Novel invariant natural killer t cells and t cell engager compositions and methods of use thereof

Abstract

Provided are novel compositions comprising natural killer T cells ("NKT cells") and T cell engagers. In certain embodiments, NKT cells are allogeneic invariant natural killer T cells ("iNKT cells") and the T cell engager is a bispecific or multispecific antibody comprising a CD3 binding domain and a tumor antigen binding domain. The invention includes methods of treating cancer comprising administering to a subject in need NKT cells (e.g., allogeneic iNKT cells) and a T cell engager.

Description

NOVEL INVARIANT NATURAL KILLER T CELLS AND T CELL ENGAGER COMPOSITIONS AND METHODS OF USE THEREOFRELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication, U.S.S.N. 63 / 588,288, filed October 5, 2023, the entire contents of which are incorporated herein by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (A132770009WO00-SEQ-LGE.xml; Size: 34,743 bytes; and Date of Creation: October 3, 2024) is herein incorporated by reference in its entirety.BACKGROUNDField of the Invention

[0003] The present invention relates to novel compositions comprising natural killerT cells, including allogeneic invariant natural killer T cells, and T cell engagers and methods of using such cells and T cell engagers for the treatment of cancer.Description

[0004] Invariant natural killer T (iNKT) cells, also known as type I or classical NKT cells, function during innate and adaptive immunity and are rare in the human blood pool, comprising just 0.01-1% of peripheral blood mononuclear cells (PBMCs). iNKT cells express a unique TCR containing an invariant Va24-Jal8 a-chain and a limited number of P chains. Unlike conventional T cells, which respond to peptide antigens in the context of MHC class I or II, iNKT cells respond and are activated by glycolipid antigens presented by the MHC class lb related molecule, CD Id. CD Id is a highly conserved, non-polymorphic molecule, expressed by many haematopoietic cells (dendritic cells, macrophages and B cells), which present lipid antigens rather than peptides to iNKT cells. The CD Id restriction identifies and defines iNKT cells from all other T cell populations.

[0005] iNKT cells are innate-like lymphocytes that promptly secrete Thl or Th2 cytokines when antigens bind with TCRs. Activated iNKT cells also regulate the adaptiveimmune response via the recruitment, activation, or modulation of the responses of natural killer cells, dendritic cells, B cells, and T cells. Many reports have suggested that the activation of iNKT cells enhances the cytotoxic T lymphocytes-mediated elimination of cancer cells or various infections. Studies focused on the activation of iNKT cells following stimulation with foreign glycolipids, such as the marine sponge-derived glycolipid a- galactosylceramide (a-GalCer), a strong and prototypical synthetic glycolipid, have suggested activation of iNKT cells enhances the cytotoxic T lymphocyte-mediated elimination of cancer cells. In addition to exerting potent cytotoxic activity upon activation, iNKT cells are important immunoregulatory cells capable of rapidly producing an array of cytokines and chemokines. The cytokines produced include interferon-y (IFN-y), interleukin-2 (IL-2), IL-3, IL-4, IL- 10, IL- 13, IL- 17, IL-21 and transforming growth factor-p.

[0006] Over the last thirty years, T cell engagers have also been developed to attack cancer cells by harnessing the killing power of cytotoxic T cells. However, first-generation T cell engagers have exhibited lackluster safety and efficacy profiles. As of January 2020, blinatumomab was the only approved T cell engager in the US. Challenges related to the use of T cell engagers include the recruitment of counterproductive CD3+ T cell subsets, the release of systemic cytokines and cytokine storm, T cell dysfunction and exhaustion due to expression of immune checkpoint molecules, the presence of an immunosuppressive tumor microenvironment, tumor antigen loss / escape, on-target off-tumor toxicity and suboptimal potency. Accordingly, a need remains for safer and more effective treatment regimens for T cell engagers.SUMMARY

[0007] The instant invention includes a pharmaceutical composition comprising a therapeutically effective amount of natural killer T cells (“NKT cells”) and a T cell engager and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the NKT cells are invariant NKT cells (“iNKT cells”). In certain embodiments, the pharmaceutical composition comprises T cell engagers that are complexed to iNKT cells. For instance, the invention includes a pharmaceutical composition comprising T cell engagers in which the CD3 binding domain of the T cell engager is bound to one or more iNKT cells in solution.

[0008] In certain embodiments, the T cell engager is a bispecific or multispecific antibody with a CD3 binding domain and a tumor antigen binding domain. In certain embodiments, the CD3 binding domain comprises a variable heavy chain with a variableheavy chain CDR1, CDR2 and CDR3. In certain embodiments, the CD3 binding domain comprises a variable light chain with a variable light chain CDR1, CDR2, and CDR3. In certain embodiments, the CD3 binding domain comprises a variable heavy chain with a variable heavy chain CDR1, CDR2 and CDR3, and a variable light chain with a variable light chain CDR1, CDR2, and CDR3. In certain embodiments, the CD3 binding domain comprises a variable light and a variable heavy chain. In certain embodiments, the CD3 binding domain is an scFv. In certain embodiments, the CD3 binding domain binds human CD3s. In certain embodiments, the CD3 binding domain binds the epitope FSEXE (SEQ ID NO: 37) and QDGNE (SEQ ID NO: 38) of CD3s. In certain embodiments, the CD3 binding domain binds human CD3 and cynomolgus monkey CD3. In certain embodiments, the CD3 binding domain is derived from a CD3 antibody that is cross -reactive to both human and to human CD3 and cynomolgus monkey CD3. In certain embodiments, the CD3 binding domain comprises a variable heavy chain and / or a variable light chain derived from HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C. In certain embodiments, the T cell engager of the pharmaceutical composition of the invention comprises a VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2, and VL CDR3 which are the complementaritydetermining regions of an anti-CD3 antibody selected from the group consisting of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C. In certain embodiments, the CD3 binding domain comprises a variable heavy chain or binding fragment thereof with an amino acid sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequences of the variable heavy chain of one ofHuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K and I2C. In certain embodiments, the CD3 binding domain comprises a variable light chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of the variable light chain of one of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C. In certain embodiments, the CD3 binding domain comprises a variable heavy chain and a variable light chain with an amino acid sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96%identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the amino acid sequences of the variable heavy chain and variable light chain of one of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C. In certain embodiments, the CD3 binding domain is an scFv or an scFv fragment. In certain embodiments, the CD3 binding domain is monovalent. In certain embodiments the CD3 binding domain bivalent. In certain embodiments, the CD3 binding domain bivalent.

[0009] In certain embodiments, the T cell engager of the pharmaceutical composition comprises a tumor antigen binding domain comprising a variable heavy chain with variable heavy chain CDR1, CDR2, and CDR3. In certain embodiments, the tumor antigen binding domain comprises a variable light chain with a variable light chain CDR1, CDR2, and CDR3. In certain embodiments of the invention, the tumor antigen binding domain comprises a ligand or receptor. In certain embodiments, the antigen binding domain binds an EGFR family molecule (e.g., HER2, HER3, and EGFR / HER1), CD19, MUC16, B7-H3, BCMA, FAP, DLL3, PSMA, P-cadherin (CDH3), mesothelin (MSLN), EGFR, claudin 6, CD 123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule-1 (CLL-1), WT1, GPRC5D, CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO, GD2, MUC1, MUC17, CEACAM5, claudin 18.2, NKG2D ligand, and / or CD22. In certain embodiments, the tumor antigen binding domain is an scFv or an scFv fragment. In certain embodiments, the antigen binding domain is monovalent. In certain embodiments the tumor antigen binding domain is bivalent. In certain embodiments, the antigen binding is trivalent.

[0010] In certain embodiments, the T cell engager of the pharmaceutical composition comprises a tumor antigen binding domain comprising a ligand or receptor that specifically binds a cell surface tumor antigen.

[0011] The T cell engager of the pharmaceutical composition and methods of the invention can be in any T cell engager format known in the art. In certain embodiments of the compositions and methods of the invention, the T cell engager is a bispecific antibody. In certain embodiments of the compositions and methods of the invention, the T cell engager is a multi- specific antibody. In In certain embodiments of the invention, the T cell engager is a heterodimer. In certain embodiments of the invention, the T cell engager is a homodimer. In certain embodiments of the invention, the T cell engager is in the knobs-in-hole antibody format. In one embodiment, the T cell engager is in the BiTE antibody format. In one embodiment of the invention, the T cell engager is in an antibody format selected from the group consisting of knobs-into-holes (including knobs-into holes with cognate light chain andknobs-into-holes with common light chain), scFv-Ig, BiTE, BiTE-Fc, DART, DART-Fc, Tetravalent DART, Diabody, BiKE, TriKE, scFv-scFv-scFv, TandAb, TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 CrossMab, DVD-Ig, IgG-IgG, and Fab-scFv-Fc. In certain embodiments of the invention, the T cell engager is bivalent. For instance, the invention includes T cell engagers which are IgG-scFv bispecific antibodies (for example, wherein B 1 is an intact IgG and B2 is an scFv attached to B 1 at the N- terminus of a light chain and / or at the C-terminus of a light chain and / or at the N-terminus of a heavy chain and / or at the C-terminus of a heavy chain of the IgG.

[0012] In certain embodiments of the invention, the pharmaceutical composition includes an iNKT activator such as a-galactosylceramide (a-GalCer) or a derivative of a- GalCer. In certain embodiments of the invention, the iNKT activator is separately administered to a patient in conjunction with administration of iNKT cells (native or engineered) and a T cell engager. In certain embodiments of the invention, the iNKT activator comprises a synthetic aminocyclitolic ceramide, such as HS44. In certain embodiments, the iNKT activator comprises glycosphingolipid, a- glyco(Gal / Glc)diacylglycerol, P-glucosylsphingosine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, diphosphati-dylglycerol, lysophosphatidylcholine, or phenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonate. In certain embodiments, the iNKT activator comprises a naturally occurring glycolipid antigen derived from a microbial pathogen that acts as an activator of iNKT cells, for instance, glycosylceramides from Sphingomonadaceae bacterial species, including, for example, GSL- 1, GSL-3 and GSL-4; galactosyl diacylglycerols isolated from Borrelia burgdorferi; galactosyl diacylglycerols from Streptococcus pneumoniae; a-Linked glucosyl DAGs and a disaccharide Gal-Glu-DAG of the protozoan Entamoeba histolytica; asperamide B from Aspergillus spp.; a-mannnosyll-3 (6'-O-acyl a-mannosyl)-l-l monoacylglycerol from Saccharopolyspora; cholesteryl 6'-O-acyl a-mannoside, found Candida albicans; and PI57 from Helicobacter pylori. In certain embodiments, the iNKT activator comprises endogenous mammalian glycolipid presented by CD Id, for example, isoglobotrihexosylceramide, P- glucosylceramide, lysophosphotidylcholine, and lysosphingomelin. In certain embodiments, the iNKT cell activator is a molecule as disclosed in US patent 9,365,496B2.

[0013] In certain embodiments, the NKT cells (e.g., iNKT cells) are allogeneic cells.In certain embodiments, the NKT cells (e.g., iNKT cells) are obtained from one or more healthy human donors. In certain embodiments, the NKT cells (iNKT cells) are harvestedfrom a single healthy human donor. In certain embodiments, the healthy donor has iNKT levels above 0.05% presence in human peripheral blood. In certain embodiments, the healthy donor has homozygous for a human leukocyte antigen matching the recipient subject or that are prevalent in the subject’s population group. For instance, in certain embodiments, the healthy human donor (or donors) are homozygous for HLA-A02, maximizing the possibility of a partial matching with any treated patients (about 40% of individuals in the US and Europe harbor an HLA-A02 allele).

[0014] In certain embodiments, the NKT cells (e.g., iNKT cells) are obtained from peripheral blood or a leukopak. In certain embodiments, the NKT cells (e.g., iNKT cells) have been isolated from peripheral blood mononuclear cells (PBMCs). In certain embodiments, the NKT cells (e.g., iNKT cells) have been isolated from other blood cells. For instance, the invention includes isolation of iNKT cells from other cell types by use of a microbead-bound monoclonal antibody to the iNKT TCR.

[0015] The invention includes a composition comprised of NKT cells (e.g., iNKT cells) which have been harvested from a donor, isolated from other cell types, and expanded. In certain embodiments of the invention, after isolation of NKT cells (e.g., iNKT cells), the cells are activated by adding humanized CD3 and CD28 agonists to cell media.

[0016] In certain embodiments, after isolation, the NKT cells (e.g., iNKT cells) are stimulated by culturing NKT cells (e.g., iNKT cells) with aGalCer-pulsed irradiated donor peripheral blood mononuclear cells (iPBMCs). In certain embodiments, NKT cells (e.g., iNKT cells) are expanded by culturing with interleukin-2 (IL-2) over one or more weeks. In certain embodiments, iNKT cells are stimulated by culturing with irradiated aGalCer loaded autologous PBMCs in the presence of human IL-2, for instance, for 10-14 days.

[0017] Lor instance, the invention includes preparing allogeneic iNKT cells from a healthy donor(s) comprising isolating iNKT cells from other cells using micro-bead bound monoclonal antibody to the iNKT TCR, stimulating iNKT cells by culturing with aGalCer- pulsed irradiated PBMCs, and expanding iNKT cells by culturing with IL-2. iNKT cells can be subsequently formulated, and optionally, cryopreserved.

[0018] In certain embodiments of the invention, after isolation, NKT cells (e.g., iNKT cells) are stimulated and expanded by culturing in the presence of (i) IL-2, IL-7, IL- 15, IL- 12, and / or TNE-alpha and (ii) irradiated PBMCs loaded with aGalCer or irradiated artificial antigen-presenting cells (aAPCs) expressing CDl-d loaded with aGalCer.

[0019] In certain embodiments, the NKT cells (e.g., iNKT cells) are purified cells.For instance, the invention includes compositions comprising about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more iNKT cells as compared to other cell types (e.g., T cells). In certain embodiments, the pharmaceutical composition comprises about 106iNKT cells, about 107iNKT cells, about 108iNKT cells, about 109iNKT cells, about IO10iNKT cells, 1O2-1O10iNKT cells, about 1O3-1O10iNKT cells, about 104-105iNKT cells, about 104- 106iNKT cells, about 104-107iNKT cells, about 104-108iNKT cells, about 105-106iNKT cells, about 105-107iNKT cells, about 105-108iNKT cells, 1O5-1O10iNKT cells, or about 106- 1O10iNKT cells. In certain embodiments of the invention, the NKT cells are iNKT cells, and at least 95% of the iNKT cells are harvested from a single donor.

[0020] In certain embodiments, the NKT cells (e.g., iNKT cells) of the pharmaceutical composition and administered pursuant to the methods described herein are engineered cells (e.g., engineered iNKT cells). For instance, the invention includes pharmaceutical compositions comprising NKT cells (e.g., iNKT) cells that are engineered to express a CAR, a TCR, and / or an armoring molecule. In certain embodiments, the iNKT cells are engineered to express a CAR and an armoring molecule. In certain embodiments, the iNKT cells are engineered to express a TCR and an armoring molecule. In certain embodiments, the CAR is a BCMA or FAP CAR. For instance, the invention includes NKT cells (e.g., iNKT cells) expressing a BCMA CAR or armored BCMA CAR as described in US provisional patent application 63 / 422,916 and PCT patent application PCT / US2023 / 078730, the entire contents of each of which is incorporated herein by reference. In certain embodiments, the invention includes NKT cells (e.g., iNKT cells) expressing a FAP CAR or armored FAP CAR as described PCT patent application PCT / US2023 / 076011, the entire contents of which is incorporated herein by reference. In certain embodiments, the TCR is a PRAME, NY-ESO-1 TCR, or MMA. In certain embodiments, the invention includes NKT cells (e.g., iNKT cells) expressing one or more TCRs as described in US provisional patent application 63 / 579,799 and US patent application 16 / 512,031, , the entire contents of each of which is incorporated herein by reference. In certain embodiments, the armoring molecule is IL- 15, including, for instance, soluble IL- 15.

[0021] In certain embodiments, the NKT cells (e.g., iNKT cells) are engineered to express another molecule (e.g., a cytokine or ligand) capable of enhancing one or more properties (e.g., survival / persistence, immune interaction with other cells such asmacrophages and / or dendritic cells, disrupt immunosuppressive tumor microenvironment) of the cells.

[0022] For certain embodiments, the NKT cells (e.g., iNKT cells) are engineered to express any suitable armoring molecule known in the art. For instance, the invention includes NKT cells (e.g., iNKT cells) engineered to express IL-15, IL-2, IL-12, CD40L, 4- 1BBL, IL- 18, IL-7, IL-33, constitutively active Akt (caAkt), hybrid IL-4 / IL-7 receptor, checkpoint inhibitors such as anti-PDl antibodies, nanobodies targeting CD47, or bispecific T-cell engagers (BiTEs).

[0023] In one embodiment, the pharmaceutical composition of the invention comprises iNKT cells that are engineered to express an exogenous TCR while also expressing its natural invariant TCR (i.e., an invariant Va24-Jal8 a-chain gene and a limited number of P chains).

[0024] The invention includes methods of treating a subject suffering from a cancer that is associated with aberrant or overexpression of the tumor antigen that is specifically targeted by the T cell engager.

[0025] The instance invention includes methods of treating a subject (i.e., a patient) suffering with cancer with a therapeutically effective amount of a pharmaceutical composition as described herein.

[0026] In certain embodiments of the invention, a subject in need of treatment is administered a therapeutically effective amount of a pharmaceutical composition comprising NKT cells (e.g., iNKT cells) and a therapeutically effective amount of a pharmaceutical composition comprising a T cell engager. For instance, when the iNKT cells and T cell engager are separately administered, the iNKT cell pharmaceutical composition comprises iNKT cells, a pharmaceutically acceptable carrier or excipient, and optionally an iNKT activator. When the iNKT cells and T cell engager are separately administered, the T cell engager pharmaceutical composition comprises a T cell engager, a pharmaceutically acceptable carrier or excipient, and optionally an iNKT activator. In certain embodiments of the invention, the NKT cells (e.g., iNKT cells) and T cell engagers are separately formulated (i.e., two separate pharmaceutical compositions) but are administered to the subject at the same time.

[0027] In certain embodiments, the iNKT cells and T cell engagers are administered sequentially to the subject. For instance, in certain embodiments, the iNKT cells (i.e., iNKTcell pharmaceutical composition) are administered on a different day that the T cell engager (i.e., T cell engager pharmaceutical composition). In certain embodiments, the iNKT cells are administered before the T cell engager. In certain embodiments, the T cell engager is administered before the iNKT cells. In certain embodiments, the subject receives a single dose of iNKT cells (e.g., iNKT cell pharmaceutical composition) followed by multiple doses over time of the T cell engager (e.g., T cell engager pharmaceutical composition). The subject may also be administered an iNKT activator either alone or formulated with the iNKT cells or T cell engager. In certain embodiments of the invention, the iNKT cells are allogeneic iNKT cells and the T cell engager is blinatumomab, catumaxomab, mosunetuzumab, tebentafusp, teclistamab, ubamatamab, enoblituzumab, linvoseltamab, odronextamab, tebentafusp, forimtamig, or runimotamab.

[0028] In certain methods of the invention, the NKT cells (e.g., iNKT cells) administered to the subject are allogeneic NKT cells (e.g., allogeneic iNKT cells). In certain embodiments, the NKT cells are not autologous NKT cells (for instance, not autologous iNKT cells). In certain methods of the invention, lymphodepletion agents are not administered to the subject prior to the administration of NKT cells (e.g., iNKT cells) and T cell engager and are not administered concurrently with the NKT cells (e.g., iNKT cells) and T cell engager. For instance in certain embodiments of the invention, the subject does not receive treatment with bendamustine, fludarabine, or cyclophosphamide prior to, or concurrent with, the treatment with allogeneic iNKT cells and T cell engager.

[0029] For instance, the invention includes methods of treating cancer in a subject comprising administering to the subject an effective amount of allogenic NKT cells (e.g., iNKT cells) and a T cell engager. In certain methods of the invention, the cancer is associated with the over-expression of an EGFR family molecule such as HER2, HER3 or EGFR / HER1. In certain embodiments, the cancer is associated with the aberrant or overexpression of CD19. In certain embodiments, the cancer is associated with the aberrant or overexpression of MUC16. In certain embodiments, the cancer is associated with the aberrant or overexpression of B7-H3. In certain embodiments, the cancer is associated with the aberrant or overexpression of BCMA. In certain embodiments, the cancer is associated with the aberrant or overexpression of FAP. In certain embodiments, the cancer is associated with the aberrant or overexpression of DLL3. In certain embodiments, the cancer is associated with the aberrant or overexpression of HER2, HER3, EGFR / HER1, CD19, MUC16, B7-H3, BCMA, FAP, DLL3, PSMA, P-cadherin (CDH3), mesothelin (MSLN),EGFR, claudin 6, CD 123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule- 1 (CLL-1), WT1, GPRC5D, CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO, GD2, MUC1, CD22, MUC17, CEACAM5, Claudin 18.2, or NKG2D ligand.

[0030] In certain methods of the invention, the cancer is a blood malignancy. In certain methods of the invention, the cancer is a B cell malignancy. In certain methods of the invention, the cancer is In some embodiments the cancer is diffuse large B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, mucosa-associated lymphatic tissue lymphoma, small lymphocytic lymphoma (also known as chronic lymphocytic leukemia, CLL), mantle cell lymphoma, primary mediastinal (thymic) large B cell lymphoma, T cell / histiocyte-rich large B-cell lymphoma, primary cutaneous diffuse large B-cell lymphoma, EBV positive diffuse large B-cell lymphoma, Burkitt’s lymphoma, lymphoplasmacytic lymphoma, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, central nervous system lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, or large B-cell lymphoma. In some embodiments, the cancer comprises a blood cancer. In some embodiments, the blood cancer comprises myeloma, leukemia, lymphoma, Non-Hodgkin lymphoma, Hodgkin lymphoma, a myeloid neoplasm, a lymphoid neoplasm, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myelogenous leukemia (CML).

[0031] In certain methods of the invention, the cancer is a carcinoma or sarcoma. In certain methods of the invention, the cancer is epithelial cancer, (e.g., breast, gastrointestinal, lung), prostate cancer, bladder cancer, lung (e.g., small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer, or a colorectal cancer. In some embodiments, the cancer to be treated or antigen is from one of the following cancers: adenocortical carcinoma, agnogenic myeloid metaplasia, AIDS-related cancers (e.g., AIDS- related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, meningiosarcoma, craniopharyngioma, hemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma,and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing's family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., nonmelanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), or Meigs' syndrome.

[0032] In one embodiment, the allogenic iNKT cells of the composition are capable of restricting immunosuppressive tumor myeloid cells via their endogenous TCR. For instance, the invention includes methods of restricting immunosuppressive tumor myeloidcells comprising contacting said immunosuppressive tumor myeloid cells with a composition comprising allogeneic iNKT cells and a T cell engager.

[0033] The invention includes methods of activating exhausted T cells in a subject comprising administering to said subject iNKT cells and a T cell engager as disclosed herein. In certain embodiments, the pharmaceutical compositions of the invention are administered to a subject to activate exhausted T cells. In one embodiment, the subject with exhausted T cells has previously received T cell engager therapy. For instance, the invention includes treating a subject with exhausted T cells caused by T cell engager therapy with an iNKT cell pharmaceutical composition of the invention. In certain embodiments, the invention includes treating a subject previously treated with T cell engager therapy by continuous infusion with an iNKT cell pharmaceutical composition of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIGURE 1 illustrates the expression of CD3s on iNKT cells, freshly isolatedT cells and expanded T cells. iNKT cells, freshly isolated T cells and expanded T cells were stained with anti-CD3s antibody. Data was analyzed by flow cytometry. Number of molecules per cell was quantified using Quantibrite and plotted. Data shown from three independent donors with three technical replicates used in the assay.

[0035] FIGURE 2A shows binding of a CD19-CD3 engager to iNKT cells (A) and T cells (B). Flow cytometric analysis was performed using anti-human IgG to confirm binding of CD3+ cells. FIGURE 2B shows binding of B7-H3 engager with iNKT cells (CD3+). iNKT cells were incubated with various concentrations of the engager followed by fluorescently tagged anti-human IgG antibody. Binding was determined by flow cytometry analysis. Secondary antibody alone was set as the negative control.

[0036] FIGURE 3 shows Mucl6-CD3 mediated iNKT activation. Assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager molecule. iNKT cells from six donors were used as effector cells. CFSE labeled OVCAR-3 cell line was used as target cells. Effector and target cells were co-cultured at 5:1, 1:1 and 1:5 E:T ratio in presence or absence of Mucl6-CD3 engager for 24h, 48h and 72h. Percentage- specific iNKT cells activation was determined using flow cytometry. Cells were acquired by FACS Canto and analyzed in Flow Jo.

[0037] FIGURE 4A shows the binding of a Mucl6-CD3 engager to iNKT cells, freshly isolated T cells and expanded T cells. A Mucl6xCD3 engager was incubated withcells at indicated concentrations and detected with anti-IgG4-Fc secondary antibody. Binding was determined by flow cytometry. MFI for each sample was plotted. Data shown using cells from one matched donor. FIGURE 4B shows the binding and retention of Mucl6-CD3 engager on iNKT, freshly isolated T cells and expanded T cells at 37oC. iNKT cells, freshly isolated T cells and expanded T cells were pre-complexed with 100 nM of Mucl6-CD3 engager. Cells were either directly stained with APC-conjugated donkey anti-human IgGl Fc and analyzed by flow cytometry, or incubated for 1, 3, 6, 24, and 48 h at 37 oC before detection of retained antibodies by flow cytometry. After subtracting the MFI of cells incubated with the secondary reagent alone for each time point, the percentage of retained engagers relative to initially bound engagers were plotted. Data shown from two independent matched donors with two replicates from each.

[0038] FIGURE 5 shows the cytolytic activity of a CD19-CD3 bispecific antibody against CD19+ and CD19- cancer cells. CFSE labeled target cells A375 (A), A375-CD19 (B), C1R (C), or Raji (D) were used for the iNKT cytotoxicity assay. In brief, expanded 1st or 2nd stim iNKT cells from two donor (effector cells) were incubated with target cells at 4: 1 E:T ratio in presence or absence of CD19-CD3 engager or HIV-CD3 for 48 h in Xuri based medium with 100 lU / ml IL-2. HIV-CD3 targets the CD4-binding site of the HIV envelope glycoprotein (heavy and light chain of VRC01) used as negative control. Percentage- specific lysis of the target cells and iNKT cells activation was determined using flow cytometry. Cells were harvested following the incubation period, stained with a live dead dye and surface markers. Cells were acquired by FACSCanto and analyzed in Flow Jo.

[0039] FIGURE 6 shows B7-H3 engager mediates cytotoxicity to B 7 -H3 -positive tumor cells in vitro. CFSE labeled target cells Hs 683 (A), A375 (B), U-87 MG (C) or A549 (D) cells were used for the iNKT cytotoxicity assay. Expanded iNKT cells from two donor (effector cells) were incubated with target cells at 4:1 E:T ratio in presence or absence of B7- H3-CD3 engager or HIV-CD3 for 48 h in RPMI-1640 based medium with 50 lU / ml IL-2. HIV-CD3 targets the CD4-binding site of the HIV envelope glycoprotein (heavy and light chain of VRC01) used as negative control. Percentage- specific lysis of the target cells was determined using flow cytometry. Cells were harvested following the incubation period, stained with a live dead dye and surface markers. Cells were acquired by FACSCanto and analyzed in Flow Jo.

[0040] FIGURE 7 shows Mucl6-CD3 mediated iNKT cytotoxicity. The assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager. iNKT cells from sixdonors were used as effector cells. CFSE labeled OVCAR-3 cell line was used as target cells. Effector and target cells ware co-cultured at 5:1, 1:1 and 1:5 E:T ratio in presence or absence of Mucl6-CD3 engager for 24h, 48h and 72h. Percentage- specific lysis of the target cells was determined using flow cytometry. Cells were acquired by FACS Canto and analyzed in Flow Jo.

[0041] FIGURE 8 demonstrates that iNKT cells exhibit better cytotoxicity against target cells than conventional T cells. The assay was performed in 2D adherent cell cultures using in-house produced B7-H3-CD3 engager molecule (knob-into-hole technology). Purified T cells and expanded iNKT cells used as effector cells and irradiated A375-GFP as target cells. Effector and target cells ware co-cultured at 1:1 E:T ratio in presence or absence of B7-H3-CD3 in media containing 50 lU / ml of IL-2. Cells were imaged every 6h for 120h using the Cytation 5 Cell Imaging Multi-Mode Reader (Biotek) driven by Gen5 software (Biotek). Between imaging reads, cells were incubated at 37 °C with 5% CO2 using the Biospa 8 Automated Incubator (Biotek).

[0042] FIGURE 9 compares killing with iNKT, freshly isolated T cells and expandedT cells combined with a MUC16-CD3 targeting bispecific antibody. The assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager molecule. iNKT cells, freshly isolated T cells, and expanded T cells from a matched donor were used as effector cells. CFSE labeled target cells used were Mucl6+ve OVCAR-3 and Mucl6-ve a375 cells. Effector and target cells ware co-cultured at 5:1, 1:1 and 1:5 E:T ratio in presence or absence of Mucl6-CD3 engager for 24h, 48h and 72h. Percentage- specific iNKT cells killing was determined using flow cytometry. Cells were acquired by FACS Canto and analyzed in FlowJo.

[0043] FIGURE 10 shows cytokine release by iNKT, freshly isolated T cells and expanded T cells upon killing with and without a MUC16xCD3 bispecific antibody. Supernatants from iNKT, freshly isolated T and expanded T cells co-cultured for 48h in the presence of 0.1 nM Mucl6xCD3 engager, or controls without engager were collected. Cytokine release was analyzed by Meso Scale Discovery Electrochemiluminescence (MSD). MDS uses sandwich ELISA method coupled with electro-chemiluminescence (ECL) detection and plate array technology to provide highly sensitive and multiplexed detection of analytes in a complex biological matrix.

[0044] FIGURES 11A-11D compare engager-mediated killing with iNKT versus T- cells. FIGURES 11A-11D show CD3-engager mediated iNKT and T cell cytotoxicity. Assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager molecule and DLL3-, Her3-, and Cldnl8.2-CD3 engager molecules (all purchased from Proteogenix). Expanded 2nd stim iNKT cells from two donors were used as effector cells. CFSE labeled OVCAR-3, SHP77, SKBR3, and PATU8988S cell lines was used as target cells. Effector and target cells ware co-cultured at 5:1, 1:1 and 1:5 E:T ratio in presence or absence of CD3 engagers in RPMI-1640 based medium with 50 lU / ml IL-2 for 24h, 48h and 72h. Percentage- specific lysis of the target cells was determined using flow cytometry. Cells were harvested following the incubation period, stained with a live dead dye and activation markers. Cells were acquired by FACS Canto and analyzed in Flow Jo.

[0045] FIGURE 12 shows that iNKT cells enhance CD3xMucl6 engager anti-tumor activity of patient immune cells. T / PBMCs were co-cultured with OVCAR3 (MUC16+) cells for 24 hours at 4:1. Engagers were added at InM. Killing was assessed via flow cytometry. iNKT cells were added at 1:5 ratio to T / PBMCs (1:20 ratio to target cells). Addition of iNKT cells even at a low number led to >2x killing improvement.

[0046] FIGURE 13 shows cytokine release by iNKT cells upon engager-mediated killing. Supernatants from iNKT cells co-cultured for 24h in the presence of 0.1 nM Mucl6xCD3 or DLL3xCD3 engager were collected. Cytokine release was analyzed using Proteome Profiler Antibody Arrays. This semiquantitative antibody array allows for the measurement of multiple proteins in a single sample. Antibody array kits contain buffers, detection antibodies, and membranes spotted in duplicate with high quality capture antibodies. The arrays utilize chemiluminescence for detection data, which was quantified using Quick Spots image analysis software designed specifically for Proteome Profiler Arrays. The results show that iNKT cells during engager-mediated killing secrete cytokines promoting activation, cytotoxicity and immunomodulation.DETAILED DESCRIPTION

[0047] The disclosure provides compositions and methods for the treatment of cancers associated with the expression of a cell surface tumor antigen. The use of T cell engagers to redirect the activity of T lymphocytes against tumor cells expressing a cell surface tumor antigen is an existing approach designed for the treatment of cancer. However,T cell depletion and the immunosuppressive nature of the tumor microenvironment limit the effectiveness of such therapies against cancer. The instant invention describes the use of allogeneic iNKT cells, both native and engineered, to overcome these limitations associated with T cell engagers.

[0048] In the instant invention, iNKT cells are capable of complexing with T cell engagers and “cross-link” tumor cells. Briefly, the T cell engager binds CD3 on an iNKT cell (via its CD3 binding domain). The T cell engager binds an overexpressed cell surface tumor antigen on the surface of the tumor cell (via its tumor antigen binding domain), and an iNKT cell binds tumor cells via its invariant T cell receptors.

[0049] iNKT cells are capable of binding stress ligands on tumor cells via NK receptors such as NKG2D or DNAM1. The binding of the iNKT cells to tumor cells activates iNKT cells, resulting in the release of cytokines and tumor cell lysis. As shown herein, the cytokine release profile of activated iNKT cells is different from that of activated T cells. Of importance, the cytokine release profile of activated iNKT cells shows less TNFa as compared to that of activated T cells. It is believed that the reduction in TNFa exhibited by activated iNKT cells compared to activated T cells may reduce the risk that the subject will experience an adverse event associated with cytokine release when iNKT cells are administered to a patient receiving T cell engager therapy (as compared to T cell engager therapy alone).

[0050] An additional benefit of the compositions and methods of the invention over traditional T cell engager therapy is the impact on exhausted T cells and tumor cell killing. The compositions and methods of the invention have the potential to activate exhausted T cells at the tumor site and attract, activate, and cause the proliferation of host immune cells at the tumor site, including natural killer and T cells. Moreover, iNKT cells are capable of restricting immunosuppressive tumor myeloid cells via their endogenous TCR.

[0051] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents define a term that contradicts that term's definition in the application, the definition that appears in this application controls. However, mention of any reference,article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0052] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. It should be understood that the terms "a" and an as used herein refer to "one or more" of the enumerated components unless otherwise indicated. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used synonymously. In addition, it should be understood that the polypeptides comprising the various combinations of the components (e.g., domains or regions) and substituents described herein, are disclosed by the present application to the same extent as if each polypeptide was set forth individually. Thus, selection of particular components of individual polypeptides is within the scope of the present disclosure.

[0053] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of 5% to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., up to 5% to 10% below) the value or range remain within the intended meaning of the recited value or range.

[0054] As used herein, “natural killer T” or “NKT” cells constitute a small population of non-conventional T lymphocytes defined by the expression of both aP T-cell receptors (TCR) and some lineage markers of NK cells. There are a number of subtypes of NKT cells, which can be determined through their T cell receptor (TCR) usage, cytokine production, expression of specific surface molecules and reactivity. The most extensively characterized subtype of NKT cells are the so-called type I or invariant natural killer T cell (iNKT cells) (See Matsuda et al. Curr Opin Immunol, 20: 358-68, 2008). The TCR repertoire expressed by iNKT cells is invariant — i.e., a canonical a-chain (Va24-Jal8 in humans; Val4-Jal8 in mice) associated with a limited spectrum of P chains (Vpi l in humans; VP8.2, VP2, VP7 in mice). This is in contrast to the polymorphic TCRs expressed by so-called nonclassical or noninvariant type II NKT cells (See Porcelli et al, J Exp Med, 178: 1-16, 1993).

[0055] As used herein, “iNKT cells” encompass both native and engineered invariant natural killer T cells. iNKT cells can be harvested from the body, for instance, isolated fromperipheral blood and expanded. In certain embodiments, iNKT cells are harvested from healthy donors. In certain embodiments, iNKT cells are isolated from the blood or PBMCs of healthy doors and subsequently activated and expanded by culturing the iNKT cells in the presence of one or more activating agents. For instance, the invention includes iNKT cells which are isolated from donor cells and cultured in the presence of aGalCer -pulsed irradiated PBMCs and interleukin-2 (IL-2).

[0056] iNKT cells constitute <0.1% of peripheral blood and <1% of bone marrow T cells in humans, but despite their relative scarcity, they exert potent immune regulation via production of IL-2, Thl-type (IFN-y, TNF-a), Th2-type (IL-4, IL-13), IL-10, and IL-17 cytokines. (See Lee et al, J Exp Med, 2002; 195: 637-641; Bendelac et al, Annu Rev Immunol, 2007; 178: 58-66; Burrows et al, Nat Immunol, 2009; 10(7): 669-71). iNKT cells are characterized by a highly restricted (invariant) T-cell receptor (TCR)-Va chain (Va24 in humans). Their TCR is unique in that it recognizes altered glycolipids of cell membranes presented in context of a ubiquitous HLA-like molecule, CD Id. (See Zajonc & Kronenberg, Immunol Rev, 2009; 230 (1): 188-200). CDld is expressed at high levels on many epithelial and hematopoietic tissues and on numerous tumor targets, and is known to specifically bind only the iNKT TCR. (See Borg et al, Nature, 2007, 448: 44-49).

[0057] Like natural killer cells (“NK cells”), iNKT cells play a major role in tumor immuno surveillance via direct cytotoxicity mediated through perforin / Granzyme B, Fas / FasL, and TRAIL pathways. However, unlike NK cells, iNKT cells are not known to be inhibited by ligands such as Class I MHC (See Brutkiewicz & Sriram, Crit Rev Oncol Hematol, 2002; 41: 287-298; Smyth et al, J. Exp. Med. 2002; 191: 661-8; Wilson & Delovitch, Nat Rev Immunol, 2003; 3: 211-222; Moiling et al, Clinical Immunology, 2008; 129: 182-194; Smyth et al, J Exp Med, 2005; 201 (12): 1973-1985; Godfrey et al, Nat Rev Immunol, 2004, 4: 231-237).

[0058] At least one contribution of iNKT cells to antitumor immunity occurs indirectly via the activation of iNKT cells by dendritic cells (“DCs”). Activated iNKT cells can initiate a series of cytokine cascades — including production of interferon gamma (IFN- y) — that helps boost the priming phase of the antitumor immune response (See Terabe &. Berzofsky, Ch 8, Adv Cancer Res, 101: 277-348, 2008). IFN-y production by iNKT cells, as well as NK cells and CD8+ effectors, has been shown to be important in tumor rejection (See Smyth et al, Blood, 99: 1259-1266, 2002).

[0059] Further, iNKT cells have been shown to specifically target the killing ofCD Id-positive tumor-associated macrophages (“TAMs”), a highly plastic subset of inflammatory cells derived from circulating monocytes that perform immunosuppressive functions (See Sica & Bronte, J Clin Invest, 117: 1155-1166, 2007). TAMs are known to be a major producer of interleukin-6 (IL-6) that promotes proliferation of many solid tumors, including neuroblastomas and breast and prostate carcinomas (See Song et al., J Clin Invest, 119: 1524-1536, 2009; Hong et al, Cancer, 110: 1911-1928, 2007). Direct CD Id-dependent cytotoxic activity of iNKT cells against TAMs suggests that important alternative indirect pathways exist by which iNKT cells can mediate antitumor immunity, especially against solid tumors that do not express CD Id.

[0060] The recognition unit, the CD Id molecule, has a structure closely resembling that of the MHC class I molecule, including the presence of beta-2 microglobulin. It is characterized by a deep cleft bordered by two alpha chains and containing highly hydrophobic residues, which accepts lipid chains. The cleft is open at both extremities, allowing it to accommodate longer chains. The canonical ligand for CD Id is the synthetic alpha galactosylceramide (“alpha GalCer”). However, many natural alternative ligands have been described, including glyco- and phospholipids, the natural lipid sulfatide found in myelin, microbial phosphoinositol mannoside and alpha-glucuronosylceramide.

[0061] The term “allogeneic” is intended to refer to NKT cells (e.g., iNKT cells) that are not isolated from the subject receiving the pharmaceutical composition comprising NKT cells (e.g., iNKT cells) and T cell engager. In some embodiments, the cells are isolated from a healthy donor and the recipient is a subject suffering from a cancer. In certain embodiments, donor cells are activated and expanded prior to administering to an unrelated subject. In certain embodiments, iNKT cells harvested from the peripheral blood of a single healthy donor can be subsequently expanded, formulated, and optionally cryopreserved, for use for treatment of several hundred subjects.

[0062] The term “autologous” is intended to refer to NKT cells (e.g., iNKT cells) that are isolated from a subject and returned to the same subject. For instance, the invention includes isolating iNKT cells from a subject, expanding the cells and using the cells in conjunction with a T cell engager to treat the subject. In one embodiment of the invention, the iNKT cells of the pharmaceutical compositions and methods are not autologous (i.e., the cells are allogeneic).

[0063] As used herein, “isolated,” for example, with respect to cells and / or nucleic acids means altered or removed from the natural state through human intervention. Isolated iNKT cells are iNKT cells that have been removed from surrounding cells, for instance, isolation of iNKT cells from a sample of PBMCs or leukopak.

[0064] As used herein, expansion of iNKT cells typically refers to an increase in number, but expansion of iNKT cells also can refer to an increase in activity or potency of the cells.

[0065] The term “iNKT activator” as used herein refers to an agent that activates iNKT cells. iNKT activators include antigens presented by CD Id that are capable of stimulating the invariant TCR expressed by iNKT cells. iNKT cells can be activated by both exogenous and a limited number of endogenous glycolipids. The glycolipid a- galactosylceramide (“a-GalCer”) is a potent and specific activator of mouse and human iNKT cells and has been used in cancer clinical trials to drive NKT cell-mediated immune responses. (See Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki K, Ueno H, Nakagawa R, Sato H, Kondo E, et al. CD Id-restricted and TCR-mediated activation of valphal4 NKT cells by glycosylceramides. Science. 1997;278:1626-1629.)

[0066] Various publications describe synthesis of a-GalCer and its derivatives. For instance, see Morita, et al., J. Med. Chem., 38:2176 (1995); Sakai, et al., J. Med. Chem., 38:1836 (1995); Morita, et al., Bioorg. Med. Chem. Lett., 5:699 (1995); Takakawa, et al., Tetrahedron, 54:3150 (1998); Sakai, et al., Org. Lett., 1:359 (1998); Figueroa-Perez, et al., Carbohydr. Res., 328:95 (2000); Plettenburg, et al., J. Org. Chem., 67:4559 (2002); Yang, et al., Angew. Chem., 116:3906 (2004); Yang, et al., Angew. Chem. Int. Ed., 43:3818 (2004); and, Yu, et al., Proc. Natl. Acad. Sci. USA, 102(9):3383-3388 (2005).

[0067] In some embodiments, the iNKT activator may be a GalCer derivative in which galactose, a key element of iNKT TCR recognition, is substituted with a nonglycosidic substitutions, for example, as described in Silk et al., Cutting Edge: Nonglycosidic CD Id Lipid Ligands Activate Human and Murine Invariant NKT Cells, J Immunol 2008; 180:6452- 6456. Exemplary GalCer variants include threitolceramide arabinitolceramide, and glycerolceramide. In one embodiment, the iNKT activator is threitolceramide modified to have restricted flexibility in the sugar headgroup, for example, by incorporating the threitol unit into a carbocycle such as six- or seven-membered ring, for example, as described inJukes et al., Non-glycosidic compounds can stimulate both human and mouse iNKT cells, Eur. J. Immunol. 2016. 46: 1224-1234.

[0068] In certain embodiments, the iNKT activator is a GalCer variant comprisingGalCer having a modification at a fatty acyl chain or a terminal benzene ring at the phytosphingosine chain, for example, glycolipid, 7DW8-5, as described by Li et al., Design of a potent CDld-binding NKT cell ligand as a vaccine adjuvant PNAS, 2010, 107: 3010- 13015.

[0069] In certain embodiments, the iNKT activator is a GalCer variant comprising carbohydrate and sphingoid base modifications in an a-galactosyl ceramide, for instance, as described in Chennamadhavuni et al., Dual Modifications of a-Galactosylceramide Synergize to Promote Activation of Human Invariant Natural Killer T Cells and Stimulate Anti-tumor Immunity, 2018, Cell Chemical Biology 25, 571-584.

[0070] In certain embodiments, the iNKT activator is a GalCer variant comprising anE-alkene linker between the sugar and lipid moieties, for example, GCK152, which has an aromatic ring in the tail of the acyl chain. (See Li et al., Invariant TCR Rather Than CD Id Shapes the Preferential Activities of C-Glycoside Analogues Against Human Versus Murine Invariant NKT Cells, J Immunol 2009; 183:4415-4421.)

[0071] The iNKT activator may encompass other glycolipid activators of iNKT cells.In certain embodiments, the iNKT activator is a synthetic aminocyclitolic ceramide, such as HS44, for example, as described in Kerzerho et al., Structural and functional characterization of a novel non-glycosidic iNKT agonist with immunomodulatory properties J Immunol. 2012 Mar. 1; 188(5): 2254-2265.

[0072] In one embodiment, the iNKT activator comprises glyco sphingolipid, a- glyco(Gal / Glc)diacylglycerol, P-glucosylsphingosine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, diphosphati-dylglycerol, lysophosphatidylcholine, or phenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonate. (See Macho-Femandez and Brigl, The extended family of CD Id-restricted NKT cells: sifting through a mixed bag of TCRs, antigens, and functions, Front. Immun. 2015, Vol. 6, Article 362.)

[0073] In some embodiments, the iNKT activator is a naturally occurring glycolipid antigen derived from a microbial pathogen that acts as an activator of iNKT cells, for instance, glycosylceramides from Sphingomonadaceae bacterial species, including, forexample, GSL-1, GSL-3 and GSL-4; galactosyl diacylglycerols isolated from Borrelia burgdorferi; galactosyl diacylglycerols from Streptococcus pneumoniae; a-Linked glucosyl DAGs and a disaccharide Gal-Glu-DAG of the protozoan Entamoeba histolytica; asperamide B from Aspergillus spp.; a-mannnosyll-3 (6'-O-acyl a-mannosyl)-l-l monoacylglycerol from Saccharopolyspora; cholesteryl 6'-O-acyl a-mannoside, found Candida albicans; and PI57 from Helicobacter pylori.

[0074] In some embodiments, the iNKT activator is an endogenous mammalian glycolipid presented by CD Id, for example, isoglobotrihexosylceramide, P-glucosylceramide, lysophosphotidylcholine, and lysosphingomelin.

[0075] In some implementations, the iNKT activator is an antibody which binds to the TCR of iNKT cells and, by such binding, causes their activation. Exemplary antibodies include the 6B11 monoclonal antibody against the iNKT TCR-alpha CDR3 loop, disclosed in Exley et al, Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR a-chain CDR3 loop, Eur. J. Immunol. 2008. 38: 1756-1766. Additional iNKT activating antibodies include NKTT320, a recombinant, humanized, monoclonal antibody that binds selectively and with high affinity to human iNKT TCR, as described in Patel et al., 2020, Cancer Immunotherapeutic Potential of NKTT320, a Novel, Invariant, Natural Killer T Cell-Activating, Humanized Monoclonal Antibody, Int J Mol Sci. 2020 June; 21: 4317.

[0076] In certain embodiments, the iNKT cell activator is a molecule as disclosed inUS patent 9,365,496B2.

[0077] As used herein, “T cell engager” refers to an engineered antibody comprising at least one tumor antigen binding domain and at least one CD3 binding domain. The invention includes bispecific and multi- specific T cell engagers. As used herein, a “tumor antigen domain” or “tumor antigen binding domain” refers to a binding domain that specifically binds a tumor antigen. As used herein, a “CD3 binding domain” refers to a binding domain that specifically binds CD3.

[0078] In certain embodiments, the T cell engager comprises a tumor antigen binding domain that binds a tumor antigen selected from the group consisting of EGFR family molecules (HER2, HER3, and EGFR / HER1), CD19, MUC16, B7-H3, BCMA, FAP, DLL3, PSMA, P-cadherin (CDH3), mesothelin (MSLN), EGFR, claudin 6, CD 123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule-1 (CLL-1), WT1, GPRC5D,CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO, GD2, MUC1, CD22, FLT3, CEACAM5, Claudin 18.2, NKG2D ligand, and MUC17, and a CD3 binding domain that binds human CD3s.

[0079] The invention includes T cell engagers with a monovalent, bivalent, or bivalent (or more) tumor antigen binding domain fused or otherwise connected to a CD3 binding domain. In certain embodiments, the T cell engager comprises a monovalent, bivalent, or bivalent (or more) CD3 binding domain fused or otherwise connected to a tumor antigen binding domain.

[0080] The T cell engagers of the invention may be configured in any bispecific or multi- specific antibody format known in the art. The T cell engagers of the invention may comprise additional domains or regions. In certain embodiments, the T cell engager is an engineered antibody selected from the group consisting of knobs-into-holes (including knobs- into holes with cognate light chain and knobs-into-holes with common light chain), scFv-Ig, BiTE, BiTE-Fc, DART, DART-Fc, Tetravalent DART, Diabody, BiKE, TriKE, scFv-scFv- scFv, TandAb, TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 CrossMab, DVD-Ig, IgG-IgG, and Fab-scFv-Fc. The invention includes T cell engagers in the antibody formats as described in Wang Q, Chen Y, Park J, Liu X, Hu Y, Wang T, McFarland K, Betenbaugh MJ. Design and Production of Bispecific Antibodies. Antibodies (Basel). 2019 Aug 2;8(3):43 and in Ma J, Mo Y, Tang M, Shen J, Qi Y, Zhao W, Huang Y, Xu Y and Qian C (2021) Bispecific Antibodies: From Research to Clinical Application. Front. Immunol. 12:626616, each of which is hereby incorporated by reference in its entirety.

[0081] T cell engagers may be bivalent bispecific antibodies, such as IgG-scFv bispecific antibodies (for instance, wherein the first binding domain is an intact IgG and the second binding domain is an scFv attached to the first binding domain at the N-terminus of the light chain and / or the at the C-terminus of a light chain and / or at the N-terminus of a heavy chain and / or at the C-terminus of a heavy chain of the IgG or vice versa.

[0082] The T cell engagers of the invention may be heterodimers or homodimers. For instance, the invention includes heterodimer T cell engagers that are in the “knob-in-hole” or “knobs-into-holes” (“KiH”) format. (See Ridgway J.B., Presta L.G., Carter P. ‘Knobs-into- holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996;9:617-621.) The KiH approach is an effective way to produce bispecific T cell engagers by driving heterodimerization through the CH3 domain of the Fc region. The“knob” is created by introducing a T366Y mutation in CH3 region of the Fc domain of the antibody or polypeptide. The ’’hole” is created in the CH3 domain by introducing the mutation (Y407T). Alternatively, the “hole” can be created in the CD3 domain by incorporating S354C and T366W mutations or Y349C, T366S, L368A, and Y407V mutations. (See Merchant A.M., Zhu Z., Yuan J.Q., Goddard A., Adams C.W., Presta L.G., Carter P. An efficient route to human bispecific IgG. Nat. Biotechnol. 1998;16:677-681.) In certain embodiments, the “hole” is created by incorporating T366S, L368A, Y407V mutations in the CH3 region of the Fc domain. The invention includes modified knob-in- hole bispecific platforms including an scFv knobs-into-hole or BiTE knob-in-hole (see Xu et al., 2015, mABs 7(l):231-242). In certain embodiments of the invention, the T cell engager is an anti-MUC16 x anti-CD3 KiH antibody, an anti-CD19 x anti-CD3 KiH antibody, or an anti-B7-H3 x anti-CD3 KiH antibody.

[0083] In certain embodiments, a heterodimer T cell engager is produced using strand-exchange engineered domain (“SEED”) heterodimerization. SEED heterodimerization utilizes complementarity of alternating sequences derived from IgG and IgA CH3 domains also known as AG SEED CH3 and GA SEED CH3. The IgG and IgA CH3 derivatives generate complementary sequences so that the two complementary heavy-chain heterodimers are assembled while excluding the assembly of homodimers lacking complementarity. (See Davis J.H., Aperlo C., Li Y., Kurosawa E., Lan Y., Lo K.M., Huston J.S. SEEDbodies: Fusion proteins based on strand-exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng. Des. Sei. PEDS. 2010;23:195-202.)

[0084] In certain embodiments, the T cell engager is a CrossMab antibody. (SeeKlein C., Schaefer W., Regula J.T., Dumontet C., Brinkmann U., Bacac M., Umana P. Engineering therapeutic bispecific antibodies using CrossMab technology. Methods. 2019;154:21-31.)

[0085] In certain embodiments, the T cell engager is comprised of asymmetric CH3IgG4 regions, in which one arm contains H435R and Y436F mutations allowing for selective isolation of the bispecific antibodies based on Protein A binding. In this antibody format, the T cell engager comprises a variable heavy chain that specifically binds to the tumor antigen, a variable heavy chain that specifically binds to CD3, and a common variable light chain. An exemplary amino acid sequence of a common variable light chain is set forth below: DIOMTOSPSSLSASVGDRVTITCRASOSISTYLNWYOOKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGOGTRLEIK (SEQ ID NO:33; CDRs underlined). (See US patent application 2018 / 0112001; Alison Crawford, Lauric Haber, Kristin Vazzana, Lauren Canova, Priyanka Ram, Jennifer Principio, Arpita Pawashe, Curtis Colleton, Marcus Kelly, Sosina Makkonen, Carlos Hickey, Paurene Duramad, Stephen Godin, John Lin, Eric Smith, Gavin Thurston, Jessica R. Kirshner. REGN4018, a novel MUC16xCD3 bispecific T-cell engager for the treatment of ovarian cancer. Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1777. )

[0086] Examples of T cell engagers designed using the asymmetric CH3 IgG4 antibody platform described above include ubamatamab, MUC-16 x CD3 T cell engager; linvoseltamab, a BCMA x CD3 T cell engager; and odronextamab, a CD20 x CD3 T cell engager. In certain embodiments of the invention, the T cell engager is ubamatamab. For instance, in certain embodiments, the T cell engager comprises a binding domain that specifically binds MUC-16 comprising the amino acid sequence as set forth in SEQ ID NO:25; a binding domain that specifically binds CD3 comprising the amino acid sequence as set forth in SEQ ID NO:29; and a binding domain comprising the common light chain amino acid sequence as set forth in SEQ ID NO:33. In certain embodiments of the invention, the T cell engager is linvoseltamab. In certain embodiments of the invention, the T cell engager is odronextamab.

[0087] In certain embodiments of the invention, the T cell engagers are in the bispecific T-cell engager (BiTE), dual-affinity re-targeting (DARTs) or Tandem diabodies (TandAbs) format.

[0088] A T cell engager in the BiTE antibody format is composed of two scFv fragments from two different monoclonal antibodies connected by a peptide linker, enabling them to retain each antibody’s binding activity when assembled. (See Mallender W.D., Voss E.W., Jr. Construction, expression, and activity of a bivalent bispecific single-chain antibody. J. Biol. Chem. 1994;269:199-206.) Blinatumomab (Blincyto®, DrugBank entry DB09052), which has been approved by the FDA for the treatment of B-cell precursor acute lymphoblastic leukemia (ALL). Blinatumomab is comprised of an anti-CD 19 scFv in the VL-VH orientation linked through a short glycine / serine (GGGGS) linker to an anti-CD3 scFv in the VH-VL orientation. (See Huehls A.M., Coupet T.A., Sentman C.L. Bispecific T- cell engagers for cancer immunotherapy. Immunol. Cell Biol. 2015;93:290-296.) In certainembodiments, the T cell engager is a BiTE antibody. In certain embodiments of the invention, the T cell engager is blinatumomab. In certain embodiments of the invention, the T cell engager is an anti-CD19 x anti-CD3 BiTE, anti-FLT3 x anti-CD3 BiTE, an anti-PSMA x anti-CD3 BiTE, and anti-DLL3 x anti-CD3 BiTE, or an anti-MUC17 x anti-CD3 BiTE.

[0089] In certain embodiments of the invention, the T cell engager is a modified BiTE antibody. For instance, the invention includes BiTE-albumin and BiTE-Fc T cell engagers. (See Arvedson T.L., Balazs M., Bogner P., Black K., Graham K., Henn A., Friedrich M., Hoffmann P., Kischel R., Kufer P., et al. Abstract 55: Generation of half-life extended anti- CD33 BiTE® antibody constructs compatible with once-weekly dosing. Cancer Res. 2017;77:55; Lorenczewski G., Friedrich M., Kischel R., Dahlhoff C., Anlahr J., Balazs M., Rock D., Boyle M.C., Goldstein R., Coxon A., et al. Generation of a Half-Life Extended Anti-CD19 BiTE® Antibody Construct Compatible with Once-Weekly Dosing for Treatment of CD19-Positive Malignancies. Blood. 2017; 130:2815.) In one embodiment of the compositions and methods of the invention, the T cell engagers is an anti-CD19 x anti-CD3 BiTE-Fc or anti-CD33 x anti-CD3 BiTE-Fc T cell engager.

[0090] In certain embodiments of the invention, the T cell engager is an antibody in the dual-affinity re-targeting format (“DART”). A DART is composed of two Fv fragments, with two unique antigen-binding sites formed when two Fv fragments heterodimerize. Specifically, Fvl consists of a VH from antibody A and a VL from antibody B, while Fv2 is made from a VH from antibody B and VL from antibody A. (See Holliger P., Prospero T., Winter G. “Diabodies”: Small bivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. USA. 1993;90:6444-6448.) In certain embodiments of the invention, the T cell engager is a DART antibody comprising a tumor antigen domain that specifically binds CD 123 and a CD3 binding domain that specifically binds CD3.

[0091] In certain embodiments of the invention, the T cell engager is a tandem diabodies antibody (“TandAbs”). TandAbs are tetravalent bispecific antibodies which provide two binding sites for each antigen to maintain the avidity of a natural bivalent antibody. (See Reusch U., Harrington K.H., Gudgeon C.J., Fucek I., Ellwanger K., Weichel M., Knackmuss S.H., Zhukovsky E.A., Fox J.A., Kunkel L.A., et al. Characterization of CD33 / CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2016;22:5829-5838; Reusch U., Burkhardt C., Fucek I., Le Gall F., Le Gall M., Hoffmann K., Knackmuss S.H., Kiprijanov S., Little M., Zhukovsky E.A. A novel tetravalent bispecific TandAb(CD30 / CD16A) efficiently recruits NK cells for the lysis of CD30+ tumor cells. MAbs. 2014;6:728-739.) In certain embodiments of the invention, the T cell engager is AFM11 (CD19xCD3). (See Kontermann R.E., Brinkmann U. Bispecific antibodies. Drug Discov. Today. 2015;20:838-847.)

[0092] As used herein, “tumor- associated antigens,” “cell surface tumor antigens,” or“tumor antigens” refers to cell surface proteins selectively expressed or over expressed on tumor cells. Examples of tumor antigens include EGFR family molecules (HER2, HER3, and EGFR / HER1), CD19, MUC16, B7-H3, BCMA, FAP, DEE3, PSMA, P-cadherin (CDH3), mesothelin (MSEN), EGFR, claudin 6, cldnl8.2, CD123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule-1 (CLL-1), WT1, GPRC5D, CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO, GD2, MUC1, MUC17, CEACAM5, Claudin 18.2, NKG2D receptor, and CD22. As used herein, a “tumor antigen binding domain” is a binding domain that binds a tumor antigen with specificity.

[0093] As used herein, the term "binding domain" or "binding region" refers to the domain, region, portion, or site of a protein, polypeptide, oligopeptide, or peptide or antibody or binding domain derived from an antibody that possesses the ability to specifically recognize and bind to a target molecule, such as an antigen, epitope, ligand, receptor, substrate, or inhibitor. For instance, a binding domain may specifically recognize and bind a tumor antigen such as CD19, MUC16, B7-H3, BCMA, FAP, HER2, HER3, DEE3, PSMA, P-cadherin (CDH3), mesothelin (MSEN), EGFR / HER1, claudin 6, CD123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule-1 (CLL-1), WT1, GPRC5D, CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO, GD2, MUC1, MUC17, CEACAM5, Claudin 18.2, NKG2D ligand, or CD22. A binding domain may also specifically bind CD3 (for instance, CD3 on the surface of a T cell or iNKT cell). Exemplary binding domains include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, scFab), receptor ectodomains, and ligands (e.g., cytokines, chemokines).

[0094] In certain embodiments, the binding domain comprises a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g., human FRs optionally comprising one or more amino acid substitutions). In certain embodiments, the binding domain comprises an scFv. A variety of assays are known for identifying binding domains of the present disclosure that specifically 1bind a particular target, including Western blot, ELISA, phage display library screening, and BIACORE interaction analysis.

[0095] A binding domain (e.g., tumor antigen binding domain, CD3 binding domain) may be derived from the variable heavy and / or light chains of a monoclonal antibody (e.g., human or humanized antibody). Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications developed by the art. Furthermore, antibodies or fragments thereof to the aforementioned antigens can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. Methods for the production of humanized antibodies are described in, e.g., EP-A1 0 239 400 and WO 90 / 07861. A further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies. The general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91 / 10741, WO 94 / 02602, WO 96 / 34096 and WO 96 / 33735.

[0096] As used herein, tumor the term tumor antigen binding domain includes nonimmunoglobulins that are capable of specifically binding a cell surface tumor antigen. Accordingly the invention includes binding domain polypeptides that are derived from polypeptide ligands such as hormones, cytokines, chemokines, and the like; cell surface or soluble receptors for such polypeptide ligands; lectins; intercellular adhesion receptors such as specific leukocyte integrins, selectins, immunoglobulin gene superfamily members, intercellular adhesion molecules (ICAM-1, -2, -3) and the like; histocompatibility antigens; etc.

[0097] Examples of cell surface receptors that may provide a binding domain polypeptide, and that may also be selected as the target molecule or antigen to which a binding domain binds, include the following, or the like: HER1 (e.g., GenBank Accession Nos. U48722, SEG_HEGFREXS, KO3193), HER2 (Yoshino et al., 1994 J. Immunol. 152:2393; Disis et al., 1994 Cane. Res. 54:16; see also, e.g., GenBank Acc. Nos. X03363, M17730, SEG_HUMHER20), HER3 (e.g., GenBank Acc. Nos. U29339, M34309), HER4 (Plowman et al., 1993 Nature 366:473; see also e.g., GenBank Acc. Nos. L07868, T64105), epidermal growth factor receptor (EGFR) (e.g., GenBank Acc. Nos. U48722, SEG_HEGFREXS, KO3193), vascular endothelial cell growth factor(e.g., GenBank No.M32977), vascular endothelial cell growth factor receptor (e.g., GenBank Acc. Nos. AF022375, 1680143, U48801, X62568), insulin-like growth factor-I (e.g., GenBank Acc. Nos. X00173, X56774, X56773, X06043, see also European Patent No. GB 2241703), insulin-like growth factor-II (e.g., GenBank Acc. Nos. X03562, X00910, SEG_HUMGFIA, SEG_HUMGFI2, M17863, M17862), transferrin receptor (Trowbridge and Omary, 1981 Proc. Nat. Acad. USA 78:3039; see also e.g., GenBank Acc. Nos. X01060, Ml 1507), estrogen receptor (e.g., GenBank Acc. Nos. M38651, X03635, X99101, U47678, M12674), progesterone receptor (e.g., GenBank Acc. Nos. X51730, X69068, M15716), follicle stimulating hormone receptor (FSH-R) (e.g., GenBank Acc. Nos. Z34260, M65085), retinoic acid receptor (e.g., GenBank Acc. Nos. L12060, M60909, X77664, X57280, X07282, X06538), MUC-1 (Barnes et al., 1989 Proc. Nat. Acad. Sci. USA 86:7159; see also e.g., GenBank Acc. Nos. SEG_MUSMUCIO, M65132, M64928) NY-ESO-1 (e.g., GenBank Acc. Nos. AJ003149, U87459), NA 17-A (e.g., European Patent No. WO 96 / 40039), Melan- A / MART-1 (Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g., GenBank Acc. Nos. U06654, U06452), tyrosinase (Topalian et al., 1994 Proc. Nat. Acad. Sci. USA 91:9461; see also e.g., GenBank Acc. Nos. M26729, SEG_HUMTYR0, see also Weber et al., J. Clin. Invest (1998) 102:1258), Gp-100 (Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g., GenBank Acc. No. S73003, see also European Patent No. EP 668350; Adema et al., 1994 J. Biol. Chem. 269:20126), MAGE (van den Bruggen et al., 1991 Science 254:1643; see also e.g, GenBank Acc. Nos. U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339, L18920, U03735, M77481), BAGE (e.g., GenBank Acc. No. U19180, see also U.S. Pat. Nos. 5,683,886 and 5,571,711), GAGE (e.g., GenBank Acc. Nos. AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143, U19142), any of the CTA class of receptors including in particular HOM-MEL-40 antigen encoded by the SSX2 gene (e.g., GenBank Acc. Nos. X86175, U90842, U90841, X86174), carcinoembyonic antigen (CEA, Gold and Freedman, 1985 J. Exp. Med. 121:439; see also e.g., GenBank Acc. Nos. SEG_HUMCEA, M59710, M59255, M29540), and PyLT (e.g., GenBank Acc. Nos. J02289, J02038).

[0098] The terms "(specifically) binds to", (specifically) recognizes", "is(specifically) directed to", and "(specifically) reacts with" mean that binding domain of a T cell engager interacts or specifically interacts with a given epitope, target or antigen on the tumor cell or CD3.

[0099] The interaction between the binding domain of the T cell engager and the epitope or the region comprising the epitope implies that a binding domain exhibits appreciable affinity for the epitope / the region comprising the epitope on a particular protein or antigen and, generally, does not exhibit significant reactivity with proteins or antigens other than the tumor antigen of interest and CD3. Whether a binding domain specifically reacts with or binds to a target (e.g., tumor antigen or CD3) can be tested readily by, inter alia, comparing the reaction of said binding domain with a target protein or antigen with the reaction of said binding domain with proteins or antigens other than the tumor antigen of interest or CD3. Preferably, a binding domain does not essentially or substantially bind to proteins or antigens other than the tumor antigen or CD3 (e.g., the first binding domain is not capable of binding to proteins other than the tumor antigen and the second binding domain is not capable of binding to proteins other than CD3).

[0100] The term "does not substantially bind" or "is not capable of binding" means that a binding domain of the T cell engager does not bind a protein or antigen other than the tumor antigen or CD3, i.e., does not show reactivity of more than 30%, preferably not more than 20%, more preferably not more than 10%, particularly preferably not more than 9%, 8%, 7%, 6% or 5% with proteins or antigens other than tumor antigen or CD3, whereby binding to the tumor antigen or CD3, respectively, is set to be 100%.

[0101] Antibodies that specifically bind to the antigen or the epitope within the antigen may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes (chimpanzee, chimp). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.

[0102] ‘Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. The T cell engagers described herein are antibodies. The term "variable" refers to the portions of the antibody or immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody (i.e., the "variabledomain(s)"). The pairing of a variable heavy chain (VH) and a variable light chain (VL) together forms a single binding domain.

[0103] The term “CDR” as used herein denotes the “complementary determining region” of an antibody. In certain embodiments, the T cell engager comprises a variable heavy chain CDR1, CDR2, and CDR3 and a variable light chain CDR1, CDR2, and CDR3 against human CD3, for instance, human CD3s, and comprises a variable heavy chain CDR1, CDR2, and CDR3 and a variable light chain CDR1, CDR2, and CDR-3 against a human cell surface tumor antigen.

[0104] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage- antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0105] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain- specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.The term "epitope" refers to a site on an antigen to which a binding domain, such as an antibody or immunoglobulin, or a derivative, fragment or variant of anantibody or an immunoglobulin, specifically binds. An "epitope" is antigenic and thus the term epitope is sometimes also referred to herein as "antigenic structure" or "antigenic determinant." Thus, the binding domain is an "antigen interaction site." Such binding / interaction is also understood to define a "specific recognition."

[0106] "Epitopes" can be formed both by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. A "linear epitope" is an epitope where an amino acid primary sequence comprises the recognized epitope. A linear epitope typically includes at least 3 or at least 4, and more usually, at least 5 or at least 6 or at least 7, for example, about 8 to about 10 amino acids in a unique sequence.

[0107] A "conformational epitope", in contrast to a linear epitope, is an epitope wherein the primary sequence of the amino acids comprising the epitope is not the sole defining component of the epitope recognized (e.g., an epitope wherein the primary sequence of amino acids is not necessarily recognized by the binding domain). Typically a conformational epitope comprises an increased number of amino acids relative to a linear epitope. With regard to recognition of conformational epitopes, the binding domain recognizes a three- dimensional structure of the antigen, preferably a peptide or protein or fragment thereof (in the context of the present invention, the antigenic structure for one of the binding domains is comprised within the target cell surface antigen protein). For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or the polypeptide backbone forming the conformational epitope become juxtaposed enabling the antibody to recognize the epitope. Methods of determining the conformation of epitopes include, but are not limited to, x-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and site-directed spin labelling and electron paramagnetic resonance (EPR) spectroscopy.

[0108] CD3" is known in the art as a multi-protein complex of six chains (see, e.g.Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178; 1999), which are subunits of the T-cell receptor complex. CD3 as used in the present disclosure can be from various animal species, including human, monkey, mouse, rat, or other mammals.

[0109] Exemplary anti-CD3 antibodies from which the CD3 binding domain can be derived include TR66, UCHT1, SP34 (See EP2982693; Zorn JA, Wheeler ML, Barnes RM, Kaberna J, Morishige W, Harris M, Huang RYC, Lohre J, Chang YC, Chau B, et al. Humanization of a strategic CD3 epitope enables evaluation of clinical T-cell engagers in afully immunocompetent in vivo model. Sei Rep. 2022; 12:3530); HuM291 (See Chau et al. (2001) Transplantation 71:941-950; BC3 monoclonal antibody (See Anasetti et al. (1990) J. Exp. Med 172:1691); OKT3 monoclonal antibody (See Perez P, Titus JA, Lotze MT, Cuttitta F, Longo DL, Groves ES, et al. Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody. J Immunol Oct. (1986) 137:2069-72; Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med.313:337) and derivatives thereof such as OKT3 ala-ala (also referred to as OKT3 AA-FL or OKT3 FL), a humanized, Fc variant with alanine substitutions at positions 234 and 235 (See Herold et al. (2003) J Clin. Invest. 11:409); visilizurnab (See Carpenter etal. (2002) Blood 99:2712), G19-4 monoclonal antibody (See Ledbetter et al., 1986, J. Immunol. 136:3945), 145-2C11 monoclonal antibody (Hirsch etal. (1988) J. Immunol. 140: 3766), L2K, 12C monoclonal antibody (See US20120244162), and the CRIS-7 monoclonal antibody (See Reinherz, E. L. et al. (eds.), Leukocyte typing IL, Springer Verlag, New York, (1986). In certain embodiments, the CD3 binding domain is one described in W0 2004 / 106380, W0 2005 / 040220A1, US 2014 / 0099318, WO 2015 / 172800, EP2155783, WO 2017 / 182672, US 7,112,324, or US 8,007,796 or derived from a CD3 binding domain thereof.

[0110] In certain embodiments of the invention, the T cell engager comprises one or more linker sequences. For instance, a binding domain of the T cell engager may comprise an scFv. In certain embodiments, the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly4Ser, or polymers thereof, i.e. (Gly4Ser)x, where x is an integer 1 or greater. The characteristics of said peptide linker, which comprise the absence of the promotion of secondary structures are known in the art and described e.g. in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Methods for preparing fused and operatively linked bispecific antibodies and expressing them in mammalian cells or bacteria are well- known in the art (e.g. WO 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).

[0111] An “immunoglobulin constant region” or “constant region” is a term defined herein to refer to a polypeptide sequence that corresponds to or is derived from part or all of one or more constant region domains. In certain embodiments, the immunoglobulin constant region corresponds to or is derived from part or all of one or more constant region domains, but not all constant region domains of a source antibody. In certain embodiments, the constant region comprises IgG CH2 and CH3 domains, e.g., IgGl CH2 and CH3 domains. Incertain embodiments, the constant region does not comprise a CHI domain. In certain embodiments, the constant region is human. In certain embodiments, the constant region is a derivative constant region that contains one or mutations. In some embodiments, the constant region is a derivative constant region that lacks or has minimal effector functions of antibodydependent cell-mediated cytotoxicity (ADCC) and complement activation and complementdependent cytotoxicity (CDC), while retaining the ability to bind some Fc receptors (such as FcRn, the neonatal Fc receptor). In certain embodiments, the constant region contains one or more mutations to reduce or eliminate FcyR binding. In certain embodiments, the constant region is mutated to eliminate glycosylation of the CH2 domain. In certain embodiments, a IgGl derivative constant region has one or more of the following amino acids mutated: glutamate at position 233(E233), leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), asparagine at position 297 (N297) glutamate at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (numbering according to EU). For example, any one or more of these amino acids can be changed to alanine. In certain embodiments, the IgGl derivative constant region has a deletion of a glycine at position 236 (G236del). In certain embodiments, the T cell engager comprises one or more of the following mutations in the constant region, N297A, E233P, F234V, L235A, G236del, and K322A. For instance, in certain embodiments, the derivative constant region comprises an N297A mutation; an N297A and K322A mutations; or E233P, F234V, L235A, and G236del mutations.

[0112] Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from the portion of a source antibody that is responsible for binding to antibody receptors on cells and the Clq component of complement. Fc stands for "fragment crystalline," the fragment of an antibody that will readily form a protein crystal. Distinct protein fragments, which were originally described by proteolytic digestion, can define the overall general structure of an immunoglobulin protein. An Fc fragment consists of the disulfide-linked heavy chain hinge regions (or portion thereof), CH2, and CH3 domains. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mot, Immunol. 31:169-217, 1994. As used herein, the term Fc region includes derivatives of naturally occurring sequences.

[0113] In order to avoid or reduce antigen-independent cytokine release syndrome (CRS) due to crosslinking of CD3 and Fey receptors followed by nonspecific activation of immune cells, T cell engagers comprising an Fc domain typically contain one or moremutations in the Fc region to reduce or eliminate FcyR binding. In certain embodiments, the T cell engager comprises one or more of the following mutations in the Fc region, N297A, E233P, F234V, L235A, G236del, and K322A. For instance, in certain embodiments, the Fc region comprises an N297A mutation; an N297A and K322A mutations; or E233P, F234V, L235A, and G236del mutations.

[0114] A skilled person will readily be able without undue burden to construct and obtain the T cell engagers described herein by utilizing established techniques and standard methods known in the art, see for example Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.; The Protein Protocols Handbook, edited by John M. Walker, Humana Press Inc. (2002); or Antibody engineering: methods and protocols / edited by Benny K.C. Lo; Benny K.C. II Series: Methods in molecular biology (Totowa, N.J.))

[0115] "Cytokine release" or "cytokine storm" or "infusion reaction" refers to the release of cytokines from T-cells. When cytokines are released into the circulation, systemic symptoms such as fever, nausea, chills, hypotension, tachycardia, asthenia, headache, rash, scratchy throat, and dyspnea can result. Some patients may experience severe, lifethreatening reactions that result from massive release of cytokines. "Reduced" cytokine release refers to the to the reduction in the release of at least one cytokine (e.g., TNFa, IFNy, IL-2, and / or IL- 10) following administration of the pharmaceutical composition or methods of invention as compared to a similar composition or methods lacking iNKT cells (e.g., a T cell engager composition).

[0116] As disclosed herein, the cytokine release profile of activated iNKT cells is different from that of activated T cells. For instance, activated T cells release more TNFa than iNKT cells. In one embodiment, “reduced” cytokine release refers to the reduction of TNFa following administration of a pharmaceutical composition of the invention as compared to an identical pharmaceutical composition that lacks NKT cells (e.g., iNKT cells).

[0117] As used herein, a "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well-known in the art (see, e.g., WO 97 / 09433, page 10, published March 13, 1997; Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975); pp.71-77; Lewin, Genes IV; Oxford University Press, NY and Cell Press;Cambridge, MA (1990), p. 8). In certain embodiments, a conservative substitution includes a leucine to serine substitution.

[0118] As used herein, the term "derivative" refers to a modification of one or more amino acid residues of a polypeptide or antibody, or a region, domain or functional unit thereof, by chemical or biological means, either with or without an enzyme, e.g.; by glycosylation, alkylation, acylation, ester formation; or amide formation. As used herein, a polypeptide or amino acid sequence "derived from a designated polypeptide or protein refers to the origin of the polypeptide. In certain embodiments, the polypeptide or amino acid sequence which is derived from a particular sequence (sometimes referred to as the "starting" or "parent" or "parental" sequence) has an amino acid sequence that is essentially identical to the starting sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or at least 50-150 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence. For example, a binding domain can be derived from an antibody, e.g., a Fab, F(ab')2, Fab', scFv, single domain antibody (sdAb), etc. As described below, in some embodiments the parent polypeptide, for example an Fc parent region, is a human wild type sequence, such as the Fc region from IgGl, IgG2, IgG3 or IgG4, although human sequences with variants can also serve as "parent polypeptides." A derivative polypeptide can refer to the variant polypeptide itself, compositions comprising the polypeptide derivative, or the DNA sequence that encodes it. Accordingly, by "antibody derivative," "derivative antibody," “antibody variant,” or “variant antibody” as used herein is meant an antibody that differs from a parent antibody by virtue of at least one amino acid modification, "IgG derivative," "derivative IgG," “IgG variant,” or “variant IgG” as used herein is meant an antibody that differs from a parent IgG (again, in many cases, from a human IgG sequence) by virtue of at least one amino acid modification, and "immunoglobulin derivative," "derivative immunoglobulin," “immunoglobulin variant,” or “variant immunoglobulin” as used herein is meant an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence by virtue of at least one amino acid modification. "Fc derivative," "derivative Fc," “Fc variant,” or “variant Fc” as used herein is meant a protein comprising an amino acid modification in an Fc domain.

[0119] For all positions discussed in the present invention that relate to antibodies, unless otherwise noted, amino acid position numbering is according to the EU index. The EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EUantibody (Edelman et al, 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference.) The modification can be an addition, deletion, or substitution. Substitutions can include naturally occurring amino acids and, in some cases, synthetic amino acids. Examples include U.S. Pat. No. 6,586,207; WO 98 / 48032; WO 03 / 073238; US2004- 0214988A1; WO 05 / 35727 A2; WO 05 / 74524A2; J. W. Chin et al, (2002), Journal of the American Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem 11 : 1135-1137; J. W. Chin, et al., (2002), PICAS United States of America 99: 11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. 1-10, all entirely incorporated by reference.

[0120] As used herein, the term “sequence identity” refers to a relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparator sequence, the sequences are said to be “identical” at that position. The percentage “sequence identity” is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of “identical” positions. The number of “identical” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of “sequence identity.” Percentage of “sequence identity” is determined by comparing two optimally aligned sequences over a comparison window. The comparison window for nucleic acid sequences can be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleic acids in length. The comparison window for polypeptide sequences can be, for instance, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300 or more amino acids in length. In order to optimally align sequences for comparison, the portion of a polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant. An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of “identical” positions between the reference and comparator sequences. Percentage “sequence identity” between two sequences can be determined using the version of the program “BLAST 2 Sequences” which was available from the National Center for Biotechnology Information as of Sep. 1, 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithmof Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing “BLAST 2 Sequences,” parameters that were default parameters as of Sep. 1, 2004, can be used for word size (3), open gap penalty (11), extension gap penalty (1), gap dropoff (50), expect value (10) and any other required parameter including but not limited to matrix option. Two nucleotide or amino acid sequences are considered to have “substantially similar sequence identity” or “substantial sequence identity” if the two sequences have 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% sequence identity relative to each other.

[0121] Polypeptides derived from another polypeptide can have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. The polypeptide can comprise an amino acid sequence which is not naturally occurring. Such derivatives necessarily have less than 100% sequence identity or similarity with the starting polypeptide. In one embodiment, the derivative will have an amino acid sequence from about 60% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide. In another embodiment, the derivative will have an amino acid sequence from about 75% to less than 100%, from about 80% to less than 100%, from about 85% to less than 100%, from about 90% to less than 100%, from about 95% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide.

[0122] As used herein, the term “promoter” refers to a region of DNA involved in binding RNA polymerase to initiate transcription.

[0123] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985)J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. As used herein, the terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.

[0124] The term “expression” refers to the biosynthesis of a product encoded by a nucleic acid. For example, in the case of nucleic acid segment encoding a polypeptide of interest, expression involves transcription of the nucleic acid segment into mRNA and the translation of mRNA into one or more polypeptides.

[0125] The terms “expression unit” and “expression cassette” are used interchangeably herein and denote a nucleic acid segment encoding a polypeptide of interest and capable of providing expression of the nucleic acid segment in a host cell. An expression unit typically comprises a transcription promoter, an open reading frame encoding the polypeptide of interest, and a transcription terminator, all in operable configuration. In addition to a transcriptional promoter and terminator, an expression unit can further include other nucleic acid segments such as, e.g., an enhancer or a polyadenylation signal.

[0126] The term “expression vector” or “vector” as used herein, refers to a nucleic acid molecule, linear or circular, comprising one or more expression units. In addition to one or more expression units, an expression vector can also include additional nucleic acid segments such as, for example, one or more origins of replication or one or more selectable markers. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both.

[0127] Any suitable vector may be used to introduce a polynucleotide that encodes a polypeptide into NKT cells (e.g., iNKT cells) or host cells. Exemplary vectors that have been described in the literature include replication deficient retroviral vectors, including but not limited to lentivirus vectors [Kim et al., J. Virol., 72(1): 811-816 (1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43 46.]; adenoassociated viral (AAV) vectors [U.S. Pat. No. 5,474,935; U.S. Pat. No. 5,139,941; U.S. Pat. No. 5,622,856; U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,773,289; U.S. Pat. No. 5,789,390; U.S. Pat. No. 5,834,441; U.S. Pat. No. 5,863,541; U.S. Pat. No. 5,851,521; U.S. Pat. No. 5,252,479; Gnatenko et al., J. Invest. Med., 45: 87 98 (1997)]; adenoviral (AV) vectors [See, e.g., U.S. Pat. No. 5,792,453;U.S. Pat. No. 5,824,544; U.S. Pat. No. 5,707,618; U.S. Pat. No. 5,693,509; U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,585,362; Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581 2584 (1992); Stratford Perricadet et al., J. Clin. Invest., 90: 626 630 (1992); and Rosenfeld et al., Cell, 68: 143 155 (1992)]; an adenoviral adenoassociated viral chimeric (see for example, U.S. Pat. No. 5,856,152) or a vaccinia viral or a herpesviral (see for example, U.S. Pat. No. 5,879,934; U.S. Pat. No. 5,849,571; U.S. Pat. No. 5,830,727; U.S. Pat. No. 5,661,033; U.S. Pat. No. 5,328,688; Lipofectin mediated gene transfer (BRL); liposomal vectors [See, e.g., U.S. Pat. No. 5,631,237 (Liposomes comprising Sendai virus proteins)]; and combinations thereof.

[0128] The term “engineered” or “recombinant” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, antibody, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In certain embodiments, a vector is engineered through recombinant nucleic acid technologies, and a cell is engineered through transfection or transduction of an engineered vector. In certain embodiments, the NKT cells (e.g., iNKT cells) are engineered to express a T cell receptor (“TCR”), chimeric antigen receptor (“CAR”) and / or armor molecule. For instance, in one embodiment of the invention, the pharmaceutical composition comprises a therapeutically effective amount of engineered iNKT cells capable of expressing an exogenous TCR, a T cell engager, and a pharmaceutically acceptable carrier. In another embodiment of the invention, the pharmaceutical composition comprises a therapeutically effective amount of engineered iNKT cells capable of expressing a CAR (e.g., BCMA CAR or FAP CAR), a T cell engager, and a pharmaceutically acceptable carrier.

[0129] In some embodiments, the nucleic acid encoding the CAR, TCR, and or armor molecule (e.g., IL- 15) is introduced into the NKT cell (e.g., iNKT cell) using a recombinant vector. In some embodiments, the recombinant vector is a viral vector. In some embodiments, the viral vector is a lentivirus, a retrovirus, an adeno-associated virus (AAV), a herpesvirus, or adenovirus. In some embodiments, the viral vector comprises a retroviral vector.

[0130] As used herein, the terms “T cell receptor” and “TCR” are used interchangeably and refer to molecules comprising CDRs or variable regions from a[3 or y5 T cell receptors. Examples of TCRs include, but are not limited to, full-length TCRs, antigenbinding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing variable regions of TCRs attached by a flexible linker, TCRchains linked by an engineered disulfide bond, single TCR variable domains, single peptide- MHC-specific TCRs, multi- specific TCRs (including bispecific TCRs), TCR fusions, TCRs comprising co-stimulatory regions, human TCRs, humanized TCRs, chimeric TCRs, recombinantly produced TCRs, and synthetic TCRs. In certain embodiments, the TCR is a full-length TCR comprising a full-length a chain and a full-length P chain. In certain embodiments, the TCR is a soluble TCR lacking transmembrane and / or cytoplasmic region(s). In certain embodiments, the TCR is a single-chain TCR (scTCR) comprising Va and VP linked by a peptide linker, such as a scTCR having a structure as described in PCT Publication No.: WO 2003 / 020763, WO 2004 / 033685, or WO 2011 / 044186, each of which is incorporated by reference herein in its entirety. In certain embodiments, the TCR comprises a transmembrane region. In certain embodiment, the TCR comprises a co-stimulatory signaling region.

[0131] As used herein, the term “full-length TCR” refers to a TCR comprising a dimer of a first and a second polypeptide chain, each of which comprises a TCR variable region and a TCR constant region comprising a TCR transmembrane region and a TCR cytoplasmic region. In certain embodiments, the full-length TCR comprises one or two unmodified TCR chains, e.g., unmodified a, P, y, or 5 TCR chains. In certain embodiments, the full-length TCR comprises one or two altered TCR chains, such as chimeric TCR chains and / or TCR chains comprising one or more amino acid substitutions, insertions, or deletions relative to an unmodified TCR chain. In certain embodiments, the full-length TCR comprises a mature, full-length TCR a chain and a mature, full-length TCR P chain. In certain embodiments, the full-length TCR comprises a mature, full-length TCR y chain and a mature, full-length TCR 5 chain.

[0132] As used herein, the term “TCR variable region” refers to the portion of a mature TCR polypeptide chain (e.g., a TCR a chain or P chain) which is not encoded by the TRAC gene for TCR a chains, either the TRBC1 or TRBC2 genes for TCR P chains, the TRDC gene for TCR 5 chains, or either the TRGC1 or TRGC2 gene for TCR y chains. In some embodiments, the TCR variable region of a TCR a chain encompasses all amino acids of a mature TCR a chain polypeptide which are encoded by a TRAV and / or TRAJ gene, and the TCR variable region of a TCR P chain encompasses all amino acids of a mature TCR P chain polypeptide which are encoded by a TRBV, TRBD, and / or TRBJ gene (see, e.g., T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated by reference herein in its entirety). TCR variable regions generallycomprise framework regions (FR) 1, 2, 3 and 4 and complementarity determining regions (CDR) 1, 2 and 3.

[0133] The term “exogenous TCR” refers to a TCR gene or TCR gene derivative that is transferred (i.e. by way of gene transfer / transduction / transfection techniques) into the cell or is the progeny of a cell that has received a transfer of a TCR gene or gene derivative. The exogenous TCR genes are inserted into the genome of the recipient cell. In some embodiments, the insertion is random insertion. Random insertion of the TCR gene is readily achieved by methods known in the art. In some embodiments, the TCR genes are inserted into an endogenous loci (such as an endogenous TCR gene loci). In some embodiments, the cells comprise one or more TCR genes that are inserted at a loci that is not the endogenous loci. In some embodiments, the cells further comprise heterologous sequences such as a marker or resistance gene.

[0134] In certain embodiments of the invention, the exogenous TCRs are able to recognize TAA antigens in a major histocompatibility complex (MHC) class I-dependent manner. “MHC class I-dependent manner,” as used herein, means that the engineered TCR elicits an immune response upon binding to TAA antigens within the context of an MHC class I molecule. In one embodiment of the invention, the MHC class I molecule is an HLA- A molecule.

[0135] The term “chimeric antigen receptor” or “CAR” refers to engineered receptors, which graft an arbitrary specificity onto an immune effector cell such as an iNKT cells. These receptors are used to graft the specificity of a monoclonal antibody onto the cell; with transfer of their coding sequence facilitated by retroviral or lentiviral vectors. The receptors are called chimeric because they are composed of parts from different sources. The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta transmembrane and endodomain; CD28 or 4 IBB intracellular domains, or combinations thereof. Such molecules result in the transmission of a signal in response to recognition by the scFv of its target. The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form a scFv. This scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (this is cleaved). A flexible spacer allows the scFv to orient in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original molecule of the signaling endodomain which protrudes into the cell and transmits the desired signal.Examples of suitable antigens to be recognized by the CARs of iNKT-CARs disclosed herein. These include FAP and BCMA.

[0136] In some embodiments, the NKT cells (e.g., iNKT cells) are engineered to express another molecule (e.g., a cytokine or ligand) capable of enhancing one or more properties (e.g., survival / persistence, immune interaction with other cells such as macrophages and / or dendritic cells, disrupt immunosuppressive tumor microenvironment) of the genetically modified cells. For instance, the NKT cells (e.g., iNKT cells) can be engineered to express any suitable armoring molecule known in the art, e.g., armoring molecule described by Yeku et al., Armored CAR T-cells: utilizing cytokines and pro- inflammatory ligands to enhance CAR T-cell anti-tumour efficacy, Biochem Soc Trans. 2016 Apr 15; 44(2): 412-418; Hawkins et al., Armored CAR T-Cells: The Next Chapter in T-Cell Cancer Immunotherapy, Biologies. 2021; 15: 95-105). Non-limiting examples of armoring molecules include IE-15, IE-2, IL-12, CD40L, 4-1BBL, IL-18, IL-7, IL-33, constitutively active Akt (caAkt), hybrid IL-4 / IL-7 receptor, checkpoint inhibitors such as anti-PDl antibodies, nanobodies targeting CD47, or bispecific T-cell engagers (BiTEs).

[0137] In certain embodiments, the NKT cells (e.g., iNKT cells) expressing the engineered TCR described herein are also engineered to express IL- 15. In some embodiments, the genetically modified cells expressing the engineered TCR described herein are also engineered to express soluble IL-15 (sIL-15).

[0138] In certain embodiments, the NKT cells (e.g., iNKT cells) expressing the CAR described herein are also engineered to express IL- 15. In some embodiments, the genetically modified cells expressing the CAR described herein are also engineered to express soluble IL-15 (sIL-15).

[0139] The role of IL- 15 in enhancing the expansion and function of T cells and natural killer T cells (e.g., iNKT cells) have been previously described (see, e.g., Lin et al., Interleukin- 15 enhances the expansion and function of natural killer T cells from adult peripheral and umbilical cord blood, Cytokine. 2015 Dec;76(2):348-355; Battram et al., IL- 15 Enhances the Persistence and Function of FAP-Targeting CAR-T Cells Compared to IL-2 or IL-15 / IL-7 by Limiting CAR-T Cell Dysfunction and Differentiation; Cancers (Basel). 2021 Jul; 13(14): 3534). There is evidence that numerous cell types are responsible for the production of IL- 15, including macrophages and DCs, and once released, IL- 15 stimulates CD8+ T-cells and NK cells which increases their proliferation and cytotoxic capacity.Administration of IL- 15 to mice has been shown to enhance anti-tumor activity of adoptively transferred CD8+ tumor-reactive T-cells, which suggests IL- 15 could also enhance antitumor activity of T-cell therapy. Further, IL- 15 can increase antigen-independent T-cell proliferation, while enabling T-cell persistence after tumor clearance. Certain previous studies used a form of IL- 15 tethered to the membrane and found that this promoted the T- cells to develop a memory phenotype. These data suggest that IL- 15 could provide long-term T-cell-mediated immunity toward the cancer antigen along with enhancing T-cell function within the tumor microenvironment.

[0140] As used herein, the term “subject” includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In one embodiment, the subject is a human. As used herein, the term “patient in need” or “subject in need” refers to a patient at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration by administration of NKT cells (e.g., iNKT cells) with a T cell engager, or a composition thereof provided herein. A patient in need may, for instance, be a patient diagnosed with a disease associated with the expression of the tumor antigen that is specifically targeted by the tumor antigen domain of the T cell engager.

[0141] The term "administered" as used herein means administration of a therapeutically effective dose of the aforementioned bispecific single chain antibody to an individual. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0142] Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by determining effective dosages and administration protocols that significantly reduce the occurrence or severity of the subject disorder in model subjects. Effective doses of the compositions of the present disclosure vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, whether treatment is prophylactic or therapeutic, as well as the specific activity of the composition itself and its ability to elicit the desired response in theindividual. Usually, the patient is a human, but in some diseases, the patient can be a nonhuman mammal. Typically, dosage regimens are adjusted to provide an optimum therapeutic response, i.e., to optimize safety and efficacy. Accordingly, a therapeutically effective amount is also one in which any undesired collateral effects are outweighed by the beneficial effects of administering NKT cells (e.g., iNKT cells, including, for instance, allogeneic iNKT cells) in combination with a T cell engager as described herein.

[0143] The attending physician and clinical factors will determine the dosage regimen. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health status, and other drugs being administered concurrently.

[0144] As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health status, and other drugs being administered concurrently. A typical dose can be, for example, in the ranges set forth in the embodiments of the invention and the appended examples; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.

[0145] As used herein, the terms “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. In some embodiments, the methods of “treatment” employ administration of iNKT cells and T cell engagers, optionally with alpha a-galactosylceramide (a-GalCer), to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0146] As used herein, the term “effective amount” or “therapeutically effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.

[0147] As used herein, the term “pharmaceutically acceptable” refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities andcompositions approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans are considered to be “pharmaceutically acceptable.Pharmaceutical Compositions Comprising NKT Cells and T Cell Engagers

[0148] The present invention includes pharmaceutical compositions comprising a therapeutically effective amount of NKT cells (e.g., iNKT cells) and T cell engager and a pharmaceutically acceptable carrier or excipient.

[0149] In certain embodiments the T cell engager binds human CD3s and is capable of binding CD3 on iNKT cells. In certain embodiments, the iNKT cells and T cell engagers form a complex in solution such that the CD3 binding domain of the T cell engagers specifically binds CD3 on the iNKT cells. As used herein, “complexed” means that the iNKT cell is specifically bound or attached to the T cell engager. For instance, the CD3 binding domain of the T cell engager may be bound to the CD3 on an iNKT cell. In certain embodiments, the CD3 domain of the T cell engager is bivalent, trivalent, or multivalent and each T cell engager molecule is capable of binding more than one CD3 (e.g., multiple CD3 on a single iNKT cell or CD3s on multiple iNKT cells). In certain embodiments, it may be desirable to modify valency to increase / decrease the binding of the CD3 binding domain of the T cell engager to CD3 on iNKT cells.

[0150] In certain embodiments, it may be desirable to modify one or more amino acids of the CD3 binding domain to increase / decrease the binding affinity of the CD3 binding domain of the T cell engager to CD3 on iNKT cells.

[0151] In certain embodiments of the invention, the pharmaceutical composition comprises a therapeutically effective amount of iNKT cells complexed to T cell engagers. The pharmaceutical composition may comprise a mixture of iNKT cells complexed to T cell engagers and free (i.e., not complexed) iNKT cells and T cell engager molecules.

[0152] In certain embodiments, the NKT cells (e.g., iNKT cells) are allogeneic cells. In certain embodiments of the invention, the NKT cells (iNKT cells) are not autologous cells. In certain embodiments, the NKT cells (e.g., iNKT cells) are obtained from a healthy human donor. In certain embodiments, the NKT cells (e.g., iNKT cells) have been isolated from PBMCs. In certain embodiments, the NKT cells (e.g., iNKT cells) have been isolated from a donor’s peripheral blood and expanded. For instance, the invention includes a composition comprised of iNKT cells which have been isolated and expanded from a human donor.

[0153] Isolation of NKT cells (e.g., iNKT cells) from biological materials such as peripheral blood may be achieved by various means. For instance, iNKT cells may be isolated by FACS selection of cells exhibiting iNKT markers, for example, CD3, CD4, and CD8. Alternatively, glycolipid-loaded CDld dimers or tetramers may be used as selection tools, for example, as described in Watarai et al., Methods for detection, isolation and culture of mouse and human invariant NKT cells, Nature Protocols, 2008, 3:70-77. Alternatively, antibodies selective for iNKT cells may be used to isolate them, for example, an anti- Va24Jal8 CDR3 loop antibody, as described in Exley et al, Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR a- chain CDR3 loop, Eur. J. Immunol. 2008. 38: 1756-1766.

[0154] Following isolation, NKT cells (e.g., iNKT) cells may be efficiently expanded ex vivo by any number of platforms known in the art. Cells may be cultured in an appropriate medium such as RPMI 1640 medium with pulsed addition of an iNKT activator to spur high rates of proliferation. Exemplary methods of expanding iNKTs are described, for example, in Chiba et al., Rapid and reliable generation of invariant natural killer T-cell lines in vitro, Immunology, 128, 324-333.

[0155] It would be appreciated that the iNKT cells can be expanded. Methods of expanding iNKT cells are known in the art. Typically, expansion would occur after the iNKT cells are obtained from the donor but before the iNKT cells are administered to the patient (i.e., ex vivo), but expansion could occur in vivo, for example, by administering one or more cytokines (e.g., IL-2, IL- 17, IL- 15). A representative compound that can cause expansion of iNKT cells is the small molecule RGI-2001 (see, for example, Duramad et al., 2011, Biol. Blood Marrow Transplant., 17(8): 1154-68). As used herein, expansion of iNKT cells typically refers to an increase in number, but expansion of iNKT cells also can refer to an increase in activity or potency of the cells.

[0156] In certain embodiments, such methods further include expanding the iNKT cells prior to the administering step. In some embodiments, such methods further include contacting the donor iNKT cells with RGI-2001 prior to the administering step.

[0157] expanding said NKT-selected cell fraction by culturing in the presence of at least: (1) IL-21 and second cytokine selected from the group consisting of IL-2, IL-7, IL- 15, IL- 12, TNE-alpha; and (2) irradiated NKT-depleted PBMCs loaded with alpha-Galactosylceramide (aGalCer) or irradiated artificial antigen-presenting cells (aAPCs) expressing CDl-d loaded with aGalCer

[0158] In certain embodiments, the NKT cells (e.g., iNKT cells) are purified cells. For instance, the invention includes compositions comprising about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more iNKT cells as compared to other cell types (e.g., T cells). In certain embodiments, the pharmaceutical composition comprises about 102iNKT cells, about 103, iNKT cells, about 104iNKT cells, about 105iNKT cells, about 106iNKT cells, about 107iNKT cells, about 108iNKT cells, about 109iNKT cells, about IO10iNKT cells, 1O2-1O10iNKT cells, about 1O3-1O10iNKT cells, about 104-105iNKT cells, about 104- 106iNKT cells, about 104-107iNKT cells, about 104-108iNKT cells, about 105-106iNKT cells, about 105-107iNKT cells, about 105-108iNKT cells, 1O5-1O10iNKT cells, or about 106- 1O10iNKT cells. In certain embodiments, the composition comprises about 1.5 x 107to 2.0 x 108allogeneic iNKT cells

[0159] In certain embodiments, the NKT cells (e.g., iNKT cells) are engineered cells. In certain embodiments, the NKT cells (e.g., iNKT cells) express a CAR. Examples of suitable antigens to be recognized by the CARs of iNKT-CARs disclosed herein. These include FAP and BCMA.

[0160] In certain embodiments, the NKT cells (e.g., iNKT cells) are engineered to express TCR. Examples of suitable antigens for expression of a TCR are PRAME and NY- ESO-1.

[0161] The NTK cells (e.g., iNKT cells) may optionally be engineered to express an armor molecule such as IL- 15. The invention includes, for instance, lentiviral transduction of CAR or TCR in iNKT cells. For instance, the invention includes lentiviral transduction of CAR or TCR in CD3 / CD28-activated iNKT cells. In certain embodiments, the invention includes lentiviral transduction of CAR or TCR in CD3 / CD28-activated iNKT cells in the presence of APC and IL- 15.

[0162] The antigen to be recognized by the engineered iNKT-CARs of the invention can be the same cell surface antigen recognized by the T cell engager.

[0163] In certain embodiments, the engineered NKT cells (e.g., engineered iNKT cells) express an exogenous TCR. For instance, iNKT cells can be harvested from PBMCs and expanded using methods known in the art. iNKT cells transfected with a polynucleotide coding for a TCR of the invention express both the engineered TCR and an iNKTendogenous TCR. For instance, in one embodiment, the iNKT cells are engineered to express a PRAME TCR (e.g., TCR targeting the PRAME425-433 peptide or NY-ESO-1 TCR. (i.e., TCR targeting the PRAME425-433 peptide) and iNKT endogenous TCR (i.e., TCR targeting glycolipids presented by CD Id).

[0164] In certain embodiments, the NKT cells (e.g., iNKT cells) expressing the engineered TCR described herein are also engineered to express IL- 15. In some embodiments, the genetically modified cells expressing the engineered TCR described herein are also engineered to express soluble IL-15 (sIL-15).

[0165] In certain embodiments, the NKT cells (e.g., iNKT cells) expressing the CAR described herein are also engineered to express IL- 15. In some embodiments, the genetically modified cells expressing the CAR described herein are also engineered to express soluble IL-15 (sIL-15).

[0166] The role of IL- 15 in enhancing the expansion and function of T cells and natural killer T cells (e.g., iNKT cells) have been previously described (see, e.g., Lin et al., Interleukin- 15 enhances the expansion and function of natural killer T cells from adult peripheral and umbilical cord blood, Cytokine. 2015 Dec;76(2):348-355; Battram et al., IL- 15 Enhances the Persistence and Lunction of LAP-Targeting CAR-T Cells Compared to IL-2 or IL-15 / IL-7 by Limiting CAR-T Cell Dysfunction and Differentiation; Cancers (Basel). 2021 Jul; 13(14): 3534). There is evidence that numerous cell types are responsible for the production of IL- 15, including macrophages and DCs, and once released, IL- 15 stimulates CD8+ T-cells and NK cells which increases their proliferation and cytotoxic capacity. Administration of IL- 15 to mice has been shown to enhance anti-tumor activity of adoptively transferred CD8+ tumor-reactive T-cells, which suggests IL- 15 could also enhance antitumor activity of T-cell therapy. Further, IL- 15 can increase antigen-independent T-cell proliferation, while enabling T-cell persistence after tumor clearance. Certain previous studies used a form of IL- 15 tethered to the membrane and found that this promoted the T- cells to develop a memory phenotype. These data suggest that IL- 15 could provide long-term T-cell-mediated immunity toward the cancer antigen along with enhancing T-cell function within the tumor microenvironment.

[0167] The T cell engager of the pharmaceutical compositions of the invention can be any T cell engager disclosed herein. For instance, the compositions (and methods) of the invention include a T cell engager that binds CD 19 and CD3, HER2 and CD3, HER3 andCD3, EGFR / HER1 and CD3, MUC16 and CD3, B7-H3 and CD3, BCMA and CD3, FAP and CD3, DLL and CD3, PSMA and CD3, P-cadherin (CDH3) and CD3, mesothelin (MSLN) and CD3, claudin-6 and CD3, CD 123 and CD3, CD20 and CD3, ENPP3 and CD3, EpCAM and CD3, TROP-2 and CD3, CD33 and CD3, human C-type lectin like molecule- 1 (CLL-1) and CD3, WT1 and CD3, GPRC5D and CD3, CD38 and CD3, FCRL5 and CD3, VEGFR and CD3, CEA and CD3, PSA and CD3, EphA2 and CD3, gplOO and CD3, GD2 and CD3, MUC1 and CD3, CD22 and CD3, FLT3 and CD3, MUC 17 and CD3, CEACAM5 and CD3, Claudin 18.2 and CD3, or an NKG2D ligand and CD3.

[0168] In certain embodiments of the invention, the pharmaceutical composition comprises a T cell engager with a CD 19 binding domain or a B7-H3 binding domain with the sequences as set forth in Table 1 below. In certain embodiments, the T cell engager of the pharmaceutical composition of the invention comprises a VH CDR1, VH CDR2, and VH CDR3 and VL CDR1, VL CDR2, and VL CDR3 which are the complementarity-determining regions of an anti-CD19 antibody or B7-H3 antibody.

[0169] In certain embodiments, the VH CDR1, VH CDR2, and VH CDR3 and the VL CDR1, VL CDR2, and VL CDR3 of the CD 19 tumor antigen binding domain of a T cell engager comprise an amino acid sequence as set forth in SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NOG, SEQ ID NOG, and SEQ ID NO:8, respectively. In certain embodiments, the CD 19 tumor antigen binding domain comprises a variable heavy chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of SEQ ID NO:1. In certain embodiments, the CD 19 tumor antigen binding domain comprises a variable light chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of SEQ ID NOG. In certain embodiments, the CD19 tumor antigen binding domain comprises a variable heavy chain and a variable light chain with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of SEQ ID NO:1 and SEQ ID NOG. In certain embodiments, the CD19 tumor antigen binding domain comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a variable light chain comprising the amino acid sequence of SEQ ID NOG.

[0170] In certain embodiments, the VH CDR1, VH CDR2, and VH CDR3 and the VL CDR1, VL CDR2, and VL CDR3 of the B7-H3 tumor antigen binding domain of a T cell engager comprise an amino acid sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. In certain embodiments, the B7-H3 tumor antigen binding domain comprises a variable heavy chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of SEQ ID NO:9. In certain embodiments, the B7-H3 tumor antigen binding domain comprises a variable light chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of SEQ ID NO: 10. In certain embodiments, the B7-H3 tumor antigen binding domain comprises a variable heavy chain and a variable light chain with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of SEQ ID NO:9 and SEQ ID NO: 10. In certain embodiments, the B7-H3 tumor antigen binding domain comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 10.Table 1: Example of Tumor Antigen Binding Domain

[0171] The T cell engager may be in any antibody format known in the art. In certain embodiments of the compositions and methods of the invention, the T cell engager is a bispecific antibody. In certain embodiments of the compositions and methods of the invention, the T cell engager is a multi- specific antibody.

[0172] In certain embodiments of the invention, the T cell engager is a heterodimer. T cell engagers in a heterodimer antibody format are known in the art and include, but are not limited to the heterodimer antibody formats disclosed herein. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in a heterodimer antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in a heterodimer antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in a heterodimer antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in a heterodimer antibody format, and a pharmaceutically acceptable carrier or excipient.

[0173] In certain embodiments of the invention, the T cell engager is in the knobs- into-holes antibody format. The “knobs” and “holes” (examples described herein) can be engineered into the CH3 of the Fc region using methods known in the art. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the knobs-into-holes antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in the knobs-into-holes antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the knobs-into-holes antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the knobs-into-holes antibody format, and a pharmaceutically acceptable carrier or excipient.

[0174] In one embodiment, the T cell engager is in the BiTE antibody or BiTE-Fc antibody format. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the BiTE or BiTE-Fc antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in the BiTE or BiTE-Fc antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the BiTE or BiTE-Fc antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the BiTE or BiTE-Fc antibody format, and a pharmaceutically acceptable carrier or excipient.

[0175] In one embodiment, the T cell engager is in the scFv-Ig antibody format. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the scFv-Ig antibody format and apharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in the scFv-Ig antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the scFv-Ig antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the scFv-Ig antibody format, and a pharmaceutically acceptable carrier or excipient.

[0176] In one embodiment, the T cell engager is in the DART, DART-Fc, or Tetravalent DART antibody format. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the DART, DART-Fc, or Tetravalent DART antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in the DART, DART-Fc, or Tetravalent DART antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the DART, DART-Fc, or Tetravalent DART antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the DART, DART-Fc or Tetravalent DART antibody format, and a pharmaceutically acceptable carrier or excipient.

[0177] In one embodiment, the T cell engager is in the diabody antibody format. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the diabody antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in the diabody antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells,native or engineered), a MUC16 x CD3 targeting T cell engager in the diabody antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the diabody antibody format, and a pharmaceutically acceptable carrier or excipient.

[0178] In one embodiment, the T cell engager is in the BiKE or TriKE antibody format. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the BiKE or TriKE antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in the BiKE or TriKE antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, for example, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the BiKE or TriKE antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the BiKE or TriKE antibody format, and a pharmaceutically acceptable carrier or excipient.

[0179] In one embodiment, the T cell engager is in the scFv-scFv-scFv antibody format. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the scFv-scFv-scFv antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD19 x CD3 targeting T cell engager in the scFv-scFv-scFv antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, for example, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the scFv-scFv-scFv antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the scFv-scFv-scFv antibody format, and a pharmaceutically acceptable carrier or excipient.

[0180] In one embodiment, the T cell engager is in the TandAb antibody format. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the TandAb antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD19 x CD3 targeting T cell engager in the TandAb antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, for example, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the TandAb antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the TandAb antibody format, and a pharmaceutically acceptable carrier or excipient.

[0181] In one embodiment, the T cell engager is in the TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 or CrossMab antibody format. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the a TrioMab, CrossMabFab, CrossMab VH-VL, 2: 1 CrossMab, 2:2 or CrossMab antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD19 x CD3 targeting T cell engager in the TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 or CrossMab antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, for example, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 or CrossMab antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 or CrossMab antibody format, and a pharmaceutically acceptable carrier or excipient.

[0182] In one embodiment, the T cell engager is in the DVD-Ig, IgG-IgG, or Fab- scFv-Fc antibody format. The invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the DVD-Ig, IgG-IgG, or Fab-scFv-Fc antibody format and a pharmaceutically acceptable carrier or excipient. For instance, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD19 x CD3 targeting T cell engager in the DVD-Ig, IgG-IgG, or Fab-scFv-Fc antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, for example, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the DVD-Ig, IgG-IgG, or Fab-scFv- Fc antibody format, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the DVD-Ig, IgG-IgG, or Fab-scFv-Fc antibody format, and a pharmaceutically acceptable carrier or excipient.

[0183] In one embodiment of the invention, the T cell engager of the pharmaceutical composition is in an antibody format selected from the group consisting of knobs-into-holes (including knobs-into holes with cognate light chain and knobs-into-holes with common light chain), scFv-Ig, BiTE, BiTE-Fc, DART, DART-Fc, Tetravalent DART, Diabody, BiKE, TriKE, scFv-scFv-scFv, TandAb, TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 CrossMab, DVD-Ig, IgG-IgG, and Fab-scFv-Fc.

[0184] In certain embodiments of the invention, the T cell engager of the pharmaceutical composition is bivalent. For instance, the invention includes T cell engagers which are IgG-scFv bispecific antibodies (for example, wherein Bl is an intact IgG and B2 is an scFv attached to B 1 at the N- terminus of a light chain and / or at the C-terminus of a light chain and / or at the N-terminus of a heavy chain and / or at the C-terminus of a heavy chain of the IgG.

[0185] In certain embodiments, the CD3 binding domain comprises a variable heavy chain with a variable heavy chain CDR1, CDR2 and CDR3. In certain embodiments, the CD3 binding domain comprises a variable light chain with a variable light chain CDR1, CDR2, and CDR3. In certain embodiments, the CD3 binding domain comprises a variable light and a variable heavy chain. In certain embodiments, the CD3 binding domain is an scFv.

[0186] In certain embodiments, the CD3 binding domain binds human CD3s. In certain embodiments, the CD3 binding domain binds the epitope FSEXE (SEQ ID NO: 37)and QDGNE (SEQ ID NO: 38) of CD3s. FSEXE (SEQ ID NO: 37) and QDGNE (SEQ ID NO: 38) is an epitope on the N terminus of CD3s. In certain embodiments, the CD3 binding domain binds human CD3 and cynomolgus monkey CD3. In certain embodiments, the CD3 binding domain is derived from a CD3 antibody that is cross-reactive to both human and to human CD3 and cynomolgus monkey CD3. In certain embodiments of the invention, the CD3 binding domain is a humanized binding domain derived from SP34. In certain embodiments of the invention, the CD3 binding domain comprises a variable heavy and light chain corresponding to anti-CD3 antibody I2C.

[0187] In certain embodiments, the CD3 binding domain of the T cell engager comprises a variable heavy chain and / or a variable light chain derived from HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C. In certain embodiments, the T cell engager of the pharmaceutical composition of the invention comprises a VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2, and VL CDR3 which are the complementaritydetermining regions of an anti-CD3 antibody selected from the group consisting of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C. In certain embodiments, the CD3 binding domain comprises a variable heavy chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of the variable heavy chain of one of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C. In certain embodiments, the CD3 binding domain comprises a variable light chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of the variable light chain of one of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C. In certain embodiments, the CD3 binding domain comprises a variable heavy chain and a variable light chain with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of the variable heavy chain and variable light chain of one of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C.

[0188] For instance, in certain embodiments, the CD3 binding domain of the T cell engager is derived from OKT3. In certain embodiments, the T cell engager of the pharmaceutical composition of the invention comprises a VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2, and VL CDR3 which are the complementarity-determiningregions of the 0KT3 derivative set forth in Table 2. In certain embodiments, the VH CDR1, VH CDR2, and VH CDR3 and the VL CDR1, VL CDR2, and VL CDR3 of the CD3 tumor antigen binding domain of a T cell engager comprise an amino acid sequence as set forth in SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively. In certain embodiments, the CD3 binding domain comprises a variable heavy chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequence of the variable heavy chain of the OKT3 derivative set forth in SEQ ID NO: 18. In certain embodiments, the CD3 binding domain comprises a variable light chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequence of the variable light chain of the OKT3 derivative set forth in SEQ ID NO: 17. In certain embodiments, the CD3 binding domain contains a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 17.Table 2: CD3 Variable Domain Examples

[0189] In certain embodiments, the CD3 binding domain is an scFv or an scFv fragment. In certain embodiments, the CD3 binding domain is monovalent. In certain embodiments the CD3 binding domain bivalent. In certain embodiments, the CD3 binding domain trivalent.

[0190] In certain embodiments of the invention, the pharmaceutical composition may further comprise a therapeutically effective amount of an iNKT activator as described herein. In certain embodiments of the invention, the iNKT activator may be administered separate from the pharmaceutical composition comprising the allogeneic iNKT cells, T cell engager and pharmaceutically acceptable carrier or excipient.

[0191] The precise dose to be employed in a composition will also depend on the route of administration, and the seriousness of the infection or disease caused by it, and should be decided according to the judgment of the practitioner and each subject's circumstances. For example, effective doses may also vary depending upon means of administration, target site, physiological state of the subject (including age, body weight, and health), whether the subject is a human or an animal, other medications administered, or whether treatment is prophylactic or therapeutic. Usually, the subject is a human but nonhuman mammals including transgenic mammals can also be treated. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0192] Those active components of the present invention are preferably used in such a pharmaceutical composition, in doses mixed with an acceptable carrier or carrier material, that the disease can be treated or at least alleviated. Such a composition can (in addition to the active component and the carrier) include filling material, salts, buffer, stabilizers, solubilizers and other materials, which are known state of the art.

[0193] The term “pharmaceutically acceptable” defines a non-toxic material, which does not interfere with effectiveness of the biological activity of the active component. The choice of the carrier is dependent on the application.

[0194] The pharmaceutical composition can contain additional components which enhance the activity of the active component or which supplement the treatment. Such additional components and / or factors can be part of the pharmaceutical composition to achieve synergistic effects or to minimize adverse or unwanted effects.

[0195] Techniques for the formulation or preparation and application / medication of active components of the present invention are published in “Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., latest edition. An appropriate application is a parenteral application, for example intramuscular, subcutaneous, intramedular injections as well as intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injections. The intravenous injection is the preferred treatment of a patient.

[0196] Pharmaceutical compositions can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a pharmaceutical composition. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.Methods of Treatment using iNKT Cells and T Cell Engagers

[0197] The instant invention includes method of treating a subject suffering from a disease or condition associated with the expression or over-expression of a tumor antigen by administering to the subject a pharmaceutical composition comprising NKT cells (e.g. allogeneic iNKT cells) and a T cell engager as described herein.

[0198] In certain embodiments, the aberrantly expressed or overexpressed tumor antigen associated with the disease or condition is specifically targeted by the antigen binding domain of the T cell engager. Treatment can be selected so that the patient with the disease or condition associated with the over-expressed cell surface tumor antigen is administered the appropriate pharmaceutical composition, i.e., a composition comprising NKT cells (e.g., allogeneic iNKT cells) and a T cell engager that specifically binds the relevant tumor antigen. For example, the invention includes a method of treating a subject suffering from a disease or condition associated with the aberrant or overexpression of CD19 by administering a pharmaceutical composition of the invention comprising iNKT cells and a T cell engager that specifically binds CD 19.

[0199] In some aspects, the disclosure provides a method for treating a cancer, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising at least 95% allogeneic invariant natural killer cells (iNKTs) isolated from a single donor and a T cell engager and a pharmaceutical composition comprising a T cell engager. In certain embodiments of the invention, the pharmaceutical composition comprising the allogeneic iNKT cells comprises at least 70%, at least 75%, 80%, at least 85%, or at least 90% or more iNKT cells as compared to other cells types. In certain embodiments of the invention, the allogeneic invariant iNKT cells are from one or more healthy donors.

[0200] In certain embodiments of the invention, the allogeneic iNKT cells comprise CD4+ and / or CD8+ iNKT cells. In certain embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, at least 85%, or at least 90% of the iNKT cells are CD4+ iNKT cells. In certain embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, at least 85%, or at least 90% of the iNKT cells are CD8+ iNKT cells.

[0201] In certain embodiments of the invention, the subject is administered between 106and 1012NKT cells (e.g., iNKT cells). The exact number of cells to be administered may be modified based on multiple factors, including, for instance, the weight and health of the subject. In certain embodiments, a 70 kg subject is administered between 108and 1012NKT cells (e.g., iNKT cells). For instance, in certain embodiments, a 70 kg subject is administered between 108and 1010allogeneic iNKT cells.

[0202] In certain embodiments, the subject is administered NKT cells (e.g., iNKT cells) and a T cell engager that are separately formulated. For instance, the invention includes treating a subject suffering from a disease or condition associated with the overexpression of a tumor antigen by administering to the subject (i) a pharmaceutical composition comprising iNKT cells and a pharmaceutically acceptable carrier or excipient and (ii) a pharmaceutical composition comprising a T cell engager and a pharmaceutically acceptable carrier or excipient. As with a pharmaceutical composition comprising both NKT cells (e.g., allogeneic iNKT cells) and a T cell engager, the T cell engager would be selected based on the over-expressed tumor antigen (i.e., a T cell engager would be selected based on its ability to specifically bind the tumor antigen).

[0203] By separately formulating the pharmaceutical composition comprising iNKT cells from the pharmaceutical composition comprising T cell engagers, it is possible toadminister the iNKT cells and T cell engagers sequentially. A pharmaceutical composition comprising allogeneic iNKT cells as described herein can be administered to a subject in need thereof prior to or after administration of a pharmaceutical composition comprising a T cell engager to the subject.

[0204] In certain embodiments, the invention includes a method of treating a subject with cancer or a disease or condition associated with an aberrantly expressed or overexpressed tumor antigen by administering the NKT cells (e.g., allogeneic iNKT cells) on a different day than the administration of the T cell engager. In this embodiment of the invention, the NKT cells (e.g., allogeneic iNKT cells) can be administered before or after the T cell engager.

[0205] In one embodiment of the invention, the NKT cells (e.g., allogeneic iNKT cells) are administered to the subject on day 1 and a T cell engager is subsequently administered once or multiple times on subsequent days. For instance, the patient may receive a single treatment with iNKT cells and subsequently be treated with a T cell engager one, two, three, four, five, six, seven, eight, nine, or more times over a period of time. In one embodiment of the invention, iNKT cells (e.g., a pharmaceutical composition comprising iNKT cells and a pharmaceutically acceptable carrier or excipient) are administered on day one of treatment, and the relevant T cell engager (e.g., a pharmaceutical composition comprising the relevant T cell engager and a pharmaceutically acceptable carrier or excipient) is administered on day two, day three, day four, day five, day six, day seven, day eight, day nine, day ten, day eleven, day twelve, day thirteen, or day fourteen.

[0206] For instance, the invention includes a method of treating a patient with subject or a condition or disease associated with the aberrant or over-expression of a tumor antigen by administering allogeneic iNKT cells (including a pharmaceutical composition comprising allogeneic iNKT cells) on day 1 and subsequently dosing the subject with multiple doses of a T cell engager over time. For instance, the T cell engager may be administered according to an established treatment schedule. For instance, the T cell engager may be administered in a pharmaceutical composition on a weekly, bi-weekly, or tri-weekly basis for some period of time (e.g., two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks). A subject receiving treatment may be administered a T cell engager weekly, bi-weekly, or tri-weekly for a set period of time followed by a break in treatment until treatments re-commence.

[0207] In certain embodiments of the invention, the T cell engager (e.g., a pharmaceutical composition comprising the T cell engager and a pharmaceutically acceptable carrier or excipient) is administered by continuous infusion after administration of allogeneic iNKT cells. For instance, T cell engager can be administered at a low dose over multiple days using an infusion pump set at a constant flow rate. Typically, a cycle of administration of a T cell engager by continuous infusion involves 1, 2, 3 or 4 weeks of treatment by continuous infusion followed by 1, 2, 3, or 4 weeks of no treatment. A patient may receive, for instance, 1-4 rounds of T cell engager by continuous infusion.

[0208] For instance, in certain embodiments, a BiTE or BiTE-Fc antibody T cell engager is administered with allogeneic iNKT cells. In certain embodiments, the BiTE or BiTE-Fc T cell engager antibody is administered by continuous infusion. In certain embodiments, the allogeneic iNKT cells are administered while the subject is also receiving the BiTE or BiTE-Fc antibody T cell engager by continuous infusion. In certain embodiments, allogeneic iNKT cells are administered to the subjection prior to administration of the BiTE or BiTE-Fc T cell engager. For instance, in certain embodiments, the allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells and a pharmaceutically acceptable buffer or excipient) are administered to the subject in need thereof, and treatment with the T cell engager by continuous infusion is initiated 1-3 days later, 2-4 days later, 1-5 days later, 5 days later, 5-10 days later, 7 days later, 10 days later, 10-15 days later, 14 days later, 15-20 days later, 20 days later, 20-25 days later, 25-30 days later, or 30 days later. In certain embodiments, the dose of the BiTE or BiTE-Fc administered by continuous infusion ranges from 5 to 40 mcg / day. In certain embodiments, the BiTE or BiTE-Fc T cell engager administered to the subject by continuous infusion specifically binds human HER2, HER3, EGFR / HER1, CD19, MUC16, B7-H3, BCMA, FAP, DLL3, PSMA, P-cadherin (CDH3), mesothelin (MSLN), claudin 6, CD 123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule-1 (CLL-1), WT1, GPRC5D, CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO, GD2, MUC1, CD22, FLT3, MUC17, CEACAM5, Claudin 18.2, or NKG2D ligand and human CD3 (e.g., human CD3s). In certain embodiments, the BiTE or BiTE-Fc T cell engager administered to the subject by continuous infusion and which specifically binds human HER2, HER3, EGFR / HER1, CD19, MUC16, B7-H3, BCMA, FAP, DLL3, PSMA, P-cadherin (CDH3), mesothelin (MSLN), claudin 6, CD 123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule- 1 (CLL-1), WT1, GPRC5D, CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO,GD2, MUC1, CD22, FLT3, MUC17, or CEACAM5, Claudin 18.2, NKG2DL or binds CD3s through a CD3 binding domain derived from OKT3, SP34, or TR66.

[0209] In certain embodiments of the invention, a subject with cancer (e.g., acute lymphoblastic leukemia) or a condition or disease associated with expression of CD19 is administered allogeneic iNKT cells and a T cell engager that specifically binds CD 19 and CD3. In certain embodiments of the invention, a subject with cancer (e.g., acute lymphoblastic leukemia) or a condition or disease associated with expression of CD19 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject commences treatment with a CD19 and CD3 T cell engager (e.g., blinatumomab) by continuous infusion. For instance, the subject may receive 15 or 28 mcg / day of the T cell engager (e.g., blinatumomab) via continuous infusion for 28 days followed by 14 days of no treatment before the next round of treatment by continuous infusion. In certain embodiments, the subject receives four rounds of treatment of blinatumomab by continuous infusion.

[0210] Human epidermal growth factor receptor 2 (HER2) is overexpressed in many cancers of epithelial origin. HER2+ cancers have been reported for bladder carcinomas, gallbladder, extrahepatic cholangiocarcinomas, cervical, uterine, testicular, breast, ovarian, lung, gastric, and colon cancers. (See Yan M, Schwaederle M, Arguello D, Millis SZ, Gatalica Z, Kurzrock R. HER2 expression status in diverse cancers: review of results from 37,992 patients. Cancer Metastasis Rev. 2015 Mar;34(l): 157-64.)

[0211] In certain embodiments of the invention, a subject with cancer (e.g., bladder carcinoma, extrahepatic cholangiocarcinoma, or a gallbladder, cervical, uterine, testicular, breast, ovarian, lung, gastric, or colon cancer) or a condition or disease associated with aberrant expression of HER2 is concurrently administered allogeneic iNKT cells and a T cell engager that specifically binds HER2 and CD3. The allogeneic iNKT cells and T cell engager may be administered as a single pharmaceutical composition as described herein. In certain embodiments of the invention, a subject with cancer (e.g., HER2+ gastric or breast cancer) or a condition or disease associated with expression of HER2 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a HER2 and CD3 T cell engager.

[0212] HER3 expression or overexpression has been described in multiple types of tumors, including breast, ovarian, lung, colon, pancreatic, melanoma, gastric, head and neck, and prostate cancers. It has been recently reported that melanomas represent the tumor type with the highest HER3 expression at the mRNA level, followed by cholangiocarcinomas and invasive breast tumors. HER3 expression has also been reported in pilocytic astrocytoma, a childhood glioma, and in rhabdomyosarcoma, a pediatric sarcoma. (See, for instance, Gandullo-Sanchez, L., Ocana, A. and Pandeilla, A. HER3 in cancer: from bench to the bedside. J Exp Clin Cancer Res 41,310 (2022).)

[0213] In certain embodiments of the invention, a subject with cancer (e.g., breast, ovarian, lung, colon, pancreatic, melanoma, gastric, head and neck, or prostate cancer or pilocytic astrocytoma or rhabdomyosarcoma) or a condition or disease associated with aberrant expression of HER3 is concurrently administered allogeneic iNKT cells and a T cell engager that specifically binds HER3 and CD3. The allogeneic iNKT cells and T cell engager may be administered as a single pharmaceutical composition as described herein. In certain embodiments of the invention, a subject with cancer (e.g., HER3+ breast, ovarian, lung, colon, pancreatic, melanoma, gastric, head and neck, or prostate cancer or pilocytic astrocytoma or rhabdomyosarcoma) or a condition or disease associated with expression of HER3 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a HER3 and CD3 T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks.

[0214] Overexpression of Epidermal Growth Factor Receptor (EGFR) and mutant hyper-active forms of EGFR are uniquely found in some brain, lung and other cancers. (See, for instance, Uribe, ML, Marrocco I, Yarden Y. EGFR in Cancer: Signaling Mechanisms, Drugs, and Acquired Resistance. Cancers (Basel). 2021 Jun 1 ; 13(11)2748.)

[0215] In certain embodiments of the invention, a subject with cancer (e.g., lung cancer such as small-cell lung cancer or brain cancer) or a condition or disease associated with aberrant expression of EGFR / HER1 is concurrently administered allogeneic iNKT cells and a T cell engager that specifically binds EGFR and CD3. The allogeneic iNKT cells and T cell engager may be administered as a single pharmaceutical composition as described herein. In certain embodiments of the invention, a subject with cancer (e.g., EGFR+ lung orbrain cancer) or a condition or disease associated with expression of EGFR is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of an EGFR and CD3 T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks.

[0216] B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily that plays an important role in regulating B-cell proliferation and survival. BCMA is expressed on the cell membrane of normal and malignant plasma cells, but not other normal tissues. (See Seckinger A, Delgado JA, Moser S, et al. Cancer Cell. 2017;31(3):396-410.) BCMA is reported as expressed in several hematologic malignancies, including multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), and Hodgkin lymphoma. (See Dogan, A., Siegel, D., Tran, N. et al. B-cell maturation antigen expression across hematologic cancers: a systematic literature review. Blood Cancer J. 10, 73 (2020).)

[0217] In certain embodiments of the invention, a subject with cancer (e.g., a hematologic malignancy such as multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), or Hodgkin lymphoma) or a condition or disease associated with aberrant expression of BCMA is concurrently administered allogeneic iNKT cells and a T cell engager that specifically binds BCMA and CD3.

[0218] The allogeneic iNKT cells and T cell engager may be administered as a single pharmaceutical composition as described herein. In certain embodiments of the invention, a subject with cancer (e.g., BCMA+ blood malignancy) or a condition or disease associated with expression of BCMA is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a BCMA targeting T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks. In certain embodiments, the BCMA T cell engager is administered twice a week or three times a week.

[0219] In certain embodiments of the invention, NTK cells, e.g., allogeneic iNKT cells, and a T cell engager comprising a BCMA binding domain and a CD3 binding domain, either concurrently or sequentially, for the treatment of relapsed or refractory multiple myeloma. For instance, the methods of the invention include administering a pharmaceutical composition comprising allogeneic iNKT cells (e.g., a pharmaceutical composition comprising IO10cells / 70 kg patient and a pharmaceutically acceptable carrier) and a T cell engager such as HPN217 (Harpoon), TNB-383 (TeneoBio), AMG701 (Amgen), AMG420 (Amgen), REGN5458 (Regeneron), Elranatamab, or CC-93269.

[0220] Prostate-Specific Membrane Antigen (PSMA) is a misnomer as expression of the protein is not limited to prostate cancer. Aberrant or overexpression of PSMA has been reported in prostate cancer; salivary gland tumors, including adenocistic carcinoma and salivary duct carcinoma; thyroid cancer, including poorly differentiated thyroid cancer and anaplastic thyroid cancer; hepatocellular carcinoma; renal cell carcinoma; glioblastoma; breast cancer; lung cancer; including small-cell lung cancer and non-small cell lung cancer; gastric cancer, including gastric adenocarcinoma; colorectal carcinomas; sarcomas, including osteosarcoma; soft tissue tumors, including myxofibrosarcomas, ganglioneuromas, extrapleural solitary fibrous tumors, inflammatory myofibroblastic tumors, desmoid-type fibromatosis, Ewing sarcomas, gastrointestinal stromal tumors, synovial sarcomas, undifferentiated pleomorphic sarcomas, and endometrial stromal sarcomas; alveolar rhabdomyosarcoma; bladder carcinoma such as urothelial carcinoma, squamous cell carcinoma, adenocarcinoma and small cell carcinoma; gynecological malignancies, including ovarian, endometrial, cervical, and vulvar carcinomas; oral squamous cell carcinomas; melanomas; and neoplasms including endolymphatic sac tumors. (See, for instance, Lauri C, Chiurchioni L, Russo VM, Zannini L, Signore A, PSMA Expression in Solid Tumors beyond the Prostate Gland: Ready for Theranostic Applications? J Clin Med. 2022 Nov 7;ll(21):6590.)

[0221] In certain embodiments of the invention, a subject with cancer (e.g., prostate cancer, hepatocellular carcinoma, renal cell carcinoma, breast cancer, lung cancer, sarcoma, soft tissue tumor) or a condition or disease associated with aberrant expression of PSMA is concurrently administered allogeneic iNKT cells and a T cell engager that specifically binds PSMA and CD3. The allogeneic iNKT cells and T cell engager may be administered as a single pharmaceutical composition as described herein. In certain embodiments of the invention, a subject with cancer (e.g., PSMA+ carcinoma or sarcoma or soft tissue cancer) ora condition or disease associated with expression of PSMA is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a PSMA T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks.

[0222] Fibroblast Activation Protein-a (FAP) is expressed at low or undetectable levels in normal tissues. FAP is overexpressed in in many tumor tissues, including breast, colorectal, pancreatic, lung, brain, intrahepatic bile duct, and ovarian cancers and has been detected in some tumors that are derived from non-epithelial tissues, such as melanoma and myeloma. (See, for instance, Xin L, Gao J, Zheng Z, Chen Y, Lv S, Zhao Z, Yu C, Yang X, and Zhang R (2021) Fibroblast Activation Protein-a as a Target in the Bench-to-Bedside Diagnosis and Treatment of Tumors: A Narrative Review. Front. Oncol. 11:648187.)

[0223] Systemic therapy targeting FAP can lead to severe cachexia, including muscle damage, osteo toxicity, and even death. (See, for instance, Roberts EW, Deonarine A, Jones JO, Denton AE, Feig C, Lyons SK, et al. Depletion of Stromal Cells Expressing Fibroblast Activation Protein-Alpha From Skeletal Muscle and Bone Marrow Results in Cachexia and Anemia. J Exp Med (2013) 210:1137-51.) Accordingly, therapeutics targeting FAP should be designed to selectively kill FAP+ fibroblasts.

[0224] In certain embodiments of the invention, a subject with cancer (e.g., breast, colorectal, pancreatic, lung, brain, intrahepatic bile duct, and ovarian cancers, melanoma and myeloma) or a condition or disease associated with aberrant expression of FAP expression is administered allogeneic iNKT cells and a T cell engager that specifically binds FAP and CD3. In certain embodiments, the allogenic iNKT cells and FAP targeting T cell engager are administered concurrently (e.g., as a single pharmaceutical composition as described herein). In certain embodiments of the invention, a subject with cancer (e.g., breast, colorectal, pancreatic, lung, brain, intrahepatic bile duct, and ovarian cancers, melanoma and myeloma) or a condition or disease associated with overexpression of FAP is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a FAP targeting T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once aweek for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks. The first dose of the T cell engager may be lower than the maximum dose.

[0225] Delta-like Canonical Notch Ligand 3 (DLL3) is a protein frequently overexpressed in small-cell lung cancer, the most aggressive phenotype within the spectrum of all lung neuroendocrine tumors. In certain embodiments of the invention, a subject with cancer (e.g., a neuroendocrine tumor, including small-cell lung cancer) or a condition or disease associated with aberrant expression of DLL3 expression is administered allogeneic iNKT cells and a T cell engager that specifically binds DLL3 and CD3. In certain embodiments, the allogenic iNKT cells and DLL3 targeting T cell engager are administered concurrently (e.g., as a single pharmaceutical composition as described herein). In certain embodiments of the invention, a subject with cancer (e.g., small-cell lung cancer or neuroendocrine carcinoma) or a condition or disease associated with overexpression of DLL3 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a DLL3 targeting T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks. The first dose of the T cell engager may be lower than the maximum dose.

[0226] Tarlatamab (AMG 757) and BI 764532 are T cell engagers that can be administered with allogeneic iNKT cells to a subject pursuant to the methods of the invention.

[0227] Numerous studies have established deregulated B7-H3 expression in a variety of malignancies, including prostate cancer, medulloblastoma, non-small cell lung cancer, pancreatic cancer, melanoma, glioma, breast, renal cell carcinoma, ovarian carcinoma, endometrial cancer, hepatocellular carcinoma, and colorectal cancer. In all studies, deregulated B7-H3 expression has been linked with more aggressive disease courses and poorer outcomes. For instance, in oral squamous cell carcinoma, B7-H3 overexpression correlated with more aggressive tumors and higher mortality and increased expression of B7- H3 in prostate cancer was strongly associated with metastasis, recurrence, post-surgical cancer progression, and death. In osteosarcoma and gastric cancer, B7-H3 deregulation reliably predicted a poorer prognosis. (See Ranjana K. Kanchan, David Doss, Parvez Khan, Mohd. Wasim Nasser, Sidharth Mahapatra. To kill a cancer: Targeting the immuneinhibitory checkpoint molecule, B7-H3, Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, Volume 1877, Issue 5, 2022.)

[0228] In certain embodiments of the invention, a subject with cancer (e.g., prostate cancer, medulloblastoma, non- small cell lung cancer, pancreatic cancer, melanoma, glioma, breast, renal cell carcinoma, ovarian carcinoma, endometrial cancer, hepatocellular carcinoma, and colorectal cancer) or a condition or disease associated with aberrant expression of B7-H3 is administered allogeneic iNKT cells and a T cell engager that specifically binds B7-H3 and CD3. In certain embodiments, the allogenic iNKT cells and B7- H3 T cell engager are administered concurrently (e.g., as a single pharmaceutical composition as described herein). In certain embodiments of the invention, a subject with cancer (e.g., prostate cancer, medulloblastoma, non-small cell lung cancer, pancreatic cancer, melanoma, glioma, breast, renal cell carcinoma, ovarian carcinoma, endometrial cancer, hepatocellular carcinoma, and colorectal cancer) or a condition or disease associated with overexpression of B7-H3 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a B7-H3 and CD3 T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks. The first dose of the T cell engager may be lower than the maximum dose.

[0229] MUC16 also known as CA125) is overexpressed in cancers including ovarian cancer, breast cancer, pancreatic cancer, non-small-cell lung cancer, intrahepatic cholangiocarcinoma- mass forming type, adenocarcinoma of the uterine cervix, and adenocarcinoma of the gastric tract, and in diseases and conditions including inflammatory bowel disease, liver cirrhosis, cardiac failure, peritoneal infection, and abdominal surgery. (See, for instance, Haridas, D. et al., 2014, FASEB J., 28:4183-4199). Expression on cancer cells is shown to protect tumor cells from the immune system. (See Felder, M. et al., 2014, Molecular Cancer, 13:129). Due to their aberrant overexpression and functional involvement, MUC16 and its ligands have emerged as potential targets for therapeutic intervention using monoclonal antibodies and immunotherapy. (See Aithal A, Rauth S, Kshirsagar P, Shah A, Lakshmanan I, Junker WM, Jain M, Ponnusamy MP, Batra SK. MUC16 as a novel target for cancer therapy. Expert Opin Ther Targets. 2018 Aug;22(8):675- 686; Haridas D, Chakraborty S, Ponnusamy MP, et al. Pathobiological implications ofMUC16 expression in pancreatic cancer. PLoS One 2011;6(10):e26839; Klug TL, Bast RC Jr., Niloff JM, et al. Monoclonal antibody immunoradiometric assay for an antigenic determinant (CA 125) associated with human epithelial ovarian carcinomas. Cancer Res 1984;44(3): 1048-53; Lakshmanan I, Salfity S, Seshacharyulu P, et al. MUC16 Regulates TSPYL5 for Lung Cancer Cell Growth and Chemoresistance by Suppressing p53. Clin Cancer Res 2017;23(14):3906-3917.)

[0230] In certain embodiments of the invention, combination therapy of NKT cells (e.g., iNKT cells) and a T cell engager that specifically targets MUC16 and CD3 can be used to treat a subject with MUC16+ cancer such as ovarian cancer, breast cancer, pancreatic cancer, non- small-cell lung cancer, intrahepatic cholangiocarcinoma- mass forming type, adenocarcinoma of the uterine cervix, and adenocarcinoma of the gastric tract. In certain embodiments of the invention, combination therapy of NKT cells (e.g., iNKT cells) and a T cell engager that specifically binds MUC6 and CD3 can be used to treat a subject with a disease or condition associated with MUC16 overexpression such as inflammatory bowel disease, liver cirrhosis, cardiac failure, peritoneal infection, and abdominal surgery.

[0231] In certain embodiments of the invention, a subject with cancer (e.g., ovarian cancer) or a condition or disease associated with expression of MUC16 is administered allogeneic iNKT cells and a T cell engager that specifically binds MUC16 and CD3. In certain embodiments of the invention, a subject with cancer (e.g., ovarian cancer) or a condition or disease associated with expression of MUC16 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a MUC16 and CD3 T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks. The first dose of the T cell engager may be lower than the maximum dose. For instance, the first dose of the MUC16 and CD3 T cell engager may be 1 mg, followed a week later with a 20 mg dose (i.e., week 2 of T cell engager dosing), and followed a week later with a 250 mg dose. In certain embodiments of the invention, NKT cells (e.g., allogeneic iNKT cells) are administered to a subject and subsequently ubamatamab is administered in weekly doses ranging from 1 mg to 800 mg.

[0232] The wide expression of NKG2D ligands (NKG2DL) on many different tumor types, with limited expression on normal tissues, makes them attractive targets forimmunotherapy. It has been estimated that more than 90% of human tumors may express at least one NKG2D ligand (See Spear P, Wu MR, Sentman ML, Sentman CL. NKG2D ligands as therapeutic targets. Cancer Immun. 2013 May 1 ; 13:8). There are two families of NKG2D ligands in humans, MHC class I chain-related proteins A (MICA) and B (MICB) as well as ULI 6 binding proteins (ULBP1 - 6). In certain embodiments of the invention, a NKG2DL is the tumor antigen target for the T cell engager. In certain embodiments of the invention, a subject with a condition or disease associated with the expression of a NKG2DL (e.g., MICA, MICB or ULBPI - 6) is administered a therapeutically effective amount of NKT cells (e.g., iNKT cells) and a T cell engager that specifically binds the NKG2DL and a CD3. For instance, NKG2DL are commonly expressed in soft tissue sarcoma (STS), but generally absent in healthy tissues. In certain embodiments of the invention, a subject with a cancer (e.g., a soft tissue sarcoma) or other condition or disease associated with the expression of a NKG2DL is administered NKT cells (e.g., iNKT cells) and a T cell engager comprising a tumor antigen binding domain that specifically binds the overexpressed NKG2DL.

[0233] MICA has broad expression on multiple tumor types, including cervical, colon, breast, lung, ovarian, prostate, renal cell, and pancreatic carcinomas as well as in cutaneous melanoma and malignant melanoma. (See Del Toro- Arreola S, Arrey gue-Garcia N, Aguilar-Lemarroy A, Cid-Arregui A, Jimenez-Perez M, Haramati J, et al. MHC class I- related chain A and B ligands are differentially expressed in human cervical cancer cell lines. Cancer Cell Int. 2011 Jun 1;11:15. 2867-11-15; Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T. Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB. Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6879-84; Shi P, Yin T, Zhou F, Cui P, Gou S, Wang C. Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K / Akt signaling pathway. BMC Cancer. 2014 May 25;14:370. 2407-14-370; Vetter CS, Groh V, thor Straten P, Spies T, Brocker EB, Becker JC. Expression of stress- induced MHC class I related chain molecules on human melanoma. J Invest Dermatol. 2002 Apr; 118(4): 600-5). The invention includes administration for iNKT cells and a T cell engager that specifically binds MICA to a subject with a disease or condition associated with the overexpression of MICA (e.g., cervical, colon, breast, lung, ovarian, prostate, renal cell, and pancreatic carcinomas and cutaneous and malignant melanomas).

[0234] In one embodiment of the invention, a subject with cancer or a condition or disease associated with expression of a NKG2DL (e.g., MICA, MICB, ULBPI - 6) isadministered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a NKG2DL and CD3 T cell engager.

[0235] CEACAM5 is a cell surface glycoprotein that is often highly expressed in gastrointestinal, breast, and lung cancer. For instance, approximately 20% of patients with NSCLC exhibit overexpression of CEACAM5. (See, for instance, Gazzah A, Bedard PL, Hierro C, et al. Safety, pharmacokinetics, and antitumor activity of the anti-CEACAM5-DM4 antibody-drug conjugate tusamitamab ravtansine (SAR408701) in patients with advanced solid tumors: first-in-human dose-escalation study. Ann Oncol. 2022;33(4):416-425; Johnson ML, Chadjaa M, Yoruk S, Besse B. Phase III trial comparing antibody-drug conjugate (ADC) SAR408701 with docetaxel in patients with metastatic non- squamous non- small cell lung cancer (NSQ NSCLC) failing chemotherapy and immunotherapy. J Clin Oncol. 2020;38(suppl 15):TPS9625.)

[0236] In certain embodiments of the invention, a subject with cancer (e.g., CEACAM5+ gastrointestinal, breast, or lung cancer) or a condition or disease associated with expression of CEACAM5 is administered allogeneic iNKT cells and a T cell engager that specifically binds CEACAM5 and CD3. In certain embodiments of the invention, a subject with cancer (e.g., gastrointestinal, breast or lung cancer) or a condition or disease associated with expression of CEACAM5 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a CEACAM5 and CD3 T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks.

[0237] Claudin 18.2, a member of the claudin family, is commonly expressed in multiple cancers, including gastric cancer and prostate cancer. (See, for instance, Tiireci O, Sahin U, Schulze-Bergkamen H, et al. A multicentre, phase Ila study of zolbetuximab as a single agent in patients with recurrent or refractory advanced adenocarcinoma of the stomach or lower oesophagus: the MONO study. Ann Oncol 2019;30:1487-95; Micke P, Mattsson JS, Edlund K, et al. Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non- small-cell lung cancer. Int J Cancer 2014;135:2206-14; Jun KH, Kim JH, Jung JH, et al.Expression of claudin-7 and loss of claudin-18 correlate with poor prognosis in gastric cancer. Int J Surg 2014;12:156-62. 10.)

[0238] In certain embodiments of the invention, a subject with cancer (e.g., Claudin 18.2+ gastrointestinal, prostate or lung cancer) or a condition or disease associated with expression of Claudin 18.2 is administered allogeneic iNKT cells and a T cell engager that specifically binds Claudin 18.2 and CD3. In certain embodiments of the invention, a subject with cancer (e.g., gastrointestinal, prostate or lung cancer) or a condition or disease associated with expression of Claudin 18.2 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a Claudin 18.2 and CD3 T cell engager. The T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks.

[0239] In certain embodiments of the invention, a subject in need thereof is administered allogeneic iNKT cells and a T cell engager such as blinatumomab, catumaxomab, mosunetuzumab, tebentafusp, teclistamab, ubamatamab, enoblituzumab, linvoseltamab, odronextamab, tebentafusp, forimtamig, or runimotamab. For instance, the invention includes administering a composition comprising about 1.5 x 107to 2.0 x 108allogeneic iNKT cells to a subject followed by administering a composition comprising blinatumomab, catumaxomab, mosunetuzumab, tebentafusp, teclistamab, ubamatamab, enoblituzumab, linvoseltamab, odronextamab, tebentafusp, forimtamig, or runimotamab.

[0240] In addition to the methods described herein, methods also are provided for increasing the frequency of endogenous iNKT cells in vivo or increasing or sustaining the frequency of infused or endogenous iNKT cells in vivo. Such methods can utilize compounds such as, without limitation, cytokines (e.g., IL-2, IL-17, IL-15) or alpha-galactosyl ceramide. Similarly, agonists of iNKT cells can be administered to a subject. Agonists of iNKT cells are known in the art and include, without limitation, synthetic agonists (see, e.g., Cerundolo et al., 2010, Curr. Opin. Immunol., 22(3):417-24), alpha-galactosylceramides and beta- mannosylceramides (see, e.g., O'Konek et al., 2011, J. Clin. Invest., 121(2):683-94; Aspeslagh et al., 2011, The EMBO J., 30(11 ):2294-305), and threitolceramide (see, e.g., Silk et al., 2008, J. Immunol., 180(10):6452-6). See, also, Cerundolo et al., 2009, Nature Rev., 9(l):28-38.

[0241] In certain embodiments, the methods relate to the combination treatment of NKT cells (e.g., iNKT cells) with a T cell engager, or compositions described herein, for the treatment of a cancer or administration to a person with a cancer. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is a B-cell cancer. In some embodiments the cancer is diffuse large B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, mucosa-associated lymphatic tissue lymphoma, small lymphocytic lymphoma (also known as chronic lymphocytic leukemia, CLL), mantle cell lymphoma, primary mediastinal (thymic) large B cell lymphoma, T cell / histiocyte-rich large B-cell lymphoma, primary cutaneous diffuse large B-cell lymphoma, EBV positive diffuse large B- cell lymphoma, burkitt's lymphoma, lymphoplasmacytic lymphoma, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, central nervous system lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, or large B-cell lymphoma. In some embodiments, the cancer comprises a blood cancer. In some embodiments, the blood cancer comprises myeloma, leukemia, lymphoma, Non-Hodgkin lymphoma, Hodgkin lymphoma, a myeloid neoplasm, a lymphoid neoplasm, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myelogenous leukemia (CML).

[0242] Certain aspects of the disclosure relate to the treatment of cancer and / or use of the NKT cells (e.g., iNKT cells) in combination with T cell engagers, and compositions of the disclosure, to treat cancer. The cancer to be treated or antigen may be an antigen associated with any cancer known in the art or, for example, epithelial cancer, (e.g., breast, gastrointestinal, lung), prostate cancer, bladder cancer, lung (e.g., small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, liver metastasis, esophageal cancer, head and neck cancer, or a colorectal cancer. In some embodiments, the cancer to be treated or antigen is from one of the following cancers: adenocortical carcinoma, agnogenic myeloid metaplasia, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, meningiosarcoma, craniopharyngioma, haemangioblastomas,medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing's family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonary lymphangiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., nonmelanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), or Meigs' syndrome.

[0243] In certain embodiments of the invention, the subject is administered antitumor therapy in addition to iNKT cells and a T cell engager. The additional anti-tumor therapy may be administered at the same time as the iNKT cells and T cell engager orsequentially (e.g., on weeks when subject does not receive iNKT and T cell engager therapy). In certain embodiments, the anti-tumor therapy comprises a PD-1 inhibitor (e.g., REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1 105, and REGN3504, as well as those disclosed in Patent Publication No. US 2015-0203580), CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T cell co-inhibitor or ligand (e.g., an antibody to LAG3, CD-28, 2B4, LY108, LAIR1 , ICOS, CD160 or VISTA), an indoleamine-2, 3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., a“VEGF-Trap” such as aflibercept or other VEGF- inhibiting fusion protein as set forth in US 7,087,41 1 , or an anti- VEGF antibody or antigen binding fragment thereof (e.g., bevacizumab, or ranibizumab) or a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib)], an Ang2 inhibitor (e.g., nesvacumab), a transforming growth factor beta (TGFp) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor ( e.g ., erlotinib, cetuximab), a CD20 inhibitor ( e.g., an anti-CD20 antibody such as rituximab), an antibody to a tumorspecific antigen [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor- M2-PK, prostate-specific antigen (PSA), pro state- specific membrane antigen (PSMA), also known as folate hydrolase 1 (FOLH1 ), mucin-1 , MART-1 , and CA19-9], a vaccine (e.g., Bacillus Calmette-Guerin, a cancer vaccine), an adjuvant to increase antigen presentation (e.g., granulocyte-macrophage colonystimulating factor), a bispecific antibody (e.g., CD3xCD20 bispecific antibody, or PSMAxCD3 bispecific antibody), a cytotoxin, a poly ADP-ribose polymerase (PARP) inhibitor, a chemotherapeutic agent (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiotherapy, an IL-6R inhibitor (e.g., sarilumab), an IL-4R inhibitor (e.g., dupilumab), an IL-10 inhibitor, a cytokine such as IL-2, IL-7, IL-21 , and IL- 15.

[0244] In certain embodiments of the invention, a subject to be treated with NKT cells (e.g., iNKT cells) and a T cell engager or a pharmaceutical composition of the invention may be pre-treated with a steroid. For instance, in certain embodiments of the invention, the subject is pre-treated with dexamethasone.

[0245] In certain embodiments of the invention, a subject to be treated with NKT cells (e.g., iNKT cells) and a T cell engager, formulated separately or formulated as a singlepharmaceutical composition as described herein, may be pre-treated with an IL-6 receptor antibody such as tocilizumab.

[0246] In certain embodiments of the invention, the methods of treating a subject do not include lymphodepletion of the subject as a pre-treatment. Lymphodepletion is frequently performed prior to immunotherapies to debulk a tumor, alter tumor phenotype, modify the tumor microenvironment, remove cytokine sinks (e.g., make IL-2, IL-7, and IL- 15 more available) and suppress the host immune system. However, lymphodepletion has multiple negative effects including, neutropenia, anemia, thrombocytopenia, and immunosuppression, and toxicities associated with lymphodepletion agents such as fludarabine and cyclophosphamide. For instance, in certain embodiments, the subject administered iNKT cells and a T cell engager is not administered a lymphodepletion agent prior to or during treatment. For instance, in certain embodiments, bendamustine, fludarabine and cyclophosphamide are not administered to the subject prior to or during treatment with iNKT cells and a T cell engager.

[0247] With particular regard to treatment of solid tumors, protocols for assessing endpoints and anti-tumor activity are well-known in the art. While each protocol may define tumor response assessments differently, the RECIST (Response evaluation Criteria in solid tumors) criteria is currently considered to be the recommended guidelines for assessment of tumor response by the National Cancer Institute (see Therasse et al., J. Natl. Cancer Inst. 92:205-216, 2000). According to the RECIST criteria tumor response means a reduction or elimination of all measurable lesions or metastases. Disease is generally considered measurable if it comprises lesions that can be accurately measured in at least one dimension as >20 mm with conventional techniques or >10 mm with spiral CT scan with clearly defined margins by medical photograph or X-ray, computerized axial tomography (CT), magnetic resonance imaging (MRI), or clinical examination (if lesions are superficial). Non- measurable disease means the disease comprises of lesions <20 mm with conventional techniques or <10 mm with spiral CT scan, and truly non-measurable lesions (too small to accurately measure). Non-measurable disease includes pleural effusions, ascites, and disease documented by indirect evidence.

[0248] The criteria for objective status are required for protocols to assess solid tumor response. Representative criteria include the following: (1) Complete Response (CR), defined as complete disappearance of all measurable disease; no new lesions; no disease related symptoms; no evidence of non-measurable disease; (2) Partial Response (PR) defined as 30%decrease in the sum of the longest diameter of target lesions (3) Progressive Disease (PD), defined as 20% increase in the sum of the longest diameter of target lesions or appearance of any new lesion; (4) Stable or No Response, defined as not qualifying for CR, PR, or Progressive Disease. (See Therasse et al., supra.)

[0249] Additional endpoints that are accepted within the oncology art include overall survival (OS), disease-free survival (DFS), objective response rate (ORR), time to progression (TTP), and progression-free survival (PFS) (see Guidance for Industry: Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologies, April 2005, Center for Drug Evaluation and Research, FDA, Rockville, Md.)

[0250] The present invention now will be illustrated by the enclosed Figures and the Examples. The following Examples further illustrate the invention but, of course, should not be construed as limiting its scope.EXAMPLESExample 1. Generation of T Cell Engager Antibodies

[0251] CD3xCD19 and CD3xB7-H3 T cell engager antibodies were constructed in the Knobs-into-Holes (KiH) antibody format. The anti-B7-H3 variable heavy and light chain sequences from enoblituzumab were used to construct the anti-B7-H3 domain (SEQ ID NO:9; SEQ ID NO: 10), and the anti-CD19 variable heavy and light chain sequences from blinatumomab were used to construct the anti-CD19 domain (SEQ ID NO: 1; SEQ ID NO:2). The anti-CD3 variable domain of the OKT3 derivative used in blinatumomab (SEQ ID NO: 17; SEQ ID NO: 18) was used for both T cell engagers.

[0252] Briefly, the anti-CD3 variable domain was cloned on a “knob” backbone, containing a T366W mutation in the CH3 of the Fc region of the antibody. The anti-B7-H3 or anti-CD19 variable domain was cloned on a “hole” backbone containing T366S, L368A, Y407V mutations in the CH3 of the Fc region. The Fc region also contained a N297A mutation to abrogate Fey receptor binding and the potential for effector cell-mediated activity, such as antibody-dependent cellular cytotoxicity (ADCC). Plasmid constructs contained a CMV promotor. Light chains were expressed under mouse IgK leader peptide and heavy chains were expressed under CD33 leader peptide.

[0253] ‘Knob” polypeptides and “hole” polypeptides were separately expressed in ExpiCHO cells and purified using a protein A column. Purified polypeptides were reduced in a IM 2-MAE, lOmM Tris pH8, lOOmM NaCl buffer for 2 hours at room temperature. The “knob” and “hole” polypeptides were then mixed in a one-to-one molar ratio and re-oxidated in a 10 mM Sodium Citrate pH 6.0, 115 mM NaCl by buffer exchange for 3 hours at room temperature. Heterodimerization was confirmed by SDS-PAGE gel and analytical SEC.

[0254] A CD3xMucl6 bispecific formatted on an IgG4 KiH was provided by Proteogenix (catalogue number PX-TA1756-100).Example 2: Isolation and Expansion of iNKT Cells and T Cells; Expression of CD3s on Cells iNKT Cells

[0255] Briefly, leucopaks from healthy adult donors were purchased from commercial vendors. iNKT cells were separated out from the peripheral mononuclear blood cell (PBMC) fraction and then expanded with irradiated aGalCer loaded autologous PBMCs in the presence of human IL-2 for 10-14 days. Cells from the flow-through fraction were cryopreserved for further expansion rounds as well as isolation of conventional T lymphocytes.T Cells

[0256] T lymphocytes were isolated from PBMCs by negative selection using EasySep™ (STEMCELL Technologies). For activation, isolated T cells were stimulated in vitro for 72 hours with ImmunoCult™ Human CD3 / CD28 T Cell Activator (STEMCELL Technologies) in presence of human IL-2 and IL-7. Afterwards, T cells were expanded for 10-14 days in media containing human IL-2 and IL-7. Medium was refreshed every third or fourth day.Expression of CD3s on iNKT and T Cells

[0257] Expanded iNKT cells, freshly isolated T cells and expanded T cells were stained with anti-CD3s antibody. Briefly, 100,000 cells were plated per well of a 96-well V bottom plate. In order to be able to quantify number of molecules per cell, one drop of Quantibrite beads was plated on one of the wells on the same plate (Quantibrite Beads PE Fluorescence Quantitation Kit, BD, Catalog number PE 340495). Beads were stained and washed in the same way as the cells. Plate was spun down for 300g for 5 minutes at room temperature. Supernatant was removed and cells were washed with 2% FBS in PBS. Cellswere spun down at 300g for 5 minutes at room temperature. Supernatant was removed and cells were incubated for 30 minutes at 4°C with anti-human CD3s antibody directly conjugated to phycoerythrin (PE) (purchased from BioLegend, catalogue number 310906). Fixable Viability Dye (eBioscience, 65-0865-14) was used to exclude dead cells. Isotype control was used as negative control for staining (mouse IgGl kappa PE isotype, BioLegend, catalog number 400112). After incubation, cells were washed with 2% FBS / PBS, resuspended in 2% FBS / PBS and analyzed by flow cytometry. Cells were gated based on forward and side scatter (height), single cells were gated based on forward scatter (width and height), and life cells were gated based on life / dead dye exclusion. Mean geometric fluorescence for PE signal was calculated for the gated cells and the beads. Specific signal was calculated by subtracting MFI of the isotype stained cells from cells stained with CD3s - PE signal.

[0258] To convert MFI values to number of molecules per cell, lot specific value for fluorophore to protein (F / P) was obtained for the CD3s -PE antibody used from the vendor. Calculations was done following the manual analysis provided by the Quantibrite Beads PE Fluorescence Quantitation Kit. Number of molecules per cell was quantified using Quantibrite and plotted. Data shown from three independent donors with three technical replicates used in the assay (FIGURE 1).Example 3: Binding and Activation of Cells

[0259] iNKT cells were incubated with various concentrations of the engager followed by fluorescently tagged anti-human IgG antibody. Binding was determined by flow cytometry analysis. Secondary antibody alone was set as the negative control. (FIGURES 2A and 2B)Mucl6-CD3 mediated iNKT activation

[0260] Assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager molecule. iNKT cells from six donors were used as effector cells. CFSE labeled OVCAR-3 cell line was used as target cells. Effector and target cells were co-cultured at 5:1, 1:1 and 1:5 E:T ratio in presence or absence of Mucl6-CD3 engager for 24h, 48h and 72h. Percentagespecific iNKT cells activation was determined using flow cytometry. Cells were acquired by FACS Canto and analyzed in FlowJo. (FIGURE 3)Mucl6-CD3 mediated activation - comparison of iNKT, freshly isolated T cells and expanded T cells

[0261] Assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager molecule. iNKT cells, freshly isolated T cells, and expanded T cells from a matched donor were used as effector cells. CFSE labeled target cells used were Mucl6+ve OVCAR-3 and Mucl6-ve a375 cells. Effector and target cells ware co-cultured at 5:1, 1: 1 and 1:5 E:T ratio in presence or absence of Mucl6-CD3 engager for 24h, 48h and 72h. Percentage- specific iNKT cells activation was determined using flow cytometry. Cells were acquired by FACS Canto and analyzed in Flow Jo. (FIGURE 4)Example 4: Cytotoxity of iNKT Cells and T Cell Engagers

[0262] CFSE labeled target cells A375 (A), A375-CD19 (B), C1R (C), or Raji (D) were used for the iNKT cytotoxicity assay. In brief, expanded 1st or 2nd stim iNKT cells from two donor (effector cells) were incubated with target cells at 4: 1 E:T ratio in presence or absence of CD19-CD3 engager or HIV-CD3 for 48 h in Xuri based medium with 100 lU / ml IL-2. HIV-CD3 targets the CD4-binding site of the HIV envelope glycoprotein (heavy and light chain of VRC01) used as negative control here. Percentage- specific lysis of the target cells and iNKT cells activation was determined using flow cytometry. Cells were harvested following the incubation period, stained with a live dead dye and activation markers. Cells were acquired by FACSCanto and analyzed in FlowJo. (FIGURE 5)

[0263] B7-H3 engager mediates cytotoxicity to B7-H3-positive tumor cells in vitro.CFSE labeled target cells Hs 683 (A), A375 (B), U-87 MG (C) or A549 (D) cells were used for the iNKT cytotoxicity assay. Expanded iNKT cells from two donor (effector cells) were incubated with target cells at 4:1 E:T ratio in presence or absence of B7-H3-CD3 engager or HIV-CD3 for 48 h in RPMI-1640 based medium with 50 lU / ml IL-2. HIV-CD3 targets the CD4-binding site of the HIV envelope glycoprotein (heavy and light chain of VRC01) used as negative control here. Percentage-specific lysis of the target cells was determined using flow cytometry. Cells were harvested following the incubation period, stained with a live dead dye and surface markers. Cells were acquired by FACSCanto and analyzed in FlowJo. (FIGURE 6)

[0264] Mucl6-CD3 mediated iNKT cytotoxicity. Assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager. iNKT cells from six donors were used as effector cells. CFSE labeled OVCAR-3 cell line was used as target cells. Effector and target cells ware co-cultured at 5:1, 1:1 and 1:5 E:T ratio in presence or absence of Mucl6-CD3 engager for 24h, 48h and 72h. Percentage- specific lysis of the target cells was determinedusing flow cytometry. Cells were acquired by FACS Canto and analyzed in FlowJo. (FIGURE 7)Example 5: Comparison of iNKT Cell Mediated Cytotoxicity to T Cell Cytotoxicity

[0265] iNKT cells showed better cytotoxicity against target cells than conventional T cell. Assay was performed in 2D adherent cell cultures using the B7-H3-CD3 engager molecule described in Example 1. Purified T cells and expanded iNKT cells used as effector cells and irradiated A375-GFP as target cells. Effector and target cells ware co-cultured at 1:1 E:T ratio in presence or absence of B7-H3-CD3 in media containing 50 lU / ml of IL-2. Cells were imaged every 6h for 120h using the Cytation 5 Cell Imaging Multi-Mode Reader (Biotek) driven by Gen5 software (Biotek). Between imaging reads, cells were incubated at 37 °C with 5% CO2 using the Biospa 8 Automated Incubator (Biotek). (FIGURE 8)

[0266] Mucl6-CD3 engager mediated killing - comparison of iNKT, freshly isolated T cells and expanded T cells. Assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager molecule. iNKT cells, freshly isolated T cells, and expanded T cells from a matched donor were used as effector cells. CFSE labeled target cells used were Mucl6+ve OVCAR-3 and Mucl6-ve a375 cells. Effector and target cells ware co-cultured at 5:1, 1:1 and 1:5 E:T ratio in presence or absence of Mucl6-CD3 engager for 24h, 48h and 72h. Percentage- specific iNKT cells killing was determined using flow cytometry. Cells were acquired by FACS Canto and analyzed in FlowJo. (FIGURE 9). FIGURE 11 compares engager-mediated killing with iNKT versus T-cells. iNKT cells from two additional donors and T cells from one additional donor were tested using the methods described above. The data shows that Mucl6-CD3 engager were able to mediate killing of target cells in a dose dependent manner by iNKT cells at 24h at all tested ratios with E:T ratio 5:1 being the most effective for iNKT cells from both doners, while T cells showed minimum killing. At 48h hour after co-culture, approximately 80% of target cells were killed by iNKT cells or T cells at 5:1 E:T ratio, and approximately 50% of target cells were killed by iNKT cells or T cells at 1:1 E:T ration. At 72 hours, both iNKT cells and T cells achieved close to 100% killing of target cells at 5:1 E:T ratio (FIG. 11 A).

[0267] Similar studies were done using iNKT cells or T cells isolated from two donors with DLL3-CD3 engagers for killing SHP333 lung cancer cell (FIGURE 11B), Her2- CD3 engagers for killing SKBR3 breast cancer cells (FIGURE 11C), or Cldnl8.2-CD3 engager for killing PATU89885 liver metastatic cancer cells (FIGURE 11D). Similar resultswere observed that the various engager mediated iNKT cell killing of the target cells faster than that of the T cells. FIGURE 12 shows a quantification of normalized cytotoxicity when iNKT cells are combined with Mucl6 engager and either patients PBMCs or T cells (e.g., to mimic the patient’s immune system), relative to PBMC or T cell monotherapy, respectively. The data shows that iNKT cells enhance CD3xMucl6 engager anti-tumor activity of patient immune cells. T / PBMCs were co-cultured with 0VCAR3 (MUC16+) cells for 24 hours at 4:1. Engagers were added at InM. Killing was assessed via flow cytometry. iNKT cells were added at 1:5 ratio to T / PBMCs (1:20 ratio to target cells). Addition of iNKT cells even at a low number led to >2x killing improvement.

[0268] Example 6: Cytokine Release by iNKT Cells and T Cells

[0269] Supernatants from iNKT cells, freshly isolated T cells and expanded T cells were co-cultured for 48h in the presence of 0.1 nM Mucl6xCD3 engager or controls without engager. Cytokine release was analyzed using Meso Scale Discovery Electrochemiluminescence (MSD). MDS uses sandwich ELISA method coupled with electro-chemiluminescence (ECL) detection and plate array technology to provide highly sensitive and multiplexed detection of analytes in a complex biological matrix. (FIGURE 10). FIGURE 13 shows cytokine release by iNKT cells upon engager- mediated killing. Supernatants from iNKT cells co-cultured for 24h in the presence of 0.1 nM Mucl6xCD3 or DLL3xCD3 engager were collected. Cytokine release was analyzed using Proteome Profiler Antibody Arrays. This semiquantitative antibody array allows for the measurement of multiple proteins in a single sample. Antibody array kits contain buffers, detection antibodies, and membranes spotted in duplicate with high quality capture antibodies. The arrays utilize chemiluminescence for detection data, which was quantified using Quick Spots image analysis software designed specifically for Proteome Profiler Arrays. The results show that iNKT cells during engager-mediated killing secrete cytokines promoting activation, cytotoxicity and immunomodulation.

Claims

CLAIMS1. A pharmaceutical composition comprising NKT cells, T cell engagers, and a pharmaceutically acceptable carrier or excipient, wherein said T cell engagers comprises a CD3 binding domain and a tumor antigen binding domain.

2. The pharmaceutical composition of claim 1, wherein the NKT cells are allogeneic NKT cells that have been isolated from cells of one or more healthy human donors.

3. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition comprises 107to 1012NKT cells.

4. The pharmaceutical composition of claim 1, wherein the NKT cells are engineered NKT cells.

5. The pharmaceutical composition of claim 4, wherein the NKT cells expresses a chimeric antigen receptor (CAR).

6. The pharmaceutical composition of claim 4, wherein the NKT cells express an exogenous T cell receptor (TCR).

7. The pharmaceutical composition of any one of claims 4-6, wherein the NKT cells express an armor molecule.

8. The pharmaceutical composition of claim 7, where in the armor molecule is IL- 15.

9. The pharmaceutical composition of any of claims 1-8, wherein the NKT cells are human iNKT cells.

10. The pharmaceutical composition of claim 9, wherein at least 90% of the human iNKT cells are from a single healthy human donor.

11. The pharmaceutical composition of any one of claim 9 or 10, wherein the human iNKT cells comprise CD8+ iNKT cells.

12. The pharmaceutical composition of any one of claims 1-11, wherein a portion of the NKT cells in the composition are complexed to the T cell engager.

13. The pharmaceutical composition of any one of claims 1-12, wherein the CD3 binding domain binds CD3s.

14. The pharmaceutical composition of claim 13, wherein the CD3 binding domain binds human CD3s.

15. The pharmaceutical composition of any one of claims 1-14, wherein the CD3 binding domains binds to human CD3 and cynomolgus monkey CD3.

16. The pharmaceutical composition of any one of claims 1-15, wherein the CD3 binding domain binds to amino acid sequence consisting of FSEXE (SEQ ID NO: 37) and / or QDGNE (SEQ ID NO: 38) of CD3s.

17. The pharmaceutical composition of any one of claims 1-16, wherein the CD3 binding domain comprises a variable heavy chain or binding fragment thereof and / or a variable light chain or a binding fragment thereof.

18. The pharmaceutical composition of any one of claims 1-17, wherein the CD3 binding domain comprises a variable heavy chain or binding fragment thereof and variable light chain or a binding fragment thereof.

19. The pharmaceutical composition of claim 17 or 18, wherein the variable heavy chain or binding fragment thereof comprises a VH CDR1, VH CDR2, VHCDR3 and wherein the variable light chain or binding fragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3.

20. The pharmaceutical composition of claim 19, wherein the VH CDR1, VH CDR2, VH CDR3, and VL CDR1, VL CDR2, and VL CDR3 are complementarity-determining regions of an anti-CD3 antibody selected from the group consisting of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, or I2C or a an anti-CD3 antibody that specifically binds to the same epitope as HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C.

21. The pharmaceutical composition of claim 20, wherein the variable heavy chain or binding fragment thereof comprises an amino acid sequence at least 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical to the amino acid sequences of the variable heavy chain of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C.

22. The pharmaceutical composition of claim 20 or 21, wherein the variable light chain or binding fragment thereof comprises an amino acid sequence at least 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical to the amino acid sequences of the variable light chain of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C.

23. The pharmaceutical composition of any one of claims 1-22, wherein the CD3 binding domain comprises a variable heavy chain and a variable light chain, and wherein the variable heavy chain and variable light chain comprise the amino acid sequence of the variable heavy chain and variable light chain of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C.

24. The pharmaceutical composition of any one of claims 17-23, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:18.

25. The pharmaceutical composition of any one of claims 17-24, wherein the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:17.

26. The pharmaceutical composition of any one of claims 17-25, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 18 and the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 17.

27. The pharmaceutical composition of claim 19, wherein the VH CDR1, VH CDR2, VHCDR3 comprise an amino acid sequence as set forth in SEQ ID NO:22, SEQ ID NO:23, andSEQ ID NO:24, respectively, and wherein the variable light chain or binding fragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprise an amino acid sequence as set forth in SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

28. The pharmaceutical composition of claim 27, wherein variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 18, and variable light chain or binding fragment thereof comprises the amino acid set forth in SEQ ID NO: 17.

29. The pharmaceutical composition of claim 19, wherein the VH CDR1, VH CDR2, VH CDR3 comprise an amino acid sequence as set forth in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, respectively, and wherein the variable light chain or binding fragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprise an amino acid sequence as set forth in SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

30. The pharmaceutical composition of claim 29, wherein variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:25, and variable light chain or binding fragment thereof comprises the amino acid set forth in SEQ ID NO: 17.

31. The pharmaceutical composition of any one of claims 1-30, wherein the CD3 binding domain is a single chain fragment variable (scFv) domain.

32. The pharmaceutical composition of any one of claims 1-30, wherein the CD3 binding domain is a fragment antigen binding (Fab) domain.

33. The pharmaceutical composition of any of claims 1-32, wherein the CD3 binding domain comprises an immunoglobulin.

34. The pharmaceutical composition of any one of claims 1-33, wherein tumor antigen binding domain specifically binds a tumor cell surface antigen selected from the group consisting of HER2, HER3, EGFR / HER1, CD19, MUC16, B7-H3, BCMA, FAP, DLL3, PSMA, P-cadherin (CDH3), mesothelin (MSLN), claudin 6, CD 123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule-1 (CLL-1), WT1, GPRC5D, CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO, GD2, MUC1, CD22, FLT3, MUC17, CEACAM5, Claudinl8.2, and NKG2D ligand.

35. The pharmaceutical composition of any one of claims 1-34, wherein the tumor antigen binding domain comprises a variable heavy chain or binding fragment thereof and / or a variable light chain or a binding fragment thereof.

36. The pharmaceutical composition of any one of claims 1-35, wherein the tumor antigen binding domain comprises a variable heavy chain or binding fragment thereof and variable light chain or a binding fragment thereof.

37. The pharmaceutical composition of claim 35 or 36, wherein the variable heavy chain or binding fragment thereof comprises a VH CDR1, VH CDR2, VHCDR3 and wherein the variable light chain or binding fragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3.

38. The pharmaceutical composition of claim 37, wherein the VH CDR1, VH CDR2, VH CDR3 comprise an amino acid sequence as set forth in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively, and wherein the variable light chain or binding fragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprise an amino acid sequence as set forth in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

39. The pharmaceutical composition of any one of claims 35-38, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:1.

40. The pharmaceutical composition of any one of claims 35-39, wherein the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:2.

41. The pharmaceutical composition of any one of claims 35-40, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:1 and the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:2.

42. The pharmaceutical composition of any one of claims 38-41, wherein the tumor antigen binding domain specifically binds human CD 19.

43. The pharmaceutical composition of any one of claim 35-37, wherein the VH CDR1, VH CDR2, VH CDR3 comprise the amino acid sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and wherein the variable light chain or binding fragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprise the amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

44. The pharmaceutical composition of claim 43, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:9.

45. The pharmaceutical composition of claim 43 or 44, wherein the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 10.

46. The pharmaceutical composition of any one of claims 43-45, wherein the variable heavy chain or binding fragment thereof comprises an amino acid as set forth in SEQ ID NO:9 and the variable light chain or binding fragment thereof comprises an amino acid as set forth in SEQ ID NO: 10.

47. The pharmaceutical composition of any one of claims 43-46, wherein the tumor antigen binding domain specifically binds human B7-H3.

48. The pharmaceutical composition of any one of claim 35-37, wherein the VH CDR1, VH CDR2, VH CDR3 comprise the amino acid sequence as set forth in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively, and wherein the variable light chain or binding fragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprise the amino acid sequence as set forth in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively.

49. The pharmaceutical composition of claim 48, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:SEQ ID NO:29.

50. The pharmaceutical composition of claim 48 or 49, wherein the variable light chain or binding fragment thereof comprises an amino acid as set forth in SEQ ID NO:33.

51. The pharmaceutical composition of any one of claims 48-50, wherein the variable heavy chain or binding fragment thereof comprises an amino acid as set forth in SEQ ID NO:29 and the variable light chain or binding fragment thereof comprises an amino acid as set forth in SEQ ID NO:33.

52. The pharmaceutical composition of any one of claims 48-51, wherein the tumor antigen binding domain specifically binds human MUC16.

53. The pharmaceutical composition of any one of claims 1-52, wherein the tumor antigen binding domain is an scFv, Fab, or immunoglobulin.

54. The pharmaceutical composition of any one of claims 1-53, wherein the T cell engager comprises a constant region or Fc region.

55. The pharmaceutical composition of claim 54, wherein the constant region or Fc region is an IgGl, IgG2, IgG3, or IgG4 Fc region.

56. The pharmaceutical composition of claim 54 or 55, wherein the constant region or Fc region contains one or more mutations as compared to an IgGl, IgG2, IgG3, or IgG4 wildtype constant region or Fc region.

57. The pharmaceutical composition of claim 56, wherein the mutated constant region or Fc region comprises one or more mutations that reduce or eliminate FcyR binding.

58. The pharmaceutical composition of claim 56 or 57, wherein the mutated constant region or Fc region comprises a N297A mutation.

59. The pharmaceutical composition of claim 56 or 57, wherein the mutated constant region or Fc region comprises N297A and K322A mutations.

60. The pharmaceutical composition of claim 56 or 57, wherein the mutated constant region or Fc region comprises E233P, F234V, L235A, and G236del mutations.

61. The pharmaceutical composition of claim of claim 56 or 57, wherein the mutated constant region or Fc region comprises a mutation at one or more amino acids selected from the group consisting of glutamate at position 233(E233), leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), asparagine at position 297 (N297) glutamate at position 318 (E318), lysine at position 320 (K320), and lysine at position 322 (K322).

62. The pharmaceutical composition of any one of claims 1-61, wherein the T cell engager is a heterodimer.

63. The pharmaceutical composition of any one of claims 1-61, wherein the T cell engager is a homodimer.

64. The pharmaceutical composition of any one of claims 1-63, wherein the T cell engager comprises a monovalent, bivalent, or trivalent CD3 binding domain.

65. The pharmaceutical composition of any one of claims 1-64, wherein the T cell engager comprises a monovalent, bivalent, or trivalent tumor antigen domain.

66. The pharmaceutical composition of any one of claims 1-62, wherein the T cell engager is a bispecific antibody.

67. The pharmaceutical composition of any one of claims 1-62, wherein the T cell engager is a multi- specific antibody.

68. The pharmaceutical composition of any one of claims 1-67, wherein the T cell engager is an antibody in a format selected from the group consisting of knobs-into-holes, knobs-into holes with cognate light chain, knobs-into-holes with common light chain, scFv- Ig, BiTE, BiTE-Fc, DART, DART-Fc, Tetravalent DART, Diabody, BiKE, TriKE, scFv- scFv-scFv, TandAb, TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 CrossMab, DVD-Ig, IgG-IgG, and Fab-scFv-Fc.

69. The pharmaceutical composition of claim 68, wherein the T cell engager is a knobs- into-holes bispecific antibody comprising a CD3 binding domain that binds CD3s and the tumor antigen binding domain comprises a variable heavy chain and a variable light chain that specifically binds human Claudin 18.2, CD19, B7-H3, or MUC16.

70. The pharmaceutical composition of any one of claims 11-69, wherein the composition further comprise an iNKT activator.

71. The pharmaceutical composition of claim 70, wherein the iNKT activator comprises a-galactosylceramide (a-GalCer) or a derivative of a-GalCer.

72. The pharmaceutical composition of any one of claims 11-71, wherein the composition comprises a therapeutically effective amount of iNKT cells and a therapeutically effective amount of T cell engager.

73. A kit comprising a container comprising the pharmaceutical composition of any one of claims 1-72.

74. A kit comprising the pharmaceutical composition of any one of claims 1-72 and written information on indications and usage.

75. Use of NKT cells and a T cell engager, such T cell engager comprising a CD3 binding domain and a tumor antigen binding domain, for the manufacture of a medicament for the treatment of a medical condition associated with the over-expression of a tumor antigen that specifically binds the tumor antigen domain of the T cell engager.

76. Use of NKT cells and a T cell engager, such T cell engager comprising a CD3 binding domain and a tumor antigen binding domain, for the manufacture of a medicament for the treatment of cancer.

77. A method of treating a subject suffering from a disease or condition associated with the over-expression of a tumor antigen, comprising administering to the subject a therapeutically effective dose of the pharmaceutical composition of claims 1-72.

78. A method of treating a subject with cancer comprising administering to the subject in need a pharmaceutical composition of any one of claims 1-72.

79. A method of treating a subject with cancer comprising administering to the subject a therapeutically effective amount of NKT cells and a T cell engager, wherein said T cell engager comprises a tumor antigen binding domain and a CD3 binding domain.

80. The method of claim 79, wherein the NKT cells and T cell engager are administered to the subject as a pharmaceutical composition comprising the NKT cells, the T cell engager, and a pharmaceutically acceptable carrier.

81. The method of claim 80, wherein said NKT cells are complexed to T cell engager.

82. The method of claims 80 or 81, wherein (i) the NKT cells are administered to the subject in an iNKT pharmaceutical composition comprising NKT cells and a pharmaceutically acceptable carrier or excipient, and (ii) the T cell engager is administered to the subject in a T cell engager pharmaceutical composition comprising the T cell engager and a pharmaceutically acceptable carrier or excipient.

83. The method of any one of claims 79-82, wherein the NKT cells and the T cell engager are administered to the subject at the same time.

84. The method of any one of claims 79-82, wherein the NKT cells and T cell engager are administered sequentially to the subject.

85. The method of claim 84, wherein the NKT cells and the T cell engager are administered on different days.

86. The method of claim 84 or 85, wherein the NKT cells are administered before the T cell engager.

87. The method of claim 81 or claim 84, wherein the T cell engager is administered before the NKT cells.

88. The method of any one of claims 84-87, wherein the NKT cells are administered on day 1 and multiple doses of T cell engager are administered on subsequent days.

89. The method of any one of claims 79-89, wherein the NKT cells are iNKT cells.

90. The method of claim 86, wherein the iNKT cells are allogeneic iNKT cells.

91. The method of claim 90, wherein the allogeneic iNKT cells comprise expanded iNKT cells obtained from donor PBMCs, leukopak, peripheral blood, or bone marrow.

92. The method of any one of claims 79-91, wherein the NKT cells express a chimeric antigen receptor.

93. The method of any one of claims 79-91, wherein the cells express an endogenous T cell receptor.

94. The method of any one of claims 79-93, wherein the cells express an armoring molecule.

95. The method of claim 94, wherein the armoring molecule is IL- 15 or soluble IL- 15.

96. The method of any one of claims 79-95, wherein a lymphodepletion agent is not administered to the subject prior to or concurrent with the administration of the NKT cells and T cell engager.

97. The method of claim 96, wherein bendamustine, fludarabine, and cyclophosphamide are not administered to the subject prior to or concurrent with the administration of the NKT cells and T cell engager.

98. The method of any one of claims 79-97, wherein the CD3 binding domain binds CD3s.

99. The method of any one of claims 79-98, wherein the CD3 binding domain binds human CD3s.

100. The method of any one of claims 79-99, wherein the CD3 binding domains binds to human CD3 and cynomolgus monkey CD3.

101. The method of any one of claims 79-100, wherein the CD3 binding domain comprises a variable heavy chain or binding fragment thereof and / or a variable light chain or a binding fragment thereof.

102. The method of any one of claims 79-101, wherein the CD3 binding domain comprises a variable heavy chain or binding fragment thereof and a variable light chain or a binding fragment thereof.

103. The method of claim 101 or 102, wherein the variable heavy chain or binding fragment thereof comprises a VH CDR1, VH CDR2, and VHCDR3.

104. The method of any one of claims 101-103, wherein the variable light chain or binding fragment thereof comprises a VL CDR1, VL CDR2, and VL CRD3.

105. The method of any one of claims 76-104, wherein the CD3 binding domain binds to epitopes FSEXE (SEQ ID NO: 37) and QDGNE (SEQ ID NO: 38) of CD3s.

106. The method of any one of claims 103-105, wherein the VH CDR1, VH CDR2, and VH CDR3 are complementarity-determining regions of an anti-CD3 antibody selected from the group consisting of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C or a an anti-CD3 antibody that specifically binds to the same epitope as HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C.

107. The method of any one of claims 103-106, wherein the VL CDR1, VL CDR2, and VL CDR3 are complementarity-determining regions of an anti-CD3 antibody selected from the group consisting of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C or a an anti-CD3 antibody that specifically binds to the same epitope as HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C.

108. The method of any one of claims 103-107, wherein the VH CDR1, VH CDR2, and VH CDR3 and VL CDR1, VL CDR2 and VL CDR 3 are complementarity-determining regions of an anti-CD3 antibody selected from the group consisting of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, or I2C or a an anti-CD3 antibody that specifically binds to the same epitope as HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C.

109. The method of any one of claims 104-108, wherein the variable heavy chain or binding fragment thereof comprises an amino acid sequence at least 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical to the amino acid sequences of the variable heavy chain of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C.

110. The method of any one of claims 101-109, wherein the variable light chain or binding fragment thereof comprises an amino acid sequence at least 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical to the amino acid sequences of the variable light chain of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C.

111. The method of any one of claims 79-110, wherein the CD3 binding domain comprises a variable heavy chain and a variable light chain, and wherein the variable heavy chain and variable light chain comprise an amino acid sequence of the variable heavy chain and variable light chain of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K or I2C.

112. The method of claim 111, wherein the VH CDR1, VH CDR2, VH CDR3 comprise an amino acid sequence as set forth in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively, and the VL CDR1, a VL CDR2, and a VL CDR3 comprise an amino acid sequence as set forth in SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

113. The methods of claim 112, wherein variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 18.

114. The method of any one of claims 111-113, wherein the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 17.

115. The method of any one of claims 111-114, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 18 and the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 17.

116. The method of claim 111, wherein the VH CDR1, VH CDR2, VH CDR3 comprise an amino acid sequence as set forth in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, respectively, and the VL CDR1, a VL CDR2, and a VL CDR3 comprise an amino acid sequence as set forth in SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

117. The method of claim 116, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:25.

118. The method of claim 116 or 117, wherein the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 17.

119. The method of any one of claims 116-118, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:25 and the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 17.

120. The method of any one of claims 79-119, wherein the CD3 binding domain comprises an scFv domain.

121. The method of any one of claims 79-119, wherein the CD3 binding domain comprises a Fab domain.

122. The method of any one of claims 79-119, wherein the CD3 binding domain comprises an immunoglobulin.

123. The method of any one of claims 79-122, wherein tumor antigen binding domain specifically binds a tumor cell surface antigen selected from the group consisting of HER2, HER3, EGFR / HER1, CD19, MUC16, B7-H3, BCMA, FAP, DLL3, PSMA, P-cadherin (CDH3), mesothelin (MSLN), claudin 6, CD123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule-1 (CLL-1), WT1, GPRC5D, CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO, GD2, MUC1, CD22, FLT3, MUC17, CEACAM5, Claudin 18.2, and NKG2D ligand.

124. The method of any one of claims 79-123, wherein the tumor antigen binding domain comprises a variable heavy chain or binding fragment thereof and / or a variable light chain or a binding fragment thereof.

125. The method of any one of claims 79-124, wherein the tumor antigen binding domain comprises a variable heavy chain or binding fragment thereof and a variable light chain or a binding fragment thereof.

126. The method of claim 124 or 125, wherein the variable heavy chain or binding fragment thereof comprises a VH CDR1, VH CDR2, and VHCDR3.

127. The method of claim any one of claims 123-126, wherein the variable light chain or binding fragment thereof comprises a VL CDR1, a VL CDR2, and a VL CDR3.

128. The method of claim 126 or 127, wherein the VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequence as set forth in SEQ ID NOG, SEQ ID NO:4, and SEQ ID NOG, respectively, and the VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequence as set forth in SEQ ID NOG, SEQ ID NOG, and SEQ ID NO:8, respectively.

129. The method claim 128, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 1.

130. The method of claim 128 or 129, wherein the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NOG.

131. The method of any one of claims 128-130, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:1 and the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:2.

132. The method of any one of claims 128-131, wherein the tumor antigen binding domain specifically binds human CD 19.

133. The method of claim 132, wherein the tumor antigen binding domain comprises blinatumomab.

134. The method of claims 126 or 127, wherein the VH CDR1, VH CDR2, and VH CDR3 comprise an amino acid sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and wherein the VL CDR1, VL CDR2, and VL CDR3 comprise an amino acid sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

135. The method of claim 134, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:9.

136. The method of claim 134 or 135, wherein the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 10.

137. The method of any one of claims 134-136, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:9 and the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO: 10.

138. The method of any one of claims 134-137, wherein the tumor antigen binding domain specifically binds human B7-H3.

139. The method of any one of claims 134-138, wherein the tumor antigen binding domain comprises enoblituzumab.

140. The method of claim 126 or 127, wherein VH CDR1, VH CDR2, and VH CDR3 comprise an amino acid sequence as set forth in SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:32, respectively, and wherein the VL CDR1, VL CDR2, and VL CDR3 comprise an amino acid sequence as set forth in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively.

141. The method of claim 140, wherein the variable heavy chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:29.

142. The method of claim 140 or 141, wherein the variable light chain or binding fragment thereof comprises the amino acid as set forth in SEQ ID NO:33.

143. The method of any one of claims 140-142, wherein the tumor antigen binding domain specifically binds human MUC16.

144. The method of any one of claims 79-143, wherein the tumor antigen binding domains comprises an scFv domain, a Fab domain, or an immunoglobulin.

145. The method of any one of claims 79-144, wherein the T cell engager comprises a constant region or Fc region.

146. The method of claim 145, wherein the constant region or Fc region is an IgGl, IgG2, IgG3, or IgG4 Fc region.

147. The method of claim 145 or 146, wherein the constant region or Fc region contains one or more mutations as compared to an IgGl, IgG2, IgG3, or IgG4 wild-type constant region or Fc region.

148. The method of claim 147, wherein the mutated constant region or Fc region comprises one or more mutations that reduce or eliminate FcyR binding.

149. The method of claim 147 or 148, wherein the mutated constant region or Fc region comprises a N297A mutation.

150. The method of claim 147 or 148, wherein the mutated constant region or Fc region comprises N297A and K322A mutations.

151. The method of claim 147 or 148, wherein the mutated constant region or Fc region comprises E233P, F234V, L235A, and G236del mutations.

152. The method of claim of claim 147 or 148, wherein the mutated constant region or Fc region comprises a mutation at one or more amino acids selected from the group consisting of glutamate at position 233(E233), leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), asparagine at position 297 (N297) glutamate at position 318 (E318), lysine at position 320 (K320), and lysine at position 322 (K322).

153. The method of any one of claims 79-152, wherein the T cell engager is a heterodimer.

154. The method of any one of claims 79-152, wherein the T cell engager is a homodimer.

155. The method of any one of claims 79-152, wherein the T cell engager comprises a monovalent, bivalent, or trivalent CD3 binding domain.

156. The method of any one of claims 79-152, wherein the T cell engager comprises a monovalent, bivalent, or trivalent tumor antigen domain.

157. The method of any one of claims 79-156, wherein the T cell engager is a bispecific antibody.

158. The method of any one of claims 79-157, wherein the T cell engager is a multispecific antibody.

159. The method of any one of claims 79-158, wherein the T cell engager is an antibody in a format selected from the group consisting of knobs-into-holes, knobs-into holes with cognate light chain, knobs-into-holes with common light chain, scFv-Ig, BiTE, BiTE-Fc, DART, DART-Fc, Tetravalent DART, Diabody, BiKE, TriKE, scFv-scFv-scFv, TandAb, TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 CrossMab, DVD-Ig, IgG- IgG, and Fab-scFv-Fc.

160. The method of any one of claims 79-159, wherein the composition further comprises an iNKT activator.

161. The method of claim 160, wherein the iNKT activator comprises a- galactosylceramide (a-GalCer) or a derivative of a-GalCer.

162. The method of any one of claims 79-161, further comprising administering to the subject a PD-1 inhibitor, a PD-L1 inhibitor, CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T cell co-inhibitor or ligand, an indoleamine-2, 3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFp) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody to a tumor- specific antigen, a cancer vaccine, an adjuvant to increase antigen presentation, a cytotoxin, a poly ADP-ribose polymerase (PARP) inhibitor, a chemotherapeutic agent, cyclophosphamide, radiotherapy, an IL-6R inhibitor, an IL-4R inhibitor, an IL- 10 inhibitor, a cytokine, and IL- 15.

163. The method of any one of claims 79-162, wherein the cancer is diffuse large B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, mucosa-associated lymphatic tissue lymphoma, small lymphocytic lymphoma (also known as chronic lymphocytic leukemia, CLL), mantle cell lymphoma, primary mediastinal (thymic) large B cell lymphoma, T cell / histiocyte-rich large B-cell lymphoma, primary cutaneous diffuse large B-cell lymphoma, EBV positive diffuse large B-cell lymphoma, burkitt's lymphoma, lymphoplasmacytic lymphoma, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, central nervous system lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, or large B-cell lymphoma. In some embodiments, the cancer comprises a blood cancer. In some embodiments, the blood cancer comprises myeloma, leukemia, lymphoma, Non-Hodgkin lymphoma, Hodgkin lymphoma, a myeloid neoplasm, a lymphoid neoplasm, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myelogenous leukemia (CML).

164. The method of any one of claims 79-162, wherein the cancer is epithelial cancer, (e.g., breast, gastrointestinal, lung), prostate cancer, bladder cancer, lung (e.g., small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, esophageal cancer, head and neck cancer, or a colorectal cancer. In some embodiments, the cancer to be treated or antigen is from one of the following cancers: adenocortical carcinoma, agnogenic myeloid metaplasia, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, meningiosarcoma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing's family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, ovarian cancer, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), pulmonarylymphangiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., nonmelanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), or Meigs' syndrome.

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