Antigen-binding proteins, antigen-binding fragments thereof, and methods of using

Abstract

Provided herein are antigen-binding proteins and antigen-binding fragments thereof comprising a first variable domain and a second variable domain that bind to an antigen. The antigen may be a fragment of NY-ESO1. Provided are nucleic acids and vectors encoding the antigen-binding proteins and antigen-binding fragments thereof as well as cells expressing the antigen-binding proteins and antigen-binding fragments thereof. Also provided are methods of making and methods of using compositions disclosed herein, including for the treatment of cancer and in adoptive cell therapy.

Description

ANTIGEN-BINDING PROTEINS, ANTIGEN-BINDING FRAGMENTS THEREOF,AND METHODS OF USINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 779,551, filed on March 28, 2025, which is hereby incorporated by reference in its entirety.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on March 12, 2026, is named SeqList-084276-00424.xml and is 124,963 bytes in size.FIELD

[0003] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, the methods and compositions herein are useful for treating disease, such as cancer.BACKGROUND

[0004] Adoptive T cell transfer (ACT) is an advanced immunotherapy technique that harnesses the body’s own immune system to combat cancer. This method can involve extracting T cells from a patient, genetically modifying or expanding them in a laboratory to enhance their cancer-fighting capabilities and then reinfusing them back into the patient. ACT has shown promising results in treating various cancers, particularly hematologic malignancies by improving the specificity and potency of the immune response against tumor cells. As such, ACT represents a promising personalized immunotherapy of cancer.

[0005] T cell-based immunotherapy can target peptide epitopes derived from tumor-associated or tumor-specific proteins, which are presented by molecules of the MHC. These tumor-associated antigens (TAAs) can be peptides derived from all protein classes, such as enzymes, receptors, transcription factors, etc., which are expressed and, as compared to unaltered cells of the same origin, usually up-regulated in cells of the respective tumor.183241445.1183465138.1

[0006] While effector CD8+T lymphocytes have previously been favored for targeting and eliminating malignant cells, the poly functional and cytotoxic subsets of CD4+T cells also play an important role in the immune response against cancer. The contributions of CD4+in the context of adoptive cell therapy can be multiple, from catalyzing the increased homing of CD8+cytotoxic lymphocytes in tumors, to providing important support to their effector functions. Furthermore, CD4+effector cells may control tumors through stromal targets or direct antitumor cytotoxic function.

[0007] Nonetheless, several hurdles must be addressed for CD4+T cells to be fully exploited for immunotherapy. Challenges for CD4+T cell therapies include the high degree of HLA polymorphism and diversity of MHC class II molecules.

[0008] Accordingly, new approaches for the use of immune cells, including CD4+T cells, in ACT are urgently needed.SUMMARY

[0009] Provided herein are antigen-binding proteins and antigen-binding fragments thereof comprising a first variable domain and a second variable domain that specifically bind to an antigen, nucleic acids encoding said antigen-binding proteins and antigen-binding fragments thereof, vectors and cells comprising such nucleic acids as well as methods of using any of the compositions disclosed herein. These antigen-binding proteins and antigen-binding fragments thereof exhibit unexpected properties, including MHC class Il-restricted recognition of tumor-associated antigens and the ability to confer enhanced cytotoxic and helper T cell functions.

[0010] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen comprises SEQ ID NO:43 (LKEFTVSGNILTIRL), wherein the first variable domain comprises complementarity determining region (CDR) al, CDRa2, and CDRa3, wherein the second variable domain comprises CDRbl, CDRb2, and CDRb3, and wherein: (a) CDRal comprises SEQ ID NO:1; (b) CDRa2 comprises SEQ ID NO:2 or SEQ ID NO:3; (c) CDRa3 comprises any one of SEQ ID NOs:4-7; (d) CDRbl comprises SEQ ID NO:8 or SEQ ID NO:9; (e) CDRb2 comprises SEQ ID NO: 10 or SEQ ID NO: 11; and (f) CDRb3 comprises any one of SEQ ID NOs: 12-15.

[0011] In some embodiments: (a) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:4; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 12; (b) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:5; CDRbl 2183465138.1comprises SEQ ID NO:9; CDRb2 comprises SEQ ID NO:11; and CDRb3 comprises SEQ ID NO: 13; (c) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:3; CDRa3 comprises SEQ IDN0:6; CDRbl comprises SEQ IDN0:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 14; or (d) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:7; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 15.

[0012] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the antigen-binding protein is a T cell receptor (TCR).

[0013] In some embodiments, provided is a TCR wherein: (a) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17; (b) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19; (c) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21; or (d) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23.

[0014] In some embodiments, (a) the first variable domain comprises SEQ ID NO: 16 and the second variable domain comprises SEQ ID NO: 17; (b) the first variable domain comprises SEQ ID NO: 18 and the second variable domain comprises SEQ ID NO: 19; (c) the first variable domain comprises SEQ ID NO:20 and the second variable domain comprises SEQ ID NO:21; or (d) the first variable domain comprises SEQ ID NO:22 and the second variable domain comprises SEQ ID NO:23.

[0015] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the first variable domain is further joined to a constant region comprising a 3183465138.1sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:75 and wherein the second variable domain is further joined to a constant region comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:76 or SEQ ID NO:77.

[0016] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the first variable domain is further joined to a sequence comprising SEQ ID NO:75 and wherein the second variable domain is further joined to a sequence comprising SEQ ID NO:76 or SEQ ID NO:77.

[0017] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the TCR is: (a) a single chain TCR; or (b) a membrane-bound TCR or soluble TCR.

[0018] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the antigen-binding protein is an antibody or antigen-binding protein fragment thereof.

[0019] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the antibody or antigen-binding protein fragment thereof is a single-chain variable fragment (scFv), fragment variable (Fv), fragment binding domain (Fab), Fab', F(ab')2, or diabody.

[0020] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the antigen-binding protein is a chimeric antigen receptor (CAR) comprising an scFv comprising the first variable domain and a second variable domain.

[0021] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the antigen-binding protein or antigen-binding protein fragment thereof binds to the antigen present in a complex with HLA-DRB3*02:02.

[0022] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the antigen-binding protein or antigen-binding protein fragment thereof is deglycosylated.

[0023] Provided herein is an antigen-binding protein or antigen-binding protein fragment thereof, wherein the antigen-binding protein or antigen-binding protein fragment thereof is conjugated to a fluorescent moiety, a detectable moiety, a purification moiety, or a combination thereof.

[0024] Provided herein is a cell expressing on its surface an antigen-binding protein or antigen-binding protein fragment thereof disclosed herein, optionally wherein the cell is isolated. In one embodiment, the cell is a lymphocyte. In one embodiment, the lymphocyte is 4183465138.1a T cell. In one embodiment, the T cell is a CD4+T cell. In one embodiment, the T cell is a CD8+T cell. In one embodiment, the lymphocyte is a natural killer (NK) cell. In one embodiment, the cell further expresses a therapeutic protein. In some embodiments, the therapeutic protein is interleukin 2 (IL-2), IL-2 mutein, interleukin 15 (IL-15), CD40 ligand (CD40L), interleukin 33 (IL-33), interleukin 12 (IL-12), a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PD-L1) inhibitor, a T cell immunoreceptor with Ig and ITIM domains (TIGIT inhibitor), or a combination thereof.

[0025] Provided herein is a cell expressing an antigen-binding protein or antigen-binding protein fragment thereof disclosed herein, optionally wherein the cell is isolated. In some embodiments, the cell is a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK) cell, optionally a HEK293 or HEK293T cell.

[0026] Provided herein is a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding protein fragment thereof disclosed herein, optionally wherein the nucleic acid or set of nucleic acids is isolated.

[0027] Provided herein is a vector or set of vectors comprising a nucleic acid or set of nucleic acids disclosed herein, optionally wherein the vector or set of vectors is isolated.

[0028] Provided herein is a pharmaceutical composition comprising (a) an antigen-binding protein or antigen-binding protein fragment thereof disclosed herein, a cell disclosed herein, a nucleic acid or set of nucleic acids disclosed herein, or a vector or set of vectors disclosed herein and (b) a pharmaceutically acceptable excipient.

[0029] Provided is a method of producing an antigen-binding protein or antigen-binding protein fragment thereof disclosed herein, the method comprising culturing a cell disclosed herein under conditions wherein the antigen-binding protein or antigen-binding protein fragment thereof is produced by the cell.

[0030] Provided is a method of increasing the cytotoxic activity of a lymphocyte against tumor cells expressing New York esophageal squamous cell carcinoma 1 (NY-ESO-1), the method comprising genetically modifying the lymphocyte to express an antigen-binding protein or antigen-binding protein fragment thereof disclosed herein.

[0031] Provided is a method of increasing cytokine secretion by a lymphocyte in the presence of tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express an antigen-binding protein or antigen-binding protein fragment thereof disclosed herein. Provided is a method of increasing the effector functions of a lymphocyte in the presence of tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express an antigen-binding protein or antigen-binding 5183465138.1protein fragment thereof disclosed herein. In one embodiment, the lymphocyte is a CD4+T cell. In one embodiment, the lymphocyte is a CD8+T cell. In one embodiment, the lymphocyte is an NK cell.

[0032] Provided is a method of reducing tumor growth in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject a cell disclosed herein, or a pharmaceutical composition comprising (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0033] Also provided is a cell disclosed herein for use in a method of reducing tumor growth in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1. Also provided is a pharmaceutical composition for use in a method of reducing tumor growth in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, wherein the pharmaceutical composition comprises (a) a cell disclosed herein and (b) pharmaceutically acceptable excipient.

[0034] Provided is a method of reducing cancer sternness in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject a cell disclosed herein, or a pharmaceutical composition comprising (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0035] Also provided is a cell disclosed herein for use in a method of reducing cancer sternness in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1. Also provided is a pharmaceutical composition for use in a method of reducing cancer sternness in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, and wherein the pharmaceutical composition comprises (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0036] Provided is a method of reducing tumor-associated fibrosis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject a cell disclosed herein, or a pharmaceutical composition comprising (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0037] Also provided is a cell disclosed herein for use in a method of reducing tumor-associated fibrosis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1. Also provided is a pharmaceutical composition for use in a method of reducing tumor-associated fibrosis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, and wherein the pharmaceutical composition comprises (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.6183465138.1

[0038] Provided is a method of reducing tumor metastasis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject a cell disclosed herein, or a pharmaceutical composition comprising (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0039] Also provided is a cell disclosed herein for use in a method of reducing tumor metastasis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1. Also provided is a pharmaceutical composition for use in a method of reducing tumor metastasis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, and wherein the pharmaceutical composition comprises (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0040] Provided is a method of reducing T cell exhaustion in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject a cell disclosed herein, or a pharmaceutical composition comprising (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0041] Also provided is a cell disclosed herein for use in a method of reducing T cell exhaustion in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1. Also provided is a pharmaceutical composition for use in a method of reducing T cell exhaustion in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, and wherein the pharmaceutical composition comprises (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0042] Provided is a method of increasing anti -tumor immunity in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject a cell disclosed herein, or a pharmaceutical composition comprising (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0043] Also provided is a cell disclosed herein for use in a method of increasing anti-tumor immunity in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1. Also provided is a pharmaceutical composition for use in a method of increasing antitumor immunity in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, and wherein the pharmaceutical composition comprises (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0044] Provided is a method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express New York esophageal squamous cell carcinoma 1 (NY-ESO-1), the method comprising administering to the subject a cell disclosed herein, or a7183465138.1pharmaceutical composition comprising (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0045] Also provided is a cell disclosed herein for use in a method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express New York esophageal squamous cell carcinoma 1 (NY-ESO-1). Also provided is a pharmaceutical composition for use in a method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express New York esophageal squamous cell carcinoma 1 (NY-ESO-1), and wherein the pharmaceutical composition comprises (a) a cell disclosed herein and (b) a pharmaceutically acceptable excipient.

[0046] Provided is a method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express NY-ESO-1, the method comprising:(a) isolating a population of immune cells from the subject or a donor, producing an isolated population of immune cells;(b) genetically modifying the isolated population of immune cells to express an antigen-binding protein or antigen-binding fragment thereof disclosed herein to produce a genetically modified population of immune cells;(c) optionally, expanding the genetically modified population of immune cells, producing an expanded population of immune cells; and(d) administering the expanded population of immune cells to the subject.

[0047] Also provided is a population of immune cells for use in a method of treating cancer in a subject in need thereof, the method comprising administering the population of immune cells to the subject, wherein at least some of the cells of the cancer express NY-ESO-1, and wherein the population of immune cells have been prepared by a method comprising:(a) isolating a population of immune cells from the subject or a donor, producing an isolated population of immune cells;(b) genetically modifying the isolated population of immune cells to express an antigenbinding protein or antigen-binding fragment thereof disclosed herein to produce a genetically modified population of immune cells; and(c) optionally, expanding the genetically modified population of immune cells, producing an expanded population of immune cells.

[0048] In one embodiment, the immune cells are lymphocytes. In one embodiment, the immune cells are innate lymphoid cells (ILCs). In one embodiment, the lymphocytes are CD4+T cells. In one embodiment, the lymphocytes are CD8+T cells. In one embodiment, the8183465138.1lymphocytes are NK cells. The method may further comprise expanding the isolated population of immune cells after step (a).

[0049] In some embodiments, the subject has one or more cancers selected from the group consisting of adrenal gland tumors, biliary cancer, bladder cancer, brain cancer, breast cancer, carcinoma, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer or hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Li-Fraumeni tumors, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumors, ovarian cancer, pancreatic cancer, pancreatic islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, urogenital cancer, and uterine cancer.

[0050] In one embodiment, the subject has a solid tumor.

[0051] In one embodiment, the method further comprises administering to the subject one or more additional therapeutic agents. The additional therapeutic agent(s) may be selected from the group consisting of taxotere, carboplatin, trastuzumab, epirubicin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, etoposide, 5-FU, gemcitabine, methotrexate, and paclitaxel, mitoxantrone, epothilone B, epidermal-growth factor receptor (EGFR)-targeting monoclonal antibody 7A7.27, vorinostat, romidepsin, docosahexaenoic acid, bortezomib, shikonin, an oncolytic virus, and a combinations thereof. The additional therapeutic agent(s) may be selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and a combinations thereof. The additional therapeutic agent(s) may be immune checkpoint inhibitor(s). In one embodiment, the immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is PD1 inhibitor, a PD-L1 inhibitor, a CD28 inhibitor, a Cytotoxic T Lymphocyte Associated Protein 4 (CTLA4) inhibitor, TIGIT inhibitor, T cell immunoglobulin and mucin-domain containing 3 (TIM3) inhibitor, or a combination thereof.

[0052] In one embodiment the subject is a human.

[0053] Provided is a method of detecting a cancer cell expressing NY-ESO-1 in a biological sample, the method comprising:(a) contacting the biological sample with an antigen-binding protein or antigen-binding fragment thereof disclosed herein; and9183465138.1(b) detecting binding of the antigen-binding protein or antigen-binding fragment thereof to the cancer cell.BRIEF DESCRIPTION OF THE FIGURES

[0054] Figs. 1A and IB illustrate the detection of antigen-specific CD4+T cells using the pMHCII multimer technology. Fig. 1A. Representative dot plots of pMHCII multimer staining of CD4+T cells specific for NY-ESO-1123-137, I1TERT916-930 and Melan-A94-io8 in Peripheral Blood Mononuclear Cells (PBMCs) and Tumor-Infiltrating Lymphocytes (TILs) / tumor infiltrating lymph nodes (TILNs) of HLA-DRB3*02:02+ / ' patients’ and healtyh donors’ (HDs’) samples. Fig. IB. Graph summarizing the antigen-specific CD4+T cell frequencies in PBMCs and TILs / TILNs from melanoma patients (n=8) and HDs (n=3).

[0055] Figs 2A, 2B, 2C, and 2D illustrate the cytotoxic potency of diverse tumor antigen-specific CD4+T cells Fig. 2A. Representative example of the T cell specific lysis as assessed by an lactate dehydrogenase (LDH) cytotoxic assay. Tumor cell lines: Class II Major Histocompatibility Complex Transactivator (CIITA)-transduced HLA-DRB3*02:02+T333A tumor cell line, HLA-DRB3*02:02+Me252 tumor cell line, and HLA-DRB3*02:02‘ GEFI tumor cell line. T cells: NY-ESO-1123-137, 11TERT916-930 and Melan-A94-io8-specific CD4+T cell clones. Top (traces from top to bottom): Me252 DRB3 *02:02 positive tumor cell line; T33A DRB3 *02:02 positive tumor cell line; GEFI DRB3 *02:02 negative tumor cell line. Lower left (traces from top to bottom): T33A DRB3 *02:02 positive tumor cell line; Me252 DRB3 *02:02 positive tumor cell line; GEFI DRB3 *02:02 negative tumor cell line. Lower right (traces from top to bottom): Me252 DRB3 *02:02 positive tumor cell line; T33A DRB3 *02:02 positive tumor cell line; GEFI DRB3*02:02 negative tumor cell line. Fig. 2B. Left panel: Cumulative analysis of LDH cytotoxic assays conducted with tumor cell lines transduced with CIITA cocultured with NY-ESO-1123-137 (n=33), I1TERT916-930 (n=l 1) and Melan-A94-io8-specific CD4+T cell clones (n=14) (E:T Ratio 30: 1). Cell types as in legend. Right panel: Cumulative analysis of LDH cytotoxic assays conducted with tumor cell lines transduced with CIITA co-cultured with (from left to right): (1) NY-ESO-1123-137-specific CD4+T cell clones from PBMC (n=22), (2) TILs (n=7), (3) TILN (n=4), and (4) PBMC from healthy donors (E:T Ratio 30:1). Statistical power was assessed using One-Way ANOVA. Fig. 2C. Cumulative analysis of LDH cytotoxic assays conducted with CIITA-transduced and untransduced tumor cells, respectively, co-cultured with NY-ESO-1123-137-specific CD4+T cell clones (n=16) (E:T Ratio 30:1). Statistical power was assessed using t test. Fig. 2D. Cumulative data of maximum values of IFN-y, TNF-a, IL-4, IL-5, IL-6, IL-9, IL-17A, IL-17F, IL-10, IL-22 and IL-13 secretion 10183465138.1obtained in peptide titration experiments using NY-ESO-li23-i37 / DRB3*02:02 CD4+T cell clones isolated from PBMC / TIL / TILN and analyzed by LegendPlex. Bars from left to right: PBMC; TIL; TILN.

[0056] Figs. 3A, 3B, 3C, and 3D illustrate the phenotypic characterization of diverse tumor antigen-specific CD4+T cells. Fig. 3A. Graphs summarizing PD-1, TIGIT, CD57 and V-domain Ig suppressor of T cell activation (VISTA) expression in NY-ESO-1123-137 PBMC (n=16) and TIL / TILN (n=ll), I1TERT916-930 PBMC (n=8) and Melan-A94-io8 PBMC (n=4)-specific CD4+T cell clones. Statistical power was assessed using One-Way ANOVA. Fig. 3B.Graphs summarizing Granzyme B (GZMB), Signaling Lymphocytic Activation Molecule Family member 7 (SLAMF7), 0X40, 4-1BB and CTLA-4 expression in NY-ESO-1123-137 PBMC (n=16) and TIL / TILN (n=l 1), I1TERT916-930 PBMC (n=8) and Melan-A94-io8 PBMC (n=4)-specific CD4+T cell clones. Statistical power was assessed using One-Way ANOVA.Fig. 3C. Cumulative analysis of the expression of Perforin-1 (PRF1), Granzyme K (GZMK), and Granzyme A (GZMA) by NY-ESO-1123-137 / hTERT9i6-93o / Melan-A94-io4-specific CD4+T cells from PBMC and TIL / TILN analyzed by flow cytometry. Fig. 3D. Graphs summarizing the correlation between the percentage of specific lysis of NY-ESO-1123-137-specific CD4+T cell clones and GZMB, SLAMF7, 4 IBB, 0X40 and CTLA-4 (n=27). Statistical power was assessed using simple linear regression.

[0057] Figs. 4A, 4B, 4C, 4D, 4E, 4F, and 4G illustrate the highly conserved TCR Va and VP usage in NY-ESO-1 i23-i37 / DRB3*02:02-specific CD4+T cells. Fig. 4A. Pie charts illustrating the relative abundance of each clonotype among all NY-ESO-1 i23-i37 / DRB3*02:02 CD4+T cell clones in melanoma patients’ PBMC (n=20), TIL / TILN (n=19) and in HDs’ PBMC (n=7), and in Melan-A94-io8 (n=9) and hTERT9i6-93o / DRB3*O2:O2 (n=9) CD4+T cell clones. Different segments of the pie charts are shown (starting from 12 o’clock) clockwise as shown in the figure legend from top to bottom. Figs. 4B, 4C, and 4D. Pie charts illustrating the distribution of each clonotype across individual patients (LAU53 (Fig.4B), LAU333 (Fig.4C), and LAU1286 (Fig. 4D)) in NY-ESO-1123-137 / DRB3 *02:02 CD4+T cell clones (top), Melan-A94-io8 / DRB3*02:02 CD4+T cell clones (middle), and hTERT9i6-93o / DRB3*O2:O2 CD4+T cell clones (bottom) in melanoma patients. Different segments of the pie charts are shown (starting from 12 o’clock) clockwise as shown in the figure legend from top to bottom. Fig. 4E. Pie charts summarizing TCR sequencing of the alpha and beta chains of multimer positive (left pies) or multimer negative (right pies) CD4+T cells sorted from in vitro expanded PBMC and TIL / TILN from patients (n=9). Different segments of the pie charts are shown (starting from 12 o’clock) clockwise as shown in the figure legend from top to bottom Fig. 4F. Pie charts 11183465138.1summarizing TCR sequencing of the alpha and beta chains of memory CD4+T cells isolated from adult blood (n=4). Different segments of the pie charts are shown (starting from 12 o’clock) clockwise as shown in the figure legend from top to bottom Fig 4G. Pie charts summarizing TCR sequencing of the alpha and beta chains of naive CD4+T cells isolated from cord blood (n=3). Different segments of the pie charts are shown (starting from 12 o’clock) clockwise as shown in the figure legend from top to bottom.

[0058] Figs. 5A, 5B, 5C, and 5D illustrate detection of NY-ESO-li23i37-specific CD4+T cells in other tumor types. Representative dot plot and pies summarizing TCR alpha and beta chains of multimer positive CD4+T cells after IVS with the NY-ESO-1123-137 peptide in (Fig. 5A) lung cancer patient samples (n=5), (Fig. 5B) ovarian cancer patient samples (n=3), (Fig. 5C) neuroblastoma pediatric cancer patient samples (n=3), and (Fig. 5D) healthy donor samples. Different segments of the pie charts are shown (starting from 12 o’clock) clockwise as shown in the figure legend from top to bottom

[0059] Figs. 6A, 6B, 6C, and 6D illustrate TCR transduction in human primary CD4+T cells. Fig. 6A. Cumulative analysis of TCR transduction in CD4+(n=12) and CD8+(n=4) T cells with or without CRISPR-editing. Statistical power was assessed using One-Way ANOVA. Fig. 6B. Cumulative analysis of percentage of live cells in TCR-transduced CD4+(n=12) or CD8+(n=4) T cells with or without CRISPR-editing. Statistical power was assessed using One-Way ANOVA. Fig.6C. Cytotoxic capacity of TCR-transduced CD4+T cells against different HLA-matched and mismatched tumor cell lines (30:1 E:T ratio) (n=4). Traces from top to bottom for 15:1 ratio: U2OS; Me252; T672E; T333A; SH-EP; SKNSH; Na8. Fig. 6D.Cytokine secretion analysis (TNF-a, IFN-y, IL-2, IL-5 and IL-13) by LegendPlex in supernatants of NY-ESO-1123-137 / TCR- transduced CD4+T cells for the higher ratio E:T 30:1 (n=6).

[0060] Figs. 7A, 7B, 7C, and 7D illustrate the adoptive transfer of NY-ESO-l / TCR T cells in tumor-bearing IL-2 -NOG mice. Fig. 7A. Schematic of experimental strategy. Fig. 7B. In vivo efficacy of adoptively transferred NY-ESO-l / TCR-transduced T cells against HLA-matched tumor xenografts (6 mice per group). Statistical power was assessed using Kruskal-Wallis test. Traces from top to bottom (by endpoint): untreated; CD8+; CD4+. Fig. 7C.Representative dot plots of human CD3 staining of PBMC of untreated mice and mice receiving NY-ESO-l / TCR- transduced CD4+T cells or NY-ESO-l / TCR-transduced CD8+T cells. Fig. 7D. Monitoring of the frequency of persisting human T cells by flow cytometry (6 mice per group). Traces from top to bottom (by endpoint): CD8+, CD4+, untreated.12183465138.1DETAILED DESCRIPTION

[0061] Provided herein are antigen-binding proteins or antigen-binding fragments thereof comprising a first variable domain and a second variable domain that bind to an antigen. The antigen may be a fragment of NY-ESO-1. Also provided herein are methods of making and methods of using the antigen-binding proteins or antigen-binding fragments thereof disclosed herein.

[0062] Antigen-binding proteins and antigen-binding fragments thereof disclosed herein demonstrate specific and high-affinity binding to the NY-ESO-1123-137 peptide (LKEFTVSGNILTIRL; SEQ ID NO:43), e.g., presented in the context of MHC class II molecule HLA-DRB3*02:02. As demonstrated in the Examples, T cells expressing the disclosed TCRs exhibit enhanced cytotoxic activity against tumor cells expressing NY-ESO-1, produce elevated levels of effector cytokines such as IFN-y, TNF-a, and IL-2, and demonstrate therapeutic efficacy in tumor models.

[0063] HLA-DRB3*02:02TCR-engineered T cells targeting NY-ESO-1123-137 / HLA-DRB3 *02:02 offer a promising and safe immunotherapeutic approach due to limited antigen expression on healthy tissues, which can be used to treat a relatively large proportion of patients with cancer. As such, the TCRs disclosed herein may serve as “quasi-universal” TCRs particularly useful for the treatment of HLA-DRB3*02:02-positive adult and pediatric patients with cancers expressing NY-ESO-1.

[0064] Antigen-binding proteins and antigen-binding fragments thereof

[0065] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein each variable domain comprises three complementarity determining regions (CDRs). Merely for the sake of convenience, the CDRs of the first variable domain are referred to as CDRal, CDRa2, and CDRa3 and the CDRs of the second variable domain are referred to as CDRbl, CDRb2, and CDRb3. A person skilled in the art will appreciate that “a” and “b” do not denote a specific relative orientation of the CDRs.

[0066] The term “variable chain” and “variable domain” may be used interchangeably.

[0067] As used herein, the term “Complementarity Determining Regions” (CDRs) refers to portions of an antigen-binding protein variable domain that are (typically) involved in antigenbinding. A person skilled in the art can identify readily the CDRs in an antigen-binding protein. In one embodiment, the Kabat numbering system, which focuses on hypervariable regions, is13183465138.1used to identify the CDRs in an antigen-binding protein. See, e.g., Kabat et al., Sequences of Immunoglobulin Chains: Tabulation Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain BP-Microglobulins, Department of Health, Education, Welfare, Public Health Service, National Institutes of Health (1979); Kabat et al., Sequences of Proteins of Immunological Interest, Diane Publishing Company (1992). The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues (primary amino acid sequence). The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or CDR, of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antigen-binding protein thereof by alignment of residues of homology in the sequence of the antigen-binding protein fragment thereof with a “standard” Kabat numbered sequence. In one embodiment, the CDRs are defined using the Chothia numbering system, which focuses on loop topologies. See Chothia & Lesk, Canonical structures for the hypervariable regions of immunoglobulins, J Mol Biol. 1987 Aug 20;196(4):901-17. Alternative methods of identifying CDRs include the methods disclosed in: (1) Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Dev Comp Immunol. 2003 Jan;27(l):55-77); (2) Abhinandan & Martin, Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains, Mol Immunol. (2008) 45:3832-9; (3) Gelfand et al., Algorithmic determination of core positions in the VL and VH domains of immunoglobulin molecules, J Comput Biol. (1998) 5:467-77; (4) Honegger & Pliickthun, Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J Mol Biol. (2001) 309:657-70. See also Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front Immunol. 2018 Oct 16;9:2278.

[0068] The term “framework regions” (FR) refers to those variable domain residues other than the CDR residues.

[0069] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of New York esophageal squamous cell carcinoma 1 (NY-ESO-1), wherein the first variable domain comprises CDRal, CDRa2, and CDRa3, wherein the second variable domain comprises CDRbl, CDRb2, and CDRb3, and wherein one or more of the CDRs are selected from the CDRs in Table 1. The fragment of NY-ESO-1 may comprise SEQ ID NO:43 (LKEFTVSGNILTIRL).14183465138.1Table 1. Selected CDR combinations (amino acid sequences).Name CDRal CDRal CDRa3 CDRbl CDRbl CDRb3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO NO NO NO NO NO TCR1 1 2 4 8 10 12 TCR2 1 2 5 9 11 13 TCR3 1 3 6 8 10 14TCR4 1 2 7 8 10 15

[0070] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein the first variable domain comprises CDRal, CDRa2, and CDRa3, wherein the second variable domain comprises CDRbl, CDRb2, and CDRb3, and wherein:(a) CDRal comprises SEQ ID NO:1;(b) CDRa2 comprises SEQ ID NO:2 or SEQ ID NO:3;(c) CDRa3 comprises any one of SEQ ID NOs:4-7;(d) CDRbl comprises SEQ ID NO:8 or SEQ ID NO:9;(e) CDRb2 comprises SEQ ID NO: 10 or SEQ ID NO: 11; and(f) CDRb3 comprises any one of SEQ ID NOs: 12-15.

[0071] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein the first variable domain comprises CDRal, CDRa2, and CDRa3, wherein the second variable domain comprises CDRbl, CDRb2, and CDRb3, and wherein:(a) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NON; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 12;(b) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:5; CDRbl comprises SEQ ID NO:9; CDRb2 comprises SEQ ID NO: 11; and CDRb3 comprises SEQ ID NO: 13;(c) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NON; CDRa3 comprises SEQ ID NO:6; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 14; or15183465138.1(d) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:7; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 15.

[0072] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein the first variable domain comprises CDRal, CDRa2, and CDRa3, wherein the second variable domain comprises CDRbl, CDRb2, and CDRb3, and wherein the antigen-binding protein or antigen-binding fragment thereof comprises the CDRs of any of the TCRs shown in Table 14 and Table 15. CDR3 sequences are provided in Table 15. A person of ordinary skill in the art can easily derive the sequences for the CDR1 and CDR2 sequences from the V segments provided in Table 14.

[0073] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of human telomerase reverse transcriptase (hTERT), wherein the first variable domain comprises CDRal, CDRa2, and CDRa3, wherein the second variable domain comprises CDRbl, CDRb2, and CDRb3, and wherein the antigen-binding protein or antigen-binding fragment thereof comprises the CDRs of any of the TCRs shown in Table 16 and Table 17. CDR3 sequences are provided in Table 17. A person of ordinary skill in the art can easily derive the sequences for the CDR1 and CDR2 sequences from the V segments provided in Table 16.

[0074] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of Melan-A, wherein the first variable domain comprises CDRal, CDRa2, and CDRa3, wherein the second variable domain comprises CDRbl, CDRb2, and CDRb3, and wherein the antigen-binding protein or antigenbinding fragment thereof comprises the CDRs of any of the TCRs shown in Table 18 and Table 19. CDR3 sequences are provided in Table 19. A person of ordinary skill in the art can easily derive the sequences for the CDR1 and CDR2 sequences from the V segments provided in Table 18.

[0075] Provided herein are also antigen-binding proteins and antigen-binding fragments thereof that comprise CDR and / or variable domain sequences that have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 16183465138.195%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of the sequences disclosed herein.

[0076] As used herein, the term “identity” or "percent identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. For example, when a position in the compared nucleotide sequence is occupied by the same base, then the molecules are identical at that position. A degree of identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. Variants having the recited percent identity to specific sequences described herein preferably at least partially retain the antigen-binding specificity and biological activity of the parent sequence. For example, polypeptides having at least 85%, 90%, 95%, 98%, or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same antigen-binding functions, as well as polynucleotides encoding such polypeptides, are contemplated. Methods and computer programs for determining both sequence identity and similarity are publicly available, including, but not limited to, the GCG program package (Devereux et al., A comprehensive set of sequence analysis programs for the VAX, Nucleic Acids Res. 1984 Jan 11; 12(1 Pt l):387-95), BLASTP, BLASTN, FASTA (Altschul et al., Basic local alignment search tool, J Mol Biol. 1990 Oct 5;215(3):403-10), and the ALIGN program (version 2.0). The well-known Smith Waterman algorithm may also be used to determine similarity. The BLAST program is publicly available from NCBI and other sources (BLAST Manual., Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at http: / / www.ncbi.nlm.nih.gov / blast / ). In comparing sequences, these methods account for various substitutions, deletions, and other modifications.

[0077] T cell receptors (TCRs) or antigen-binding fragments thereof

[0078] In one embodiment, the antigen-binding protein is a TCR or an antigen-binding fragment thereof.

[0079] As used herein, the term “T cell receptor” or “TCR” refers to a heterodimer comprising a first variable domain and a second variable domain. In one embodiment, the TCR comprises a variable alpha and a variable beta chain. In one embodiment, the TCR comprises a variable gamma chain and a variable delta chain. TCR variable chains usually comprise a variable region, a constant region, a transmembrane region, and a short cytoplasmic tail. Each variable region of the TCR usually has three hypervariable regions (CDRs), facilitating recognition of a cognate antigen. Alpha / beta T cells, which recognize peptides from degraded proteins bound to major histocompatibility complex (MHC) molecules at the cell surface, are 17183465138.1significantly more abundant as compared to gamma / delta T cells. The latter are not MHC-restricted and do not appear to recognize peptide antigens. The terms “antigen-binding portion” or “antigen-binding fragment” as used herein may refer to a region on a TCR that binds to its antigen. TCRs disclosed do not necessarily comprise all elements of a classic TCR (for example, the TCR may be lacking the constant region, the transmembrane region, and / or the cytoplasmic tail).

[0080] The TCR may be bound to the membrane / surface of a cell. The TCR may be soluble.

[0081] A “TCR complex,” as used herein, comprises the TCR and additional signaling molecules that are not variable chains. Preferably, the TCR complex comprises six CD3 signaling modules (2x CD3 epsilon, CD3 delta, CD3 gamma, and 2x CD3 zeta), which form three CD3 dimers: CD3 epsilon / gamma, CD3 epsilon / delta, and CD3 zeta / zeta.

[0082] The variable TCR chains allow recognition of peptide fragments that have been generated by proteolytic degradation of foreign or self-proteins within cells expressing MHC class I or class II molecules. The peptides are usually presented to the TCR by the MHC molecules located on antigen-presenting cells (APCs). When the TCR engages with an antigenic peptide and MHC (together, a peptide / MHC complex or an antigen / MHC complex), the T cell is activated through signal transduction, mediated by the CD3 subunits in the TCR complex. Antigen / MHC complexes are, however, not the only molecules capable of interaction with TCRs. Non-peptide antigens such as lipids can interact with TCRs via some of the five isoforms of CD1 (a-e) (Mori & Libero, T cells specific for lipid antigens, Immunol Res. 2012 Sep;53(l-3): 191-9, incorporated herein by reference in its entirety). Further, several studies describe TCRs binding to metabolic intermediates bound to the MHC like molecule MR1 (Reantragoon et al., Structural insight into MRl-mediated recognition of the mucosal associated invariant T cell receptor, J Exp Med. 2012 Apr 9;209(4):761-74, incorporated herein by reference in its entirety).

[0083] TCR diversity arises through somatic gene rearrangement, which creates a range of receptors from limited genes. At the TCR-a locus (tra), discrete variable (V) and junctional (J) gene segments are recombined and joined with a constant (C) segment. Recombination at the TCR-b locus (trb) locus occurs in a similar fashion but includes adding a diversity (D) segment and choosing one of two C segments. TCR variability is mainly in the three CDR loops on each chain, which form the antigen-binding site at the TCR’s membrane-distal end. The variable CDR1 and 2 are encoded in the germline by the T cell receptor alpha variable (TRAV) and T cell receptor beta variable (TRBV) gene segments. In contrast, the hypervariable CDR3 loops are generated by random deletion and addition of template and non-template nucleotides at the 18183465138.1junction between recombining V, (D) and J gene segments. Theoretically, gene rearrangement utilizing V-(D)-J recombination can produce about 1018different TCRs in humans and about 1015different TCRs in the mouse.

[0084] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein the first and / or the second variable domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the sequences in Table 2. Provided is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein the first and / or the second variable domain comprises any of the sequences in Table 2.Table 2. Selected variable chain combinations (amino acid sequences).Name First variable chain Second variable chain SEQ ID NO SEQ ID NO TCR1 16 17TCR2 18 19TCR3 20 21TCR4 22 23

[0085] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein:(a) the first variable domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16 and the second variable domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17;19183465138.1(b) the first variable domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18 and the second variable domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19;(c) the first variable domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20 and the second variable domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21; or(d) the first variable domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22 and the second variable domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23.

[0086] Provided herein is an antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein:(a) the first variable domain comprises SEQ ID NO: 16 and the second variable domain comprises SEQ ID NO: 17;(b) the first variable domain comprises SEQ ID NO: 18 and the second variable domain comprises SEQ ID NO: 19;20183465138.1(c) the first variable domain comprises SEQ ID NO:20 and the second variable domain comprises SEQ ID NO:21; or(d) the first variable domain comprises SEQ ID NO:22 and the second variable domain comprises SEQ ID NO:23.

[0087] In some embodiments, the first and / or the second TCR variable domain is further joined to a constant region, optionally, through a linker. In some embodiments, the first and / or the second TCR variable domain is joined to a constant region comprising a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs:75-77. In some embodiments, the first and / or the second TCR variable domain is joined to a constant region comprising any one of SEQ ID NOs:75-77.

[0088] Provided is an antigen-binding protein or antigen-binding fragment thereof comprising a first domain and a second domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein the first and / or the second domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of the sequences in Table 3. Provided is an antigen-binding protein or antigen-binding fragment thereof comprising a first domain and a second domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein the first and / or the second domain comprises any of the sequences in Table 3.Table 3. Selected combinations of TCR chains comprising a variable and a constant region.Name First chain Second chainSEQ ID NO SEQ ID NO TCR1 24 25TCR2 26 27TCR3 28 29TCR4 30 31

[0089] Provided is an antigen-binding protein or antigen-binding fragment thereof comprising a first domain and a second domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein:21183465138.1(a) the first domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24 and the second domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:25; (b) the first domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:26 and the second domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:27; (c) the first domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:28 and the second domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:29; or (d) the first domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:30 and the second domain comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 31.22183465138.1

[0090] Provided is an antigen-binding protein or antigen-binding fragment thereof comprising a first domain and a second domain, wherein the antigen-binding protein or antigen-binding fragment thereof binds to a fragment of NY-ESO-1, wherein:(a) the first domain comprises SEQ ID NO:24 and the second domain comprises SEQ ID NO:25;(b) the first domain comprises SEQ ID NO:26 and the second domain comprises SEQ ID NO:27;(c) the first domain comprises SEQ ID NO:28 and the second domain comprises SEQ ID NO:29; or(d) the first domain comprises SEQ ID NO:30 and the second domain comprises SEQ ID NO:31.

[0091] In one embodiment, the TCR is a single chain TCR. In a “single chain TCR,” the two variable domains, e.g., the V-alpha and the V-beta chains, are covalently linked through a suitable linker. The linker may be a polypeptide linker. In one embodiment, the TCR V-a chain is covalently linked to the V-0 chain through a suitable linker sequence fused to the C-terminus of the V-a chain and the N-terminus of the V-0 chain. In another embodiment, the orientation of the chains is reversed.

[0092] In some embodiments, the antigen-binding protein or antigen-binding fragment thereof comprises one or more amino acid modifications that enhance stability, reduce immunogenicity, or improve expression. Such modifications may include, but are not limited to, substitution of residues prone to oxidation, deamidation, or isomerization; introduction of disulfide bonds to stabilize the structure; or modification of potential T cell epitopes to reduce immunogenicity. In some embodiments, the TCR comprises modifications in the constant region to enhance pairing of the introduced TCR chains and reduce mispairing with endogenous TCR chains, such as introduction of an additional disulfide bond between the constant regions.

[0093] Antibodies and antigen-binding fragments thereof

[0094] In one embodiment, the antigen-binding protein or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof.

[0095] The term “antibody” is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and antigen-binding fragments thereof (e.g., paratopes, CDRs), so long as they exhibit the desired biological activity and specificity. The terms “antigen-binding portion” or 23183465138.1“antigen-binding fragment” as used herein may refer to a region on an antibody that binds to its antigen.

[0096] In one embodiment, the antibody fragment is a Fab fragment, which comprises or consists essentially of a variable (VL) and constant (CL) domain of the light chain and a variable domain (VH) and the first constant domain (CHI) of the heavy chain.

[0097] In one embodiment, the antibody fragment is a Fab’ fragment, which refers to a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain.

[0098] In one embodiment, the antibody fragment is an Fd fragment comprising or consisting essentially of VH and CHI domains.

[0099] In some embodiments of the aspects described herein, the antibody portion is an Fd’ fragment comprising VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain.

[0100] Single-chain Fv or scFv antibody fragments comprise or consist essentially of the VH and VL domains of antibody, such that these domains are present in a single polypeptide chain. Generally, an Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen-binding. See, e.g., Pluckthun, 113 Pharmacology Monoclonal Antibodies 269 (Rosenburg & Moore, eds., Springer-Verlag, New York, 1994). Accordingly, in some embodiments of the aspects described herein, the antibody fragment is a Fv fragment comprising or consisting essentially of the VL and VH domains of a single arm of an antibody.

[0101] In one embodiment, the antibody portion is a diabody comprising two antigenbinding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain.

[0102] In one embodiment, the antibody portion is a dAb fragment comprising or consisting essentially of a VH domain.

[0103] In one embodiment, the antibody portion is a F(ab’)2 fragment, which comprises a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region.

[0104] Linear antibodies refer to the antibodies as described in Zapata et al., Engineering linear F(ab’)2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity, Protein Eng. 1995 Oct;8(10): 1057-62. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. In some embodiments of the aspects described 24183465138.1herein, the antibody fragment is a linear antibody comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions.

[0105] Various techniques have been developed and are available for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies. See, e.g., Morimoto & Inouye K, Single-step purification of F(ab’)2 fragments of mouse monoclonal antibodies (immunoglobulins Gl) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW, J Biochem Biophys Methods. 1992 Mar;24(l-2): 107-17; Brennan et al., Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin Gl fragments, Science. 1985 Jul 5;229(4708):81-3. However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab’-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab’)2 fragments. According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody fragment of choice is a single chain Fv fragment (scFv). See, e.g., PCT Publication No. WO1993016185 entitled “Biosynthetic binding protein for cancer marker”.

[0106] Contemplated antibodies or antigen-binding fragments may have any of constant regions, including IgAl, IgA2, IgM, IgG, IgD, and IgE, and any isotype, including IgGl, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgGl is used. In one embodiment, the human isotype IgG4 is used. Light chain constant regions can be X or K. The antibody or antigen-binding fragment thereof may comprise sequences from more than one class or isotype.

[0107] Purification of antigen-binding proteins or antigen-binding fragments thereof

[0108] In some embodiments, the antigen-binding protein or antigen-binding fragment thereof is an isolated antigen-binding protein or antigen-binding fragment thereof. The terms “purified” or “isolated” antigen-binding protein, peptide, polypeptide, or protein refer to a antigen-binding protein, peptide, polypeptide, or protein that has been separated from other proteins, lipids, and / or nucleic acids with which it may be naturally associated. The polypeptide / protein can constitute at least 10% (i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0109] Modifications of antigen-binding proteins and antigen-binding fragments thereof 25183465138.1

[0110] In some embodiments, amino acid sequence modification(s) of the antigen-binding proteins or antigen-binding fragments thereof described herein are contemplated. Amino acid sequence variants of the antigen-binding protein or antigen-binding fragment thereof can be prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antigen-binding protein or antigen-binding fragment thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequences of the antigen-binding protein or antigen-binding fragment thereof. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., with respect to binding specificity, binding affinity, biological activity, etc.[OHl] One type of antigen-binding protein variant is a conservative amino acid substitution variant. These variants have at least one amino acid residue in the antigen-binding protein or antigen-binding fragment thereof replaced by a different residue that has similar side chain properties. Amino acids can be grouped according to similarities in the properties of their side chains (see Lehninger, BIOCHEMISTRY (2nded., Worth Publishers, New York, 1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M);(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q);(3) acidic: Asp (D), Glu (E);(4) basic: Lys (K), Arg (R), His (H).

[0112] As such, a non-limiting example for a conservative amino acid substitution is one that replaces a non-polar amino acid with another non-polar amino acid.

[0113] Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties:(1) hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M);(2) neutral hydrophilic: Ser (S), Thr (T), Cys (C), Asn (N), Gin (Q);(3) acidic: Asp (D), Glu (E);(4) basic: Lys (K), Arg (R), His (H);(5) residues that influence chain orientation: Gly (G), Pro (P);(6) aromatic: Phe (F), Trp (W), Tyr (Y).

[0114] As such, a non-limiting example for a conservative amino acid substitution is one that replaces a hydrophobic amino acid with another hydrophobic amino acid.

[0115] In some embodiments, one or more of the CDRs of an antigen-binding protein or antigen-binding fragment thereof disclosed herein have a conservative amino acid substitution.26183465138.1

[0116] Provided herein is an antigen-binding protein or antigen-binding fragment thereof that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions as compared to another antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0117] Provided herein is an antigen-binding protein or antigen-binding fragment thereof that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to another antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0118] Further contemplated are amino acid sequence insertions, which can include amino-and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a dozen or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0119] Any cysteine residue not involved in maintaining the proper conformation of the antigen-binding protein or antigen-binding fragments thereof also can be substituted, for example with a serine or an alanine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0120] Conversely, cysteine bond(s) can be added to the antigen-binding protein or antigenbinding fragment thereof to improve its stability (particularly where the antigen-binding protein or antigen-binding fragment thereof is an antibody fragment such as an Fv fragment).

[0121] In some embodiments, the antigen-binding protein or antigen-binding fragment thereof has one or more amino acid alterations that alter the original glycosylation pattern of the antigen-binding protein or antigen-binding fragment thereof. By “altering the original glycosylation pattern” is meant deleting one or more carbohydrate moieties found in the antigen-binding protein or antigen-binding fragment thereof, and / or adding one or more glycosylation sites that are not present in the antibody or antigen-binding fragment thereof disclosed herein. Glycosylation of antigen-binding proteins is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Addition of glycosylation sites to the antigen-binding proteins or antigen-binding fragments thereof is accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide 27183465138.1sequences (for N-linked glycosylation sites). The alteration can also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody or antigen-binding fragment thereof (for O-linked glycosylation sites).

[0122] In some embodiments, the antigen-binding proteins or antigen-binding fragments thereof provided herein are deglycosylated or aglycosylated.

[0123] Where the antigen-binding protein or antigen-binding fragment thereof comprises an Fc region, the carbohydrate(s) attached thereto can be altered. For example, antibodies with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody or antigen-binding fragment thereof have been described. See, e.g., U.S. Patent Publication No.2003 / 0157108 (entitled “Glycoprotein compositions”; U.S. Patent Publication No.2004 / 0093621 (entitled “Antibody composition which specifically binds to CD20”). Antibodies with a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody or antigen-binding fragment thereof are referenced in PCT Publication No. W02003 / 011878 (entitled “Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity”); U.S. Patent No. 6,602,684 (entitled “Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity”). Antibodies with at least one galactose residue in the oligosaccharide attached to an Fc region of the antibody or antigen-binding fragment thereof are reported in PCT Publication No. WO1997 / 30087 (entitled “Preparation of glycosylated antibodies”). See also PCT Publication Nos. WO1998 / 58964 (entitled “Methods and compositions for galactosylated glycoproteins”) and WO 1999 / 22764 (entitled “Methods and compositions comprising glycoprotein glycoforms”) concerning antibodies with altered carbohydrate attached to the Fc region thereof.

[0124] The contemplated antigen-binding proteins and antigen-binding fragments thereof also feature humanized frameworks for reduced immunogenicity. In some embodiments, the CDRs of the contemplated antigen-binding protein or antigen-binding fragment thereof are located in frameworks obtained from a human antigen-binding protein or antigen-binding fragment thereof. In other embodiments, surface-exposed framework residues of the contemplated antigen-binding protein or antigen-binding fragment thereof are replaced with framework residues of a human antigen-binding protein or antigen-binding fragment thereof. The CDRs may also be located in rabbit or humanized frameworks linked to human constant regions (i.e., chimeric antigen-binding proteins).

[0125] Techniques for humanization of antigen-binding proteins are known to one of ordinary skill in the art and are generally reviewed in Safdari et al., Antibody humanization 28183465138.1methods - a review and update, Biotechnol Genet Eng Rev. 2013;29:175-86, hereby incorporated by reference in its entirety. Humanization of antigen-binding proteins generally comprises grafting of CDRs (such as the CDRs disclosed herein) or conservative substituted variants thereof into an appropriate human variable region framework, for example, as disclosed in Jones et al., Replacing the complementarity-determining regions in a human antibody with those from a mouse, Nature. 1986 May 29-Jun 4;321(6069):522-5, hereby incorporated by reference in its entirety. Common methods used include, but are not limited to, framework-homology-based humanization, germline humanization, CDR-homology-based humanization and specificity determining residues (SDR) grafting.

[0126] In one embodiment, the CDRs of a contemplated antigen-binding protein or antigenbinding fragment thereof are located in a framework that is a composite of two or more human antibodies. In such embodiments, the contemplated antigen-binding protein or antigenbinding fragment thereof comprise two or more sequence segments (“composites”) derived from V-regions of unrelated human antibodies that are selected to maintain monoclonal antibody sequences important for antigen-binding of the starting precursor antigen-binding protein, and which have all been filtered for the presence of potential T cell epitopes using “in silico tools” (Holgate & Baker, Circumventing immunogenicity in the development of therapeutic antibodies, Idrugs. 2009 Apr; 12(4): 233 -7). The close fit of human sequence segments with all sections of the starting antibody V regions and the elimination of CD4+T cell epitopes prior to synthesis of the antigen-binding protein or antigen-binding fragment thereof allow this technology to circumvent immunogenicity while maintaining optimal affinity and specificity through the prior analysis of sequences necessary for antigenspecificity (Holgate & Baker, 2009).

[0127] Antibodies with improved binding to the neonatal Fc receptor (FcRn), and increased half-lives, are described in PCT Publication No. W02000 / 42072 (entitled “Polypeptide variants with altered effector function”) and U.S. Patent Publication No. 2005 / 0014934 (entitled “Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis”). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. For example, the Fc region can have substitutions at one or more of positions 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428 or 434 (Eu numbering of residues). An Fc region with improved FcRn binding may comprise amino acid substitutions at one, two or three of positions 307, 380 and 434 of the Fc region thereof (Eu numbering of residues). In one embodiment, the antibody or antigen-binding fragment thereof has 307 / 43429183465138.1mutations. Engineered antibodies that bind to FcRn with three or more (e.g., four) functional antigen-binding sites are also contemplated. See, e.g., U.S. Patent Pub. No. US 2002 / 0004587.

[0128] Antibodies binding pMHC are also referred to as TCR-like antibodies (or TCR mimic (TCRm) antibodies by some groups). They combine the ability to target specific pMHC complexes with the favorable properties of antibodies. See, e.g., Hoydahl et al., Targeting the MHC Ligandome by Use of TCR-Like Antibodies, Antibodies (Basel). 2019 May 9;8(2):32.

[0129] Chimeric antigen receptors (CARs)

[0130] In one embodiment, the antigen-binding protein or antigen-binding fragment thereof is a CAR.

[0131] In a “chimeric antigen receptor (CAR),” an antigen-binding domain is fused to an intracellular signaling domain capable of activating an immune cell (e.g., a T cell). A CAR’s extracellular binding domain may be composed of a single chain variable fragment (scFv) derived from fusing the variable heavy and light regions of a murine or humanized monoclonal antibody. Alternatively, scFvs may be used that are derived from Fabs (instead of from an antibody, e.g., obtained from Fab libraries). A CAR may comprise the CDRs of a TCR. The scFv may be fused to a transmembrane domain and then to an intracellular signaling domain. The CAR can be a first-generation, second generation or third-generation CAR. “First-generation” CARs generally include those that provide CD3(^ signals upon antigen binding. “Second-generation” CARs generally include those that provide both co-stimulation (e.g. CD28 or CD 137) and activation (CD3Q. “Third-generation” CARs generally include those that provide multiple co-stimulation (e.g. CD28 and CD137) and activation (CD3Q.

[0132] Heterodimeric fusions

[0133] The antigen-binding proteins or antigen-binding fragments thereof disclosed herein are compatible with the heterodimeric fusion proteins disclosed in PCT Application No. PCT / EP2024 / 088236, filed December 20, 2024, entitled HETERODIMERIC PROTEINS COMPRISING DIMERIZATION MOTIFS AND METHODS OF USING, incorporated herein by reference in its entirety.

[0134] Provided herein is heterodimeric protein comprising a TCR disclosed herein and a dimerization motif. As used herein, “heterodimeric protein” refers to a protein formed by the combination of two different monomeric proteins. Dimerization sequences can facilitate the dimerization of polypeptide sequences that the dimerization sequences are fused to. As used herein, a “dimerization motif’ comprises two subunits (such as a first dimerization domain and a second dimerization domain), which interact with each other and cause the formation of a dimer. In some embodiments, the heterodimeric fusion proteins comprise a dimerization motif 30183465138.1that is derived from the human gamma-aminobutyric acid (GABA)-receptor. See Burmakina et al., Heterodimeric coiled-coil interactions of human GABAB receptor, Proc Natl Acad Sci U S A. 2014 May 13; 11 l(19):6958-63, incorporated herein in its entirety by reference.

[0135] Provided herein is a heterodimeric protein comprising:(a) (1) a first polypeptide comprising (i) a first variable domain as disclosed herein, (ii) optionally, a linker, and (iii) a first dimerization domain comprising a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:78 (STNNNEEEKSRLLEKENRELEKIIAEKEERVSELRHQLQSR) and (2) a second polypeptide comprising (i) a second variable domain as disclosed herein, (ii) optionally, a linker, and (iii) a second dimerization domain comprising a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 79 (SVNQASTSRLEGLQSENHHLRMKITELDKDLEEVTMQLQDT); or(b) (1) a first polypeptide comprising (i) a first variable domain as disclosed herein, (ii) optionally, a linker, and (iii) a first dimerization domain comprising a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:79 and (2) a second polypeptide comprising (i) a second variable domain as disclosed herein, (ii) optionally, a linker, and (iii) a second dimerization domain comprising a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:78.

[0136] Provided herein is a heterodimeric protein comprising:(a) (1) a first polypeptide comprising (i) a first variable domain as disclosed herein and (ii) a first dimerization domain comprising a sequence comprising SEQ ID NO:78 and (2) a second polypeptide comprising (i) a second variable domain as disclosed herein and (ii) a second dimerization domain comprising SEQ ID NO:79; or31183465138.1(b) (1) a first polypeptide comprising (i) a first variable domain as disclosed herein and (ii) a first dimerization domain comprising SEQ ID NO:79 and (2) a second polypeptide comprising (i) a second variable domain as disclosed herein and (ii) a second dimerization domain comprising SEQ ID NO:78.

[0137] The heterodimeric proteins may further comprise an ER-retention motif. In some embodiments, (1) the first polypeptide comprises a sequence that has 1, 2, 3, or 4 amino substitutions as compared to SEQ ID NO:80 (QQLRSRRH) and the second polypeptide comprises a sequence that has 1, 2, 3, or 4 amino substitutions as compared to SEQ ID NO:81 (PEKTTYIK); or (2) the first polypeptide comprises a sequence that has 1, 2, 3, or 4 amino substitutions as compared to SEQ ID NO:81 and the second polypeptide comprises a sequence that has 1, 2, 3, or 4 amino substitutions as compared to SEQ ID NO:80.

[0138] In some embodiments, (1) the first polypeptide comprises SEQ ID NO:80 (QQLRSRRH) and the second polypeptide SEQ ID NO: 81 (PEKTTYIK); or (2) the first polypeptide further comprises SEQ ID NO:81 and the second polypeptide SEQ ID NO:80.

[0139] Conjugates of antigen-binding proteins and antigen-binding fragments thereof

[0140] In some embodiments described herein, the antigen-binding protein or antigenbinding fragment thereof disclosed herein is conjugated to a functional moiety. Examples of useful functional moieties include, but are not limited to, a blocking moiety, a detectable moiety, a diagnostic moiety, a therapeutic moiety, or a moiety for purification.

[0141] Illustrative blocking moieties include moieties of sufficient steric bulk and / or charge such that reduced glycosylation occurs, for example, by blocking the ability of a glycosidase to glycosylate the antigen-binding protein or antigen-binding fragment thereof. The blocking moiety may additionally or alternatively, reduce effector function, for example, by inhibiting the ability of the Fc region to bind a receptor or complement protein. Blocking moieties may include cysteine adducts and PEG moieties.

[0142] In one embodiment, the blocking moiety is a cysteine, preferably a cysteine that has associated with a free cysteine, e.g., during or subsequent to the translation of the Fc containing polypeptide, e.g., in cell culture. Other blocking cysteine adducts include cystine, mixed disulfide adducts, or disulfide linkages.

[0143] In one embodiment, the blocking moiety is a polyalkylene glycol moiety, for example, a PEG moiety and preferably a PEG-maleimide moiety. Pegylation moieties (or related polymers) can be, for example, polyethylene glycol (“PEG”), polypropylene glycol (“PPG”), polyoxyethylated glycerol (“POG”) and other polyoxyethylated polyols, polyvinyl alcohol (“PVA”) and other polyalkylene oxides, polyoxyethylated sorbitol, or 32183465138.1polyoxyethylated glucose. The polymer can be a homopolymer, a random or block copolymer, a terpolymer based on the monomers listed above, straight chain or branched, substituted or unsubstituted as long as it has at least one active sulfone moiety. The polymeric portion can be of any length or molecular weight, but these characteristics can affect the biological properties. Polymer average molecular weights particularly useful for decreasing clearance rates in pharmaceutical applications are in the range of 2,000 to 35,000 Daltons. In addition, if two groups are linked to the polymer, one at each end, the length of the polymer can impact upon the effective distance, and other spatial relationships, between the two groups. Thus, one skilled in the art can vary the length of the polymer to optimize or confer the desired biological activity. PEG is useful in biological applications for several reasons. PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze, and is nontoxic. Pegylation can improve pharmacokinetic performance of a molecule by increasing the molecule’ s apparent molecular weight. The increased apparent molecular weight reduces the rate of clearance from the body following subcutaneous or systemic administration. In many cases, pegylation can decrease antigenicity and immunogenicity. In addition, pegylation can increase the solubility of a biologically active molecule.

[0144] Examples of moieties useful for purification (and / or for detection) include, but are not limited to, Albumin-binding protein (ABP), Alkaline Phosphatase (AP), AU1 epitope, AU5 epitope, Bacteriophage T7 epitope (T7-tag), Bacteriophage V5 epitope (V5-tag), Biotincarboxy carrier protein (BCCP), Bluetongue virus tag (B-tag), Calmodulin binding peptide (CBP), Chloramphenicol Acetyl Transferase (CAT), Cellulose binding domain (CBP), Chitin binding domain (CBD), Choline-binding domain (CBD), Dihydrofolate reductase (DHFR), E2 epitope, FLAG epitope, Galactose-binding protein (GBP), Green fluorescent protein (GFP), Glu-Glu (EE-tag), Glutathione S-transferase (GST), Human influenza hemagglutinin (HA), HaloTag®, Histidine affinity tag (HAT), Horseradish Peroxidase (HRP), HSV epitope, Ketosteroid isomerase (KSI), KT3 epitope, LacZ, Luciferase, Maltose-binding protein (MBP), Myc epitope, NusA, PDZ domain, PDZ ligand, Polyarginine (Arg-tag), Polyaspartate (Asp-tag), Polycysteine (Cys-tag), Polyhistidine (His-tag), Polyphenylalanine (Phe-tag), Profinity eXact, Protein C, SI -tag, S-tag, Streptavadin-binding peptide (SBP), Staphylococcal protein A (Protein A), Staphylococcal protein G (Protein G), Strep-tag, Streptavadin, Small Ubiquitin-like Modifier (SUMO), Tandem Affinity Purification (TAP), T7 epitope, Thioredoxin (Trx), TrpE, Ubiquitin, Universal, and VSV-G.

[0145] Examples of detectable moieties which are useful for the antigen-binding protein or antigen-binding fragment thereof disclosed herein include fluorescent moieties or labels,33183465138.1imaging agents, radioisotopic moi eties, radiopaque moi eties, and the like, e.g., detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels. Illustrative fluorophores include fluorescent dyes (e.g. fluorescein, rhodamine, and the like) and other luminescent molecules (e.g. luminal). A fluorophore may be environmentally-sensitive, such that its fluorescence changes if it is located close to one or more residues in the modified protein that undergo structural changes upon binding a substrate (e.g., dansyl probes). Illustrative radiolabels include small molecules containing atoms with one or more low sensitivity nuclei (13C,15N,2H,1251,123I, "TC,43K,52Fe,67Ga,68Ga,inIn and the like). Other useful moieties are known in the art.

[0146] Examples of diagnostic moieties include detectable moieties suitable for revealing the presence of a disease or disorder. Typically, a diagnostic moiety allows for determining the presence, absence, or level of a molecule, for example, a target peptide, protein, or proteins, that is associated with a disease or disorder. Such diagnostics are also suitable for prognosing and / or diagnosing a disease or disorder and its progression.

[0147] Examples of therapeutic moieties include, but are not limited to, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, or anti-infective agents.

[0148] Non-limiting examples of therapeutic moieties include radionuclides with high-energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA, and therefore suitable for inducing cell death (e.g., of a cancer cell). Non-limiting examples of high-energy radionuclides include:90Y,125I,131I,123I,inIn,105Rh,153Sm,67Cu,67Ga,166Ho,177LU,186Re and188Re. These isotopes typically produce high-energy a- or P-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells and are essentially non-immunogenic.

[0149] Non-limiting examples of therapeutic moieties also include cytotoxic agents such as cytostatics (e.g., alkylating agents, DNA synthesis inhibitors, DNA-intercalators or crosslinkers, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenesis agents, and the like.

[0150] Non-limiting examples of therapeutic moieties also include alkylating agents such as the anthracy cline family of drugs (e.g., 34axotere34e, carminomycin, cyclosporin- A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenediones, and aziridines). In another embodiment, the chemotherapeutic moiety is a cytostatic agent such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited 34183465138.1to, methotrexate and di chloromethotrexate, 3-amino-l,2,4-benzotriazine 1,4-di oxide, aminopterin, cytosine P-D-arabinofuranoside, 5-fluoro-5 '-deoxyuridine, 5 -fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Non-limiting examples of DNA-intercalators or cross-linkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0151] Non-limiting examples of therapeutic moieties also include transcription regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other non-limiting examples of cytostatic agents that are compatible with the embodiments disclosed herein include ansamycin benzoquinones, quinonoid derivatives (e.g., quinolones, genistein, bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0152] Non-limiting examples of therapeutic moieties also include cytotoxic nucleosides such as, for example, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine; tubulin binding agents such as 35axoter (e.g., paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, dolastatins (e.g., Dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD6126), combretastatins (e.g., Combretastatin A-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); anti-angiogenesis compounds such as Angiostatin Kl-3, DL-a-difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0153] Non-limiting examples of therapeutic moieties also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens, (e.g., di ethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminogluthetimide), 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-l,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-a, rapamycin, sex hormone-binding globulin, and thapsigargin.

[0154] Non-limiting examples of therapeutic moieties also include enzyme inhibitors such as, S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenz-imidazole 1-P-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-l-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.35183465138.1

[0155] Non-limiting examples of therapeutic moieties also include gene regulators such as 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0156] Non-limiting examples of therapeutic moieties also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and the podophyllotoxins. Particularly useful members of those classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, leurosidine, vindesine, leurosine and the like.

[0157] Still other cytotoxins that are compatible with the teachings herein include auristatins (e.g., auristatin E and monomethylauristan E), calicheamicin, gramicidin D, maytansanoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, 36axotere36e, dihydroxy anthracindione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0158] The functional moiety may also have one or more of the herein-mentioned functions.

[0159] Techniques for conjugating moieties to an antigen-binding protein or antigenbinding fragment thereof are well known, see, e.g., Amon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., Antibodies For Drug Delivery, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, Immunol. Rev., 62:119-58 (1982).

[0160] To increase the half-life of an antigen-binding protein or antigen-binding fragment thereof, one can attach a salvage receptor binding epitope to the antigen-binding protein or antigen-binding fragment thereof (especially an antibody fragment), as described, e.g., in U.S. Patent. No. 5,739,277. The term “salvage receptor binding epitope” may refer to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for 36183465138.1increasing the in vivo serum half-life of the IgG molecule (e.g, Ghetie & Ward ES, Multiple roles for the major histocompatibility complex class I- related receptor FcRn, Annu Rev Immunol. 2000; 18:739-66). Antibodies with substitutions in an Fc region thereof and increased serum half-lives are also described in PCT Publications Nos. W02000 / 42072 (entitled “Polypeptide variants with altered effector function”), W02002 / 060919 (entitled “Molecules with extended half-lives, compositions and uses thereof’); Shields et al., High resolution mapping of the binding site on human IgGl for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgGl variants with improved binding to the Fc gamma R, J Biol Chem.2001 Mar 2;276(9):6591-604; Hinton et al., Engineered human IgG antibodies with longer serum half-lives in primates, J Biol Chem. 2004 Feb 20;279(8):6213-6. For example, a nucleic acid molecule encoding the salvage receptor binding epitope can be linked in frame to a nucleic acid encoding an antigen-binding protein or antigen-binding fragment thereof described herein so that the fusion protein expressed by the engineered nucleic acid molecule comprises the salvage receptor binding epitope and a antigen-binding protein or antigen-binding fragment thereof described herein. In another embodiment, the serum half-life can also be increased, for example, by attaching other polypeptide sequences. For example, the antigen-binding protein or antigen-binding fragment thereof can be attached to serum albumin or a portion of serum albumin that binds to the FcRn receptor or a serum albumin binding peptide so that serum albumin binds to the antibody or antigen-binding fragment thereof, e.g., such polypeptide sequences are disclosed in WO2001 / 045746. In one embodiment, the half-life of a Fab is increased by these methods. See also Dennis et al., Albumin binding as a general strategy for improving the pharmacokinetics of proteins, J Biol Chem. 2002 Sep 20;277(38):35035-43 for additional serum albumin binding peptide sequences.

[0161] Other types of functional moieties are known in the art and can be readily used in the methods and compositions of disclosed herein based on the teachings contained herein. The functional moiety may also have one or more of the above-mentioned functions.

[0162] Antigens

[0163] Provided herein are antigen-binding proteins and antigen-binding fragments thereof that bind to its cognate antigen.

[0164] As used herein, an “antigen” is a target molecule that an antigen-binding protein or antigen-binding fragment thereof binds to. An antigen may be, for example, a peptide, a protein, a polysaccharide, a lipid, or a nucleic acid. An antigen may be found on the surface of a pathogen such as a bacterium, virus, or fungus. Antigens can be derived from healthy cells 37183465138.1or diseased cells e.g., a cancer cell). Antigens can be naturally occurring or artificial. An antigen can be located on a target cell that a person skilled in the art wishes to target using the compositions and methods disclosed herein. Encompassed within the definition of an antigen are fragments of the antigen that an antigen-binding protein or antigen-binding fragment thereof binds to. An antigen is the molecule that is specifically recognized and bound by the binding site of the antigen-binding protein or antigen-binding fragment thereof.

[0165] As used herein, “binding” of an antigen-binding protein or antigen-binding fragment thereof to the antigen includes the selective interaction of the antigen-binding protein or antigen-binding fragment thereof with the antigen. Binding therefore includes, e.g., primary and secondary interactions including hydrogen bonds, ionic interactions, salt bridges, as well as hydrophilic and hydrophobic interactions.

[0166] As used herein, “affinity”, represented by the equilibrium constant for the dissociation (KD) of an antigen with an antigen-binding protein, is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein. The smaller the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 / KD). AS will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest.

[0167] In some embodiments, the antigen-binding proteins or antigen-binding fragments thereof described herein bind to their antigen with a KD of 10'5to 10'12mol / 1, 10'6to 10'12mol / 1, 10'7to IO’12mol / 1, 10'8to IO’12mol / 1, 10'9to 10'12mol / 1, IO'10to 10'12mol / 1, or 10'11to 10'13mol / 1. In other embodiments, the antigen-binding proteins or antigen-binding fragments thereof described herein bind to their antigen with a KD of 10'5to 1 O'12mol / 1, 10'6to 10'12mol / 1, 10'7to IO’12mol / 1, 10'8to IO’12mol / 1, 10'9to 1 O'12mol / 1, 10'10to 1 O'12mol / 1, 10'11to 1 O'12mol / 1.

[0168] In some embodiments, the antigen-binding proteins and antigen-binding fragments thereof provided herein bind specifically to their antigen. The term “specificity” herein refers to the ability of an antigen-binding protein or antigen-binding fragment thereof to bind to its antigen, while only having little or no detectable reactivity with other epitopes. Specificity can be relatively determined by competition assays or by epitope identification / characterization techniques described herein or their equivalents known in the art.

[0169] The “epitope” typically refers to the specific portion of an antigen that is recognized by an antigen-binding protein. As used herein, an “epitope” can be formed both from contiguous amino acids (a linear epitope), or noncontiguous amino acids juxtaposed by tertiary folding of a protein (a conformational epitope). Epitopes formed from contiguous amino acids 38183465138.1are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a particular spatial conformation. An “epitope” includes the unit of structure conventionally bound by, e.g., an immunoglobulin Vu / VLpair or by TCR Va / VP chains. Epitopes generally define the minimum binding site for an antigen-binding protein or antigen-binding fragment thereof and thus represent the target of specificity of an antigen-binding protein or antigen-binding fragment thereof.

[0170] In some embodiments, the antigen comprises a portion or fragment of any of the full-length proteins listed in Table 4. In some embodiments, the antigen comprises a protein fragment disclosed in Table 4.Table 4. Selected antigens and the proteins from which the antigens are derived from. Protein Fragment (amino Fragment (amino acid sequence) SEQ ID acid range in NO protein)MAGE-A1 151-165 LQLVFGIDVKEADPT 32165-179 TGHSYVLVTCLGLSY 33 MAGE-A3 198-212 KAGLLIIVLAIIARE 34287-301 LHHMVKISGGPHISY 35 MAGE-A12 198-212 KTGLLIIVLAIIAKE 36287-301 LHHLLKISGGPHISY 37 Mel an A 94-108 EPVVPNAPPAYEKLS 38 hTERT 916-930 GTAFVQMPAHGLFPW 3946-60 APLDGVL ANPPNIS S 40 Mesothelin 121-135 DLLLFLNPDAFSGPQ 41499-513 EYFVKIQSFLGGAPT 42NY-ESO-1 123-137 LKEFTVSGNILTIRL 43

[0171] In one embodiment, the antigen is derived from New York esophageal squamous cell carcinoma 1 (NY-ESO-1). The NY-ESO-1 antigen, which is also known as cancer-testis antigen IB (CTAG1B), exhibits high mRNA levels in a large number of epithelial cancer types, but very low levels in normal tissue aside from the testis and ovaries. In one embodiment, the antigen comprises residues 123-137 of NY-ESO-1 (SEQ IDNO:43 (LKEFTVSGNILTIRL)).

[0172] Without wishing to be bound by theory, the antigen-binding proteins disclosed herein that recognize the NY-ESO-1123-137 epitope in complex with an HLA may engage CD4+T cells to provide helper functions that enhance the anti-tumor immune response, including39183465138.1recruitment and activation of CD8+cytotoxic T cells, promotion of B cell antibody responses, and modulation of the tumor microenvironment through cytokine secretion.

[0173] In one embodiment, antigen is derived from human telomerase reverse transcriptase (hTERT), also referred to as telomerase catalytic subunit. In one embodiment, the antigen comprises residues 916-930 of hTERT.

[0174] In one embodiment, antigen is derived from Melan-A, also referred to as melanoma antigen recognized by T cells 1. In one embodiment, the antigen comprises residues 94-108 of Melan-A.

[0175] In one embodiment, antigen is derived from Mesothelin. In one embodiment, antigen is derived from melanoma-associated antigen (MAGE). In some embodiments, the antigen is derived from MAGE-A1, MAGE- A3, or MAGE-A12.

[0176] Major Histocompatibility Complexes (MHCs)

[0177] In some embodiments, the antigen-binding protein or antigen-binding fragment thereof binds to the antigen while the antigen is in a complex with a Major Histocompatibility Complex (MHC).

[0178] MHC molecules are generally classified into two categories: class I and class II MHCs. MHC I and II molecules present protein fragments to CD8+and CD4+T cells, respectively.

[0179] Class I MHCs

[0180] MHC class I molecules present peptides from proteins synthesized within cells, allowing CD8+T cells to detect and destroy abnormal cells expressing mutant sequences, microbial genes, or foreign polymorphic genes. MHC class I molecules generally are integral membrane proteins comprising a glycoprotein heavy chain, also referred to herein as the alpha chain, which has three extracellular domains (e.g., alphal, alpha2 and alpha3), a transmembrane domain and a cytoplasmic (intracellular) domain. The heavy chain is noncovalently associated with a soluble subunit called p2-microglobulin (also referred to herein as beta2m or beta 2 microglobulin). Generally, class I molecules bind peptides of about 8-12 amino acids in length. Humans have between three and six different class I molecules, which can each bind many different types of peptides. The polymorphic nature of MHC class I molecules, with over 10,000 different alleles, allows for a diverse repertoire of peptides to be presented.40183465138.1

[0181] Naturally occurring MHC heavy chain molecules are encoded by a cluster of genes on human chromosome 6 or mouse chromosome 17. Said MHC molecules are referred to as H-2 in mice and HLA (Human Leucocyte Antigen) in humans.

[0182] In contrast to the class I MHC heavy chain, beta2m is a relatively non-polymorphic component of class I major histocompatibility complex proteins. Only seven beta2m alleles are known in the mouse and only one has been identified in humans so far. Examples of naturally occurring beta2m include beta2m encoded on human chromosome 15 and mouse chromosome 2.

[0183] Class II MH Cs

[0184] MHC II molecules are found on immune cells such as B cells, monocytes, macrophages, dendritic cells, and on epithelial cells when triggered by inflammatory signals. In contrast, MHC I molecules are expressed more ubiquitously. Dendritic cells use MHC II molecules to present antigens to naive CD4+T cells, causing CD4+T cell activation. Subsequently, MHC II molecules facilitate interactions between B cells, macrophages, and these specific CD4+effector T cells. Peptide fragments presented by MHC class II complexes are generally larger than those presented by MHC class I complexes, since the peptide-binding groove of MHC class II is open, allowing peptides to extend out of this site. The MHC class II associated peptides are usually derived from extracellular proteins and from self-proteins that are degraded in the endosomal pathway.

[0185] MHC class II molecules are heterodimers composed of two chains: an alpha (a) chain and a beta (P) chain. Each chain has two extracellular domains, a transmembrane domain, and a cytoplasmic tail. The al and pi domains form the peptide-binding groove, which is open at both ends, allowing it to accommodate longer peptides, typically 15-24 amino acids in length. The a2 and P2 domains are membrane-proximal and have an immunoglobulin-like structure. The CD4+molecule on T cells interacts with the P2 domain of the MHC class II molecule. This interaction stabilizes the binding of the T cell receptor (TCR) to the antigen-MHC complex, enhancing the activation signal. The CD4+-MHC class II interaction is important for the formation of the immunological synapse, a specialized junction between the T cell and the APC that facilitates effective communication and signal transduction.

[0186] Humans have three MHC II loci (named HLA-DR, HLA-DQ and HLA-DP). MHC II molecules are polymorphic (>3,000 alleles known). Their polymorphic amino acids similarly cluster in and around the peptide-binding groove, shaping the peptide-binding pockets. Consequently, different MHC II alleles bind different peptides by virtue of their different anchor residues.41183465138.1

[0187] The MHC -molecules of the human are also designated as human leukocyte-antigens (HLA).

[0188] In one embodiment, the antigen-binding protein or antigen-binding fragment thereof binds to the antigen in the context of the class II MHC complex HLA-DRB3*02:02. HLA-DRB3 *02:02 is a common allele, present in half of the Caucasian population.

[0189] In some embodiments, the antigen-binding protein or antigen-binding fragment thereof disclosed herein binds to the NY-ESO-1123-137 peptide (SEQ ID NO:43) presented in the context of HLA-DRB3*02:02 with a binding affinity (KD) of less than 10'6M, less than 10'7M, less than 10'8M, or less than 10'9M. The binding affinity may be determined using surface plasmon resonance (SPR), biolayer interferometry (BLI), isothermal titration calorimetry (ITC), or other biophysical methods known in the art.

[0190] Nucleic acids and vectors

[0191] Provided herein are nucleic acids encoding the antigen-binding proteins or antigenbinding fragments thereof disclosed herein. The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural., chemically or biochemically modified, non-natural., or derivatized nucleotide bases.

[0192] Provided herein are vectors comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein. “Vector,” as used herein, means a vehicle that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo. Non-limiting examples of vectors include a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA mini-circle, or a virus (including virus-derived sequences). A viral particle is a vector herein.

[0193] Also, provided herein are viral genomes comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0194] Provided herein is a nucleic acid sequence or pair of nucleic acid sequences that comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the sequences in Table 5 or Table 6. Provided herein is a nucleic acid sequence that comprises any one of the sequences in Table 5 or Table 6.42183465138.1

[0195] Provided herein is a nucleic acid sequence or pair of nucleic acid sequences comprising the CDR encoding sequences of a TCR shown in Table 5.

[0196] Provided herein is a nucleic acid sequence or pair of nucleic acid sequences comprising the variable chain encoding sequences of a TCR shown in Table 6.

[0197] Provided herein is a nucleic acid sequence or pair of nucleic acid sequences comprising the variable region and constant region encoding sequences of a TCR shown in Table 6Table 5. Selected CDR combinations (nucleic acid sequences).Name CDRla CDR2a CDR3a CDRlb CDR2b CDR3b SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO NO NO NO NO NO TCR1 44 45 47 51 53 55 TCR2 44 45 48 52 54 56 TCR3 44 46 49 51 53 57TCR4 44 45 50 51 53 58 Table 6. Selected chain combinations (nucleic acid sequences).Name Chain 1 (variable Chain 1 Chain 2 (variable + Chain 2 (variable + constant (variable constant regions) region) regions) region)SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO TCR1 59 60 61 62TCR2 63 64 65 66TCR3 67 68 69 70TCR4 71 72 73 74

[0198] Provided herein is an expression construct comprising a nucleic acid sequence encoding antigen-binding protein or antigen-binding fragment thereof disclosed herein. The nucleic acid sequence encoding the antigen-binding protein or antigen-binding fragment thereof is usually operatively linked to an expression control sequence.

[0199] In some embodiments, the AAV genome further comprises an expression control sequence, including one that is operably linked to a transgene. As used herein, “operatively linked” refers to a first molecule joined to a second molecule, wherein the molecules are so arranged that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, an expression control sequence is operatively linked to a transcribable polynucleotide molecule if the expression control sequence modulates transcription of the transcribable43183465138.1polynucleotide molecule of interest in a cell. Additionally, two portions of an expression control sequence are operatively linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion. Two sequences may be operatively linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operatively linked to one another with no intervening nucleotides present.

[0200] Expression control sequences can include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (z.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein processing and / or secretion.

[0201] A great number of expression control sequences, e.g., native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized to drive expression of the transgene, depending upon the type of expression desired. For eukaryotic cells, expression control sequences commonly used include a promoter, an enhancer, and a polyadenylation sequence which may include splice donor and acceptor sites. The polyadenylation sequence generally is inserted following the transgene.

[0202] As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.

[0203] A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions.

[0204] An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g., by the application of a chemical inducer.

[0205] A wide variety of vectors can be used for the expression of an antigen-binding protein or antigen-binding fragment thereof disclosed herein. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into a host cell genome and express viral genes stably and efficiently have made them attractive candidates for 44183465138.1the transfer of foreign nucleic acids into cells. Accordingly, in some embodiments, a viral vector is used to introduce one or more nucleotide sequences encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein into a host cell for expression. The viral vector may comprise a nucleotide sequence encoding an antigen-binding protein or antigen-binding fragment thereof operably linked to one or more expression control sequences, for example, a promoter. Alternatively, the viral vector may not contain an expression sequence and will instead rely on a control sequence within the host cell to drive expression of the antigen-binding protein or antigen-binding fragment thereof. Non-limiting examples of viral vectors that may be used to deliver a nucleic acid include adenoviral vectors, AAV vectors, and retroviral vectors.

[0206] For example, an adeno-associated virus (AAV) can be used to introduce one or more nucleotide sequences encoding an antigen-binding protein or antigen-binding fragment thereof into a host cell for expression. AAV systems have been described previously and are generally well known in the art (Kelleher & Vos, Long-term episomal gene delivery in human lymphoid cells using human and avian adenoviral-assisted transfection, Biotechniques. 1994 Dec; 17(6): 1110-7; Cotten et al., High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles, Proc Natl Acad Sci U S A. 1992 Jul l;89(13):6094-8; Curiel, High-efficiency gene transfer employing adenovirus-polylysine-DNA complexes, Nat Immun.1994 Mar-Jun; 13(2-3): 141-64; Muzyczka, Use of adeno-associated virus as a general transduction vector for mammalian cells, Curr Top Microbiol Immunol. 1992;158:97-129). Details concerning the generation and use of rAAV vectors are described, for example, in U.S. Pat. Nos. 5,139,941 (entitled “AAV transduction vectors”) and 4,797,368 (entitled “Adeno-associated virus as eukaryotic expression vector”), each incorporated herein by reference in its entirety.

[0207] In some embodiments, a retroviral expression vector can be used to introduce one or more nucleotide sequences encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein into a host cell for expression. These systems have been described previously and are generally well known in the art (Nicolas and Rubinstein, In, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986). Examples of vectors for eukaryotic expression in mammalian cells include AD5, pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated45183465138.1viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and beta-actin.

[0208] In some embodiments, expression of the antigen-binding protein or antigen-binding fragment thereof is regulated by a constitutively activated promoter. In some embodiments, expression of the antigen-binding protein or antigen-binding fragment thereof is regulated by an inducible promoter. In some embodiments, expression of the antigen-binding protein or antigen-binding fragment thereof in a lymphocyte is induced upon activation of the lymphocyte.

[0209] Combinations of retroviruses and an appropriate packaging line may also find use, where the capsid proteins will be functional for infecting the target cells. Usually, the cells and viruses will be incubated for at least about 24 hours in the culture medium. The cells are then allowed to grow in the culture medium for short intervals in some applications, e.g., 24-72 hours, or for at least two weeks, and may be allowed to grow for five weeks or more, before analysis. Commonly used retroviral vectors are “defective,” z.e., unable to produce viral proteins required for productive infection. Replication of the vector requires growth in the packaging cell line. The host cell specificity of the retrovirus is determined by the envelope protein, env (pl20). The envelope protein is provided by the packaging cell line. Envelope proteins are of at least three types, ecotropic, amphotropic and xenotropic. Retroviruses packaged with ecotropic envelope protein, e.g., MMLV, are capable of infecting most murine and rat cell types. Ecotropic packaging cell lines include BOSC23. Retroviruses bearing amphotropic envelope protein, e.g., 4070A, are capable of infecting most mammalian cell types, including human, dog, and mouse. Amphotropic packaging cell lines include PA12 and PA317. Retroviruses packaged with xenotropic envelope protein, e.g., AKR env, are capable of infecting most mammalian cell types, except murine cells. The vectors may include genes that can later be removed, e.g., using a recombinase system such as Cre / Lox, or the cells that express them destroyed, e.g., by including genes that allow selective toxicity such as herpesvirus TK, BCL-xs, etc. Suitable inducible promoters are activated in a desired target cell type, either the transfected cell or progeny thereof.

[0210] Non-limiting examples of the vectors useful for the genetically engineered cells comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein include retroviral vector SFG.MCS, and helper plasmids RD114, Peg-Pam3, lentiviral vector pRRL, and helper plasmids R8.74 and pMD2G (e.g., Addgene Plasmid #12259). In some embodiments, the Sleeping Beauty transposon system can be used (Deniger et al., Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T- 46183465138.1cell Receptors Using the Sleeping Beauty Transposon / Transposase System, Mol Ther. 2016 Jun;24(6): 1078-1089). In some embodiments, nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof can be introduced into cells via deforming a cell as it passes through a small opening, disrupting the cell membrane and allowing material to be inserted into the cell, for example, electroporation, or the Cell Squeeze® method. Such electroporation methods of an RNA encoding a transgene allow for transient expression of such transgene in cells which can limit toxicity and other undesirable effects of engineered cells.

[0211] In some embodiments, genome-editing techniques, such as CRISPR / Cas9 systems, designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available to induce expression of the antigen-binding protein or antigen-binding fragment thereof disclosed herein in a cell, including in an immune cell. In general, “CRISPR / Cas9 system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an47axotere47es CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. One or more elements of a CRISPR system may be derived from a type I, type II, or type III CRISPR system. Alternatively, one or more elements of a CRISPR system may be derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).

[0212] In some embodiments, cells, such as lymphocytes, disclosed herein are genetically modified by transfecting the cell with a vector (e.g., lentiviral vector) encoding the antigenbinding protein or antigen-binding fragment thereof and CA9 or a functional fragment thereof. In some embodiments, the nucleic acid(s) encoding the antigen-binding protein or antigenbinding fragment thereof and CA9 (or functional fragment thereof) or can be introduced into the cell using one, two, or more vectors.

[0213] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising exogenous vectors and / or nucleic acids47183465138.1are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory, New York).

[0214] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An illustrative colloidal system for use as an in vitro and in vivo release vehicle is a liposome (e.g., an artificial membrane vesicle).

[0215] In the case where a non-viral delivery system is used, an illustrative delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, bound to a liposome via a binding molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, in a complex with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, content or in a complex with a micelle, or associated otherwise with a lipid. The compositions associated with lipids, lipids / DNA or lipids / expression vector are not limited to any particular structure in solution. For example, they can be present in a bilayer structure, as micelles, or with a “collapsed” structure. They can also be simply interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0216] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; Dicetylphosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); Cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the sole solvent since it evaporates more easily than methanol. “Liposome” is a generic term that encompasses a variety of unique and multilamellar lipid vehicles formed by the generation of bilayers or closed lipid aggregates. Liposomes can be characterized as having vesicular structures with a bilayer membrane of 48183465138.1phospholipids and an internal aqueous medium. Multilamellar liposomes have multiple layers of lipids separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and trap dissolved water and solutes between the lipid bilayers. However, compositions that have different structures in solution than the normal vesicular structure are also included. For example, lipids can assume a micellar structure or simply exist as nonuniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0217] Regardless of the method used to introduce exogenous nucleic acids into a host cell, the presence of the recombinant DNA sequence in the host cell can be confirmed by a series of tests. Such assays include, for example, “molecular biology” assays well known to those skilled in the art, such as Southern and Northern blot, RT-PCR and PCR; biochemical assays, such as the detection of the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blot) or by assays described herein to identify agents that are within the scope of the disclosure.

[0218] Cells

[0219] Provided are cells that express an antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0220] The antigen-binding protein or antigen-binding fragment thereof may be expressed on the surface of the cell. For example, an immune cell may be genetically engineered to express on its surface an antigen-binding protein or antigen-binding fragment thereof disclosed herein, wherein the antigen-binding protein is a TCR.

[0221] The antigen-binding protein or antigen-binding fragment thereof may be secreted by the cell. For example, a cell may be genetically engineered to produce an antigen-binding protein or antigen-binding fragment thereof disclosed herein, wherein the antigen-binding protein is an antibody or antigen-binding fragment thereof.

[0222] Provided herein are cells comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0223] Provided herein are cells comprising one or more nucleic acids encoding an antigenbinding protein or antigen-binding fragment thereof disclosed herein. Provided herein are cells comprising one or more nucleic acids, pairs of nucleic acids, expression constructs, pairs of expression constructs, vectors, or pairs of vectors disclosed herein.49183465138.1

[0224] In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In one embodiment, the cell is a human cell. The cell may be isolated. The cell may be any cell disclosed herein.

[0225] The cell may be derived from a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human).

[0226] In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. Lymphocytes are one of the subtypes of a white blood cell in a vertebrate’s immune system and include T cells, B cells, and natural killer (NK) cells. In cancer immunotherapy, lymphocytes can be used in adoptive cell therapy (ACT) and immune checkpoint blockade (ICB) to enhance antitumor responses. The lymphocytes can be engineered to express CARs or expanded ex vivo for reinfusion into patients. Provided herein are methods of using genetically modified lymphocytes in ACT. Provided herein are genetically modified lymphocytes that bind to certain antigens in the context of an MHC.

[0227] T cells (T lymphocytes)

[0228] In one embodiment, the cell is a T cell. As used herein, the term “T cell” has its general meaning in the art. T cells are an important component of the immune system and play a central role in cell-mediated immunity. T cells are known as conventional lymphocytes as they recognize the antigen with their TCR (T cell receptor for the antigen) with presentation or restriction by molecules of the HMC. There are several subsets of T cells each having a distinct function such as CD8+T cells, CD4+T cells, Gamma delta T cells, and Regulatory T cells (Tregs). See also Masopust et al., Guidelines for T cell nomenclature, Nat Rev Immunol.2025 Nov 18.

[0229] Provided is a T cell expressing a TCR disclosed herein (this includes any variant of a TCR disclosed herein). Provided is a T cell expressing a TCR or a CAR comprising the CDRs of a TCR disclosed herein (this includes any CDR variants of a TCR disclosed herein).

[0230] TILs

[0231] As used herein, the term “tumor infiltrating T cells” or “tumor infiltrating lymphocytes (TILs)” refers to the pool of T cells of a patient that have left the blood stream and have migrated into a tumor.

[0232] Provided is a TIL expressing a TCR disclosed herein (this includes any variant of a TCR disclosed herein). Provided is a TIL expressing TCR or a CAR comprising the CDRs of a TCR disclosed herein (this includes any CDR variants of a TCR disclosed herein).

[0233] Innate lymphoid cells (ILCs)50183465138.1

[0234] Innate lymphoid cells (ILCs) are a recently discovered family of immune cells that play important roles in maintaining tissue homeostasis and immunity. They contribute to the body’s defense against pathogens by secreting signaling molecules and regulating both innate and adaptive immune cells. ILCs are particularly abundant at mucosal surfaces, where they play a key role in mucosal immunity. Additionally, ILCs are involved in tissue repair and regeneration, helping to maintain the integrity of epithelial tissues and supporting tissue morphogenesis and metabolism.

[0235] ILCs are classified into three main groups based on their functions and cytokine production. Group 1 ILCs, which include NK cells and ILCls, produce interferon-y (IFNy) and tumor necrosis factor alpha (TNF-a), and are involved in immunity against intracellular pathogens. Group 2 ILCs, or ILC2s, release cytokines associated with T-helper 2 responses (e.g., IL-4, IL-5, IL-9, IL-13) and are important for immune responses to helminths. Group 3 ILCs, or ILC3s, produce IL- 17 A, IL-22, and lymphotoxin, and are important for immunity against extracellular bacteria.

[0236] Provided is an ILC TIL expressing a TCR disclosed herein (this includes any variant of a TCR disclosed herein). Provided is an ILC expressing a TCR or a CAR comprising the CDRs of a TCR disclosed herein (this includes any CDR variants of a TCR disclosed herein).

[0237] CD8+T cells

[0238] CD8+T cells, also known as cytotoxic T lymphocytes (CTL), T-killer cells, cytolytic T cells, or killer T cells, are essential components of the adaptive immune system. Their primary function is to identify and eliminate cells infected by viruses, bacteria, or transformed into cancer cells. These cells express the CD8 receptor, which allows them to recognize antigens presented by MHC class I molecules on the surface of target cells. Upon activation, CD8+T cells release cytotoxic granules containing perforin and granzymes, leading to the destruction of the infected or abnormal cells. Additionally, CD8+T cells can produce cytokines that enhance the immune response and recruit other immune cells to the site of infection. Their ability to specifically target and kill infected or cancerous cells makes them important for maintaining immune surveillance and combating diseases. As used herein, the term “CD8+T cell” has its general meaning in the art and refers to a subset of T cells which express CD8+on their surface.

[0239] Provided is a CD8+T cell comprising a TCR or a CAR disclosed herein, wherein the TCR binds to an antigen that is in the context of an MHC class I complex.

[0240] CD4+T cells51183465138.1

[0241] CD4+T cells, also known as helper T cells, are important components of the immune system, playing versatile roles in coordinating and enhancing immune responses against cancer and chronic infections. CD4+T cells support CD8+T cells through cytokine production (such as IL-2 and IL-21) and direct cell interactions, and they help dendritic cells (DCs) present antigens more effectively. CD4+T cells produce effector cytokines like IFN-y and TNF-a, which have direct antiviral and antitumor effects, and can acquire cytotoxic functions to kill target cells. CD4+T cells are important for B cell help, promoting antibody production and germinal center reactions through interactions involving CD40L and IL-21. Regulatory T cells (Tregs), a subset of CD4+T cells, maintain immune tolerance and prevent autoimmunity by producing immunosuppressive cytokines like IL-10 and TGF-p. As used herein, the term “CD4+T cells” (also called T helper cells or TH cells) refers to T cells which express the CD4 glycoprotein on their surfaces and which assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. CD4+T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH 17, TH9, TFH or Treg, which secrete different cytokines to facilitate different types of immune responses. Signaling from the APC directs T cells into particular subtypes. In addition to CD4+, the TH cell surface biomarkers known in the art include CXCR3 (Thl), CCR4, Crth2 (Th2), CCR6 (Thl7), CXCR5 (Tfh) and as well as subtype-specific expression of cytokines and transcription factors including T-bet, GAT A3, EOMES, RORyT, BCL6 and FoxP3.

[0242] Provided is a CD4+T cell comprising a TCR or a CAR disclosed herein, wherein the TCR binds to an antigen that is in the context of an MHC class II complex.

[0243] Provided is a CD4+T cell comprising a TCR or a CAR disclosed herein, wherein the TCR binds to a peptide in the context of the class II MHC complex HLA-DRB3*02:02. Since a large number of patients express the HLA-DRB3*02:02 complex, a therapy using such a CD4+T cell can be used in a large number of cancer patients in need of treatment.

[0244] NK cells

[0245] Natural Killer (NK) cells are innate lymphoid cells that play an important role in the immune system by mediating cytotoxicity and cytokine production without the need for prior antigen sensitization. They are equipped with a variety of activating and inhibitory receptors that allow them to recognize and respond to conserved molecular patterns, making them a first 52183465138.1line of defense against infections and cellular transformations. The cytotoxic function of NK cells is primarily executed through the formation of an immunological synapse, which facilitates the directed secretion of lytic granules containing perforin and granzymes to lyse target cells. NK cells can also kill target cells through death receptor-mediated interactions, such as Fas-FasL and TRAIL, which activate apoptotic signaling in the target cells.

[0246] In addition to their cytotoxic capabilities, NK cells are rapid producers of cytokines, including type II interferon (IFNy), tumor necrosis factor-alpha (TNFa), and various interleukins and chemokines. These cytokines play a significant role in promoting host defense by activating other immune cells, recruiting them to infection sites, and facilitating T cell recognition. The production of cytokines by NK cells is induced by the same activation pathways that promote cytotoxicity, involving engagement of activating receptors and cytokine stimulation.

[0247] NKT cells also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. NKT cells include NK1.1+and NK1. G, as well as CD4+, CD4+, CD8+, and CD8+cells. NK cell can refer to a differentiated lymphocyte with a CD16+CD56+and / or CD57+TCR' phenotype.

[0248] CAR-T cells

[0249] Provided is a CAR-T cell expressing a chimeric antigen receptor (CAR) disclosed herein (this includes any variant of a CAR disclosed herein). Provided is a T cell expressing a CAR comprising the CDRs of an antigen-binding protein or antigen-binding fragment thereof disclosed herein (this includes any CDR variants of an antigen-binding protein or antigenbinding fragment thereof disclosed herein). As used herein the term “CAR-T cell” refers to a T lymphocyte that has been genetically engineered to express a CAR. The definition of CAR T cells encompasses all classes and subclasses of T lymphocytes including CD4+, CD8+T cells, gamma delta T cells as well as effector T cells, memory T cells, regulatory T cells, and the like.

[0250] The immune cells that are genetically modified may be “derived” or “obtained” from the subject who will receive the treatment using the genetically modified immune cells. Alternatively, the genetically modified immune cells may “derived” or “obtained” from a different subject.

[0251] Cell preparations

[0252] Generally, adoptive T cell therapy relies on the in vitro expansion of endogenous, cancer-reactive T cells. These T cells can be harvested from cancer patients, manipulated, and then reintroduced into the same or a different patient as a mechanism for generating productive tumor immunity.53183465138.1

[0253] In one embodiment, the cell that is genetically modified to express an antigenbinding protein or antigen-binding fragment thereof disclosed herein is autologous to a patient. In one embodiment, the cell that is genetically modified to express an antigen-binding protein or antigen-binding fragment thereof disclosed herein is allogeneic to a patient.

[0254] Provided herein are methods of genetically modifying cells to express an antigenbinding protein or antigen-binding fragment thereof disclosed herein.

[0255] Provided herein are methods of genetically modifying cells to express an antigenbinding protein or antigen-binding fragment thereof disclosed herein on their surface. The antigen-binding protein or antigen-binding fragment thereof may be a TCR or a CAR.

[0256] In some embodiments, the lymphocytes disclosed herein are derived from CD34+hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. In some embodiments, the lymphocytes disclosed herein are autologous, allogeneic, syngeneic, or xenogeneic. In a preferred embodiment, the lymphocytes disclosed herein are autologous. In some embodiments, the lymphocytes disclosed herein are human.

[0257] Prior to the expansion and genetic modification of the lymphocytes described herein, a source of lymphocytes from a subject may be obtained. Lymphocytes can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumors. As described herein, any number of lymphocyte lines available in the art can be used. Lymphocytes can be obtained from a unit of blood collected from a subject using any number of techniques known to the person skilled in the art, such as the Ficoll™ separation. Circulating blood cells of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T lymphocytes, monocytes, granulocytes, B lymphocytes, other nucleated white blood cells, red blood cells, and platelets. The cells harvested by apheresis can be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for the subsequent processing steps. The cells may be washed with phosphate-buffered saline (PBS). Alternatively, the wash solution may lack calcium and may lack magnesium or may lack many, if not all, divalent cations. As those of ordinary skill in the art would readily appreciate, a washing step can be achieved by methods known to those skilled in the art, such as using a semiautomatic continuous flow centrifuge (e.g., the Cobe 2991 cell processor, the Baxter CytoMate, or elHaemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+free, PBS free Mg2+, PlasmaLyte54183465138.1A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample can be removed, and the cells resuspended directly in a culture medium.

[0258] As described herein, lymphocytes may be isolated from peripheral blood by lysis of red blood cells and depletion of monocytes, for example, by centrifugation through a PERCOLL™ or Ficoll™ gradient or by countercurrent centrifugal elutriation. Lymphocytes may also be isolated from the spleen. If needed, specific subpopulation lymphocytes, such as T lymphocytes (e.g., CD3+, CD28+, CD4+, CD8+, CD4+5RA+or CD4+5RO+T lymphocytes) can be further isolated by positive or negative selection techniques. For example, T lymphocytes may be isolated by incubation with conjugated anti-CD3 beads for a sufficient period of time (z.e., 30 minutes to 24 hours) for positive selection of the desired T lymphocytes. For the isolation of T lymphocytes from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cellular performance. Longer incubation times can be used to isolate T lymphocytes in any situation where there are few T lymphocytes compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. The person skilled in the art will recognize that multiple rounds of selection may also be used. It may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also undergo new rounds of selection.

[0259] Enrichment of a population of lymphocytes (e.g., T lymphocytes) by negative selection can be performed with a combination of antibodies directed to unique surface markers for the negatively selected cells. One method is the sorting and / or selection of cells by negative magnetic immune adherence or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present in the negatively selected cells. For example, to enrich CD4+cells by negative selection, a monoclonal antibody typically includes antibodies against CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. Alternatively, the regulatory T lymphocytes are depleted by anti-C25 conjugate beads or other similar selection method.

[0260] Lymphocytes for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, freezing and the following thawing step provide a more uniform product by eliminating granulocytes and, to some extent, monocytes in the cell population. After the washing step that removes the plasma and platelets, the cells can be suspended in a freezing solution. Although many solutions and freezing parameters are known in the art and will be useful in this context, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose human albumin and 7.5% DMSO or 31.25% Plasmalyte A, 31.25% dextrose 5%, 0.45% NaCl, 10%55183465138.1dextran 40 and 5% of dextrose, 20% serum of human albumin and 7.5% of DMSO or other suitable cell freezing medium containing for example Hespan and PlasmaLyte A. The cells may then be frozen at -80 °C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used, as well as uncontrolled freezing immediately at -20 °C or in liquid nitrogen.

[0261] The cryopreserved cells may be thawed and washed as described herein and allowed to stand for one hour at room temperature before activation using the methods of the present disclosure. As described herein, lymphocytes can be expanded, frozen, and used later. As described herein, samples may be collected from a patient shortly after the diagnosis of a particular disease as described herein, but before any treatment. The cells may be isolated from a blood sample or an apheresis of a subject before any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablatories such as CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit calcium-dependent calcineurin phosphatase (e.g., ciclosporin and FK506) or inhibit p70S6 kinase that is important for signaling induced by the growth factor (rapamycin). The cells may be isolated from a patient and frozen for later use together with (e.g., before, simultaneously or after) bone marrow or stem cell transplant, therapy with T lymphocyte ablation using chemotherapeutic agents such as fludarabine, radiotherapy external beam (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. As described herein, the cells may be isolated before and can be frozen for later use in the treatment after therapy with ablation of B lymphocytes, such as agents that react with CD20, for example, Rituxan.

[0262] Either before or after the genetic modification of lymphocytes (e.g., T lymphocytes) to express a desirable transgene (such as a gene encoding a heterodimeric protein or fragment thereof disclosed herein), lymphocytes can be activated and expanded generally using methods such as those described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and the publication of US patent application. No. 20060121005.

[0263] The expansion may take place in vitro (e.g., for production of cells for adoptive T cell or NK cell therapy) and in vivo e.g., for durability of adoptive cell therapy). For example, cells may be cultured in media optionally containing one or more growth factors, i.e., a growth 56183465138.1factor cocktail. In some embodiments, the cell culture medium is a defined cell culture medium. The cell culture medium may include neoantigen peptides. Stable cell lines may be established to allow for the continued propagation of cells.

[0264] “Activation of a T cell” may refer to induction of signal transduction pathways in the T cell resulting in production of cellular products (e.g., interleukin-2) by that T cell. Methods of inducing and measuring T cell activation are known in the art. See, e.g., Phetsouphanh et al., Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells. Int J Mol Sci. 2015 Aug 12; 16(8): 18878-93, which is incorporated herein by reference in its entirety. T cell activation can be measured, for example, by detecting activation marker CD25 by flow cytometry. Activation can also be measured by, for example, measuring the amount of IL-2 produced by a T cell after a peptide / MHC complex has bound to the TCR. T cell activation may be determined, e.g., by measuring changes in the level of expression of cytokines and / or T cell activation markers, and / or the induction of antigen-specific proliferating cells. Techniques known to those of skill in the art, including, but not limited to, immunoprecipitation followed by Western blot analysis, ELISAs, flow cytometry, Northern blot analysis, and RT-PCR can be used to measure the expression cytokines and T cell activation markers. Cytokine release may be measured by measuring secretion of cytokines including, but not limited to, Interleukin-2 (IL-2), Interleukin- 4 (IL-4), Interleukin-6 (IL-6), Interleukin- 12 (IL-12), Interleukin- 16 (IL-16), PDGF, TGF-alpha, TGF-beta, TNF-alpha, TNF-beta, GCSF, GM-CSF, MCSF, IFN-alpha, IFN-beta, IFN-gamma, TFN-gamma, IGF-I, and IGF-II.

[0265] Additional modi fications o f cells

[0266] In some embodiments, the lymphocytes disclosed herein further engineered to secrete therapeutic proteins, including, but not limited to IL-2, IL-2 mutein, IL-15, CD40L, IL-33, and IL- 12, a PD1 inhibitor, an PD-L1 inhibitor, a TIGIT inhibitor variants thereof. The therapeutic protein may be any therapeutic protein disclosed herein. In some embodiments, the lymphocytes disclosed herein are modified using the Genetic Engineering for the Enhanced Performance of T cells technology described in PCT Publication No. WO2021 / 097278 (entitled “Compositions and methods for immunotherapy”), which is incorporated herein by reference in its entirety. In some embodiments, the lymphocytes disclosed herein are further engineered to express a transgene described in WO2021 / 097278, including, but not limited to the transgenes recited in the claims of WO2021 / 097278.

[0267] In some embodiments, lymphocytes are genetically modified to express therapeutic proteins by introducing nucleic acid sequences encoding the therapeutic proteins using viral 57183465138.1vectors (e.g., lentiviral or retroviral vectors) or non-viral methods (e.g., electroporation of mRNA or DNA, CRISPR-mediated integration). The therapeutic protein-encoding sequences may be operably linked to constitutive promoters (e.g., EFla, CMV, PGK) or inducible promoters to control expression levels. In some embodiments, the nucleic acid encoding the therapeutic protein is integrated into a safe harbor locus such as AAVS1 or CCR5 to ensure stable expression without disrupting essential genes.

[0268] In some embodiments, the therapeutic protein is secreted by the lymphocyte to act on surrounding cells in the tumor microenvironment. For example, IL-2, IL-2 mutein, IL-15, IL-33, and IL- 12 can enhance T cell proliferation and activation. CD40L can activate antigen-presenting cells and enhance anti-tumor immunity. In some embodiments, the IL-2 mutein comprises mutations that reduce binding to IL-2Ra while maintaining or enhancing binding to IL-2RPy, thereby reducing toxicity while preserving therapeutic efficacy. Examples of IL-2 muteins include, but are not limited to, those described in Levin et al., Exploiting a natural conformational switch to engineer an interleukin-2 'superkine', Nature. 2012 Mar 25;484(7395):529-33, which is incorporated herein by reference in its entirety.

[0269] In some embodiments, the PD-1 inhibitor expressed by the lymphocyte is a dominant-negative PD-1 polypeptide that lacks the intracellular signaling domain or comprises mutations in the immunoreceptor tyrosine-based inhibitory motif (ITIM) or immunoreceptor tyrosine-based switch motif (ITSM). Such dominant-negative PD-1 can compete with endogenous PD-1 for binding to PD-L1 / PD-L2 without transmitting inhibitory signals. In some embodiments, the PD-L1 inhibitor is a soluble PD-L1 -binding antibody fragment (e.g., scFv) or a PD-L1 -binding receptor fragment expressed on the cell surface or secreted by the lymphocyte. In some embodiments, the TIGIT inhibitor is a dominant-negative TIGIT polypeptide, a soluble TIGIT receptor fragment, or an anti-TIGIT antibody fragment.

[0270] In some embodiments, the lymphocytes disclosed herein are further engineered to express a protein that inhibits, blocks, or antagonizes the interaction of immunosuppressive polypeptides and / or their ligands. Immunosuppressive polypeptides that known to suppress or decrease an immune response via their binding include CD47, PD-1, CTLA-4, and their corresponding ligands, including SIRPalpha, PD-L1, PD-L2, B7-1, B7-2, and TIGIT. Such polypeptides are present in the tumor microenvironment and inhibit immune responses to neoplastic cells.

[0271] In some embodiments, lymphocytes disclosed herein are engineered to express a PD-1 variant (a PD-1 decoy) that is designed compete with endogenous PD-1. In some embodiments, the PD-1 decoy is lacking the cytoplasmic domain of PD-1. In some 58183465138.1embodiments, the PD-1 transmembrane and intracellular signaling domains in the PD-1 decoy are replaced with a co-stimulating signaling domain of CD28 or a constitutively active IL-7 receptor, to convert possible inhibitory signal to improved T cell function.

[0272] In some embodiments, the lymphocytes disclosed herein are engineered to express one or more co-stimulatory polypeptides to stimulate or increase an immune response via their binding. Non-limiting examples of such co-stimulatory polypeptides include CD28, OX-40, 4-1BB, CD27, and NKG2D and their corresponding ligands, including B7-1, B7-2, OX-40L, 4-1BBL, CD70, and NKG2D ligands.

[0273] Expression systems for antigen-binding proteins and antigen-binding fragments thereof

[0274] The antigen-binding proteins or antigen-binding fragments thereof disclosed herein are typically produced by recombinant expression. Nucleic acids encoding light and heavy chain variable regions, optionally linked to constant regions, can be inserted into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding the antigen-binding protein or antigen-binding fragment thereof disclosed can be operably linked to control sequences in the expression vector(s) that ensure the expression of antigen-binding protein or antigen-binding fragment thereof. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the antigenbinding protein or antigen-binding fragment thereof.

[0275] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resi stance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362).

[0276] Signal peptides or signal sequence for the expression of an antigen-binding protein or antigen-binding fragment thereof are known in the art.

[0277] The expression of the antigen-binding protein or antigen-binding fragment thereof contemplated herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include 59183465138.1bacterial or eukaryotic hosts, including yeast, insects, fungi, bird, and mammalian cells either in vivo, or in situ, or host cells of mammalian, insect, bird or yeast origin. The mammalian cell or tissue can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell may be used.

[0278] E. coli is one prokaryotic host particularly useful for cloning the polynucleotides (e.g., DNA sequences). Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other 60axotere60e60riaceae, such as Salmonella, Serratia, and various Pseudomonas species.

[0279] Other microbes, such as yeast, are also useful for expression. Saccharomyces and Pichia are illustrative yeast hosts, with suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired. Typical promoters include 3 -phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.

[0280] Further, by use of, for example, the yeast ubiquitin hydrolase system, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be accomplished. The fusion proteins so produced can be processed in vivo or purified and processed in vitro, allowing synthesis of an antigen-binding protein or antigen-binding fragment thereof disclosed herein with a specified amino terminus sequence. Moreover, problems associated with retention of initiation codon-derived methionine residues in direct yeast (or bacterial) expression maybe avoided. See Sabin et al. High-Level Expression and In Vivo Processing of Chimeric Ubiquitin Fusion Proteins in Saccharomyces Cerevisiae, Nat Biotechnol 7, 705-709 (1989); Miller, Cloning and Expression of a Yeast Ubiquitin-Protein Cleaving Activity in Escherichia Coli, Nat Biotechnol 7, 698-704 (1989).

[0281] Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast cells are grown in mediums rich in glucose can be utilized to an antigen-binding protein or antigen-binding fragment thereof disclosed herein. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

[0282] Production of an antigen-binding protein or antigen-binding fragment thereof in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express a transmembrane polypeptide by methods known to those of skill. See, e.g., Saleem 60183465138.1et al., Production Technologies for Recombinant Antibodies: Insights into Eukaryotic, Prokaryotic, and Transgenic Expression Systems. Biochem Genet. 2025 Oct;63(5):3928-3967.

[0283] In addition to microorganisms, mammalian tissue culture may also be used to express and produce the antigen-binding proteins or antigen-binding fragments thereof disclosed herein. See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Eukaryotic cells might be preferred, because a number of suitable host cell lines capable of secreting heterologous proteins have been developed in the art, and include CHO cell lines, various COS cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed B-cells, and hybridomas. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Expression control sequences may be promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co MS, Avdalovic NM, Caron PC, Avdalovic MV, Scheinberg DA, Queen C. et al., Chimeric and humanized antibodies with specificity for the CD33 antigen, J Immunol. 1992 Feb 15;148(4):1149-54.

[0284] Alternatively, nucleotide sequences encoding an antigen-binding protein or antigenbinding fragment thereof can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for the two chains of the antigen-binding protein or antigen-binding fragment thereof in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.

[0285] Additionally, plants have emerged as convenient, safe and economical alternative main-stream expression systems for recombinant production of an antigen-binding protein or antigen-binding fragment thereof, which are based on large scale culture of microbes or animal cells. Antigen-binding proteins or antigen-binding fragments thereof can be expressed in plant cell culture, or plants grown conventionally. The expression in plants may be systemic, limited to sub-cellular plastids, or limited to seeds (endosperms). See, e.g., U.S. Patent Pub. No.2003 / 0167531; U.S. Patent Nos. 6,080,560 and 6,512,162; and WO 0129242.

[0286] The vectors containing the polynucleotide sequences of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection 61183465138.1may be used for other cellular hosts. See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nded., 1989). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. See generally Sambrook et al. (1989). For production of transgenic animals, transgenes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

[0287] The antigen-binding protein or antigen-binding fragment thereof can be expressed using a single vector or two vectors. When the two chains of the antigen-binding protein or antigen-binding fragment thereof are cloned on separate expression vectors, the vectors can be co-transfected to obtain expression and assembly of an intact antigen-binding protein or antigen-binding fragment thereof.

[0288] Once expressed, the antigen-binding protein or antigen-binding fragment thereof can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis and the like. See generally Scopes, Protein Purification (Springer-Verlag, N. Y., (1982)). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity can be preferred, and 98 to 99% or more homogeneity can be most preferred, for pharmaceutical uses.

[0289] Pharmaceutical compositions

[0290] In one embodiment, provided herein is a pharmaceutical composition comprising (a) an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell expressing an antigenbinding protein or antigen-binding fragment thereof disclosed herein and (b) a pharmaceutically acceptable carrier.

[0291] Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. The pharmaceutical compositions may generally be formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0292] The terms “pharmaceutically acceptable,” “physiologically tolerable,” as referred to compositions, carriers, diluents, and reagents, are used interchangeably and include materials 62183465138.1are capable of administration to or upon a subject without the production of undesirable physiological effects to the degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” includes an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.

[0293] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the compositions disclosed herein, use of the media or compound in the compositions disclosed herein is contemplated. In some embodiments, a second therapeutic agent, such as an anti-cancer or anti-tumor agent, can also be incorporated into pharmaceutical compositions.

[0294] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate-buffered saline (PBS). The composition may be sterile and fluid to the extent that easy syringeability exists. In some embodiments, the compositions disclosed herein are stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0295] In some embodiments, the pharmaceutical composition further includes a cryoprotectant (e.g., glycerol, DMSO, PEG).

[0296] In some embodiments, the pharmaceutical composition will be suitable for administration to a subject, e.g., will be sterile. For example, in some embodiments, a subject pharmaceutical composition will be suitable for administration to a human subject, e.g., where the composition is sterile and is free of detectable pyrogens and / or other toxins and / or such detectable pyrogens and / or other toxins are below permissible limits.

[0297] Where a composition disclosed herein is administered as an injectable (e.g., subcutaneously, intraperitoneally, intramuscularly, and / or intravenously) directly into a tissue,63183465138.1a formulation can be provided as a ready -to-use dosage form, a non-aqueous form e.g., a reconstitutable storage-stable powder) or an aqueous form, such as liquid composed of pharmaceutically acceptable carriers and excipients. The formulations comprising an antigenbinding protein or antigen-binding fragment thereof disclosed herein may also be provided so as to enhance serum half-life of the subject protein following administration. For example, the antigen-binding protein or antigen-binding fragment thereof may be provided in a liposome formulation, prepared as a colloid, or other conventional techniques for extending serum halflife. A variety of methods are available for preparing liposomes are known in the art. The preparations may also be provided in controlled release or slow-release forms.

[0298] Other examples of formulations suitable for parenteral administration include isotonic sterile injection solutions, anti-oxidants, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. For example, a pharmaceutical composition can be present in a container, e.g., a sterile container, such as a syringe. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0299] The concentration of an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein in a formulation can vary widely (e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight) and will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected and the patient’s needs.

[0300] Methods

[0301] Provided herein are methods of making and methods of using the compositions disclosed herein, including the antigen-binding proteins or antigen-binding fragments thereof disclosed herein. The methods disclosed herein may be performed in vitro, in vivo, or ex vivo, as appropriate.64183465138.1

[0302] Discussion of various features of the methods of the invention and preferred embodiments apply mutatis mutandis to compositions for use of the invention, as appropriate.

[0303] Methods of producing genetically modified immune cells

[0304] Provided herein are methods that involve the genetic modification of immune cells, such lymphocytes.

[0305] The disclosure further provides a method of preparing the genetically modified lymphocytes disclosed herein. In one aspect, provided is a method comprising: (a) providing a plurality of lymphocytes; (b) introducing into the plurality of lymphocytes one or more nucleic acid molecules encoding an antigen-binding protein or antigen-binding fragment thereof to obtain a plurality of genetically modified lymphocytes; and (c) optionally, expanding the plurality of genetically modified lymphocytes in a cell culture medium. Alternatively viewed, provided is a method comprising: (a) introducing into a plurality of lymphocytes one or more nucleic acid molecules encoding an antigen-binding protein or antigen-binding fragment thereof to obtain a plurality of genetically modified lymphocytes; and (b) optionally, expanding the plurality of genetically modified lymphocytes in a cell culture medium. The lymphocytes may further be activated before or after genetic modification.

[0306] The term “culturing” or “expanding” refers to maintaining or cultivating cells under conditions in which they can proliferate and avoid senescence. For example, cells may be cultured in media optionally containing one or more growth factors, i.e., a growth factor cocktail. In some embodiments, the cell culture medium is a defined cell culture medium. The cell culture medium may include neoantigen peptides. Stable cell lines may be established to allow for the continued propagation of cells.

[0307] Cells may be further purified or enriched prior to use or storage.

[0308] Methods of producing an antigen-binding protein or antigen-binding fragment thereof

[0309] Provided herein is a method of making an antigen-binding protein or antigen-binding fragment thereof disclosed herein, the method comprising: (i) providing a cell comprising one or more nucleic acid molecules encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein; (ii) expressing in the cell the antigen-binding protein or antigen-binding fragment thereof; and (iii) collecting the antigen-binding protein or antigenbinding fragment thereof. Alternatively viewed, the present invention provides a method of making an antigen-binding protein or antigen-binding fragment thereof disclosed herein, the method comprising: (i) expressing in a cell the antigen-binding protein or antigen-binding fragment thereof, wherein the cell comprises one or more nucleic acid molecules encoding an 65183465138.1antigen-binding protein or antigen-binding fragment thereof disclosed herein; and (ii) collecting the antigen-binding protein or antigen-binding fragment thereof. In some embodiments, the antigen-binding protein or antigen-binding fragment thereof is further purified. The antigen-binding protein or antigen-binding fragment thereof may be secreted by the cell and collected from the cell medium. The antigen-binding protein or antigen-binding fragment thereof may be produced intracellularly and obtained after cell lysis.

[0310] In some embodiments, the cell is a Chinese Hamster Ovary (CHO) cell or a variant thereof like CHO-K1, CHO-S, CHO-DXB11, or CHO-DG44. In one embodiment, the cell is a Human Embryonic Kidney (HEK), e.g., a HEK293 or HEK293T, cell. In one embodiment, the cell is a Mouse Myeloma cell (e.g., NSO or Sp2 / 0).

[0311] Methods of detecting an antigen in a biological sample

[0312] Provided herein are kits for an antigen disclosed herein present in a sample, including in a biological or clinical sample. The antigen may be an NY-ESO-1 fragment, an hTERT fragment, or a Melan-A fragment. The antigen may be a fragment disclosed in Table 4.The antigen may comprise SEQ ID NO:43. The antigen may be presented in an HLA-DRB3*02:02 complex.

[0313] These kits may comprise an antigen-binding protein or antigen-binding fragment thereof disclosed herein and various reagents, for example, reagents that aid in detection of binding between the antigen-binding protein or antigen-binding fragment thereof and the antigen.

[0314] The term “biological sample” as used herein may refer to a sample obtained from an organism (e.g., patient) or from components (e.g., cells) of an organism. The sample may be of any biological tissue, cell(s) or fluid. The sample may be a “clinical sample” which is a sample derived from a subject, such as a human patient. Such samples include, but are not limited to, saliva, sputum, blood, blood cells (e.g., white cells), bodily fluids, lavages, pancreatic juices, gastric juices, discharges, CSF, lymph amniotic fluid, plasma, semen, bone marrow, and tissue or fine needle biopsy samples, urine, stool, peritoneal fluid, and pleural fluid, or cells therefrom, and any combinations thereof. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. A biological sample may also be referred to as a “patient sample.” A biological sample may also include a substantially purified or isolated protein, membrane preparation, or cell culture.

[0315] The kits may be used in vitro assays, such as immunoassays, e.g. enzyme immune assays (EIA), enzyme linked immunosorbent assay (ELISA), ELISPOT (enzyme-linked immunospot), radioimmunoassays (RIAs), immunofluorescence, and other assays known in 66183465138.1the art, including but not limited to Western Blot analysis and / or immunoprecipitation methods. The in vitro assays may be competitive, or indirect, such as in a sandwich assay, or may be an antibody capture method.

[0316] An illustrative, but non-limiting, direct ELISA protocol for detecting an antigen (including, but not limited to, NY-ESO-1) present in a sample is provided. For example, a buffered solution of an antigen, e.g., a sample containing NY-ESO-1 or an antigenic fragment thereof (e.g., a biological sample containing or suspected of containing NY-ESO-1) is added to a well of a microtiter plate, e.g. a 96-well plate. A solution of non-reacting protein, e.g. bovine serum albumin or casein is then added to the well. An antibody or antigen-binding fragment thereof disclosed herein conjugated to a reporter molecule enzyme is added, e.g. conjugated to horse-radish peroxidase, although that is not necessarily the enzyme, as other common enzymes include alkaline phosphatase, or P-D-galactosidase, although other enzymes are conceivable and considered embodied by the present disclosure. A substrate for the enzyme is then added, which leads to a detectable signal. For example, adding TMB to horseradish peroxidase leads to a colored product, in which case the ELISA is a colorimetric assay. ELISAs may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical.

[0317] An illustrative, but non-limiting, sandwich ELISA protocol for detecting an antigen (including, but not limited to, NY-ESO-1) present in a sample is provided. The capture antibody or antigen-binding fragment thereof (including, but not limited to, an antibody or antigen-binding fragment thereof disclosed herein) is bound to (i.e. “immobilized”) on a substrate, e.g. a microtiter plate. Antigen-containing sample (i.e. sample containing the antigen or an antigenic fragment thereof, is then added to the substrate at which point it is captured by the antibody or antigen-binding fragment thereof. The substrate is then washed to remove unbound antigen. A second antibody or antigen-binding fragment thereof targeting the same antigen (including, but not limited to, an antibody or antigen-binding fragment thereof disclosed herein) is added, which binds to a different epitope on the antigen. The second antibody or antigen-binding fragment thereof is bound to a reporter molecule, e.g., an enzyme, although the reporter molecule may be any molecule that leads to a detectable signal. The plate may be washed a second time, and in those instances where the reporter molecule is an enzyme, a substrate may be added, e.g., TMB, that results in a detectable signal (also a colorimetric assay).67183465138.1

[0318] Another type of common ELISA is competitive ELISA. An illustrative, but nonlimiting, competitive ELISA protocol for detecting an antigen (including, but not limited to, NY-ESO-1) present in a sample is provided. In these embodiments, an unlabeled antibody or antigen-binding fragment thereof targeting the antigen to be detected (including, but not limited to, an antibody or antigen-binding fragment thereof disclosed herein) is incubated in the presence of an antigen-containing sample (e.g.., a sample containing NY-ESO-1 or an antigenic fragment thereof), which is then added to an antigen-coated well. The plate is washed to remove unbound antibodies. A secondary antibody is added that is specific to the primary antibody or antigen-binding fragment thereof. The secondary antibody is bound to a reporter molecule, as described herein, such as an enzyme (or any other molecule that may lead to a detectable signal). Some competitive ELISAs utilize labeled antigens rather than labeled antibodies; the less antigen in the sample, the more labeled antigen is retained, and the stronger a detectable signal results.

[0319] Other forms of common in vitro assays include radioimmunoassays (RIAs). Typically, a known quantity of an antigen is linked to a radioactive tracer, e.g., 1-125 although others are suitable for use, which is then mixed with a known amount of antigen-binding protein or antigen-binding fragment thereof specific for the antigen. Then, a sample containing unknown quantity of an antigen is added, (e.g., a biological sample that contains or is suspected of containing the antigen) is added. This is a direct competitive for specific binding; as the concentration of unlabeled antigen is increased, the binding between the antigen-binding protein or antigen-binding fragment thereof and the labeled standard is decreased, which is directly measurable by measuring radioactivity. Other assays are known and a person of ordinary skill in the art would readily recognize their applicability.

[0320] In some embodiments, provided is a method of detecting an antigen in a sample. Such methods may utilize any of the assays described herein, or others that are known in the art. Several of the assays described herein are capable of quantifying the amount of antigen present in a sample, and so accordingly, in some embodiments, the present disclosure is directed to methods of quantifying the amount of antigen present in a sample, e.g., a biological sample.

[0321] Provided herein is a method of detecting an antigen in a sample, the method comprising contacting the sample with an antigen-binding protein or antigen-binding fragment thereof disclosed herein, wherein the antigen-binding protein or antigen-binding fragment thereof disclosed herein binds to the antigen.68183465138.1

[0322] Provided herein is a method of detecting a peptide antigen comprising SEQ ID NO:43 in a sample, the method comprising contacting the sample with an antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0323] Provided is a method of detecting the presence of antigen (including, but not limited to, NY-ESO-1), or an antigenic fragment thereof, in a sample, the method comprising:(a) obtaining a sample containing, or suspected of containing, the antigen (including, but not limited to, NY-ESO-1), or an antigenic fragment thereof;(b) contacting the sample with an antibody or antigen-binding fragment thereof disclosed herein; and(c) detecting the presence of specific binding of the antibody or antigen-binding fragment thereof to the antigen (including, but not limited to, NY-ESO-1), or an antigenic fragment thereof.

[0324] Also provided are kits or articles of manufacture for use in the methods described herein. In some embodiments, the kits comprise the compositions described herein in suitable packaging. Suitable packaging for compositions described herein is known in the art and includes, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and / or sealed.

[0325] Also provided are kits comprising the compositions described herein. These kits may further comprise instruction(s) on methods of using the composition, such as uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing the administration of the composition or performing any methods described herein. For example, in some embodiments, the kit comprises an antibody or antigen-binding fragment thereof, a pharmaceutically acceptable carrier suitable for injection, and one or more of: a buffer, a diluent, a filter, a needle, a syringe, and a package insert with instructions for performing the injections.

[0326] Methods of treatment

[0327] Provided are methods of treating a subject having a disease or disorder by administering a therapeutically effective amount of an antigen-binding protein or antigenbinding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigen- 69183465138.1binding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigenbinding fragment thereof disclosed herein. The cell may be an immune cell (such as a lymphocyte).

[0328] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of treatment. Also provided herein is a pharmaceutical composition for use in a method of treatment, wherein the pharmaceutical composition comprises an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigenbinding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein. The cell may be an immune cell (such as a lymphocyte). The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0329] In some embodiments, the cell or population of cells is washed or otherwise purified to remove cytokines before administration to the subject.

[0330] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (z.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically 70183465138.1significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0331] The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development.

[0332] An “effective amount” or “therapeutically effective amount” refers to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. In relation to ATC, an “effective amount” or “therapeutically effective amount” may also be expressed a certain or a minimal number of cells that are administered to a subject and that are capable of producing a medically desirable result in the treated subject.

[0333] As used herein, the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has undergone treatment in the past, is currently undergoing any form of treatment, or is receiving treatment in the future. As used herein, the terms “subject” and “subjects” may refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human). The subject may be a human or a non-human. In some embodiments, the subject is a human. In some embodiments, the subject is immune-depleted.

[0334] Provided herein is a method of treating cancer, the method comprising administering to a subject in need thereof an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigenbinding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein. In 71183465138.1some embodiments, the cancer comprises cells that express NY-ESO-1. In some embodiments, treating cancer comprises reducing tumor burden, inhibiting tumor growth, reducing tumor metastasis, inducing tumor cell death, or any combination thereof.

[0335] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of treating cancer, the method comprising administering to a subject in need thereof the antigen-binding protein or antigen-binding fragment thereof, the nucleic acid or set of nucleic acids, the vector, or the cell (or the population of cells). Also provided is a pharmaceutical composition for use in a method of treating cancer, wherein the pharmaceutical composition comprises an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein, and wherein the method comprises administering to a subject in need thereof the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent. In some embodiments, the cancer comprises cells that express NY-ESO-1. In some embodiments, treating cancer comprises reducing tumor burden, inhibiting tumor growth, reducing tumor metastasis, inducing tumor cell death, or any combination thereof.

[0336] The term “cancer” refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Accordingly, the term “cancer” as used herein refers to an uncontrolled growth of cells, which interferes with the normal functioning of the bodily organs and systems, including cancer stem cells and tumor vascular niches. A subject that has a cancer is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hematopoietic cancers, such as leukemia, are able to out- 72183465138.1compete the normal hematopoietic compartments in a subject, thereby leading to hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.

[0337] Provided herein is a method of reducing the growth of a tumor, the method comprising administering to a subject in need thereof an antigen-binding protein or antigenbinding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigenbinding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigenbinding fragment thereof disclosed herein. When reducing tumor growth, the rate of the growth of the tumor can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by 100%.

[0338] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of reducing the growth of a tumor (or for use in a method of treating cancer by reducing the growth of a tumor), the method comprising administering to a subject in need thereof the antigen-binding protein or antigen-binding fragment thereof, the nucleic acid or set of nucleic acids, the vector, or the cell (or the population of cells). Also provided is a pharmaceutical composition for use in a method of reducing the growth of a tumor (or for use in a method of treating cancer by reducing the growth of a tumor) wherein the pharmaceutical composition comprises an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein, and wherein 73183465138.1the method comprises administering to a subject in need thereof the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent When reducing tumor growth, the rate of the growth of the tumor can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by 100%.

[0339] Provided herein is a method of reducing cancer sternness, the method comprising administering to a subject in need thereof an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigenbinding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein. The term “reducing” may refer to reducing by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by 100%.

[0340] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of reducing cancer sternness (or for use in a method of treating cancer by reducing the cancer sternness), the method comprising administering to a subject in need thereof the antigen-binding protein or antigen-binding fragment thereof, the nucleic acid or set of nucleic, the vector, or the cell (or the population of cells). Also provided herein is a pharmaceutical composition for use in a method of reducing cancer sternness (or for use in a method of treating cancer by reducing the cancer sternness), wherein the pharmaceutical composition comprises an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or 74183465138.1antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein, and wherein the method comprises administering to a subject in need thereof the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent. The term “reducing” may refer to reducing by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or by 100%.

[0341] Methods for assessing cancer sternness are known in the art and include, but are not limited to: (1) flow cytometric analysis of cancer stem cell markers such as CD44, CD133, CD24, ALDH1, EpCAM, and lineage-specific markers; (2) in vitro sphere formation assays (tumorsphere or mammosphere assays) to assess self-renewal capacity; (3) in vivo limiting dilution assays to measure tumor-initiating capacity; (4) analysis of sternness-associated transcription factors such as SOX2, OCT4, NANOG, and KLF4 by qRT-PCR, Western blot, or immunohistochemistry; (5) functional assays measuring ALDH enzymatic activity using ALDEFLUOR reagent; and (6) gene expression profiling to assess sternness signatures. Reducing cancer sternness can be measured as a decrease in any of these parameters by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control.

[0342] Provided herein is a method of reducing tumor-associated fibrosis, the method comprising administering to a subject an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0343] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof 75183465138.1disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of reducing tumor-associated fibrosis (or for use in a method of treating cancer by reducing tumor-associated fibrosis), the method comprising administering to a subject the antigen-binding protein or antigen-binding fragment thereof, the nucleic acid or set of nucleic acids, the vector, or the cell (or the population of cells). Also provided is a pharmaceutical composition for use in a method of reducing tumor-associated fibrosis (or for use in a method of treating cancer by reducing tumor-associated fibrosis), wherein the pharmaceutical composition comprises an antigenbinding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein, and wherein the method comprises administering to a subject the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent

[0344] Methods for assessing tumor-associated fibrosis are known in the art and include, but are not limited to: (1) histological staining methods such as Masson's trichrome staining, Sirius red staining, or picrosirius red staining to visualize collagen deposition; (2) immunohistochemical or immunofluorescent staining for fibrosis markers including a-smooth muscle actin (a-SMA), fibroblast activation protein (a-FAP), vimentin, and collagen isoforms (collagen I, III, IV); (3) gene expression analysis (qRT-PCR or RNA-seq) of fibrosis-associated genes including COL1A1, COL3A1, FN1, ACTA2, TGFB1, and CTGF; (4) quantitative analysis of collagen content using hydroxyproline assays; (5) imaging methods such as second harmonic generation (SHG) microscopy to quantify fibrillar collagen; and (6) measurement of tissue stiffness using atomic force microscopy or rheological methods. Reducing tumor-associated fibrosis can be measured as a decrease in any of these parameters by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control.

[0345] Provided herein is a method of reducing tumor metastasis, the method comprising administering to a subject in need thereof an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigenbinding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof 76183465138.1disclosed herein, a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0346] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of reducing tumor metastasis (or for use in a method of treating cancer by reducing tumor metastasis), the method comprising administering to a subject in need thereof the antigen-binding protein or antigenbinding fragment thereof, the nucleic acid or set of nucleic acids, the vector, or the cell (or the population of cells). Also provided herein is a pharmaceutical composition for use in a method of reducing tumor metastasis (or for use in a method of treating cancer by reducing tumor metastasis), wherein the pharmaceutical composition comprises an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigenbinding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein, and wherein the method comprises administering to a subject in need thereof the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0347] Methods for assessing tumor metastasis are known in the art and include, but are not limited to: (1) in vivo metastasis models including spontaneous metastasis models (orthotopic implantation followed by monitoring of distant organ metastasis) and experimental metastasis models (intravenous or intracardiac injection of tumor cells followed by analysis of organ colonization); (2) quantification of metastatic nodules on organ surfaces (e.g., lung surface nodule counting); (3) histological analysis of tissue sections to identify micrometastases; (4) bioluminescence or fluorescence imaging of tumor cells engineered to express luciferase or 77183465138.1fluorescent proteins; (5) detection and quantification of circulating tumor cells (CTCs) in blood using flow cytometry, immunomagnetic separation, or microfluidic devices; (6) detection of disseminated tumor cells (DTCs) in bone marrow or other organs; (7) molecular methods such as qRT-PCR for tumor-specific transcripts in distant organs; and (8) non-invasive imaging methods such as MRI, CT, or PET scans. Reducing tumor metastasis can be measured as a decrease in any of these parameters by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control.

[0348] Provided herein is a method of increasing cytokine production in the tumor microenvironment, the method comprising administering to a subject in need thereof an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigenbinding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein. Methods of measuring cytokines are known in the art. The cytokines may be pro-inflammatory cytokines. For example, pro-inflammatory cytokines include, but are not limited, to IL-1, IL-2, IL-6, IL-8, IL- 12, IL- 17, IL- 18, IFN-gamma, TNF-alpha, perforin, and granzyme B. Expression of these cytokines can be measured on the nucleic acid level (e.g., by RT-PCT) or on the protein level (e.g., by ELISA).

[0349] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of increasing cytokine production in the tumor microenvironment (or for use in a method of treating cancer by increasing cytokine production in the tumor microenvironment), the method comprising administering to a subject in need thereof the antigen-binding protein or antigen-binding fragment thereof, the nucleic acid or set of nucleic acids, the vector, or the cell (or the 78183465138.1population of cells). Also provided is a pharmaceutical composition for use in a method of increasing cytokine production in the tumor microenvironment (or for use in a method of treating cancer by increasing cytokine production in the tumor microenvironment), wherein the pharmaceutical composition comprises an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein, and wherein the method comprises administering to a subject in need thereof the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent. Methods of measuring cytokines are known in the art. The cytokines may be pro-inflammatory cytokines. For example, pro-inflammatory cytokines include, but are not limited, to IL-1, IL-2, IL-6, IL-8, IL- 12, IL- 17, IL- 18, IFN-gamma, TNF-alpha, perforin, and granzyme B. Expression of these cytokines can be measured on the nucleic acid level (e.g., by RT-PCT) or on the protein level (e.g., by ELISA).

[0350] Provided is a method of reducing T cell exhaustion in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to a subject in need thereof an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigenbinding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0351] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or 79183465138.1antigen-binding fragment thereof disclosed herein for use in a method of reducing T cell exhaustion in a subject with cancer (or for use in a method of treating cancer by reducing T cell exhaustion in a subject with cancer), the method comprising administering to a subject in need thereof the antigen-binding protein or antigen-binding fragment thereof, the nucleic acid or set of nucleic acids, the vector, or the cell (or the population of cells). Also provided is a pharmaceutical composition for use in a method of reducing T cell exhaustion in a subject with cancer (or for use in a method of treating cancer by reducing T cell exhaustion in a subject with cancer), wherein the pharmaceutical composition comprises an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigenbinding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein, and wherein the method comprises administering to a subject in need thereof the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0352] Methods for assessing T cell exhaustion are known in the art and include, but are not limited to: (1) flow cytometric analysis of exhaustion markers including PD-1, TIM-3, LAG-3, TIGIT, 2B4, CD 160, CTLA-4, and BTLA, with co-expression of multiple inhibitory receptors being indicative of advanced exhaustion; (2) functional assays measuring T cell proliferative capacity, cytokine production (particularly IFN-y, TNF-a, and IL-2), and cytotoxic activity; (3) transcriptional profiling to assess expression of exhaustion-associated genes such as PDCD1, HAVCR2, LAG3, TIGIT, TOX, and NR4A1; (4) epigenetic analysis to assess chromatin accessibility and DNA methylation patterns associated with exhaustion; (5) assessment of metabolic dysfunction characteristic of exhausted T cells, including impaired glycolysis and mitochondrial function; and (6) evaluation of T cell differentiation state and loss of memory potential. Reducing T cell exhaustion can be measured as a decrease in exhaustion markers or an increase in functional capacity by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control.

[0353] As used herein, the terms “increasing” and “decreasing” (or “reducing”) are meant in reference to a control. For example, the increase or decrease could be by at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 9-fold, at least 8-fold, at least 10-fold, at least 15-fold, or at 80183465138.1least 20-fold. A person skilled in the art can readily choose an appropriate control for a given application. For example, a control could refer to a sample, cell, tissue, or patient that is not exposed to composition disclosed herein. A control could refer to the same sample, cell, tissue, or patient but at an earlier time. A control value may be derived from a single sample, cell, tissue, or patient. Alternatively, a control value may be calculated for a population of samples, cells, tissues, or patients. Depending on the application, the control may be a sample, cell, or tissue derived from a patient with a disease or disorder. The control patient may be a person who has a disease or disorder. The control may be a sample, cell, or tissue derived from a healthy patient. The control patient may be a healthy patient.

[0354] Provided herein is a method of increasing anti-tumor immunity, the method comprising administering to a subject in need thereof an antigen-binding protein or antigenbinding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigenbinding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigenbinding fragment thereof disclosed herein.

[0355] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of increasing anti -tumor immunity (or for use in a method of treating cancer by increasing anti-tumor immunity), the method comprising administering to a subject in need thereof the antigen-binding protein or antigen-binding fragment thereof, the nucleic acid or set of nucleic acids, the vector, or the cell (or the population of cells). Also provided is a pharmaceutical composition for use in a method of increasing anti-tumor immunity (or for use in a method of treating cancer by increasing antitumor immunity), wherein the pharmaceutical composition comprises an antigen-binding 81183465138.1protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein, wherein the method comprises administering to a subject in need thereof the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0356] Provided herein is a method of increasing infiltration of a tumor with immune cells, the method comprising administering to a subject in need thereof an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigenbinding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigenbinding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0357] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of increasing infiltration of a tumor with immune cells (or for use in a method of treating cancer by increasing infiltration of a tumor with immune cells), the method comprising administering to a subject in need thereof the antigen-binding protein or antigen-binding fragment thereof, the nucleic acid or set of nucleic acids, the vector, or the cell (or the population of cells). Also provided is a pharmaceutical composition for use in a method of increasing infiltration of a tumor with immune cells (or for use in a method of treating cancer by increasing infiltration of a tumor with immune cells), wherein the pharmaceutical composition comprises an antigen-binding 82183465138.1protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein, and wherein the method comprises administering to a subject in need thereof the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0358] Provided herein is a method of reducing T cell tolerance, the method comprising administering to a subject in need thereof an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigenbinding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein or a pharmaceutical composition comprising an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein.

[0359] Also provided herein is an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigen-binding protein or antigen-binding fragment thereof disclosed herein for use in a method of reducing T cell tolerance (or for use in a method of treating cancer by reducing T cell tolerance), the method comprising administering to a subject in need thereof the antigen-binding protein or antigenbinding fragment thereof, the nucleic acid or set of nucleic acids, the vector, or the cell (or the population of cells). Also provided is a pharmaceutical composition for use in a method of reducing T cell tolerance (or for use in a method of treating cancer by reducing T cell tolerance), wherein the pharmaceutical composition comprises an antigen-binding protein or antigenbinding fragment thereof disclosed herein, a nucleic acid or set of nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, a vector 83183465138.1comprising one or more nucleic acids encoding an antigen-binding protein or antigen-binding fragment thereof disclosed herein, or a cell (or a population of cells) expressing an antigenbinding protein or antigen-binding fragment thereof disclosed herein, and wherein the method comprises administering to a subject in need thereof the pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, excipient or diluent.

[0360] In one embodiment, the subject has a cancer, wherein at least some of the cancer cells express NY-ESO1. In one embodiment, the subject has a cancer, wherein at least some of the cancer cells present an epitope derived from NY-ESO1 on their surface. In one embodiment, the subject has a cancer, wherein at least some of the cancer cells present an epitope comprising SEQ ID NO:43 on their surface. In one embodiment, the subject has a cancer, wherein at least some of the cancer cells present an epitope comprising SEQ ID NO:43 on their surface in an HLA-DRB3*02:02 complex. A person skilled in the art can easily detect whether the subject has a cancer, wherein at least some of the cancer cells present an epitope derived from NY-ESO1 on their surface, using for example, qPCR or any of the methods for the detection of NY-ESO1 disclosed herein.

[0361] Provided is a method of treating cancer in a subject in need thereof, the method comprising:(a) isolating a population of immune cells from the subject or a donor, producing an isolated population of immune cells;(b) genetically modifying the isolated population of immune cells to express an antigen-binding protein or antigen-binding protein fragment thereof disclosed herein to produce a genetically modified population of immune cells;(c) optionally, expanding the genetically modified population of immune cells, producing an expanded population of immune cells; and(d) administering the expanded population of immune cells to the subject.

[0362] Also provided is a population of immune cells for use in a method of treating cancer in a subject in need thereof, the method comprising administering the population of immune cells to the subject, wherein the population of immune cells has been prepared by a method comprising:(a) isolating a population of immune cells from the subject or a donor, producing an isolated population of immune cells;84183465138.1(b) genetically modifying the isolated population of immune cells to express an antigen-binding protein or antigen-binding fragment thereof disclosed herein to produce a genetically modified population of immune cells; and(c) optionally, expanding the genetically modified population of immune cells, producing an expanded population of immune cells.

[0363] In one embodiment, at least some of the cells of the cancer express NY-ESO-1.

[0364] In some embodiments, the population of immune cells isolated from the subject or donor comprises CD34+hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells.

[0365] In some embodiments, the population of immune cells isolated from the subject or donor comprises lymphocytes or ILCs.

[0366] In some embodiments, the population of immune cells isolated from the subject or donor are obtained from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, or tumors.

[0367] Provided herein is a method of increasing the cytotoxic activity of a lymphocyte against tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express an antigen-binding protein or antigen-binding protein fragment thereof disclosed herein. The cytotoxic activity may be increased by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold compared to an unmodified lymphocyte or a lymphocyte modified to express a control antigen-binding protein, as measured by assays known in the art including, but not limited to, lactate dehydrogenase (LDH) release assays, chromium release assays, or real-time cytotoxicity monitoring.

[0368] Provided herein is a method of increasing cytokine secretion by a lymphocyte in the presence of tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express the antigen-binding protein or antigen-binding protein fragment thereof disclosed herein. Cytokine secretion may be increased by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold compared to an unmodified lymphocyte or a lymphocyte modified to express a control antigen-binding protein, as measured by assays known in the art.

[0369] Provided herein is a method of increasing the effector functions of a lymphocyte in the presence of tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express the antigen-binding protein or antigen-binding protein fragment thereof disclosed herein. Effector function(s) may be increased by at least 1.5-fold,85183465138.1at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold compared to an unmodified lymphocyte or a lymphocyte modified to express a control antigen-binding protein, as measured by assays known in the art.

[0370] As used herein, "effector functions" of lymphocytes include, but are not limited to, cytotoxic activity (killing of target cells), cytokine secretion (e.g., IFN-y, TNF-a, IL-2, IL-4, IL-5, IL-13), chemokine secretion, proliferative capacity, degranulation (release of perforin, granzymes), expression of activation markers (e.g., CD25, CD69, CD 107a), and metabolic activity. Effector functions can be measured using assays known in the art, including but not limited to: (1) cytotoxicity assays such as LDH release assays, chromium release assays, or real-time cytotoxicity assays; (2) cytokine / chemokine measurements by ELISA, multiplex assays (e.g., Legendplex), or intracellular cytokine staining followed by flow cytometry; (3) degranulation assays measuring CD 107a surface expression by flow cytometry; (4) proliferation assays using CFSE dilution, thymidine incorporation, or Ki-67 staining; and (5) metabolic assays measuring glucose uptake, oxygen consumption, or extracellular acidification rate.

[0371] Cancers that can be treated by the compositions and methods disclosed herein include tumors that are not vascularized or are not substantially vascularized, as well as vascularized tumors. Cancers may comprise non-solid tumors (such as hematologic tumors, e.g., leukemias and lymphomas) or may comprise solid tumors. The types of cancers to be treated with the pharmaceutical compositions disclosed herein include, but are not limited to, carcinoma, blastoma and sarcoma, and certain leukemias or malignant lymphoid tumors, benign and malignant tumors and malignancies, e.g., sarcomas, carcinomas, and melanomas. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0372] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or haematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous myelogenous leukemia, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma (indolent and high-grade forms), myeloma Multiple, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0373] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. The different types of solid tumors are named 86183465138.1for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovium, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, carcinoma of the sweat gland, medullary thyroid carcinoma, papillary thyroid carcinoma, sebaceous gland carcinoma of pheochromocytomas, carcinoma papillary, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as glioma) (such as brainstem glioma and mixed gliomas), glioblastoma (also astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis).

[0374] In some embodiments, the tumor / cancer to be treated is selected from adrenal gland tumors, biliary cancer, bladder cancer, brain cancer, breast cancer, carcinoma, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer or hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Li-Fraumeni tumors, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumors, ovarian cancer, pancreatic cancer, pancreatic islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, urogenital cancer, and uterine cancer.

[0375] In some embodiments, the cancer is a cancer known to express NY-ESO-1, including but not limited to melanoma, synovial sarcoma, myxoid / round cell liposarcoma, neuroblastoma, hepatocellular carcinoma, esophageal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, non-small cell lung cancer, and multiple myeloma. The expression of NY-ESO-1 in the subject’s cancer may be confirmed prior to treatment using diagnostic methods known in the art.

[0376] The pharmaceutical compositions, as described, can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of 87183465138.1administration will be determined by factors such as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages can be determined by clinical trials. The precise amount of the compositions disclosed herein to be administered can be determined by a physician having account for individual differences in age, weight, tumor size, extent of infection or metastasis, and patient’s condition (subject).

[0377] In some embodiments, the subject is selected for treatment based on expression of NY-ESO-1 in the subject's cancer cells. Methods for determining NY-ESO-1 expression include, but are not limited to, immunohistochemistry, quantitative RT-PCR, RNA sequencing, Western blot analysis, and flow cytometry. In some embodiments, the subject is selected for treatment based on HLA genotyping, wherein the subject expresses HLA-DRB3*02:02 or another MHC class II allele that can present NY-ESO-1 -derived peptides.

[0378] In some embodiments, the amount of a composition disclosed herein administered is in the range of about 0.001 mg / kg to about 1000 mg / kg of patient body weight, and any range in between. Depending on the type and severity of the infection, about 0.1 mg / kg to about 50 mg / kg body weight (for example, about 0.1-15 mg / kg / dose) of a composition disclosed herein can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The compositions disclosed herein can be delivered relatively low volume rates, for example but not necessarily from about 0.001 ml / day to 10 ml / day so as to minimize tissue disturbance or trauma near the site where the formulation is released. The formulation may be released at a rate of, depending on the specific biological agent(s), at a low dose, e.g., from about 0.01 pg / hr or 0.1 pg / hr, 0.25 pg / hr, 1 pg / hr, generally up to about 200 pg / hr, or the formulation is delivered at a low volume rate e.g., a volume rate of from about 0.001 ml / day to about 1 ml / day, for example, 0.01 micrograms per day up to about 20 milligrams per day. Dosage depends on a number of factors such as potency, bioavailability, and toxicity of the active ingredient used and the requirements of the subject. The progress of this therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0379] In some embodiments, the composition disclosed herein is administered to the mammal by intravenous infusion, z.e., introduction of the composition disclosed herein into the vein of a mammal over a certain period of time. In some embodiments, the period of time is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.88183465138.1

[0380] In some embodiments, a dose of a composition disclosed herein is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, once every two weeks, or once a month. In other embodiments, two, three or four doses of a composition disclosed herein is administered to a subject every day, every couple of days, every third day, once a week, once every two weeks or once a month. In some embodiments, a dose(s) of a composition disclosed herein is administered for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days or 28 days. In some embodiments, a dose of a composition disclosed herein is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, or 6 months.

[0381] It can generally be stated that a pharmaceutical composition comprising ells described herein can be administered at a dose of 104to 109cells / kg body weight, e.g., 105to 106cells / kg body weight, including all values integers within these intervals. The compositions can also be administered several times at these dosages. The genetically modified cells disclosed herein can be administered using infusion techniques that are commonly known in immunotherapy see, e.g., Rosenberg et al., Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report, N Engl J Med. 1988 Dec 22;319(25): 1676-80). The optimal dose and treatment regimen for a particular patient can be determined by one skilled in the art of medicine by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0382] In some embodiments, the genetically modified cells are administered after lymphodepleting chemotherapy. Non-limiting examples of lymphodepleting agents include cyclophosphamide, fludarabine, and a combinations thereof. Without wishing to be bound by theory, lymphodepletion may enhance the engraftment, expansion, and persistence of adoptively transferred cells by reducing competition for homeostatic cytokines and eliminating immunosuppressive regulatory T cells.

[0383] The administration of the present compositions can be carried out in any convenient way, including infusion or injection (i.e., intravenous, intrathecal., intramuscular, intraluminal., intratracheal., intraperitoneal., or subcutaneous), transdermal administration, or other methods known in the art. Administration can be once every two weeks, once a week, or more often, but the frequency may be decreased during a maintenance phase of the disease or disorder. In some embodiments, the composition is administered by intravenous infusion.

[0384] Combination therapies89183465138.1

[0385] In certain cases, the compositions described herein are administered to a patient together with (e.g., before, simultaneously, or consecutively) any number of relevant treatment modalities. In some embodiments, the method may further include administering to the subject a second therapeutic agent. In some embodiments, the second therapeutic agent is an anticancer or anti-tumor agent. In some embodiments, the composition is administered to the subject before, after, or concurrently with the second therapeutic agent.

[0386] “ Combination” therapy, as used herein, unless otherwise clear from the context, is meant to encompass administration of two or more therapeutic agents in a coordinated fashion, and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time.

[0387] Also described herein, the compositions disclosed herein can be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablating agents such as CAMPATH, anti-cancer antibodies. CD3 or other antibody therapies, cytoxine, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.

[0388] The compositions described herein can also be administered to a patient together with (e.g., before, simultaneously or after) bone marrow transplantation, therapy with T lymphocyte ablation using chemotherapy agents such as fludarabine, radiation therapy external beam (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. Also described herein, the compositions can be administered after ablative therapy of B lymphocytes, such as agents that react with CD20, for example, Rituxan. For example, subjects may undergo standard treatment with high-dose chemotherapy followed by transplantation of peripheral blood stem cells. In certain cases, after transplantation, the subjects receive an infusion of the expanded lymphocytes, or the expanded lymphocytes are administered before or after surgery.

[0389] In some embodiments, the method further comprises administering a therapeutically effective amount of an immune checkpoint modulator. Accordingly, in some embodiments, the pharmaceutical compositions disclosed herein are administered with a checkpoint inhibitor. Checkpoint proteins interact with specific ligands that send a signal into the T cell and switch 90183465138.1off or inhibit T cell function. By expressing high levels of checkpoint proteins on their surface, cancer cells can control the function of T cells that enter the tumor microenvironment, thus suppressing the anticancer immune response. Examples of immune checkpoint modulators include PD1, PDL1, CTLA4, TIM3, LAG3, and TRAIL. The immune checkpoint protein Programmed Death-1 (PD-1) is a key immune checkpoint receptor ex-pressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, Programmed Death Ligand-1 (PD-L1) and Programmed Death Ligand-2 (PD-L2), that are expressed on antigen-presenting cells as well as many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1. Inhibition of the PD-1 / PD-L1 interaction can promote potent antitumor activity. Examples of PD-1 inhibitors include, but are not limited to, Pembrolizumab (MK-3475), Nivolumab (MDX-1106), Cemiplimab-rwlc (REGN2810), Pidilizumab (CT-011), Spartalizumab (PDR001), tislelizumab (BGB-A317), PF-06801591, AK105, BCD-100, BI 754091, JS001, LZM009, MEDI0680, MGA012, Sym021, TSR-042. Examples of PD-L1 inhibitors include, but are not limited to, Atezolizumab (MPDL3280A), Durvalumab (MEDI4736), Avelumab (MSB0010718C), BGB-A333, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316. The checkpoint modulators may be administered simultaneously, separately, or concurrently with the pharmaceutical compositions disclosed herein.

[0390] In some embodiments, the method further comprises administering a therapeutically effective amount of a “chemotherapeutic agent,” which is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, methyldopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBLTMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin,91183465138.1phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotics chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; 92axotere92e; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®.; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2 2’’ -tri chlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 92axoter, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; 92axote; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal 92183465138.1agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, 93axote, gemcitabine, KRAS mutation covalent inhibitors and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Additional examples include irinotecan, oxaliplatinum, and other standard colon cancer regimens.

[0391] In some embodiments, the second therapeutic agent is selected from the group consisting of taxotere, carboplatin, trastuzumab, epirubicin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, etoposide, 5-FU, gemcitabine, methotrexate, and paclitaxel, mitoxantrone, epothilone B, epidermal-growth factor receptor (EGFR)-targeting monoclonal antibody 7A7.27, vorinostat, romidepsin, docosahexaenoic acid, bortezomib, shikonin, an oncolytic virus, and a combinations thereof.

[0392] In some embodiments, the second therapeutic agent is selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and a combinations thereof.

[0393] Numbered embodiments

[0394] Also provided herein are numbered embodiments to illustrate, but not to limit, the invention.

[0395] Embodiment 1. An antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen comprises SEQ ID NO:43 (LKEFTVSGNILTIRL), wherein the first variable domain comprises complementarity determining region (CDR) al, CDRa2, and CDRa3, wherein the second variable domain comprises CDRbl, CDRb2, and CDRb3, and wherein: (a) CDRal comprises SEQ ID NO:1; (b) CDRa2 comprises SEQ ID NO:2 or SEQ ID NO:3; (c) CDRa3 comprises any one of SEQ ID NOs:4-7; (d) CDRbl comprises SEQ ID NO:8 or SEQ ID NO:9; (e) CDRb2 comprises SEQ ID NO: 10 or SEQ ID NO: 11; and (f) CDRb3 comprises any one of SEQ ID NOs: 12-15.

[0396] Embodiment 2. The antigen-binding protein or antigen-binding fragment thereof of embodiment 1, wherein: (a) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:4; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 12; (b) CDRal comprises SEQ ID NO:1;93183465138.1CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:5; CDRbl comprises SEQ ID NO:9; CDRb2 comprises SEQ ID NO: 11; and CDRb3 comprises SEQ ID NO: 13; (c) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:3; CDRa3 comprises SEQ ID NO:6; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 14; or (d) CDRal comprises SEQ ID NO:1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:7; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 15.

[0397] Embodiment 3. The antigen-binding protein or antigen-binding protein fragment thereof of embodiment 1 or 2, wherein the antigen-binding protein is a T cell receptor (TCR).

[0398] Embodiment 4. The antigen-binding protein or antigen-binding protein fragment thereof of embodiment 3, wherein: (a) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17; (b) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19; (c) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21; or (d) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23.

[0399] Embodiment 5. The antigen-binding protein or antigen-binding protein fragment thereof of embodiment 4, wherein: (a) the first variable domain comprises SEQ ID NO: 16 and the second variable domain comprises SEQ ID NO: 17; (b) the first variable domain comprises SEQ ID NO: 18 and the second variable domain comprises SEQ ID NO: 19; (c) the first variable domain comprises SEQ ID NO:20 and the second variable domain comprises SEQ ID NO:21;94183465138.1or (d) the first variable domain comprises SEQ ID NO:22 and the second variable domain comprises SEQ ID NO:23.

[0400] Embodiment 6. The antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 3-5, wherein the first variable domain is further joined to a constant region comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:75 and wherein the second variable domain is further joined to a constant region comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:76 or SEQ ID NO:77.

[0401] Embodiment 7. The antigen-binding protein or antigen-binding protein fragment thereof of embodiment 6, wherein the first variable domain is further joined to a sequence comprising SEQ ID NO:75 and wherein the second variable domain is further joined to a sequence comprising SEQ ID NO:76 or SEQ ID NO:77.

[0402] Embodiment 8. The antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 3-7, wherein the TCR is: (a) a single chain TCR; or (b) a membrane-bound TCR or soluble TCR.

[0403] Embodiment 9. The antigen-binding protein or antigen-binding protein fragment thereof of embodiment 1 or 2, wherein the antigen-binding protein is an antibody or antigenbinding protein fragment thereof.

[0404] Embodiment 10. The antigen-binding protein or antigen-binding protein fragment thereof of embodiment 9, wherein the antibody or antigen-binding protein fragment thereof is a single-chain variable fragment (scFv), fragment variable (Fv), fragment binding domain (Fab), Fab', F(ab')2, or diabody.

[0405] Embodiment 11. The antigen-binding protein or antigen-binding protein fragment thereof of embodiment 1 or 2, wherein the antigen-binding protein is a chimeric antigen receptor (CAR), optionally comprising a scFv comprising the first variable domain and a second variable domain.

[0406] Embodiment 12. The antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 1-11, wherein the antigen-binding protein or antigenbinding protein fragment thereof binds to the antigen present in a complex with HLA-DRB3*02:02.

[0407] Embodiment 13. The antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 1-12, wherein the antigen-binding protein or antigenbinding protein fragment thereof is deglycosylated.95183465138.1

[0408] Embodiment 14. The antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 1-13, wherein the antigen-binding protein or antigenbinding protein fragment thereof is conjugated to a fluorescent moiety, a detectable moiety, a purification moiety, or a combination thereof.

[0409] Embodiment 15. A cell expressing on its surface the antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 3-8 or 11-14, optionally wherein the cell is isolated.

[0410] Embodiment 16. The cell of embodiment 15, wherein the cell is a lymphocyte.

[0411] Embodiment 17. The cell of embodiment 16, wherein the lymphocyte is a T cell.

[0412] Embodiment 18. The cell of embodiment 17, wherein the T cell is a CD4+T cell.

[0413] Embodiment 19. The cell of embodiment 17, wherein the T cell is a CD8+T cell.

[0414] Embodiment 20. The cell of embodiment 16, wherein the lymphocyte is a natural killer (NK) cell.

[0415] Embodiment 21. The cell of any one of embodiments 15-20, wherein the cell further expresses a therapeutic protein.

[0416] Embodiment 22. The cell of embodiment 21, wherein therapeutic protein is IL-2, IL-2 mutein, IL- 15, CD40L, IL-33, IL- 12, a PD1 inhibitor, an PD-L1 inhibitor, a TIGIT inhibitor, or a combination thereof.

[0417] Embodiment 23. A cell expressing the antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 9, 10 or 12-14, optionally wherein the cell is isolated.

[0418] Embodiment 24. The cell of embodiment 23, wherein the cell is a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK) cell, optionally, a HEK293 or HEK293T cell.

[0419] Embodiment 25. A nucleic acid or set of nucleic acids encoding the antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 1-14, optionally wherein the nucleic acid or set of nucleic acids are isolated.

[0420] Embodiment 26. A vector or set of vectors comprising the nucleic acid or set of nucleic acids of embodiment 25, optionally wherein the vector or set of vectors are isolated.

[0421] Embodiment 27. A pharmaceutical composition comprising (a) the antigen-binding protein or antigen-binding protein fragment thereof of embodiments 9, 10, or 12-14, a cell of any one of embodiments 15-22, the nucleic acid or set of nucleic acids of embodiment 25, or the vector or set of vectors of embodiment 26 and (b) a pharmaceutically acceptable excipient.96183465138.1

[0422] Embodiment 28. A method of producing an antigen-binding protein or antigenbinding protein fragment thereof, the method comprising culturing a cell of embodiment 23 or 24 under conditions wherein the antigen-binding protein or antigen-binding protein fragment thereof is produced by the cell.

[0423] Embodiment 29. A method of increasing the cytotoxic activity of a lymphocyte against tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express the antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 3-8 or 11-14. A suitable control may include a lymphocyte that is not genetically modified, a lymphocyte that does not express the antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 3-8 or 11-14, or a lymphocyte that expresses a control antigen-binding protein or antigen-binding protein fragment thereof.

[0424] Embodiment 30. A method of increasing cytokine secretion by a lymphocyte in the presence of tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express the antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 3-8 or 11-14. A suitable control may include a lymphocyte that is not genetically modified, a lymphocyte that does not express the antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 3-8 or 11-14, or a lymphocyte that expresses a control antigen-binding protein or antigen-binding protein fragment thereof.

[0425] Embodiment 31. A method of increasing the effector functions of a lymphocyte in the presence of tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express the antigen-binding protein or antigen-binding protein fragment thereof of any one of embodiments 3-8 or 11-14. A suitable control may include a lymphocyte that is not genetically modified, a lymphocyte that does not express the antigenbinding protein or antigen-binding protein fragment thereof of any one of embodiments 3-8 or 11-14, or a lymphocyte that expresses a control antigen-binding protein or antigen-binding protein fragment thereof.

[0426] Embodiment 32. A method of any one of embodiments 29-30, wherein the lymphocyte is a CD4+T cell.

[0427] Embodiment 33. A method of any one of embodiments 29-30, wherein the lymphocyte is a CD8+T cell.

[0428] Embodiment 34. A method of any one of embodiments 29-30, wherein the lymphocyte is an NK cell.97183465138.1

[0429] Embodiment 35. A method of reducing tumor growth in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient.

[0430] Embodiment 36. A method of reducing cancer sternness in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject the cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient.

[0431] Embodiment 37. A method of reducing tumor-associated fibrosis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient.

[0432] Embodiment 38. A method of reducing tumor metastasis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient.

[0433] Embodiment 39. A method of reducing T cell exhaustion in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject the cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient.

[0434] Embodiment 40. A method of increasing anti -tumor immunity in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient.

[0435] Embodiment 41. A method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express New York esophageal squamous cell carcinoma 1 (NY-ESO-1), the method comprising administering to the subject the cell of any one of98183465138.1embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient.

[0436] Embodiment 42. A method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express NY-ESO-1, the method comprising: (a) isolating a population of immune cells from the subject or a donor, producing an isolated population of immune cells; (b) genetically modifying the isolated population of immune cells to express the antigen-binding protein or antigen-binding fragment thereof of any one of embodiments 3-8 or 11-14 to produce a genetically modified population of immune cells; (c) expanding the genetically modified population of immune cells, producing an expanded population of immune cells; and (d) administering the expanded population of immune cells to the subject.

[0437] Embodiment 43. The method of embodiment 42, wherein the immune cells are lymphocytes.

[0438] Embodiment 44. The method of embodiment 42, wherein the immune cells are innate lymphoid cells (ILCs).

[0439] Embodiment 45. The method of embodiment 43, wherein the lymphocytes are CD4+T cells.

[0440] Embodiment 46. The method of embodiment 43, wherein the lymphocytes are CD8+T cells.

[0441] Embodiment 47. The method of embodiment 43, wherein the lymphocytes are NK cells.

[0442] Embodiment 48. The method of any one of embodiments 42-47, the method further comprising expanding the isolated population of immune cells after step (a).

[0443] Embodiment 49. The method of any one of embodiments 35-48, wherein the subject has one or more cancers selected from the group consisting of adrenal gland tumors, biliary cancer, bladder cancer, brain cancer, breast cancer, carcinoma, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer or hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Li-Fraumeni tumors, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumors, ovarian cancer, pancreatic cancer, pancreatic islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, urogenital cancer, and uterine cancer.99183465138.1

[0444] Embodiment 50. The method of any one of embodiments 35-48, wherein the subject has a solid tumor.

[0445] Embodiment 51. The method of any one of embodiments 35-50, the method further comprising administering to the subject an additional therapeutic agent.

[0446] Embodiment 52. The method of embodiment 51, wherein the additional therapeutic agent is selected from the group consisting of taxotere, carboplatin, trastuzumab, epirubicin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, etoposide, 5-FU, gemcitabine, methotrexate, and paclitaxel, mitoxantrone, epothilone B, epidermal-growth factor receptor (EGFR)-targeting monoclonal antibody 7A7.27, vorinostat, romidepsin, docosahexaenoic acid, bortezomib, shikonin, an oncolytic virus, and a combinations thereof.

[0447] Embodiment 53. The method of embodiment 51, wherein the additional therapeutic agent is selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and a combinations thereof.

[0448] Embodiment 54. The method of embodiment 51, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

[0449] Embodiment 55. The method of embodiment 54, wherein the immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof.

[0450] Embodiment 56. The method of embodiment 54 or 55, wherein the immune checkpoint inhibitor is a PD1 inhibitor, a PD-L1 inhibitor, a CD28 inhibitor, a CTLA4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, or a combination thereof.

[0451] Embodiment 57. The method of any one of embodiments 35-56, wherein the subject is a human.

[0452] Embodiment 58. The cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing tumor growth in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

[0453] Embodiment 59. The cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing cancer sternness in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.100183465138.1

[0454] Embodiment 60. The cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing tumor-associated fibrosis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

[0455] Embodiment 61. The cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing tumor metastasis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

[0456] Embodiment 62. The cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing T cell exhaustion in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

[0457] Embodiment 63. The cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of increasing anti-tumor immunity in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

[0458] Embodiment 64. The cell of any one of embodiments 15-22, or a pharmaceutical composition comprising (a) the cell of any one of embodiments 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express New York esophageal squamous cell carcinoma 1 (NY-ESO-1), the method comprising administering to the subject the cell or the pharmaceutical composition.

[0459] Embodiment 65. A population of immune cells for use in a method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express NY-ESO-1, and wherein the population of immune cells has been prepared by a method comprising: (a) isolating a population of immune cells from the subject or a donor, producing an isolated population of immune cells; (b) genetically modifying the isolated population of immune cells to express the antigen-binding protein or antigen-binding fragment thereof of any one of embodiments 3-8 or 11-14 to produce a genetically modified population of immune cells; and 101183465138.1(c) expanding the genetically modified population of immune cells, producing an expanded population of immune cells; and wherein the method of treating cancer comprises administering the expanded population of immune cells to the subject.

[0460] Embodiment 66. The population of immune cells for use of embodiment 65, wherein the immune cells are lymphocytes.

[0461] Embodiment 67. The population of immune cells for use of embodiment 65, wherein the immune cells are innate lymphoid cells (ILCs).

[0462] Embodiment 68. The population of immune cells for use of embodiment 66, wherein the lymphocytes are CD4+T cells.

[0463] Embodiment 69. The population of immune cells for use of embodiment 66, wherein the lymphocytes are CD8+T cells.

[0464] Embodiment 70. The population of immune cells for use of embodiment 66, wherein the lymphocytes are NK cells.

[0465] Embodiment 71. The population of immune cells for use of any one of embodiments 66-70, the method further comprising expanding the isolated population of immune cells after step (a).

[0466] Embodiment 72. The cell, pharmaceutical composition or population of immune cells for use of any one of embodiments 58-71, wherein the subject has one or more cancers selected from the group consisting of adrenal gland tumors, biliary cancer, bladder cancer, brain cancer, breast cancer, carcinoma, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer or hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Li-Fraumeni tumors, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumors, ovarian cancer, pancreatic cancer, pancreatic islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, urogenital cancer, and uterine cancer.

[0467] Embodiment 73. The cell, pharmaceutical composition or population of immune cells for use of any one of embodiments 58-71, wherein the subject has a solid tumor.

[0468] Embodiment 74. The cell, pharmaceutical composition or population of immune cells for use of any one of embodiments 58-73, the method further comprising administering to the subject an additional therapeutic agent.102183465138.1

[0469] Embodiment 75. The cell, pharmaceutical composition or population of immune cells for use of embodiment 74, wherein the additional therapeutic agent is selected from the group consisting of taxotere, carboplatin, trastuzumab, epirubicin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, etoposide, 5-FU, gemcitabine, methotrexate, and paclitaxel, mitoxantrone, epothilone B, epidermal-growth factor receptor (EGFR)-targeting monoclonal antibody 7A7.27, vorinostat, romidepsin, docosahexaenoic acid, bortezomib, shikonin, an oncolytic virus, and a combinations thereof.

[0470] Embodiment 76. The cell, pharmaceutical composition or population of immune cells for use of embodiment 74, wherein the additional therapeutic agent is selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and a combinations thereof.

[0471] Embodiment 77. The cell, pharmaceutical composition or population of immune cells for use of embodiment 74, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

[0472] Embodiment 78. The cell, pharmaceutical composition or population of immune cells for use of embodiment 77, wherein the immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof.

[0473] Embodiment 79. The cell, pharmaceutical composition or population of immune cells for use of embodiment 77 or 78, wherein the immune checkpoint inhibitor is a PD1 inhibitor, a PD-L1 inhibitor, a CD28 inhibitor, a CTLA4 inhibitor, TIGIT inhibitor, TIM3 inhibitor, or a combination thereof.

[0474] Embodiment 80. The cell, pharmaceutical composition or population of immune cells for use of any one of embodiments 58-79, wherein the subject is a human.

[0475] Embodiment 8E A method of detecting a cancer cell expressing NY-ESO1 in a biological sample, the method comprising: (a) contacting the biological sample with the antigen-binding protein or antigen-binding fragment thereof of any one of embodiments 1-7 and 12-14; and (b) detecting binding of the antigen-binding protein or antigen-binding fragment thereof to the cancer cell.

[0476] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Regarding any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise.103183465138.1

[0477] In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0478] It is to be understood that this invention is not limited to the particular molecules, constructs, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. It is further to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments of the invention, and in the invention generally.

[0479] All referenced patents, patent applications, book chapters, scientific publications, etc., are incorporated herein by reference in their entireties, whether explicitly stated or not. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0480] To facilitate a better understanding of the present invention, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the invention.

[0481] Selected sequences disclosed herein can be found in the following tables.Table 7. Selected CDR sequences (amino acid sequences).SEQ ID Name Description Amino acid sequenceNO1 TCR1 CDRla SSVPPY1 TCR2 CDRla SSVPPY1 TCR3 CDRla SSVPPY1 TCR4 CDRla SSVPPY2 TCR1 CDR2a YTTGATLV2 TCR2 CDR2a YTTGATLV2 TCR4 CDR2a YTTGATLV3 TCR3 CDR2a YTSAATLV4 TCR1 CDR3a CAVSKGGGNKLTFG5 TCR2 CDR3a CAVRGASAGNKLTFG6 TCR3 CDR3a CAAVKGGGNKLTFG7 TCR4 CDR3a CAVSGQGAQKLVFG104183465138.1SEQ ID Name Description Amino acid sequenceNO8 TCR1 CDRlb DFQATT8 TCR3 CDRlb DFQATT8 TCR4 CDRlb DFQATT9 TCR2 CDRlb MNHEY10 TCR1 CDR2b SNEGSKA10 TCR3 CDR2b SNEGSKA10 TCR4 CDR2b SNEGSKA11 TCR2 CDR2b SVGAGI12 TCR1 CDR3b CSVPEGARGLGTEAFFG 13 TCR2 CDR3b CASSTNRVSTDTQYFG 14 TCR3 CDR3b CSARRGGNTGELFFG15 TCR4 CDR3b CSERGQGFAEAFFGTable 8. Selected CDR sequences (nucleic acid sequences).SEQ Name Description Nucleic acid sequenceID NO44 TCR1 CDRla TCGTCTGTTCCACCATAT44 TCR2 CDRla TCGTCTGTTCCACCATAT44 TCR3 CDRla TCGTCTGTTCCACCATAT44 TCR4 CDRla TCGTCTGTTCCACCATAT45 TCR1 CDR2a TACACAACAGGGGCCACCCTGGTT45 TCR2 CDR2a TACACAACAGGGGCCACCCTGGTT45 TCR4 CDR2a TACACAACAGGGGCCACCCTGGTT46 TCR3 CDR2a TACACATCAGCGGCCACCCTGGTT47 TCR1 CDR3a TGTGCTGTGAGTAAGGGAGGAGGAAACAAACT CACCTTTGGG48 TCR2 CDR3a TGTGCTGTGAGGGGCGCGTCTGCAGGCAACAA GCTAACTTTTGGA49 TCR3 CDR3a TGTGCTGCGGTGAAGGGAGGAGGAAACAAACT CACCTTTGGG50 TCR4 CDR3a TGTGCTGTGAGTGGTCAGGGAGCCCAGAAGCT GGTATTTGGC51 TCR1 CDRlb GACTTTCAGGCCACAACT51 TCR3 CDRlb GACTTTCAGGCCACAACT51 TCR4 CDRlb GACTTTCAGGCCACAACT52 TCR2 CDRlb ATGAACCATGAATAC53 TCR1 CDR2b TCCAATGAGGGCTCCAAGGCC53 TCR3 CDR2b TCCAATGAGGGCTCCAAGGCC53 TCR4 CDR2b TCCAATGAGGGCTCCAAGGCC54 TCR2 CDR2b TCAGTTGGTGCTGGTATC55 TCR1 CDR3b TGCAGTGTACCTGAGGGGGCGCGGGGGCTGGG CACTGAAGCTTTCTTTGGA56 TCR2 CDR3b TGTGCCAGCAGTACGAACAGGGTTAGCACAGA TACGCAGTATTTTGGC57 TCR3 CDR3b TGCAGTGCCAGAAGGGGGGGCAACACCGGGGAGCTGTTTTTTGGA105183465138.1SEQ Name Description Nucleic acid sequenceID NO58 TCR4 CDR3b TGCAGTGAGAGAGGGCAGGGTTTCGCTGAAGCTTTCTTTGGATable 9. Variable chain sequences (amino acid sequences) for selected TCRs that were isolated in the screen discussed in the Examples and that recognize a fragment from NY-ESO-1.SEQ Name Description Amino acid sequenceID NO16 TCR1 Alpha variable MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVS chain VSEGALVLLRCNYSSSVPPYLFWYVQYPNQ GLQLLLKYTTGATLVKGINGFEAEFKKSETS CDRs in bold FHLTKPSAHMSDAAEYFCAVSKGGGNKLT FGTGTQLKVEL17 TCR1 Beta variable MLLLLLLLGPGSGLGAVVSQHPSRVICKSGT chain SVKIECRSLDFQATTMFWYRQFPKQSLMLM ATSNEGSKATYEQGVEKDKFLINHASLTLST CDRs in bold LTVTSAHPEDSSFYICSVPEGARGLGTEAFF GQGTRLTVV18 TCR2 Alpha variable MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVS chain VSEGALVLLRCNYSSSVPPYLFWYVQYPNQ GLQLLLKYTTGATLVKGINGFEAEFKKSETS CDRs in bold FHLTKPSAHMSDAAEYFCAVRGASAGNKL TFGGGTRVLVKP19 TCR2 Beta variable MSIGLLCCAALSLLWAGPVNAGVTQTPKFQ chain VLKTGQSMTLQCAQDMNHEYMSWYRQDP GMGLRLIHYSVGAGITDQGEVPNGYNVSRS CDRs in bold TTEDFPLRLLSAAPSQTSVYFCASSTNRVST DTQYFGPGTRLTVL20 TCR3 Alpha variable MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVS chain VSEGALVLLRCNYSSSVPPYLFWYVQYPNQ GLQLLLKYTSAATLVKGINGFEAEFKKSETS CDRs in bold FHLTKPSAHMSDAAEYFCAAVKGGGNKLT FGTGTQLKVEL21 TCR3 Beta variable MLLLLLLLGPGSGLGAVVSQHPSRVICKSGT chain SVKIECRSLDFQATTMFWYRQFPKQSLMLM ATSNEGSKATYEQGVEKDKFLINHASLTLST CDRs in bold LTVTSAHPEDSSFYICSARRGGNTGELFFGE GSRLTVL22 TCR4 Alpha variable MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVS chain VSEGALVLLRCNYSSSVPPYLFWYVQYPNQ GLQLLLKYTTGATLVKGINGFEAEFKKSETS CDRs in bold FHLTKPSAHMSDAAEYFCAVSGQGAQKLV FGQGTRLTINP23 TCR4 Beta variable MLLLLLLLGPGSGLGAVVSQHPSRVICKSGT chain SVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTCDRs in bold106183465138.1SEQ Name Description Amino acid sequenceID NO LTVTSAHPEDSSFYICSERGQGFAEAFFGQGTRLTVVTable 10. Chain sequences (amino acid sequences) for selected TCRs that were isolated in the screen discussed in the Examples and that recognize a fragment from NY-ESO-1. SEQ Name Description Amino acid sequenceID NO24 TCR1 Alpha chain MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVS VSEGALVLLRCNYSSSVPPYLFWYVQYPNQ CDRs in bold; GLQLLLKYTTGATLVKGINGFEAEFKKSETS constant region in FHLTKPSAHMSDAAEYFCAVSKGGGNKLT italics FGYGYQLKVELNIQNPDPAVYQLRDSKSSDKS VCLFTDFDSQTNVSQSKDSDVYITDKTVLDMR SMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT FFPSPESSCDVKLVEKSFETDTNLNFQNLSVIG FRILLLKVAGFNLEMTLRL WSS25 TCR1 Beta chain MLLLLLLLGPGSGLGAVVSQHPSRVICKSGT SVKIECRSLDFQATTMFWYRQFPKQSLMLM CDRs in bold; ATSNEGSKATYEQGVEKDKFLINHASLTLST constant region in LTVTSAHPEDSSFYICSVPEGARGLGTEAFF italics GQGTRLT NNEDLNKVFPPEVA VFEPSEAEISH TQKA TL VCLA TGFFPDHVELSWWVNGKEVHS GVSTDPQPLKEQPALNDSRYCLSSRLRVSA TFW QNPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVSAEA WGRADCGFTSVSYQQGVLSA TILYEIL LGKA TLYAVLVSAL VEMAMVKRKDF26 TCR2 Alpha chain MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVS VSEGALVLLRCNYSSSVPPYLFWYVQYPNQ CDRs in bold; GLQLLLKYTTGATLVKGINGFEAEFKKSETS constant region in FHLTKPSAHMSDAAEYFCAVRGASAGNKL italics YVGGGYWLVNWPNIQNPDPAVYQLRDSKSSDK SVCLFTDFDSQTNVSQSKDSDVYITDKTVLDM RSMDFKSNSAVAWSNKSDFACANAFNNSIIPED TFFPSPESSCDVKLVEKSFETDTNLNFQNLSVI GFRILLLKVAGFNLLMTLRL WSS27 TCR2 Beta chain MSIGLLCCAALSLLWAGPVNAGVTQTPKFQ VLKTGQSMTLQCAQDMNHEYMSWYRQDP CDRs in bold; GMGLRLIHYSVGAGITDQGEVPNGYNVSRS constant region in TTEDFPLRLLSAAPSQTSVYFCASSTNRVST italics DTQYFGPGTRLTVLEDZXVFFPPEE4 VFEPS EAEISHTQKA TL VCLA TGFYPDHVELSWWVNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV SATFWQNPRNHFRCQVQFYGLSENDEWTQDR AKPVTQIVSAEAWGRADCGFTSESYQQGVLSA TILYEILLGKA TLYAVLVSAL VLMAMVKRKDSRG28 TCR3 Alpha chain MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVSVSEGALVLLRCNYSSSVPPYLFWYVQYPNQ107183465138.1SEQ Name Description Amino acid sequenceID NO CDRs in bold; GLQLLLKYTSAATLVKGINGFEAEFKKSETS constant region in FHLTKPSAHMSDAAEYFCAAVKGGGNKLT italics FGTGTQLKVELNIQNPDPAVYQLRDSKSSDK SVCLFTDFDSQTNVSQSKDSDVYITDKTVLD MRSMDFKSNSAVAWSNKSDFACANAFNNSII PEDTFFPSPESSCDVKLVEKSFETDTNLNFQN LSVIGFRILLLKVAGFNLLMTLRLWSS29 TCR3 Beta chain MLLLLLLLGPGSGLGAVVSQHPSRVICKSGT SVKIECRSLDFQATTMFWYRQFPKQSLMLM CDRs in bold; ATSNEGSKATYEQGVEKDKFLINHASLTLST constant region in LTVTSAHPEDSSFYICSARRGGNTGELFFGE italics GSRLTVLEDWVFEPPEE4 VFEPSEAEISHTQK A TL VCLA TGFYPDHVELSWWVNGKEVHSGVST DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPR NHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA EA WGRADCGFTSESYQQGVLSATILYEILLGKA TLYAVLVSALVLMAMVKRKDSRG30 TCR4 Alpha chain MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVS VSEGALVLLRCNYSSSVPPYLFWYVQYPNQ CDRs in bold; GLQLLLKYTTGATLVKGINGFEAEFKKSETS constant region in FHLTKPSAHMSDAAEYFCAVSGQGAQKLV italics ^GQGY^EYWPNIQNPDPAVYQLRDSKSSDKSV CLFTDFDSQTNVSQSKDSDVYITDKTVLDMRS MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTF FPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF RILLLKVAGFNLEMTLRLWSS31 TCR4 Beta chain MLLLLLLLGPGSGLGAVVSQHPSRVICKSGT SVKIECRSLDFQATTMFWYRQFPKQSLMLM CDRs in bold; ATSNEGSKATYEQGVEKDKFLINHASLTLST constant region in LTVTSAHPEDSSFYICSERGQGFAEAFFGQG italics TRLT NNEDLNKVFPPEVA VFEPSEAEISHTQKA TLVCLATGFFPDHVELSWWVNGKEVHSGVST DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPR NHFRCQVQFYGLSENDEWTQDRAKPVTQIVSA EA WGRADCGFTSVSYQQGVLSATILYEILLGKA TLYAVLVSALVLMAMVKRKDF75 Alpha chain NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQ constant region TNVSQ SKD SD VYITDKTVLDMRSMDFKSNS AVAWSNKSDFACANAFNNSIIPEDTFFPSPES SCDVKLVEKSFETDTNLNFQNLSVIGFRILLL KVAGFNLLMTLRLWS S76 Beta chain EDLKNVFPPEVAVFEPSEAEISHTQKATLVCL constant region 1 ATGFYPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSESYQQGVLSATILYEILLGI<ATLYAVLVSALVLMAMVI<RI<DSRG108183465138.1SEQ Name Description Amino acid sequenceID NO77 Beta chain EDLNKVFPPEVAVFEPSEAEISHTQKATLVCL constant region 2 ATGFFPDHVELSWWVNGKEVHSGVSTDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNH FRCQVQFYGLSENDEWTQDRAKPVTQIVSA EAWGRADCGFTSVSYQQGVLSATILYEILLGI<ATLYAVLVSALVLMAMVI<RI<DFTable 11. Variable chain sequences and chain sequences comprising a constant region for selected TCRs that were isolated in the screen discussed in the Examples and that recognize a fragment from NY-ESO-1. In the nucleic acid sequence, the constant region does not start at the beginning of a codon. Specifically, the amino acid at the junction between the J and the C region is encoded by the last nucleotide of the J and the first 2 nucleotides of the constant nucleic acid regions. For the sake of clarity, in the correspondence acid sequence (see Table 9 and Table 10), the last amino acid of the variable region is encoded by the penultimate nucleotide of the J nucleic acid region (J regions are underlined in this table). _SEQ Name Description Nucleic acid sequenceID NO59 TCR1 Alpha chain ATGCTCCTGCTGCTCGTCCCAGTGCTCGAG GTGATTTTTACCCTGGGAGGAACCAGAGC CDRs in bold; CCAGTCGGTGACCCAGCTTGGCAGCCACG hTRAJlO TCTCTGTCTCTGAGGGAGCCCTGGTTCTGC underlined; TGAGGTGCAACTACTCATCGTCTGTTCCA constant region in CCATATCTCTTCTGGTATGTGCAATACCCC italics AACCAAGGACTCCAGCTTCTCCTGAAGTA CACAACAGGGGCCACCCTGGTTAAAGG CATCAACGGTTTTGAGGCTGAATTTAAGAA GAGTGAAACCTCCTTCCACCTGACGAAAC CCTCAGCCCATATGAGCGACGCGGCTGAG TACTTCTGTGCTGTGAGTAAGGGAGGAG GAAACAAACTCACCTTTGGGACAGGCAC TCAGCTAAAAGTGGAACTCAATATCCAGAA CCCTGACCCTGCCGTGTACCAGCTGAGAGAC TCTAAATCCAGTGACAAGTCTGTCTGCCTATT CA CCGA TTTTGA TTCTCAAA CAAA TG TG TCA C AAAGTAAGGATTCTGATGTGTATATCACAGAC AAAACTGTGCTAGACA TGAGGTCTA TGGACTT CAAGAGCAACAGTGCTGTGGCCTGGAGCAA CAAA TCTGACTTTGCA TGTGCAAA CGCCTTCA ACAACAGCATTATTCCAGAAGACACCTTCTTC CCCAGCCCAGAAAGTTCCTGTGA TGTCAAGC TGGTCGAGAAAAGCTTTGAAACAGATACGAA CCTAAACTTTCAAAACCTGTCAGTGATTGGGT TCCGAATCCTCCTCCTGAAAGTGGCCGGGTT TAA TCTGCTCA TGACGCTGCGGCTGTGGTCC AGC60 TCR1 Alpha variable ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGchain GTGATTTTTACCCTGGGAGGAACCAGAGC109183465138.1SEQ Name Description Nucleic acid sequenceID NO CCAGTCGGTGACCCAGCTTGGCAGCCACG CDRs in bold; TCTCTGTCTCTGAGGGAGCCCTGGTTCTGC hTRAJlO TGAGGTGCAACTACTCATCGTCTGTTCCA underlined CCATATCTCTTCTGGTATGTGCAATACCCC AACCAAGGACTCCAGCTTCTCCTGAAGTA CACAACAGGGGCCACCCTGGTTAAAGG CATCAACGGTTTTGAGGCTGAATTTAAGAA GAGTGAAACCTCCTTCCACCTGACGAAAC CCTCAGCCCATATGAGCGACGCGGCTGAG TACTTCTGTGCTGTGAGTAAGGGAGGAG GAAACAAACTCACCTTTGGGACAGGCAC TCAGCTAAAAGTGGAACTCA61 TCR1 Beta chain ATGCTGCTGCTTCTGCTGCTTCTGGGGCCA GGCTCCGGGCTTGGTGCTGTCGTCTCTCAA CDRs in bold; CATCCGAGCAGGGTTATCTGTAAGAGTGG hTRAJlO AACCTCTGTGAAGATCGAGTGCCGTTCCCT underlined; GGACTTTCAGGCCACAACTATGTTTTGGT constant region in ATCGTCAGTTCCCGAAACAGAGTCTCATGC italics TGATGGCAACTTCCAATGAGGGCTCCAA GGCCACATACGAGCAAGGCGTCGAGAAG GACAAGTTTCTCATCAACCATGCAAGCCTG ACCTTGTCCACTCTGACAGTGACCAGTGC CCATCCTGAAGACAGCAGCTTCTACATCTG CAGTGTACCTGAGGGGGCGCGGGGGCT GGGCACTGAAGCTTTCTTTGGACAAGGC ACCAGACTCACAGTTGTAGAGGACCTGAAC AAGGTGTTCCCACCCGAGGTCGCTGTGTTTG AGCCATCAGAAGCAGAGATCTCCCACACCCA AAAGGCCACACTGGTGTGCCTGGCCACAGG CTTCTTCCCCGACCACGTGGAGCTGAGCTGG TGGGTGAA TGGGAAGGAGGTGCACAGTGGG GTCAGCACGGACCCGCAGCCCCTCAAGGAG CAGCCCGCCCTCAATGACTCCAGATACTGCC TGAGCAGCCGCCTGAGGGTCTCGGCCACCT TCTGGCAGAACCCCCGCAACCACTTCCGCTG TCAAGTCCAGTTCTACGGGCTCTCGGAGAAT GACGAGTGGACCCAGGATAGGGCCAAACCC GTCACCCAGA TCGTCAGCGCCGAGGCCTGG GGTAGAGCAGACTGTGGCTTTACCTCGGTGT CCTACCAGCAAGGGGTCCTGTCTGCCACCAT CCTCTATGAGATCCTGCTAGGGAAGGCCACC CTGTA TGCTGTGCTGGTCAGCGCCCTTGTGT TGA TGGCCA TGGTCAA GA GAAA GGA TTTC62 TCR1 Beta variable ATGCTGCTGCTTCTGCTGCTTCTGGGGCCA chain GGCTCCGGGCTTGGTGCTGTCGTCTCTCAA CATCCGAGCAGGGTTATCTGTAAGAGTGG CDRs in bold; AACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGT110183465138.1SEQ Name Description Nucleic acid sequenceID NOhTRBJl-1 ATCGTCAGTTCCCGAAACAGAGTCTCATGC underlined TGATGGCAACTTCCAATGAGGGCTCCAA GGCCACATACGAGCAAGGCGTCGAGAAG GACAAGTTTCTCATCAACCATGCAAGCCTG ACCTTGTCCACTCTGACAGTGACCAGTGC CCATCCTGAAGACAGCAGCTTCTACATCTG CAGTGTACCTGAGGGGGCGCGGGGGCT GGGCACTGAAGCTTTCTTTGGACAAGGC ACCAGACTCACAGTTGTAG63 TCR2 Alpha chain ATGCTCCTGCTGCTCGTCCCAGTGCTCGAG GTGATTTTTACCCTGGGAGGAACCAGAGC CDRs in bold; CCAGTCGGTGACCCAGCTTGGCAGCCACG hTRAJ17 TCTCTGTCTCTGAGGGAGCCCTGGTTCTGC underlined; TGAGGTGCAACTACTCATCGTCTGTTCCA constant region in CCATATCTCTTCTGGTATGTGCAATACCCC italics AACCAAGGACTCCAGCTTCTCCTGAAGTA CACAACAGGGGCCACCCTGGTTAAAGG CATCAACGGTTTTGAGGCTGAATTTAAGAA GAGTGAAACCTCCTTCCACCTGACGAAAC CCTCAGCCCATATGAGCGACGCGGCTGAG TACTTCTGTGCTGTGAGGGGCGCGTCTGC AGGCAACAAGCTAACTTTTGGAGGAGGAA CCAGGGTGCTAGTTAAACCAA4Z47CG4G4 ACCCTGACCCTGCCGTGTACCAGCTGAGAGA CTCTAAATCCAGTGACAAGTCTGTCTGCCTAT TCACCGA TTTTGA TTCTCAAACAAA TGTGTCA CAAAGTAAGGATTCTGATGTGTATATCACAGA CAAAACTGTGCTAGACATGAGGTCTATGGACT TCAAGAGCAACAGTGCTGTGGCCTGGAGCAA CAAA TCTGACTTTGCA TGTGCAAA CGCCTTCA ACAACAGCATTATTCCAGAAGACACCTTCTTC CCCAGCCCAGAAAGTTCCTGTGA TGTCAAGC TGGTCGAGAAAAGCTTTGAAACAGATACGAA CCTAAACTTTCAAAACCTGTCAGTGATTGGGT TCCGAA TCCTCCTCCTGAAAGTGGCCGGGTT TAA TCTGCTCA TGACGCTGCGGCTGTGGTCC AGC64 TCR2 Alpha variable ATGCTCCTGCTGCTCGTCCCAGTGCTCGAG chain GTGATTTTTACCCTGGGAGGAACCAGAGC CCAGTCGGTGACCCAGCTTGGCAGCCACG CDRs in bold; TCTCTGTCTCTGAGGGAGCCCTGGTTCTGC hTRAJ17 TGAGGTGCAACTACTCATCGTCTGTTCCA underlined; CCATATCTCTTCTGGTATGTGCAATACCCC const...

Claims

CLAIMS1. An antigen-binding protein or antigen-binding fragment thereof comprising a first variable domain and a second variable domain, wherein the antigen comprises SEQ ID NO:43 (LKEFTVSGNILTIRL), wherein the first variable domain comprises complementarity determining region (CDR) al, CDRa2, and CDRa3, wherein the second variable domain comprises CDRbl, CDRb2, and CDRb3, and wherein:(a) CDRal comprises SEQ ID NO:1;(b) CDRa2 comprises SEQ ID NO:2 or SEQ ID NO:3;(c) CDRa3 comprises any one of SEQ ID NOs:4-7;(d) CDRbl comprises SEQ ID NO:8 or SEQ ID NO:9;(e) CDRb2 comprises SEQ ID NO: 10 or SEQ ID NO: 11; and(f) CDRb3 comprises any one of SEQ ID NOs: 12-15.

2. The antigen-binding protein or antigen-binding fragment thereof of claim 1, wherein:(a) CDRal comprises SEQ ID NO: 1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:4; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 12;(b) CDRal comprises SEQ ID NO: 1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:5; CDRbl comprises SEQ ID NO:9; CDRb2 comprises SEQ ID NO: 11; and CDRb3 comprises SEQ ID NO: 13;(c) CDRal comprises SEQ ID NO: 1; CDRa2 comprises SEQ ID NO:3; CDRa3 comprises SEQ ID NO:6; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 14; or(d) CDRal comprises SEQ ID NO: 1; CDRa2 comprises SEQ ID NO:2; CDRa3 comprises SEQ ID NO:7; CDRbl comprises SEQ ID NO:8; CDRb2 comprises SEQ ID NO: 10; and CDRb3 comprises SEQ ID NO: 15.

3. The antigen-binding protein or antigen-binding protein fragment thereof of claim 1 or 2, wherein the antigen-binding protein is a T cell receptor (TCR).

4. The antigen-binding protein or antigen-binding protein fragment thereof of claim 3, wherein: (a) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ 145183241445.1183465138.1ID NO: 16 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO: 17;(b) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO: 18 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO: 19;(c) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:20 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:21; or(d) the first variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:22 and the second variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to identical to SEQ ID NO:23.

5. The antigen-binding protein or antigen-binding protein fragment thereof of claim 4, wherein: (a) the first variable domain comprises SEQ ID NO: 16 and the second variable domain comprises SEQ ID NO: 17;(b) the first variable domain comprises SEQ ID NO: 18 and the second variable domain comprises SEQ ID NO: 19;(c) the first variable domain comprises SEQ ID NO:20 and the second variable domain comprises SEQ ID NO:21; or(d) the first variable domain comprises SEQ ID NO:22 and the second variable domain comprises SEQ ID NO:23.

6. The antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 3-5, wherein the first variable domain is further joined to a constant region comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:75 and wherein the second variable domain is further joined to a constant region146183465138.1comprising a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:76 or SEQ ID NO:77.

7. The antigen-binding protein or antigen-binding protein fragment thereof of claim 6, wherein the first variable domain is further joined to a sequence comprising SEQ ID NO:75 and wherein the second variable domain is further joined to a sequence comprising SEQ ID NO:76 or SEQ IDNO:77.

8. The antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 3-7, wherein the TCR is:(a) a single chain TCR; or(b) a membrane-bound TCR or soluble TCR.

9. The antigen-binding protein or antigen-binding protein fragment thereof of claim 1 or 2, wherein the antigen-binding protein is an antibody or antigen-binding protein fragment thereof.

10. The antigen-binding protein or antigen-binding protein fragment thereof of claim 9, wherein the antibody or antigen-binding protein fragment thereof is a single-chain variable fragment (scFv), fragment variable (Fv), fragment binding domain (Fab), Fab’, F(ab’)2, or diabody.

11. The antigen-binding protein or antigen-binding protein fragment thereof of claim 1 or 2, wherein the antigen-binding protein is a chimeric antigen receptor (CAR), optionally comprising a scFv comprising the first variable domain and a second variable domain.

12. The antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 1-11, wherein the antigen-binding protein or antigen-binding protein fragment thereof binds to the antigen present in a complex with HLA-DRB3*02:02.

13. The antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 1-12, wherein the antigen-binding protein or antigen-binding protein fragment thereof is deglycosylated.147183465138.

114. The antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 1-13, wherein the antigen-binding protein or antigen-binding protein fragment thereof is conjugated to a fluorescent moiety, a detectable moiety, a purification moiety, or a combination thereof.

15. A cell expressing on its surface the antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 3-8 or 11-14, optionally wherein the cell is isolated.

16. The cell of claim 15, wherein the cell is a lymphocyte.

17. The cell of claim 16, wherein the lymphocyte is a T cell.

18. The cell of claim 17, wherein the T cell is a CD4+T cell.

19. The cell of claim 17, wherein the T cell is a CD8+T cell.

20. The cell of claim 16, wherein the lymphocyte is a natural killer (NK) cell.

21. The cell of any one of claims 15-20, wherein the cell further expresses a therapeutic protein.

22. The cell of claim 21, wherein the therapeutic protein is interleukin 2 (IL-2), IL-2 mutein, interleukin 15 (IL-15), CD40 ligand (CD40L), interleukin 33 (IL-33), interleukin 12 (IL-12), a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PD-L1) inhibitor, a T cell immunoreceptor with Ig and ITIM domains (TIGIT inhibitor), or a combination thereof.

23. A cell expressing the antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 9, 10 or 12-14, optionally wherein the cell is isolated.

24. The cell of claim 23, wherein the cell is a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK) cell, optionally, wherein the cell is a HEK293 or HEK293T cell.148183465138.

125. A nucleic acid or set of nucleic acids encoding the antigen-binding protein or antigenbinding protein fragment thereof of any one of claims 1-14, optionally wherein the nucleic acid or set of nucleic acids are isolated.

26. A vector or set of vectors comprising the nucleic acid or set of nucleic acids of claim 25, optionally wherein the vector or set of vectors are isolated.

27. A pharmaceutical composition comprising (a) the antigen-binding protein or antigenbinding protein fragment thereof of claims 9, 10, or 12-14, a cell of any one of claims 15-22, the nucleic acid or set of nucleic acids of claim 25, or the vector or set of vectors of claim 26 and (b) a pharmaceutically acceptable excipient.

28. A method of producing an antigen-binding protein or antigen-binding protein fragment thereof, the method comprising culturing a cell of claim 23 or 24 under conditions wherein the antigen-binding protein or antigen-binding protein fragment thereof is produced by the cell.

29. A method of increasing the cytotoxic activity of a lymphocyte against tumor cells expressing New York esophageal squamous cell carcinoma 1 (NY-ESO-1), the method comprising genetically modifying the lymphocyte to express the antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 3-8 or 11-14.

30. A method of increasing cytokine secretion by a lymphocyte in the presence of tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express the antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 3-8 or 11-14.

31. A method of increasing the effector functions of a lymphocyte in the presence of tumor cells expressing NY-ESO-1, the method comprising genetically modifying the lymphocyte to express the antigen-binding protein or antigen-binding protein fragment thereof of any one of claims 3-8 or 11-14.

32. A method of any one of claims 29-30, wherein the lymphocyte is a CD4+T cell.

33. A method of any one of claims 29-30, wherein the lymphocyte is a CD8+T cell.149183465138.

134. A method of any one of claims 29-30, wherein the lymphocyte is an NK cell.

35. A method of reducing tumor growth in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient.

36. A method of reducing cancer sternness in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject the cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient.

37. A method of reducing tumor-associated fibrosis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient.

38. A method of reducing tumor metastasis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient.

39. A method of reducing T cell exhaustion in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject the cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient.

40. A method of increasing anti -tumor immunity in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient.150183465138.

141. A method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express NY-ESO-1, the method comprising administering to the subject the cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient.

42. A method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express NY-ESO-1, the method comprising:(a) isolating a population of immune cells from the subject or a donor, producing an isolated population of immune cells;(b) genetically modifying the isolated population of immune cells to express the antigenbinding protein or antigen-binding fragment thereof of any one of claims 3-8 or 11-14 to produce a genetically modified population of immune cells;(c) optionally, expanding the genetically modified population of immune cells, producing an expanded population of immune cells; and(d) administering the expanded population of immune cells to the subject.

43. The method of claim 42, wherein the immune cells are lymphocytes.

44. The method of claim 42, wherein the immune cells are innate lymphoid cells (ILCs).

45. The method of claim 43, wherein the lymphocytes are CD4+T cells.

46. The method of claim 43, wherein the lymphocytes are CD8+T cells.

47. The method of claim 43, wherein the lymphocytes are NK cells.

48. The method of any one of claims 42-47, the method further comprising expanding the isolated population of immune cells after step (a).

49. The method of any one of claims 35-48, wherein the subject has one or more cancers selected from the group consisting of adrenal gland tumors, biliary cancer, bladder cancer, brain cancer, breast cancer, carcinoma, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer or hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Li-151183465138.1Fraumeni tumors, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumors, ovarian cancer, pancreatic cancer, pancreatic islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, urogenital cancer, and uterine cancer.

50. The method of any one of claims 35-48, wherein the subject has a solid tumor.

51. The method of any one of claims 35-50, the method further comprising administering to the subject an additional therapeutic agent.

52. The method of claim 51, wherein the additional therapeutic agent is selected from the group consisting of taxotere, carboplatin, trastuzumab, epirubicin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, etoposide, 5-FU, gemcitabine, methotrexate, and paclitaxel, mitoxantrone, epothilone B, epidermal-growth factor receptor (EGFR)-targeting monoclonal antibody 7A7.27, vorinostat, romidepsin, docosahexaenoic acid, bortezomib, shikonin, an oncolytic virus, and a combinations thereof.

53. The method of claim 51, wherein the additional therapeutic agent is selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and a combinations thereof.

54. The method of claim 51, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

55. The method of claim 54, wherein the immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof.

56. The method of claim 54 or 55, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD1 inhibitor, a PD-L1 inhibitor, a CD28 inhibitor, a Cytotoxic T Lymphocyte Associated Protein 4 (CTLA4) inhibitor, TIGIT inhibitor, T cell immunoglobulin and mucin-domain containing 3 (TIM3) inhibitor, or a combination thereof.152183465138.

157. The method of any one of claims 35-56, wherein the subject is a human.

58. The cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing tumor growth in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

59. The cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing cancer sternness in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

60. The cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing tumor-associated fibrosis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

61. The cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing tumor metastasis in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

62. The cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of reducing T cell exhaustion in a subject with cancer, wherein at least some of the cancer cells express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.153183465138.

163. The cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of increasing anti -turn or immunity in a subject with cancer, wherein at least some of the cells of the tumor express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

64. The cell of any one of claims 15-22, or a pharmaceutical composition comprising (a) the cell of any one of claims 15-22 and (b) a pharmaceutically acceptable excipient, for use in a method of treating cancer in a subject in need thereof, wherein at least some of the cells of the cancer express NY-ESO-1, the method comprising administering to the subject the cell or the pharmaceutical composition.

65. A population of immune cells for use in a method of treating cancer in a subject in need thereof, the method comprising administering the population of immune cells to the subject, wherein at least some of the cells of the cancer express NY-ESO-1, and wherein the population of immune cells has been prepared by a method comprising:(a) isolating a population of immune cells from the subject or a donor, producing an isolated population of immune cells;(b) genetically modifying the isolated population of immune cells to express the antigenbinding protein or antigen-binding fragment thereof of any one of claims 3-8 or 11-14 to produce a genetically modified population of immune cells; and(c) optionally, expanding the genetically modified population of immune cells, producing an expanded population of immune cells.

66. The population of immune cells for use of claim 65, wherein the immune cells are lymphocytes.

67. The population of immune cells for use of claim 65, wherein the immune cells are innate lymphoid cells (ILCs).

68. The population of immune cells for use of claim 66, wherein the lymphocytes are CD4+T cells.154183465138.

169. The population of immune cells for use of claim 66, wherein the lymphocytes are CD8+T cells.

70. The population of immune cells for use of claim 66, wherein the lymphocytes are NK cells.

71. The population of immune cells for use of any one of claims 66-70, the method further comprising expanding the isolated population of immune cells after step (a).

72. The cell, pharmaceutical composition or population of immune cells for use of any one of claims 58-71, wherein the subject has one or more cancers selected from the group consisting of adrenal gland tumors, biliary cancer, bladder cancer, brain cancer, breast cancer, carcinoma, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer or hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Li-Fraumeni tumors, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple neuroendocrine type I and type II tumors, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma tumors, ovarian cancer, pancreatic cancer, pancreatic islet cell cancer, parathyroid cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, urogenital cancer, and uterine cancer.

73. The cell, pharmaceutical composition or population of immune cells for use of any one of claims 58-71, wherein the subject has a solid tumor.

74. The cell, pharmaceutical composition or population of immune cells for use of any one of claims 58-73, the method further comprising administering to the subject an additional therapeutic agent.

75. The cell, pharmaceutical composition or population of immune cells for use of claim 74, wherein the additional therapeutic agent is selected from the group consisting of taxotere, carboplatin, trastuzumab, epirubicin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, etoposide, 5-FU, gemcitabine, methotrexate, and paclitaxel, mitoxantrone, epothilone B, epidermal-growth factor receptor (EGFR)-targeting monoclonal antibody 7A7.27, vorinostat,155183465138.1romidepsin, docosahexaenoic acid, bortezomib, shikonin, an oncolytic virus, and a combinations thereof.

76. The cell, pharmaceutical composition or population of immune cells for use of claim 74, wherein the additional therapeutic agent is selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and a combinations thereof.

77. The cell, pharmaceutical composition or population of immune cells for use of claim 74, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

78. The cell, pharmaceutical composition or population of immune cells for use of claim 77, wherein the immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof.

79. The cell, pharmaceutical composition or population of immune cells for use of claim 77 or 78, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD1 inhibitor, a PD-L1 inhibitor, a CD28 inhibitor, a Cytotoxic T Lymphocyte Associated Protein 4 (CTLA4) inhibitor, TIGIT inhibitor, T cell immunoglobulin and mucin-domain containing 3 (TIM3) inhibitor, or a combination thereof.

80. The cell, pharmaceutical composition or population of immune cells for use of any one of claims 58-79, wherein the subject is a human.

81. A method of detecting a cancer cell expressing NY-ESO1 in a biological sample, the method comprising:(a) contacting the biological sample with the antigen-binding protein or antigen-binding fragment thereof of any one of claims 1-7 and 12-14; and(b) detecting binding of the antigen-binding protein or antigen-binding fragment thereof to the cancer cell.156183465138.1

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