Antigen-specific t cell compositions and methods of making the same

Abstract

Provided are antigen-specific T cell compositions of making the same. In some embodiments, the present technology comprises T cell compositions that are expresses to one or more of CD2, CD3, or CD28. In some embodiments, the T cell compositions of the present technology are donor derived. In some embodiments, the T cell compositions are generated by the steps of (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

Description

Attorney Docket No. 184P-413376-WOANTIGEN-SPECIFIC T CELL COMPOSITIONS AND METHODS OF MAKING THE SAMECROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 713,539, filed October 29, 2024, the contents of which are incorporated herein by reference in their entirety.BACKGROUND

[0002] Immunotherapy aims to leverage the immune system to alter the course of diseases such as cancer. Cancer immunotherapy has evolved in various directions, including vaccines that boost immune responses against tumor antigens, ex vivo expansion of autologous cytotoxic T-cells with or without engineered receptors for tumor recognition to release inhibitory receptor-ligand interactions on immune cells. In vitro preparation of T cells for adoptive cell transfer (ACT) of cancer allows for the selection of cells with high-avidity recognition of the tumor, expansion of tumor-specific T cells away from the suppressive properties of the tumor microenvironment, and manipulation of the T-cells to enhance their anti-tumor activity, such as the introduction of endogenous TCR or chimeric antigen receptors (CARs). ACT also provides the ability to manipulate the host microenvironment to increase favorable conditions for recruiting transferred T-cells.

[0003] Although ACT with autologous tumor-infiltrating lymphocytes (TILs) has been successful in metastatic melanoma, the overall success rate remains low. Despite the advancements, there remains a significant gap in the field, where some ACT therapies for solid tumors may fail to discriminate healthy cells from malignant cells. As such, immunotherapies having enhanced efficacy and cancer antigen recognition are needed.SUMMARY

[0004] The present technology comprises antigen-specific T cell compositions.Attorney Docket No. 184P-413376-WO

[0005] In some embodiments, the present technology comprises T cell compositions generated by the steps of (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0006] In some embodiments, the present technology comprises T cell compositions generated by the steps of (a) obtaining a T cell population from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0007] In some embodiments, the present technology comprises methods of generating T cell compositions comprising one or more receptors specific to at least a portion of one or more cancer antigens, the methods comprising (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti- CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0008] In some embodiments, the present technology comprises methods of generating T cell compositions comprising one or more receptors specific to at least a portion of one or more cancer antigens, the methods comprising (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines comprising or consisting of IL-2; (c) culturing the T cell population with one or more antibodies comprising or consisting of (i) anti-CD3 antibody, (ii) an anti-CD3 antibody and an anti-CD28 antibody, or (iii) an anti-CD3 antibody and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0009] In some embodiments, the present technology comprises methods of generating T cell compositions comprising one or more receptors specific to at least a portion of one or more cancer antigens, the methods comprising (a) isolating a T cell populationAttorney Docket No. 184P-413376-WO from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti- CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0010] In some embodiments, the present technology comprises methods of generating T cell compositions comprising one or more receptors specific to at least a portion of one or more cancer antigens, the methods comprising (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines comprising or consisting of IL-2; (c) culturing the T cell population with one or more antibodies comprising or consisting of (i) anti-CD3 antibody, (ii) an anti-CD3 antibody and an anti-CD28 antibody, (iii) an anti-CD3 antibody and an anti-CD2 antibody, or (iv) an anti-CD3 antibody, an anti- CD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0011] In some embodiments, isolating the T cell population comprises obtaining a T cell population.

[0012] In some embodiments, (a) comprises or consists of purifying or selecting for a T cell population.

[0013] In some embodiments, (a) comprises purifying or selecting for a single cell suspension.

[0014] In some embodiments, the single cell suspension is purified from a tumor or a tumor-draining lymph node.

[0015] In some embodiments, (b) is optional.

[0016] In some embodiments, (c) and (d) are optional.

[0017] In some embodiments, the T cell population is an autologous T cell population.

[0018] In some embodiments, the T cell population is an allogenic T cell population.

[0019] In some embodiments, the one or more cytokines comprise or consist of IL-2,IL-4, IL-7, IL-15, IL-12, and IL-18.Attorney Docket No. 184P-413376-WO

[0020] In some embodiments, the period of time comprises at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 28 days, 30 days, 35 days, or 40 days.

[0021] In some embodiments, (b) and (c) occur simultaneously.

[0022] In some embodiments, the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody.

[0023] In some embodiments, the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody and an anti-CD28 antibody.

[0024] In some embodiments, the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody and an anti-CD2 antibody.

[0025] In some embodiments, the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody, an anti-CD2 antibody, and an anti-CD23 antibody.

[0026] In some embodiments, the anti-CD3 antibody is selected from the group consisting of muromonab, teplizumab, otelixizumab, visilizumab, foralumab, and UCHT1.

[0027] In some embodiments, the anti-CD28 antibody is theralizumab.

[0028] In some embodiments, the T cell composition has an increase in one or more gene expression levels selected from the group consisting of a perforin-1 (PRF1 ) gene expression level, a granzyme B (GZMB) gene expression level, an interferon gamma (INFy) gene expression level, a tumor necrosis factor alpha (TNFa) gene expression level, a human leukocyte antigen class II histocompatibility antigen, DR alpha chain (HLA-DRA) gene expression level, a CD25 gene expression level, a CD69 gene expression level, and an IL- 2 gene expression level, relative to a control.

[0029] In some embodiments, the T cell composition has an increase in one or more protein levels selected from the group consisting of a PRF1 protein level, a GZMB protein level, an INFy protein level, a TNFa protein level, an HLD-DRA protein level, a CD25 protein level, a OD69 protein level, and an IL-2 protein level, relative to a control.Attorney Docket No. 184P-413376-WO

[0030] In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the T cells in the T cell composition are specific to the one or more cancer antigens.

[0031] In some embodiments, the T cell composition comprises T cells that are derived from a subject having a cancer.

[0032] In some embodiments, the T cells that are derived from a subject having cancer comprises T cells that were isolated or obtained from the subject having cancer.

[0033] In some embodiments, the cancer comprises a solid tumor.

[0034] In some embodiments, the cancer comprises a metastatic cancer.

[0035] In some embodiments, the cancer is a breast cancer.

[0036] In some embodiments, the breast cancer is a HER2 positive breast cancer.

[0037] In some embodiments, the breast cancer is a HER2 negative breast cancer.

[0038] In some embodiments, the breast cancer is a HER2 / neu-negative breast cancer.

[0039] In some embodiments, the breast cancer is a triple negative breast cancer.

[0040] In some embodiments, the breast cancer is selected from the group consisting of a stage I breast cancer, a stage II breast cancer, a stage III breast cancer, and a stage IV breast cancer.

[0041] In some embodiments, the T cell composition is formulated for subcutaneous, intravenous, intramuscular, intracranial, intra-arterial, intraventricular, intranodal, intratumor, or intraperitoneal administration.

[0042] In some embodiments, the T cell composition is formulated for subcutaneous, intravenous, intramuscular, intracranial, intra-arterial, intraventricular, intranodal, intratumor, or intraperitoneal administration.

[0043] In some embodiments, one or more of the T cell compositions is formulated in a vaccine.Attorney Docket No. 184P-413376-WO

[0044] In some embodiments, one or more of the T cell compositions is formulated in a nanoparticle.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG.1 illustrates an exemplary dosing scheme of a T cell composition (ACT) in mice in accordance with the methods of the present technology. TDLNs: tumor-draining lymph nodes.

[0046] FIGS. 2A -2C illustrate the change in tumor volume in mice inoculated with 4T 1 mammary carcinoma tumor that were administered a T cell composition (ACT), in accordance with the methods of the present technology.

[0047] FIG. 3 illustrates survival rates in the mice of FIGS. 2A-2C (N= 8 groups, 12 mice per group).

[0048] FIGS. 4A and 4B illustrate change in tumor volume in mice administered T cell compositions that were cultured with an anti-CD2 antibody and anti-CD3 antibody (CD3 / CD2), an anti-CD3 antibody and an anti-CD28 antibody (CD3 / CD28), or an anti-CD3 antibody alone (CD3), relative to controls. 2-WAY ANOVA, Turkey multiple comparisons; * 0.05, ** 0.001 , *** <0.0001 ; mean ± standard deviation.

[0049] FIGS. 5A-5F illustrate in vitro killing capacity of mammary carcinoma cells (4T 1 ) and renal carcinoma cells (RENCA) and specificity of T cell compositions of the present technology.

[0050] FIG. 6 illustrates lung metastasis in untreated 4T1 -tumour bearing mice compared to non-tumor bearing control mice. Arrows indicate macrometastatic lesions on the lung ventricles.

[0051] FIGS. 7A-7C illustrate average changes in tumor volume (FIG. 7A), changes in tumor volume in individual mice (FIG. 7B), and survival fraction (FIG. 7C) in mice inoculated with B cell lymphoma cells (A20) that were administered a T cell composition (ACT) compared with controls in accordance with the methods of the present technology.Attorney Docket No. 184P-413376-WO

[0052] FIG. 7 illustrates in vitro cytotoxicity of T cell compositions (ACT) prepared with two different activation protocols (anti-CD3 alone activation and anti-CD3,CD2 activation) in A20 B cell lymphoma cells.

[0053] FIG. 8 illustrates in vitro cytotoxicity of anti-CD19 CAR T cells and mock CAR T cells in A20 B cell lymphoma cells.

[0054] FIG. 9 illustrates A20 B cell tumor volume in mice upon treatment with Mock CAR T cells and anti-CD19 CAR T cells compared with controls. Mock CAR T cells and anti-CD19 CAR T cells were transferred to mice on day 14.

[0055] FIG. 10 illustrates changes in tumor volume in mice after A20 tumor inoculation with mock CAR T cell treatment and anti-CD19 CAR T cell treatment compared with control.DETAILED DESCRIPTION

[0056] The present technology comprises cancer antigen-specific T cell compositions. The T cell compositions may be donor derived.T Cell Compositions

[0057] The present technology comprises T cell compositions that are specific to at least a portion of one or more cancer antigens. The T cell compositions may comprise one or more T cells that bind to and / or recognize at least a portion of one or more cancer antigens. The portion of the one or more cancer antigens may comprise a protein, a peptide, or an amino acid motif that is associated with a cancer cell.

[0058] In some embodiments, the T cell compositions comprise one or more T cells having one or more receptors that are specific to at least the portion of the one or more cancer antigens. Nonlimiting examples of the one or more cancer antigens include Melanoma-associated antigen 4 (MAGEA4; National Center for Biotechnology Information (NCBI) Accession: NP_001373125.1 ), Melanoma-associated antigen 1 (MAGEA1 ; NCBI Accession: NP_004979.3); New York esophageal squamous cell carcinoma 1 (NY-ESO-1 ; NCBI Accession: CAA05908.1 ), Kita-Kyushu lung cancer antigen-1 (KK-LC-1 ; NCBI Accession: NP 001017978.1 ), Preferentially Expressed Antigen in Melanoma (PRAME; NCBI Accession: CAG30435.1 ), B7-H3 (NCBI Accession: Q5ZPR3.1 ); B7-H4 (NCBIAttorney Docket No. 184P-413376-WOAccession(s): AAZ17406.1 ; Q7Z7D3.1 ), Cluster of Differentiation 19 (0D19; NOBI Accession(s): AAB60697.1 ; AAA69966.1 ), and Cluster of Differentiation 22 (CD22; NCBI Accession(s): NP_001762.2; NP_001172028.1 ; NP_001 172029.1 ; NP_001 172030.1 ; NP_001265346.1 ), KRAS protein (NCBI Accession(s): NP_001356715; NP_001356716) including its various isoforms and mutated forms (i.e., KRAS G12D), Fibroblast Growth Factor Receptor 3 (NCBI Accession(s): XP_054205236) including its various isoforms and mutated forms (i.e., FGFR3 Y373C), Melanoma Antigen Recognized by T cells (MART-1 ) (NCBI Accession(s): NP_005502), and Glycoprotein 100 (gp100) (NCBI Accession(s): AAC60634).

[0059] In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the T cells in the T cell composition are specific to the one or more cancer antigens.

[0060] In some embodiments, about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the T cells in the T cell composition are specific to the one or more cancer antigens.

[0061] In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the T cells in the T cell composition are specific to the one or more cancer antigens.

[0062] The T cell compositions may comprise an increase in one or more gene expression levels relative to a control. In some embodiments, the T cell compositions comprise an increase in one or more gene expression levels selected from the group consisting of a perforin-1 (PRF1 ; NCBI Gene ID: 5551 ) gene expression level, a granzyme B (GZMB; NCBI Gene ID: 3002) gene expression level, an interferon gamma (INFy; NCBI Gene ID: 3458) gene expression level, a tumor necrosis factor (TNF; NCBI Gene ID: 7124) gene expression level, a human leukocyte antigen class II histocompatibility antigen, DR alpha chain (HLA-DRA; NCBI Gene ID: 3122) gene expression level, a CD25 (NCBI Gene ID: 3559) gene expression level, a CD69 level (NCBI Gene ID: 969) gene expression, and an interleukin-2 (IL-2; NCBI Gene ID: 3558) gene expression level, relative to a control.Attorney Docket No. 184P-413376-WO

[0063] The T cell compositions may comprise an increase in one or more protein levels, relative to a control. In some embodiments, the T cell compositions comprise an increase in one or more protein levels selected from the group consisting of a PRF1 protein level (NCBI Accession(s): KAI4076276.1 ; KAI4076275.1 ), a GZMB protein level (NCBI Accession(s): P10144.2; NP_001332940.1 ; NP_004122.2; EAW66003.1 ; EAW66002.1 ; AAH30195.1 ), an INFy protein level (NCBI Accession(s): AAB59534.1 ; AAA53230.1 ; AAM28885.1 ), a TNFa protein level, (NCBI Accession(s): CAA78745.1 ; BAF31279.1 ; P01375.1 ; NP_000585.2), an HLA-DRA protein level (NCBI Accession(s): LIQL51 198.1 ; NP_061984.2), a CD25 protein level (NCBI Accession(s): NP_000408.1 ; NP_001295171 .1 ; NP_001295172.1 ), a CD69 protein level (NCBI Accession(s): AAH20057.1 ; NP_391988.1 ; EAW85959.1 ; EAW85956.1 ; EAW85952.1 ; EAW85951.1 ; EAW85949.1 ), and an IL-2 protein level (NCBI Accession(s): P60568.1 ; EAX05227.1 ; NP_000577.2; AAA70092.1 ; AAA59140.1 ), relative to a control.

[0064] The T cell composition may comprise T cells that are derived from a subject having a cancer. The T cells that are derived from the subject having a cancer may comprise T cells that were isolated or obtained from the subject.

[0065] The cancer may comprise a solid tumor and / or a metastatic cancer. In some embodiments, the cancer is a breast cancer (e.g., a HER2 positive breast cancer, a HER2 negative breast cancer, a HER2 / neu-negative breast cancer, or a triple negative breast cancer) or a B cell malignancy. The breast cancer may be selected from the group consisting of a stage I breast cancer, a stage II breast cancer, a stage III breast cancer, and a stage IV breast cancer. The B cell malignancy may comprise leukemia (e.g., B-cell acute lymphoblastic leukemia) or lymphoma.Methods of Generating T Cell Compositions

[0066] The T cell compositions of the present technology may be generated by one or more steps of (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody (e.g., of muromonab, teplizumab, otelixizumab, visilizumab, foralumab, and UCHT1 ), an anti-CD28 antibody (e.g., theralizumab), and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.Attorney Docket No. 184P-413376-WO

[0067] The T cell population in may comprise an autologous T cell population or an allogenic T cell population. In some embodiments, isolating the T cell population in (a) comprises obtaining a T cell population. The isolating or obtaining the T cell population may comprise purifying or selecting for a single cell suspension. The single cell suspension may be purified from a tumor or a tumor-draining lymph node.

[0068] In some embodiments, the isolating or obtaining of the T cell composition comprises isolating or obtaining T cells from peripheral blood (e.g., whole blood isolate or a peripheral blood mononuclear cell isolation). The T cells may be isolated using any means known in the art, including, but not limited to magnetic columns and / or beads (e.g., negative or positive selection) or cell sorting (e.g., fluorescence-activated cell sorting).

[0069] In some embodiments, the one or more cytokines in (b) may comprise or consist of IL-2. In some embodiments, the one or more cytokines in (b) comprise or consist of IL-2, interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 4 (IL-4), interleukin 12 (IL-12), and interleukin 18 (IL-18), or any combination thereof. In some embodiments, the culturing the T cell population with one or more cytokines in (b) is an optional step.

[0070] The one or more antibodies in (c) may comprise or consist of (i) anti-CD3 antibody, (ii) an anti-CD3 antibody and an anti-CD28 antibody, (iii) an anti-CD3 antibody and an anti-CD2 antibody, or (iv) an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody. In some embodiments, step (c) occurs simultaneously with step (b). In some embodiments, step (c) is optional.

[0071] In some embodiments, the period of time in (d) comprises about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 28 days, 30 days, 35 days, or 40 days. Step (d) may occur simultaneously to step (c), or step (d) may be optional.

[0072] In some embodiments, the period of time in (d) comprises at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 28 days, 30 days, 35 days, or 40 days. Step (d) may occur simultaneously to step (c), or step (d) may be optional.Attorney Docket No. 184P-413376-WO

[0073] In some embodiments, the period of time in (d) comprises at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 28 days, 30 days, 35 days, or 40 days. Step (d) may occur simultaneously to step (c), or step (d) may be optional.

[0074] In some embodiments, the T cell population in (c) or (d) comprises one or more pre-rapid expansion protocol (pre-REP) T cells. The pre-REP T cells may comprise a tumorinfiltrating lymphocyte (TIL) that has been dissociated from a tumor fragment.

[0075] In some embodiments, (d) comprises one or more rapid expansion protocol (REP). The REP may comprise stimulating with one or more T cells in the T cell population with one or more costimulatory agents. Nonlimiting examples of costimulatory agents include a cytokine (e.g., IL-2), an antibody (e.g., an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD2 antibody), a ligand or cancer antigen (e.g., CD40 ligand (CD40L) (NCBI Accession: NP 000065.1 ), 4-1 BB (NCBI Accession: AAA53133.1 ), 0X40 (NCBI Accession: CAE1 1757.1 ) ICOS-L (NCBI Accession(s): XP_01 1527816.1 ; XP_047296685.1 ; XP_011527818.1 ; XP_047296687.1 ; XP_047296688.1 ; XP_054180385.1 )), or one or more irradiated feeder cells.

[0076] One or more of steps (a)-(d) may comprise expanding the T cell population according to any means known in the art. In some embodiments, one or more of steps (a)- (d) are conducted according to the methods disclosed in Zhao Y et al., High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Mol Ther. 2006 Jan;13(1 ):151 -9 or Kverneland AH, et al., Adoptive cell therapy in combination with checkpoint inhibitors in ovarian cancer. Oncotarget. 2020 Jun 2;1 1 (22):2092-2105, each incorporated herein by reference in their entireties.Engineering T cells

[0077] The T cells in the T cell compositions of the present technology may be engineered to increase or decrease expression of one or more genes and / or levels of one or more proteins, relative to baseline.

[0078] In some embodiments, one or more T cells in the T cell compositions are engineered to express an exogenous T cell receptor or increase expression of anAttorney Docket No. 184P-413376-WO endogenous T cell receptor, relative to baseline. In some embodiments, the T cell receptor is a receptor specific to a cancer antigen selected from the group consisting of MAGE-A4, MAGEA1 , NY-ESO-1 , KK-LC-1 , FRAME, B7-H3, B7-H4, CD19, and CD22. In some embodiments, the T cell receptor comprises a chimeric antigen receptor (CAR) and / or a CC- chemokine receptor (COR). In some embodiments, the T cell receptor is specific to a cancer antigen disclosed in Xiao, J. et al., Expression of four cancer-testis antigens in TNBC indicating potential universal immunotherapeutic targets. J Cancer Res Clin Oncol 149, 15003-1501 1 (2023) or Wescott, Elizabeth C et al., Epithelial Expressed B7-H4 Drives Differential Immunotherapy Response in Murine and Human Breast Cancer. Cancer research communications vol. 4,4 (2024): 1 120-1134, each incorporated herein by reference in their entireties.

[0079] In some embodiments, one or more T cells in the T cell compositions are engineered to express an exogenous cytokine or to increase expression of an endogenous cytokine, relative to baseline. The cytokines may comprise any of those disclosed in Zhang L et al., Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther. 201 1 Apr;19(4):751 -9 or in Stephan MT et al., T herapeutic cell engineering with surface-conjugated synthetic nanoparticles. Nat Med. 2010 Sep;16(9):1035-41 . doi: 10.1038 / nm.2198, each incorporated herein by reference in their entireties.

[0080] One or more T cells in the T cell compositions may be engineered to reduce or remove expression of an inhibitory receptor (e.g., PD-1 ) or a transcript of a gene encoding an inhibitory receptor. In some embodiments, The T cells in the T cell compositions are engineered to reduce or remove expression of T-cell receptor a constant (TRAC) transcript or a TRAC protein level, relative to baseline.

[0081] Exemplary CA Rs:

[0082] The CARs of the present technology may comprise one or more of an antigen recognition domain, a hinge region, a transmembrane domain, and an endodomain (e.g., an intracellular signaling domain), each of which may be derived from one or more sources, including naturally occurring and synthetic sources. In some embodiments, the CARs areAttorney Docket No. 184P-413376-WO donor-derived and / or cord blood derived. In some embodiments, the CAR is a humanized CAR.

[0083] The antigen recognition domain may bind and / or specifically recognize a target antigen (e.g., a cancer antigen). The antigen recognition domain may be an extracellular antigen recognition domain. The antigen recognition domain may comprise a single-chain variable fragment (scFv) derived from one or more antibodies or antigen binding fragments thereof. In some embodiments, the antigen recognition domain may comprise or consist of at least a portion of a nanobody, a ligand (e.g., a natural ligand or a receptor portion thereof, such as NKG2D, or a PD-1 extracellular domain), or an alternative binding protein (i.e., a protein which acts as a binding agent but is not a traditional antibody). In some embodiments, the antigen recognition domain may be an anti-CD19 antibody, such as an human anti-CD19 scFv. In some embodiments, the antigen recognition domain may be an FMC63 monoclonal antibody that binds human CD19.

[0084] The hinge region may provide flexibility and / or enhance spatial arrangement between the antigen recognition domain and the transmembrane domain, relative to a control. The hinge region may be at least partially derived from an IgG, a CD8 peptide, or a CD28 peptide. The hinge region may also enhance binding of the CAR to a target antigen, relative to a control.

[0085] The transmembrane domain may promote or enhance anchoring of the CAR to the membrane of one or more T cells in the T cell composition. The transmembrane domain may be derived from a transmembrane region of an immunological protein, including those selected from the group consisting of CD3 , CD8, CD28, and CD4.

[0086] The endodomain may comprise or consist of an intracellular signaling domain. In some embodiments, the CARs comprise two or more endodomains. The endodomain may promote or stimulate one or more T cells in the T cell composition during or after cancer antigen binding. The endodomain may comprise, consist of, or be derived from an intracellular signaling domain from CD3^ (zeta chain), which may act as part of the T cell receptor complex and delivers the activation signal required for T cell function. The endodomain may also comprise one or more co-stimulatory signaling domains (such as CD2, CD28, 4-1 BB [CD137], 0X40, or IGOS) (e.g., in addition to CD3 . The co-stimulatoryAttorney Docket No. 184P-413376-WO domains may enhance an activation, survival, proliferation, and / or cytotoxic function of the T cell composition.

[0087] In some embodiments, the CAR specifically binds and / or recognizes CD19 (e.g., a CD19 antigen) or at least a portion thereof (e.g., two or more amino acids). In some embodiments, the CAR is an anti-CD19 CAR. In some embodiments, the CAR comprises or consists of a CAR of a therapy selected from the group consisting of tisagenlecleucel, axicabtagene ciloleucel, lisocabtagene maraleucel, brexucabtagene autoleucel, ciltacabtagene autoleucel, and idecabtagene vicleuce. In some embodiments, the CAR comprises or consists of a CAR of a therapy selected from the group consisting of (i) NXC- 201 , HBI0101 , IM19, CAR-GPRC5D, CDH17 / GUCY2C CAR-T, SL1904B, AIC100, SCT- 001 , GD2CAR, Senl-h19 CAR-T, LMY-920, SNC-109, t aPD1 -MSLN-CAR T Cells, CD19x22 CAR T, IL13Ra2 CAR-T, LMP1 CAR- T, IM96, FL-33, KD-025, GPC3 CAR, WL276, NKG2D CAR-T, Anti-CEA CAR-T, CD7 CAR-T, CD318-CAR, CT125B, CD19 / CD22 CAR-T, Claudin18.2-Targeted CAR, APRIL CAR-T, BCMA CAR-T, ADGRE2 CAR-T, CAIX-targeted CAR-T, EphA2-targeted CAR, CLL1 CAR, CD38 CAR, Anti-FcRL5 CAR, GPC3-CAR, CD70 CAR, CD123 CAR, Anti-CD56-CAR-T, Anti-CLL1 / +CD33 CAR T Cells, those of (ii) NCT06758713, NCT07143929, NCT06828341 , NCT06010862, NCT05618041 ,NCT06215950, NCT04572308, NCT04546906, NCT03258047, or those (iii) disclosed in Kfir-Erenfeld S, et al, Feasibility of a Novel Academic BCMA-CART (HBI0101) for the Treatment of Relapsed and Refractory AL Amyloidosis. Clin Cancer Res. 2022 Dec 1 ;28(23):5156-5166; Asherie N, et al, Development and manufacture of novel locally produced anti-BCMA CAR T cells for the treatment of relapsed / refractory multiple myeloma: results from a phase I clinical trial. Haematologica. 2023 Jul 1 ;108(7) :1827-1839, each of which are incorporated herein by reference in their entireties.

[0088] Tumor-infiltrating lymphocytes (TILs): In some embodiments, the T cell compositions comprise or consist of TILs. In some embodiments, the T cell compositions comprise two or more TILs. The TILs may comprise immune cells, such as T cells, derived from a tumor or otherwise cancerous tissue. The TILs may be isolated from the tumor or cancerous tissue, selected for based on a cancer antigen recognition associated with the tumor or cancerous tissue, and may be optionally expanded (e.g., ex vivo expansion). TheAttorney Docket No. 184P-413376-WOTILs may specifically bind and / or recognize the cancer antigen. Additionally, the TILs may be used to target a cancer of the present technology without genetically modifying the TILs. The TILs may be derived from a donor, such as an autologous donor. In some embodiments, the donor is the subject in need thereof of the present technology. In some embodiments, the TILs comprise LN-144 or LN-145.

[0089] T Cell Receptor (TCR)-transgenic T Cells: In some embodiments, the T cell compositions comprise of consist of TCR-transgenic T cells. In some embodiments the TCR transgenic T cells comprise two or more TCR-transgenic T cells. The TCR transgenic T cells may be engineered to express a cancer antigen-specific TCR that specifically bind to and / or recognizes the cancer antigen, such as when the cancer antigen is presented by a human leukocyte antigen (HLA) molecule. The TCR of the TCR-transgenic T cell may comprise a TCR that is a non-native TCR. In some embodiments, the TCR specifically binds to and / or recognizes an intracellular antigen associated with a tumor or otherwise cancerous tissue, which may be presented by the HLA molecule.

[0090] Tumor-Draining Lymph Nodes (TDLNs): In some embodiments, the T cell compositions are derived from tumor-draining lymph nodes (TDLNs) as TDLNs may contain cancer-specific T cells. In some embodiments, the T cell compositions are derived from a combination of TDLNs and tumor tissue to enrich for tumor-specific T cells.Controls

[0091] In some embodiments, the controls of the present technology comprise a T cell composition that (1 ) lacks one or more features of the T cell compositions of the present technology, (2) is synthesized by a method lacking one or more steps of the methods by which the T cell compositions of the present technology are generated, or (3) is synthesized by a method having one or more steps in an alternate order relative to those of the methods by which the T cell compositions of the present technology are generated.Dosing

[0092] The T cell compositions of the present technology may be present in a dose comprising about of 5 x 104T cells to about 15 x 1 O10cells. In some embodiments, the T cell compositions are administered at a dose comprising about 5 x 104T cells, about 6 xAttorney Docket No. 184P-413376-WO104T cells, about 7 x 104T cells, about 8 x 104T cells, about 9x 104T cells, about 10 x 104T cells, about 1 x 105T cells, about 5x 105T cells, about 10x 105T cells, about 1 x 106T cells, about 5x 106T cells, about 10x 106T cells, about 1 x 107T cells, about 5x 107T cells, about 10x 107T cells, about 1 x 108T cells, about 5x 108T cells, about 10x 108T cells, about 1 x 109T cells, about 5x 109T cells, about 10x 109T cells, about 1 x 1 O10T cells, about 2 x 1 O10T cells, about 3 x 1 O10T cells, about 4 x 1 O10T cells, about 5 x 1 O10T cells, about 6 x 1 O10T cells, about 7 x 1 O10T cells, about 8 x 1 O10T cells, about 9 x 1 O10T cells, about 10 x 1010T cells, about 11 x 1010T cells, about 12 x 1010T cells, about 13 x 1010T cells, about 14 x 1010T cells, or about 15 x 1010T cells.

[0093] The T cell compositions of the present technology may be present in a dose comprising at least of 5 x 104T cells to at least 15 x 1010cells. In some embodiments, the T cell compositions are administered at a dose comprising at least 5 x 104T cells, at least 6 x 104T cells, at least 7 x 104T cells, at least 8 x 104T cells, at least 9x 104T cells, at least 10 x 104T cells, at least 1 x 105T cells, at least 5x 105T cells, at least 10x 105T cells, at least 1 x 106T cells, at least 5x 106T cells, at least 10x 106T cells, at least 1 x 107T cells, at least 5x 107T cells, at least 10x 107T cells, at least 1 x 108T cells, at least 5x 108T cells, at least 10x 108T cells, at least 1 x 109T cells, at least 5x 109T cells, at least 10x 109T cells, at least 1 x 1 O10T cells, at least 2 x 1 O10T cells, at least 3 x 1 O10T cells, at least 4 x 1 O10T cells, at least 5 x 1 O10T cells, at least 6 x 1 O10T cells, at least 7 x 1 O10T cells, at least 8 x 1010T cells, at least 9 x 1010T cells, at least 10 x 1010T cells, at least 11 x 1010T cells, at least 12 x 1010T cells, at least 13 x 1010T cells, at least 14 x 1010T cells, or at least 15 x 101° T cells.

[0094] The T cell compositions of the present technology may be present in a dose comprising at least about of 5 x 104T cells to at least about 15 x 1 O10cells. In some embodiments, the T cell compositions are administered at a dose comprising at least about 5 x 104T cells, at least about 6 x 104T cells, at least about 7 x 104T cells, at least about 8 x 104T cells, at least about 9x 104T cells, at least about 10 x 104T cells, at least about 1 x105T cells, at least about 5x 105T cells, at least about 10x 105T cells, at least about 1 x 106T cells, at least about 5x 106T cells, at least about 10x 106T cells, at least about 1 x 107T cells, at least about 5x 107T cells, at least about 10x 107T cells, at least about 1 x 108TAttorney Docket No. 184P-413376-WO cells, at least about 5x 108T cells, at least about 10x 108T cells, at least about 1 x 109T cells, at least about 5x 109T cells, at least about 10x 109T cells, at least about 1 x 1010T cells, at least about 2 x 1 o10T cells, at least about 3 x 1 o10T cells, at least about 4 x 1 o10T cells, at least about 5 x 1010T cells, at least about 6 x 1010T cells, at least about 7 x 1010T cells, at least about 8 x 1010T cells, at least about 9 x 1010T cells, at least about 10 x 1010T cells, at least about 11 x 1 o10T cells, at least about 12 x 1 o10T cells, at least about 13 x 1010T cells, at least about 14 x 1010T cells, or at least about 15 x 1010T cells.Formulations

[0095] The T cell compositions of the present technology may be formulated for administration to a subject by any means known in the art. In some embodiments, one or more of the T cell compositions are formulated for subcutaneous, intravenous, intramuscular, intracranial, intra-arterial, intraventricular, intranodal, intratumor, intraperitoneal, or oral administration.

[0096] The T cell compositions may be formulated in a vaccine or a nanoparticle.EXAMPLESExample 1 : Generation of T Cell Compositions

[0097] T cell compositions of the present technology were generated and assessed in a murine cancer model. The T cell compositions (ACT) were generated from a 4T1 mammary carcinoma model (4T 1 -ACT). For the generation of the 4T 1 -ACT model, female BALB / c mice (7 to 8 weeks old) were subcutaneously injected with 5 x 1044T1 cells and allowed to develop tumors until they reached -100 mm3in size. 10-13 days post-tumor engraftment tumor-bearing mice (Donors) were sacrificed, and their tumor-draining lymph nodes (TDLNs) were harvested. For the adoptive cellular therapy (ACT), T cells (Tc) were isolated from TDLNs, by a negative selection process of a TDLN single cell suspension. T cells were activated in vitro using activating antibodies to either CD3 or CD3 in combination to CD28 or CD2 for 48-72 hours in the presence of IL-2 (50IU / ml). Following removal of stimulation, T cells where further expanded in the presence of IL-2 for an additional 4 days. T cell activation was assessed via flow cytometric analysis of CD69 and CD25 expressionAttorney Docket No. 184P-413376-WO at 48-72hrs post activation. Expanded T cells were phenotyped by flow cytometry to determine the % of T cell subsets.

[0098] T cell compositions of the present technology were also generated and assessed in an A20 B cell lymphoma cancer model. The T cell compositions (ACT) were generated from an A20 B cell lymphoma cancer model. For the generation of the A20-ACT model, female BALB / c mice (6 to 8 weeks old) were ectopically implanted with 4 x 105A20 B cells and allowed to develop tumors until they reached -100 mm3in size. 10-13 days posttumor engraftment tumor-bearing mice (Donors) were sacrificed, and their tumor-draining lymph nodes (TDLNs) and tumor were harvested and processed together for downstream T cell isolation. For the adoptive cellular therapy (ACT), T cells (Tc) were isolated from TDLNs and tumor by a negative selection process of a TDLN single cell suspension. T cells were activated in vitro using activating antibodies to either CD3 or CD3 in combination to CD28 or CD2 for 48-72 hours. Following removal of stimulation, T cells where further expanded in the presence of IL-2 (50IU / ml) from day 3 onwards. T cell activation was assessed via flow cytometric analysis of CD69 and CD25 expression at 48-72hrs post activation. Expanded T cells were phenotyped by flow cytometry to determine the % of T cell subsets.Example 2: Impact of T Cell Compositions on Tumor Growth and Survival4T1 ModelAssessing Tumor Size

[0099] For the in vivo 4T1 -ACT study, T cell compositions were intravenously infused into 4T1 tumor-bearing recipient mice (1 x 106T-cells / mouse) and tumor growth was monitored throughout the experiment; mice were sacrificed when tumor size reached humane endpoint (1200mm3) Tumor lung metastasis was also monitored when mice were sacrificed at day 28 or humane endpoint.Assessing Efficacy of T Cell Composition Therapies

[0100] Tumor Volume'. To improve the efficacy of the ACT, 4T1 -mice were treated with T cell compositions as a monotherapy (ACT) and their corresponding single treatment controls (FIGS. 2A-2C). The corresponding single treatment controls followed the same dosages and timings. Statistical significance in tumour volume growth between groups wasAttorney Docket No. 184P-413376-WO determined with a t test at day 28. A 2-way repeat ANOVA between days 21 -28 was performed. Tumor growth was monitored throughout the study and compared to controls (FIG. 6).

[0101] Survival: Mouse survival was monitored further up to day 109 (FIG. 3). Survival was assessed on 8 groups with 12 mice per group. The surviving mice were re-challenged with 5 x 1044T1 cells and the tumor growth and survival were monitored throughout the duration of the experiment over a period of 33 days of tumor inoculation. This was done in parallel to 4T 1 tumor cell line primary inoculation (5 x 1044T1 cells) of tumor-inexperienced, untreated aged-, gender-matched mice (control). Mice were sacrificed at day of termination of experiment or when tumors reached a humane endpoint of 1200mm3.

[0102] T cell composition specificity: Tumor volume was separately assessed in mice treated with T cell compositions that were cultured with an anti-CD2 antibody and anti-CD3 antibody (CD3 / CD2), an anti-CD3 antibody and an anti-CD28 antibody (CD3 / CD28), or an anti-CD3 antibody alone (CD3), relative to controls (FIGS. 4A and 4B). Each of the T cell composition-treated groups demonstrated reduced tumor volume over time, relative to untreated controls, with the CD3 / CD2 group having statistically significant tumor volume reduction by day 28, relative to the untreated control (FIG. 4B) (2-WAY ANOVA, Turkey multiple comparisons; * 0.05, ** 0.001 , “* <0.0001 ; Mean ± standard deviation (SD)).

[0103] Tumor killing capacity of 4T1 cells and renal carcinoma (RENCA) was also assessed in vitro using these T cell compositions (FIGS. 5A-5F). The RENCA cells used were a renal cancer mouse cell line synergistic to 4T 1 and MHC-compatible with 4T 1 (MHC haplotype: H-2Kd). The T cell compositions were derived from TDLNs of 4T 1 tumors. Killing capacity of the T cell compositions were assessed in two replicates (FIGS. 5A-5C and FIGS. 5D-5F, respectively). Within 24 to 48 hours, the T cell compositions showed minimal killing of the RENCA cell line above background, as opposed to about 50-95% killing of the 4T1 cells.A20 Model

[0104] Tumor Volume: To assess efficacy of the ACT therapy, an A20 lymphoma mouse model was assessed. Mice were injected under the mammary fat pad with B cellAttorney Docket No. 184P-413376-WO lymphoma (A20) cells. Mice were treated with ACT as a monotherapy compared with controls (FIGs. 7A-7B). ACT comprised tumor specific T cells obtained and expanded ex vivo from tumor-draining A20 tumors and tumor draining lymph nodes. Tumor volume was assessed over 28 days following A20 tumor inoculation.

[0105] Survival: Mouse survival was also monitored in these mice (FIG. 7C). Survival was assessed in the ACT treatment group and A20 inoculated controls.Example 3: In Vitro Killing Assays (A20 B Cell Lymphoma)

[0106] ACT: T cells isolated from A20 tumor draining lymph nodes were cocultured in with A20 B cell lymphoma cells following in vitro activation and expansion with two different activation protocols (anti-CD3 alone activation vs anti-CD3,CD2 activation). T cell cytotoxicity in A20 B cells was quantified using Lactate dehydrogenase release assay 48 hrs post-co-culture of Target- effector cells. The data was pooled from four independent repeats. Results of the in vitro cell killing assay are shown in FIG. 8.

[0107] Anti-CD 19 CAR T Cells:

[0108] Generation of CAR T Cells: Retroviral vectors encoding murine or human CAR constructs were produced using Phoenix Eco or / and HEK293T packaging cells, respectively, maintained in IMDM supplemented with 10% FCS and 1 % Glutamax or DMEM supplemented or DMEM + 10% FBS + 2 mM L-glutamine + 1 % Pen / Strep + 1 mM sodium pyruvate. Cells were transfected with envelope, Gag-Pol, and transfer plasmids using GeneJuice transfection reagent, and viral supernatants were harvested 48-72hr posttransfection, clarified by centrifugation and filtration (0.45 pm), and used fresh or stored at - 80 °C.

[0109] Murine T cells were isolated from splenocytes by mechanical dissociation and red blood cell lysis with ACK buffer, followed by overnight culture in complete RPMI-1640 medium. Cells were activated using plate-bound anti-CD3 and anti-CD2 antibodies or anti- CD3 and anti-CD28 antibodies.

[0110] Non-tissue culture-treated plates were coated overnight at 4 °C with Retronectin (10-20 pg / mL) to facilitate viral binding. For transduction, activated T cells (2 xAttorney Docket No. 184P-413376-WO106cells / 0.5ml) were added to wells containing retroviral supernatant (1.5ml per well) and centrifuged at 1 ,000xg for 90 min at 32 °C (spinoculation), then incubated at 37 °C for 12 h.

[0111] Following transduction, cells were cultured in complete medium supplemented with IL-2 (50 IU / mL) and maintained at 1 -2 x 106cells / mL, with cytokine replenishment every 2 days. CAR expression was confirmed by flow cytometry before downstream applications or in vivo transfer.

[0112] In vitro killing assays: In vitro killing assays of anti-CD19 CAR T cells against A20 B cell lymphoma were carried out to confirm the specificity of CAR T cells to the CD19 expressing A20 B cell line (FIG. 9). Following generation of CAR and MOCK CAR expressing mouse T cells in vitro (using anti-CD3 / CD2 activation of splenocyte-derived T cells from naive mice (non-tumour bearing mice)), T cells were co-cultured with A20 B cell lymphoma cells at 1 :1 and 1 :5 target to effector ratios and T cell cytotoxicity was quantified using Lactate dehydrogenase release assay of the culture supernatant at 48hrs post-co- culture. FIG. 9 represents pooled data from three independent experiments. Mock antihuman CD19 CAR T cells were used as a negative control, and anti-mouse CD19 CAR T cells were used as a positive control. Table 1 below provides the domains of the anti-CD19 CAR and Mock CAR constructs used in the present example. The domains of Table 1 are listed in the N-terminal to C-terminal direction.Table 1 . CAR constructs (N-term to C-term)Example 4: Anti-CD19 CAR T Cell Therapy in A20 B Cell LymphomaAttorney Docket No. 184P-413376-WO

[0114] Changes in tumor volume in mice after A20 tumor inoculation with mock CAR T cell treatment and anti-CD19 CAR T cell treatment compared with control is shown in FIG. 10. Anti-CD19 CAR T cells and mock CAR T cells were transferred to mice on day 14.Additional Embodiments

[0115] Various embodiments of the present technology are set forth below in paragraphs

[0116] to

[0162] :

[0116] 1. A T cell composition generated by the steps of (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0117] 2. A T cell composition generated by the steps of (a) obtaining a T cell population from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0118] 3. The T cell composition of embodiment 1 or 2, wherein (a) comprises or consists of purifying or selecting for a T cell population.

[0119] 4. The T cell composition of embodiment 1 or 2, wherein (a) comprises purifying or selecting for a single cell suspension.

[0120] 5. The T cell composition of embodiment 4, wherein the single cell suspension is purified from a tumor or a tumor-draining lymph node.

[0121] 6. The T cell composition of any one of embodiments 1 -5, wherein (b) is optional.

[0122] 7. The T cell composition of any one of embodiments 1 -6, wherein (c) and(d) are optional.Attorney Docket No. 184P-413376-WO

[0123] 8. A method of generating a T cell composition comprising one or more receptors specific to at least a portion of one or more cancer antigens, comprising (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0124] 9. A method of generating a T cell composition comprising one or more receptors specific to at least a portion of one or more cancer antigens, comprising (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines comprising or consisting of IL-2; (c) culturing the T cell population with one or more antibodies comprising or consisting of (i) anti-CD3 antibody, (ii) an anti-CD3 antibody and an anti-CD28 antibody, or (iii) an anti-CD3 antibody and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0125] 10. A T cell composition comprising one or more receptors specific to at least a portion of one or more cancer antigens, the T cell composition generated by a method comprising the steps of: (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines; (c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-OD28 antibody, and an anti-CD2 antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0126] 11. A T cell composition comprising one or more receptors specific to at least a portion of one or more cancer antigens, the T cell composition generated by a method comprising the steps of: (a) isolating a T cell population from a donor; (b) culturing the T cell population with one or more cytokines comprising or consisting of IL-2; (c) culturing the T cell population with one or more antibodies comprising or consisting of (i) anti-CD3 antibody, (ii) an anti-CD3 antibody and an anti-CD28 antibody, (iii) an anti-CD3 antibody and an anti- CD2 antibody, or (iv) an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2Attorney Docket No. 184P-413376-WO antibody; and (d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

[0127] 12. The T cell composition of any one of embodiments 8-11 , wherein the one or more cancer antigens are selected from the group consisting of MAGE-A4, MAGEA1 , NY-ESO-1 , KK-LC-1 , PRAME, B7-H3, B7-H4, CD19, and CD22.

[0128] 13. The T cell composition or the method of any one of embodiments 8-12, wherein isolating the T cell population comprises obtaining a T cell population.

[0129] 14. The T cell composition or the method of any one of embodiments 8-13, wherein (a) comprises or consists of purifying or selecting for a T cell population.

[0130] 15. The T cell composition or the method of any one of embodiments 8-12, wherein (a) comprises or consists of purifying or selecting for a T cell population.

[0131] 16. The T cell composition or the method of any one of embodiments 8-12, wherein (a) comprises purifying or selecting for a single cell suspension.

[0132] 17. The T cell composition or the method of embodiment 16, wherein the single cell suspension is purified from a tumor or a tumor-draining lymph node.

[0133] 18. The T cell composition or the method of any one of embodiments 8-17, wherein (b) is optional.

[0134] 19. The T cell composition or the method of any one of embodiments 8-18, wherein (c) and (d) are optional.

[0135] 20. The T cell composition or the method of any one of embodiments 1 -19, wherein the T cell population is an autologous T cell population.

[0136] 21 . The T cell composition or the method of any one of embodiments 1 -20, wherein the T cell population is an allogenic T cell population.

[0137] 22. The T cell composition or the method of any one of embodiments 1 -21 , wherein the one or more cytokines comprise or consist of IL-2, IL-4, IL-7, IL-15, IL-12, andIL-18.Attorney Docket No. 184P-413376-WO

[0138] 23. The T cell composition or the method of any one of embodiments 1 -22, wherein the period of time comprises at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 28 days, 30 days, 35 days, or 40 days.

[0139] 24. The T cell composition or the method of any one of embodiments 1 -23, wherein (b) and (c) occur simultaneously.

[0140] 25. The T cell composition or the method of any one of embodiments 1 -24, wherein the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody.

[0141] 26. The T cell composition or the method of any one of embodiments 1 -24, wherein the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody and an anti-CD28 antibody.

[0142] 27. The T cell composition or the method of any one of embodiments 1 -24, wherein the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody and an anti-CD2 antibody.

[0143] 28. The T cell composition or the method of any one of embodiments 1 -24, wherein the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody, an anti-CD2 antibody, and an anti-CD23 antibody.

[0144] 29. The T cell composition or the method of any one of embodiments 1 -28, wherein the anti-CD3 antibody is selected from the group consisting of muromonab, teplizumab, otelixizumab, visilizumab, foralumab, and UCHT1.

[0145] 30. The T cell composition or the method of any one of embodiments 1 -29, wherein the anti-CD28 antibody is theralizumab.

[0146] 31 . The T cell composition or the method of any one of embodiments 1 -30, wherein the T cell composition has an increase in one or more gene expression levels selected from the group consisting of a perforin-1 (PRF1 ) gene expression level, a granzyme B (GZMB) gene expression level, an interferon gamma (INFy) gene expression level, a tumor necrosis factor alpha (TNFa) gene expression level, a human leukocyte antigen class II histocompatibility antigen, DR alpha chain (HLA-DRA) gene expression level, a OD25Attorney Docket No. 184P-413376-WO gene expression level, a CD69 gene expression level, and an IL-2 gene expression level, relative to a control.

[0147] 32. The T cell composition or the method of any one of embodiments 1 -31 , wherein the T cell composition has an increase in one or more protein levels selected from the group consisting of a PRF1 protein level, a GZMB protein level, an INFy protein level, a TNFa protein level, an HLD-DRA protein level, a CD25 protein level, a CD69 protein level, and an IL-2 protein level, relative to a control.

[0148] 33. The T cell composition or the method of any one of embodiments 1 -32, wherein at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the T cells in the T cell composition are specific to the one or more cancer antigens.

[0149] 34. The T cell composition or the method of any one of embodiments 1 -33, wherein the T cell composition comprises T cells that are derived from a subject having a cancer.

[0150] 35. The T cell composition or the method of embodiment 34, wherein the T cells that are derived from a subject having cancer comprises T cells that were isolated or obtained from the subject having cancer.

[0151] 36. The T cell composition or the method of embodiment 34 or 35, wherein the cancer comprises a solid tumor.

[0152] 37. The T cell composition or the method of any one of embodiments 34-36, wherein the cancer comprises a metastatic cancer.

[0153] 38. The T cell composition or the method of any one of embodiments 34-37, wherein the cancer is a breast cancer.

[0154] 39. The T cell composition or the method of embodiment 38, wherein the breast cancer is a HER2 positive breast cancer.

[0155] 40. The T cell composition or the method of embodiment 38, wherein the breast cancer is a HER2 negative breast cancer.Attorney Docket No. 184P-413376-WO

[0156] 41. The T cell composition or the method of embodiment 38, wherein the breast cancer is a HER2 / neu-negative breast cancer.

[0157] 42. The T cell composition or the method of any one of embodiments 38,40, or 41 , wherein the breast cancer is a triple negative breast cancer.

[0158] 43. The T cell composition or the method of any one of embodiments 38-42, wherein the breast cancer is selected from the group consisting of a stage I breast cancer, a stage II breast cancer, a stage III breast cancer, and a stage IV breast cancer.

[0159] 44. The T cell composition or the method of any one of embodiments 1 -43, wherein the T cell composition is formulated for subcutaneous, intravenous, intramuscular, intracranial, intra-arterial, intraventricular, intranodal, intratumor, or intraperitoneal administration.

[0160] 45. The T cell composition or the method of any one of embodiments 1 -44, wherein the T cell composition is formulated for subcutaneous, intravenous, intramuscular, intracranial, intra-arterial, intraventricular, intranodal, intratumor, or intraperitoneal administration.

[0161] 46. The T cell composition or the method of embodiment 44 or 45, wherein one or more of the T cell compositions is formulated in a vaccine.

[0162] 47. The T cell composition or the method of embodiment 44 or 45, wherein one or more of the T cell compositions is formulated in a nanoparticle.

Claims

Attorney Docket No. 184P-413376-WOCLAIMS l / We claim:1 . A T cell composition generated by the steps of(a) isolating a T cell population from a donor;(b) culturing the T cell population with one or more cytokines;(c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and(d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

2. A T cell composition generated by the steps of(a) obtaining a T cell population from a donor;(b) culturing the T cell population with one or more cytokines;(c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and(d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

3. The T cell composition of claim 1 or 2, wherein (a) comprises or consists of purifying or selecting for a T cell population.

4. The T cell composition of claim 1 or 2, wherein (a) comprises purifying or selecting for a single cell suspension.

5. The T cell composition of claim 4, wherein the single cell suspension is purified from a tumor or a tumor-draining lymph node.Attorney Docket No. 184P-413376-WO6. The T cell composition of any one of claims 1 -5, wherein (b) is optional.

7. The T cell composition of any one of claims 1 -6, wherein (c) and (d) are optional.

8. A method of generating a T cell composition comprising one or more receptors specific to at least a portion of one or more cancer antigens, comprising(a) isolating a T cell population from a donor;(b) culturing the T cell population with one or more cytokines;(c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and(d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

9. A method of generating a T cell composition comprising one or more receptors specific to at least a portion of one or more cancer antigens, comprising(a) isolating a T cell population from a donor;(b) culturing the T cell population with one or more cytokines comprising or consisting of IL-2;(c) culturing the T cell population with one or more antibodies comprising or consisting of(i) anti-CD3 antibody, (ii) an anti-CD3 antibody and an anti-CD28 antibody, or (iii) an anti-CD3 antibody and an anti-CD2 antibody; and(d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

10. A T cell composition comprising one or more receptors specific to at least a portion of one or more cancer antigens, the T cell composition generated by a method comprising the steps of:(a) isolating a T cell population from a donor;Attorney Docket No. 184P-413376-WO(b) culturing the T cell population with one or more cytokines;(c) culturing the T cell population with one or more antibodies selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and(d) expanding the T cell population for a period of time sufficient to generate the T cell composition.1 1. A T cell composition comprising one or more receptors specific to at least a portion of one or more cancer antigens, the T cell composition generated by a method comprising the steps of:(a) isolating a T cell population from a donor;(b) culturing the T cell population with one or more cytokines comprising or consisting of IL-2;(c) culturing the T cell population with one or more antibodies comprising or consisting of(i) anti-CD3 antibody, (ii) an anti-CD3 antibody and an anti-OD28 antibody, (iii) an anti-CD3 antibody and an anti-CD2 antibody, or (iv) an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody; and(d) expanding the T cell population for a period of time sufficient to generate the T cell composition.

12. The T cell composition of any one of claims 8-1 1 , wherein the one or more cancer antigens are selected from the group consisting of MAGE-A4, MAGEA1 , NY-ESO-1 , KK-LC-1 , FRAME, B7-H3, B7-H4, CD19, and CD22.

13. The T cell composition or the method of any one of claims 8-12, wherein isolating the T cell population comprises obtaining a T cell population.

14. The T cell composition or the method of any one of claims 8-13, wherein (a) comprises or consists of purifying or selecting for a T cell population.Attorney Docket No. 184P-413376-WO15. The T cell composition or the method of any one of claims 8-12, wherein (a) comprises or consists of purifying or selecting for a T cell population.

16. The T cell composition or the method of any one of claims 8-12, wherein (a) comprises purifying or selecting for a single cell suspension.

17. The T cell composition or the method of claim 16, wherein the single cell suspension is purified from a tumor or a tumor-draining lymph node.

18. The T cell composition or the method of any one of claims 8-17, wherein (b) is optional.

19. The T cell composition or the method of any one of claims 8-18, wherein (c) and (d) are optional.

20. The T cell composition or the method of any one of claims 1 -19, wherein the T cell population is an autologous T cell population.21 . The T cell composition or the method of any one of claims 1 -20, wherein the T cell population is an allogenic T cell population.

22. The T cell composition or the method of any one of claims 1 -21 , wherein the one or more cytokines comprise or consist of IL-2, IL-4, IL-7, IL-15, IL-12, and IL-18.

23. The T cell composition or the method of any one of claims 1 -22, wherein the period of time comprises at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 28 days, 30 days, 35 days, or 40 days.Attorney Docket No. 184P-413376-WO24. The T cell composition or the method of any one of claims 1 -23, wherein (b) and (c) occur simultaneously.

25. The T cell composition or the method of any one of claims 1 -24, wherein the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody.

26. The T cell composition or the method of any one of claims 1 -24, wherein the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody and an anti- CD28 antibody.

27. The T cell composition or the method of any one of claims 1 -24, wherein the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody and an anti- CD2 antibody.

28. The T cell composition or the method of any one of claims 1 -24, wherein the one or more antibodies in (c) comprises or consists of an anti-CD3 antibody, an anti-CD2 antibody, and an anti-CD23 antibody.

29. The T cell composition or the method of any one of claims 1 -28, wherein the anti-CD3 antibody is selected from the group consisting of muromonab, teplizumab, otelixizumab, visilizumab, foralumab, and UCHT1 .

30. The T cell composition or the method of any one of claims 1 -29, wherein the anti-OD28 antibody is theralizumab.31 . The T cell composition or the method of any one of claims 1 -30, wherein the T cell composition has an increase in one or more gene expression levels selected from the group consisting of a perforin-1 (PRF1 ) gene expression level, a granzyme B (GZMB) gene expression level, an interferon gamma (INFy) gene expression level, a tumor necrosis factor alpha (TNFa) gene expression level, a human leukocyte antigen class II histocompatibilityAttorney Docket No. 184P-413376-WO antigen, DR alpha chain (HLA-DRA) gene expression level, a OD25 gene expression level, a CD69 gene expression level, and an IL-2 gene expression level, relative to a control.

32. The T cell composition or the method of any one of claims 1 -31 , wherein the T cell composition has an increase in one or more protein levels selected from the group consisting of a PRF1 protein level, a GZMB protein level, an INFy protein level, a TNFa protein level, an HLD-DRA protein level, a CD25 protein level, a CD69 protein level, and an IL-2 protein level, relative to a control.

33. The T cell composition or the method of any one of claims 1 -32, wherein at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the T cells in the T cell composition are specific to the one or more cancer antigens.

34. The T cell composition or the method of any one of claims 1 -33, wherein the T cell composition comprises T cells that are derived from a subject having a cancer.

35. The T cell composition or the method of claim 34, wherein the T cells that are derived from a subject having cancer comprises T cells that were isolated or obtained from the subject having cancer.

36. The T cell composition or the method of claim 34 or 35, wherein the cancer comprises a solid tumor.

37. The T cell composition or the method of any one of claims 34-36, wherein the cancer comprises a metastatic cancer.

38. The T cell composition or the method of any one of claims 34-37, wherein the cancer is a breast cancer.Attorney Docket No. 184P-413376-WO39. The T cell composition or the method of claim 38, wherein the breast cancer is a HER2 positive breast cancer.

40. The T cell composition or the method of claim 38, wherein the breast cancer is a HER2 negative breast cancer.41 . The T cell composition or the method of claim 38, wherein the breast cancer is a HER2 / neu-negative breast cancer.

42. The T cell composition or the method of any one of claims 38, 40, or 41 , wherein the breast cancer is a triple negative breast cancer.

43. The T cell composition or the method of any one of claims 38-42, wherein the breast cancer is selected from the group consisting of a stage I breast cancer, a stage II breast cancer, a stage III breast cancer, and a stage IV breast cancer.

44. The T cell composition or the method of any one of claims 1 -43, wherein the T cell composition is formulated for subcutaneous, intravenous, intramuscular, intracranial, intra-arterial, intraventricular, intranodal, intratumor, or intraperitoneal administration.

45. The T cell composition or the method of any one of claims 1 -44, wherein the T cell composition is formulated for subcutaneous, intravenous, intramuscular, intracranial, intra-arterial, intraventricular, intranodal, intratumor, or intraperitoneal administration.

46. The T cell composition or the method of claim 44 or 45, wherein one or more of the T cell compositions is formulated in a vaccine.

47. The T cell composition or the method of claim 44 or 45, wherein one or more of the T cell compositions is formulated in a nanoparticle.

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