Humanized Anti-mesothelin scfvs

Abstract

The disclosure provides scFv molecule that specifically bind mesothelin, chimeric antigen receptors that comprise antigen-binding domains comprising the scFv, nucleotides that encode the same, cells comprising the same, and methods of using the same in the treatment of cancer.

Description

HUMANIZED ANTI-MESOTHELIN SCFVSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No.63 / 738,245, filed December 23, 2024, which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

[0002] The content of the electronically submitted sequence listing (Name: 4285_028PC01_SequenceListing_ST26.xml; Size: 21,405 bytes; and Date of Creation: December 17, 2025) is incorporated herein by reference in its entirety.BACKGROUND OF THE DISCLOSURE

[0003] Immunotherapy has emerged as a powerful tool in the fight against various types of diseases, including cancer. Immunotherapies harness the power of the patient's own immune system to combat various types of tumors.

[0004] Chimeric antigen receptor (CAR) T cell therapy is a specific form of cell-based immunotherapy that uses engineered T cells to fight cancer. In CAR-T cell therapy, T cells are harvested from a patient’s blood, engineered ex vivo to express CARs containing both antigenbinding and T cell-activating domains, expanded into a larger population, and administered to the patient. The CAR-T cells act as a living drug, binding to cancer cells and bringing about their destruction. When successful, the effects of CAR-T cell treatment tend to be long lasting, as evidenced by detection of CAR-T cell persistence and expansion in the patients long after clinical remission.

[0005] Though CAR T cell therapy has been transformative in treating subsets of hematological malignancies, its efficacy in solid tumors remains limited. Poor CAR-T cell persistence has been associated with a lack of response in solid tumors. As such, there remains a need in the art for improved CAR constructs for treating various types of cancer.SUMMARY OF THE DISCLOSURE

[0006] Some aspects of the present disclosure are directed to a single chain variable fragment (scFv) that specifically binds human mesothelin comprising: (a)(i) a heavy chain complementarity determining region 1 (HC-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) a light chain (LC) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b)(i) an HC framework region 1 (HC-FR1), (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. In some aspects, the scFv comprises an amino acid sequence having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.

[0007] In some aspects, at least two of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least three of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least four of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least five of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least six of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least seven of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

[0008] In some aspects, the HC-FR1 comprises a human immunoglobulin heavy chain (hIGHV) FR1. In some aspects, the HC-FR2 comprises an hIGHV-FR2. In some aspects, the HC-FR3 comprises an hIGHV-FR3. In some aspects, the HC-FR4 comprises an hIGHV-FR4. In some aspects, (i) the HC-FR1 comprises an hIGHV-FRI, (ii) the HC-FR2 comprises an hIGHV-FR2, (iii) the HC-FR3 comprises an hIGHV-FR3, and (iv) the HC-FR4 comprises an hIGHV-FR4. In some aspects, the LC-FR1 comprises a human immunoglobulin light chain(hIGKV) FR1. In some aspects, the LC-FR2 comprises an hIGKV-FR2. In some aspects, the LC-FR3 comprises an hIGKV-FR3. In some aspects, the LC-FR4 comprises an hIGKV-FR4. In some aspects, (i) the LC-FR1 comprises an hIGKV-FRI, (ii) the LC-FR2 comprises an hIGKV-FR2, (iii) the LC-FR3 comprises an hIGKV-FR3, and (iv) the LC-FR4 comprises an hIGKV-FR4.

[0009] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV-FRI, (ii) the HC-FR2 comprises an hIGHV-FR2, (iii) the HC-FR3 comprises an hIGHV-FR3, and (iv) the HC-FR4 comprises an hIGHV-FR4; and (b)(i) the LC-FR1 comprises an hIGKV-FRI, (ii) the LC-FR2 comprises an hIGKV-FR2, (iii) the LC-FR3 comprises an hIGKV-FR3, and (iv) the LC-FR4 comprises an hIGKV-FR4. In some aspects, the hIGHV comprises hIGHVl-45, hIGHV3-73, hIGHV5-51, or hIGHV7-4-l. In some aspects, the hIGKV comprises IGKV3-11 or IGKV6-21. In some aspects, the hIGHV comprises hIGHVl-45 and the hIGKV comprises hIGKV3-11.

[0010] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV 1-45 -FR1, (ii) the HC-FR2 comprises an hIGHVl-45-FR2, (iii) the HC-FR3 comprises an hIGHVl-45-FR3, and (iv) the HC-FR4 comprises an hIGHVl-45-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-ll-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-11-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4. In some aspects, the hIGHV comprises hIGHV3-73 and the hIGKV comprises IGKV3-11.

[0011] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV3-73-FRl, (ii) the HC-FR2 comprises an hIGHV3-73-FR2, (iii) the HC-FR3 comprises an hIGHV3-73-FR3, and (iv) the HC-FR4 comprises an hIGHV3-73-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-ll-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-11-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4. In some aspects, the hIGHV comprises hIGHV5-51 and the hIGKV comprises IGKV3-11.

[0012] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV5-51-FR1, (ii) the HC-FR2 comprises an hIGHV5-51-FR2, (iii) the HC-FR3 comprises an hIGHV5-51-FR3, and (iv) the HC-FR4 comprises an hIGHV5-51-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-ll-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-11-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4. In some aspects, the hIGHV comprises hIGHV7-4-l and the hIGKV comprises IGKV3-11.

[0013] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV7-4-l-FRl, (ii) the HC-FR2 comprises an hIGHV7-4-l-FR2, (iii) the HC-FR3 comprises an hIGHV7-4-l-FR3, and (iv) the HC-FR4 comprises an hIGHV7-4-l-FR4; and (b)(i) the LC-FR1 comprises anhIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-l 1-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4. In some aspects, the hIGHV comprises hIGHVl-45 and the hIGKV comprises hIGKV6-21.

[0014] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV 1-45 -FR1, (ii) the HC-FR2 comprises an hIGHVl-45-FR2, (iii) the HC-FR3 comprises an hIGHVl-45-FR3, and (iv) the HC-FR4 comprises an hIGHVl-45-FR4; and (b)(i) the LC-FR1 comprises an hIGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4. In some aspects, the hIGHV comprises hIGHV3-73 and the hIGKV comprises hIGKV6-21.

[0015] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV3-73-FRl, (ii) the HC-FR2 comprises an hIGHV3-73-FR2, (iii) the HC-FR3 comprises an hIGHV3-73-FR3, and (iv) the HC-FR4 comprises an hIGHV3-73-FR4; and (b)(i) the LC-FR1 comprises an hIGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4. In some aspects, the hIGHV comprises hIGHV5-51 and the hIGKV comprises hIGKV6-21.

[0016] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV5-51-FR1, (ii) the HC-FR2 comprises an hIGHV5-51-FR2, (iii) the HC-FR3 comprises an hIGHV5-51-FR3, and (iv) the HC-FR4 comprises an hIGHV5-51-FR4; and (b)(i) the LC-FR1 comprises an hIGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4. In some aspects, the hIGHV comprises hIGHV7-4-l and the hIGKV comprises hIGKV6-21.

[0017] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV7-4-l-FRl, (ii) the HC-FR2 comprises an hIGHV7-4-l-FR2, (iii) the HC-FR3 comprises an hIGHV7-4-l-FR3, and (iv) the HC-FR4 comprises an hIGHV7-4-l-FR4; and (b)(i) the LC-FR1 comprises an MGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

[0018] In some aspects, the scFv comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 5. In someaspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0019] Some aspects of the present disclosure are directed to a chimeric antigen receptor (CAR) comprising a scFv disclosed herein. In some aspects, the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, (iv) an intracellular signaling domain, or (v) any combination thereof. In some aspects, the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, and (iv) an intracellular signaling domain.

[0020] In some aspects, the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin. In some aspects, the immunoglobulin is selected from of IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, IgM, and any combination thereof. In some aspects, the hinge region is derived from a CD28 hinge region.

[0021] In some aspects, the transmembrane domain comprises a transmembrane domain derived from CD28, CD8 alpha, CD8 beta, KIR2DS2, 0X40, CD2, CD4, CD45, PD-1, CTLA-4 (CD152), CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, interleukin-2 receptor (IL2R) beta, IL2R gamma, IL7R alpha, ITGA1 (VLA1, CD49a), ITGA4 (IA4, CD49D), ITGA6 (VLA-6, CD49f), ITGAD (CD lid), ITGAE (CD 103), ITGAL (CDlla, LFA-1), ITGAM (CDllb), ITGAX (CDllc), ITGB1 (CD29), ITGB2 (CD18, LFA-1), ITGB7, TNFR2, DNAM-1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL-1 (SELPLG, CD162), CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), LTBR, PAG / Cbp, NKG2D, NKG2C, or any combination thereof. In some aspects, the transmembrane domain is derived from a CD28 transmembrane domain. In some aspects, the transmembrane domain is derived from a CD8 alpha transmembrane domain.

[0022] In some aspects, the costimulatory domain comprises a costimulatory domain derived from a cytoplasmic / intracellular domain of 4-1BB (CD137), interleukin-2 receptor (IL-2R), IL-12R, IL-7R, IL-21R, IL-23R, IL-15R, CD2, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, 0X40, DAP 10, B7-H3, CD28 deleted for Lek binding, CTLA-4, BTLA, GITR, HVEM, PD-1, TLR2, TLR4, TLR7, TLR9, Fc receptor gamma chain, Fc receptor epsilon chain, a ligand that specifically binds with CD83, or any combination thereof. In some aspects, the costimulatory domain is derived from a CD28 costimulatory domain.

[0023] In some aspects, the intracellular signaling domain comprises a CD3 zeta (CD247) activating domain, a CD3 gamma activating domain, a CD3 delta activating domain, a CD3 epsilon activating domain, a CD3 eta activating domain, a CD79A activating domain, a CD79B activating domain, a DAP12 activating domain, a FCER1G activating domain, a FCGR2A activating domain, aFCGR2C activating domain, or any combination thereof. In some aspects, the intracellular signaling domain comprises a CD3 zeta activating domain.

[0024] In some aspects, the CAR has a lower immunogenicity when administered to a human subject than a reference scFv, wherein the reference scFv does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, or LC-FR4. In some aspects, the CAR has a lower tonic signaling when expressed on the surface of an immune cell than a reference CAR, wherein the reference CAR comprises an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, or LC-FR4.

[0025] Some aspects of the present disclosure are directed a nucleic acid molecule encoding an scFv disclosed herein.

[0026] Some aspects of the present disclosure are directed a nucleic acid molecule encoding a CAR, wherein the CAR comprises an scFv disclosed herein. In some aspects, the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, (iv) an intracellular signaling domain, or (v) any combination thereof. In some aspects, the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, and (iv) an intracellular signaling domain.

[0027] In some aspects, the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin. In some aspects, the immunoglobulin is selected from of IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, IgM, and any combination thereof. In some aspects, the hinge region is derived from a CD28 hinge region.

[0028] In some aspects, the transmembrane domain comprises a transmembrane domain derived from CD28, CD8 alpha, CD8 beta, KIR2DS2, 0X40, CD2, CD4, CD45, PD-1, CTLA-4 (CD152), CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, interleukin-2 receptor (IL2R) beta, IL2R gamma, IL7R alpha, ITGA1 (VLA1, CD49a), ITGA4 (IA4, CD49D), ITGA6 (VLA-6, CD49f), ITGAD (CD lid), ITGAE (CD 103), ITGAL (CDlla, LFA-1), ITGAM (CDllb), ITGAX (CDllc), ITGB1 (CD29), ITGB2 (CD18, LFA-1), ITGB7, TNFR2, DNAM-1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL-1 (SELPLG, CD162), CD100(SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), LTBR, PAG / Cbp, NKG2D, NKG2C, or any combination thereof. In some aspects, the transmembrane domain is derived from a CD28 transmembrane domain. In some aspects, the transmembrane domain is derived from a CD8 alpha transmembrane domain.

[0029] In some aspects, the costimulatory domain comprises a costimulatory domain derived from a cytoplasmic / intracellular domain of 4-1BB (CD137), interleukin-2 receptor (IL-2R), IL-12R, IL-7R, IL-21R, IL-23R, IL-15R, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, 0X40, DAP10, B7-H3, CD28 deleted for Lek binding, CTLA-4, BTLA, GITR, HVEM, PD-1, TLR2, TLR4, TLR7, TLR9, Fc receptor gamma chain, Fc receptor epsilon chain, a ligand that specifically binds with CD83, or any combination thereof. In some aspects, the costimulatory domain is derived from a CD28 costimulatory domain.

[0030] In some aspects, the intracellular signaling domain comprises a CD3 zeta activating domain, a CD3 gamma activating domain, a CD3 delta activating domain, a CD3 epsilon activating domain, a CD3 eta activating domain, a CD79A activating domain, a CD79B activating domain, a DAP12 activating domain, a FCER1G activating domain, a FCGR2A activating domain, aFCGR2C activating domain, or any combination thereof. In some aspects, the intracellular signaling domain comprises a CD3 zeta activating domain.

[0031] Some aspects of the present disclosure are directed to a vector comprising a nucleic acid molecule disclosed herein. In some aspects, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated viral vector (AAV), a lentiviral vector, or any combination thereof.

[0032] Some aspects of the present disclosure are directed to an engineered immune cell comprising an scFv disclosed herein, a CAR disclosed herein, a nucleic acid molecule disclosed herein, or a vector disclosed herein. In some aspects, the engineered immune cell is a T cell, an NK cell, a B cell, or any combination thereof. In some aspects, the T cell is an aP T cell, a y5 T cell, or any combination thereof. In some aspects, the engineered immune cell is a tumor infiltrating lymphocyte (TIL).

[0033] Some aspects of the present disclosure are directed to a population of engineered immune cells comprising an engineered immune cell disclosed herein.

[0034] In some aspects, fewer engineered immune cells of the population of engineered immune cells express one or more markers of T cell exhaustion following administration to asubject, as compared to a population of engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, or LC-FR4. In some aspects, the one or more markers of T cell exhaustion comprise PD-1, TIM-3, or both.

[0035] In some aspects, following administration to a subject, the engineered immune cells have a diminished capacity for trogocytosis, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, or LC-FR4. In some aspects, the engineered immune cells have increased cytokine secretion capacity after chronic stimulation with a target antigen, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, or LC-FR4. In some aspects, the engineered immune cells have enhanced anti-tumor activity, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, or LC-FR4. In some aspects, the engineered immune cells exhibit enhanced proliferation following repeated antigen stimulation, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, orLC-FR4.

[0036] Some aspects of the present disclosure are directed to a method of treating a subject in need thereof comprising administering to the subject an scFv disclosed herein, a CAR disclosed herein, a nucleic acid molecule disclosed herein, a vector disclosed herein, an engineered immune cell disclosed herein, or a population of engineered immune cells disclosed herein.

[0037] In some aspects, the subject is afflicted with a cancer. In some aspects, the cancer comprises a lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer ofthe kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof. In some aspects, the subject is afflicted with a cancer comprising one or more tumor cells that express mesothelin. In some aspects, the subject is afflicted an ovarian cancer. In some aspects, the subject is afflicted with mesothelioma. In some aspects, the cancer is locally advanced. In some aspects, the cancer is metastatic. In some aspects, the cancer is refractory or relapsed.

[0038] In some aspects, the method further comprises administering to the subject an additional anti-cancer agent. In some aspects, the additional anti-cancer therapy comprises a chemotherapy, an immunotherapy, a radiotherapy, a surgery, or any combination thereof. In some aspects, the additional anti-cancer therapy comprises a chemotherapy. In some aspects, the additional anti-cancer therapy comprises an immune-checkpoint inhibitor. In some aspects, the additional anti-cancer therapy comprises a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a GITR agonist, or any combination thereof. In some aspects, the additional anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds and inhibits PD-1. In some aspects, the anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds and inhibits PD-L1.BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0039] FIGs. 1A-1H are images of a multiple sequence amino acid alignments of humanized scFvs #13 (FIG. 1 A; SEQ ID NO: 1), #16 (FIG. IB; SEQ ID NO: 2), #33 (FIG. 1C; SEQ ID NO: 3), #36 (FIG. ID; SEQ ID NO: 4), #53 (FIG. IE; SEQ ID NO: 5), #56 (FIG. IF; SEQ ID NO: 6), #73 (FIG. 1G; SEQ ID NO: 7), and #76 (FIG. 1H; SEQ ID NO: 8) vs the LMB scFv (FIGs. 1 A-1H; SEQ ID NO: 10). Mismatched residues are highlighted.

[0040] FIGs. 2A-2H are images of multiple sequence amino acid alignments of humanized scFvs #13 (FIG. 2A; SEQ ID NO: 1), #16 (FIG. 2B; SEQ ID NO: 2), #33 (FIG. 2C; SEQ ID NO: 3), #36 (FIG. 2D; SEQ ID NO: 4), #53 (FIG. 2E; SEQ ID NO: 5), #56 (FIG. 2F; SEQ ID NO: 6), #73 (FIG. 2G; SEQ ID NO: 7), and #76 (FIG. 2H; SEQ ID NO: 8) vs the SSI scFv (FIGs. 2A-2H; SEQ ID NO: 9). Mismatched residues are highlighted.

[0041] FIGs. 3A-3M present data showing that humanized scFvs maintain anti-tumor functionality. FIG. 3A is a schematic of scFv humanization. FIG. 3B is a graphicalrepresentation of mesothelin (MSLN) expression on NCI-H226 cells analyzed by flow cytometry. Staining with anti-human mesothelin antibody (light) and isotype control (dark) are shown (FIG. 3B). FIGs. 3C-3M are graphical representations of cytotoxicity of CAR-T cells comprising the humanized scFvs and controls (as indicated) against NCI-H226 (solid lines) or HEK-293T (dotted lines). Mean ± s.e.m. are shown.

[0042] FIGs. 4A-4D present data showing that humanized scFvs have reduced xenogenicity. Xenogenicity of scFvs was determined by flow cytometry. The MFI of goat antimouse IgGF(ab’)2 normalized by goat anti-human IgGF(ab’)2 (FIG. 4A), goat anti-human IgG (H+L) (FIG. 4B), or protein L (FIG. 4C) are shown. Mean ± s.e.m. are shown. FIG. 4D shows representative flow cytometry dot plots for the CAR staining.

[0043] FIGs. 5A-5C present data showing that scFv humanization modulates functional avidity. Normalized cytotoxicity of CAR-T cells against NCI-H226 in the presence of a blocking antibody at concentrations of 1 mg / mL (FIG. 5 A), 10 mg / mL (FIG. 5B), or 4 mg / mL (FIG. 5C) relative to isotype control added at the same concentrations. Mean ± s.e.m. are shown and p-values were calculated using one-way ANOVA with Dunnett’ s multiple comparison test.

[0044] FIGs. 6A-6H present data showing that framework regions of scFvs affect the strength of antigen-independent tonic signaling. Expression of CD69 (FIGs. 6A, 6C, 6D, and 6F) and CD25 (FIGs. 6B-6C and 6E-6F) analyzed by flow cytometry for CD4+(FIGs. 6A-6C) and CD8+(FIGs. 6D-6F) CAR-T cells. The MFI for each marker (FIGs. 6A-6B and 6D-6E) and the percentage of CD25+CD69+cells (FIGs. 6C and 6F) are shown. Mean ± s.e.m. are shown and p-values were calculated using one-way ANOVA with Sidak’s multiple comparison test. FIGs. 6G-6H are representative flow cytometry dot plots for CD4+(FIG. 6G) and CD8+(FIG. 6H) CAR-T cells.

[0045] FIGs. 7A-7H present data showing that framework regions of scFvs affect the strength of antigen-independent tonic signaling. Expression of PD-1 (FIGs. 7A, 7C, 7D, and 7F) and Tim-3 (FIGs. 7B-7C and 7E-7F) analyzed by flow cytometry for CD4+(FIGs. 7A-7C) and CD8+(FIGs. 7D-7F) CAR-T cells. The MFI for each marker (FIGs. 7A-7B and 7D-7E) and the percentage of PD-1+Tim-3+cells (FIGs. 7C and 7F) are shown. Mean ± s.e.m. are shown and p-values were calculated using one-way ANOVA with Sidak’s multiple comparison test. FIGs. 7G-7H are representative flow cytometry dot plot for CD4+(FIG. 7G) and CD8+(FIG. 7H) CAR-T cells.

[0046] FIGs. 8 A-8H present data showing that framework regions of scFvs affect the CAR-T cell metabolic phenotype through antigen-independent tonic signaling. 2-NBDG uptake by CD8+(FIG. 8A) and CD4+(FIG. 8B) CAR-T cells analyzed by flow cytometry. FIGs. 8C-8Hshow metabolic phenotype of CAR-T cells analyzed by Seahorse ATP Rate Assay, including a representative ECAR plot (FIG. 8C), a stacked bar graph ATP production by glycolysis vs mitochondrial oxidative phosphorylation (FIG. 8D), ATP Production Rate by glycolysis (FIG.8E), total ATP Production Rate (FIG. 8F), and proportions of total ATP production by glycolysis (FIG. 8G) and mitochondrial oxidative phosphorylation (FIG. 8H). Mean ± s.e.m. are shown (FIGs. 8A, 8B, and 8D-8H). p-values were calculated using one-way ANOVA with Sidak’s multiple comparison test (FIGs. 8 A, 8B, and 8E-8H).

[0047] FIGs. 9A-9J provide data showing that framework regions of scFvs affect CAR-T cell phenotype after prolonged exposure to tumor cells. FIGs. 9A-9F show expression of PD-1 (FIG. 9A, 9C, 9D, and 9F) and Tim-3 (FIGs. 9B-9C and 9E-9F) analyzed by flow cytometry for CD4+(FIGs. 9A-9C) and CD8+(FIGs. 9D-9F) CAR-T cells. The MFI for each marker (FIGs. 9A-9B and 9D-9E) and the percentage of PD-1+Tim-3+cells (FIGs. 9C and 9F) are shown. FIG. 9G provides a representative flow cytometry dot plot for CD4+and CD8+CAR-T cells. FIG. 9H is a schematic of trogocytosis. Acquired target antigen on CAR-T cell surface can lead to chronic stimulation (top) or fratricide (bottom) to drive T cell dysfunction. FIG. 91 is a graphical representation of trogocytosis by CAR-T cells as analyzed by flow cytometry. Fraction of MSLN+cells in CD3+T cells are shown. FIG. 9J provides a representative flow cytometry dot plot for the trogocytosis assay. Mean ± s.e.m. are shown and p-values were calculated using one-way ANOVA with Dunnett’s multiple comparison test (FIGs. 9A-9F and 91).

[0048] FIGs. 10A-10H provide data showing that framework engineering improves antitumorfunction of CAR-T cells. FIGs. 10A-10B are graphical representation of normalized fold expansion of CAR-T cells stimulated with 0.1 pg (FIG. 10A) or 1 pg (FIG. 10B) plate-bound recombinant human mesothelin. FIG. 10C is a graphical representation of normalized cell index of NCI-H226 analyzed using xCELLigence real-time cell analysis platform. CAR-T cells after chronic stimulation were added at 24h post seeding of NCI-H226. FIGs. 10D-10F are graphs showing cytokine secretion profiles analyzed by flow cytometry for CAR-T cells. CAR-T cells after chronic stimulation were re-stimulated by NCI-H226. fFN-y+TNF-oC cells (% of CD8+CAR-T cells) (FIG. 10D) and IFN-y TNF-oc' IL-2' cells (% of CD8+or CD4+CAR-T cells) (FIG. 10E-10F) are shown. FIGs. 10G-10H are representative pie graphs showing the cytokine secretion profile. Mean ± s.e.m. are shown (FIGs. 10A, 10B, and 10D-10F) and p-values were calculated using two-way ANOVA with Dunnett’ s multiple comparison test (FIGs.10A-10B) and two-tailed t-test (FIGs. 10D-10F).

[0049] FIGs. 11A-11E present data showing that framework engineering improves antitumor function of CAR-T cells in a 3D-spheroid model. FIGs. 11 A-l IB show results of a 3D spheroid cytotoxicity assay for NCI-H226 analyzed using xCELLigence real-time analysis platform. Normalized spheroid area (FIG. 11 A) and representative images (FIG. 11B) are shown. FIGs. 11C-11D are graphical representations of a flow cytometry analysis of primary cancer cells derived from the ascites of an ovarian cancer patient. FIG. 11C is a flow cytometry dot plot of CD45 vs EpCAM used to distinguish cancer cells from non-cancerous cells, performed on ascites-derived cells from an ovarian cancer patient. FIG. 1 ID shows staining with anti-human mesothelin (light) and isotype control (dark) on EpCAM+cells. FIG. 1 IE is a graphical representation of the results of a 3D spheroid cytotoxicity assay for primary cancer cells derived from the ascites of an ovarian cancer patient. Normalized spheroid area is shown. Mean ± s.e.m. are shown and p-values were calculated using two-way ANOVA with Sidak’s multiple comparison test.

[0050] FIGs. 12A-12B present data showing that framework engineering improves antitumor function of CAR-T cells in an in vivo mouse model of a solid tumor. NSG mice were injected subcutaneously with AsPC-1 and injected intravenously with the indicated CAR-T cells or PBS after one week. 7-10 mice in each treatment pooled from two independent experiments are shown. Tumor volume (FIG. 12A) and percent survival (FIG. 12B) are shown. Mean ± s.e.m. are shown (FIG. 12A), and p-values were calculated using two-way ANOVA with Sidak’s multiple comparison test (FIG. 12A) and log-rank test (FIG. 12B).DETAILED DESCRIPTION OF DISCLOSURE

[0051] Some aspects of the present disclosure relate to a humanized single chain variable fragment (scFv) that specifically binds human mesothelin. In some aspects, the scFv comprises: (a) (i) a heavy chain complementarity determining region 1 (HC-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) a light chain (LC) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC framework region 1 (HC-FR1), (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.

[0052] Some aspects of the present disclosure are direct to a CAR comprising a humanized scFv disclosed herein. Some aspects of the present disclosure are direct to polynucleotides comprising a nucleotide sequence encoding a humanized scFv disclosed herein, e.g., a CAR comprising a humanized scFv disclosed herein. Some aspects of the present disclosure are directed to a host cell comprising the polynucleotide. Other aspects of the present disclosure are directed to antibodies comprising a humanized scFv disclosed herein. Further aspects of the present disclosure are directed to methods of treating a subject in need thereof comprising administering the scFv, the polynucleotide, the cell, and / or the antibody to the subject. In some aspects, the subject is afflicted with a cancer.I. Terms

[0053] In order that the present description can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0054] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence," is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0055] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0056] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of are also provided. As used herein, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,” “includes,” and “including”) will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms“comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms, while retaining their ordinary meanings.

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0058] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0059] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).

[0060] The term "antibody" refers, in some aspects, to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In some antibodies, e.g., naturally-occurring IgG antibodies, the heavy chain constant region is comprised of a hinge and three domains, CHI, CH2 and CH3. In some antibodies, e.g., naturally-occurring IgG antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chainscontain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. A heavy chain may have the C-terminal lysine or not. Unless specified otherwise herein, the amino acids in the variable regions are numbered using the Kabat numbering system and those in the constant regions are numbered using the EU system.

[0061] An immunoglobulin can be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. The IgG isotype is divided in subclasses in certain species: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice. In some aspects, the antibodies described herein are of the IgGl subtype. Immunoglobulins, e.g., IgGl, exist in several allotypes, which differ from each other in at most a few amino acids. "Antibody" includes, by way of example, both naturally-occurring and non-naturally-occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and nonhuman antibodies and wholly synthetic antibodies.

[0062] The term "single chain Fv" or "scFv" refers to a polypeptide comprising two domains of an Fv fragment of an antibody, VL and VH, joined by a linker. An scFv is made as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. scFv can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0063] The term "chimeric antigen receptor" or "CAR," as used herein, refers to an engineered antigen-binding polypeptide, comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Expression of a CAR on the surface of a cell, e.g., an immune cell, allows the cell to target and bind a particular antigen. In some aspects, the CAR is expressed by an immune cell, e.g., a T cell. In some aspects, the antigen binding domain comprises an Fab, Fab', F(ab')2, Fd, Fv, single-chain fragment variable (scFv), single chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof. In some aspects, the CAR comprises an scFv disclosed herein. In some aspects, the transmembrane domain comprises a transmembrane domain derived from a CD28 or CD8 alpha transmembrane domain. In some aspects, the intracellular domain comprises a costimulatory domain or a portion thereof. In some aspects, the intracellular domain comprises a costimulatory domain derived from a CD28 costimulatory domain. In some aspects, theintracellular domain comprises a costimulatory domain derived from a CD3 zeta costimulatory domain. A CAR can further comprise a "hinge" or "spacer" domain. Non-limiting examples of hinge / spacer domains include immunoglobulin hinge / spacer domains, such as an IgGl hinge domain, and IgG2 hinge domain, an IgG3 hinge domain, or an IgG4 hinge domain; a CD28 hinge domain; or a CD8 hinge domain.

[0064] As used herein, the term “affinity” refers to a measure of the strength of the binding of an antigen or target (such as an epitope) to its cognate binding domain (such as a paratope). As used herein, the term “avidity” refers to the overall stability of the complex between a population of epitopes and paratopes (z.e., antigens and antigen binding domains).

[0065] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds," refer to an scFv binding to an epitope on a predetermined antigen. Typically, the scFv (i) binds with an equilibrium dissociation constant (KD) of approximately less than 10'7M, such as approximately less than 10'8M, 10'9M or IO'10M or even lower when determined by, e.g., surface plasmon resonance (SPR) technology in a BI AC ORE® 2000 instrument using the predetermined antigen, e.g., mesothelin, as the analyte and the antibody as the ligand, or Scatchard analysis of binding of the antibody to antigen positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least twofold greater than its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely-related antigen. Accordingly, an scFv that "specifically binds to mesothelin" refers to an scFv that binds to mesothelin with a KD of 10'7M or less, such as approximately less than 10'8M, 10'9M or 10'10M or even lower.

[0066] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in the protein can contain a modification such as, but not limited to, glycosylation, phosphorylation or disulfide bond formation. A "protein" can comprise one or more polypeptides.

[0067] The term "nucleic acid molecule," as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single- stranded or doublestranded, and can be cDNA.

[0068] Conservative amino acid substitutions" refer to substitutions of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine,threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0069] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0070] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0071] The nucleic acid and protein sequences described herein can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0072] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0073] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell that comprises a nucleic acid that is not naturally present in the cell, and can be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny cannot, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0074] An "immune response" is as understood in the art, and generally refers to a biological response within a vertebrate against foreign agents or abnormal, e.g., cancerous cells, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4+cell, a CD8+T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell.

[0075] "Immunotherapy" refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying the immune system or an immune response.

[0076] As used herein, the term "linked" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage also can be genetic (i.e., recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.

[0077] As used herein, the terms “treat,” “treatment,” or “treatment of’ when used in the context of treating cancer refer to reducing disease pathology, reducing or eliminating disease symptoms, promoting increased survival rates, and / or reducing discomfort. For example, treating can refer to the ability of a therapy when administered to a subject, to reduce disease symptoms, signs, or causes. Treating also refers to mitigating or decreasing at least one clinical symptom and / or inhibition or delay in the progression of the condition and / or prevention or delay of the onset of a disease or illness.

[0078] As used herein, "cancer" refers a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can result in the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0079] As used herein, the term an “effective amount” or a “therapeutically effective amount” of an administered therapeutic substance, such as an scFv or a CAR-T cell, is an amount sufficient to carry out a specifically stated or intended purpose, such as treating or treatment of cancer. An “effective amount” can be determined empirically in a routine manner in relation to the stated purpose.

[0080] As used herein, the terms “subject,” “individual,” or “patient,” refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.

[0081] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.

[0082] Various aspects described herein are described in further detail in the following subsections.IL Compositions of the DisclosureILA. scFv

[0083] Some aspects of the present disclosure are directed to a humanized scFv that specifically binds human mesothelin. In some aspects, the scFv comprises: (a) (i) a HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC framework region 1 (HC-FR1), (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. In some aspects, the scFv comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.Table 1: scFv Amino Acid Sequences

[0084] In some aspects, at least two of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least three of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least four of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least five of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least six of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least seven of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

[0085] In some aspects, at least the HC-FR1 and HC-FR2 are humanized. In some aspects, at least the HC-FR1 and HC-FR3 are humanized. In some aspects, at least the HC-FR1 and HC-FR4 are humanized. In some aspects, at least the HC-FR1 and LC-FR1 are humanized. In some aspects, at least the HC-FR1 and LC-FR2 are humanized. In some aspects, at least the HC-FR1 and LC-FR3 are humanized. In some aspects, at least the HC-FR1 and LC-FR4 are humanized. In some aspects, at least the HC-FR2 and HC-FR3 are humanized. In some aspects, at least the HC-FR2 and HC-FR4 are humanized. In some aspects, at least the HC-FR2 and LC-FR1 are humanized. In some aspects, at least the HC-FR2 and LC-FR2 are humanized. In some aspects, at least the HC-FR2 and LC-FR3 are humanized. In some aspects, at least the HC-FR2 and LC-FR4 are humanized. In some aspects, at least the HC-FR3 and HC-FR4 are humanized. In some aspects, at least the HC-FR3 and LC-FR1 are humanized. In some aspects, at least the HC-FR3 and LC-FR2 are humanized. In some aspects, at least the HC-FR3 and LC-FR3 are humanized. In some aspects, at least the HC-FR3 and LC-FR4 are humanized. In some aspects, at least the HC-FR4 and LC-FR1 are humanized. In some aspects, at least the HC-FR4 and LC-FR2 are humanized. In some aspects, at least the HC-FR4 and LC-FR3 are humanized. In some aspects, at least the HC-FR4 and LC-FR4 are humanized. In some aspects, at least the LC-FR1 and LC-FR2 are humanized. In some aspects, at least the LC-FR1 and LC-FR3 are humanized. In some aspects, at least the LC-FR1 and LC-FR4 are humanized. In some aspects, at least the LC-FR2 and LC-FR3 are humanized. In some aspects, at least the LC-FR2 and LC-FR4 are humanized. In some aspects, at least the LC-FR3 and LC-FR4 are humanized.

[0086] In some aspects, the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

[0087] In some aspects, the HC-FR1 comprises a human immunoglobulin heavy chain (hIGHV) FR1. In some aspects, the HC-FR2 comprises an hIGHV-FR2. In some aspects, the HC-FR3 comprises an hIGHV-FR3. In some aspects, the HC-FR4 comprises an hIGHV-FR4. In some aspects, (i) the HC-FR1 comprises an hIGHV-FRI, (ii) the HC-FR2 comprises an hIGHV-FR2, (iii) the HC-FR3 comprises an hIGHV-FR3, and (iv) the HC-FR4 comprises an hIGHV-FR4.

[0088] In some aspects, the LC-FR1 comprises a human immunoglobulin light chain (hIGKV) FR1. In some aspects, the LC-FR2 comprises an hIGKV-FR2. In some aspects, the LC-FR3 comprises an hIGKV-FR3. In some aspects, the LC-FR4 comprises an hIGKV-FR4. In some aspects, (i) the LC-FR1 comprises an hIGKV-FRI, (ii) the LC-FR2 comprises an hIGKV-FR2, (iii) the LC-FR3 comprises an hIGKV-FR3, and (iv) the LC-FR4 comprises an hIGKV-FR4.

[0089] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV-FRI, (ii) the HC-FR2 comprises an hIGHV-FR2, (iii) the HC-FR3 comprises an hIGHV-FR3, and (iv) the HC-FR4 comprises an hIGHV-FR4; and (b) (i) the LC-FR1 comprises an hIGKV-FRI, (ii) the LC-FR2 comprises an hIGKV-FR2, (iii) the LC-FR3 comprises an hIGKV-FR3, and (iv) the LC-FR4 comprises an hIGKV-FR4.

[0090] In some aspects, the hIGHV comprises hIGHVl-45. In some aspects, the hIGHV comprises hIGHV3-73. In some aspects, the hIGHV comprises hIGHV5-51. In some aspects, the hIGHV comprises hIGHV7-4-l. In some aspects, the hIGKV comprises IGKV3-11. In some aspects, the hIGKV comprises IGKV6-21.

[0091] In some aspects, the hIGHV comprises hIGHVl-45 and the hIGKV comprises hIGKV3-ll. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV 1-45 -FR1, (ii) the HC-FR2 comprises an hIGHVl-45-FR2, (iii) the HC-FR3 comprises an hIGHVl-45-FR3, and (iv)the HC-FR4 comprises an hIGHVl-45-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-11-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4.

[0092] In some aspects, the hIGHV comprises hIGHV3-73 and the hIGKV comprises IGKV3-11. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV3-73-FRl, (ii) the HC-FR2 comprises an hIGHV3-73-FR2, (iii) the HC-FR3 comprises an hIGHV3-73-FR3, and (iv) the HC-FR4 comprises an hIGHV3-73-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-11-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4.

[0093] In some aspects, the hIGHV comprises hIGHV5-51 and the hIGKV comprises IGKV3-11. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV5-51-FRl, (ii) the HC-FR2 comprises an hIGHV5-51-FR2, (iii) the HC-FR3 comprises an hIGHV5-51-FR3, and (iv) the HC-FR4 comprises an hIGHV5-51-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-11-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4.

[0094] In some aspects, the hIGHV comprises hIGHV7-4-l and the hIGKV comprises IGKV3-11. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV7-4-l-FRl, (ii) the HC-FR2 comprises an hIGHV7-4-l-FR2, (iii) the HC-FR3 comprises an hIGHV7-4-l-FR3, and (iv) the HC-FR4 comprises an hIGHV7-4-l-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-l 1-FR3, and (iv) the LC-FR4 comprises an MGKV3-11-FR4.

[0095] In some aspects, the hIGHV comprises hIGHVl-45 and the hIGKV comprises hIGKV6-21. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV 1-45 -FR1, (ii) the HC-FR2 comprises an hIGHVl-45-FR2, (iii) the HC-FR3 comprises an hIGHVl-45-FR3, and (iv) the HC-FR4 comprises an hIGHVl-45-FR4; and (b)(i) the LC-FR1 comprises an hIGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

[0096] In some aspects, the hIGHV comprises hIGHV3-73 and the hIGKV comprises hIGKV6-21. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV3-73-FRl, (ii) the HC-FR2 comprises an hIGHV3-73-FR2, (iii) the HC-FR3 comprises an hIGHV3-73-FR3, and (iv) the HC-FR4 comprises an hIGHV3-73-FR4; and (b)(i) the LC-FR1 comprises an hIGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

[0097] In some aspects, the hIGHV comprises hIGHV5-51 and the hIGKV comprises hIGKV6-21. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV5-51-FRl, (ii) the HC-FR2 comprises an hIGHV5-51-FR2, (iii) the HC-FR3 comprises an hIGHV5-51-FR3, and (iv) the HC-FR4 comprises an hIGHV5-51-FR4; and (b)(i) the LC-FR1 comprises an hIGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

[0098] In some aspects, the hIGHV comprises hIGHV7-4-l and the hIGKV comprises hIGKV6-21. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV7-4-l-FRl, (ii) the HC-FR2 comprises an hIGHV7-4-l-FR2, (iii) the HC-FR3 comprises an hIGHV7-4-l-FR3, and (iv) the HC-FR4 comprises an hIGHV7-4-l-FR4; and (b)(i) the LC-FR1 comprises an MGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

[0099] In some aspects, the scFv comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.

[0100] In some aspects, the scFv comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC framework region 1 (HC-FR1), (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0101] In some aspects, the scFv comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0102] In some aspects, the scFv comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises an amino acid sequence having at least 98%sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0103] In some aspects, the scFv comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0104] In some aspects, the scFv comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forthin SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0105] In some aspects, the scFv comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and theLC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0106] In some aspects, the scFv comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFvcomprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0107] In some aspects, the scFv comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, thescFv comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0108] In some aspects, the scFv has a lower immunogenicity when administered to a human subject than a reference scFv, wherein the reference scFv does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, or LC-FR4. In some aspects, the scFv has a lower tonic signaling when expressed on the surface of an immune cell than a reference CAR, wherein the reference CAR comprises an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, or LC-FR4.II.B. Anti-mesothelin CAR Constructs

[0109] Some aspects of the present disclosure are directed to CAR that specifically binds mesothelin, wherein the CAR comprises an antigen-binding domain, wherein the antigenbinding domain comprises a humanized scFv disclosed herein. In some aspects, the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, (iv) an intracellular signaling domain, or (v) any combination thereof.II.B.l. Antigen-Binding Domain

[0110] In some aspects, the antigen-binding domain of the CAR comprises: (a)(i) a HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acidsequence set forth in SEQ ID NO: 16; and (b)(i) an HC framework region 1 (HC-FR1), (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (v) an LC-FR2, (v) an LC-FR3, and (v) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.

[0111] In some aspects, at least two of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least three of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least four of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least five of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least six of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized. In some aspects, at least seven of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

[0112] In some aspects, at least the HC-FR1 and HC-FR2 are humanized. In some aspects, at least the HC-FR1 and HC-FR3 are humanized. In some aspects, at least the HC-FR1 and HC-FR4 are humanized. In some aspects, at least the HC-FR1 and LC-FR1 are humanized. In some aspects, at least the HC-FR1 and LC-FR2 are humanized. In some aspects, at least the HC-FR1 and LC-FR3 are humanized. In some aspects, at least the HC-FR1 and LC-FR4 are humanized. In some aspects, at least the HC-FR2 and HC-FR3 are humanized. In some aspects, at least the HC-FR2 and HC-FR4 are humanized. In some aspects, at least the HC-FR2 and LC-FR1 are humanized. In some aspects, at least the HC-FR2 and LC-FR2 are humanized. In some aspects, at least the HC-FR2 and LC-FR3 are humanized. In some aspects, at least the HC-FR2 and LC-FR4 are humanized. In some aspects, at least the HC-FR3 and HC-FR4 are humanized. In some aspects, at least the HC-FR3 and LC-FR1 are humanized. In some aspects, at least the HC-FR3 and LC-FR2 are humanized. In some aspects, at least the HC-FR3 and LC-FR3 are humanized. In some aspects, at least the HC-FR3 and LC-FR4 are humanized. In some aspects, at least the HC-FR4 and LC-FR1 are humanized. In some aspects, at least the HC-FR4 and LC-FR2 are humanized. In some aspects, at least the HC-FR4 and LC-FR3 are humanized. In some aspects, at least the HC-FR4 and LC-FR4 are humanized. In some aspects, at least theLC-FR1 and LC-FR2 are humanized. In some aspects, at least the LC-FR1 and LC-FR3 are humanized. In some aspects, at least the LC-FR1 and LC-FR4 are humanized. In some aspects, at least the LC-FR2 and LC-FR3 are humanized. In some aspects, at least the LC-FR2 and LC-FR4 are humanized. In some aspects, at least the LC-FR3 and LC-FR4 are humanized.

[0113] In some aspects, the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

[0114] In some aspects, the HC-FR1 comprises a human immunoglobulin heavy chain (hIGHV) FR1. In some aspects, the HC-FR2 comprises an hIGHV-FR2. In some aspects, the HC-FR3 comprises an hIGHV-FR3. In some aspects, the HC-FR4 comprises an hIGHV-FR4. In some aspects, (i) the HC-FR1 comprises an hIGHV-FRI, (ii) the HC-FR2 comprises an hIGHV-FR2, (iii) the HC-FR3 comprises an hIGHV-FR3, and (iv) the HC-FR4 comprises an hIGHV-FR4.

[0115] In some aspects, the LC-FR1 comprises a human immunoglobulin light chain (hIGKV) FR1. In some aspects, the LC-FR2 comprises an hIGKV-FR2. In some aspects, the LC-FR3 comprises an hIGKV-FR3. In some aspects, the LC-FR4 comprises an hIGKV-FR4. In some aspects, (i) the LC-FR1 comprises an hIGKV-FRI, (ii) the LC-FR2 comprises an hIGKV-FR2, (iii) the LC-FR3 comprises an hIGKV-FR3, and (iv) the LC-FR4 comprises an hIGKV-FR4.

[0116] In some aspects, (a)(i) the HC-FR1 comprises an hIGHV-FRI, (ii) the HC-FR2 comprises an hIGHV-FR2, (iii) the HC-FR3 comprises an hIGHV-FR3, and (iv) the HC-FR4 comprises an hIGHV-FR4; and (b) (i) the LC-FR1 comprises an hIGKV-FRI, (ii) the LC-FR2 comprises an hIGKV-FR2, (iii) the LC-FR3 comprises an hIGKV-FR3, and (iv) the LC-FR4 comprises an hIGKV-FR4.

[0117] In some aspects, the hIGHV comprises hIGHVl-45. In some aspects, the hIGHV comprises hIGHV3-73. In some aspects, the hIGHV comprises hIGHV5-51. In some aspects, the hIGHV comprises hIGHV7-4-l. In some aspects, the hIGKV comprises IGKV3-11. In some aspects, the hIGKV comprises IGKV6-21.

[0118] In some aspects, the hIGHV comprises hIGHVl-45 and the hIGKV comprises hIGKV3-ll. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV 1-45 -FR1, (ii) the HC-FR2 comprises an hIGHVl-45-FR2, (iii) the HC-FR3 comprises an hIGHVl-45-FR3, and (iv) the HC-FR4 comprises an hIGHVl-45-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-11-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4.

[0119] In some aspects, the hIGHV comprises hIGHV3-73 and the hIGKV comprises IGKV3-11. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV3-73-FRl, (ii) the HC-FR2 comprises an hIGHV3-73-FR2, (iii) the HC-FR3 comprises an hIGHV3-73-FR3, and (iv) the HC-FR4 comprises an hIGHV3-73-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-11-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4.

[0120] In some aspects, the hIGHV comprises hIGHV5-51 and the hIGKV comprises IGKV3-11. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV5-51-FRl, (ii) the HC-FR2 comprises an hIGHV5-51-FR2, (iii) the HC-FR3 comprises an hIGHV5-51-FR3, and (iv) the HC-FR4 comprises an hIGHV5-51-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-11-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4.

[0121] In some aspects, the hIGHV comprises hIGHV7-4-l and the hIGKV comprises IGKV3-11. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV7-4-l-FRl, (ii) the HC-FR2 comprises an hIGHV7-4-l-FR2, (iii) the HC-FR3 comprises an hIGHV7-4-l-FR3, and (iv) the HC-FR4 comprises an hIGHV7-4-l-FR4; and (b)(i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-l 1-FR3, and (iv) the LC-FR4 comprises an MGKV3-11-FR4.

[0122] In some aspects, the hIGHV comprises hIGHVl-45 and the hIGKV comprises hIGKV6-21. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV 1-45 -FR1, (ii) the HC-FR2 comprises an hIGHVl-45-FR2, (iii) the HC-FR3 comprises an hIGHVl-45-FR3, and (iv) the HC-FR4 comprises an hIGHVl-45-FR4; and (b)(i) the LC-FR1 comprises an hIGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

[0123] In some aspects, the hIGHV comprises hIGHV3-73 and the hIGKV comprises hIGKV6-21. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV3-73-FRl, (ii) the HC-FR2 comprises an hIGHV3-73-FR2, (iii) the HC-FR3 comprises an hIGHV3-73-FR3, and (iv) the HC-FR4 comprises an hIGHV3-73-FR4; and (b)(i) the LC-FR1 comprises an hIGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

[0124] In some aspects, the hIGHV comprises hIGHV5-51 and the hIGKV comprises hIGKV6-21. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV5-51-FRl, (ii) the HC-FR2 comprises an hIGHV5-51-FR2, (iii) the HC-FR3 comprises an hIGHV5-51-FR3, and (iv) the HC-FR4 comprises an hIGHV5-51-FR4; and (b)(i) the LC-FR1 comprises an hIGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

[0125] In some aspects, the hIGHV comprises hIGHV7-4-l and the hIGKV comprises hIGKV6-21. In some aspects, (a)(i) the HC-FR1 comprises an hIGHV7-4-l-FRl, (ii) the HC-FR2 comprises an hIGHV7-4-l-FR2, (iii) the HC-FR3 comprises an hIGHV7-4-l-FR3, and (iv) the HC-FR4 comprises an hIGHV7-4-l-FR4; and (b)(i) the LC-FR1 comprises an MGKV6-21-FR1, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

[0126] In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.

[0127] In some aspects, the antigen-binding domain of the CAR comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, theantigen-binding domain of the CAR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0128] In some aspects, the antigen-binding domain of the CAR comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, theantigen-binding domain of the CAR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0129] In some aspects, the antigen-binding domain of the CAR comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, theantigen-binding domain of the CAR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0130] In some aspects, the antigen-binding domain of the CAR comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, theantigen-binding domain of the CAR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0131] In some aspects, the antigen-binding domain of the CAR comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, theantigen-binding domain of the CAR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0132] In some aspects, the antigen-binding domain of the CAR comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, theantigen-binding domain of the CAR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0133] In some aspects, the antigen-binding domain of the CAR comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, theantigen-binding domain of the CAR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0134] In some aspects, the antigen-binding domain of the CAR comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 81% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 82% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 83% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 84% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, theantigen-binding domain of the CAR comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 8.I.B.2. Hinge Region

[0135] The CAR further comprises a hinge region. In some aspects, the CAR comprises a CD28 hinge region. In some aspects, the hinge region is positioned between the antigenbinding domain (z.e., the scFv) and the transmembrane domain. In some aspects, the CAR comprises a CD8 hinge region. In some aspects, the CAR comprises a hinge region derived from an immunoglobulin. The hinge region from any immunoglobulin can be used in the CAR constructs of the present disclosure. In some aspects, the CAR comprises an IgAl hinge region. In some aspects, the CAR comprises an IgA2 hinge region. In some aspects, the CAR comprises an IgGl hinge region. In some aspects, the CAR comprises an IgG2 hinge region. In some aspects, the CAR comprises an IgG3 hinge region. In some aspects, the CAR comprises an IgG4 hinge region. In some aspects, the CAR comprises an IgD hinge region. In some aspects, the CAR comprises an IgE hinge region. In some aspects, the CAR comprises an IgM hinge region.

[0136] In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and(b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 1, and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the hinge region comprises a CD28 hinge region. In some aspects, the hinge region comprises a CD8 hinge region. In some aspects, the hinge region comprises an immunoglobulin hinge region disclosed herein.

[0137] In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 2, and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the hinge region comprises a CD28 hinge region. In some aspects, the hinge region comprises a CD8 hinge region. In some aspects, the hinge region comprises an immunoglobulin hinge region disclosed herein.

[0138] In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 3, and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the hinge region comprises a CD28 hinge region. In some aspects, the hinge region comprises a CD8 hinge region. In some aspects, the hinge region comprises an immunoglobulin hinge region disclosed herein.

[0139] In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the CARcomprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 4, and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the hinge region comprises a CD28 hinge region. In some aspects, the hinge region comprises a CD8 hinge region. In some aspects, the hinge region comprises an immunoglobulin hinge region disclosed herein.

[0140] In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5; and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 5, and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the hinge region comprises a CD28 hinge region. In some aspects, the hinge region comprises a CD8 hinge region. In some aspects, the hinge region comprises an immunoglobulin hinge region disclosed herein.

[0141] In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) anLC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6; and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 6, and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the hinge region comprises a CD28 hinge region. In some aspects, the hinge region comprises a CD8 hinge region. In some aspects, the hinge region comprises an immunoglobulin hinge region disclosed herein.

[0142] In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 7, and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the hinge region comprises a CD28 hinge region. In some aspects, the hinge region comprises a CD8 hinge region. In some aspects, the hinge region comprises an immunoglobulin hinge region disclosed herein.

[0143] In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acidsequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8; and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the CAR comprises an antigen-binding domain and a hinge region, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 8, and wherein the hinge region comprises a CD28 hinge region, a CD8 hinge region, or a hinge region of an immunoglobulin. In some aspects, the hinge region comprises a CD28 hinge region. In some aspects, the hinge region comprises a CD8 hinge region. In some aspects, the hinge region comprises an immunoglobulin hinge region disclosed herein.ILB.3. Transmembrane Domain

[0144] The CAR further comprises a transmembrane domain. Any transmembrane domain of any protein can be used in the CAR constructs of the present disclosure. In some aspects, the transmembrane domain comprises or is derived from the transmembrane domain of CD28, CD8 alpha, CD8 beta, KIR2DS2, 0X40, CD2, CD4, CD45, PD-1, CTLA-4 (CD 152), CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, interleukin-2 receptor (IL2R) beta, IL2R gamma, IL7R alpha, ITGA1 (VLA1, CD49a), ITGA4 (IA4, CD49D), ITGA6 (VLA-6, CD49f), ITGAD (CD1 Id), ITGAE (CD 103), ITGAL (CD1 la, LFA-1), ITGAM (CDllb), ITGAX (CDllc), ITGB1 (CD29), ITGB2 (CD18, LFA-1), ITGB7, TNFR2, DNAM-1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL-1 (SELPLG, CD162), CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), LTBR, PAG / Cbp, NKG2D, NKG2C, or any combination thereof. In some aspects, the transmembranedomain comprises or is derived from the transmembrane domain of CD28. In some aspects, the transmembrane domain comprises or is derived from the transmembrane domain of CD 8 alpha.

[0145] In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigenbinding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain. In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain. In some aspects, the transmembrane domain comprises a CD8 alpha transmembrane domain.

[0146] In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigenbinding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain. In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain. In some aspects, the transmembrane domain comprises a CD8 alpha transmembrane domain.

[0147] In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigenbinding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain. In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain. In some aspects, the transmembrane domain comprises a CD8 alpha transmembrane domain.

[0148] In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigenbinding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain. In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain. In some aspects, the transmembrane domain comprises a CD8 alpha transmembrane domain.

[0149] In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigenbinding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5; and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain. In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain,wherein the antigen-binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain. In some aspects, the transmembrane domain comprises a CD8 alpha transmembrane domain.

[0150] In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigenbinding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6; and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain. In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain. In some aspects, the transmembrane domain comprises a CD8 alpha transmembrane domain.

[0151] In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence setforth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigenbinding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain. In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain. In some aspects, the transmembrane domain comprises a CD8 alpha transmembrane domain.

[0152] In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigenbinding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8; and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain. In some aspects, the CAR comprises an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, and wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain. In some aspects, the transmembrane domain comprises a CD28 transmembranedomain. In some aspects, the transmembrane domain comprises a CD8 alpha transmembrane domain.II.B.4. Costimulatory Domain

[0153] The CAR further comprises costimulatory domain. Any costimulatory domain can be used in the CAR constructs of the present disclosure. In some aspects, the costimulatory domain comprises a costimulatory domain derived from a cytoplasmic / intracellular domain of 4-1BB (CD137), interleukin-2 receptor (IL-2R), IL-12R, IL-7R, IL-21R, IL-23R, IL-15R, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, 0X40, DAP10, B7-H3, CD28 deleted for Lek binding, CTLA-4, BTLA, GITR, HVEM, PD-1, TLR2, TLR4, TLR7, TLR9, Fc receptor gamma chain, Fc receptor epsilon chain, a ligand that specifically binds with CD83, or any combination thereof. In some aspects, the costimulatory domain comprises a CD28 costimulatory domain.

[0154] In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0155] In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the aminoacid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0156] In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0157] In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0158] In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0159] In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80%sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0160] In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0161] In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the CAR comprises an antigen-binding domain and a CD28 costimulatory domain, wherein the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 8.I I.B.5. Intracellular Signaling Domain

[0162] The CAR further comprises an intracellular signaling domain. Any intracellular signaling domain can be used in the CAR constructs of the present disclosure. In some aspects, the intracellular signaling domain comprises a CD3 zeta activating domain, a CD3 gamma activating domain, a CD3 delta activating domain, a CD3 epsilon activating domain, a CD3 eta activating domain, a CD79A activating domain, a CD79B activating domain, a DAP12 activating domain, a FCER1G activating domain, a FCGR2A activating domain, a FCGR2C activating domain, or any combination thereof. In some aspects, the CAR comprises a CD3 zeta activating domain.

[0163] In some aspects, the CAR comprises an antigen-binding domain and an intracellular signaling domain comprising a CD3 zeta activating domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 1 and an intracellular signaling domain comprising a CD3 zeta activating domain.

[0164] In some aspects, the CAR comprises an antigen-binding domain and an intracellular signaling domain comprising a CD3 zeta activating domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii)an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 2 and an intracellular signaling domain comprising a CD3 zeta activating domain.

[0165] In some aspects, the CAR comprises an antigen-binding domain and an intracellular signaling domain comprising a CD3 zeta activating domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 3 and an intracellular signaling domain comprising a CD3 zeta activating domain.

[0166] In some aspects, the CAR comprises an antigen-binding domain and an intracellular signaling domain comprising a CD3 zeta activating domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; whereinone or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 4 and an intracellular signaling domain comprising a CD3 zeta activating domain.

[0167] In some aspects, the CAR comprises an antigen-binding domain and an intracellular signaling domain comprising a CD3 zeta activating domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 5 and an intracellular signaling domain comprising a CD3 zeta activating domain.

[0168] In some aspects, the CAR comprises an antigen-binding domain and an intracellular signaling domain comprising a CD3 zeta activating domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an aminoacid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 6 and an intracellular signaling domain comprising a CD3 zeta activating domain.

[0169] In some aspects, the CAR comprises an antigen-binding domain and an intracellular signaling domain comprising a CD3 zeta activating domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 7 and an intracellular signaling domain comprising a CD3 zeta activating domain.

[0170] In some aspects, the CAR comprises an antigen-binding domain and an intracellular signaling domain comprising a CD3 zeta activating domain, wherein the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; and wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the CAR comprises an antigen-binding domain comprisingthe amino acid sequence set forth in SEQ ID NO: 8 and an intracellular signaling domain comprising a CD3 zeta activating domain.II.C. Nucleic Acid Molecules

[0171] Some aspects of the present disclosure are directed to nucleic acid molecules comprising a nucleotide sequence encoding an scFv disclosed herein. Some aspects of the present disclosure are directed to nucleic acid molecules comprising a nucleotide sequence encoding a CAR disclosed herein. In some aspects, the CAR comprises (i) an antigen-binding domain comprising an scFv disclosed herein, (ii) a transmembrane domain disclosed herein, and (iii) an intracellular signaling domain disclosed herein. In some aspects, the CAR further comprises a hinge region disclosed herein.

[0172] In some aspects, the nucleic acid molecule further comprises a nucleotide sequence encoding an armoring molecule. In some aspects, the nucleotide sequence encoding the CAR and the nucleotide sequence encoding the armoring moiety are expressed under the control of the same promoter. In some aspects, the nucleotide sequence encoding the CAR and the nucleotide sequence encoding the armoring moiety are expressed as a single contiguous polypeptide. In some aspects, the CAR and the nucleotide sequence encoding the armoring moiety are linked by a nucleotide sequence encoding a linker. In some aspects, the linker is a peptide linker. In some aspects, the linker is a cleavable linker. In some aspects, the linker is a self-cleaving peptide linker.

[0173] In some aspects, the nucleic acid molecule encodes a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acidsequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 1; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0174] In some aspects, the nucleic acid molecule encodes a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 2; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alphatransmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0175] In some aspects, the nucleic acid molecule encodes a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 3; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0176] In some aspects, the nucleic acid molecule encodes a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 4; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0177] In some aspects, the nucleic acid molecule encodes a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5; wherein the hinge region comprises a hinge region derived from CD28, CD8, or animmunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 5; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0178] In some aspects, the nucleic acid molecule encodes a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 6; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0179] In some aspects, the nucleic acid molecule encodes a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 7; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0180] In some aspects, the nucleic acid molecule encodes a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprisingthe amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 8; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0181] Some aspects of the present disclosure are directed to a vector comprising a nucleic acid molecule disclosed herein. In some aspects, the vector comprises a viral vector. In some aspects, the vector comprises a DNA vector. In some aspects, the vector comprises a plasmid. In some aspects, the vector comprises an RNA vector. In some aspects, the vector comprises a retroviral vector, a murine leukemia virus vector, an SFG vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated viral vector (AAV), a lentiviral vector, or any combination thereof. In some aspects, the vector comprises a lentiviral vector.II.D. Engineered Cells

[0182] Some aspects of the present disclosure are directed to cells comprising a polynucleotide or a polypeptide disclosed herein. Some aspects of the present disclosure are directed to a cell comprising a polynucleotide encoding a CAR disclosed herein (e.g., comprising an scFv disclosed herein). As used herein, a cell comprising a polynucleotide encoding a CAR is also referred to as an "engineered cell". In some aspects, the cell is animmune cell e.g., an engineered immune cell). In some aspects, the cell is a T cell, an NK cell, a B cell, or any combination thereof. In some aspects, the cell is a T cell. In some aspects, the T cell is an aP T cell. In some aspects, the T cell is a y5 T cell. In some aspects, the cell is a tumor infiltrating lymphocyte.

[0183] The cell of the present disclosure can be obtained through any source. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained from, e.g., 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 addition, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation and / or apheresis. In certain aspects, the cells collected by apheresis are washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent processing. In some aspects, the cells are washed with PBS. As will be appreciated, a washing step can be used, such as by using a semiautomated flowthrough centrifuge, e.g., the COBE™ 2991 cell processor, the Baxter CYTOMATE™, or the like. In some aspects, the washed cells are resuspended in one or more biocompatible buffers, or other saline solution with or without buffer. In certain aspects, the undesired components of the apheresis sample are removed. Additional methods of isolating T cells for a T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is herein incorporated by references in its entirety.

[0184] In certain aspects, T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, e.g., by using centrifugation through a PERCOLL TM gradient. In some aspects, a specific subpopulation of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells is further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. In some aspects, cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected can be used. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDllb, CD16, HLA-DR, and CD8. In certain aspects, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the present disclosure.

[0185] In some aspects, PBMCs are used directly for genetic modification with the immune cells (such as CARs) using methods as described herein. In certain aspects, after isolating the PBMCs, T lymphocytes are further isolated, and both cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion.

[0186] In some aspects, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of these types of CD8+ cells. In some aspects, the expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and are negative for granzyme B. In some aspects, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some aspects, effector T cells are negative for CD62L, CCR7, CD28, and CD 127 and positive for granzyme B and perforin. In certain aspects, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.

[0187] In some aspects, the immune cells, e.g., T cells, are genetically modified following isolation using known methods, or the immune cells are activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In another aspect, the immune cells, e.g., T cells, are genetically modified with the CARs described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then are activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, e.g., in U.S. Patent Nos. 6,905,874; 6,867,041; and 6,797,514; and PCT Publication No. WO 2012 / 079000, the contents of which are hereby incorporated by reference in their entirety. Generally, such methods include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC). One example is The Dynabeads® system, a CD3 / CD28 activator / stimulator system for physiological activation of human T cells. In other aspects, the T cells are activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent Nos.6,040,177 and 5,827,642 and PCT Publication No. WO 2012 / 129514, the contents of which are hereby incorporated by reference in their entirety.

[0188] In some aspects, the T cells are obtained from a donor subject. In some aspects, the donor subject is human patient afflicted with a cancer or a tumor. In other aspects, the donor subject is a human patient not afflicted with a cancer or a tumor.

[0189] In some aspects, the engineered cell (e.g., T cell) comprises a nucleic acid molecule encoding a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 1; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0190] In some aspects, the engineered cell (e.g., T cell) comprises a nucleic acid molecule encoding a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forthin SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 2; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0191] In some aspects, the engineered cell (e.g., T cell) comprises a nucleic acid molecule encoding a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3; wherein the hinge region comprises a hinge region derivedfrom CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 3; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0192] In some aspects, the engineered cell (e.g., T cell) comprises a nucleic acid molecule encoding a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 4; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derivedfrom a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0193] In some aspects, the engineered cell (e.g., T cell) comprises a nucleic acid molecule encoding a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 5; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0194] In some aspects, the engineered cell (e.g., T cell) comprises a nucleic acid molecule encoding a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ IDNO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 6; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0195] In some aspects, the engineered cell (e.g., T cell) comprises a nucleic acid molecule encoding a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises aCD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 7; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0196] In some aspects, the engineered cell (e.g., T cell) comprises a nucleic acid molecule encoding a CAR comprising (i) an antigen-binding domain, (ii) a hinge region, (iii) a transmembrane domain, (iv) a costimulatory domain, and (v) an intracellular signaling domain, wherein: the antigen-binding domain comprises (a) (i) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, (iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, (iv) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, (v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and (vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and (b) (i) an HC-FR1, (ii) an HC-FR2, (iii) an HC-FR3, (iv) an HC-FR4, (v) an LC-FR1, (vi) an LC-FR2, (vii) an LC-FR3, and (viii) an LC-FR4; and (c) a linker between the HC-FR4 and the LC-FR1; wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; wherein the antigen-binding domain of the CAR comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain. In some aspects, the CAR comprises an antigen-binding domain comprising the amino acid sequence set forth in SEQ ID NO: 8; wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin; wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 alpha transmembrane domain; wherein the costimulatory domain is derived from a CD28 costimulatory domain; and wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

[0197] Some aspects of the present disclosure are directed to a population of engineered immune cells comprising more than one engineered immune cell disclosed herein. In some aspects, fewer engineered immune cells of the population of engineered immune cells express one or more markers of T cell exhaustion following administration to a subject, as compared to a population of engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, or LC-FR4. In some aspects, the one or more markers of T cell exhaustion comprise PD-1, TIM-3, or both. In some aspects, following administration to a subject, the engineered immune cells have a diminished capacity for trogocytosis, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, or LC-FR4. In some aspects, the engineered immune cells have increased cytokine secretion capacity after chronic stimulation with a target antigen, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, or LC-FR4. In some aspects, the engineered immune cells have enhanced anti-tumor activity (e.g., after chronic stimulation with a cancer-derived cell line expressing a target antigen), as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, or LC-FR4. In some aspects, the engineered immune cells exhibit enhanced proliferation following repeated antigen stimulation, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, orLC-FR4.III. Methods of the Disclosure

[0198] Some aspects of the present disclosure are directed to methods of treating a subject in need thereof, comprising administering to the subject a composition (e.g., an scFv, a nucleic acid molecule, a vector, an engineered immune cell, or a population of engineered immune cells) disclosed herein. In some aspects, the subject is afflicted with a disease or condition. In some aspects, the subject is afflicted with a cancer. In some aspects, the cancer comprises a lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof. In some aspects, the subject is afflicted with a cancer comprising one or more tumor cells that express mesothelin. In some aspects, the subject is afflicted with mesothelioma. In some aspects, the subject is afflicted with an ovarian cancer. In some aspects, the cancer comprises a tumor derived from mesothelioma (e.g., a tumor arising from the metastasis of a mesothelioma). In some aspects, the cancer comprises a tumor derived from an ovarian cancer (e.g., a tumor arising from the metastasis of an ovarian cancer).

[0199] In some aspects, the cancer is locally progressed. In some aspects, the cancer is metastatic. In some aspects, the cancer is recurrent. In some aspects, the cancer is relapsed.

[0200] The compositions disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, can be used in combination with other anti-cancer therapies. In some aspects, the additional anti-cancer therapy comprises a chemotherapy. In some aspects, the additional anti-cancer therapy comprises one or more additional immunotherapies. In some aspects, the additional anti-cancer therapy comprises a radiotherapy. In some aspects, the additional anti-cancer therapy comprises a surgery. In some aspects, the compositions disclosed herein are administered concurrently with the additional anti-cancer agent. In some aspects, the compositions disclosed herein and the additional anti-cancer agent are administered sequentially (e.g, on the same day or on different days).

[0201] Generally, the compositions disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, can be combined with (i) an agonist of a stimulatory (e.g, co-stimulatory) molecule (e.g., receptor or ligand) and / or (ii) an antagonist of an inhibitory signal or molecule (e.g., receptor or ligand) on immune cells, such as T cells, both of which result in amplifying immune responses, such as antigen-specific T cell responses. In some aspects, an immuno-oncology agent is (i) an agonist of a stimulatory (including a costimulatory) molecule (e.g., receptor or ligand) or (ii) an antagonist of an inhibitory (includinga co-inhibitory) molecule (e.g., receptor or ligand) on cells, e.g., those inhibiting T cell activation or those involved in innate immunity, e.g., NK cells, and wherein the immunooncology agent enhances innate immunity. Such immuno-oncology agents are often referred to as immune checkpoint regulators, e.g., immune checkpoint inhibitor or immune checkpoint stimulator.

[0202] In some aspects, a composition disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, is administered with an agent that targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin super family (IgSF). For example, the compositions disclosed herein can be administered to a subject with an agent that targets a member of the IgSF family to increase an immune response. For example, the compositions disclosed herein can be administered with an agent that targets (or binds specifically to) a member of the B7 family of membrane -bound ligands that includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6 or a co-stimulatory or co-inhibitory receptor or ligand binding specifically to a B7 family member.

[0203] A composition disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, can also be administered with an agent that targets a member of the TNF and TNFR family of molecules (ligands or receptors), such as CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn 14, TWEAK, BAFFR, ED AR, XEDAR, TACI, APRIL, BCMA, LTpR, LIGHT, DcR3, HVEM, VEGI / TLI A, TRAMP / DR3, EDAI, EDA2, TNFR1, Lymphotoxin a / TNFp, TNFR2, TNFa, LTpR, Lymphotoxin a 1 [32, FAS, FASL, RELT, DR6, TROY, and NGFR (see, e.g, Tansey (2009) Drug Discovery Today 00: 1).

[0204] In some aspects, a composition disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, is administered in combination with and one or more of an antagonist (inhibitor or blocking agent) of a protein that inhibits T cell activation e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, TIM-3, and TIM-4.

[0205] In some aspects, a composition disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, is administered in combination with and one or more of an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28,4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, 0X40, OX40L, CD70, CD27, CD40, DR3 and CD28H.

[0206] Exemplary agents that modulate one of the above proteins include: YERVOY® (ipilimumab) or Tremelimumab (to CTLA-4), galiximab (to B7.1), BMS-936558 (to PD-1), MK-3475 (to PD-1), atezolizumab (TECENTRIQ®), AMP224 (to B7DC), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3), IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566 (to CD137), CDX-1127 (to CD27), anti-OX40 (Providence Health Services), huMAbOX40L (to OX40L), Atacicept (to TACI), CP-870893 (to CD40), Lucatumumab (to CD40), Dacetuzumab (to CD40), Muromonab-CD3 (to CD3); anti-GITR antibodies MK4166, TRX518, Medil873, INBRX-110, LK2-145, GWN-323, GITRL-Fc, or any combination thereof.

[0207] Other molecules that can be combined with the compositions disclosed herein for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells, e.g., antagonists of KIR (e.g., lirilumab).

[0208] In some aspects, a composition disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, is administered to a subject together with an antibody that specifically binds PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, 0X40, ICOS, CD137, KIR, TGFp, IL-10, IL-8, IL-2, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, VISTA, CD96, GITR or any combination thereof. In some aspects, a composition disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, is administered in combination with an antibody or antigen-binding portion thereof that specifically binds and inhibits PD-1 (e.g, nivolumab or pembrolizumab). In some aspects, a composition disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, is administered in combination with an antibody or antigen-binding portion thereof that specifically binds and inhibits PD-L1.

[0209] In some aspects, a composition disclosed herein, e.g. a T-cell comprising a polynucleotide encoding a CAR disclosed herein, is administer in combination (e.g, simultaneously or separately) with an additional treatment, such as irradiation and / or chemotherapy, e.g., using camptothecin (CPT-11), 5 -fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, or camptothecin + apo21 / TRAIL (a 6X combo)), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3L2' (bcl-xl inhibitor), indoleamine dioxygenase- 1 inhibitor (e.g., INCB24360, indoximod, NLG-919, or F001287), AT-101 (R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070(obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein- 1) antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g, smac7, smac4, small molecule smac mimetic, synthetic smac peptides (see Fulda et al., Nat Med 2002;8:808-15), ISIS23722 (LY2181308), or AEG-35156 (GEM-640)), HD AC (histone deacetylase) inhibitors, anti-CD20 antibodies e.g, rituximab), angiogenesis inhibitors e.g, bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g., Avastin), synthetic triterpenoids (see Hyer et al, Cancer Research 2005;65:4799-808), c-FLIP (cellular FLICE-inhibitory protein) modulators e.g., natural and synthetic ligands of PPARy (peroxisome proliferator-activated receptor y), 5809354 or 5569100), kinase inhibitors e.g., Sorafenib), Trastuzumab, Cetuximab, Temsirolimus, mTOR inhibitors such as rapamycin and temsirolimus, Bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, Lenalildomide, GSK3P inhibitors, IAP inhibitors and / or genotoxic drugs.

[0210] In some aspects, the additional anti-cancer agent comprises one or more antiproliferative cytotoxic agents. In some aspects, the additional anti-cancer agent comprises an alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes). In some aspects, the additional anti-cancer agent comprises uracil mustard, chlormethine, cyclophosphamide (CYTOXAN®) fosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, temozolomide, and any combination thereof.

[0211] In some aspects, the additional anti-cancer agent comprises an antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors). In some aspects, the additional anti-cancer agent comprises methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine, gemcitabine, and any combination thereof.

[0212] In some aspects, the additional anti-cancer agent comprises a taxane, paclitaxel e.g, TAXOL™), docetaxel, discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D, desoxyepothilone Bl,

[0017] -dehydrodesoxyepothilone B,

[0018] dehydrodesoxy epothilones B, C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone BIO, discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389), Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoid immunoconjugates (DM-1), MKC-1, ABT-751, Tl-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin, 17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-l,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, and cryptothilone 1, a microtubuline stabilizing, and any combination thereof.

[0213] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-FV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); ); Crooks, Antisense drug Technology: Principles, strategies and applications, 2ndEd. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0214] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1

[0215] Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer immunotherapy, demonstrating remarkable efficacy in treating subsets of hematologicalmalignancies. However, this success has yet to be fully translated into solid tumors. Induction of T cell dysfunction through chronic antigen exposure and the immunosuppressive tumor microenvironment, both of which compromise CAR-T cell persistence and restrict therapeutic efficacy, pose significant challenges to treating solid tumors. Chronic T cell activation is known to induce a state of exhaustion and has been observed in unresolved chronic infection as well as in solid tumors. Notably, signatures associated with T cell dysfunction have been observed in preclinical models of solid tumors, in pre-infusion CAR-T cell products of non-responding patients, and in post-treatment samples from non-responders. Furthermore, analyses from clinical trials have underscored a positive correlation between CAR-T cell persistence and favorable clinical outcomes, highlighting the critical need to mitigate antigen-dependent exhaustion to enhance CAR-T cell longevity and improve therapeutic response.

[0216] The scFv sequence is a key determinant of CAR-T cell functionality and clinical efficacy. Humanization of mouse-derived scFvs represents one approach to improving CAR-T cell performance.

[0217] Mesothelin is a promising target for immunotherapy due to its limited expression in normal tissues, being restricted to the serous membranes, and its overexpression in many cancer types including mesothelioma and ovarian cancer, both of which are associated with poor prognoses. Despite this, mesothelin-targeted therapies have struggled to deliver durable clinical success. SSI, a mouse-derived anti -human mesothelin scFv, has been evaluated in clinical trials as both antibody-toxin conjugates and CAR-T cells; however, the latter demonstrated limited efficacy due to poor persistence and the generation of a host anti-CAR immune response. The emergence of adaptive immune responses against CAR components has been documented in several clinical trials, with poor CAR-T cell persistence and severe allergic reactions upon reinfusion linked to CAR immunogenicity.

[0218] In this Example, we conducted a comprehensive analysis of the impact of scFv framework modifications on CAR-T cell phenotype, aiming to generate an scFv sequence that confers sustained persistence and a durable anti-tumor response in solid tumors.

[0219] Methods

[0220] Humanization of mouse-derived scFv

[0221] Alignments of the amino acid sequence of mouse-derived anti-human MSLN scFv SSI against human immunoglobulin heavy (IGHV) and light (IGKV) chains were generated using IMGT. SSI CDR sequences were grafted onto four IGHV alleles (IGHV1-45, IGHV3-73, IGHV5-51, and IGHV7-4-1) and two IGKV alleles (IGKV3-11, IGKV6-21) after the introduction of selected amino acid mutations. Humanized heavy and light chains werecombined using a linker sequence (GVGGSGGGGSGGGGS; SEQ ID NO: 18) to generate humanized anti-MSLN scFvs.

[0222] Generation of anti-MSLN CAR constructs

[0223] Anti-MSLN CAR constructs were generated by linking the anti-MSLN scFv sequences to those encoding the extracellular hinge, transmembrane, and intracellular domains of CD28 and the intracellular domain of CD3^ (SEQ ID NO: 17). The CAR-encoding genes were linked to a NGFR gene using a furin cleavage site followed by a Ser-Gly-Ser-Gly linker and a P2A ribosome skipping sequence. These constructs were subsequently cloned into the pMX retroviral vector.Table 2: CD3 Zeta Amino Acid Sequence

[0224] Primary human T cell culture

[0225] Peripheral blood mononuclear cells were isolated from the blood of healthy donors using Lymphoprep and SepMate-50 (STEMCELL Technologies). CD3+T cells were isolated using Pan T Cell Isolation Kit, human (Miltenyi Biotec). T cells were activated by culturing with gamma-irradiated K562-based artificial antigen presenting cells expressing membranebound anti-human CD3 antibody (clone OKT3) and co-stimulatory molecules CD80 and CD83. T cells were cultured in RPMI 1640 with 25 mM HEPES (Gibco) supplemented with 10% human AB serum (Gemini Bio-Products), 50 mg / mL gentamicin (Bioshop), 100 lU / mL human IL-2, and 10 ng / mL human IL-15 (PeproTech). Cytokines were replenished every 2-3 days.

[0226] Generation of CAR-T cells from primary human T cells

[0227] The pMX retroviral vector was transfected into the 293 GPG packaging cell line, which expresses the VSV-G envelope protein, using TransIT-293 Transfection Reagent (Minis Bio). The resulting VSV-G pseudotyped virus was then used to generate a stable PG13 packaging cell line expressing the gibbon ape leukemia virus (GaLV) envelope protein. Primary human T cells were transduced with GaLV pseudotyped virus in the presence of RetroNectin (Takara Bio), following the manufacturer’s protocol. Briefly, non-tissue culture treated plates were coated with RetroNectin at 50 pg / mL, followed by the addition of GaLV pseudotyped virus and centrifugation at 2,000 x g for 2 hours. The viral supernatant was thenremoved and activated T cells were added on day 2 post-activation. Transduction efficiency was assessed by the expression of truncated NGFR. Transduced T cells were subsequently purified using CD271 MicroBead Kits (Miltenyi Biotec) and used for downstream experiments.

[0228] Cell lines

[0229] PG13, HEK293T, and NCI-H226 were obtained from American Type Culture Collection (ATCC). 293GPG was a kind gift. K562-based artificial antigen presenting cells were cultured in RPMI 1640 (Gibco), 293 GPG, PG13, and HEK293T in DMEM, high glucose (Gibco), and NCI-H226 in RPMI 1640 (ATCC modification). All media were supplemented with 10% FBS (Gibco) and 50 mg / mL gentamicin (Bioshop).

[0230] Flow cytometry

[0231] The following antibodies were used: APC-CD3 (UCHT1, BioLegend); BV421-CD3 (UCHT1, BioLegend); BV605-CD4 (RPA-T4, BioLegend); PerCP-CD8 (RPA-T8, BioLegend); FITC-CD271 (NGFR) (ME20.4, BioLegend); AF700-CD271 (NGFR) (ME20.4, BioLegend); Purified-Mesothelin (MN, BioLegend); APC-Mesothelin (420411, R&D Systems); PE-mouse IgG2a (RMG2a-62, BioLegend); Purified-mouse IgG2a K isotype control (MOPC-173, BioLegend); APC-CD25 (BC96, BioLegend); FITC-CD69 (FN50, BioLegend); PE-PD-1 (EH12.2H7, BioLegend); BV421-Tim-3 (F38-2E2, BioLegend); APC-mouse IgGl K isotype control (MOPC-21, BioLegend); FITC-mouse IgGl K isotype control (MOPC-21, BioLegend); PE-mouse IgGl K isotype control (MOPC-21, BioLegend); BV421-mouse IgGl K isotype control (MOPC-21, BioLegend); FITC-IL-2 (MQ1-17H12, BioLegend); APC-TNF-oc (MAbll, BioLegend); PE-IFN-y (B27, BioLegend); FITC-CD45 (HI30, BioLegend); AF647-CD326 (EpCAM) (9C4, BioLegend); PE-AffiniPure F(ab')2Fragment Goat AntiMouse IgG, F(ab')2fragment specific (Jackson ImmunoResearch); PE-AffiniPure F(ab')2Fragment Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch); PE-AffiniPure F(ab')2Fragment Goat Anti-Human IgG, F(ab')2fragment specific (Jackson ImmunoResearch); Biotin-protein L (GenScript). Cells were blocked using Human TruStain FcX (BioLegend). Dead cells were labeled with 7-AAD Viability Staining Solution (BioLegend) or LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Cells were analyzed using CytoFLEX S Flow Cytometer (Beckman Coulter) and data analysis was performed using FlowJo (BD).

[0232] Cytotoxicity assay

[0233] Target cells were labeled with the Vybrant DiO Cell-Labeling Solution (Thermo Fisher Scientific) and seeded at a density of 20,000 cells per well in a 96-well plate. T cells were added at the indicated E:T ratios. Next day, both suspension and adherent cells were collected by trypsinization and stained with TO-PRO-3 (Thermo Fisher Scientific) to assess viability. The percentage of TO-PRO-3 -positive cells among the DiO-labeled target cells was used to determine cytotoxicity. To correct for spontaneous target cell death, the percentage of TO-PRO-3 -positive cells in wells containing only the target cells was subtracted to calculate Acytotoxicity. For the functional avidity assay, DiO-labeled target cells were incubated with either an anti-human mesothelin antibody (clone MN) or an isotype control antibody at concentrations of 10, 4, or 1 pg / mL for 2 hours at 37 °C prior to performing the cytotoxicity assay. Functional avidity was calculated as the ratio of Acytotoxicity in the presence of the anti-mesothelin antibody to the Acytotoxicity in the presence of the isotype control antibody.

[0234] Tonic signaling assay

[0235] Purified CAR-T cells were seeded at a concentration of 400,000 cells / mL in RPMI 1640 with 25 mM HEPES (Gibco) supplemented with 10% human AB serum (Gemini BioProducts), 50 mg / mL gentamicin (Bioshop), and 200 lU / mL human IL-2. Cells were collected every 2-3 days for flow cytometry analysis and re-seeding.

[0236] Metabolic assay

[0237] To assess glucose uptake, purified CAR-T cells were incubated in glucose-free media for 4 hours at 37 °C, followed by staining with 25 mM 2-NBDG (Thermo Fisher Scientific) for 30 minutes at 37 °C. Cells were then analyzed by flow cytometry. Metabolic phenotyping was performed using the Seahorse ATP Real-Time Rate Assay (Agilent Technologies) according to the manufacturer’s instructions. Seahorse cell plates were precoated with 50 pg / mL poly-D-lysine (MilliporeSigma). Purified CAR-T cells were resuspended in Seahorse XF assay medium (RPMI, pH 7.4, 10 mmol / L glucose, 2 mmol / L L-glutamine, and 1 mmol / L sodium pyruvate) and 360,000 cells were seeded per well. The cells were incubated at 37 °C in a CO2-free incubator for 60 minutes before analysis using the XFe96 extracellular analyzer (Agilent Technologies). Data analysis was performed with Seahorse Wave software (Agilent Technologies).

[0238] Chronic stimulation assay

[0239] Purified CAR-T cells were co-cultured with NCLH226 at E:T ratio of 1 :5 in RPMI 1640 with 25 mM HEPES (Gibco) supplemented with 10% human AB serum (Gemini Bio-Products), and 50 mg / mL gentamicin (Bioshop). Cells were collected every 4-6 days for flow cytometry analysis, functional assays, and re-seeding.

[0240] Generation of recombinant human mesothelin

[0241] The sequence encoding soluble recombinant human mesothelin was fused to a 6x His tag on the C-terminus and cloned into the pcDNA3.1 vector. Recombinant mesothelin protein was produced using the Expi293 Expression System Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Protein was purified with a HisTrap excel column (Cytiva) and concentrated using Amicon Ultra Centrifugal Filter, 10 kDa MWCO (Millipore Sigma) following the manufacturers’ protocols. Protein concentration was determined with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), and purity was confirmed by SDS-PAGE with Oriole Fluorescent Gel Stain (Bio-Rad).

[0242] Proliferation assay

[0243] Wells of a 96 well plate were coated with 1 pg of anti-His antibody isolated from a hybridoma (MediMabs) then with 1 or 0.1 pg of purified recombinant mesothelin. Purified CAR-T cells were seeded at a density of 50,000 cells per well. Cells were collected and counted every 2-3 days and used for flow cytometry analysis, functional assays, and re-seeding.

[0244] xCELLigence real-time cell analysis

[0245] For the 2D xCELLigence assay, target cells were seeded at a density of 5,000 cells per well in an RTCA E-Plate and incubated overnight before adding CAR-T cells at an E:T ratio of 4:1. Cell index values were normalized to those measured immediately prior to CAR-T cell addition. In the 3D spheroid assay, target cells were labeled using the CellTrace Violet Cell Proliferation Kit (Thermo Fisher Scientific) and seeded at a density of 5,000 cells (NCI-H226) or 10,000 cells (primary ovarian cancer cells) per well in 96-well low-attachment roundbottom plates. Following a 2-day incubation, CAR-T cells were added at an E:T ratio of 2:1 (NCLH226) or 1 : 1 (primary ovarian cancer cells). Spheroid area was measured and normalized to control wells containing only target cells. All measurements were obtained using the xCELLigence RTCA eSight instrument (Agilent Technologies).

[0246] Cytokine secretion assay

[0247] Target cells were seeded at a density of 20,000 cells per well in a 96 well plate and CAR-T cells were added at an E:T ratio of 1:1. After 1 hour of incubation, Brefeldin A (BioLegend) was added. Cells were collected 5 hours later and stained for cell surface makers before performing intracellular cytokine staining using Cyto-Fast Fix / Perm Buffer Set (BioLegend).

[0248] Primary ovarian cancer cell culture

[0249] Cryopreserved ascites from ovarian cancer patients were thawed and seeded in DMEM, high glucose (Gibco), supplemented with 10% FBS (Gibco) and 50 mg / mL gentamicin (Bioshop), in tissue-culture treated flasks. The following day, non-adherent cells were removed by replacing the supernatant with fresh media. Once the adherent cells reached confluence, they were used for flow cytometry analysis, functional assays, or further expansion.

[0250] Mouse Studies

[0251] Male NSG mice bred at the Princess Margaret Cancer Centre Animal Resource Centre were used. Mice were injected with 1 x 106AsPC-1 cells subcutaneously and with 4 x 106CAR-T cells or PBS after one week. CAR-T cells were generated as described above and expanded ex vivo with plate-bound mesothelin for 9 days prior to infusion. Tumor volumes were measured every 2-3 days and monitored until the mice reached the humane endpoint, at which point they were euthanized by CO2 inhalation in accordance with humane endpoint guidelines. Mice were monitored at least once daily throughout the experiments.

[0252] Results

[0253] Humanized scFvs maintain antigen-specific recognition while displaying variable functional avidity

[0254] To investigate the role of framework regions on CAR-T cell functionality, humanization through CDR grafting was performed on mouse anti-human mesothelin scFv SSI. The amino acid sequence of SSI was aligned against a database containing the sequences of human immunoglobulins and CDR grafting was performed onto selected alleles. The heavy chain and the light chain were grafted onto four or two framework regions derived from human antibodies, respectively, generating eight humanized anti-mesothelin scFvs (FIGs. 1A-1H, 2A-2H, and 3A; Table 1; and SEQ ID NOs: 1-10). These scFvs were used to generate second-generation CAR constructs containing the hinge, transmembrane, and intracellular domains derived from CD28, a T cell co- stimulatory molecule, and the intracellular domain derived from CD3 zeta (SEQ ID NO: 17), a protein comprising the T cell receptor complex. In addition to the humanized scFvs, CAR constructs were also generated using parental SSI scFv and a humanized SSI scFv.

[0255] To validate mesothelin-recognition, primary human T cells were transduced with genes encoding the anti-mesothelin CAR molecules using an amphotropic retroviral system. By performing a flow cytometry -based cytotoxicity assay, we demonstrated that all eight scFvs recognized mesothelin and induced cytotoxicity against a mesothelioma-derived cell line, NCI-H226, comparable to parental SSI (SEQ ID NO: 9) and LMB (SEQ ID NO: 10), without incurring non-specific cytotoxicity against a mesothelin-negative cell line (FIGs. 3B-3M).

[0256] In order to assess whether the humanization process lowered CAR immunogenicity, we evaluated the recognition of humanized scFvs by polyclonal anti-mouse IgG F(ab’)2 fragments. The results demonstrated reduced recognition compared to parental SSI, indicating decreased xenogenicity achieved through CDR grafting (FIGs. 4A-4D).

[0257] Given that antibody humanization affects the CDR conformation, and therefore the antigen-antibody binding, we explored whether our humanized scFv-based CAR-T cells also had altered binding strength to mesothelin. This is pertinent because it has been shown that CAR-T cells with lower affinity are functionality superior to high-affinity equivalents. To measure the binding strength of our scFvs to mesothelin, we conducted cytotoxicity assays in the presence of anti-mesothelin blocking antibodies with overlapping epitope as SSI. By masking the mesothelin protein on the target cell surface, cytotoxicity was measured against target cells presenting fewer antigen molecules. The CDR grafting process resulted in a series of scFvs exhibiting variable functional avidity, where six (#13, #16, #33, #36, #73, #76) out of eight showed significantly reduced avidity, and four (#33, #36, #73, #76) exhibited markedly low functional avidity. This highlighted the contributions of the framework regions towards antigen recognition in the context of CAR-T cells (FIGs. 5A-5C).

[0258] Framework regions of scFvs affect strength of antigen-independent tonic signaling

[0259] CAR molecules can aggregate on the cell surface in the absence of the cognate antigen and deliver an activation signal, an effect known as antigen-independent tonic signaling. The sustained T cell activation delivered through CARs exhibiting excessive tonic signaling promotes T cell exhaustion and terminal differentiation, resulting in a lack of durable clinical response. To investigate the role of the framework regions following CDR grafting, we quantified the strength of tonic signaling in our humanized scFvs. When the CAR-T cells were cultured in the absence of target cells, the expression of surface markers associated with T cell activation and exhaustion (CD69, CD25, PD-1, and Tim-3) revealed that framework sequences have a major role in determining the intensity of tonic signaling, where certain scFvs such as #16 and #56 displayed significantly higher levels of tonic signaling (FIGs. 6A-6H and 7A-7H).

[0260] Activated T cells, as well as CAR-T cells exhibiting high tonic signaling, preferentially utilize the glycolytic pathway as opposed to oxidative phosphorylation. Analysis of CAR-T cells from clinical trials has also shown that glycolysis is a hallmark of CAR-T cells with limited durable responses. To this end, we measured the uptake of 2-NBDG, a fluorescent glucose analog, by our CAR-T cells. Consistent with the surface marker phenotypes, scFvs #16and #56 showed the highest uptake, indicating more intense tonic signaling (FIGs. 8A-8B). To further characterize the metabolic phenotype, we performed the Seahorse ATP Rate assay on the scFvs with the highest tonic signaling (#16 and #56) and low tonic signaling (#33). In line with previous results, scFvs with high tonic signaling preferentially utilized the glycolytic pathway and were more metabolically active, producing higher amounts of ATP in the basal state (FIGs. 8C-8H). Overall, framework sequences profoundly affect the intensity of tonic signaling, and our evaluation of tonic signaling prompted us to exclude #16 and #56 with the highest tonic signaling from further analysis.

[0261] Framework regions of scFvs affect CAR-T cell phenotype after prolonged exposure to tumor cells

[0262] To study how the framework engineering can affect the exhaustion phenotype of CAR-T cells upon prolonged exposure to tumor cells, we characterized the expression of markers associated with T cell exhaustion (PD-1 and Tim-3) on CAR-T cells after recurrent co-culture with mesothelin-expressing tumor cells. Parental SSI CAR-T cells exhibited elevated expression of PD-1 and Tim-3, consistent with the acquisition of phenotypes characteristic of terminally differentiated and exhausted T cells. Meanwhile, four humanized scFvs (#33, #36, #73, #76) exhibited reduced expression of both markers, suggesting that these cells were more resistant to tumor-driven exhaustion (FIG. 9A-9G).

[0263] CAR-T cells can acquire parts of cell membranes from tumor cells upon antigen recognition, a process known as trogocytosis. After acquiring the target protein on the T cell surface, the binding of CAR molecules to the cognate antigen can trigger T cell exhaustion and fratricide (FIG. 9H). Because we have shown previously that modifications to the framework regions reduced functional avidity (FIGs. 5A-5C), we investigated whether this also translated to reduced trogocytosis. Consistent with the earlier results, humanized scFvs (#13, #33, #36, #53, #73, #76) displayed limited trogocytosis, measured by mesothelin staining on CAR-T cell surface upon co-culture with mesothelin-expressing tumor cells (FIGs. 9I-9J). Altogether, framework engineering generated scFvs with a diminished capacity for trogocytosis, contributing to their reduced propensity for tumor-induced exhaustion.

[0264] Framework engineering improves anti-tumor function of CAR-T cells

[0265] Terminally differentiated and exhausted T cells can display blunted cell proliferation. Given this, we investigated the proliferative capacity of CAR-T cells upon repeated antigen stimulation. Relative to parental SSI, four of the humanized scFvs (#33, #36, #73, #76) exhibited enhanced proliferation, in line with reduced antigen-driven exhaustion,thereby maintaining proliferative capacity over a prolonged period of exposure to the cognate antigen (FIGs. 10A-10B).

[0266] A key determinant of an effective CAR-T cell therapy is the durability of response. To test this, we performed a real-time cytotoxicity assay after rounds of co-culture with tumor cells. Framework engineering generated an scFv which maintained anti-tumor functionality after continued exposure to tumor cells, suggesting its enhanced durability (FIG. 10C). Cytokine polyfunctionality, defined by the ability to secrete multiple cytokines, is considered another important characteristic of functional CAR-T cells. Based on this observation, we analyzed the cytokine secretion capacity after chronic stimulation with the tumor cells. In addition to enhanced cytotoxicity, the humanized scFv, #33, also displayed stronger cytokine secretion profiles with an increased fraction of polyfunctional T cells and a reduced proportion of dysfunctional T cells that lacked cytokine production (FIGs. 10D-10H).

[0267] Three-dimensional tumor models are increasingly recognized for their ability to more accurately replicate the physiological characteristics of tumors in preclinical studies. Building on this, we evaluated the efficacy of the humanized scFv in a 3D spheroid platform. Consistent with our earlier findings, the humanized scFv exhibited enhanced anti-tumor activity in a spheroid model using NCI-H226 cells (FIGs. 11A-11B). Since ovarian cancer frequently overexpresses mesothelin, we established an additional 3D spheroid model using primary ovarian cancer cells isolated from the ascitic fluid of a patient with ovarian cancer. Flow cytometry confirmed mesothelin expression in the primary cancer cells (EpCAM+CD45 ) (FIGs. 11C-11D). In this patient-derived spheroid model, the humanized scFv, #33, similarly demonstrated superior cytotoxicity (FIG. 1 IE).

[0268] Finally, in a humanized mouse model of solid tumors, CAR-T cells with the humanized scFv #33 showed better tumor control over the parental scFv (FIGs. 12A and 12B). Altogether, these findings underscore that framework engineering resulted in an scFv with improved proliferation, cytokine production, and sustained anti-tumor efficacy.

[0269] Although CAR-T cell therapies now constitute a significant share of the therapeutic landscape for hematological malignancies, their efficacy in solid tumors remains limited. Even in hematologic settings, CAR-T cell therapies targeting CD 19 or BCMA often face relapse challenges, where malignant cells responsible for recurrence may still express the target antigen, implicating dysfunctional CAR-T cells in treatment failure. This dysfunction is partly driven by the inherent self-regulatory mechanisms of T cells, which upregulate inhibitory surface molecules upon activation to maintain immune tolerance under homeostatic conditions.

[0270] In normal physiological contexts, sustained T cell activation signals either autoreactivity or unresolved chronic infection, prompting immune regulatory mechanisms to protect host tissues from targeted cytotoxicity and prolonged inflammation. This selfregulation becomes a significant obstacle in CAR-T cell therapy, where cells can undergo sustained activation through either antigen-independent tonic signaling or chronic antigendependent stimulation. CAR molecules, being synthetic, lack the evolutionary refinement of T cell receptors, which utilize low levels of tonic signaling as part of their natural development process. In addition, within the tumor microenvironment, persistent antigen exposure drives CAR T cell exhaustion and subsequent dysfunction. Efforts to improve CAR design, such as optimizing hinge domain length and intracellular signaling domains, have sought to address these challenges by enhancing CAR functionality and persistence. In this study, we extend these strategies by exploring the critical role of scFv framework regions in modulating CAR-T cell function.

[0271] Our comprehensive analysis of CAR T cell phenotypes underscores the pivotal role of the scFv framework region in dictating CAR T cell behavior, both in antigen-dependent and antigen-independent contexts. Using the mouse-derived anti-human mesothelin scFv SSI as a template, we performed CDR grafting onto a series of human antibody-derived framework regions to generate a panel of scFvs displaying a broad spectrum of functional phenotypes. We observed that framework sequences modulate the intensity of tonic signaling. Importantly, while framework engineering preserved antigen recognition, it significantly reduced functional avidity in six of the eight instances. This is of particular interest, as earlier reports suggest that fine-tuning the strength of antigen recognition is beneficial in improving CAR-T cell function in addition to widening the therapeutic window and reducing on-target off-tumor toxicity. CAR T cells have been shown to engage in a serial killing behavior, wherein an effector cell efficiently traverses multiple target cells, executing cytotoxicity sequentially58. The ideal scFv would deliver a strong enough activation signal to induce cytotoxicity while minimizing activation-induced exhaustion and prolonged tumor cell binding. This aspect may have contributed significantly to the limited antigen-dependent exhaustion and improved persistence of anti-tumor function observed in our humanized scFvs.

[0272] Our approach to mitigating excessive CAR-T cell activation through framework engineering parallels strategies combining CAR-T therapy with immune checkpoint inhibitors, such as PD-1 blockade, which aim to rescue exhausted CAR-T cells. Altogether, our findings emphasize that engineering of the scFv framework region is an indispensable step in developing effective CAR-T cell therapies and that both experimental and in silico analyses ofscFv function hold substantial promise for enhancing the efficacy of CAR-T cells targeting solid tumors.***

[0273] The foregoing description of the specific aspects will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0274] Other aspects of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.

[0275] All publications, patents, and patent applications disclosed herein are incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. What is claimed:

1. A single chain variable fragment (scFv) that specifically binds human mesothelin comprising:(a) (i) a heavy chain complementarity determining region 1 (HC-CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 11, (ii) an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12,(iii) an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13,(iv) a light chain (LC) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14,(v) an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and(vi) an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and(b) (i) an HC framework region 1 (HC-FR1),(ii) an HC-FR2,(iii) anHC-FR3,(iv) an HC-FR4,(v) anLC-FRl,(vi) an LC-FR2,(vii) anLC-FR3, and(viii) an LC-FR4; and(c) a linker between the HC-FR4 and the LC-FR1 ;wherein one or more of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized; andwherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.

2. The scFv of claim 1, which comprises an amino acid sequence having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.

3. The scFv of claim 1 or 2, wherein at least two of the HC-FR1, HC-FR2, HC-FR3, HC- FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

4. The scFv of claim 1 or 2, wherein at least three of the HC-FR1, HC-FR2, HC-FR3, HC- FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

5. The scFv of claim 1 or 2, wherein at least four of the HC-FR1, HC-FR2, HC-FR3, HC- FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

6. The scFv of claim 1 or 2, wherein at least five of the HC-FR1, HC-FR2, HC-FR3, HC- FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

7. The scFv of claim 1 or 2, wherein at least six of the HC-FR1, HC-FR2, HC-FR3, HC- FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

8. The scFv of claim 1 or 2, wherein at least seven of the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

9. The scFv of claim 1 or 2, wherein the HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, and LC-FR4 are humanized.

10. The scFv of any one of claims 1 to 9, wherein the HC-FR1 comprises a human immunoglobulin heavy chain (hIGHV) FR1.

11. The scFv of any one of claims 1 to 10, wherein the HC-FR2 comprises an hIGHV-FR2.

12. The scFv of any one of claims 1 to 11, wherein the HC-FR3 comprises an hIGHV-FR3.

13. The scFv of any one of claims 1 to 12, wherein the HC-FR4 comprises an hIGHV-FR4.

14. The scFv of any one of claims 1 to 13, wherein: (i) the HC-FR1 comprises an hlGHV- FR1, (ii) the HC-FR2 comprises an hIGHV-FR2, (iii) the HC-FR3 comprises an hIGHV-FR3, and (iv) the HC-FR4 comprises an hIGHV-FR4.

15. The scFv of any one of claims 1 to 14, wherein the LC-FR1 comprises a human immunoglobulin light chain (hIGKV) FR1.

16. The scFv of any one of claims 1 to 15, wherein the LC-FR2 comprises an hIGKV-FR2.

17. The scFv of any one of claims 1 to 16, wherein the LC-FR3 comprises an hIGKV-FR3.

18. The scFv of any one of claims 1 to 17, wherein the LC-FR4 comprises an hIGKV-FR4.

19. The scFv of any one of claims 1 to 18, wherein: (i) the LC-FR1 comprises an hlGKV- FR1, (ii) the LC-FR2 comprises an hIGKV-FR2, (iii) the LC-FR3 comprises an hIGKV-FR3, and (iv) the LC-FR4 comprises an hIGKV-FR4.

20. The scFv of any one of claims 1 to 19, wherein:(a) (i) the HC-FR1 comprises an hIGHV-FRI, (ii) the HC-FR2 comprises an hIGHV-FR2, (iii) the HC-FR3 comprises an hIGHV-FR3, and (iv) the HC-FR4 comprises an hIGHV-FR4; and(b) (i) the LC-FR1 comprises an hIGKV-FRI, (ii) the LC-FR2 comprises an hIGKV-FR2, (iii) the LC-FR3 comprises an hIGKV-FR3, and (iv) the LC-FR4 comprises an hIGKV-FR4.

21. The scFv of any one of claims 10 to 20, wherein the hIGHV comprises hIGHVl-45, hIGHV3-73, hIGHV5-51, or MGHV7-4-1.

22. The scFv of any one of claims 15 to 21, wherein the hIGKV comprises IGKV3-11 or IGKV6-21.

23. The scFv of claim 21 or 22, wherein the hIGHV comprises hIGHV 1 -45 and the hIGKV comprises hIGKV3-l 1.

24. The scFv of any one of claims 1 to 19, wherein:(a) (i) the HC-FR1 comprises an hIGHV 1-45 -FR1, (ii) the HC-FR2 comprises an MGHV1-45-FR2, (iii) the HC-FR3 comprises an MGHV1-45-FR3, and (iv) the HC-FR4 comprises an hIGHVl-45-FR4; and(b) (i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-l 1-FR3, and (iv) the LC-FR4 comprises an MGKV3-11-FR4.

25. The scFv of claim 21 or 22, wherein the hIGHV comprises hIGHV3-73 and the hIGKV comprises IGKV3-11.

26. The scFv of any one of claims 1 to 19, wherein:(a) (i) the HC-FR1 comprises an hIGHV3-73-FRl, (ii) the HC-FR2 comprises an MGHV3-73-FR2, (iii) the HC-FR3 comprises an MGHV3-73-FR3, and (iv) the HC-FR4 comprises an hIGHV3-73-FR4; and(b) (i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-l 1-FR3, and (iv) the LC-FR4 comprises an MGKV3-11-FR4.

27. The scFv of claim 21 or 22, wherein the hIGHV comprises hIGHV5-51 and the hIGKV comprises IGKV3-11.

28. The scFv of any one of claims 1 to 19, wherein:(a) (i) the HC-FR1 comprises an hIGHV5-51-FRl, (ii) the HC-FR2 comprises an hIGHV5-51-FR2, (iii) the HC-FR3 comprises an hIGHV5-51-FR3, and (iv) the HC-FR4 comprises an hIGHV5-51-FR4; and(b) (i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-l 1-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4.

29. The scFv of claim 21 or 22, wherein the hIGHV comprises hIGHV7-4- 1 and the hIGKV comprises IGKV3-11.

30. The scFv of any one of claims 1 to 19, wherein:(a) (i) the HC-FR1 comprises an hIGHV7-4-l-FRl, (ii) the HC-FR2 comprises an hIGHV7-4-l-FR2, (iii) the HC-FR3 comprises an hIGHV7-4-l-FR3, and (iv) the HC-FR4 comprises an hIGHV7-4-l-FR4; and(b) (i) the LC-FR1 comprises an hIGKV3-l 1-FR1, (ii) the LC-FR2 comprises an hIGKV3-l 1-FR2, (iii) the LC-FR3 comprises an hIGKV3-l 1-FR3, and (iv) the LC-FR4 comprises an hIGKV3-l 1-FR4.

31. The scFv of claim 21 or 22, wherein the hIGHV comprises hIGHV 1 -45 and the hIGKV comprises hIGKV6-21.

32. The scFv of any one of claims 1 to 19, wherein:(a) (i) the HC-FR1 comprises an hIGHV 1-45 -FR1, (ii) the HC-FR2 comprises an MGHV1-45-FR2, (iii) the HC-FR3 comprises an MGHV1-45-FR3, and (iv) the HC-FR4 comprises an hIGHVl-45-FR4; and(b) (i) the LC-FR1 comprises an hIGKV6-21-FRl, (ii) the LC-FR2 comprises an MGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an MGKV6-21-FR4.

33. The scFv of claim 21 or 22, wherein the hIGHV comprises hIGHV3-73 and the hIGKV comprises hIGKV6-21.

34. The scFv of any one of claims 1 to 19, wherein:(a) (i) the HC-FR1 comprises an hIGHV3-73-FRl, (ii) the HC-FR2 comprises an hIGHV3-73-FR2, (iii) the HC-FR3 comprises an hIGHV3-73-FR3, and (iv) the HC-FR4 comprises an hIGHV3-73-FR4; and(b) (i) the LC-FR1 comprises an hIGKV6-21-FRl, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

35. The scFv of claim 21 or 22, wherein the hIGHV comprises hIGHV5-51 and the hIGKV comprises hIGKV6-21.

36. The scFv of any one of claims 1 to 19, wherein:(a) (i) the HC-FR1 comprises an hIGHV5-51-FRl, (ii) the HC-FR2 comprises an hIGHV5-51-FR2, (iii) the HC-FR3 comprises an hIGHV5-51-FR3, and (iv) the HC-FR4 comprises an hIGHV5-51-FR4; and(b) (i) the LC-FR1 comprises an hIGKV6-21-FRl, (ii) the LC-FR2 comprises an hIGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

37. The scFv of claim 21 or 22, wherein the hIGHV comprises hIGHV7-4-l and the hIGKV comprises hIGKV6-21.

38. The scFv of any one of claims 1 to 19, wherein:(a) (i) the HC-FR1 comprises an hIGHV7-4-l-FRl, (ii) the HC-FR2 comprises an hIGHV7-4-l-FR2, (iii) the HC-FR3 comprises an hIGHV7-4-l-FR3, and (iv) the HC-FR4 comprises an hIGHV7-4-l-FR4; and(b) (i) the LC-FR1 comprises an hIGKV6-21-FRl, (ii) the LC-FR2 comprises an MGKV6-21-FR2, (iii) the LC-FR3 comprises an hIGKV6-21-FR3, and (iv) the LC-FR4 comprises an hIGKV6-21-FR4.

39. The scFv of any one of claims 1 to 38, comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.

40. The scFv of any one of claims 1 to 39, comprising the amino acid sequence set forth in SEQ ID NO: 1.

41. The scFv of any one of claims 1 to 39, comprising the amino acid sequence set forth in SEQ ID NO: 2.

42. The scFv of any one of claims 1 to 39, comprising the amino acid sequence set forth in SEQ ID NO: 3.

43. The scFv of any one of claims 1 to 39, comprising the amino acid sequence set forth in SEQ ID NO: 4.

44. The scFv of any one of claims 1 to 39, comprising the amino acid sequence set forth in SEQ ID NO: 5.

45. The scFv of any one of claims 1 to 39, comprising the amino acid sequence set forth in SEQ ID NO: 6.

46. The scFv of any one of claims 1 to 39, comprising the amino acid sequence set forth in SEQ ID NO: 7.

47. The scFv of any one of claims 1 to 39, comprising the amino acid sequence set forth in SEQ ID NO: 8.

48. A chimeric antigen receptor (CAR) comprising the scFv of any one of claims 1 to 47.

49. The CAR of claim 48, further comprising (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, (iv) an intracellular signaling domain, or (v) any combination thereof.

50. The CAR of claim 48, further comprising (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, and (iv) an intracellular signaling domain.

51. The CAR of claim 49 or 50, wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin.

52. The CAR of claim 51, wherein the immunoglobulin is selected from of IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, IgM, and any combination thereof.

53. The CAR of any one of claims 49 to 51, wherein the hinge region is derived from a CD28 hinge region.

54. The CAR of any one of claims 49 to 53, wherein the transmembrane domain comprises a transmembrane domain derived from CD28, CD8 alpha, CD8 beta, KIR2DS2, 0X40, CD2, CD4, CD45, PD-1, CTLA-4 (CD 152), CD27, LFA-1 (CD 11 a, CD 18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, interleukin-2 receptor (IL2R) beta, IL2R gamma, IL7R alpha, ITGA1 (VLA1, CD49a), ITGA4 (IA4, CD49D), ITGA6 (VLA-6, CD49f), ITGAD (CD lid), ITGAE (CD 103), ITGAL (CD1 la, LFA-1), ITGAM (CD1 lb), ITGAX (CD11c), ITGB1 (CD29), ITGB2 (CD18, LFA-1), ITGB7, TNFR2, DNAM-1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL-1 (SELPLG, CD 162), CD 100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), LTBR, PAG / Cbp, NKG2D, NKG2C, or any combination thereof.

55. The CAR of any one of claims 49 to 54, wherein the transmembrane domain is derived from a CD28 transmembrane domain.

56. The CAR of any one of claims 49 to 54, wherein the transmembrane domain is derived from a CD8 alpha transmembrane domain.

57. The CAR of any one of claims 49 to 56, wherein the costimulatory domain comprises a costimulatory domain derived from a cytoplasmic / intracellular domain of 4- IBB (CD137), interleukin-2 receptor (IL-2R), IL-12R, IL-7R, IL-21R, IL-23R, IL-15R, CD2, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), LIGHT, NKG2C, 0X40, DAP 10, B7-H3, CD28 deleted for Lek binding, CTLA-4, BTLA, GITR, HVEM, PD-1, TLR2, TLR4, TLR7, TLR9, Fc receptor gamma chain, Fc receptor epsilon chain, a ligand that specifically binds with CD83, or any combination thereof.

58. The CAR of any one of claims 49 to 57, wherein the costimulatory domain is derived from a CD28 costimulatory domain.

59. The CAR of any one of claims 49 to 58, wherein the intracellular signaling domain comprises a CD3 zeta (CD247) activating domain, a CD3 gamma activating domain, a CD3 delta activating domain, a CD3 epsilon activating domain, a CD3 eta activating domain, a CD79A activating domain, a CD79B activating domain, a DAP 12 activating domain, a FCER1G activating domain, a FCGR2A activating domain, a FCGR2C activating domain, or any combination thereof.

60. The CAR of any one of claims 49 to 59, wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

61. The CAR of any one of claims 48 to 60, which has a lower immunogenicity when administered to a human subject than a reference scFv, wherein the reference scFv does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, or LC-FR4.

62. The CAR of any one of claims 48 to 61, which has a lower tonic signaling when expressed on the surface of an immune cell than a reference CAR, wherein the reference CAR comprises an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC- FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, orLC-FR4.

63. A nucleic acid molecule encoding the scFv of any one of claims 1 to 47.

64. A nucleic acid molecule encoding a CAR, wherein the CAR comprises the scFv of any one of claims 1 to 47.

65. The nucleic acid molecule of claim 64, wherein the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, (iv) an intracellular signaling domain, or (v) any combination thereof.

66. The nucleic acid molecule of claim 64, wherein the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, and (iv) an intracellular signaling domain.

67. The nucleic acid molecule of claim 65 or 66, wherein the hinge region comprises a hinge region derived from CD28, CD8, or an immunoglobulin.

68. The nucleic acid molecule of claim 67, wherein the immunoglobulin is selected from of IgAl, IgA2, IgGl, IgG2, IgG3, IgG4, IgD, IgE, IgM, and any combination thereof.

69. The nucleic acid molecule of any one of claims 65 to 68, wherein the hinge region is derived from a CD28 hinge region.

70. The nucleic acid molecule of any one of claims 65 to 69, wherein the transmembrane domain comprises a transmembrane domain derived from CD28, CD8 alpha, CD8 beta, KIR2DS2, 0X40, CD2, CD4, CD45, PD-1, CTLA-4 (CD152), CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, interleukin-2 receptor (IL2R) beta, IL2R gamma, IL7R alpha, ITGA1 (VLA1, CD49a), ITGA4 (IA4, CD49D), ITGA6 (VLA-6, CD49f), ITGAD (CD lid), ITGAE (CD 103), ITGAL (CD1 la, LFA-1), ITGAM (CD1 lb), ITGAX (CD11c), ITGB1 (CD29), ITGB2 (CD18, LFA-1), ITGB7, TNFR2, DNAM-1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL-1 (SELPLG, CD 162), CD 100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), LTBR, PAG / Cbp, NKG2D, NKG2C, or any combination thereof.

71. The nucleic acid molecule of any one of claims 65 to 70, wherein the transmembrane domain is derived from a CD28 transmembrane domain.

72. The nucleic acid molecule of any one of claims 65 to 70, wherein the transmembrane domain is derived from a CD8 alpha transmembrane domain.

73. The nucleic acid molecule of any one of claims 65 to 72, wherein the costimulatory domain comprises a costimulatory domain derived from a cytoplasmic / intracellular domain of 4-1BB (CD137), interleukin-2 receptor (IL-2R), IL-12R, IL-7R, IL-21R, IL-23R, IL-15R, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, 0X40, DAP10, B7-H3, CD28 deleted for Lek binding, CTLA-4, BTLA, GITR, HVEM, PD-1, TLR2, TLR4, TLR7, TLR9, Fc receptor gamma chain, Fc receptor epsilon chain, a ligand that specifically binds with CD83, or any combination thereof.

74. The nucleic acid molecule of any one of claims 65 to 73, wherein the costimulatory domain is derived from a CD28 costimulatory domain.

75. The nucleic acid molecule of any one of claims 65 to 74, wherein the intracellular signaling domain comprises a CD3 zeta activating domain, a CD3 gamma activating domain, a CD3 delta activating domain, a CD3 epsilon activating domain, a CD3 eta activating domain, a CD79A activating domain, a CD79B activating domain, aDAP12 activating domain, a FCER1G activating domain, a FCGR2A activating domain, a FCGR2C activating domain, or any combination thereof.

76. The nucleic acid molecule of any one of claims 65 to 75, wherein the intracellular signaling domain comprises a CD3 zeta activating domain.

77. A vector comprising the nucleic acid molecule of any one of claims 63 to 76.

78. The vector of claim 77, which is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated viral vector (AAV), a lentiviral vector, or any combination thereof.

79. An engineered immune cell comprising the scFv of any one of claims 1 to 47, the CAR of any one of claims 48 to 62, the nucleic acid molecule of any one of claims 63 to 76, or the vector of claim 77 of 78.

80. The engineered immune cell of claim 79, which is a T cell, an NK cell, a B cell, or any combination thereof.

81. The engineered immune cell of claim 80, wherein the T cell is an aP T cell, a y5 T cell, or any combination thereof.

82. The engineered immune cell of any one of claims 79 to 81, which is a tumor infiltrating lymphocyte (TIL).

83. A population of engineered immune cells comprising the engineered immune cell of any one of claims 79 to 82.

84. The population of engineered immune cells of claim 83, wherein fewer engineered immune cells of the population of engineered immune cells express one or more markers of T cell exhaustion following administration to a subject, as compared to a population of engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC- FR2, LC-FR3, or LC-FR4.

85. The population of engineered immune cells of claim 84, wherein the one or more markers of T cell exhaustion comprise PD-1, TIM-3, or both.

86. The population of engineered immune cells of any one of claims 83 to 85, wherein following administration to a subject, the engineered immune cells have a diminished capacity for trogocytosis, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, orLC-FR4.

87. The population of engineered immune cells of any one of claims 83 to 86, wherein the engineered immune cells have increased cytokine secretion capacity after chronic stimulation with a target antigen, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC- FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, or LC-FR4.

88. The population of engineered immune cells of any one of claims 83 to 87, wherein the engineered immune cells have enhanced anti-tumor activity, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LCFR3, or LC- FR4.

89. The population of engineered immune cells of any one of claims 83 to 87, wherein the engineered immune cells exhibit enhanced proliferation following repeated antigenstimulation, as compared to engineered immune cells that express a CAR comprising an scFv that does not comprise a humanized HC-FR1, HC-FR2, HC-FR3, HC-FR4, LC-FR1, LC-FR2, LC-FR3, orLC-FR4.

90. A method of treating a subject in need thereof comprising administering to the subject the scFv of any one of claims 1 to 47, the CAR of any one of claims 48 to 62, the nucleic acid molecule of any one of claims 63 to 76, the vector of claim 77 of 78, the engineered immune cell of any one of claims 79 to 82, or the population of engineered immune cells of any one of claims 83 to 89.

91. The method of claim 90, wherein the subject is afflicted with a cancer.

92. The method of claim 91, wherein the cancer comprises a lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, or any combination thereof.

93. The method of any one of claims 90 to 92, wherein the subj ect is afflicted with a cancer comprising one or more tumor cells that express mesothelin.

94. The method of any one of claims 90 to 93, wherein the subject is afflicted an ovarian cancer.

95. The method of any one of claims 90 to 94, wherein the subject is afflicted with mesothelioma.

96. The method of any one of claims 91 to 95, wherein the cancer is locally advanced.

97. The method of any one of claims 91 to 95, wherein the cancer is metastatic.

98. The method of any one of claims 91 to 97, wherein the cancer is refractory or relapsed.

99. The method of any one of claims 90 to 98, further comprising administering to the subject an additional anti-cancer agent.

100. The method of claim 99, wherein the additional anti-cancer therapy comprises a chemotherapy, an immunotherapy, a radiotherapy, a surgery, or any combination thereof.

101. The method of claim 99 or 100, wherein the additional anti-cancer therapy comprises a chemotherapy.

102. The method of any one of claims 99 to 101, wherein the additional anti-cancer therapy comprises an immune-checkpoint inhibitor.

103. The method of any one of claims 99 to 102, wherein the additional anti-cancer therapy comprises a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a GITR agonist, or any combination thereof.

104. The method of any one of claims 99 to 103, wherein the additional anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds and inhibits PD-1.

105. The method of any one of claims 99 to 104, wherein the anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds and inhibits PD- Ll.

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