracellular domain.

[0151] Non-limiting examples of transmembrane domains of particular use in this disclosure may be derived or modified from transmembrane region(s) of linker of activated T cells (LAT), Ebola virus fusogenic glycoprotein (GP), the ^, ^ or ^ chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154.

[0152] In certain embodiments, the transmembrane domain is derived from CD28, CD8^, CD4, CD3^, or CD86.

[0153] In a specific embodiment, the transmembrane domain is derived from CD8^. In certain embodiments, the CD8^ transmembrane domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 101, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 101. In certain embodiments, the nucleotide sequence that encodes the CD8^ transmembrane domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 100, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 100. In certain embodiments, the CD8^ transmembrane domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 101. In certain embodiments, the nucleotide sequence that encodes the CD8^ transmembrane domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 100.

[0154] In certain embodiments, the CAR described herein comprises a hinge and transmembrane domain derived from CD8^. The CD8^ hinge and transmembrane domain comprises or consists essentially of the amino acid sequence set forth in any one of SEQ ID NO: 15, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 15. In certain embodiments, the nucleotide sequence that encodes the CD8^ hinge and transmembrane domain comprises or consists essentially of the nucleotide sequence set forthAttorney Docket No: 251609.000137 in SEQ ID NO: 14, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with any one of SEQ ID NO: 14. In certain embodiments, the CD8^ hinge and transmembrane domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the nucleotide sequence that encodes the CD8^ hinge and transmembrane domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 14.

[0155] In certain embodiments, the transmembrane domain is derived from CD28. The CD28 transmembrane domain comprises or consists essentially of the amino acid sequence set forth in any one of SEQ ID NO: 103, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 103. In certain embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 102, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with any one of SEQ ID NO: 102. In certain embodiments, the CD28 transmembrane domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 103. In certain embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 102.

[0156] In certain embodiments, the CAR described herein comprises a hinge and transmembrane domain derived from CD28. The CD28 hinge and transmembrane domain comprises or consists essentially of the amino acid sequence set forth in any one of SEQ ID NO: 25, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 25. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge and transmembrane domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 24, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with any one of SEQ ID NO: 24. In certain embodiments, the CD28 hinge and transmembrane domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the nucleotide sequence that encodes theAttorney Docket No: 251609.000137 CD28 hinge and transmembrane domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 24.

[0157] Additionally, transmembrane domains can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex. In other instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to avoid binding of proteins naturally associated with the transmembrane domain. In certain embodiments, the transmembrane domain includes additional amino acids to allow for flexibility and / or optimal distance between the domains connected to the transmembrane domain. Intracellular Domain

[0158] In certain aspects, CARs of the present disclosure comprise an intracellular domain, which comprises a primary signaling domain and optionally one or more co-stimulatory signaling domains. The intracellular domain, which comprises a primary signaling domain and optionally one or more co-stimulatory signaling domains, is responsible for activation of at least one of the normal effector functions of the lymphocyte in which the CAR has been placed in. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire signaling domain is present, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the signaling domain sufficient to transduce the effector function signal.

[0159] Non-limiting examples of signaling domains which can be used in the CARs of the present disclosure include, e.g., signaling domains derived from DAP10, DAP12, Fc epsilon receptor I ^ chain (FCER1G), FcR ^, CD3^, CD3^, CD3^, CD3^, CD5, CD22, CD226, CD66d, CD79A, CD79B, and Megf10.

[0160] In certain embodiments, the CAR of the present disclosure comprises a primary signaling domain derived from CD3^. In certain embodiments, the primary signaling domain derived from CD3^ comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 19, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO:Attorney Docket No: 251609.000137 19. In certain embodiments, the nucleotide sequence that encodes the CD3^ signaling domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID: 18, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity SEQ ID NO: 18. In certain embodiments, the CD3^ signaling domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the nucleotide sequence that encodes the CD3^ signaling domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID: 18.

[0161] Non-limiting examples of co-stimulatory signaling domains which can be used in the CARs of the present disclosure include, those derived from CD28, CD27, CD40, CD134 (OX- 40), 4-1BB (CD137), CD226, CD79A, ICOS, CD19, MyD88, BTLA, CD30, GITR, and HVEM.

[0162] In certain embodiments, the CAR of the present disclosure comprises one co- stimulatory domain. In certain embodiments, the CAR of the present disclosure comprises a co-stimulatory domain derived from CD28. In certain embodiments, the CAR of the present disclosure comprises a co-stimulatory domain derived from 4-1BB (CD137). In certain embodiments, the CAR of the present disclosure comprises two co-stimulatory domains. In certain embodiments, the CAR of the present disclosure comprises a co-stimulatory domain derived from CD28 and a co-stimulatory domain derived from 4-1BB (CD137). In certain embodiments, the CAR of the present disclosure comprises more than two co-stimulatory domains.

[0163] In certain embodiments, the primary signaling domain(s) and co-stimulatory domain(s) can be in any order. In certain embodiments, the primary signaling domain is upstream of the co-stimulatory domains. In certain embodiments, the primary signaling domain is downstream from the co-stimulatory domains. In the cases where two or more co-stimulatory domains are used, the order of the co-stimulatory domains could be switched.

[0164] In certain embodiments, the co-stimulatory domain derived from 4-1BB (CD137) comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 17. In certain embodiments, the nucleotide sequence that encodes the 4-1BB (CD137) co-stimulatory domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 16, or aAttorney Docket No: 251609.000137 nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 16. In certain embodiments, the 4-1BB (CD137) co-stimulatory domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the nucleotide sequence that encodes the 4-1BB (CD137) co-stimulatory domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 16.

[0165] In certain embodiments, the co-stimulatory domain derived from CD28 comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 27, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 27. In certain embodiments, the nucleotide sequence that encodes the CD28 co-stimulatory domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 26, or a nucleotide sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with SEQ ID NO: 26. In certain embodiments, the CD28 co-stimulatory domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 27. In certain embodiments, the nucleotide sequence that encodes the CD28 co-stimulatory domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 26.

[0166] In certain embodiments, the co-stimulatory domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 17 and / or 27, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 17 and / or 27. In certain embodiments, the nucleotide sequence that encodes the co-stimulatory domain comprises or consists essentially of the nucleotide sequence of SEQ ID NO: 16 and / or 26, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 16 and / or 26. In certain embodiments, the co- stimulatory domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 17 and / or 27. In certain embodiments, the nucleotide sequence that encodes the co-stimulatory domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 16 and / or 26.Attorney Docket No: 251609.000137

[0167] In certain embodiments, the co-stimulatory domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 17 and 27, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 17 and 27. In certain embodiments, the nucleotide sequence that encodes the co-stimulatory domain comprises or consists essentially of the nucleotide sequence of SEQ ID NO: 16 and 26, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 16 and / or 26. In certain embodiments, the co-stimulatory domain comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 17 and 27. In certain embodiments, the nucleotide sequence that encodes the co-stimulatory domain comprises or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 16 and 26. Additional Genes

[0168] In addition to the components described above, the CAR may further comprise at least one additional gene that encodes an additional peptide. Non-limiting examples of additional genes can include a transduced host cell selection marker, an in vivo tracking marker, a cytokine, a suicide gene, or some other functional gene. In certain embodiments, the CAR comprises at least one additional gene. In certain embodiments, the CAR comprises one additional gene. In other embodiments, the CAR comprises two additional genes. In yet another embodiment, the CAR comprises three additional genes.

[0169] For example, the CAR construct may comprise an additional gene which is a GFP (e.g., superfold GFP). The GFP can be used as a tag for host cell selection. As another example, the CAR construct may comprise an additional gene which is truncated CD19 (tCD19). The tCD19 can be used as a tag. Expression of tCD19 may also help determine transduction efficiency. Other examples of additional genes include genes that encode polypeptides with a biological function; examples include, but are not limited to, cytokines, chimeric cytokine receptors, dominant negative receptors, and safety switches (CD20, truncated EGFR or HER2, inducible caspase 9 molecules).

[0170] Non-limiting examples of classes of additional genes that can be used to increase the effector function of CAR containing host cells, include (a) secretable cytokines (e.g., but not limited to, IL-7, IL-12, IL-15, IL-18), (b) membrane bound cytokines (e.g., but not limited to, IL-15), (c) chimeric cytokine receptors (e.g., but not limited to, IL-2 / IL-7, IL-4 / IL-7), (d)Attorney Docket No: 251609.000137 constitutive active cytokine receptors (e.g., but not limited to, C7R), (e) dominant negative receptors (DNR; e.g., but not limited to TGFRII DNR), (f) ligands of co-stimulatory molecules (e.g., but not limited to, CD80, 4-1BBL), (g) nuclear factor of activated T-cells (NFATs) (e.g., NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5), (h) antibodies, including fragments thereof and bispecific antibodies (e.g., but not limited to, bispecific T-cell engagers (BiTEs)), or (i) a second CAR.

[0171] In certain embodiments, the sequence encoding an additional gene is operably linked to the sequence encoding CAR via a sequence encoding a self-cleaving peptide (e.g., a 2A peptide) and / or an Internal Ribosome Entry Site (IRES) as disclosed herein. Non-Limited Examples of CARs

[0172] In one aspect, the present disclosure provides a CAR polypeptide. In some embodiments, a CAR polypeptide of the present disclosure comprises or consists essentially of the amino acid sequence SEQ ID NO: 21, 31, 43, 45, 55, 57, 67, 69, 81, 83, or 85, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any one of SEQ ID NO: 21, 31, 43, 45, 55, 57, 67, 69, 81, 83, or 85.

[0173] In some embodiments, a CAR polypeptide of the present disclosure comprises a hinge and TM domain derived from CD8^, a co-stimulatory signaling domain derived from 4-1BB (CD137), and a CD3^ signaling domain.

[0174] In some embodiments, a CAR polypeptide of the present disclosure comprises or consists essentially of the amino acid sequence SEQ ID NO: 21, 43, 55, 67, or 81, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with any one of SEQ ID NO: 21, 43, 55, 67, or 81.

[0175] In some embodiments, a CAR polypeptide of the present disclosure comprises or consists essentially of the amino acid sequence SEQ ID NO: 21 or 43, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 21 or 43.

[0176] In some embodiments, a CAR polypeptide of the present disclosure comprises or consists essentially of the amino acid sequence SEQ ID NO: 21, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90,Attorney Docket No: 251609.000137 at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 21.

[0177] In some embodiments, a CAR polypeptide of the present disclosure comprises or consists essentially of the amino acid sequence SEQ ID NO: 43, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 43.

[0178] In one aspect, the present disclosure provides a polynucleotide encoding a CAR described herein. In certain embodiments, the CAR can be encoded by one polynucleotide chain. In certain embodiments, the nucleotide sequence encoding a CAR described herein comprises the nucleotide sequence of SEQ ID NO: 20, 30, 42, 44, 54, 56, 66, 68, 90, 82, or 84, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 20, 30, 42, 44, 54, 56, 66, 68, 90, 82, or 84.

[0179] In certain embodiments, the nucleotide sequence encoding a CAR described herein comprises the nucleotide sequence of SEQ ID NO: 20, 42, 54, 66, or 90, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 20, 42, 54, 66, or 90.

[0180] In certain embodiments, the nucleotide sequence encoding a CAR described herein comprises the nucleotide sequence of SEQ ID NO: 20 or 42, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 20 or 42.

[0181] In certain embodiments, the nucleotide sequence encoding a CAR described herein comprises the nucleotide sequence of SEQ ID NO: 20, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 20.

[0182] In certain embodiments, the nucleotide sequence encoding a CAR described herein comprises the nucleotide sequence of SEQ ID NO: 42, or a sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91,Attorney Docket No: 251609.000137 at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 42.

[0183] In various embodiments, the polynucleotide encoding a CAR is a DNA molecule. In various embodiments, the polynucleotide encoding a CAR is an RNA molecule. In some embodiments, the polynucleotide encoding a CAR is an mRNA molecule.

[0184] In one aspect, the present disclosure provides CAR polypeptides encoded by a polynucleotide described above. Vectors

[0185] In one aspect, the present disclosure provides recombinant vectors comprising a polynucleotide encoding a CAR comprising polynucleotides encoding the proteins disclosed above. The terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to genetically modify the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, synthesized RNA and DNA molecules, phages, viruses, etc.

[0186] The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become produced, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. An expression product can be characterized as intracellular, extracellular or transmembrane.

[0187] In certain embodiments, recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 20, 30, 42, 44, 54, 56, 66, 68, 90, 82, or 84, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 20, 30, 42, 44, 54, 56, 66, 68, 90, 82, or 84. In certain embodiments, recombinant vectors comprise a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 21, 31, 43, 45, 55, 57, 67, 69, 81, 83, or 85, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 91, at least 92, at least 93, at least 94,Attorney Docket No: 251609.000137 at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 21, 31, 43, 45, 55, 57, 67, 69, 81, 83, or 85.

[0188] In certain embodiments, the recombinant vector comprises a polynucleotide encoding a CAR, wherein the polynucleotide is operatively linked to at least one additional gene.

[0189] In certain embodiments, the vector is a viral vector. Viral vectors that can encode a CAR of the present disclosure include, but are not limited to, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, and a vaccinia virus vector. In certain embodiments, the viral vector is a retroviral vector. In certain embodiments, the viral vector is a lentiviral vector. In certain embodiments, the viral vector is a third generation lentiviral vector.

[0190] In certain embodiments, the vector is a non-viral vector. The non-viral vector may be a plasmid or a transposon (such as, for example, a Sleeping Beauty or a PiggyBac transposon).

[0191] In certain embodiments, the polynucleotide encoding the CAR is operably linked to at least a regulatory element for expression of the chimeric antigen receptor. The regulatory element can mediate expression of the CAR in the host cell. Regulatory elements include, but are not limited to, promoters, enhancers, initiation sites, polyadenylation (polyA) tails, IRES elements, response elements, and termination signals. In certain embodiments, the regulatory element regulates CAR expression. In certain embodiments, the regulatory element increases the expression of the CAR. In certain embodiments, the regulatory element increases the expression of the CAR once the host cell is activated. In certain embodiments, the regulatory element decreases expression of the CAR. In certain embodiments, the regulatory element decreases expression of the CAR once the host cell is activated.

[0192] Generally speaking, a suitable vector may contain an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers. For example, these promoters may include, but are not limited to, the lac or trp promoters of Escherichia coli (E. coli), the lambda phage PL promoter, eukaryotic promoters (including CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, methanol oxidase promoter of Pichia pastoris) and other well-known promoters that are capable of controlling gene expression in prokaryotic cells or eukaryotic cells or viruses. Marker genes may be used to provide phenotypic characters for the selection of transformed host cells. For example, marker genes may include but are not limited to dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline resistance or ampicillin resistance for E. coli. When the polynucleotide is expressed, the expression vector may further include an enhancerAttorney Docket No: 251609.000137 sequence. The transcription will be enhanced if the enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, typically containing about 10-300 base pairs. Enhancers act on promoters to enhance gene transcription.

[0193] In one embodiment, the vector described herein is a lentiviral vector and the promoter used herein is a CMV promoter.

[0194] In one embodiment, the vector described herein is a mRNA and the promoter used herein is a T7 promoter. Cell Compositions and Methods of Preparation

[0195] In one aspect, the present disclosure provides an isolated host cell comprising a polynucleotide or a recombinant vector described herein. In one aspect, the present disclosure provides an isolated host cell comprising a CAR described herein.

[0196] The term “host cell” refers any cell that contains a heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). For example, a host cell can be a cell from any organism that is selected, modified, transformed, grown, used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. An appropriate host may be determined. For example, the host cell may be selected based on the vector backbone and the desired result. By way of example, a plasmid or cosmid can be introduced into a prokaryote host cell for replication of several types of vectors. Bacterial cells such as, but not limited to DH5^, JM109, and KCB, SURE® Competent Cells, and SOLOPACK Gold Cells, can be used as host cells for vector replication and / or expression. Additionally, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to yeast (e.g., YPH499, YPH500 and YPH501), insects and mammals. Examples of mammalian eukaryotic host cells for replication and / or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12. In certain embodiments, the host cell is autologous. In certain embodiments, the host cell is allogenic.

[0197] Host cells of the present disclosure include immune cells (e.g., T cells and natural killer cells) that contain the DNA or RNA sequences encoding the CAR and express the CAR on the cell surface. Host cells may be used for enhancing T-cell activity, natural killer cell activity, treatment of tumors, and / or treatment of autoimmune diseases.Attorney Docket No: 251609.000137

[0198] In various embodiments, the host cell is an immune cell. The immune cell may be a T cell, a NK cell, an iNKT cell, or a macrophage. The immune cell may also be monocytes or granulocytes, such as bone marrow cells, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophilic Granulocytes.

[0199] In various embodiments, the host cell is a T cell. In certain embodiments, the T cell is selected from a CD8+ T cell, a CD4+ T cell, a cytotoxic T cell, an ^^ T cell receptor (TCR) T cell, an invariant natural killer T (iNKT) cell, a ^^ T cell, a memory T cell including memory stem T cell (TSCM), a naïve T cell, an effector T cell, a T-helper cell, and a regulatory T cell (Treg).

[0200] In various embodiments, the host cell is a NK cell. The terms “natural killer cell” and “NK cell” are used interchangeable and used synonymously herein. As used herein, NK cell refers to a differentiated lymphocyte with a CD16+ CD56+ and / or CD57+ TCR- phenotype. NKs are characterized by their ability to bind to and kill cells that fail to express “self” MHC / HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0201] In various embodiments, the host cell has been activated and / or expanded ex vivo.

[0202] In various embodiments, the host cell is an allogeneic cell. In various embodiments, the host cell is an autologous cell.

[0203] In certain embodiments, the host cell is isolated from a subject having a cancer. In certain embodiments, the cancer comprises one or more cells expressing MET. Non-limiting examples of cancers that express MET include breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, prostate cancer, pancreatic cancer, kidney cancer, bladder cancer, testicular cancer, ovarian cancer, endometrial cancer, cervical cancer, neuroendocrine cancer, head and neck cancer, brain cancer, sarcoma, melanoma, and lymphoma. In certain embodiments, the cancer is a solid tumor.

[0204] In certain embodiments, the host cell is derived from a blood, marrow, tissue, or a tumor sample.

[0205] In one aspect, the present disclosure provides a method of generating an isolated host cell described herein. The method includes genetically modifying the host cell with a polynucleotide encoding a CAR, or the recombinant vector comprising the polynucleotide encoding a CAR. The genetically modifying step may be conducted in vivo or ex vivo. In certain embodiments, the genetically modifying step is conducted ex vivo. The method may furtherAttorney Docket No: 251609.000137 include activation and / or expansion of the host cell ex vivo before, after and / or during the genetic modification.

[0206] The term “genetic modification” or “genetically engineering” means the introduction of a “foreign” (i.e., extrinsic or extracellular) genetic material typically in the form of a polynucleotide (e.g., DNA or RNA) sequence into a host cell, so that the host cell can express the introduced genetic material to produce a desired substance, typically a polypeptide or protein (e.g., chimeric antigen receptor) coded by the introduced genetic material. The introduced genetic material may also include regulatory or control sequences operably linked to polynucleotide encoding the chimeric antigen receptor, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “genetic modified” or “genetically engineered.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0207] Polynucleotides of the present disclosure may be introduced into the host cells via viral or non-viral gene methods. Suitable methods for polynucleotide delivery for use with the current methods include any method known by those of skill in the art, by which a polynucleotide can be introduced into an organelle, cell, tissue or organism.

[0208] In certain embodiments, polynucleotides of the present disclosure are transferred to the host cell via viral transduction. The term “transduction” means the introduction of a foreign nucleic acid into a cell using a viral vector. In certain embodiments, the host cells can be transduced via retroviral transduction. References describing retroviral transduction of genes are Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell 33:153 (1983); Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263; International Patent Publication No. WO 95 / 07358, published Mar. 16, 1995, by Dougherty et al.; and Kuo et al., Blood 82:845 (1993), each of which is hereby incorporated by reference in its entirety. In certain embodiments, the host cells can be transduced via lentiviral transduction.

[0209] In certain embodiments, polynucleotides of the present disclosure are transferred to the host cell via transfection. The term “transfection” means the introduction of a “foreign” (i.e., extrinsic or extracellular) nucleic acid into a cell using recombinant DNA technology. For example, polynucleotides may be introduced into the host cell using transfection reagents such as Lipofectamine or Superfect, or a liposome (e.g., polynucleotide entrapped in a lipid complex suspended in an excess of aqueous solution. See e.g., Ghosh and Bachhawat, (1991) In: LiverAttorney Docket No: 251609.000137 Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. pp.87-104). As another example, polynucleotides may be selectively taken up by a host cell via receptor- mediated endocytosis using cell type-specific receptors (see e.g., Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; Perales et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994; Myers, EPO 0273085; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993; Nicolau et al., (1987) Methods Enzymol., 149, 157-176, each of which is hereby incorporated by reference in its entirety).

[0210] Electroporation is another method of polynucleotide transfer (see e.g., Potter et al., (1984) Proc. Nat'l Acad. Sci. USA, 81, 7161-7165 and Tur-Kaspa et al., (1986) Mol. Cell Biol., 6, 716-718, both of which are incorporated herein in their entirety for all purposes). Methods of electroporation for use with this invention include, for example, Sardesai NY., and Weiner DB, Current Opinion in Immunotherapy 23:421-9 (2011) and Ferraro B. et al., Human Vaccines 7:120-127 (2011), both of which are hereby incorporated by reference in their entirety for all purposes. Electroporation may be carried out ex vivo or in vivo. Ex vivo electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. Cell wall-degrading enzymes, such as pectin-degrading enzymes, may be used to render the host cells more susceptible to genetic modification by electroporation than untreated cells. See e.g., U.S. Pat. No. 5,384,253, which is incorporated herein by reference in its entirety for all purposes. In vivo electroporation may involve a basic injection technique in which a vector is injected intradermally in a subject. Electrodes then apply electrical pulses to the intradermal site causing the cells localized there (e.g., resident dermal dendritic cells), to take up the vector. These tumor antigen-expressing dendritic cells activated by local inflammation can then migrate to lymph-nodes.

[0211] In further embodiments, polynucleotides of the present disclosure may be transferred to the host cell via calcium phosphate precipitation (see e.g., Graham and van der Eb, (1973) Virology, 52, 456-467; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987), and Rippe et al., Mol. Cell Biol., 10:689-695, 1990); DEAE-dextran-based method (e.g., a polynucleotide is delivered into a cell using DEAE-dextran followed by polyethylene glycol. See e.g., Gopal TV, Mol Cell Biol. 1985 May; 5(5):1188-90); sonication loading (introduction of a polynucleotide by direct sonic loading. See e.g., Fechheimer et al., (1987) Proc. Nat'l Acad. Sci. USA, 84, 8463-8467); and microprojectile bombardment (e.g., one or more particles may be coated with at least one polynucleotide and delivered into cells by a propelling force. See e.g., U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042; and PCT Application WO 94 / 09699; Klein et al., (1987) Nature, 327, 70-73, Yang et al., (1990) Proc.Attorney Docket No: 251609.000137 Nat'l Acad. Sci. USA, 87, 9568-9572); each reference cited here is incorporated by reference in its entirety for all purposes.

[0212] Prior to expansion and genetic modification of the host cells, a source of host cells can be obtained from a subject through a variety of non-limiting methods. For example, host cells can be obtained from a number of non-limiting 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, any number of T cell lines available and known to those skilled in the art, may be used. In another embodiment, host cells can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In another embodiment, host cells are part of a mixed population of cells which present different phenotypic characteristics. Modified cells resistant to an immunosuppressive treatment and susceptible to be obtained by for example, inactivating one component of the TCR and / or one or more endogenous MHC molecules, are also encompassed in the scope of the present invention.

[0213] The host cells can be genetically modified after stimulation / activation. The terms “activation” or “stimulation” means to induce a change in their biologic state by which the host cells (e.g., T-cells and NK cells) express activation markers, produce cytokines, proliferate and / or become cytotoxic to target cells. All these changes can be produced by primary stimulatory signals. Co-stimulatory signals can amplify the magnitude of the primary signals and suppress cell death following initial stimulation resulting in a more durable activation state and thus a higher cytotoxic capacity. A “co-stimulatory signal” refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T-cell and / or NK cell proliferation and / or upregulation or downregulation of key molecules.

[0214] Activation or stimulation of immune cells (e.g., T cells or NK cells) may be carried out using methods known by those skilled in the art. For example, T cells can be activated generally using methods as described, for example, in U.S. Patents 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; and 6,867,041, each of which is incorporated herein by reference in its entirety. Generally, T cells are expanded by contact with an agent that stimulates a CD3 TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T-cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.Attorney Docket No: 251609.000137

[0215] NK cells can be activated by, for example and not limitation, inhibition of inhibitory receptors on NK cells (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E or LILRB5 receptor) or by feeder cells (e.g., native K562 cells or K562 cells that are genetically modified to express 4-1BBL and cytokines such as IL15 or IL21).

[0216] Polynucleotides of the present disclosure may be transferred to the host T cells or NK cells such as described above. Following gene transfer the cells may be immediately infused. In certain aspects, following gene transfer, the cells may be propagated for days, weeks, or months ex vivo as a bulk population within about 1, 2, 3, 4, 5 days or more. In a further aspect, following gene transfer, the cells are cloned and a clone demonstrating presence of a single integrated or episomally maintained expression cassette or plasmid, and expression of the chimeric receptor is expanded ex vivo. The clone selected for expansion demonstrates the capacity to specifically recognize and lyse antigen-expressing target cells. The recombinant T cells or T cell precursor may be expanded by stimulation with IL-2, or other cytokines that bind the common gamma-chain (e.g., IL-7, IL-15, IL-21 and others). The recombinant T cells or T cell precursor may be expanded by stimulation with artificial antigen presenting cells. The recombinant T cells may be expanded on artificial antigen presenting cell or with an antibody. Subsets of the recombinant T cells may be deleted on artificial antigen presenting cell or with an antibody. In a further aspect, the genetically modified host cells may be cryopreserved.

[0217] In some embodiments, host cells are genetically modified using gene editing with homology-directed repair (HDR). Homology-directed repair (HDR) is a mechanism used by cells to repair double strand DNA breaks. In HDR, a donor polynucleotide with homology to the site of the double strand DNA break is used as a template to repair the cleaved DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the cellular DNA. As such, new nucleic acid material may be inserted or copied into a target DNA cleavage site. Double strand DNA breaks in host cells may be induced by a site-specific nuclease. The term “site-specific nuclease” as used herein refers to a nuclease capable of specifically recognizing and cleaving a nucleic acid (DNA or RNA) sequence. Suitable site- specific nucleases for use in the present disclosure include, but are not limited to, RNA-guided endonucleases (e.g., CRISPR-associated (Cas) proteins such as Cas9 or Cas12), zinc finger nucleases, TALEN nucleases, or mega-TALEN nucleases.

[0218] In some embodiments, a site-specific nuclease (e.g., a Cas9 or Cas12 in complex with a guide RNA) capable of inducing a double strand break in a target DNA sequence is introduced to a host cell, along with a donor polynucleotide encoding a polypeptide of a CAR of the presentAttorney Docket No: 251609.000137 disclosure. In some embodiments, the donor polynucleotide encoding a polypeptide of a CAR of the present disclosure is inserted into a genomic locus, such as the TCR alpha constant (TRAC) locus, or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) locus. In some embodiments, the donor polynucleotide encoding a polypeptide of a CAR of the present disclosure is inserted into a genomic locus such that the expression of the CAR is driven by the endogenous promoter. In some embodiments, insertion of the donor polynucleotide encoding a polypeptide of a CAR of the present disclosure into a genomic locus results in the inactivation of the gene at the genomic locus. In such embodiments, the nucleotide sequence encoding the CAR is flanked by 5' and 3' arms homologous to the genomic locus being targeted.

[0219] In some embodiments, a nucleotide sequence encoding a CAR of the present disclosure is flanked by 5' and 3' arms homologous to the TRAC locus. In one embodiment, the 5' homology arm comprises the sequence of SEQ ID NO: 104 or a sequence having at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 104. In one embodiment, the 3' homology arm comprises the sequence of SEQ ID NO: 112 or a sequence having at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 112. In one embodiment, the insertion site within the TRAC locus comprises the target sequence TCAGGGTTCTGGATATCTGT (SEQ ID NO: 105). In one embodiment, the insertion is mediated by a Cas9 endonuclease complexed with a gRNA comprising the targeting sequence UCAGGGUUCUGGAUAUCUGU (SEQ ID NO: 106).

[0220] In some embodiments, a nucleotide sequence encoding a CAR of the present disclosure is flanked by 5' and 3' arms homologous to the GAPDH locus. In one embodiment, the 5' homology arm comprises the sequence of SEQ ID NO: 107 or a sequence having at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 107. In one embodiment, the 3' homology arm comprises the sequence of SEQ ID NO: 113 or a sequence having at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 113. In one embodiment, the insertion site within the GAPDH locus comprises the target sequence ATCTTCTAGGTATGACAACGA (SEQ ID NO: 108). In one embodiment, the insertion is mediated by a Cas12 endonuclease complexed with a gRNA comprising the targeting sequence AUCUUCUAGGUAUGACAACGA (SEQ ID NO: 109). In one embodiment, the insertion site within the GAPDH locus comprises the target sequence TCTAGGTATGACAACGAATT (SEQ ID NO: 110). In one embodiment, theAttorney Docket No: 251609.000137 insertion is mediated by a Cas9 endonuclease complexed with a gRNA comprising the targeting sequence UCUAGGUAUGACAACGAAUU (SEQ ID NO: 111).

[0221] In another embodiment, methods here further comprise a step of genetically modifying host cells by inactivating at least one gene expressing one component of the TCR and / or one or more endogenous MHC molecules. In certain embodiments, the gene is selected from the group consisting of TCR^ (TRAC), TCR^ (TCRB), CD3, MHC, ^2M, TAP1, TAP2, CIITA, RFX5, RFXAP and / or RFXANK. In certain embodiments, the method further comprises introducing into modified host cells a site-specific nuclease capable of selectively inactivate a target gene by DNA cleavage of the genes. In a preferred embodiment, the site- specific nuclease is a Cas9 endonuclease or TALE-nuclease. Pharmaceutical Compositions

[0222] In another aspect, the present disclosure provides pharmaceutical composition comprising the CAR-modified host cells described herein and a pharmaceutically acceptable carrier and / or excipient.

[0223] In one aspect, the present disclosure provides a pharmaceutical composition comprising a polynucleotide or a recombinant vector described herein, and a pharmaceutically accepted carrier and / or excipient.

[0224] Examples of pharmaceutical carriers include but are not limited to sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

[0225] Compositions disclosed herein may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0226] Compositions disclosed herein may comprise one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidantsAttorney Docket No: 251609.000137 such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0227] The pharmaceutical compositions of the invention may comprise the compounds described herein and a pharmaceutically acceptable carrier or excipient. Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of glycopeptides, or excipients or other stabilizers and / or buffers. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, e.g., phenol and ascorbic acid. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. Pharmaceutically acceptable carriers and formulations are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. ("Remington's"). One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the composition, and on its particular physio-chemical characteristics.

[0228] In certain embodiments, the compositions are formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural or intramuscular administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile. In certain embodiments, the composition is reconstituted from a lyophilized preparation prior to administration.

[0229] In certain embodiments, the pharmaceutical compositions described herein may comprise one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents comprise an immunomodulatory agent, a chemotherapeutic agent, an anti-angiogenic agent, an anti-inflammatory agent, or a cell death inducer, or a combination thereof. Non-limiting examples of such therapeutic agents include those described elsewhere herein.

[0230] In certain embodiments, the pharmaceutical compositions described herein comprise one or more cell death inducers. The cell death inducers described herein may be a B-cellAttorney Docket No: 251609.000137 lymphoma 2 (BCL-2) family inhibitor, such as a B-cell lymphoma-extra large (BCL-XL) inhibitor. Non-limiting examples of BCL-2 family inhibitors include BCL-XL inhibitors such as Navitoclax (ABT-263), A-1331852, WEHI-539, A-1155463, and ABT-737; BCL-2 inhibitors such as ABT-199 (Venetoclax); and MCL-1 inhibitors such as S-63845. Therapeutic Applications

[0231] In conjunction with the compounds and / or compositions described herein, provided herein are methods of treatment using such compositions. In one aspect, the present disclosure provides a method for treating a cancer in a subject in need thereof. A therapeutically effective amount of the CAR-modified host cells described herein or the pharmaceutical composition comprising the host cells is administered to the subject.

[0232] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The term “cancer” includes, for example, the soft tissue tumors (e.g., lymphomas), and tumors of the blood and blood-forming organs (e.g., leukemias), and solid tumors, which is one that grows in an anatomical site outside the bloodstream (e.g., carcinomas). Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (e.g., osteosarcoma or rhabdomyosarcoma), and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), adenosquamous cell carcinoma, lung cancer (e.g., including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (e.g., including gastrointestinal cancer, pancreatic cancer), cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, primary or metastatic melanoma, multiple myeloma and B-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, brain (e.g., high grade glioma, diffuse pontine glioma, ependymoma, neuroblastoma, or glioblastoma), as well as head and neck cancer, and associated metastases. Additional examples of cancer can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, § on Hematology and Oncology, published by Merck Sharp & DohmeAttorney Docket No: 251609.000137 Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online edition at internet website of Merck Manuals); and SEER Program Coding and Staging Manual 2016, each of which are incorporated by reference in their entirety for all purposes.

[0233] In certain embodiments, the cancer that is treated with the compositions and / or methods of the present disclosure is selected from breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, prostate cancer, pancreatic cancer, kidney cancer, bladder cancer, testicular cancer, ovarian cancer, endometrial cancer, cervical cancer, neuroendocrine cancer, head and neck cancer, brain cancer, sarcoma, melanoma, and lymphoma.

[0234] In certain embodiments, host cells modified with a MET CAR, or pharmaceutical compositions thereof, are administered to a subject to treat a cancer expressing MET. Cancers expressing MET include, but are not limited to, breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, prostate cancer, pancreatic cancer, kidney cancer, bladder cancer, testicular cancer, ovarian cancer, endometrial cancer, cervical cancer, neuroendocrine cancer, head and neck cancer, brain cancer, sarcoma, melanoma, and lymphoma.

[0235] In certain embodiments, the cancer is a solid tumor.

[0236] In certain embodiments, therapeutic methods of the present disclosure include one or more of the following steps: a) isolating T cells, NK cells, iNKT cells or macrophages from the subject or generating T cells, NK cells, iNKT cells or macrophages from stem cells including induced pluripotent stem cells (IPS cells); b) genetically modifying said T cells, NK cells, iNKT cells, macrophages or stem cells including IPS cells ex vivo with the polynucleotide or the vector; c) optionally, expanding and / or activating said T cells, NK cells, iNKT cells or macrophages before, after or during step (b); and d) introducing the genetically modified T cells, NK cells, iNKT cells or macrophages into the subject.

[0237] In certain embodiments of any of the therapeutic methods described above, the composition is administered in a therapeutically effective amount. The terms "therapeutically effective amount" and "effective amount" are used interchangeably herein to refer to the administration of an agent to a subject, either alone or as part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount capable of having any detectable, positive effect on any symptom, aspect, or characteristic of a disease, disorder or condition when administered to the subject. The therapeutically effective amount can be ascertained by measuring relevant physiological effects, and it can be adjusted in connection with the dosing regimen and diagnostic analysis of the subject's condition, and the like.

[0238] The dosages of the composition administered in the methods of the invention may vary widely, depending upon the subject’s physical parameters, the frequency ofAttorney Docket No: 251609.000137 administration, the manner of administration, the clearance rate, and the like. The initial dose may be larger, and might be followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level. It is contemplated that a variety of doses will be effective to achieve in vivo persistence of modified host cells. It is also contemplated that a variety of doses will be effective to improve in vivo effector function of modified host cells.

[0239] In certain embodiments, composition comprising the modified host cells manufactured by the methods described herein may be administered at a dosage of 102to 1010cells / kg body weight, 105to 109cells / kg body weight, 105to 108cells / kg body weight, 105to 107cells / kg body weight, 107to 109cells / kg body weight, or 107to 108cells / kg body weight, including all integer values within those ranges. The number of modified host cells will depend on the therapeutic use for which the composition is intended for.

[0240] Modified host cells may be administered multiple times at dosages listed above. The modified host cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.

[0241] The compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for cancer, such as antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.

[0242] It is also contemplated that when used to treat various diseases / disorders, the compositions and methods of the present disclosure can be utilized with other therapeutic methods / agents suitable for the same or similar diseases / disorders. Such other therapeutic methods / agents can be co-administered (simultaneously or sequentially) to generate additive or synergistic effects. Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0243] In certain embodiments of any of the above therapeutic methods, the method further comprises administering to the subject one or more additional compounds such as immuno- suppressives, biologicals, probiotics, prebiotics, and cytokines (e.g., IFN or IL-2).

[0244] As a non-limiting example, the invention can be combined with other therapies that block inflammation (e.g., via blockage of IL1, INF^ / ^, IL6, TNF, IL23, etc.).

[0245] The methods and compositions of the invention can be combined with other immunomodulatory treatments such as, e.g., therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agentsAttorney Docket No: 251609.000137 that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 4-1BB, OX40, etc.). The methods of the invention can be also combined with other treatments that possess the ability to modulate NKT function or stability, including but not limited to CD1d, CD1d-fusion proteins, CD1d dimers or larger polymers of CD1d either unloaded or loaded with antigens, CD1d-chimeric antigen receptors (CD1d-CAR), or any other of the five known CD1 isomers existing in humans (CD1a, CD1b, CD1c, CD1e). The methods of the invention can also be combined with other treatments such as midostaurin, enasidenib, or a combination thereof.

[0246] Therapeutic methods of the invention can be combined with additional immunotherapies and therapies. For example, when used for treating cancer, the compositions of the invention can be used in combination with conventional cancer therapies, such as, e.g., surgery, radiotherapy, chemotherapy or combinations thereof, depending on type of the tumor, patient condition, other health issues, and a variety of factors. In certain aspects, other therapeutic agents useful for combination cancer therapy with the inhibitors of the invention include anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including, e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000). In one embodiment, the modified host cells of the invention can be used in combination with a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof (e.g., anti- hVEGF antibody A4.6.1, bevacizumab or ranibizumab).

[0247] Non-limiting examples of chemotherapeutic compounds which can be used in combination treatments of the present disclosure include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacitidine, bcg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole,Attorney Docket No: 251609.000137 leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0248] In further embodiments, the cell compositions of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In a further embodiment, the cell compositions described herein are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells described herein. In an additional embodiment, expanded cells are administered before or following surgery.

[0249] In certain embodiments, the method described herein comprise administering the cell compositions described herein with one or more cell death inducers. The cell death inducers described herein may be a B-cell lymphoma 2 (BCL-2) family inhibitor, such as a B-cell lymphoma-extra large (BCL-XL) inhibitor. Non-limiting examples of BCL-2 family inhibitors include BCL-XL inhibitors such as Navitoclax (ABT-263), A-1331852, WEHI-539, A- 1155463, and ABT-737; BCL-2 inhibitors such as ABT-199 (Venetoclax); and MCL-1 inhibitors such as S-63845.

[0250] In various embodiments of the methods described herein, the subject is a human. The subject may be a juvenile or an adult, of any age or sex. EXAMPLES

[0251] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.Attorney Docket No: 251609.000137 Example 1. Determination of the in vitro and in vivo cytotoxicity of novel MET-targeting CAR constructs

[0252] An overarching goal of the experiments described herein was to improve the efficacy of MET-targeting chimeric antigen receptor T cell (MET CAR-T) in solid tumors. Whether CRISPR / Cas9-edited MET CAR-T cells would show improved efficacy as compared to conventional lentivirus / retrovirus-based CAR-T therapy in solid tumors was unknown. The lowest level of MET expression that would activate MET CAR-T was also unknown. Determination of the MET CAR-T activation threshold can inform potential off-tumor cytotoxicity in normal tissues, and also if MET CAR-T can potentially kill tumors with low MET expression. The in vivo efficacy of MET CAR-T had not yet been established, which is required before clinical translation.

[0253] Diverse tumor cell lines strongly express MET protein. A range of tumor cell lines of various tissue origins, including triple negative breast cancer (MDA-MB-231) (Figure 1A), diffuse large B-cell lymphoma (Karpas-422) (Figure 1B), lung adenocarcinoma (A549) (Figure 1C), hepatocellular carcinoma (HepG2) (Figure 1D), and renal cell carcinoma (786- O) (Figure 1E) each exhibited MET expression by flow cytometry with the monoclonal anti- MET antibody (Roche, clone SP44) in comparison with the IgG negative control. Strong MET expression was also demonstrated in a human biopsy of a papillary renal cell carcinoma (PRCC) with negative expression in the neighboring background tissues (Figure 1F). The above-described findings confirmed strong MET expression in diverse tumor types supporting its use as a target for CAR-T therapy.

[0254] Engineered MET CAR-T inhibited proliferation of MET-positive solid tumors. To test the potential for CAR-T to target MET positive tumors, a second generation CAR was constructed, the structure of which is depicted in Figure 2A. Eight antigen binding motifs were screened with either CD28 or 4-1BB co-stimulatory domains, totaling 16 constructs. The sequences of the VHH or scFv-based constructs are provided in Tables 1-7 below. To express the MET CAR in primary human T cells, in vitro transcribed messenger RNA (mRNA) encoding the various CARs was electroporated into primary human T cells. The target A549 cells express the nuclear red fluorescent protein mKate2. Monitoring the mKate2 signal using the real-time imager CellCyte showed reduced A549 cell proliferation in the presence of MET CAR-T cells. A549 cells treated with mock CD3+ T cells served as negative control and showed continuous proliferation (Figure 2B).Attorney Docket No: 251609.000137 Table 1. Amino Acid and Nucleotide Sequences for PC15 CAR constructsAttorney Docket No: 251609.000137Attorney Docket No: 251609.000137Attorney Docket No: 251609.000137Table 2. Amino Acid and Nucleotide Sequences for PC16 CAR constructsAttorney Docket No: 251609.000137Attorney Docket No: 251609.000137Attorney Docket No: 251609.000137Table 3. Amino Acid and Nucleotide Sequences for PC17 CAR constructsAttorney Docket No: 251609.000137Attorney Docket No: 251609.000137Attorney Docket No: 251609.000137Table 4. Amino Acid and Nucleotide Sequences for PC18 CAR constructsAttorney Docket No: 251609.000137Attorney Docket No: 251609.000137Attorney Docket No: 251609.000137Table 5. Amino Acid and Nucleotide Sequences for PC19 CAR constructsAttorney Docket No: 251609.000137Attorney Docket No: 251609.000137Attorney Docket No: 251609.000137Table 6. Amino Acid and Nucleotide Sequences for PC11 CAR constructAttorney Docket No: 251609.000137Attorney Docket No: 251609.000137Table 7. Amino Acid and Nucleotide Sequences for 5D5 CAR constructAttorney Docket No: 251609.000137

[0255] MET CARs comprising camelid-derived nanobodies as the antigen binding motif were most effective. Nanobodies are single chain antibodies that are significantly smaller in size than conventional mouse or human antibodies. Nanobody-based MET CARs, referred to as PC15 and PC16 CAR (or PC15 nanobody-CAR and P16 nanobody-CAR, respectively) herein, exhibited significantly improved activity in comparison to a published 5D5 CAR9(Figure 2B). Quantification of cytotoxicity using flow cytometry showed that PC15 and PC16 CARs induced ~80-90% of target cell death after 24 hours, significantly improved from either published or other MET CARs from earlier tests (see, e.g., 5D5 CAR and PC11 CAR in Figure 2C).

[0256] Novel nanobody MET CAR-T showed reduced tonic signaling. Tonic signaling, a state in which T cells are constantly activated even without antigen, can contribute to reduced CAR-T efficacy. To test if PC15 and PC16 show reduced tonic signaling, the T-cell activation marker CD69 was monitored in response to increasing antigen (decreasing E:T ratio in Figure 3). PC11 showed high basal CD69 expression in the absence of antigen, and no antigen- dependent increase in CD69 expression. PC15 and PC16, however, showed low basal CD69 expression in the absence of target cells and were responsive to increasing target stimulation. CD19 CAR served as a positive control for non-tonic signaling CAR.

[0257] Cell lysis of MET-positive tumor was specific to MET CAR-T. To establish that MET CAR-T cytotoxicity was specifically due to MET recognition on tumor cells, whether CD19 CAR-T could lyse the breast cancer cell lines MDA-MB-231 and its variants LM2 and BoM, all of which do not express CD19, was tested. As expected, CD19 CAR-T only mediated basal cytotoxicity against all three cell lines as a result of allogeneic killing. In contrast, PC16 MET CAR-T in the same experiment achieved up to 70% of target tumor lysis (Figure 4).

[0258] Determination of the efficacy of CRISPR / Cas9 engineered MET CAR-T. Building on the novel PC15 and PC16 nanobody-CARs described herein, MET CAR-T performance was further improved by applying CRISPR / Cas9 technology to place PC16 CAR under the control of the T-cell receptor promoter. CAR expression driven by a T-cell receptor promoter can lead to homogeneous CAR expression and avoid random viral integration seen in viral- transduced CAR-T manufacturing.

[0259] Engineering of the PC16 CAR to the T-cell Receptor Alpha Constant (TRAC) locus. To test if placing MET CAR under a T-cell receptor promoter improves solid tumor killing,Attorney Docket No: 251609.000137 knock-in of PC16 CAR was directed to the T-cell Receptor Alpha Constant (TRAC) locus (Figure 5A). Approximately 90% targeting efficiency was found for knock-out at the TRAC locus as demonstrated by only 10% of the remaining T cells expressing native T-cell receptor (Figure 5B). Knock-in efficiency based on CAR expression was approximately 20% (Figure 5B). The TRAC-PC16 CAR-T exhibited significant cytotoxicity against the MET-positive Karpas-422 lymphoma cell line (Figure 5C). Example 2. In vivo efficacy of PC16 mRNA CAR against triple-negative breast cancer (TNBC) metastatic to

[0260] Forkhead N1 athymic mice were intravenously (IV) injected with the LM2 human cancer cell line. This is a triple-negative breast cancer (TNBC) cell line selected to metastasize to the lung and engineered to express luciferase. As depicted in Figure 6, after approximately 10 days, PBS, 5 million mock electroporated T cells (“Mock T”), or 5 million MET CAR-T cells (“PC16”) were IV injected on days 1, 5, and 14 (arrows). Luminescence was measured as Normalized Total Flux. MET CAR-T cells resulted in reduced luminescence as compared to PBS and mock T cell conditions. Example 3. Combination of MET CAR-T cells with BCL-2 family inhibitors

[0261] Select BCL-2 family inhibitors, Navitoclax, ABT-199 (Venetoclax), S-63845, and A-1331852, were incubated with MDA-MB-231 cells, using a serial dilution starting from 1024 nM. The respective protein targets of the tested molecules are shown in Figure 7B. As shown in Figure 7C, two of the tested molecules, Navitoclax and A-1331852, show prominent dose-dependent killing of MDA-MB-231 cells.

[0262] Target cells, MDA-MB-231 or A549 cells, were mixed with MET CAR-T cells at a CAR-T:target ratio of 0.5:1 and a BCL-2 family inhibitor (Navitoclax or Venetoclax), and incubated for 6 hours (Figure 8A). The CAR-T cells were engineered by CRISPR knock-in of the PC16 CAR construct at the TRAC locus. The concentrations of Navitoclax or Venetoclax used in the experiments are as indicated in Figure 8B. Following the 6-hour incubation, flow cytometry was performed to probe cell death using the apoptosis marker Annexin V. Figure 8B shows improved tumor cell killing using navitoclax in combination with the MET CAR-T cells (first and third graphs). No improved killing for MDA-MB-231 cells was observed when the MET CAR-T cells were combined with Venetoclax (middle graph). For A549 cells, a synergist effect was observed when the MET CAR-T cells were combined with Navitoclax, with a Synergy score of 12 as determined by SynergyFinder (third graph). These data suggest that BCL-XL inhibition enhances MET CAR-T killing in solid tumors.Attorney Docket No: 251609.000137

[0263] In a similar set of experiments, MDA-MB-231 cells were mixed with MET CAR- T cells at a CAR-T:target ratio of 0.5:1 and a different BCL-2 family inhibitor (ABT-199, S- 63845, or A-1331852), and incubated for 6 hours. The CAR-T cells were engineered by transfecting T cells with an mRNA encoding the PC16 CAR. The concentrations of ABT-199, S-63845, or A-1331852 used in the experiments are as indicated in Figure 8C. The results show improvement of tumor killing when the MET CAR-T cells were combined with A- 1331852. These data suggest that BCL-XL inhibition may cooperate with CAR-T for tumor killing in MDA-MB-231. Example 4. Avidity of nanobody MET CAR-T cells

[0264] Target cells (786-O) were plated as a monolayer on a glass slide. T cells with no CAR (untransduced) or expressing one of the three MET CARs (PC11, 5D5, and PC16) were added to the monolayer of target cells. An acoustic force was applied to the cells, and as the force increased, the T cells were detached from the target cells. The percentages of T cells remained bound to the target cells were measured. This measurement provides insight into the interaction strength, or avidity, between the CAR-T cells and the target cells. The data show that the PC16 CAR exhibits the greatest avidity (Figures 9A-9B). Figure 9B presents the same data as in Figure 9A, but at the endpoint of 1000 pN force and relative to the untransduced T cells. References 1. Dees, S., Ganesan, R., Singh, S., and Grewal, I.S. (2020). Emerging CAR-T Cell Therapy for the Treatment of Triple-Negative Breast Cancer. Mol. Cancer Ther.19, 2409–2421. https: / / doi.org / 10.1158 / 1535-7163.MCT-20-0385. 2. Zhang, Y., Du, Z., and Zhang, M. (2016). Biomarker development in MET-targeted therapy. Oncotarget 7, 37370–37389. https: / / doi.org / 10.18632 / oncotarget.8276. 3. Yang, X., Liao, H.-Y., and Zhang, H.-H. (2022). Roles of MET in human cancer. Clin. Chim. Acta 525, 69–83. https: / / doi.org / 10.1016 / j.cca.2021.12.017. 4. Graveel, C.R., Tolbert, D., and Vande Woude, G.F. (2013). MET: A Critical Player in Tumorigenesis and Therapeutic Target. Cold Spring Harb. Perspect. Biol.5, a009209. https: / / doi.org / 10.1101 / cshperspect.a009209. 5. Fernandes, M., Jamme, P., Cortot, A.B., Kherrouche, Z., and Tulasne, D. (2021). When the MET receptor kicks in to resist targeted therapies. Oncogene 40, 4061–4078. https: / / doi.org / 10.1038 / s41388-021-01835-0. 6. Wilson, T.R., Fridlyand, J., Yan, Y., Penuel, E., Burton, L., Chan, E., Peng, J., Lin, E., Wang, Y., Sosman, J., et al. (2012). Widespread potential for growth-factor-drivenAttorney Docket No: 251609.000137 resistance to anticancer kinase inhibitors. Nature 487, 505–509. https: / / doi.org / 10.1038 / nature11249. 7. Engelman, J.A., Zejnullahu, K., Mitsudomi, T., Song, Y., Hyland, C., Park, J.O., Lindeman, N., Gale, C.-M., Zhao, X., Christensen, J., et al. (2007). MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316, 1039–1043. https: / / doi.org / 10.1126 / science.1141478. 8. Capmatinib in MET Exon 14–Mutated or MET-Amplified Non–Small-Cell Lung Cancer | NEJM https: / / www.nejm.org / doi / full / 10.1056 / nejmoa2002787. 9. Shah, P.D., Huang, A.C., Xu, X., Orlowski, R., Amaravadi, R.K., Schuchter, L.M., Zhang, P., Tchou, J., Matlawski, T., Cervini, A., et al. (2023). Phase I Trial of Autologous RNA- electroporated cMET-directed CAR T Cells Administered Intravenously in Patients with Melanoma and Breast Carcinoma. Cancer Res Commun 3, 821–829. https: / / doi.org / 10.1158 / 2767-9764.CRC-22-0486. * * *

[0265] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0266] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.Attorney Docket No: 251609.000137 List of Sequences SEQ ID NO: 1 PC15 FR1 Protein sequence EVQLVESGGGLVQPGGSLRLSCAASGFILD SEQ ID NO: 2 PC15 CDR1 Protein sequence YYAIG SEQ ID NO: 3Protein sequence WFRQAPGKEREGVL SEQ ID NO: 4 PC15 CDR2 Protein sequence CIDASDDITYYADSVKG SEQ ID NO: 5 PC15 FR3 Protein sequence RFTISRDNAKNTVYLQMNSLKPEDTGVYYCAT SEQ ID NO: 6 CDR3 Protein sequence PIGLSSSCLLEYDYDY SEQ ID NO: 7 PC15, PC17, PC18 FR4 Protein sequence WGQGTLVTVSS SEQ ID NO: 8 PC15 VHH DNA sequence gaggtgcaattggtggagtctgggggaggcttggtgcagcctggggggtccctgagactctcctgtgcagcctctggattcattttgga ttattatgccataggctggttccgccaggccccagggaaggagcgcgagggggtcttatgtattgatgctagtgatgatattacatactat gcagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgag gacacgggcgtttattactgtgcgacccccatcggactgagtagtagctgcctacttgaatatgattatgactactggggccaggggac cctggtcacggtctcctcc SEQ ID NO: 9 Entire PC15 VHH sequence Protein sequence EVQLVESGGGLVQPGGSLRLSCAASGFILDYYAIGWFRQAPGKEREGVLCIDASDDIT YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCATPIGLSSSCLLEYDYDY WGQGTLVTVSS SEQ ID NO: 10 CD8^ signal peptide DNA sequence atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggccc SEQ ID NO: 11 CD8^ signal peptide Protein sequence MALPVTALLLPLALLLHAARP SEQ ID NO: 12 MYC tag DNA sequence gagcagaagctgatcagcgaggaggacctg SEQ ID NO: 13 MYC tag Protein sequence EQKLISEEDL SEQ ID NO: 14 CD8^ hinge-TM DNA sequenceAttorney Docket No: 251609.000137 acaacaacaccagctcctcgaccaccaacacctgctcctaccattgcttctcagcctctgtccctgcgcccagaggcgtgccggccag ctgctggaggagcagtgcacacgagggggctggacttcgcctgtgatatttacatctgggcgcccttggccgggacttgtggggtcct tctcctgtcactggttatcaccctttactgc SEQ ID NO: 15 CD8^ hinge-TM Protein sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV LLLSLVITLYC SEQ ID NO: 16 4-1BB DNA sequence aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctg ccgatttccagaagaagaagaaggaggatgtgaactg SEQ ID NO: 17 4-1BB Protein sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 18 CD3^ DNA sequence agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaa gagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaag gcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaagg ggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 19 CD3^ Protein sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO: 20 Entire PC15-CAR sequence DNA sequence atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggcccgagcagaagctgatcagcgagg aggacctggaggtgcaattggtggagtctgggggaggcttggtgcagcctggggggtccctgagactctcctgtgcagcctctggatt cattttggattattatgccataggctggttccgccaggccccagggaaggagcgcgagggggtcttatgtattgatgctagtgatgatatt acatactatgcagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctga aacctgaggacacgggcgtttattactgtgcgacccccatcggactgagtagtagctgcctacttgaatatgattatgactactggggcc aggggaccctggtcacggtctcctccacaacaacaccagctcctcgaccaccaacacctgctcctaccattgcttctcagcctctgtcc ctgcgcccagaggcgtgccggccagctgctggaggagcagtgcacacgagggggctggacttcgcctgtgatatttacatctgggc gcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattc aaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgt gaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctag gacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctc aggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggg gcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccct cgc SEQ ID NO: 21 Entire PC15-CAR sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASG FILDYYAIGWFRQAPGKEREGVLCIDASDDITYYADSVKGRFTISRDNAKNTVYLQM NSLKPEDTGVYYCATPIGLSSSCLLEYDYDYWGQGTLVTVSSTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAttorney Docket No: 251609.000137 SEQ ID NO: 24 CD28 hinge-TM DNA sequence gcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctt tgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaac agtggcctttattattttctgggtg SEQ ID NO: 25 CD28 hinge-TM Protein sequence AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWV SEQ ID NO: 26 CD28 costimulatory domain DNA sequence aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagc cctatgccccaccacgcgacttcgcagcctatcgctcc SEQ ID NO: 27 CD28 costimulatory domain Protein sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 28 CD3^ DNA sequence agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaa gagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaag gcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaagg ggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 29 CD3^ Protein sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO: 30 Entire PC15.1 sequence DNA sequence atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggcccgagcagaagctgatcagcgagg aggacctggaggtgcaattggtggagtctgggggaggcttggtgcagcctggggggtccctgagactctcctgtgcagcctctggatt cattttggattattatgccataggctggttccgccaggccccagggaaggagcgcgagggggtcttatgtattgatgctagtgatgatatt acatactatgcagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctga aacctgaggacacgggcgtttattactgtgcgacccccatcggactgagtagtagctgcctacttgaatatgattatgactactggggcc aggggaccctggtcacggtctcctccgcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaacc attatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggag tcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacat gaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcca gagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaag agaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaagg cctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggg gcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 31 Entire PC15.1 sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASG FILDYYAIGWFRQAPGKEREGVLCIDASDDITYYADSVKGRFTISRDNAKNTVYLQM NSLKPEDTGVYYCATPIGLSSSCLLEYDYDYWGQGTLVTVSSAAAIEVMYPPPYLDN EKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQAttorney Docket No: 251609.000137 LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 32 PC11 ScFv DNA sequence caggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaggatctcctgtcagggttctggatacagttttc ccacccactggatcacctgggtgcgccagatgcccgggaaaggcctggagtggatgggaacgattgatcctactgactcttacaattt ctatggaccgtcgttccaaggccacgtcaccatctcagccgacagctccagcagcaccgcctacctgcagtggagcagcctgaagg cctcggacaccgccatgtattactgtgcgagagatggcaactactatgatagtcgcggttattactacgatacttttgatatgtggggcca agggacactggtcaccgtctcctcaggactaggaggactaggaggaggaggaagtggaggaggaggaagtggaggaagtagtgg agtgggatccgacatccagatgacccagtctccatccttcctctctgcatctgtcggagacagagtcaccatcacttgccgggccagtc agggcatcagtacttatttagcctggtatcaacaaaaaccagggacagcccctaaactcctgatctattctgcatccactttggaaagtgg ggtcccatcgcgattcagcggaagtggatccgggacagatttcactctcaccatcagcagcctgcagcctgaagattctgcaacttact attgtcaacaggctgacagtttcccgctcactttcggcggagggaccaaggtggagatcaaa SEQ ID NO: 33 PC16 FR1 Protein sequence EVQLVESGGGLVQPGGSLRLSCAASGFSLD SEQ ID NO: 34 PC16 CDR1 Protein sequence YFAIG SEQ ID NO: 35 PC16 FR2 Protein sequence WFRQAPGKEREEIS SEQ ID NO: 36 PC16 CDR2 Protein sequence CISNSDGSTYYANSVKG SEQ ID NO: 37 PC16 FR3 Protein sequence RFTISIDNAKNTVYLQMTSLKPEDTAVYYCAT SEQ ID NO: 38 PC16 CDR3 Protein sequence PVGLGPFCKTTNDYDY SEQ ID NO: 39 PC16 FR4 Protein sequence SGQGTLVTVSS SEQ ID NO: 40 PC16 VHH DNA sequence gaggtgcaattggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattctctttggat tattttgccataggctggttccgccaggccccagggaaggagcgcgaggaaatctcatgtattagtaacagtgatggtagcacatacta tgcaaactccgtgaagggccgattcaccatctccatagacaatgccaagaacacggtgtatctgcaaatgacaagcctgaaacctgag gacacggccgtttattactgtgcgacccccgtggggttggggccattctgtaagacgaccaatgactatgactacagcggccagggg accctggtcacggtctcctcc SEQ ID NO: 41 Entire PC16 VHH sequence Protein sequence EVQLVESGGGLVQPGGSLRLSCAASGFSLDYFAIGWFRQAPGKEREEISCISNSDGST YYANSVKGRFTISIDNAKNTVYLQMTSLKPEDTAVYYCATPVGLGPFCKTTNDYDY SGQGTLVTVSS SEQ ID NO: 42 Entire PC16-CAR sequence DNA sequence atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggcccgagcagaagctgatcagcgagg aggacctggaggtgcaattggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattAttorney Docket No: 251609.000137 ctctttggattattttgccataggctggttccgccaggccccagggaaggagcgcgaggaaatctcatgtattagtaacagtgatggtag cacatactatgcaaactccgtgaagggccgattcaccatctccatagacaatgccaagaacacggtgtatctgcaaatgacaagcctg aaacctgaggacacggccgtttattactgtgcgacccccgtggggttggggccattctgtaagacgaccaatgactatgactacagcg gccaggggaccctggtcacggtctcctccacaacaacaccagctcctcgaccaccaacacctgctcctaccattgcttctcagcctctg tccctgcgcccagaggcgtgccggccagctgctggaggagcagtgcacacgagggggctggacttcgcctgtgatatttacatctgg gcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatat tcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggat gtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatcta ggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccct caggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggagg ggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccc tcgc SEQ ID NO: 43 Entire PC16-CAR sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASG FSLDYFAIGWFRQAPGKEREEISCISNSDGSTYYANSVKGRFTISIDNAKNTVYLQMT SLKPEDTAVYYCATPVGLGPFCKTTNDYDYSGQGTLVTVSSTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 44 Entire PC16.1 sequence DNA sequence atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggcccgagcagaagctgatcagcgagg aggacctggaggtgcaattggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggatt ctctttggattattttgccataggctggttccgccaggccccagggaaggagcgcgaggaaatctcatgtattagtaacagtgatggtag cacatactatgcaaactccgtgaagggccgattcaccatctccatagacaatgccaagaacacggtgtatctgcaaatgacaagcctg aaacctgaggacacggccgtttattactgtgcgacccccgtggggttggggccattctgtaagacgaccaatgactatgactacagcg gccaggggaccctggtcacggtctcctccgcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatgg aaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttggg ggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgact acatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgct ccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacg aagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcagga aggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaa ggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 45 Entire PC16.1 sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASG FSLDYFAIGWFRQAPGKEREEISCISNSDGSTYYANSVKGRFTISIDNAKNTVYLQMT SLKPEDTAVYYCATPVGLGPFCKTTNDYDYSGQGTLVTVSSAAAIEVMYPPPYLDNE KSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRS RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 46 PC17 FR1 Protein sequence EVQLVESGGGLVQAGGSLRLSCAASGRTIS SEQ ID NO: 47 PC17 CDR1 Protein sequenceAttorney Docket No: 251609.000137 RYTMG SEQ ID NO: 48 PC17 FR2 Protein sequence WFRQAPGKEREFVA SEQ ID NO: 49 PC17 CDR2 Protein sequence AISWSGDNTNYADSVKG SEQ ID NO: 50 PC17 FR3 Protein sequence RFTISRPNTKNTMYLQMNSLKPEDTAVYYCAA SEQ ID NO: 51 PC17 CDR3 Protein sequence DYRSGSYYQASEWTRPSGYDY SEQ ID NO: 52 PC17 VHH DNA sequence gaggtgcaattggtggagtctgggggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgcaccatc agtaggtataccatgggctggttccgccaggctccagggaaggagcgtgagtttgtagcagctattagctggagtggtgataacacaa actatgcagactccgtgaagggccgattcaccatctccagacccaacaccaagaacacgatgtatctgcaaatgaacagcctgaaac ctgaggacacggccgtttattactgtgcagcagattaccgaagtggtagttactaccaggcatcagagtggacacggccatcggggta tgactactggggccaggggaccctggtcacggtctcctcc SEQ ID NO: 53 Entire PC17 VHH sequence Protein sequence EVQLVESGGGLVQAGGSLRLSCAASGRTISRYTMGWFRQAPGKEREFVAAISWSGD NTNYADSVKGRFTISRPNTKNTMYLQMNSLKPEDTAVYYCAADYRSGSYYQASEW TRPSGYDYWGQGTLVTVSS SEQ ID NO: 54 Entire PC17-CAR sequence DNA sequence ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCG CCCGGCCCGAGCAGAAGCTGATCAGCGAGGAGGACCTGgaggtgcaattggtggagtctgggg gaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgcaccatcagtaggtataccatgggctggttccg ccaggctccagggaaggagcgtgagtttgtagcagctattagctggagtggtgataacacaaactatgcagactccgtgaagggccg attcaccatctccagacccaacaccaagaacacgatgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtg cagcagattaccgaagtggtagttactaccaggcatcagagtggacacggccatcggggtatgactactggggccaggggaccctg gtcacggtctcctccacaacaacaccagctcctcgaccaccaacacctgctcctaccattgcttctcagcctctgtccctgcgcccaga ggcgtgccggccagctgctggaggagcagtgcacacgagggggctggacttcgcctgtgatatttacatctgggcgcccttggccg ggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccattt atgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagt gaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagaga ggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcct gtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggca cgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 55 Entire PC17-CAR sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQAGGSLRLSCAASG RTISRYTMGWFRQAPGKEREFVAAISWSGDNTNYADSVKGRFTISRPNTKNTMYLQ MNSLKPEDTAVYYCAADYRSGSYYQASEWTRPSGYDYWGQGTLVTVSSTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRAttorney Docket No: 251609.000137 SEQ ID NO: 56 Entire PC17.1 sequence DNA sequence atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggcccgagcagaagctgatcagcgagg aggacctggaggtgcaattggtggagtctgggggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggac gcaccatcagtaggtataccatgggctggttccgccaggctccagggaaggagcgtgagtttgtagcagctattagctggagtggtga taacacaaactatgcagactccgtgaagggccgattcaccatctccagacccaacaccaagaacacgatgtatctgcaaatgaacagc ctgaaacctgaggacacggccgtttattactgtgcagcagattaccgaagtggtagttactaccaggcatcagagtggacacggccat cggggtatgactactggggccaggggaccctggtcacggtctcctccgcggccgcaattgaagttatgtatcctcctccttacctagac aatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgg gtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagca ggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgc gacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataa cgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgaga aggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcg agcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgca ggccctgccccctcgc SEQ ID NO: 57 Entire PC17.1 sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQAGGSLRLSCAASG RTISRYTMGWFRQAPGKEREFVAAISWSGDNTNYADSVKGRFTISRPNTKNTMYLQ MNSLKPEDTAVYYCAADYRSGSYYQASEWTRPSGYDYWGQGTLVTVSSAAAIEVM YPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 58 PC18 FR1 Protein sequence EVQLVESGGGLVQPGGSLRLSCAASGFTLD SEQ ID NO: 59 PC18 CDR1 Protein sequence YYAIN SEQ ID NO: 60 PC18 FR2 Protein sequence WFRQAPGKEREGVS SEQ ID NO: 61 PC18 CDR2 Protein sequence CISGGDGSTYYADSVKG SEQ ID NO: 62 PC18 FR3 Protein sequence RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT SEQ ID NO: 63 PC18 CDR3 Protein sequence ALGLSSSCHGDGDY SEQ ID NO: 64 PC18 VHH DNA sequence gaggtgcaattggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttgga ttattatgccataaactggttccgccaggccccagggaaggagcgcgagggggtctcatgtattagtggtggtgatggtagcacatact atgcagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaaacctg aggacacggccgtttattactgtgcgacagccttaggattatcaagtagctgccacggagacggatatgactactggggccagggga ccctggtcacggtctcctccAttorney Docket No: 251609.000137 SEQ ID NO: 65 Entire PC18 VHH sequence Protein sequence EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAINWFRQAPGKEREGVSCISGGDGS TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATALGLSSSCHGDGYDY WGQGTLVTVSS SEQ ID NO: 66 Entire PC18-CAR sequence DNA sequence ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCG CCCGGCCCGAGCAGAAGCTGATCAGCGAGGAGGACCTGgaggtgcaattggtggagtctgggg gaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttggattattatgccataaactggttccgcca ggccccagggaaggagcgcgagggggtctcatgtattagtggtggtgatggtagcacatactatgcagactccgtgaagggccgatt caccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcg acagccttaggattatcaagtagctgccacggagacggatatgactactggggccaggggaccctggtcacggtctcctccacaaca acaccagctcctcgaccaccaacacctgctcctaccattgcttctcagcctctgtccctgcgcccagaggcgtgccggccagctgctg gaggagcagtgcacacgagggggctggacttcgcctgtgatatttacatctgggcgcccttggccgggacttgtggggtccttctcct gtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactact caagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcag acgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaag agacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagat aagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctc agtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 67 Entire PC18-CAR sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASG FTLDYYAINWFRQAPGKEREGVSCISGGDGSTYYADSVKGRFTISRDNAKNTVYLQ MNSLKPEDTAVYYCATALGLSSSCHGDGYDYWGQGTLVTVSSTTTPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 68 Entire PC18.1 sequence DNA sequence atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggcccgagcagaagctgatcagcgagg aggacctggaggtgcaattggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggatt cactttggattattatgccataaactggttccgccaggccccagggaaggagcgcgagggggtctcatgtattagtggtggtgatggta gcacatactatgcagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcct gaaacctgaggacacggccgtttattactgtgcgacagccttaggattatcaagtagctgccacggagacggatatgactactggggc caggggaccctggtcacggtctcctccgcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaac cattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttggggga gtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactaca tgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcca gagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaag agaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaagg cctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggg gcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 69 Entire PC18.1 sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQPGGSLRLSCAASG FTLDYYAINWFRQAPGKEREGVSCISGGDGSTYYADSVKGRFTISRDNAKNTVYLQAttorney Docket No: 251609.000137 MNSLKPEDTAVYYCATALGLSSSCHGDGYDYWGQGTLVTVSSAAAIEVMYPPPYL DNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 70 PC11 scFv Protein sequence QVQLVQSGAEVKKPGESLRISCQGSGYSFPTHWITWVRQMPGKGLEWMGTIDPTDS YNFYGPSFQGHVTISADSSSSTAYLQWSSLKASDTAMYYCARDGNYYDSRGYYYDT FDMWGQGTLVTVSSGLGGLGGGGSGGGGSGGSSGVGSDIQMTQSPSFLSASVGDRV TITCRASQGISTYLAWYQQKPGTAPKLLIYSASTLESGVPSRFSGSGSGTDFTLTISSLQ PEDSATYYCQQADSFPLTFGGGTKVEIK SEQ ID NO: 71 PC19 FR1 Protein sequence EVQLVESGGGLVQAGGSLRLSCAASGFTFD SEQ ID NO: 72 PC19 CDR1 Protein sequence DYAIG SEQ ID NO: 73 PC19 FR2 Protein sequence WFRQAPGEEREGVS SEQ ID NO: 74 PC19 CDR2 Protein sequence SISSTYGLTYYADSVKG SEQ ID NO: 75 PC19 FR3 Protein sequence RFTISSSNAKNTVYLQMNNLKPEDTAVYYCAA SEQ ID NO: 76 PC19 CDR3 Protein sequence TPIERLGLDAYEYDY SEQ ID NO: 77 PC19 FR4 Protein sequence WGQGTQVTVSS SEQ ID NO: 78 PC19 VHH DNA sequence GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCGGCGGCAGCCTG AGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATCGGCTGGT TCAGGCAGGCCCCCGGCGAGGAGAGGGAGGGCGTGAGCAGCATCAGCAGCACC TACGGCCTGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGC AGCAACGCCAAGAACACCGTGTACCTGCAGATGAACAACCTGAAGCCCGAGGAC ACCGCCGTGTACTACTGCGCCGCCACCCCCATCGAGAGGCTGGGCCTGGACGCCT ACGAGTACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC SEQ ID NO: 79 Entire PC19 VHH sequence Protein sequence EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGEEREGVSSISSTYGL TYYADSVKGRFTISSSNAKNTVYLQMNNLKPEDTAVYYCAATPIERLGLDAYEYDY WGQGTQVTVSS SEQ ID NO: 80 Entire PC19-CAR sequence DNA sequenceAttorney Docket No: 251609.000137 atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggcccgagcagaagctgatcagcgagg aggacctgGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCGGCGGC AGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATCG GCTGGTTCAGGCAGGCCCCCGGCGAGGAGAGGGAGGGCGTGAGCAGCATCAGC AGCACCTACGGCCTGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATC AGCAGCAGCAACGCCAAGAACACCGTGTACCTGCAGATGAACAACCTGAAGCCC GAGGACACCGCCGTGTACTACTGCGCCGCCACCCCCATCGAGAGGCTGGGCCTG GACGCCTACGAGTACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC acaacaacaccagctcctcgaccaccaacacctgctcctaccattgcttctcagcctctgtccctgcgcccagaggcgtgccggccag ctgctggaggagcagtgcacacgagggggctggacttcgcctgtgatatttacatctgggcgcccttggccgggacttgtggggtcct tctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaa actactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggag cgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttgg acaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcag aaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccag ggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 81 Entire PC19-CAR sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQAGGSLRLSCAASG FTFDDYAIGWFRQAPGEEREGVSSISSTYGLTYYADSVKGRFTISSSNAKNTVYLQM NNLKPEDTAVYYCAATPIERLGLDAYEYDYWGQGTQVTVSSTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 82 Entire PC19.1 sequence DNA sequence atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggcccgagcagaagctgatcagcgagg aggacctgGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCGGCGGC AGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATCG GCTGGTTCAGGCAGGCCCCCGGCGAGGAGAGGGAGGGCGTGAGCAGCATCAGC AGCACCTACGGCCTGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATC AGCAGCAGCAACGCCAAGAACACCGTGTACCTGCAGATGAACAACCTGAAGCCC GAGGACACCGCCGTGTACTACTGCGCCGCCACCCCCATCGAGAGGCTGGGCCTG GACGCCTACGAGTACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC gcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctt tgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaac agtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccggg cccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcaga cgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaaga gacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagata agatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctca gtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 83 Entire PC19.1 sequence Protein sequence MALPVTALLLPLALLLHAARPEQKLISEEDLEVQLVESGGGLVQAGGSLRLSCAASG FTFDDYAIGWFRQAPGEEREGVSSISSTYGLTYYADSVKGRFTISSSNAKNTVYLQM NNLKPEDTAVYYCAATPIERLGLDAYEYDYWGQGTQVTVSSAAAIEVMYPPPYLDN EKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQAttorney Docket No: 251609.000137 LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 84 Entire PC11 sequence DNA sequence atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgcccggccccaggtgcagctggtgcagtctgg agcagaggtgaaaaagcccggggagtctctgaggatctcctgtcagggttctggatacagttttcccacccactggatcacctgggtg cgccagatgcccgggaaaggcctggagtggatgggaacgattgatcctactgactcttacaatttctatggaccgtcgttccaaggcca cgtcaccatctcagccgacagctccagcagcaccgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattactg tgcgagagatggcaactactatgatagtcgcggttattactacgatacttttgatatgtggggccaagggacactggtcaccgtctcctca ggactaggaggactaggaggaggaggaagtggaggaggaggaagtggaggaagtagtggagtgggatccgacatccagatgac ccagtctccatccttcctctctgcatctgtcggagacagagtcaccatcacttgccgggccagtcagggcatcagtacttatttagcctgg tatcaacaaaaaccagggacagcccctaaactcctgatctattctgcatccactttggaaagtggggtcccatcgcgattcagcggaag tggatccgggacagatttcactctcaccatcagcagcctgcagcctgaagattctgcaacttactattgtcaacaggctgacagtttccc gctcactttcggcggagggaccaaggtggagatcaaacgtggaggagccagcctcgtggaagcggccgcaattgaagttatgtatc ctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggac cttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtga ggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagcc ctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggc cagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagat ggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtga gattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctac gacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 85 Entire PC11 sequence Protein sequence MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGESLRISCQGSGYSFPTHWITW VRQMPGKGLEWMGTIDPTDSYNFYGPSFQGHVTISADSSSSTAYLQWSSLKASDTA MYYCARDGNYYDSRGYYYDTFDMWGQGTLVTVSSGLGGLGGGGSGGGGSGGSSG VGSDIQMTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKPGTAPKLLIYSASTLE SGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQADSFPLTFGGGTKVEIKRGGASLV EAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 86 PC11 HCDR1 Protein sequence THWIT SEQ ID NO: 87 PC11 HCDR2 Protein sequence TIDPTDSYNFYGPSFQG SEQ ID NO: 88 PC11 HCDR3 Protein sequence DGNYYDSRGYYYDTFDM SEQ ID NO: 89 PC11 VH Protein sequence QVQLVQSGAEVKKPGESLRISCQGSGYSFPTHWITWVRQMPGKGLEWMGTIDPTDS YNFYGPSFQGHVTISADSSSSTAYLQWSSLKASDTAMYYCARDGNYYDSRGYYYDT FDMWGQGTLVTVSS SEQ ID NO: 90 PC11 LCDR1 Protein sequence RASQGISTYLAAttorney Docket No: 251609.000137 SEQ ID NO: 91 PC11 LCDR2 Protein sequence SASTLES SEQ ID NO: 92 PC11 LCDR3 Protein sequence QQADSFPLT SEQ ID NO: 93 PC11 VL Protein sequence DIQMTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKPGTAPKLLIYSASTLESGV PSRFSGSGSGTDFTLTISSLQPEDSATYYCQQADSFPLTFGGGTKVEIKRGGASLVE SEQ ID NO: 94 Entire 5D5 sequence DNA sequence atgctgctgctggtgaccagcctgctgctgtgtgagctgccccaccccgcctttctgctgatccccgacatccagatgacccagagccc cagcagcgtgagcgccagcgtgggcgaccgggtgaccatcacctgccgggccagccagggcatcaacacctggctggcctggtat cagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgaagagcggcgtgcccagccggtttagcggct ctggctctggcgccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactactgccagcaggccaacagct tccccctgacctttggcggcggaacaaaggtggagatcaagggcagcacctccggcagcggcaagcctggcagcggcgagggca gcaccaagggccaggtgcagctggtgcagagcggagccgaggtgaagaagcctggcgcctccgtcaaggtgtcctgcgaggcca gcggctacaccttcaccagctacggcttcagctgggtgcggcaggcaccaggccagggcctcgaatggatgggctggatcagcgc cagcaacggcaacacctactacgcccagaagctgcagggcagggtcaccatgaccaccgacaccagcaccagcagcgcctacat ggaactgcggagcctgagaagcgacgacaccgccgtgtactactgcgccagggtgtacgccgactacgccgattactggggccag ggcaccctggtgaccgtgagcagcgaaagcaagtatggccccccatgccctccctgccccatttacatctgggcgcccttggccggg acttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatg agaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaa gttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagagga gtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtac aatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgat ggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO: 95 Entire 5D5 sequence Protein sequence MLLLVTSLLLCELPHPAFLLIPDIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWY QQKPGKAPKLLIYAASSLKSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQANSFP LTFGGGTKVEIKGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGASVKVSCEASG YTFTSYGFSWVRQAPGQGLEWMGWISASNGNTYYAQKLQGRVTMTTDTSTSSAYM ELRSLRSDDTAVYYCARVYADYADYWGQGTLVTVSSESKYGPPCPPCPIYIWAPLA GTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO: 96 CD8^ hinge DNA sequence Acaacaacaccagctcctcgaccaccaacacctgctcctaccattgcttctcagcctctgtccctgcgcccagaggcgtgccggcca gctgctggaggagcagtgcacacgagggggctggacttcgcctgtgat SEQ ID NO: 97 CD8^ hinge Protein sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 98 CD28 hinge DNA sequenceAttorney Docket No: 251609.000137 Gcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacct ttgtccaagtcccctatttcccggaccttctaagccc SEQ ID NO: 99 CD28 hinge Protein sequence AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP SEQ ID NO: 100 CD8^ TM domain DNA sequence Atttacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc SEQ ID NO: 101 CD8^ TM domain Protein sequence IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 102 CD28 TM domain DNA sequence Ttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg SEQ ID NO: 103 CD28 TM domain Protein sequence FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 104 TRAC 5’ homology sequence Gcggccgctcggtccgctagcgatgtaaggagctgctgtgacttgctcaaggccttatatcgagtaaacggtagtgctggggcttaga cgcaggtgttctgatttatagttcaaaacctctatcaatgagagagcaatctcctggtaatgtgatagatttcccaacttaatgccaacatac cataaacctcccattctgctaatgcccagcctaagttggggagaccactccagattccaagatgtacagtttgctttgctgggcctttttcc catgcctgcctttactctgccagagttatattgctggggttttgaagaagatcctattaaataaaagaataagcagtattattaagtagccct gcatttcaggtttccttgagtggcaggccaggcctggccgtgaacgttcactgaaatcatggcctcttggccaagattgatagcttgtgc ctgtccctgagtcccagtccatcacgagcagctggtttctaagatgctatttcccgtataaagcatgagaccgtgacttgccagccccac agagccccgcccttgtccatcactggcatctggactccagcctgggttggggcaaagagggaaatgagatcatgtcctaaccctgga attggatcctcttgtcttacagatggatctgga SEQ ID NO: 105 TRAC gRNA sequence DNA sequence TCAGGGTTCTGGATATCTGT SEQ ID NO: 106 TRAC gRNA sequence RNA sequence UCAGGGUUCUGGAUAUCUGU SEQ ID NO: 107 GAPDH 5’ homology sequence TCGAGGAATTCCTGGCTTGTTGTCCACAACCATTAAACCTTAAAAGCTTTAAAAG CCTTATATATTCTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTT GCTGATTTATATTAATTTTATTGTTCAAACATGAGAGCTTAGTACGTGAAACATG AGAGCTTAGTACATTAGCCATGAGAGCTTAGTACATTAGCCATGAGGGTTTAGTT CATTAAACATGAGAGCTTAGTACATTAAACATGAGAGCTTAGTACATACTATCAA CAGGTTGAACTGCTGATCTGTACAGTAGAATTGGTAAAGAGAGTTGTGTAAAAT ATTGAGTTCGCACATCTTGTTGTCTGATTATTGATTTTTGGCGAAACCATTTGATC ATATGACAAGATGTGTATCTACCTTAACTTAATGATTTTGATAAAAATCATTAGG TACCGAATTCACGCGTATTGGGATGAAGACTGTGGATGGCCCCTCCGGGAAACT GTGGCGTGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCT GCCAAGGCTGTGGGCAAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATG GCCTTCCGTGTCCCCACTGCCAACGTGTCAGTGGTGGACCTGACCTGCCGTCTAG AAAAACCTGCCAAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTCGGAG GGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACT TCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCAAAttorney Docket No: 251609.000137 CGACCACTTTGTCAAGCTCATTTCCTGGTATGTGGCTGGGGCCAGAGACTGGCTC TTAAAAAGTGCAGGGTCTGGCGCCCTCTGGTGGCTGGCTCAGAAAAAGGGCCCT GACAACTCTTTACATCTTCTAGGTATGACAACGAGTTCGGATATAGCAATAGAGT GGTCGATCTGATGGCTCATATGGCTAGCAAAGAG SEQ ID NO: 108 GAPDH gRNA1 sequence (for Cas12) DNA sequence ATCTTCTAGGTATGACAACGA SEQ ID NO: 109 GAPDH gRNA1 sequence (for Cas12) RNA sequence AUCUUCUAGGUAUGACAACGA SEQ ID NO: 110 GAPDH gRNA2 sequence (for Cas9) DNA sequence TCTAGGTATGACAACGAATT SEQ ID NO: 111 GAPDH gRNA2 sequence (for Cas9) RNA sequence UCUAGGUAUGACAACGAAUU SEQ ID NO: 112 TRAC 3’ homology sequence Aatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctc aaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaaca gtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcc caggtaagggcagctttggtgccttcgcaggctgtttccttgcttcaggaatggccaggttctgcccagagctctggtcaatgatgtctaa aactcctctgattggtggtctcggccttatccattgccaccaaaaccctctttttactaagaaacagtgagccttgttctggcagtccagag aatgacacgggaaaaaagcagatgaagagaaggtggcaggagagggcacgtggcccagcctcagtctctccaactgagttcctgc ctgcctgcctttgctcagactgtttgccccttactgctcttctaggcctcattctaagccccttctccaagttgacgcgtgcggccgc SEQ ID NO: 113 GAPDH 3’ homology sequence GCGGCCGCGTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGT GCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCC TGGAAGGTGCCACCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTG TCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGG GGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGA TTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGAG TAAGACCCCTGGACCACCAGCCCCAGCAAGAGCACAAGAGGAAGAGAGAGACC CTCACTGCTGGGGAGTCCCTGCCACACTCAGTCCCCCACCACACTGAATCTCCCC TCCTCACAGTTGCCATGTAGACCCCTTGAAGAGGGGAGGGGCCTAGGGAGCCGC ACCTTGTCATGTACCATCAATAAAGTACCCTGTGCTCAACCAGTTACTTGTCCTGT CTTATTCTAGGGTCTGGGGCAGAGGGGAGGGAAGCTGGGCTTGTGTCAAGGTGA GACATTCTTGCTGGGGAGGGACCTGGTATGTTCTCCTCAGACTGAGGGTAGGGCC TCCAAACAGCCTTGCTTGCTTCGAGAACCATTTGCTTCCCGCTCAGACGTCTTGA GTGCTACAGGAAGCTGGCACCACTACTTCAGAGAACAAGGCCTTTTCCTCTCCTC GCTCCAGTATCCCAATGGCGCGCCGAGCTTGGC

Claims

Attorney Docket No: 251609.000137 Claims 1. A chimeric antigen receptor (CAR) comprising: a) an extracellular domain which specifically binds to Mesenchymal Epithelial Transition factor (MET) and comprises at least (i) a VHH domain comprising a complementarity determining region (CDR) 1, CDR2, and CDR3, and wherein the CDR1, CDR2, and CDR3 comprise: 1) SEQ ID NOs: 2, 4, and 6, respectively; 2) SEQ ID NOs: 34, 36, and 38, respectively; 3) SEQ ID NOs: 47, 49, and 51, respectively; 4) SEQ ID NOs: 59, 61, and 63, respectively; or 5) SEQ ID NOs: 72, 74, and 76, respectively, or (ii) a scFv comprising a light chain variable domain (VL) comprising a light chain CDR1 (LCDR1), LCDR2, and LCDR3 comprising SEQ ID NOs: 90, 91, and 92, respectively, and a heavy chain variable domain (VH) comprising a heavy chain CDR1 (HCDR1), HCDR2, and HCDR3 comprising SEQ ID NOs: 86, 87, and 88, respectively; b) a transmembrane (TM) domain; c) a co-stimulatory signaling domain; and d) a primary intracellular signaling domain.

2. The CAR of claim 1, wherein the extracellular domain comprises at least a VHH domain comprising the amino acid sequence of SEQ ID NO: 9, 41, 53, 65, or 79, or a sequence having at least 80% identity thereto.

3. The CAR of claim 2, wherein the nucleotide sequence encoding the at least a VHH domain comprises the nucleotide sequence of SEQ ID NO: 8, 40, 52, 64, or 78, or a sequence having at least 80% identity thereto.

4. The CAR of any one of claims 1-3, wherein the extracellular domain comprises two or more VHH domains.

5. The CAR of claim 1, wherein the extracellular domain comprises an scFv comprising a VL comprising the amino acid sequence of SEQ ID NO: 93, or a sequence having at least 80% identity thereto; and a VH comprising the amino acid sequence of SEQ ID NO: 89, or aAttorney Docket No: 251609.000137 sequence having at least 80% identity thereto.

6. The CAR of claim 5, wherein the extracellular domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 70, or a sequence having at least 80% identity thereto. The CAR of claim 6, wherein the nucleotide sequence encoding the scFv comprises the nucleotide sequence of SEQ ID NO: 32, or a sequence having at least 80% identity thereto.

8. The CAR of any one of claims 1-7, wherein the primary intracellular signaling domain is CD3^ signaling domain.

9. The CAR of claim 8, wherein the CD3^ signaling domain comprises the amino acid sequence of SEQ ID NO: 19, or a sequence having at least 80% identity thereto.

10. The CAR of claim 9, wherein the nucleotide sequence encoding the CD3^ signaling domain comprises the nucleotide sequence of SEQ ID NO: 18, or a sequence having at least 80% identity thereto.

11. The CAR of any one of claims 1-10, wherein the TM domain is derived from CD8^ or CD28.

12. The CAR of any one of claims 1-11, further comprising a hinge domain.

13. The CAR of claim 11 or claim 12, comprising a hinge and TM domain derived from CD8^.

14. The CAR of claim 13, wherein the hinge and TM domain derived from CD8^ comprises the amino acid sequence of SEQ ID NO: 15, or a sequence having at least 80% identity thereto.

15. The CAR of claim 14, wherein the nucleotide sequence encoding the hinge and TM domain derived from CD8^ comprises the nucleotide sequence of SEQ ID NO: 14, or a sequence having at least 80% identity thereto.

16. The CAR of claim 11 or claim 12, comprising a hinge and TM domain derived fromAttorney Docket No: 251609.000137 17. The CAR of claim 16, wherein the hinge and TM domain derived from CD28 comprises the amino acid sequence of SEQ ID NO: 25, or a sequence having at least 80% identity thereto.

18. The CAR of claim 17, wherein the nucleotide sequence encoding the hinge and TM domain derived from CD28 comprises the nucleotide sequence of SEQ ID NO: 24, or a sequence having at least 80% identity thereto.

19. The CAR of any one of claims 1-18, wherein the co-stimulatory signaling domain is derived from 4-1BB (CD137) or CD28.

20. The CAR of claim 19, wherein the co-stimulatory signaling domain derived from 4- 1BB (CD137) comprises the amino acid sequence of SEQ ID NO: 17, or a sequence having at least 80% identity thereto.

21. The CAR of claim 20, wherein the nucleotide sequence encoding the co-stimulatory signaling domain derived from 4-1BB (CD137) comprises the nucleotide sequence of SEQ ID NO: 16, or a sequence having at least 80% identity thereto.

22. The CAR of claim 19, wherein the co-stimulatory signaling domain derived from CD28 comprises the amino acid sequence of SEQ ID NO: 27, or a sequence having at least 80% identity thereto.

23. The CAR of claim 22, wherein the nucleotide sequence encoding the co-stimulatory signaling domain derived from 4-1BB (CD137) comprises the nucleotide sequence of SEQ ID NO: 26, or a sequence having at least 80% identity thereto.

24. The CAR of claim 1, comprising a hinge and TM domain derived from CD8^, a co- stimulatory signaling domain derived from 4-1BB (CD137), and a CD3^ signaling domain.

25. The CAR of claim 1, comprising the amino acid sequence SEQ ID NO: 21, 31, 43, 45, 55, 57, 67, 69, 81, 83, or 85, or a sequence having at least 80% identity thereto.Attorney Docket No: 251609.000137 26. The CAR of claim 1, comprising the amino acid sequence SEQ ID NO: 21 or 43, or a sequence having at least 80% identity thereto.

27. A polynucleotide molecule comprising a nucleotide sequence encoding the CAR of any one of claims 1-26.

28. The polynucleotide molecule of claim 27, wherein the nucleotide sequence encoding the CAR comprises SEQ ID NO: 20, 30, 42, 44, 54, 56, 66, 68, 90, 82, or 84, or a sequence having at least 80% identity thereto.

29. The polynucleotide molecule of claim 28, wherein the nucleotide sequence encoding the CAR comprises SEQ ID NO: 20 or 42, or a sequence having at least 80% identity thereto.

30. The polynucleotide molecule of any one of claims 27-29, wherein the nucleotide sequence encoding the CAR is operably linked to a promoter.

31. The polynucleotide molecule of claim 30, wherein the promoter is a cytomegalovirus (CMV) or a T7 promoter.

32. The polynucleotide molecule of any one of claims 27-29, wherein the nucleotide sequence encoding the CAR is flanked by 5’ and 3’ homology arms for genomic insertion.

33. The polynucleotide molecule of any one of claims 27-31, wherein the nucleic acid molecule is mRNA.

34. A recombinant vector comprising the polynucleotide molecule of any one of claims 27- 33.

35. The vector of claim 34 which is a viral vector.

36. The vector of claim 35 which is a lentiviral vector.

37. An isolated host cell comprising the CAR of any one of claims 1-26.Attorney Docket No: 251609.000137 38. An isolated host cell comprising the polynucleotide molecule of any one of claims 27- 39. An isolated host cell comprising the vector of any one of claims 34-36.

40. The host cell of any one of claims 37-39, wherein the nucleotide sequence encoding the CAR has been inserted into the T-cell Receptor Alpha Constant (TRAC) locus or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) locus.

41. The host cell of any one of claims 40, wherein the TRAC gene is knocked out.

42. The host cell of any one of claims 37-41, which is a mammalian cell.

43. The host cell of any one of claims 37-42, which is selected from a cytotoxic cell, a T cell, a stem cell, a progenitor cell, and a cell derived from a stem cell or a progenitor cell.

44. The host cell of claim 43, wherein the T cell is selected from CD8+ T cells, CD4+ T cells, T-helper cells, cytotoxic T cells, T-regulatory cells (Treg), and gamma-delta T cells.

45. The host cell of claim 43, wherein the cytotoxic cell is a cytotoxic T cell or a cytotoxic NK cell.

46. The host cell of any one of claims 37-45, wherein the host cell has been activated and / or expanded ex vivo.

47. The host cell of any one of claims 37-46, wherein the host cell is an allogeneic cell.

48. The host cell of any one of claims 37-46, wherein the host cell is an autologous cell.

49. The host cell of claim 48, wherein the host cell has been isolated from a subject having a cancer.

50. The host cell of claim 49, wherein the subject is human.Attorney Docket No: 251609.000137 51. A pharmaceutical composition comprising the host cell of any one of claims 37-50 and a pharmaceutically acceptable carrier and / or excipient.

52. The pharmaceutical composition of claim 51, wherein the pharmaceutical composition further comprises one or more additional therapeutic agents.

53. The pharmaceutical composition of claim 52, wherein the one or more additional therapeutic agents comprise an immunomodulatory agent, a chemotherapeutic agent, an anti- angiogenic agent, an anti-inflammatory agent, or a cell death inducer, or a combination thereof.

54. The pharmaceutical composition of claim 53, wherein the cell death inducer is a^BCL- 2 family inhibitor.

55. The pharmaceutical composition of claim 54, wherein the BCL-2 family inhibitor is selected from Navitoclax (ABT-263), Venetoclax, A-1331852, WEHI-539, A-1155463, and ABT-737, or a combination thereof.

56. A method for producing the host cell of any one of claims 37-50 comprising genetically modifying said cell with the polynucleotide molecule of any one of claims 27-33 or the vector of any one of claims 34-36.

57. The method of claim 56, wherein the genetic modification is conducted ex vivo.

58. The method of claim 56 or claim 57, wherein the method further comprises activation and / or expansion of the cell ex vivo.

59. A method for stimulating elimination of a MET-expressing cell in a subject in need thereof, said method comprising administering to the subject an effective amount of the host cells of any one of claims 37-50 or the pharmaceutical composition of any one of claims 51- 55.

60. A method for treating a MET-positive cancer in a subject in need thereof, said method comprising administering to the subject an effective amount of the host cells of any one of claims 37-50 or the pharmaceutical composition of any one of claims 51-55.Attorney Docket No: 251609.000137 61. The method of claim 60, wherein the cancer is a solid tumor.

62. The method of claim 60 or claim 61, wherein the cancer is selected from a breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, prostate cancer, pancreatic cancer, kidney cancer, bladder cancer, testicular cancer, ovarian cancer, endometrial cancer, cervical cancer, neuroendocrine cancer, head and neck cancer, brain cancer, sarcoma, melanoma, and lymphoma.

63. The method of claim 62, wherein the breast cancer is a triple negative breast cancer.

64. The method of claim 62, wherein the lung cancer is a non-small cell lung cancer (NSCLC).

65. The method of claim 62, wherein the lung cancer is a small cell lung cancer (SCLC).

66. The method of any one of claims 59-65, wherein the method comprising administering to the subject one or more additional therapeutic agents.

67. The method of claim 66, wherein the one or more additional therapeutic agents comprise an immunomodulatory agent, a chemotherapeutic agent, an anti-angiogenic agent, an anti-inflammatory agent, a cell death inducer, a surgery, a radiotherapy, a bone marrow transplantation, or a combination thereof.

68. The method of claim 67, wherein the cell death inducer is a^BCL-2 family inhibitor.

69. The method of claim 68, wherein the BCL-2 family inhibitor is selected from Navitoclax (ABT-263), Venetoclax, A-1331852, WEHI-539, A-1155463, and ABT-737, or a combination thereof.

Images