Infiltrative immune cells

Abstract

Embodiments of the disclosure include a cell engineered to comprise heterologous expression of a matrix metalloproteinase (MMP) and / or an osteopontin (OPN). Cells of the present disclosure may be used in methods of treating a disease associated with expression of a tumor antigen in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of the cells of the present disclosure.

Description

INFILTRATIVE IMMUNE CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 63 / 582,392 filed September 13, 2023, all of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on 12 September 2024, is named BAYMP0405WO_Sequence_Listing.xml and is 178,707 bytes in size.TECHNICAL FIELD

[0003] Embodiments of the disclosure include at least the fields of molecular biology, cell biology, cell therapy, and medicine, including immunotherapy.BACKGROUND

[0004] Although chimeric antigen receptor T (CAR-T) cell therapy has been successful at treating select hematologic malignancies, success in the solid tumor setting has been limited. A significant factor in the failure of CAR-T therapy in this setting is the low infiltration of CAR-Ts into tumors due to the exclusionary nature of the tumor-specific extracellular matrix (ECM) and basement membrane (BM), which encapsulate the tumor. Tumor encapsulation occurs when cancer associated fibroblasts (CAFs) recruited to the tumor secrete large volumes of ECM fibers, including type I and type IV collagen, while simultaneously secreting enzymes that crosslink these fibers into a dense, rigid structure. Unlike the ECM and BM of healthy tissues, which has a loose basket-weave pattern that T cells easily traverse, tumor-specific matrices are stiff and characterized by small pores, leading to T cell exclusion.

[0005] The present disclosure provides solutions to the long-felt need in the art for improving immune cell infiltration of solid tumor environments.BRIEF SUMMARY OF THE DISCLOSURE

[0006] Embodiments of the disclosure concerns methods and compositions related to immunotherapy for a medical condition.

[0007] In this disclosure, immune cells, including at least T cells and CAR-T cells, may be engineered to express one or more heterologous matrix metalloproteinases (MMPs) and / or one or more osteopontins (OPN), such as for the purpose of increasing their infiltration into solid tumors. Solid tumors can be encapsulated by modified basement membranes (BMs) and extracellular matrices (ECMs) that prevent efficient infiltration of immunotherapeutic cells during therapy (including at least T cells during CAR-T cell therapy), hindering patient responses. MMPs are proteases that break down ECM fibers, while OPN anchors the cellular cytoskeleton to mediate cell migration. The secretion of these proteins significantly enhanced cell infiltration (including at least immune cells, e.g., T cells and CAR-T cells) through BMs and ECMs in vitro and in vivo. Because tumors can be surrounded by different ECM fibers, and MMPs each degrade a different ECM fiber, the present disclosure provides cells that may be modified to secrete the correct MMP to target a subject’s specific tumor profile, in some embodiments. In some embodiments, a cell comprises, consists of, or consists essentially of a leukocyte. In some embodiments, a cell comprises, consists of, or consists essentially of a T cell, an NK cell, or a B cell. In specific embodiments, a cell comprises, consists of, or consists essentially of an allogeneic, autologous, syngeneic, or xenogeneic cell with respect to an individual. In specific embodiments, a cell comprises, consists of, or consists essentially of a mammalian cell. In specific embodiments, a cell comprises, consists of, or consists essentially of a human cell. In specific embodiments, an immune cell, such as a T cell or NK cell expresses one or more MMPs and / or one or more OPN and / or one or more therapeutic genes, such as a CAR and / or TCR. In certain embodiments, the MMP comprises MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and / or MMP28. In specific embodiments, the OPN comprises OPNa, OPNb, OPNc, OPN4 and / or OPN5. In some embodiments, a CAR of the disclosure comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 39 or 91. In some embodiments, a cell comprises, consists of, or consists essentially of heterologous OPNa, OPNb, OPNc, OPN4, OPN-I, OPN5, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11,MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and / or MMP28. In some embodiments, an MMP of the disclosure, comprises MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and / or MMP28. In some embodiments, an OPN of the disclosure comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 19-22, or 76-79. In some embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2, EGFR, EGFRvIII, HER2, PSMA, CEA, IL13Ra2, Gpc3, CAIX, LI -CAM, CA 125, CD 133, CD 19, FAP, CTAG1B, Muc-1, FR-a, CD70, 5T4, 8H9, avp6integrin, BCMA, B7-H3, B7-H6, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD47, CD123, CD138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, , EGFR family including ErbB2 (HER2), EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FBP, fetal AchR, GD3, HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-l lRa, Lambda, Lewis-Y, Kappa, KDR, MCSP, Mesothelin, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, ROR1, SP17, Survivin, TAG72, TIM3, TIGIT, TROP2, TEMs, HMW-MAA, VEGFR2, or any combination thereof. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous OPN. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous OPN-B. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous OPN-A. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous OPN-C. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous OPN-4. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous OPN-5. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous OPN-I. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous MMP. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous MMP-7. In specific embodiments, a cell comprises, consists of, or consists essentially of a CAR that targets GD2 and a heterologous MMP14. In specific embodiments, aplurality of cells comprises, consists of, or consists essentially of one or more cells of the disclosure. In some embodiments, a plurality of cells is frozen.

[0008] Some embodiments relate to methods of treating a disease associated with expression of a tumor antigen in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a cell or plurality of cells of the disclosure. In some embodiments, a disease comprises, consists of, or consists essentially of cancer, asthma, allergies, an autoimmune disorder, inflammation, and / or an infection. In some embodiments, a disease comprises, consists of, or consists essentially of a solid tumor. In some embodiments, a solid tumor comprises, consists of, or consists essentially of a solid tumor of the pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and / or breast. In some embodiments, a cancer or solid tumor comprises, consists of, or consists essentially of expression of GD2, EGFR, EGFRvIII, HER2, PSMA, CEA, IL13Ra2, Gpc3, CAIX, Ll-CAM, CA 125, CD133, CD19, FAP, CTAG1B, Muc-1, FR-a, CD70, 5T4, 8H9, avp6integrin, BCMA, B7-H3, B7-H6, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD47, CD 123, CD 138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, , EGFR family including ErbB2 (HER2), EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FBP, fetal AchR, GD3, HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-l lRa, Lambda, Lewis- Y, Kappa, KDR, MCSP, Mesothelin, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, ROR1, SP17, Survivin, TAG72, TIM3, TIGIT, TROP2, TEMs, HMW-MAA, VEGFR2, or any combination thereof. In some embodiments, a therapeutically effective amount of a cell or plurality of cells are delivered intravenously, intramuscularly, subcutaneously, transdermally, intraperitoneally, intraorbitally, by implantation, intrathecally, intraventricularly, and / or intratumorally. In some embodiments, a method further comprises treatment with at least one or more co-therapies. In some embodiments, administering of the therapeutically effective amount of the cell or plurality of cells occurs before administration of the at least one or more co-therapies. In some embodiments, administering of the therapeutically effective amount of the cell or plurality of cells occurs simultaneously to administration of the at least one or more co-therapies. In some embodiments, administering of the therapeutically effective amount of the cell or plurality of cells occurs after administration of the at least one or more co-therapies. In some embodiments, a cell or plurality of cells are delivered via a same route of administration as the at least one or more co-therapies. In some embodiments, a cell or plurality of cells are delivered via a different route of administration as the at least one or more co-therapies. In some embodiments, a cell or plurality of cells are delivered together with the at least one or more co-therapies. In some embodiments, a cell or plurality of cells are delivered separately from the at least one or more co-therapies. In some embodiments, at least one or more therapeutic agents comprise, consist of, or consist essentially of chemotherapy, radiotherapy, immunotherapy, surgery, and / or drug therapy. Some embodiments relate to methods of enhancing infiltration of an immune cell into a tumor, the method comprising the steps of: a) first engineering the immune cell to comprise a heterologous CAR and / or TCR; b) second engineering the immune cell to comprise a heterologous MMP and / or OPN; c) producing a multi-engineered immune cell from steps (a) and (b); d) administering the multiengineered cell to an individual. In some embodiments, a methods of enhancing infiltration of an immune cell into a tumor comprises, consists of, or consists essentially of a an immune cell engineered to comprise a CAR and / or TCR that targets GD2, EGFR, EGFRvIII, HER2, PSMA, CEA, IL13Ra2, Gpc3, CAIX, Ll-CAM, CA 125, CD133, CD19, FAP, CTAG1B, Muc-1, FR- a, CD70, 5T4, 8H9, avp6integrin, BCMA, B7-H3, B7-H6, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD47, CD 123, CD 138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, , EGFR family including ErbB2 (HER2), EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FBP, fetal AchR, GD3, HLA- A1+MAGE1, HLA-A1+NY-ES0-1, IL-l lRa, Lambda, Lewis- Y, Kappa, KDR, MCSP, Mesothelin, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, R0R1, SP17, Survivin, TAG72, TIM3, TIGIT, TROP2, TEMs, HMW-MAA, VEGFR2, or any combination thereof. In some embodiments, a methods of enhancing infiltration of an immune cell into a tumor comprises, consists of, or consists essentially of an immune cell engineered to comprise OPNa, OPNb, OPNc, 0PN4, OPN-I, and / or 0PN5. In some embodiments, a methods of enhancing infiltration of an immune cell into a tumor comprises, consists of, or consists essentially of an immune cell engineered to comprise MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and / or MMP28. In some embodiments, a methods of enhancing infiltration of an immune cell into a tumor comprises, consists of, or consists essentially of an immune cell engineered to comprise OPNa, OPNb, OPNc, 0PN4, OPN-I, 0PN5, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and / or MMP28. In some embodiments, a method comprises the step of a) first engineering animmune cell to comprise a heterologous CAR and / or TCR, that precedes a step of b) second engineering the immune cell to comprise a heterologous MMP and / or OPN. In some embodiments, a method comprises the step of b) second engineering the immune cell to comprise a heterologous MMP and / or OPN, that precedes a step of a) first engineering an immune cell to comprise a heterologous CAR and / or TCR. In some embodiments, a methods of enhancing infiltration of an immune cell into a tumor comprises, consists of, or consists essentially of an immune cell that comprises, consists of, or consists essentially of a T cell, NK cell, or B cell. In some embodiments, a cell of the disclosure comprises a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NOs: 1-57. In some embodiments, a cell of the disclosure comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 58-100. In some embodiments, a method of enhancing infiltration of an immune cell into a tumor comprises, consists of, or consists essentially of administering a cell of the disclosure to an individual that has been diagnosed, or is suspected of having a tumor. In some embodiments, a method of enhancing infiltration of an immune cell into a tumor comprises, consists of, or consists essentially of administering a cell of the disclosure to an individual that has been diagnosed, or is suspected of having a solid tumor. In some embodiments, a method of enhancing infiltration of an immune cell into a tumor comprises, consists of, or consists essentially of administering a cell of the disclosure to an individual that has been diagnosed, or is suspected of having a tumor of the pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and / or breast. In some embodiments, a method of enhancing infiltration of an immune cell into a tumor comprises, consists of, or consists essentially of administering a cell of the disclosure to an individual that has been diagnosed, or is suspected of having neuroblastoma.

[0009] Some embodiments relate to a nucleic acid, comprising a heterologous MMP and / or heterologous OPN, and a reporter gene and / or a CAR and / or TCR. In some embodiments, an MMP comprises MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP1 1, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and / or MMP28. In some embodiments, an MMP comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-18, or 58-75. In some embodiments, an OPN comprises OPNa, OPNb, OPNc, OPN4, OPN-I, and / or OPN5. In some embodiments, an OPN comprises an amino acidsequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 19-22, or 76-79. In some embodiments, a CAR and / or TCR targets GD2, EGFR, EGFRvIII, HER2, PSMA, CEA, IL13Ra2, Gpc3, CAIX, Ll-CAM, CA 125, CD133, CD19, FAP, CTAG1B, Muc-1, and / or FR- a. In some embodiments, a CAR comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 39 or 91. In some embodiments, a reporter comprises GFP or tCD19. In some embodiments, a the reporter comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 34 or 81. In some embodiments, a nucleic acid comprises a nucleic acid sequence and / or encodes an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-100.

[0010] Some embodiments relate to a kit comprising nucleic acids, polypeptides, and / or cells of the disclosure. In some embodiments, a kit comprises instructions for performing a method of the disclosure. In some embodiments, a kit is housed in a suitable container.

[0011] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter which form the subject of the claims of the disclosure. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present disclosure. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the disclosure as set forth in the appended claims. The novel features which are believed to be characteristic of the disclosure, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] For a more complete understanding of the present disclosure, reference is now made to the following description taken in conjunction with the accompanying drawings.

[0013] FIGs. 1A-1B. Solid tumors are encapsulated by a basement membrane (BM) and extracellular matrices (ECM) that exclude T cells. FIG. 1A depicts how BM and ECM are secreted by cancer associated fibroblasts (CAFs) around a tumor. Crosslinking of BM or ECM fibers can increase their density and stiffness, and reduce pore size surrounding the tumor, encapsulating the tumor, and preventing T cell infiltration. FIG. IB depicts immune cells trapped in the ECM (arrow) surrounding a tumor (area enclosed by dotted line) in a xenograft mouse model of neuroblastoma (LAN-1). Tissue sections from the xenograft neuroblastoma mouse model were stained for type I collagen (red, left panel, arrow), and CD3 (brown, right panel, arrow).

[0014] FIGs. 2A-2C. T cells were successfully transduced with OPN and MMP constructs. FIG 2A depicts transduction efficiency (y-axis) in activated T cells (ATCs) from 3 donors with different isoforms of OPN (e.g., OPN-A, OPN-B, OPN-C, or OPN-I). Transduction was measured as the expression of tCD19 or green fluorescent protein (GFP) by flow cytometry (x-axis). FIG. 2B depicts transduction efficiency (y-axis) in ATCs from 3 donors with different MMPs (e.g., MMP-7 or MMP-14). Transduction was measured as the expression of tCD19 or GFP by flow cytometry (x-axis). FIG. 2C demonstrates transduction efficiency of cells from donors 1 and 3 with MMP-7 via detection of a reporter (GFP) by flow cytometry.

[0015] FIGs. 3A-3B. Transduced T cells secreted more OPN and MMP than controls. FIG. 3A depicts the concentration of OPN (y-axis, ng / mL) secreted by untransduced T cells, second-generation GD2-CAR-T cells, and T cells transduced with OPN. FIG. 3B depicts the concentration of MMP-7 (y-axis, ng / mL) secreted by untransduced T cells, second- generation GD2-CAR-T cells, and T cells transduced with an MMP-7.

[0016] FIGs. 4A-4D. Infiltration of various T cells through Matrigel. FIG. 4 A depicts proportional invasion of ATCs (y-axis, % invasion) from 4 donors in increasing concentrations of solubilized basement membrane matrix (Matrigel; x-axis, ug / mL). FIG. 4B depicts proportional invasion of T cells from 4 donors in 3 mg / mL of solubilized basement membrane matrix (Matrigel; x-axis) that were untransduced, transduced with a GD2 CARcomprising a 41BBZ co-stimulatory domain (“GD2-41BBZ”), or transduced with a GD2 CAR comprising a 28Z co-stimulatory domain (“GD2-28Z”). FIG. 4C depicts proportional invasion of T cells from 3 donors in 3 mg / mL Matrigel (x-axis) that were untransduced or transduced with an OPN (e.g. OPN-A, OPN-B, OPN-C, or OPN-I). FIG. 4D depicts proportional invasion of T cells from 3 donors in 3 mg / mL Matrigel (x-axis) that were untransduced or transduced with an MMP.

[0017] FIGs. 5A-5B. T cells were successfully double-transduced with a CAR and an OPN or MMP construct. FIGs. 5A-5B depict proportion of untransduced, single transduce, and double-transduced ATCs, transduced with a CAR (e.g., GD2-41BBZ or GD2- 28Z) and either an OPN (e.g. OPN-B) or an MMP (e.g. MMP-7 or MMP-14).

[0018] FIGs. 6A-6B. OPN and MMP-7 expression in double-transduced CAR-T cells, as confirmed by ELISA (n=3) on day 10. FIG. 6A depicts the concentration of secreted MMP (e.g. MMP-7) by untransduced cells, cells transduced with a CAR, or cells transduced with a CAR and an MMP on day 10 post-activation. FIG. 6B depicts the concentration of secreted OPN by untransduced cells, cells transduced with a CAR, or cells transduced with a CAR and an OPN on day 10 post-activation.

[0019] FIGs. 7A-7E. Overexpression of MMP and / or OPN did not affect CAR-T cell expansion, toxicity, or phenotype. FIG. 7A depicts cell expansion (y-axis) across 12 days between untransduced (UnTx) and engineered cells. FIG. 7B depicts cell expansion (y-axis) across 11 days between cells engineered to comprise expression of GD2 CAR and MMP7, MMP14, or OPN-B. FIG. 7C depicts specific lysis by untransduced or engineered T cells against LAN-1 cells at various effector to T cell (E:T) ratios in a five-hour51Cr-release assay. FIGs. 7D-7E depict cellular phenotype profiles 7 days (FIG. 7E) and 8 days (FIG. 7D) after T cell activation. For each bar of FIG. 7D, the groups from top to bottom are: TN, TEFF, TCM, and TEM. For each bar of FIG. 7E, the groups from top to bottom are: TN, TEFF, TCM, and TEM.

[0020] FIGs. 8A-8G. MMP and OPN restored infiltrative ability of CAR-T cells in solid tumor environments. FIGs. 8A-8B depict proportional invasion of T cells from 4 donors in 3 mg / mL Matrigel (x-axis) that were untransduced or engineered (e.g., with a CAR, an OPN, or a CAR and an MMP or an OPN). FIGs. 8C depicts a schematic of a Halo assay used to model T-cell extravasation and infiltration of T-cells into a tumor. FIG. 8D depicts a real-time Incucyte image captured at t = 0 hours of the Halo assay area. FIG. 8E depicts a real-timeIncucyte image captured at t = 24 hours of the Halo assay area. FIGs. 8F-8G depict total green and red object area of (y-axis, um2 / image) after the Halo assay.

[0021] FIGs. 9A-9B. OPN.GD2-28Z CAR-T cells reduced tumor burden in a xenograft model of neuroblastoma. FIG. 9A shows tumor size, as measured by tumor bioluminescence, at 7 and 10 days post CAR T cell infusion. FIG. 9B shows tumor burden over 28 days, as measured by tumor bioluminescence.

[0022] FIGs. 10A-10D. Treatment with OPN-GD2.CAR T cells led to rapid tumor clearance and increased survival in mice with neuroblastoma (NB). FIG. 10A shows a schematic of in vivo tumor experiments. FIGs. 10B-10C show tumor burden, measured by tumor luminescence, in mice treated with GD2.CAR, MMP7-GD2.CAR, or OPN.GD2.CAR across time (FIG. 10B), and at day 29 (FIG. 10C) post CAR T cell infusion; n=9-12 mice, median, unpaired t test, *P < .05. FIG. 10D shows a Kaplan-Meier survival curve of mice without treatment (tumor only), treated with untransduced T cells (UnTd), or treated with engineered T cells (e g., GD2.CAR, MMP7-GD2.CAR, or OPN.GD2.CAR T cells); *P < .05, determined by log-rank test (Mantel-Cox).

[0023] FIG. 11A-11D. OPN expression in GD2.CAR T cells dramatically increased infiltration into NB tumors. FIG. HA shows a schematic of in vivo experiments to assess infiltration of tumors by engineered T cells. FIG 11B shows CAR T cell luminescence signal measured at tumor site of mice treated with GD2.CAR, MMP7-GD2.CAR, or OPN.GD2.CAR T cells. For each time-point, the groups from left to right represent: GD2.CAR, MMP7- GD2.CAR, and OPN.GD2.CAR; n= 7, mean, two-way ANOVA. FIG. 11C shows the number of CAR T cells per field in tumor sections; n=10-l l, median, one-way ANOVA, *P < .05, *** < 001, *** < 0001. FIG. HD shows representative 20X microscopy images of tumor sections stained for collagen (light gray) and CD3 (dark gray).

[0024] FIG. 12 shows schematics of illustrative nucleic acid constructs of the disclosure.

[0025] FIG. 13 shows schematics of illustrative nucleic acid constructs of the disclosure.

[0026] FIG. 14 shows a schematic of an illustrative nucleic acid construct of the disclosure.

[0027] FIG. 15 shows a schematic of an illustrative nucleic acid construct of the disclosure.

[0028] FIG. 16 shows a schematic of an illustrative nucleic acid construct of the disclosure.

[0029] FIG. 17 shows a schematic of an illustrative nucleic acid construct of the disclosure.

[0030] FIG. 18 shows a schematic of an illustrative nucleic acid construct of the disclosure.

[0031] FIG. 19 shows a schematic of an illustrative nucleic acid construct of the disclosure.DETAILED DESCRIPTION OF THE DISCLOSUREI. Definitions

[0032] As used herein, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0033] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0034] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0035] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0036] The compositions and methods fortheir use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the disclosure.

[0037] Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0038] The term “disease associated with expression of a tumor antigen” as used herein refers to any medical condition associated with tumor antigens, including cancer, asthma, allergies, autoimmune disorders, inflammation, and / or infections.

[0039] In some embodiments, the disclosed methods are directed to methods for treating cancer. The cancer may be a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may be recurrent, metastatic, relapsed, or of a Stage I, II, III, or IV.

[0040] As used herein, the term “overexpression” means expression of a gene at a level higher than naturally-occurring levels. In some cases, overexpression may refer to expression of a gene in a cell that normally does not express said gene or expresses the gene at a low level.

[0041] As used herein, the term “therapeutically effective amount” is synonymous with “effective amount”, “therapeutically effective dose”, and / or “effective dose” and refers to the amount of a compound that will elicit the biological, cosmetic or clinical response being sought by the practitioner in an individual in need thereof. The appropriate effective amount to be administered for a particular application of the disclosed methods can be determined by those skilled in the art, using the guidance provided herein. For example, an effective amount can be extrapolated from in vitro and in vivo assays as described in the present specification. One skilled in the art will recognize that the condition of the individual can be monitored throughout the course of therapy and that the effective amount of an immune cell or composition disclosed herein that is administered can be adjusted accordingly.

[0042] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0043] As used herein, the term “individual” or “subject” generally refers to an individual in need of a therapy for a condition, e.g., cancer. The individual can be a mammal, such as a human, dog, cat, horse, pig or rodent. The individual can be one that has or is suspected of having or at risk for having a disease or medical condition, e.g., cancer. The individual may have a disease or be suspected of having the disease. The individual may be asymptomatic. The individual may be of any gender or biological sex.

[0044] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0045] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, 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 specific embodiments, a vector is engineered through recombinant nucleic acid technologies, and a cell is engineered through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.

[0046] Embodiments of the disclosure include methods and compositions for the treatment of a disease associated with expression of a tumor antigen by improving immune cell infiltration through basement membranes (BMs) and / or extracellular matrices (ECMs) surrounding a tumor environment. In some embodiments, a heterologous MMP and / or heterologous OPN is expressed in a cell to improve infiltration through BM and / or ECMs.

[0047] In some embodiments, an MMP of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 1-18.

[0048] In some embodiments, an MMP of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 58- 75.

[0049] In some embodiments, an OPN of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 19- 22.

[0050] In some embodiments, an OPN of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,8%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 4%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 76- 9.II. Solid Tumor Environments

[0051] Solid tumors are complex structures that comprise not only tumor cells but also vasculature, extracellular matrix (ECM), stromal cells, and immune cells. In tumors, the ECM can be remodeled by cancer cells to modulate intratumoral signaling, nutrient availability, metabolism, oxygenation, and immunogenicity, for example. Exerting this regulatory control, the ECM can influence the tumor’s malignancy, growth, and response to therapy.

[0052] Many solid tumors express high levels of various ECM molecules like fibrillar collagens, fibronectin, elastin, polysaccharides, and laminins (Provenzano et al., 2008; Mammoto et al., 2013). Some cancers, e.g., pancreatic ductal adenocarcinomas (PDACs), are particularly rich in hyaluronan, a polysaccharide (Provenzano and Hingorani, 2013). In many cases, the ECM compromises up to 60% of the tumor mass. Although cancer cells can produce ECM molecules, cancer-associated fibroblasts (CAFs) are believed to be the major contributors of the ECM found in the tumor microenvironment (Casey et al., 2009; Naba et al., 2012). Indeed, the infiltration of fibroblasts / myofibroblasts and the subsequent accumulation of significant amounts of collagenous ECM is observed in many solid tumors. This process, called desmoplasia, is strongly linked to poor prognosis and resistance to systemic therapy (Conti et al., 2008; Schober et al., 2014).

[0053] By supporting tumor cells via paracrine stromal cell-derived factor-1 (SDF1) and transforming growth factor beta (TGF / ? ) signals, CAFs further contribute not only to a more malignant tumor phenotype by driving epithelial-to-mesenchymal transition (EMT) but also induce production of collagen and other ECM molecules (Zode et al., 2009; Porsch et al., 2013; Garcia et al., 2016).

[0054] All components of the TME vary significantly from their respective counterparts in non-malignant tissues. Consequently, also the tumor ECM diverges strongly not only in amount of deposition but also in composition, organization, and post-translational modification from the ECM in surrounding normal tissue. In invasive ductal carcinomas, collagen production is shifted toward Col I and Col III compared to benign mammary lesions (Deak et al., 1991; Kauppila et al., 1998). The increased expression is mainly observed in the stromal part of the tumor. As the benign lesions also consist mainly of cells with fibroblastic characteristics, this demonstrated the tendency and capacity of breast tumor cells to shift the secreted matrisome of its stroma. In the desmoplastic stroma of breast carcinomas, up to 15% of the collagenous matrix consists of Col V, a collagen isoform of low abundance in normal and fibrocystic (<0.1%) breast tissue (Barsky et al., 1982).

[0055] Using Second Harmonic Generation microscopy, observation of fiber formation indicated that increasing the Col V / Col I ratio reduces length and organization of collagen fibers (Ajeti et al., 2011). At higher ratios, fiber formation can be completely inhibited, resulting in a gel-like ECM (Pucci-Minafra and Luparello, 1991). Col IV, on the other hand, is downregulated in ovarian carcinomas compared to benign tissue, and the expression isinversely correlated with stage and markers of malignancy (Bar et al., 2004). The same pattern of enhanced Col I / Col III expression and reduced Col IV expression is observed in human lung tumors (Fang et al., 2019). In melanoma, increased Col I expression is observed and correlated with invasiveness, angiogenesis, and reduced survival (van Kempen et al., 2008; Miskolczi et al., 2018). High collagen messenger RNA (mRNA) expression, the aberrant form of fibrous collagen spindles, and the increased expression of matrix proteases are signs of an fast turnover of the collagen in tumors (Kauppila et al., 1998).III. Immune Cells and Methods of Production

[0056] The cells and methods provided herein may comprise cell engineering which improves immune cell infiltration through basement membranes (BMs) and / or extracellular matrices (ECMs).

[0057] Certain embodiments relate to cells comprising polypeptides or nucleic acids of the disclosure. Cells of the present disclosure may comprise one or more therapeutic polypeptides or polynucleotides. In some embodiments, disclosed is a cell comprising one or more complexes. In some embodiments, the disclosed is a cell that further comprises CAR polypeptides. In some embodiments, a cell comprises a CAR polypeptide comprising a tumor antigen binding domain. Cells comprising a CAR polypeptide may, in certain embodiments, further comprise one or more additional therapeutic polypeptides and / or polynucleotides. Cells comprising a therapeutic polypeptide or polynucleotide of the present disclosure may further comprise one or more additional genetic modifications (e.g., genetic mutations, gene deletions, gene additions, etc.) which, in some embodiments, improve the efficacy or safety of a therapeutic cell. Certain non-limiting examples of such genetic modifications are described in: Puig-Saus and Ribas. Gene editing: towards the third generation of adoptive T cell transfer therapies. Immuno-Oncology Technology. 2019 June 13; 1 : 19-26, incorporated by reference herein in its entirety.

[0058] In some embodiments, a cell comprises one or more endopeptidases (e.g., matrix metalloproteinases), one or more bone sialoproteins (e.g., osteopontins), one or more genetically engineered receptors (e.g., a CAR), or a combination thereof.

[0059] In some embodiments, the cell is an immune cell, such as a granulocyte, myeloid derived suppressor cell, dendritic cell, macrophage, monocyte, T cell, natural killer (NK) cell, B cell, and / or plasma cell.

[0060] The cells of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the treatment is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.1. T Cells

[0061] In some embodiments, the immune cell is a T cell. “T cell” includes all types of immune cells expressing CD3 including, but not limited to, helper T cells (Th), invariant natural killer T (iNKT) cells, cytotoxic T cells, regulatory T cells (Tregs), alpha-beta (aP) T cells, and gamma-delta (y5) T cells. The T cell may refer to a CD4+or CD8+T cell.2. NK Cells

[0062] In some embodiments, the immune cell is a natural killer (NK) cell. “NK cell” includes any cell along the NK-cell hematopoietic lineage, including, for example, CD56bright, CD56dim(CD16+KIRs+), CD57+, CD56brightCD16-, and / or CD56dimCD16+NK cells. NK cells may be identified morphologically, e.g., by the presence of cytoplasmic lytic granules, or by the use of markers, such as, but not limited to, T-BET, EOMES, perforin, granzyme, CD56, CD57, CD16, CD94, KIR, and / or NKG2C.3. Mononuclear Phagocytes

[0063] In some embodiments, the immune cell is a mononuclear phagocyte. “Mononuclear phagocytes” includes any type of monocytes, macrophages, or dendritic cells (DC). Mononuclear phagocyte subtypes may be identified morphologically or by the use of markers. For example, macrophages may express CSF1, CSF2, and / or IL-34; monocytes may express CSF1, CSF2, CD14, CD16, NR4A1, CEBPp, KLF2, CD209a, and / or Ly6C; dendritic cells may express CD8, CD103, CDl lb, CD172a, CD141, BDCA1, BATF3, IRF4, and / or E2- 2. Monocytes may be CD14+CD16‘ (“classical”), CD14+CD16+(“intermediate”), or CD14lowCD16+(“non-classical”). DC may be conventional / classical (eDC) or plamacytoid DC (pDC), CD141+(“cDCl”), or BDAC1+(“cDC2”).4. Other Cells

[0064] Other suitable mammalian cells may include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC®) No. CCL- 2), CHO cells (e.g., ATCC® Nos. CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC® No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC® No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC® No. CCL10), PC12 cells (ATCC® No. CRL1721), COS cells, COS-7 cells (ATCC® No. CRL1651), RATI cells, mouse L cells (ATCC® No. CCLI.3), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0065] In some instances, the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell (e.g., peripheral blood stem cell) or progenitor cell obtained from an individual. The cell may be allogeneic, autologous, syngeneic, or xenogeneic with respect to an individual, including a recipient individual of the cell.IV. Nucleic Acids

[0066] Aspects of the disclosure include nucleic acids that may encode one or more endopeptidases (e.g., matrix metalloproteinases), one or more bone sialoproteins (e.g., osteopontins), one or more genetically engineered receptors (e.g., a CAR), or a combination thereof. In certain embodiments, nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, chimeric antigen receptor, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing described herein. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0067] The term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or noncoding sequences may, but need not, be present within a polynucleotide.

[0068] As will be understood by those in the art, the term “nucleic acid” or “polynucleotide” encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.

[0069] In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

[0070] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / orcan comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

[0071] In some embodiments, a nucleic acid of the disclosure comprises a sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 1-57.1. Polycistronic Expression Constructs

[0072] Polycistronic expression constructs allow the expression of a plurality of gene products from a single nucleic acid and are useful in many basic research and therapeutic applications. One beneficial feature of polycistronic expression constructs is the possibility to express two or more gene products from the same nucleic acid construct, achieving simultaneous expression of the two or more gene products in a target cell. In some aspects, a nucleic acid of the disclosure comprises a polycistronic expression construct.

[0073] To co-express multiple gene in the same polycistronic transcription unit, the polycistronic transcription unit may comprise an internal ribosome entry site (IRES) or a sequence coding for at least one protease cleavage site and / or self-cleaving peptide for polycistronic transcription. In some embodiments, a nucleic acid or polypeptide of the disclosure comprises an IRES, self-cleaving peptide sequence, and / or a protease cleavage site. a. Internal Ribosomal Entry Site (IRES)

[0074] In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819, each herein incorporated by reference). b. Self-cleaving Peptides

[0075] The self-cleaving peptides are 18-22 amino acids long 2A peptides that mediate ‘ribosomal skipping’ between the proline and glycine residues and inhibit peptide bond formation without affecting downstream translation. These peptides allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use ofthe term “self-cleaving” is not intended to imply proteolytic cleavage reaction. Self-cleaving peptides are found in members of the Picomaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV) and porcine teschovirus-1 (PTV-1) (Donnelly, M L, et al., J. Gen. Virol., 82, 1027-101 (2001); Ryan, M D, et al., J. Gen. Virol., 72, 2727-2732 (2001) and cardioviruses such as Theilovirus (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis viruses. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are sometimes referred to herein as “F2A”, “E2A”, “P2A”, and “T2A”, respectively. Aphthovirus 2A polypeptides are typically about 18-22 amino acids long and contain a DxlEx2NPG, where xl is often valine or isoleucine. As noted above, the 2A sequence is believed to mediate ‘ribosomal skipping’ between the proline and glycine, impairing normal peptide bond formation between the P and G without affecting downstream translation. The C terminus of cardiovirus 2A peptides is conserved, shows a high degree of similarity with FMDV 2A peptide, and has been shown to also mediate self-cleavage (Donnelly, M L, et al., J. Gen. Virol., 78, 13-21 (1997). FDMV 2A peptide has been shown to mediate cleavage of an artificial polyprotein (Ryan, M D and Drew, J., EMBO J., 13, 928-933 (1994). The ability to express four proteins efficiently and stoichiometrically from one polycistron in vivo was demonstrated using self-processing 2A peptides to express the four CD3 proteins (Szymczak et al., Nature Biotech. 5, 589-594, 2004). Polycistronic transgenes in which the individual cDNAs are separated by 2A peptides have been shown to promote polycistronic gene expression in transfected cells including human embryonic stem cells (Hasegawa, K., et al., Stem Cells. 2007 July; 25(7): 1707-12, 2007).

[0076] In some embodiments, a self-cleaving peptide of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 32.

[0077] In some embodiments, a self-cleaving peptide of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 80.c. Protease Cleavage Site

[0078] In certain embodiments of the invention, the use of one or more protease cleavage sites are used to create multiple proteins or protein fragments from one larger protein.

[0079] Furin protease cleavage site: As used herein, the term “furin protease cleavage site” (also referred to as “furin cleavage site” or “furin cleavage sequence”) refers to the amino acid sequence of a peptide or protein that serves as a recognition sequence for enzymatic protease cleavage by furin or furin-like proteases. Typically, a furin protease cleavage site has a consensus sequence Arg-X-X-Arg, wherein X is any amino acid. The cleavage site is positioned after the carboxy -terminal arginine (Arg) residue in the sequence. A furin cleavage site may have a consensus sequence Lys / Arg-X-X-X-Lys / Arg-Arg, wherein X is any amino acid. The cleavage site is positioned after the carboxy-terminal arginine (Arg) residue in the sequence.

[0080] Furin: As used herein, the term “furin” refers to any protease that can recognize and cleave the furin protease cleavage site as defined herein, including furin or furin-like protease. Furin is also known as paired basic amino acid cleaving enzyme (PACE). Furin belongs to the subtili sin-like proprotein convertase family. The gene encoding furin was known as FUR (FES Upstream Region).2. Expression

[0081] The nucleic acid molecules may be used to express recombinant polypeptides. Expression systems suitable for carrying out the present invention are presented herein, merely as example, and should not be understood to constitute the only possible or contemplated means of expression. One skilled in the art will recognize that a variety of different expression systems may be employed to produce the same or substantively similar results as those described herein.In some aspects, nucleic acids of the disclosure comprise a vector. In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of a desired sequence or a portion thereof (e.g., a fragment containing one or more MMPs, CARs, TCRs, and / or OPNs). a. Vectors

[0082] To express the polypeptides or peptides of the disclosure, DNAs encoding the polypeptides or peptides are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences. In some aspects, a vector that encodes a functionally sequence with appropriate restriction sites engineered so that any variable region sequences can be easily inserted and expressed. In some aspects, a vector that encodes a functionally complete MMP, OPN, and / or chimeric receptor can be easily inserted and expressed. Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using the same are well known in the art.

[0083] In some aspects, contemplated are expression vectors comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., an endopeptidases (e.g., matrix metalloproteinases), a bone sialoproteins (e.g., osteopontins), a genetically engineered receptor (e.g., a CAR), or a combination thereof.). Expression vectors comprising the nucleic acid molecules may encode the heavy chain, light chain, or the antigenbinding portion thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, cytokines, or combinations thereof. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0084] To express the polypeptides, DNAs encoding partial or polypeptides are inserted into expression vectors such that the gene area is operatively linked to transcriptional and translational control sequences. Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” typically include one or more of the following operatively linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, long-terminal repeat sequences, and a selectable marker element. Such sequences and methods of using the same are well known in the art.

[0085] The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters may include cisand trans-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and / or rate of transcription of a gene. For example, a promoter may be a cis-acting transcriptional control element, including an enhancer, a repressor binding sequence and the like. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on / off, regulate, modulate, etc.) gene transcription. Most often the core promoter sequences lie within 1-2 kilo-basepairs (kbp) of the translation start site, more often within 1 kbp and often within 500 basepairs (bp) or 200 bp or fewer, of the translation start site. By convention, promoter sequences are usually provided as the sequence on the coding strand of the gene it controls. In the context of this application, a promoter is typically referred to by the name of the gene for which it naturally regulates expression. Reference to a promoter by name includes a wild type, native promoter as well as variants of the promoter that retain the ability to induce expression. Reference to a promoter by name is not restricted to a particular species, but also encompasses a promoter from a corresponding gene in other species. A large number of promoters including constitutive, inducible and repressible promoters from a variety of different sources, are well known in the art (and identified in databases such as GenBank) and are available as or within cloned polynucleotides (from, e.g., repositories such as ATCC, and / or Addgene as well as other commercial or individual sources).

[0086] As used herein, a “constitutive promoter” refers to an unregulated promoter that allows for continual transcription of its associated gene in any suitable host cell or organism.

[0087] As used herein, an “inducible promoter” refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region.

[0088] In some embodiments, a promoter comprises a polynucleotide sequence derived from Moloney Murine Leukemia Virus (MMLV) promoter. In some embodiments, a nucleic acid of the disclosure comprises a promoter sequence.

[0089] In some embodiments, a promoter of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 33.

[0090] In some embodiments, a nucleic acid of the disclosure comprises a reporter gene. Examples of reporter genes include, but are not limited to, glutathione-5- transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP) and variations (e.g., emerald), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein(YFP), autofluorescent proteins (e.g., blue fluorescent protein (BFP)), and truncated CD 19 (tCD19).

[0091] In some embodiments, a reporter of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 34.

[0092] In some embodiments, a reporter of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 81.b. Expression Systems

[0093] Numerous expression systems exist that comprise at least a part or all of the expression vectors discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include in but are not limited to bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system. c. Methods of Gene Transfer

[0094] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolauet al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation / inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.(1) Transduction

[0095] As used herein the term "transduce" or "transduction" refers to the process whereby a foreign nucleotide sequence is introduced into a cell by means of a virus or viral vector. In some embodiments, this transduction is done via a viral vector.

[0096] In certain aspects, the term "viral vector" intends a recombinant vector that retains the ability to infect and transduce nondividing and / or slowly-dividing cells and may integrate into the target cell's genome. In some aspects, the vector is derived from or based on a wild-type virus. In some aspects, the vector is derived from or based on a wild-type lentivirus. Examples of such include without limitation, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (Hy). Alternatively, it is contemplated that other retrovirus can be used as a basis for a vector backbone such murine leukemia virus (MLV). It will be evident that a viral vector according to the disclosure need not be confined to the components of a particular virus. The viral vector may comprise components derived from two or more different viruses, and may also comprise synthetic components. Viral vector components may be manipulated to obtain desired characteristics, such as target cell specificity.

[0097] The recombinant viral vectors of this disclosure may be derived from primates and non-primates. Examples of primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype "slow virus" visna / maedi virus (VMV), as well as the related caprine arthritis-encephalitisvirus (CAEV), equine infectious anemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). Prior art recombinant lenti-viral vectors are known in the art, e.g., see U.S. Pat. Nos. 6,924,123; 7,056,699; 7,419,829 and 7,442,551, incorporated herein by reference.

[0098] In some embodiments, a nucleic acid of the disclosure comprises one or more viral vector components comprising a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 35-38.(2) Transfection

[0099] As used herein, the term "transfection" is defined as the introduction of an extracellular nucleic acid into a host cell by any means known in the art, including calcium phosphate co-precipitation, viral transduction, liposome fusion, microinjection, microparticlebombardment, electroporation, etc. The terms "uptake of nucleic acid by a host cell", "taking up of nucleic acid by a host cell", "uptake of particles comprising nucleic acid by a host cell", and "taking up of particles comprising nucleic acid by a host cell" denote any process wherein an extracellular nucleic acid, with or without accompanying material, enters a host cell.

[0100] A variety of methods are known in the art and suitable for transfection of nucleic acid into a cell. The polynucleotides of the present invention may be formulated, using the methods described herein. The formulations may contain polynucleotides which may be modified and / or unmodified. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and / or a sustained-release delivery depot.

[0101] The formulated polynucleotides may be delivered to the cell using routes of administration known in the art and described herein. Examples of typical methods include, but are not limited to, naked delivery, lipidoid mediate transfer, liposome-, lipoplexes, and / or lipid nanoparticle-mediated transfer, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. d. Host Cells

[0102] In another aspect, contemplated are the use of host cells into which a recombinant expression vector has been introduced. An expression construct encoding a polypeptide can be transfected into cells according to a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. In certain aspects, the polypeptide expression construct can be placed under control of a promoter that is linked to immune-cell activation, such as one that is controlled by NFAT-1 or NF-KB, both of which are transcription factors that can be activated upon immune-cell activation. In other embodiments, a promoter may be used to direct expression of the polypeptide in only those cell in which the promoter is active. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale productionof vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

[0103] For stable transfection of mammalian cells, it is known, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods known in the arts. e. Mutation

[0104] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antibody or antibody derivative, chimeric antigen receptor, etc.) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0105] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, e.g., Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.V. Polypeptide Expression

[0106] In some aspects, there are nucleic acid molecules encoding polypeptides or peptides of the disclosure, including one or more endopeptidases (e.g., matrixmetalloproteinases), one or more bone sialoproteins (e.g., osteopontins), one or more genetically engineered receptors (e.g., a CAR), or a combination thereof. In some aspects, there are nucleic acid molecules encoding polypeptides, endopeptidases (e.g., matrix metalloproteinases), bone sialoproteins (e.g., osteopontins), fusion binding proteins of any kind, including chimeric receptors of any kind, or antigen binding fragments of the disclosure. The nucleic acid molecules may be used to express polypeptides, including endopeptidases, bone sialoproteins, and / or chimeric polypeptides.

[0107] As used herein, a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, a “peptide” refers to a molecule comprising at least three amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects. In some aspects, a modified / variant protein or polypeptide may be a chimeric polypeptide, i.e., a combination of two or more domains of wild-type proteins.

[0108] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed or inserted. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., a chimeric antigen receptor (CAR), a chimeric T-cell receptor (cTCR), an endopeptidase (e.g., matrix metalloproteinases), a bone sialoprotein (e.g., osteopontins), or a combination thereof). The term “recombinant” or “heterologous” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and thisgenerally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0109] In certain aspects the size of a protein or polypeptide (wild-type or modified) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization (e.g., a marker, such as dsRed mono), for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.

[0110] The polypeptides or proteins of the disclosure may comprise, consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids, or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361,362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399,400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750, or more contiguous amino acids, or any range derivable therein, of SEQ ID NOs: 58-100.[OHl] In some aspects, a protein or polypeptide may comprise, consist essentially of, or consist of amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306,307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383,384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421,422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 (or any derivable range therein) of SEQ ID NOs: 58-100.

[0112] In some aspects, a protein or polypeptide may comprise, consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425,450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 (or any derivable range therein) contiguous amino acids of SEQ ID NOs: 58-100.

[0113] In some aspects, the polypeptide or protein may comprise, consist essentially of, or consist of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 (or any derivable range therein) contiguous amino acids of SEQ ID NOs: 58-100 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with one of SEQ ID NOs: 58-100.

[0114] In some aspects there is a polypeptide starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, , 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, , 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, , 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, , 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, , 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, , 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, , 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, , 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, , 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, , 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, , 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, , 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, , 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, , 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, , 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, , 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 of any of SEQ ID NOs: 58-100 and comprising at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, , 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, , 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, , 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, , 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, , 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, , 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, , 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, , 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, , 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, , 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318. , 319, 320, 321, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 (or any derivable range therein) contiguous amino acids of any of SEQ ID NOs: 58-100.

[0115] In some embodiments, provided herein are nucleotide as well as the protein, polypeptide, and peptide sequences for various genes. In certain embodiments, wild type copies of certain proteins, polypeptides, and / or nucleotides described herein have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank® and GenPept® databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

[0116] It is contemplated that in compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per mL. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / mL or more (or any range derivable therein).A. Variant Polypeptides

[0117] The following is a discussion of changing the amino acid subunits of a protein to create an equivalent, or even improved, second-generation variant polypeptide or peptide. For example, certain amino acids may be substituted for other amino acids in a protein or polypeptide sequence with or without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and nevertheless produce a protein with similar or desirable properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes which encode proteins without appreciable loss of their biological utility or activity.

[0118] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.

[0119] Amino acid sequence variants of the disclosure can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the disclosure may affect 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more noncontiguous or contiguous amino acids of the protein or polypeptide, as compared to a wildtype protein or a protein sequence provided herein. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0120] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3'sequences, respectively, and yet still be essentially identical as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0121] Deletion variants typically lack one or more residues of the native, wild type, or reference protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein.

[0122] Insertional mutants typically involve the addition of amino acid residues at a non-terminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0123] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein or polypeptide, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar chemical properties. “Conservative amino acid substitutions” may involve exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted forms of amino acid moieties.

[0124] Alternatively, substitutions may be “non-conservative”, such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting an amino acid residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.B. Considerations for Substitutions

[0125] One skilled in the art can determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan will also be able to identify amino acid residues and portions of the molecules that are conserved among similar proteins or polypeptides. In further embodiments, areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without significantly altering the biological activity or without adversely affecting the protein or polypeptide structure.

[0126] In making such changes, the hydropathy index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (—0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathy amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and others. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain embodiments, the substitution of amino acids whose hydropathyindices are within ±2 is included. In some aspects of the present disclosure, those that are within ±1 are included, and in other aspects of the present disclosure, those within ±0.5 are included.

[0127] It also is understood in the art that the substitution of like amino acids can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding, that is, as a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (—0.4); proline (-0.5+1); alanine (—0.5); histidine (—0.5); cysteine (—1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other embodiments, those which are within ±1 are included, and in still other embodiments, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitopic core regions.” It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0128] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0129] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since suchresidues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and / or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the world wide web at expasy . org / proteomi cs / protein_structure .

[0130] In some embodiments of the disclosure, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinities, and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native antibody).VI. Genetically Engineered Receptors

[0131] Immune cells of the present disclosure can be genetically engineered to express one or more antigen-binding receptors that target a cancer antigen, such as engineered CARs or, alternatively, engineered TCRs, or both. For example, the immune cells may be immune cells that are modified to express a CAR and / or TCR having antigenic specificity for GD2. Other CARs and / or TCRs may be expressed by the same cells as the GD2 antigen receptorexpressing cells, and they may be directed to different antigens. In some aspects, the immune cells are engineered to express the GD2-specific CAR and / or GD2-specific TCR by knock-in of the CAR or TCR using, for example, CRISPR / Cas technology.

[0132] Illustrative antigen receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers W0200014257,WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, W02013 / 123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and / or those described by Sadelain et al., 2013; Davila et al, 2013; Turtle et al, 2012; Wu et al, 2012. In some aspects, the genetically engineered antigen receptors include a CAR as described in U.S. Patent No. : 7,446, 190, and those described in International Patent Application Publication No. : WO / 2014055668 Al.

[0133] While CARs are encouraging therapies against cancer, CARs are limited to surface antigens and may have limited stimulatory capabilities. TCRs have properties that may help overcome some of the deficiencies of CARs. For example, TCRs can recognize any peptide that is processed for antigen presentation, and TCR signaling is 10 to 100 times more sensitive than CAR signaling (Harris et al., J Immunol. 2018 Feb l;200(3): 1088-1100.). For at least these reasons, TCR-based immunotherapy is a promising anti-cancer therapy, especially towards solid tumors.

[0134] TCRs are composed of aP (or y5) chain heterodimers that assemble at the cell membrane with the CD3 signaling complex (CD3sy, CD3s5, and CD3 Q. The a and P chains each comprise an extracellular immunoglobulin (Ig)-like domain comprising a variable region that provides antigen binding specificity and a constant domain, a transmembrane domain, and a short cytoplasmic region that lacks intracellular signaling motifs. Upon TCR binding to an antigen, immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 complex undergo phosphorylation and activate a downstream T cell signaling cascade.

[0135] Chimeric TCRs (cTCRs) combine the specificity of antigen binding domains, e.g., antibody scFv, with the ability of TCRs to engage endogenous signaling complexes (e.g., CD3 complex) to activate immune cells, e.g., T cells. cTCRs may comprise various structural configurations. In general, the endogenous variable region of a and / or p chains are replaced with an antigen binding domain of interest (e.g., a scFv targeting a cancer antigen) that is linked directly to the constant region of the a or P chains. In some instances, the entire antigen binding domain may be linked to one or both a and P chains, although the antigen binding domain may also be split amongst the two chains (e.g., an antibody VL chain may be linked to the a-chain while the VH may be linked to the P chain, or vice versa). In some instances, the a and / or pchains may be engineered to comprise one or more cysteine residues and / or one or more hydrophobic substitutions in the constant domain to enhance heterodimer stability and complex formation, such as those described in Cohen et al., Cancer Res. 2007 Apr 15; 67(8): 3898- 3903. and / or Jin et al., JCI Insight. 2018 Apr 19;3(8):e99488., both hereby incorporated by reference in their entirety. The antigen binding domain may also be linked to other components of the TCR complex, such as the y5 chains or the CD3 complex subunits, including the CD3y, CD35, and / or CD3s subunits.

[0136] Genetically engineered receptors of the disclosure may comprise one or more of: a signal peptide, an antigen binding domain, a peptide space, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, and / or a TCR chain.

[0137] In some embodiments, a CAR of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 39.

[0138] In some embodiments, a CAR of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 91.1. Signal peptide

[0139] Polypeptides and / or nucleic acids of the present disclosure may comprise a signal peptide. A “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g., to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface. In some embodiments, a signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if a receptor is to be glycosylated and anchored in the cell membrane. Generally, the signal peptide natively attached to the aminoterminal most component is used (e.g., in an scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).

[0140] In some embodiments, the signal peptide is cleaved after passage of the endoplasmic reticulum (ER), i.e., is a cleavable signal peptide. In some embodiments, a restriction site is at the carboxy end of the signal peptide to facilitate cleavage. In some aspects, a signal peptide is derived from CD8, CD27, granulocyte-macrophage colony-stimulating factor receptor (GMSCFR), 1g heavy chain, a killer cell immunoglobulin-like receptor (KIR), CD3, or CD4.

[0141] In some embodiments, a signal peptide of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 40-43.

[0142] In some embodiments, a signal peptide of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 92-95.2. Antigen binding domain

[0143] Polypeptides and / or nucleic acids of the present disclosure may comprise one or more antigen binding domains. An “antigen binding domain” describes a region of a polypeptide capable of binding to an antigen under appropriate conditions. In some embodiments, the antigen binding domain comprises a nanobody, truncated extracellular domain of a ligand, antibody, Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, triabody, tetrabody, multispecific antibody formed from antibody fragments, single-domain antibody (sdAb), single chain comprising complementary scFvs (tandem scFvs), bispecific tandem scFvs, trispecific tandem scFvs, Fv construct, disulfide-linked Fv, dual variable domainimmunoglobulin (DVD-Ig) binding protein, nanobody, aptamer, affibody, affilin, affilin based on ubiquitin, affilin based on gamma-B-crystalin, D-domain based on alpha3D, affitin, affimer, alphabody, anticalin, avimer, ADNECTIN™, Knottin, designed ankyrin repeat proteins, Nanofitin, DARPin, centyrin, Fynomer, Kunitz domain peptide, monobody, or a combination thereof.

[0144] In some embodiments, an antigen binding domain is a single-chain variable fragment (scFv) based on one or more antibodies (e.g., GD2 antibodies). In some embodiments, an antigen binding domain comprise a variable heavy (VH) region and a variable light (VL) region, with the VH and VL regions being on the same polypeptide. In some embodiments, the antigen binding domain comprises a linker between the VH and VL regions. A linker may enable the antigen binding domain to form a desired structure for antigen binding.

[0145] The variable regions of the antigen-binding domains of the polypeptides of the disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term “CDR” refers to a complementarity-determining region that is based on a part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B cells and T cells respectively, where these molecules bind to their specific antigen. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.

[0146] Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence.

[0147] It is also contemplated that the antigen binding domain may be multi-specific or multivalent by multimerizing the antigen binding domains, e.g., VH and VL region pairs that bind either the same antigen (multi -valent) or a different antigen (multi-specific).- I l l -

[0148] The binding affinity of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10'5M, 10'6M, 10'7M, 10'8M, 10'9M, 10'10M, 10-11M, 10'12M, or 10'13M. In some embodiments, the Ko of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10’5M, 10’6M, 10’7M, 10’8M, 10’9M, 10’10M, lO^M, 10'12M, or 10'13M (or any derivable range therein).

[0149] Binding affinity, KA, or KD can be determined by methods known in the art such as by surface plasmon resonance (SRP)-based biosensors, by kinetic exclusion assay (KinExA), by optical scanner for microarray detection based on polarization-modulated oblique-incidence reflectivity difference (OI-RD), or by ELISA.

[0150] In some embodiments, the antigen binding domain targets disialoganglioside 2 (GD2), epidermal growth factor receptor (EGFR), variant III of the epidermal growth factor receptor (EGFRvIII), human epidermal growth factor receptor 2(HER2), mesothelin, prostatespecific membrane antigen (PSMA), carcinoembryonic antigen (CEA), Interleukin- 13Ra2 (IL- 13Ra2), Glypican-3 (Gpc3), carbonic anhydrase IX (CAIX), LI cell adhesion molecule (LI- CAM), cancer antigen 125 (CA 125), Cluster of differentiation 133 (CD 133), CD19, fibroblast activation protein (FAP), cancer / testis antigen IB(CTAGIB), mucin 1 (Muc-1), Folate receptor-a (FR-a), CD70, 5T4, 8H9, avp6integrin, BCMA, B7-H3, B7-H6, CA9, CD5, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD47, CD 123, CD 138, CD171, CEA, CSPG4, CS1, CLL1, CD99, DLL3, , EGFR family including ErbB2 (HER2), EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, FBP, fetal AchR, GD3, HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-l lRa, Lambda, Lewis- Y, Kappa, KDR, MCSP, Mesothelin, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSC1, PSCA, ROR1, SP17, Survivin, TAG72, TIM3, TIGIT, TROP2, TEMs, HMW-MAA, VEGFR2, or any combination thereof.

[0151] In some embodiments, an antigen binding domain of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 44.

[0152] In some embodiments, an antigen binding domain of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 96.3. Hinge

[0153] Polypeptides and / or nucleic acids of the present disclosure may comprise one or more hinge domains. As used herein, the term “hinge” refers to a flexible polypeptide connector region (also referred to herein as “hinge region”) providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides. A “hinge” derived from an immunoglobulin (e.g., IgGl) is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton (1985) Molec. Immunol., 22: 161- 206). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions. The hinge region may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region as described in U.S. Pat. No. 5,677,425, incorporated by reference herein. The hinge region can include a complete hinge region derived from an antibody of a different class or subclass from that of the CHI domain. The term “hinge” can also include regions derived from CD8 and other receptors that provide a similar function in providing flexibility and spacing to flanking regions. In some embodiments, a hinge is flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen binding. In some aspects, the hinge comprises a region derived from CD30, IgGl, IgG2, IgG3, IgG4, or CD28. In some embodiments, the spacer comprises or further comprises the CH2- CH3 region of immunoglobulin and portions of CD3. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res.

[0154] The length of a hinge may have effects on the CAR’s signaling activity and / or the CAR-T cells’ expansion properties in response to antigen-stimulated CAR signaling. In some embodiments, a shorter spacer such as less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids is used. In some embodiments, a longer spacer, such as one that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,250, 260, 270, 280, or 290 amino acids may have the advantage of increased expansion in vivo or in vitro. In some embodiments, a hinge can have a length of at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120,130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280,290, 300, 325, 350, or 400 amino acids (or any derivable range therein).

[0155] When the hinge comprises multiple parts, there may be anywhere from 0-50 amino acids in between the various parts. For example, there may be at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any derivable range therein) between the hinge and the CH2 or CH3 region or between the CH2 and CH3 region when both are present. In some embodiments, the hinge comprises a CH2, and / or CH3 region.

[0156] In some embodiments, a hinge of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 45.

[0157] In some embodiments, a hinge of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 97.4. Transmembrane domain

[0158] Polypeptides and / or nucleic acids of the present disclosure may comprise a transmembrane domain. In some embodiments, a transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0159] In some embodiments, the transmembrane domain is interposed between the hinge or the antigen binding domain and the intracellular region. In some embodiments, the transmembrane domain is interposed between the hinge or antigen binding domain and a costimulatory domain.

[0160] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some embodiments, the transmembrane domain is derived from CD28, CD8, CD4, CD3-zeta (CD3Q, CD134, or CD7.

[0161] In some embodiments, a transmembrane domain of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 46.

[0162] In some embodiments, a transmembrane domain of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 98.5. Cytoplasmic region

[0163] Polypeptides and / or nucleic acids of the present disclosure may comprise one or more cytoplasmic regions. After antigen recognition, receptors of the present disclosure may cluster and a signal transmitted to the cell through a cytoplasmic region. In some embodiments, a cytoplasmic region comprises one or more costimulatory domains and / or one or more intracellular signaling domains (e.g., ITAM-comprising intracellular signaling domain). In some embodiments, the cytoplasmic region comprises an intracellular signaling domain.

[0164] Co-stimulatory domains and / or intracellular signaling domains suitable for use in the polypeptides of the disclosure include any domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation, e.g., by way of binding of the antigen to the antigen binding domain. In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) IT AM motif as described herein.

[0165] Intracellular signaling domains suitable for use in the polypeptides of the disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. An ITAM motif is YX1X2(L / I), where XI and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e.g., (YXlX2(L / I))(X3)n(YXlX2(L / I)), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.

[0166] A suitable intracellular signaling domains may be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3-zeta; and CD79A (antigen receptorcomplex-associated protein alpha chain). Suitable intracellular signaling domains are known in the art.

[0167] In some embodiments, a suitable intracellular signaling domain can comprise an ITAM motif-containing portion of DAP12, FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain, Fc receptor gamma-chain, fc-epsilon R1 -gamma, fcRgamma, fceRI gamma, high affinity immunoglobulin epsilon receptor subunit gamma, immunoglobulin E receptor, high affinity gamma chain, T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD35; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). In some embodiments, an intracellular signaling domain is derived from CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD32, Fc receptor common chain, CD27, CD28, CD79, 4-1BB, DAP12, NKG2D, OX-40 (CD134), DAP10, CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46, or NKp80.

[0168] In some embodiments, an intracellular signaling domain of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 47.

[0169] In some embodiments, an intracellular signaling domain of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 99.6. Costimulatory domain

[0170] Polypeptides and / or nucleic acids of the present disclosure may comprise one or more costimulatory domains. Non-limiting examples of suitable costimulatory regions, such as those included in the cytoplasmic region, include, but are not limited to, polypeptides from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0171] A costimulatory region may have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids or any range derivable therein. In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA; etc.). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, 0X40, TXGP1L). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, T14, TNFRSF7, and Tp55). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some embodiments, the costimulatory region is derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). In some embodiments, the costimulatory region derived from anintracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3- 395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2).

[0172] In some embodiments, a co-stimulatory domain of the disclosure comprises a nucleic acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 48.

[0173] In some embodiments, a costimulatory domain of the disclosure comprises an amino acid sequence at least or equal to, exactly or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NO: 100.7. TCR chains

[0174] Polypeptides and / or nucleic acids of the present disclosure may comprise one or more TCR chains. A TCR chain, such as the a, P, y, or 5 chains, may be used to generate cTCRs. In some embodiments, the TCR chains are derived from mouse TCR chains. The TCR chains may be humanized. In some embodiments, the TCR chains are a and P chains. In some embodiments, the chains are engineered to comprise one or more cysteine residues and / or one or more hydrophobic substitutions in the constant domain to enhance heterodimer stability and complex formation, such as those described in Cohen et al., Cancer Res. 2007 Apr 15; 67(8):3898-3903. and / or Jin et al., JCI Insight. 2018 Apr 19;3(8):e99488., both hereby incorporated by reference in their entirety.VII. Cancer Therapy

[0175] In some aspects, the disclosed methods comprise administering a cancer therapy to a subject, individual, or patient. In some embodiments, the disclosed methods are not limited to use as a cancer therapeutic, but may also be utilized in any immunotherapy in which modulation of the activation of an immune effector cell is desired, such as but not limited to, autoimmune disease and / or infectious disease (e.g., bacterial, viral, protozoan, etc.). The cancer therapy may be chosen based on an expression level measurement, alone or in combination with a clinical risk score calculated for the subject. The cancer therapy may be chosen based on a genotype of a subject. In some aspects, the cancer therapy comprises a local cancer therapy. In some aspects, the cancer therapy excludes a systemic cancer therapy. In some aspects, the cancer therapy excludes a local therapy. In some aspects, the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy. In some aspects, the cancer therapy comprises an immunotherapy, which may be a checkpoint inhibitor therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.

[0176] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain aspects, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some aspects, the cancer is a Stage I cancer. In some aspects, the cancer is a Stage II cancer. In some aspects, the cancer is a Stage III cancer. In some aspects, the cancer is a Stage IV cancer.

[0177] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinomain adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchi olo-alveol ar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing’s sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0178] In some aspects, the cancer is breast cancer. In some aspects, the cancer is HER2+ breast cancer.

[0179] In some aspects the cancer is brain cancer. In some aspects the cancer is a GD2 positive brain cancer. In some aspects, the cancer is glioblastoma, neuroblastoma, and / or medulloblastoma.

[0180] In some aspects the cancer is a blood cancer. In some aspects, the cancer is leukemia, acute myeloid leukemia, chronic myeloid leukemia, lymphoma, Non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, myeloma, myelodysplastic syndromes, myelodysplastic neoplasms.

[0181] Methods may involve the determination, administration, or selection of an appropriate cancer “management regimen” and predicting the outcome of the same. As used herein the phrase “management regimen” refers to a management plan that specifies the type of examination, screening, diagnosis, surveillance, care, and treatment (such as dosage, schedule and / or duration of a treatment) provided to a subject in need thereof (e.g., a subject diagnosed with cancer).A. Radiotherapy

[0182] In some aspects, a radiotherapy, such as ionizing radiation, is administered to a subject. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). A non-limiting example of ionizing radiation is x- radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.

[0183] In some aspects, the radiotherapy can comprise external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT). In some aspects, the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy. In some aspects, the internal radiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy. In some aspects, the radiotherapy is administered to a primary tumor.

[0184] In some aspects, the amount of ionizing radiation is greater than 20 gray (Gy) and is administered in one dose. In some aspects, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some aspects, the amount of ionizing radiation is at least, at most, or exactly 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range therein). In some aspects, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein). When more than one dose is administered, the dose may be about1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.

[0185] In some aspects, the amount of radiotherapy administered to a subject may be presented as a total dose of radiotherapy, which is then administered in fractionated doses. For example, in some aspects, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some aspects, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some aspects, the total dose of radiation is at least, at most, or about 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 Gy (or any derivable range therein). In some aspects, the total dose is administered in fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein). In some aspects, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionated doses are administered (or any derivable range therein). In some aspects, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some aspects, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.B. Cancer Immunotherapy

[0186] In some aspects, the methods comprise administration of a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can, in some cases, be categorized as active, passive or hybrid (i.e., active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and / or cytokines. Various immunotherapies are known in the art, and certain examples are described below.C. Checkpoint Inhibitors and Combination Treatment

[0187] Aspects of the disclosure may include administration of immune checkpoint inhibitors, examples of which are further described below. As disclosed herein, “checkpoint inhibitor therapy” (also “immune checkpoint blockade therapy,” “checkpoint blockade therapy,” “immune checkpoint therapy,” “ICT,” “checkpoint blockade immunotherapy,” or “CBI”), refers to cancer therapy comprising providing one or more immune checkpoint inhibitors to a subject suffering from or suspected of having cancer.1. PD-1, PDL1, and PDL2 inhibitors

[0188] PD-1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role ofPD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0189] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc, and CD273. In some aspects, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0190] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another aspect, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U. S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0191] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some aspects, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP -224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 andWO201 1 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0192] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0193] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.2. CTLA-4, B7-1, and B7-2

[0194] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4 or CTLA4), also known as CD 152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number LI 5006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA- 4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some aspects, the inhibitor blocks theCTLA-4 and B7-1 interaction. In some aspects, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0195] In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0196] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0197] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WO 01 / 14424).

[0198] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7- 2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.3. LAG3

[0199] Another immune checkpoint that can be targeted in the methods provided herein is the lymphocyte-activation gene 3 (LAG3), also known as CD223 and lymphocyte activating3. The complete mRNA sequence of human LAG3 has the Genbank accession number NM 002286. LAG3 is a member of the immunoglobulin superfamily that is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG3’s main ligand is MHC class II, and it negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1, and has been reported to play a role in Treg suppressive function. LAG3 also helps maintain CD8+T cells in a tolerogenic state and, working with PD-1, helps maintain CD8 exhaustion during chronic viral infection. LAG3 is also known to be involved in the maturation and activation of dendritic cells. Inhibitors of the disclosure may block one or more functions of LAG3 activity.

[0200] In some aspects, the immune checkpoint inhibitor is an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0201] Anti-human-LAG3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-LAG3 antibodies can be used. For example, the anti-LAG3 antibodies can include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781. The anti-LAG3 antibodies disclosed in: US 9,505,839 (BMS-986016, also known as relatlimab); US 10,711,060 (IMP-701, also known as LAG525); US 9,244,059 (IMP731, also known as H5L7BW); US 10,344,089 (25F7, also known as LAG3.1); WO 2016 / 028672 (MK-4280, also known as 28G-10); WO 2017 / 019894 (BAP050); Burova E., et al., J. ImmunoTherapy Cancer, 2016; 4(Supp. 1):P195 (REGN3767); Yu, X., et al., mAbs, 2019; 11 :6 (LBL-007) can be used in the methods disclosed herein. These and other anti-LAG-3 antibodies useful in the claimed invention can be found in, for example: WO 2016 / 028672, WO 2017 / 106129, WO 2017062888, WO 2009 / 044273, WO 2018 / 069500, WO 2016 / 126858, WO 2014 / 179664, WO 2016 / 200782, WO 2015 / 200119, WO 2017 / 019846, WO 2017 / 198741, WO 2017 / 220555, WO 2017 / 220569, WO 2018 / 071500, WO2017 / 015560; WO 2017 / 025498, WO 2017 / 087589 , WO 2017 / 087901, WO 2018 / 083087, WO 2017 / 149143, WO 2017 / 219995, US 2017 / 0260271, WO 2017 / 086367, WO2017 / 086419, WO 2018 / 034227, and WO 2014 / 140180. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 also can be used.

[0202] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-LAG3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.4. TIM-3

[0203] Another immune checkpoint that can be targeted in the methods provided herein is the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2) and CD366. The complete mRNA sequence of human TIM-3 has the Genbank accession number NM_032782. TIM-3 is found on the surface IFNy- producing CD4+Thl and CD8+Tel cells. The extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of variable length located closer to the membrane. TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, it mediates T-cell exhaustion. TIM-3 has also been shown as a CD4+Thl-specific cell surface protein that regulates macrophage activation. Inhibitors of the disclosure may block one or more functions of TIM-3 activity.

[0204] In some aspects, the immune checkpoint inhibitor is an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0205] Anti-human-TIM-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-TIM-3 antibodies can be used. For example, anti-TIM-3 antibodies including: MBG453, TSR-022 (also known as Cobolimab), and LY3321367 can be used in the methods disclosed herein. These and other anti-TIM-3 antibodies useful in the claimed invention can be found in, for example: US 9,605,070, US 8,841,418, US2015 / 0218274, and US 2016 / 0200815. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to TIM-3 also can be used.

[0206] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-TIM-3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range or value therein) variable region amino acid sequence identity with the above-mentioned antibodies.D. Activator of co-stimulatory molecules

[0207] In some aspects, the immunotherapy comprises an activator (also “agonist”) of a co-stimulatory molecule. In some aspects, the agonist comprises an agonist of CD3, B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Agonists include activating antibodies, polypeptides, compounds, and nucleic acids.E. Chemotherapies

[0208] In some aspects, a therapy of the present disclosure comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5 -fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhy diazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some aspects, cisplatin is a particularly suitable chemotherapeutic agent.

[0209] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be deliveredvia other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection.

[0210] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g.. Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain aspects, appropriate intravenous doses for an adult include about 60 mg / m2to about 75 mg / m2at about 21-day intervals or about 25 mg / m2to about 30 mg / m2on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg / m2once a week.

[0211] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria, is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, intravenous doses include, for example, initially about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days or about 3 mg / kg to about 5 mg / kg twice a week or about 1.5 mg / kg / day to about 3 mg / kg / day. Because of adverse gastrointestinal effects, the intravenous route is preferred in certain cases. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0212] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5 -fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode-oxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0213] The amount of the chemotherapeutic agent delivered to a patient may be variable. In one suitable aspect, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other aspects, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than thechemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.F. Hormone therapy

[0214] In some aspects, a cancer therapy of the present disclosure is a hormone therapy.In particular aspects, a prostate cancer therapy comprises hormone therapy. Various hormone therapies are known in the art and contemplated herein. Examples of hormone therapies include, but are not limited to, luteinizing hormone-releasing hormone (LHRH) analogs, LHRH antagonists, androgen receptor antagonists, and androgen synthesis inhibitors.G. Surgery

[0215] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present aspects, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0216] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.H. Additional cancer therapies

[0217] Therapeutic methods disclosed herein may comprise one or more additional cancer therapies. A cancer therapy of the disclosure may comprise, for example, cryoablative therapy, high-intensity ultrasound (also “high-intensity focused ultrasound”), photodynamic therapy, laser ablation, and / or irreversible electroporation. A cancer therapy of the disclosure may comprise 1, 2, 3, 4, 5, or more distinct therapeutic methods.

[0218] It is contemplated that a cancer treatment may exclude any of the cancer treatments described herein. Furthermore, aspects of the present disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein. In some aspects, the patient is one that has been determined to be resistant to a therapy described herein. In some aspects, the patient is one that has been determined to be sensitive to a therapy described herein.VIII. Kits

[0219] Certain aspects of the present disclosure also concern kits comprising compositions of the disclosure or compositions to implement methods of the disclosure. In particular embodiments, the kit comprises cells, fresh or frozen, and that may or may not have been pre-activated or expanded. The cells may or may not already express one or more components of the elements encompassed herein, such as cells, vectors, buffers, primers, enzymes, salts, and so forth. The kit may comprise one or more reagents for transfection or transduction of cells, including reagents such as vectors that express the component s), primers for amplification of the component(s), and so forth. In some cases, the cells may or may not also express one or more complex as defined herein, and when they do not, the kit may comprise vectors that express the complex(es), primers for amplification of the complex(es), and so forth. In specific embodiments, the kit comprises one or more polynucleotides that express one or more MMPs, one or more OPNs, and / or one or more chimeric receptors, including CARs and / or TCRs. The kit may comprise cells that express one or more MMPs, one or more OPNs, and / or one or more chimeric receptors, including CARs and / or TCRs.

[0220] Kits may comprise components that may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means. Individual components may also be provided in a kit in concentrated amounts; in some embodiments, acomponent is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more, as examples.EXAMPLES

[0221] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow present techniques discovered by the inventors to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.EXAMPLE 1 - ENHANCING INFILTRATION OF CAR-T CELLS INTO SOLID TUMORSBackground

[0222] While chimeric antigen receptor T cell (CAR-T) therapy has been successful at treating select hematologic malignancies, success in the solid tumor setting has been limited. A significant factor in the failure of CAR-T therapy in this setting is the low infiltration of CAR-Ts into tumors due to the exclusionary nature of the tumor-specific extracellular matrix (ECM) and basement membrane (BM), which encapsulate the tumor (FIG. 1A). Tumor encapsulation occurs when cancer associated fibroblasts (CAFs) recruited to the tumor secrete large volumes of ECM fibers, including type I and type IV collagen, while simultaneously secreting enzymes that crosslink these fibers into a dense, rigid structure. Unlike the ECM and BM of healthy tissues, which has a loose basket-weave pattern that T cells easily traverse, tumor-specific matrices are stiff and characterized by small pores, leading to T cell exclusion (FIG. 1A)

[0223] T cell infiltration requires cell movement and enzymatic matrix degradation: T cell infiltration into tumors relies upon three main factors: 1) adhesion molecule-mediated cell movement, 2) cytoskeletal rearrangements, and 3) the enzymatic degradation of matrix fibers. T cells move to the site of a tumor through the circulation, then use rolling, firm adhesion, andtransmigration to extravasate through the BM into the interstitial space. Within tissues, T cells change shape by rapidly reorganizing their cortical actin cytoskeletons to travel to the tumor by “flowing and squeezing” or crawling along ECM fibers.

[0224] In tumor tissue sections, the inventors observed that T cells were aggregated within the ECM surrounding the tumor (blue, dotted line) and did not exhibit efficient infiltration (FIG. IB).

[0225] The inventors analyzed data sets from three clinical studies sampling different tumor types for gene expression. The results showed that SPP1, a gene that encodes the adhesion molecule osteopontin, was the most differentially expressed gene in tumor-infiltrating leukocytes compared to those that are tumor-adjacent. Further, OPN expression in solid tumors was positively correlated with increased invasion and metastasis. In addition, preliminary geneexpression data showed that MMP-7 and MMP-14 were highly upregulated in tumorinfiltrating leukocytes. Taken together, these data suggested that OPN and / or MMPs play an important role in the movements required for travel through the tumor-specific ECM and BM.

[0226] The inventors engineered vectors to express heterologous OPN isoforms or MMPs in T cells (e.g., SEQ ID NOs: 23-31, 49-57; e.g., constructs depicted in FIG. 12). Vectors comprised a protein of interest (e.g., OPN isoforms or MMPs) were operatively linked by a self-cleaving sequence (e.g., T2A) to a reporter gene (e.g., GFP or tCD19). The results showed that T cells can be engineered with vectors comprising expression of OPN-A, OPN-B, OPN-C, or OPN-I (FIG. 2A). The results also showed that T cells can be engineered with vectors comprising expression of MMP7 or MMP14 (FIG. 2B). Flow cytometry of engineered cells showed a high proportion of engineered cells expressing the reporter gene (FIG. 2C).

[0227] The inventors used enzyme linked immunosorbent assay (ELISA) to assess the expression levels of OPN or MMP by engineered T cells. Cells were plated at le6 cells per well and incubated for 24 hours. The results showed that cells engineered to express OPN secreted more OPN compared to controls (FIGs. 3A). The results showed that cells engineered to express MMP secreted more MMP compared to controls (FIGs. 3B). Statistical significance was determined using an unpaired t-test, p<0.05.

[0228] To assess the tumor infiltration capabilities of T cells, the inventors used a solubilized basement membrane matrix (Matrigel), which mimics type IV collagen, and a permeable cell support (Transwell) to measure migration in vitro. A mixture of T cellsresuspended in solubilized basement membrane matrix, was layered on top of a permeable cell support and allowed to polymerize. The T cells were resuspended in increasing concentrations of solubilized basement membrane matrix (e.g., 300 ug / mL, 400 ug / mL, 500 ug / mL, and 700 ug / mL). After polymerization, a chemoattractant was added to the bottom chamber to activate the T cells. Twenty-four hours later, activated T cells (ATC) that invaded the bottom chamber were collected for counting by flow cytometry using counting beads. The results showed that as the density of solubilized basement membrane matrix increased, the invasion of ATCs decreased (FIG. 4A).

[0229] The inventors then tested the invasion capabilities of T cells engineered to comprise a CAR (e.g., GD2-41BBZ, or GD2-28Z). In this assay, non-engineered cells (e.g., untransduced) and cells engineered to comprise a CAR (CAR-T cells) were resuspended in 3 mg / mL of solubilized basement membrane matrix (e.g., Matrigel) — a concentration that mimics the ECM density in a solid tumor. After 24 hours, the proportional invasion of the CAR-T cells through the solubilized basement membrane matrix was compared to the invasion of the non-engineered T cells (FIG. 4B; y-axis, proportional invasion). Proportional invasion represents a fold-change of CAR-T cells over non-engineered cells from the same donor. The results showed that the CAR-T cells did not invade as well as the non-engineered cells (FIG. 4B) Statistical significance was determined using an unpaired t-test, p<0.05.

[0230] Next, the inventors assessed the effect of heterologous expression of OPN and MMP in invasion, as aforementioned. After 24 hours, the proportional invasion of the engineered cells through the solubilized basement membrane matrix (e.g., Matrigel) was compared to the invasion of the non-engineered T cells (FIGs. 4C-4D; y-axis, proportional invasion). The results showed that heterologous expression of OPN-A, OPN-B, OPN-C, OPN- I, or MMP7 improved the invasion capabilities of T cells compared to the controls (FIGs. 4C- 4D). 4D

[0231] Next, the inventors generated genetically engineered cells that expressed both a CAR and heterologous OPN or MMP (using, for example, nucleic acids of SEQ ID NOs: 54- 57; e.g., constructs depicted in FIG. 12). To this end, ATC were transduced with both construct by mixing viral supernatants. Transduction was measured by flow cytometry 5 days postactivation. GD2-CAR expression was measured using the anti -idiotype antibody 1A7, while OPN (e.g., OPN-B) and MMP (e.g., MMP-7, or MMP-14) transduction was measured by the presence of a reported gene (e.g., tCD19, or GFP). The results showed that T cells engineeredto comprise heterologous expression of both a CAR and an OPN or MMP can be achieved (FIGs. 5A-5B)

[0232] The inventors measured the production of OPN or MMP in T cells engineered to comprise heterologous expression of both a CAR and OPN or MMP (double engineered cells) using ELISA. The results showed that double engineered cells produced higher levels of OPN or MMP than single engineered cells (e.g., CAR T cells) or non-engineered cells (e.g., untransduced) (FIGs. 6A-6B).

[0233] To assess the effects of engineering T cells to express a CAR, OPN, MMP or a combination of a CAR with OPN or MMP, the inventors measured expansion, toxicity, and T cell phenotype of the engineered cells. The results showed that engineering to express heterologous OPN or MMP in CAR T cells did not affect expansion (FIGs. 7A-7B), cytotoxicity (FIG. 7C), or cellular phenotype (FIGs. 7D-7E), compared to CAR T cells.

[0234] The invasion of double engineered T cells (e.g., CAR T cells with heterologous expression of an OPN or an MMP) was assessed against controls (e.g., CAR T cells or nonengineered cells), as previously discussed. After 24 hours, the proportional invasion of the double engineered T cells through the solubilized basement membrane matrix (e.g., Matrigel) was compared to the invasion of the control cells (FIG. 8A-8B; y-axis, proportional invasion). The results showed that CAR T cells engineered to express heterologous MMP7, MMP14, or OPN-B were more invasive than CAR T cells by about 4-fold (FIG. 8A). The results also showed that T cells engineered to express heterologous OPN-A, OPN-B, OPN-C, or OPN-I were more invasive than non-engineered T cells (FIG. 8B). These results suggested that heterologous expression of OPN and / or MMP may confer an infiltration advantage into solid tumors in vivo that would translate to increased tumor clearance and survival. To further investigate this question, the inventors used an in-vitro model of invasion called a Halo assay, in which a core of tumor cells is plated with a ring of T cells separated by a space (FIG. 8C). This assay models extravasation and tumor infiltration. Live imaging shows a separation between tumor cells and T cells at plating (FIG. 8D) and cell movement from the basement membrane to the tumor droplet and tumor infiltration after at least 24 hours (FIG. 8E). Quantification of cells across the object area over time showed increased migration of the CAR-T cells engineered to express a heterologous OPN or MMP compared to CAR T cells or non-engineered cells (FIGs. 8F-8G).

[0235] Encouraged by the in vitro studies, the inventors assessed the effect of CAR T cells engineered to express a heterologous OPN or MMP in tumor growth in vivo using a xenograft mouse model of cancer. NSG mice were injected subcutaneously with 2e6 FFLuc- LAN-1 tumor cells, and 0.8 x 0.8 cm tumors were established. Five mice per group were then injected intravenously with 3.5e6 of T cells comprising: a GD2 CAR (GD2-28Z), a GD2 CAR and heterologous MMP7 (MMP7.GD2-28Z), a GD2 CAR and heterologous MMP14 (MMP14.GD2-28Z), or a GD2 CAR and heterologous OPN-B (OPN-B.GD2-28Z), and tumors were measured every other day with calipers. Tumor growth was measured at days 7 and 10 (FIG. 9A) or at days 1, 4, 7, 11, 14, 18, 21, 25, and 28 (FIG. 9B) using bioluminescence imaging. Bioluminescence imaging of the three tumor-free mice shows radiance of 1-2 logs lower than mice in the GD2-28Z condition (FIG. 9A). When tumors reached 1.5 cm in diameter, mice were sacrificed and tumors were collected, measured, and fixed for staining. The results showed that CAR T cells engineered to express heterologous OPN-B had increased antitumor activity compared to all other groups (FIGs. 9A-9B).

[0236] In addition, the inventors assessed the antitumor effects of CAR T cells engineered to express a heterologous OPN (OPN-GD2.CAR) or MMP (MMP7-GD2.CAR) compared to CAR T cells (GD2.CAR) in a xenograft mouse model of neuroblastoma in which NSG mice were subcutaneously injected with 3 x 106LAN-1 FFLuc tumor cells, and approximately ten days later they were treated with 3.5 x 106CAR T cells by tail vein injection (FIG. 10A). The results showed a decrease in tumor burden over time (FIG. 10B), a significant decrease in tumor burden at day 29 post T cell infusion (FIG. 10C), and an extension in survival (FIG. 10D) for mice treated with our OPN-GD2.CAR T cells compared to the other groups.

[0237] To assess the expansion and migration of engineered T cells in vivo, the inventors used a xenograft mouse model of neuroblastoma in which NSG mice were subcutaneously injected with 3 x 106LAN-1 WT tumor cells, and approximately ten days later they were treated by tail vein injection with 3.5 x 106of CAR T cells (GD2.CAR), CAR T cells comprising heterologous expression of MMP7 (MMP7-GD2.CAR), or CAR T cells comprising heterologous expression of OPN (OPN-GD2.CAR), and firefly luciferase (FIG. 11 A). The results showed that T cells from all groups expanded at similar rates (FIG. 11B), but more OPN-GD2.CAR T cells and MMP7-GD2.CAR T cells were found in the tumor compared to GD2.CAR by flow cytometry (FIG. 11C), and tissue immunohistochemistry(FIG. HD) These results showed that heterologous expression of MMP7 or OPN increased the invasion properties of CAR T cells (e.g., anti-GD2 CAR T cells). Examples of Methods

[0238] T cell engineering. Activated T cells were retrovirally transduced to express a GD2-28Z CAR and MMP-7 (MMP7.GD2-28Z), a GD2-28Z CAR and MMP-14 (MMP14.GD2-28Z), or a GD2-28Z CAR and OPN (OPN.GD2-28Z). The expansion, phenotype and cytotoxicity of gene-modified GD2 CAR-Ts were evaluated using flow cytometry quantification of CD45RO and CCR7 expression. A high rate of engineering (e.g., double-transduction) was achieved with a mean of 68% or higher in all conditions.

[0239] Infiltration assays. Infiltration was assessed with permeable cell support (Transwell) invasion assays. The solubilized basement membrane matrix (Matrigel) invasion assay used high-concentration Matrigel (type IV collagen) to measure extravasation through the tumor-specific Basement Membrane (BM). T cells were resuspended in different concentrations of solubilized basement membrane matrix (Matrigel) and layered in the top of the permeable cell support (Transwell). T cells migrated to a chemoattractant (FBS) at the bottom of the well, and the number of cells that infiltrated through the matrix into the bottom of the well was measured after 24 hours.

[0240] Halo invasion assays combined BM extravasation and ECM infiltration. The innovative Halo assay was recently developed by a group at St. Jude and combined aspects of movement through the tumor-specific BM and ECM in the same well. In FIG. 8D, fluorescently labeled T cells (green, outer ring) can be seen embedded in solubilized basement membrane matrix (Matrigel) and plated on the perimeter of the well in a 96 well plate. Fluorescently labeled tumor cells (red, inner core) embedded in type I collagen were then plated in the center of the well. The matrices were allowed to polymerized, covered in medium and imaged every hour using a 3D cell imaging apparatus (Incucyte). The data produced represented T cell infiltration in terms of the area that T cells (green) overlapped with tumor (red). A representative image, taken at least 24 hours after plating, is shown in FIG. 8E.

[0241] Xenograft model of neuroblastoma. NSG mice were injected subcutaneously with 2e6 FFLuc-LAN-1 tumor cells, and 0.8 x 0.8 cm tumors were established. Five mice per group were then injected intravenously with 3.5e6 GD2-28Z, MMP7.GD2-28Z, MMP14.GD2- 28Z or OPN.GD2-28Z CAR T cells, and tumors were measured every other day with calipers. When tumors reached 1.5 cm in diameter, mice were sacrificed and tumors were collected,measured, and fixed for staining. In the cohort of mice treated with GD2-28Z CAR-Ts, one mouse was sacrificed on day five due to tumor burden, and the remaining mice all had palpable tumors. In contrast, while two of the mice treated with OPN.GD2-28Z had palpable tumors, the remaining three had no measurable tumor.Discussion

[0242] Solid tumors have historically posed considerable challenges to CAR-T cell therapy, primarily due to their complex tumor microenvironment, poor infiltration, and immunosuppressive factors. If CAR-T cell infiltration can be improved with proven enhancement in tumor control, it would open up new possibilities for treating a broader range of malignancies beyond hematological cancers, where CAR-T therapy has already demonstrated remarkable success. Enhanced CAR-T infiltration into solid tumors could potentially lead to more effective and durable responses, improving patient outcomes and expanding the therapeutic options available to clinicians. Ultimately, this would also spur further research into refining CAR-T engineering and combination therapies, paving the way for more personalized and targeted cancer treatments.EXAMPLE 2 - CAR T CELLS EXPRESSING HETEROLOGOUS OPN AND MMP

[0243] To assess whether the effects of MMP (e.g., MMP-7) and OPN (e.g., OPN-B) may be synergistic, the inventors engineer CAR T cells to express heterologous OPN and MMP. Ten mice per group are treated with GD2-28Z CAR-Ts that have been engineering to express both MMP-7 and OPN (MMP7.OPN.GD2-28Z). This process is repeated in additional donors or additional xenograft tumor models (e.g. 143B osteosarcoma model).

[0244] To test the effects of synergism in vivo, a xenograft neuroblastoma infiltration assay is used in which NSG mice are injected subcutaneously with 2e6 FFLuc-LAN-1 tumors cells, and 1 cm x 1 cm tumors are established. Five mice per group are injected with 10e6 GD2- 28Z, MMP7.GD2-28Z, OPN.GD2-28Z, or MMP7.OPN.GD2-28Z CAR-T cells. Five days later, mice are sacrificed and tumors collected, sectioned and stained for CD45 and type I collagen. The number of infiltrating CAR T cells is compared to establish how MMP and OPN expression may synergize for CAR-T cell infiltration of tumors in vivo.

[0245] A screening database (e.g. the TCGA) is used to determine which diseases and / or cancers may be most susceptible to treatment with cells of the present disclosure. In vitro and in vivo cell-killing assays are performed using different human cancer cell lines.

[0246] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosure as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. “Strategies for improving the efficacy of CAR T cells in solid cancers,” Kyte, Jon A. Cancers (2022), 14(3): 571 : (DOI: 10.3390 / cancersl4030571).“Recent advances in CAR-based solid tumor immunotherapy,” Shin et al.“Osteopontin Blockade Immunotherapy Increases Cytotoxic T Lymphocyte Lytic Activity and Suppresses Colon Tumor Progression,” Klement et al., Cancers (2021), 73(5), 1006 (https : / / doi . org / 10.3390 / cancers 13051006).“Compositions and methods for TCR reprogramming using fusion proteins,” US17 / 604,857.“Current Research & Development Status of Chimeric Antigen Receptor (CAR) T-Cell Therapy,” Jan. 2022 (https: / / www.bccresearch.com / market-research / biotechnology / chimeric- antigen-receptor-car-t-cell-therapy . html) .

Claims

CLAIMSWhat is claimed is:

1. An engineered immune cell comprising: a) one or more heterologous chimeric antigen receptors (CAR) and / or one or more heterologous T-cell receptors (TCR); and b) one or more heterologous matrix metalloproteinases (MMP) and / or one or more osteopontins (OPN).

2. The cell of claim 1, wherein the immune cell comprises a leukocyte.

3. The cell of any one of claims 1 or 2, wherein the immune cell comprises a B cell, T cell, and / or NK cell.

4. The cell of any one of claims 1-3, wherein the cell is allogeneic, autologous, syngeneic, or xenogeneic with respect to an individual.

5. The cell of any one of claims 1-4, wherein the cell is a mammalian cell.

6. The cell of any one of claims 1-5, wherein the cell is a human cell.

7. The cell of any one of claims 1-6, wherein the CAR and / or TCR targets GD2, EGFR,EGFRvIII, HER2, PSMA, CEA, IL13Ra2, Gpc3, CAIX, LI -CAM, CA 125, CD 133, CD 19, FAP, CTAG1B, Muc-1, and / or FR-a.

8. The cell of claims 7, wherein the CAR comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 39 or 91.

9. The cell of any one of claims 1-8, wherein the MMP comprises MMP 1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and / or MMP28.

10. The cell of claims 9, wherein the MMP comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-18, or 58-75.

11. The cell of any one of claims 1-10, wherein the OPN comprises OPNa, OPNb, OPNc, OPN4, OPN-I, and / or OPN5.

12. The cell of claims 11, wherein the OPN comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 19-22, or 76-79.

13. The immune cell of any one of claims 1-12, wherein the CAR targets GD2 and the OPN comprises OPNb.

14. The immune cell of any one of claims 1-11, wherein the CAR targets GD2 and the MMP comprises MMP14.

15. The immune cell of any one of claims 1-11, wherein the CAR targets GD2 and the MMP comprises MMP7.

16. The immune cell of any one of claims 1-11, wherein the CAR targets GD2 and the OPN comprises OPNa.

17. The immune cell of any one of claims 1-11, wherein the CAR targets GD2 and the OPN comprises OPNb.

18. The immune cell of any one of claims 1-11, wherein the CAR targets GD2 and the OPN comprises OPNc.

19. The immune cell of any one of claims 1-11, wherein the CAR targets GD2 and the OPN comprises 0PN4.

20. The immune cell of any one of claims 1-11, wherein the CAR targets GD2 and the OPN comprises OPN5.

21. The immune cell of any one of claims 1-11, wherein the CAR targets GD2 and the OPN comprises OPN-I.

22. A plurality of cells, comprising the cell of any one of claims 1-21.

23. The plurality of cells of claim 22, further comprising a pharmaceutically acceptable excipient.

24. The plurality of cells of claim 22 or 23, wherein the cells are frozen.

25. A method of treating a disease associated with expression of a tumor antigen in a subj ect in need thereof, the method comprising administering to the subject a therapeutically effective amount of a cell or plurality of cells of any one of claims 1-24.

26. The method of claim 25, wherein the disease comprises cancer, asthma, allergies, an autoimmune disorder, inflammation, and / or an infection.

27. The method of claim 26, wherein the disease comprises cancer.

28. The method of claim 27, wherein the cancer comprises a solid tumor.

29. The method of any one of claims 26-28, wherein the cancer comprises a tumor of the pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and / or breast.

30. The method of any one of claims 26-29, wherein the cancer comprises expression of GD2, EGFR, EGFRvIII, HER2, PSMA, CEA, IL13Ra2, Gpc3, CAIX, Ll-CAM, CA 125, CD133, CD19, FAP, CTAG1B, Muc-1, and / or FR-a.

31. The method of any one of claims 25-30, wherein the therapeutically effective amount of the cell or plurality of cells is delivered intravenously, intramuscularly, subcutaneously, transdermally, intraperitoneally, intraorbitally, by implantation, intrathecally, intraventricularly, and / or intratum orally.

32. The method of any one of claims 25-31, further comprising treatment with at least one or more co-therapies.

33. The method of claim 32, wherein administering of the therapeutically effective amount of the cell or plurality of cells occurs before administration of the at least one or more cotherapies.

34. The method of claim 32, wherein administering of the therapeutically effective amount of the cell or plurality of cells occurs simultaneously to administration of the at least one or more co-therapies.

35. The method of claim 32, wherein administering of the therapeutically effective amount of the cell or plurality of cells occurs after administration of the at least one or more co- therapies.

36. The method of any one of claims 32-35, wherein the cell or plurality of cells are delivered via a same route of administration as the at least one or more co-therapies.

37. The method of any one of claims 32-35, wherein the cell or plurality of cells are delivered via a different route of administration as the at least one or more co-therapies.

38. The method of any one of claims 32-37, wherein the cell or plurality of cells are delivered together with the at least one or more co-therapies.

39. The method of any one of claims 32-37, wherein the cell or plurality of cells are delivered separately from the at least one or more co-therapies.

40. The method of any one of claims 32-39, wherein the at least one or more therapeutic agents comprise chemotherapy, radiotherapy, immunotherapy, surgery, and / or drug therapy.

41. A method of enhancing infiltration of an immune cell into a tumor, the method comprising the steps of: a) engineering the immune cell to comprise a heterologous CAR and / or TCR; b) engineering the immune cell to comprise a heterologous MMP and / or OPN; c) producing a multi-engineered immune cell from steps (a) and (b); d) administering the multi-engineered cell to an individual.

42. The method of claim 41, wherein the CAR and / or TCR targets GD2, EGFR, EGFRvIII, HER2, PSMA, CEA, IL13Ra2, Gpc3, CAIX, Ll-CAM, CA 125, CD133, CD19, FAP, CTAG1B, Muc-1, and / or FR-a.

43. The method of claim 41 or 42, wherein the CAR comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 39 or 91.

44. The method of any one of claims 41-43, wherein the OPN comprises OPNa, OPNb, OPNc, OPN4, OPN-I, and / or OPN5.

45. The method of any one of claims 41-44, wherein the OPN comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 19-22, or 76-79.

46. The method of any one of claims 41-45, wherein the MMP comprises MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and / or MMP28.

47. The method of any one of claims 41-46, wherein the MMP comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-18, or 58-75.

48. The method of any one of claims 41-47, wherein step (a) occurs before step (b).

49. The method of any one of claims 41-46, wherein step (b) occurs before step (a).

50. The method of any one of claims 41-49, wherein the cell is a T cell, NK cell, or B cell.

51. The method of any one of claims 41-50, wherein the cell comprises a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NOs: 1-57.

52. The method of any one of claims 41-51, wherein the cell comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 58-100.

53. The method of any one of claims 41-52, wherein the individual has been diagnosed, or is suspected of having a tumor.

54. The method of claim 53, wherein the tumor comprises a solid tumor.

55. The method of claim 54, wherein the solid tumor comprises a tumor of the pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and / or breast.

56. The method of claim 54 or 55, wherein the solid tumor comprises a neuroblastoma.

57. A nucleic acid, comprising: a heterologous MMP and / or heterologous OPN, and a reporter gene and / or a CAR and / or TCR.

58. The nucleic acid of claim 57, wherein the MMP comprises MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, and / or MMP28.

59. The nucleic acid of claim 57 or 58, wherein the MMP comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-18, or 58-75.

60. The nucleic acid of any one of claims 57-59, wherein the OPN comprises OPNa, OPNb, OPNc, OPN4, OPN-I, and / or OPN5.

61. The nucleic acid of any one of claims 57-60, wherein the OPN comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 19-22, or 76-79.

62. The nucleic acid of any one of claims 57-61, wherein the CAR and / or TCR targets GD2, EGFR, EGFRvIII, HER2, PSMA, CEA, IL13Ra2, Gpc3, CAIX, Ll-CAM, CA 125, CD133, CD19, FAP, CTAG1B, Muc-1, and / or FR-a.

63. The nucleic acid of any one of claims 57-62, wherein the CAR comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 39 or 91.

64. The nucleic acid of any one of claims 57-63, wherein the reporter comprises GFP or tCD19.

65. The nucleic acid of any one of claims 57-64, wherein the reporter comprises an amino acid sequence and / or is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 34 or 81.

66. The nucleic acid of any one of claims 57-65, comprising a nucleic acid sequence and / or encoding an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-100.

67. A cell comprising the nucleic acid of any one of claims 57-66.

68. The cell of claim 67, wherein the cell is a T cell, NK cell, or B cell.

69. A kit, housed in a suitable container, comprising the composition of any one of claims1-24 and / or 48-59.

70. The kit of claim 69, further comprising instructions for performing the method of any one of claims 25-56.

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