Methods and uses of modulating CD160 function in antigen-specific immune cells

Abstract

The present invention provides modified antigen-specific immune cells that express exogenous CD160 protein. In some embodiments, the modified antigen-specific immune cells further comprise a functional exogenous receptor, such as an engineered TCR or CAR. The present invention also provides a method for modulating CD160 activity in antigen-specific immune cells. The present invention also provides methods and pharmaceutical compositions for cancer treatment using the modified antigen-specific immune cells and CD160 activity modulators described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 812,897, filed March 1, 2019, the entire contents of each of which are incorporated herein by reference. Submitting a sequence listing as an ASCII text file

[0002] The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 756592000240SEQLIST.TXT, Recorded: February 24, 2020, Size: 17KB).

[0003] The present invention relates to methods and uses of modulating CD160 function in antigen-specific immune cells, including natural and engineered antigen-specific αβ T cells, and other immune cells such as natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, γδ T cells, and macrophages, with or without engineered antigen recognition. The present invention also relates to antigen-specific immune cells that express exogenous CD160 protein and methods of using same to modulate antigen-specific immune cell function in the treatment of cancer. [Background technology]

[0004] T cells are natural agents that protect our bodies against infection and cancer. In particular, checkpoint blockade and chimeric antigen receptor (CAR) T cell therapies for treating cancer patients can efficiently reengage tumor-targeted T cells. The success of these immunotherapies has clearly established the effectiveness of T cells in cancer treatment and sparked the exploration of diverse approaches to activate and engage T cells in the fight against cancer. Over the past half century, pioneering researchers and physicians have explored the adoptive transfer of tumor-targeted T cells to treat human solid tumors. Various tumor-targeting T cells, including in vitro-activated T cells, tumor-infiltrating lymphocytes (TILs), T cells bearing specific T cell receptors (TCRs) that recognize tumor antigens or tumor-associated antigens (TCR-T), and T cells derived from tumor antigen-loaded dendritic cells (DC-T), have been tested and shown promising efficacy in preclinical models and human patients. More recently, T cells recognizing neoantigens derived from mutated cancer cells have been used in adoptive T cell therapy for lung and breast cancer, demonstrating efficacy in some cases. Nevertheless, even with the aid of other therapies such as radiation, vaccination, chemotherapy, and co-infusion of the antitumor cytokine IL-2, it has been difficult to reproducibly achieve sustained control and elimination of solid tumors using adoptive transfer of tumor-targeted T cells.

[0005] The relatively low response rates and eventual loss of tumor control by infused tumor-targeting T cells can be attributed to a variety of intrinsic and extrinsic barriers to T cells. These barriers stem from homeostatic immune tolerance mechanisms and tumor-induced immunosuppressive mechanisms. In particular, these tolerance forces create barriers that prevent effective tumor control by T cells. For example, naturally occurring antitumor T cells, controlled by central tolerance mechanisms including positive and negative thymic selection, often possess T cell receptors (TCRs) with low or intermediate affinity for non-mutated tumor antigens or tumor-associated antigens. Furthermore, evolving tumors reduce the expression of class I or II tumor histocompatibility complex (MHC) molecules, effectively limiting tumor antigen presentation on tumor cells and, as a result, limiting their recognition by T cells bearing the cognate TCR. Much effort has been made in this field to overcome these barriers, with varying degrees of success. CARs enable efficient engagement of T cells with tumor cells, helping to overcome insufficient or lost tumor recognition by T cells. Notably, CAR-T cells targeting CD19 or BCMA antigens can eliminate B-cell leukemia / lymphoma or multiple myeloma, respectively. Other approaches have focused on overcoming the recognition barrier by boosting the affinity of tumor-specific TCRs through in vitro evolution or by selecting TCRs that recognize neoantigens, showing modest improvements in efficacy.

[0006] Although these strategies are promising, there are additional barriers beyond antigen recognition that must be overcome for tumor-targeted T cells to eliminate or sustainably control established tumors. In particular, cold tumors, which do not activate intratumoral T cells, correlate with poor prognosis and poor response to immunotherapy. It has been postulated that T cell exhaustion as a result of checkpoint inhibition, shutting down of T cell-specific chemoattractants, unfavorable nutritional intake, and hypoxia, as well as many other unknown factors in the tumor microenvironment, may contribute to the cold tumor phenomenon. Various genetic engineering strategies have been explored to enhance T cell trafficking and activation in tumors. Ectopic expression of chemokine receptors or CARs that recognize VEGFR in tumor-specific T cells has been shown to boost tumor control by transferred T cells. Furthermore, inactivation of negative T cell regulators, such as PD-1, CBLB, or adenosine 2A receptor, in tumor-specific T cells enhanced the antitumor function of T cells. Interestingly, signals that enhance mitochondrial biogenesis and oxidative phosphorylation, such as ectopic expression of PGC1alpha or OPA1, or CARs with the CD278 signaling domain, can also boost the antitumor function of transferred T cells. Although T cell function in tumor control can be enhanced by a variety of molecular and cellular processes, these strategies are generally insufficient to enable sustained tumor control by T cells or to eliminate established tumors. Thus, it is important to search for molecules that can be used to reprogram tumor-targeting T cells for sustained control and elimination of solid tumors.

[0007] CD160 is a 27-kDa glycoprotein first identified on human natural killer cells using the monoclonal antibody BY55 (Maiza et al., 1993, J Exp Med. 178(3):1121-6). Later, the dominant form of CD160 was found to be a glycosyl-phosphatidylinositol (GPI)-anchored immunoglobulin (Ig)-like cell membrane receptor found on the major CD16+ NK cell subset, NK-T cells, γδ-T cells, some subsets of CD4 T cells and CD8+ cytolytic T cells, as well as activated endothelial cells (Le Bouteiller et al., 2011, Immunol Lett., 138(2):93-6). The cDNA sequence of human CD160 encodes a 181-amino acid, cysteine-rich, glycosylphosphatidylinositol-anchored protein with a single Ig-like domain. Subsequently, additional isoforms containing transmembrane domains and / or lacking extracellular Ig-like domains were identified. CD160 is expressed on the cell surface as a tightly disulfide-bonded multimer. CD160 is a ligand for HVEM, and binding of CD160 to HVEM results in T cell inhibition and anergy (Cai et al., 2009, Nat Immunol., 9(2):176-185). CD160, along with anti-PD-1 antibodies, is often considered an immune checkpoint inhibitor with anti-cancer activity (Stecher et al., 2017, Front Immunol., 8:572). It has been proposed that CD160 competes with BTLA (CD272) for binding to HVEM (Kojima et al., 2011, J Mol Biol., 413(4):762-72). Murine and human CD160 on NK cells and some human T cell subsets has low affinity for MHC class Ia and Ib and may play a role in NK and T cell activation (Maeda et al., 2005, J Immunol., 175(7):4426-32; Agrawal et al., 1999, J Immunol., 162(3):1223-6).CD160 is also expressed by endothelial cells and has been proposed as a potential new target in cases of pathological ocular and tumor angiogenesis in humans that are unresponsive to or resistant to current antiangiogenic drugs (Chabot et al., 2011, J Exp Med., 208(5):973-86). Loss of CD160 function analysis in mice indicated that CD160 is important for NK-mediated IFN-γ production but not for the cytolytic activity of NK cells, and is apparently not required for T lymphocyte development and function (Tu et al., 2015, J Exp Med., 212(3):415-29). To date, there is no published evidence for the function of CD160 on antigen-specific T cells in the control and elimination of established tumors. The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Maiza et al., 1993, J Exp Med.. 178(3):1121-6 [Non-patent document 2] Le Bouteiller et al., 2011, Immunol Lett., 138(2):93-6 Summary of the Invention [Means for solving the problem]

[0009] The present application provides modified antigen-specific immune cells comprising exogenous CD160 protein on their surface and methods of using same to treat cancer. The present invention also provides methods of modulating CD160 activity in antigen-specific immune cells.

[0010] One aspect of the present application provides modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, and wherein the immune cells are T cells. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by antigen-presenting cells (APCs), and natural killer T cells (NK-T cells). In some embodiments, the modified antigen-specific immune cells are cytotoxic T cells. In some embodiments, the modified antigen-specific immune cells are tumor-infiltrating T cells or anti-tumor T cells activated by APCs. In some embodiments, the anti-tumor T cells activated by APCs are anti-tumor T cells activated by dendritic cells (DCs). In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, gamma delta T cells, and macrophages. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, or a variant thereof having at least about 90% identity to any one of SEQ ID NOs: 1-4.

[0011] In some embodiments of any one of the above modified antigen-specific immune cells, the exogenous CD160 protein is membrane-bound. In some embodiments, the exogenous CD160 protein is membrane-bound via a GPI linker. In some embodiments, the exogenous CD160 protein is bound to the modified antigen-specific immune cell via an immune cell binding moiety. In some embodiments, the immune cell binding moiety binds to a surface molecule of the immune cell. In some embodiments, the exogenous CD160 protein comprises a transmembrane domain. In some embodiments, the exogenous CD160 protein further comprises an intracellular domain. In some embodiments, the exogenous CD160 protein further comprises an intracellular domain derived from a CD160 splice variant. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from a signaling subunit of a TCR complex. In some embodiments, the signaling subunit of a TCR complex is selected from the group consisting of CD3 gamma, CD3 delta, and CD3 epsilon.

[0012] In some embodiments of any one of the above modified antigen-specific immune cells, the exogenous CD160 protein is membrane-bound and comprises an intracellular domain. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, or both. In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain. In some embodiments, the intracellular domain comprises a primary signaling domain. In some embodiments, the primary signaling domain comprises a CD3ζ domain. In some embodiments, the intracellular domain does not comprise a primary signaling domain.

[0013] In some embodiments of any one of the modified antigen-specific immune cells described above, the modified antigen-specific immune cell further comprises a functional exogenous receptor. In some embodiments, the functional exogenous receptor is an engineered T cell receptor (TCR). In some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR).

[0014] One aspect of the present application provides a method for producing modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, the method comprising producing the modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, and the immune cells are T cells. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells). In some embodiments, the modified antigen-specific immune cells are cytotoxic T cells. In some embodiments, the modified antigen-specific immune cells are tumor-infiltrating T cells or anti-tumor T cells activated by APCs. In some embodiments, the anti-tumor T cells activated by APCs are anti-tumor T cells activated by DCs. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, gamma delta T cells, and macrophages.

[0015] In some embodiments of any one of the above production methods, the method comprises contacting precursor antigen-specific immune cells with an exogenous CD160 protein. In some embodiments, the exogenous CD160 protein comprises an immune cell binding moiety that binds to a surface molecule of the immune cell. In some embodiments, the production method comprises introducing a nucleic acid encoding the exogenous CD160 protein into the precursor antigen-specific immune cells. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is introduced into the precursor antigen-specific immune cells by transfection. In some embodiments, the nucleic acid is introduced into the precursor antigen-specific immune cells by transduction or electroporation. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, or a variant thereof having at least about 90% identity to any one of SEQ ID NOs: 1-4.

[0016] In some embodiments of any one of the above production methods, the exogenous CD160 protein is membrane-bound. In some embodiments, the exogenous CD160 protein is membrane-bound via a GPI linker. In some embodiments, the immune cell binding moiety binds to a surface molecule of an immune cell. In some embodiments, the exogenous CD160 protein is bound to the modified antigen-specific immune cell via the immune cell binding moiety. In some embodiments, the exogenous CD160 protein comprises a transmembrane domain. In some embodiments, the exogenous CD160 protein further comprises an intracellular domain. In some embodiments, the exogenous CD160 protein further comprises an intracellular domain derived from a CD160 splice variant. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from a signaling subunit of a TCR complex. In some embodiments, the signaling subunit of a TCR complex is selected from the group consisting of CD3 gamma, CD3 delta, and CD3 epsilon.

[0017] In some embodiments of any one of the above production methods, the exogenous CD160 protein is membrane-bound and comprises an intracellular domain. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, or both. In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain. In some embodiments, the intracellular domain comprises a primary signaling domain. In some embodiments, the primary signaling domain comprises a CD3ζ domain. In some embodiments, the intracellular domain does not comprise a primary signaling domain.

[0018] In some embodiments of any one of the above production methods, the precursor antigen-specific immune cells comprise a second nucleic acid encoding a functional exogenous receptor. In some embodiments, the production method further comprises contacting the precursor antigen-specific immune cells with the second nucleic acid encoding the functional exogenous receptor. In some embodiments, the functional exogenous receptor is an engineered T cell receptor (TCR). In some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to the same promoter. In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to separate promoters. In some embodiments, the first nucleic acid and the second nucleic acid are on the same vector. In some embodiments, the first nucleic acid and / or the second nucleic acid are on separate vectors. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, an episomal vector expression vector, a herpes simplex viral vector, and derivatives thereof. In some embodiments, the vector is a non-viral vector.

[0019] In some embodiments of any one of the above methods of production, the method further comprises isolating or enriching immune cells comprising the first and / or second nucleic acid. In some embodiments, the method further comprises formulating the modified antigen-specific immune cells that express CD160 with at least one pharmaceutically acceptable carrier.

[0020] Also provided are modified antigen-specific immune cells produced by any one of the above production methods.

[0021] Further provided is a pharmaceutical composition comprising a modified antigen-specific immune cell according to any one of the above modified immune cells and a pharmaceutically acceptable carrier.

[0022] Another aspect of the present application provides a method of treating a disease in an individual, the method comprising administering to the individual an effective amount of a modified antigen-specific immune cell described in any one of the above modified antigen-specific immune cells or a pharmaceutical composition described in any one of the above pharmaceutical compositions. In some embodiments, the modified antigen-specific immune cell is derived from the individual. Yet another aspect of the present application provides a method of treating a disease in an individual, the method comprising administering to the individual an effective amount of an exogenous CD160 protein or a nucleic acid encoding an exogenous CD160 protein, wherein the exogenous CD160 protein comprises a binding moiety that recognizes a surface molecule on an immune cell in the individual.

[0023] In some embodiments of any one of the above methods of treatment, the administration is intratumoral administration. In some embodiments, the administration is to a lymph node. In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, lung cancer, esophageal cancer, pancreatic cancer, breast cancer, liver cancer, brain cancer, and ovarian cancer. In some embodiments, the individual is a human.

[0024] One aspect of the present invention provides a method of activating the immunostimulatory activity of CD160 in antigen-specific immune cells, the method comprising contacting the antigen-specific immune cells with an effective amount of an agent that activates the immunostimulatory activity of CD160 in the antigen-specific immune cells. In some embodiments, the method comprises enhancing the endogenous immunostimulatory activity of CD160 in the antigen-specific immune cells, wherein the agent enhances the endogenous immunostimulatory activity of CD160 in the antigen-specific immune cells.

[0025] One aspect of the present invention provides a method of treating an immune disease in an individual, comprising administering to the individual a therapeutically effective amount of an agent that modulates the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells. In some embodiments, the immune disease is an autoimmune disease or an inflammatory disease, and the agent inhibits the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0026] One aspect of the invention provides a method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells. Another aspect of the invention provides a method of treating infection in an individual, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0027] Compositions, uses, kits and articles of manufacture comprising any one of the modified antigen-specific immune cells are also provided. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1A shows ectopic expression of mouse CD160 in Pmel T cells compared to control Pmel T cells analyzed by FACS, and Figure 1B shows the median fluorescence intensity (MFI) of CD160 staining for Pmel T cells modified with exogenous CD160 compared to control.

[0029] [Figure 2-1] Figure 2A shows the effect of exogenous CD160 expression on granzyme A and perforin levels in T cells, as determined by intracellular staining and FACS analysis. Figure 2B shows the effect of exogenous CD160 expression on INF-γ and TNFα levels in T cells, as determined by intracellular staining and FACS analysis. Figure 2C shows the cytolytic activity analysis of Pmel T cells infected with control or CD160 viruses when cocultured with B16F0 melanoma tumor cells. [Figure 2-2] Same as above.

[0030] [Figure 3]Figure 3A shows the effect on tumor size in B16F0-bearing mice administered control Pmel T cells (T cells specific for tumor antigens in B16F0) or 100,000, 200,000, 300,000, or 400,000 CD160-modified Pmel T cells. Figure 3B shows the effect on tumor size in B16F0-bearing mice administered control Pmel T cells, CD160-modified splenic T cells (non-specific for B16F0 antigens), and CD160-modified Pmel T cells.

[0031] [Figure 4-1] Figure 4A shows the relative changes in tumor size in B16F0-bearing mice treated with cyclophosphamide (CYP) preconditioning and (a) control Pmel T cells; or (b) CD160-modified Pmel T cells. Figure 4B shows growth curves and selected tumor images at various time points for a representative mouse treated with CD160-modified Pmel T cells. Figure 4C shows Kaplan-Meier survival analysis of B16F0 melanoma-bearing mice treated with CYP preconditioning and (a) control Pmel T cells; or (b) CD160-modified Pmel T cells. [Figure 4-2] Same as above.

[0032] [Figure 5-1] Figure 5A shows the relative change in tumor size in B16F0-bearing mice administered CYP preconditioning and (a) PBS; (b) 150,000 control Pmel T cells; or (c) 150,000 control CD160-modified Pmel T cells. Figure 5B shows the relative change in tumor size in B16F0-bearing mice administered CYP preconditioning and (a) PBS; (b) 300,000 control Pmel T cells; or (c) 300,000 control CD160-modified Pmel T cells. Figure 5C shows Kaplan-Meier survival analysis of B16F0 melanoma-bearing mice administered CYP preconditioning and (a) PBS; (b) control Pmel T cells; (c) 150,000 control CD160-modified Pmel T cells; or (d) 300,000 control CD160-modified Pmel T cells. [Figure 5-2] Same as above.

[0033] [Figure 6-1] Figure 6A shows the effect on mean tumor size in metastatic B16F10-bearing mice left untreated or administered CYP preconditioning and (a) PBS; (b) control Pmel T cells; or (c) CD160-modified Pmel T cells. Figure 6B shows the effect on individual tumor size in metastatic B16F10-bearing mice left untreated or administered CYP preconditioning and (a) PBS; (b) control Pmel T cells; or (c) CD160-modified Pmel T cells. [Figure 6-2] Same as above.

[0034] [Figure 7] Figure 7A shows the relative changes in tumor size in B16F10-bearing mice treated with CYP preconditioning and (a) control Pmel T cells; or (b) CD160-modified Pmel T cells. Figure 7B shows Kaplan-Meier survival analysis of B16F10 melanoma-bearing mice treated with CYP preconditioning and (a) PBS; (b) control Pmel T cells; or (c) CD160-modified Pmel T cells.

[0035] [Figure 8-1] Figures 8A-C show the relative changes in tumor size in B16F0-bearing mice administered CYP preconditioning and PBS, mCD160-modified Pmel T cells, or Pmel T cells modified with one of the CD160 chimeras as indicated (GEM124, 125 in Figure 8A; GEM126, 127 in Figure 8B; and GEM123, 128 in Figure 8C). [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0036] [Figure 9-1]Figure 9A is a schematic diagram showing, from left to right, GPI-anchored mCD160, GPI-anchored hCD160 isoform, transmembrane hCD160 isoform, and transmembrane hCD160 isoform containing an intracellular domain. Figure 9B shows the nucleotide sequences of mouse and human CD160 isoforms and the degree of conservation between them. [Figure 9-2] Same as above.

[0037] [Figure 10-1] Figure 10A shows the relative changes in tumor size in B16F0-bearing mice administered CYP preconditioning and (a) control Pmel T cells (VECTOR); or (b) Pmel T cells modified with exogenous GPI-anchored mCD160, GPI-anchored hCD160 isoform, transmembrane hCD160 isoform, or transmembrane hCD160 isoform containing the extracellular domain, respectively. Figure 10B shows Kaplan-Meier survival analysis of B16F0 melanoma-bearing mice administered CYP preconditioning and (a) control Pmel T cells (VECTOR); or (b) Pmel T cells modified with exogenous GPI-anchored hCD160 isoform, transmembrane hCD160 isoform, or transmembrane hCD160 isoform containing the extracellular domain, respectively. [Figure 10-2] Same as above.

[0038] [Figure 11-1]Figure 11A shows the cytolytic activity analysis of tumor-infiltrating T cells (TILs) extracted from Lewis lung carcinoma (LLC) cells infected with control or mCD160 viruses. Figure 11B shows a representative schematic diagram of an in vivo experiment on the LLC tumor-control ability of LLC TILs modified with exogenous CD160 or CD160 chimera (GEM124). Figure 11C shows Kaplan-Meier survival analysis of LLC-bearing mice left untreated or administered CYP preconditioning and (a) PBS; (b) control TILs; (c) mCD160-modified TILs; or (d) TILs modified with CD160 chimera GEM124. [Figure 11-2] Same as above.

[0039] [Figure 12-1] Figure 12A shows a schematic diagram representing CD19-CAR-T cells overexpressing human CD160 or a variant thereof, such as CD160TC. Figure 12B shows a lentiviral vector construct designed to overexpress human CD160 or a variant thereof together with a CAR-recognized tumor-specific antigen, such as CD19. [Figure 12-2] Same as above.

[0040] [Figure 13-1]Figure 13A shows the proliferation of CD19-CAR-T cells overexpressing huCD160TC and CD19-CAR-T cells that do not overexpress CD160 (wild-type CD19-CAR-T). Figure 13B shows the viability of CD19-CAR-T cells overexpressing huCD160TC and wild-type CD19-CAR-T cells after two weeks of culture. Figure 13C shows the cytolytic activity analysis of CD19-CAR-T cells overexpressing huCD160TC and wild-type CD19-CAR-T cells at various effector to target (E:T) ratios. Figure 13D shows IFN-γ production in CD19-CAR-T cells overexpressing huCD160TC and wild-type CD19-CAR-T cells. Figure 13E shows a topograph depicting tumor size in Nalm6 tumor-bearing mice administered (a) no CAR-T cells (None); (b) control CD19-CAR-T cells (19CAR), or (c) CD19-CAR-T cells overexpressing huCD160TC (CD160TC 19CAR). Figure 13F shows Kaplan-Meier survival analysis of Ramos tumor-bearing mice administered (a) no CAR-T cells (--); (b) wild-type CD19-CAR-T cells, or (c) CD19-CAR-T cells overexpressing huCD160TC (CD160TC). [Figure 13-2] Same as above.

[0041] [Figure 14-1] Figure 14A shows a schematic diagram representing NY-ESO-1-specific TCR-T cells overexpressing human CD160 or a variant thereof, such as huCD160TC. Figure 14B shows a lentiviral vector construct designed to overexpress human CD160 or a variant thereof together with a TCR-recognizing a tumor-specific antigen, such as a NY-ESO-1-specific TCR. [Figure 14-2] Same as above.

[0042] [Figure 15-1]Figure 15A shows FACS analysis of wild-type 1G4-TCR T cells and 1G4-TCR T cells expressing huCD160TC, demonstrating NY-ESO-1-specific 1G4-TCR levels as determined by tetramer analysis. Figure 15B shows proliferation of 1G4-TCR T cells overexpressing huCD160TC and 1G4-TCR T cells not overexpressing CD160 (wild-type 1G4-TCR T). Figure 15C shows the percentage of stem / memory T cells among wild-type 1G4-TCR T cells and 1G4-TCR T cells expressing huCD160TC. Figure 15D shows cytolytic activity analysis of 1G4-TCR T cells overexpressing huCD160TC and wild-type 1G4-TCR T cells at various effector to target (E:T) ratios. Figure 15E shows IFN-γ production in 1G4-TCR-T cells overexpressing huCD160TC and wild-type 1G4-TCR-T cells. Figure 15F shows tumor size in A375 melanoma-bearing mice administered (a) no TCR-T cells (none); (b) control 1G4-TCR-T cells; or (c) 1G4-TCR-T cells overexpressing huCD160TC. [Figure 15-2] Same as above. [Figure 15-3] Same as above.

[0043] [Figure 16] Figure 16 shows an experimental schematic of autologous TIL therapy in patient-derived xenograft (PDX) mouse tumor models. Tumor tissues resected from cancer patients with various cancers, such as lung, esophageal, colon, gastric, or pancreatic cancer, were implanted and passaged into NSG immunodeficient mice to generate PDX models bearing human tumors. Autologous tumor-infiltrating leukocytes (TILs) were extracted from the resected tumors for CD160 modification and subsequent functional testing in autologous human tumors in the PDX models.

[0044] [Figure 17-1]Figure 17A shows a schematic diagram representing anti-tumor TILs overexpressing human CD160 or its variants, such as huCD160TC. Figure 17B shows a lentiviral vector construct overexpressing human CD160 or its variants, along with GFP as a co-expressed reporter. [Figure 17-2] Same as above.

[0045] [Figure 18] Figure 18A shows the cytolytic activity analysis of tumor-specific TILs overexpressing huCD160TC (CD160TC) and corresponding TILs that do not overexpress CD160 (None; vector) at various effector to target (E:T) ratios. Figure 18B shows tumor size in autologous esophageal tumor-bearing mice administered (a) control TILs that do not overexpress CD160 (None) or (c) TILs that overexpress huCD160TC. GEM indicates genetic enhancer regulators expressed by corresponding TILs. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present application provides methods and compositions for modulating the immunostimulatory activity of CD160. The present application is based on the surprising discovery that CD160, previously thought to function primarily as an inhibitory checkpoint molecule for T cells, can be responsible for stimulating immune responses in antigen-specific immune cells, such as T cells.

[0047] We demonstrated that tumor-specific T cells can be reprogrammed with CD160 to control and eliminate established solid tumors in immunocompetent mice. Furthermore, CD160-programmed T cells were shown to be highly effective in controlling metastatic melanoma and lung cancer in mouse models where these treatments are highly difficult. Ectopic expression of CD160 enhanced the function of tumor-specific T cells bearing a TCR specifically recognizing GP100 and polyclonal lung cancer tumor-infiltrating T cells ("TILs") that recognize multiple antigens. CD160-modified tumor-specific T cells provided effective control of solid tumors of different tissue origins, regardless of their metastatic potential. Furthermore, by creating a CD160 chimera with a TCR and costimulatory signaling domain, we demonstrated that CD28 costimulatory signals synergize with CD160, further enhancing antigen-specific T cell function.

[0048] Importantly, human and mouse CD160 have conserved functions in enhancing the function of antigen-specific T cells in tumor control and elimination in vivo, suggesting that CD160 may control a highly conserved pathway in regulating antigen-specific T cell function. These findings demonstrate for the first time that CD160 can be used to transform antigen-specific T cells into powerful therapeutic agents for the control and elimination of cancers, including established solid tumors. Furthermore, these findings strongly suggest that CD160-based immune cell (e.g., T cell) reprogramming may be broadly applicable to all tumor-targeting immune cells (e.g., T cells) and tumors of different tissue origins.

[0049] The findings discussed above further suggest that CD160 may be an important target for exogenous modulation to boost or suppress the function of antigen-specific T cells. For example, endogenous CD160 in antigen-specific immune cells can be targeted to boost the activity of antigen-specific T cells and activate inflammatory responses to viral and bacterial infections. Conversely, CD160 can be targeted to inhibit the activity of antigen-specific T cells during unwanted immune responses, such as in inflammatory and autoimmune diseases. Given the important function of CD160 on antigen-specific T cells, CD160 expression levels may correlate with the effective and functional status of antigen-specific T cells at inflammatory sites, including tumors or inflamed tissues. Higher CD160 expression on these cells may indicate an activated state of antigen-specific T cells, while low levels or the absence of CD160 expression may indicate an inactive state of antigen-specific T cells. Thus, CD160 can be used as a biomarker to predict the functional status of antigen-specific T cells and, therefore, the effectiveness of immunotherapy.

[0050] Thus, in one aspect, modified antigen-specific immune cells (e.g., T cells) are provided that comprise (e.g., on their surface) an exogenous CD160 protein, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein. In some embodiments, the exogenous CD160 protein is attached to the cell membrane of the modified antigen-specific immune cells via a GPI linker. In some embodiments, the exogenous CD160 protein is a transmembrane protein comprising a transmembrane domain. In some embodiments, the exogenous CD160 protein comprises a transmembrane domain and an intracellular signaling domain derived from a costimulatory molecule. In some embodiments, the exogenous CD160 protein is attached to the modified antigen-specific immune cells via an immune cell binding moiety, such as an antibody that recognizes and activates a T cell surface molecule. In some embodiments, the modified antigen-specific immune cells further comprise a functional exogenous receptor, such as a modified T cell receptor, an engineered T cell receptor, or a chimeric antigen receptor (CAR). Also provided are methods for producing the modified antigen-specific immune cells described above.

[0051] In another aspect, methods are provided for modulating the immunostimulatory activity of CD160 in antigen-specific immune cells, for example, to treat immune diseases such as autoimmune and inflammatory diseases. In some embodiments, methods are provided for activating (e.g., enhancing) the immunostimulatory activity of CD160 in antigen-specific immune cells by contacting the antigen-specific immune cells with an agent that activates (e.g., enhances) the activity of CD160 (e.g., an agonist anti-CD160 antibody). In some embodiments, methods are provided for inhibiting (e.g., downregulating) the immunostimulatory activity of CD160 by contacting the antigen-specific immune cells with an agent that inhibits (e.g., downregulates) the activity of CD160 (e.g., an antagonist anti-CD160 antibody).

[0052] Also provided are compositions (such as pharmaceutical compositions), kits and articles of manufacture comprising the modified antigen-specific immune cells, as well as methods of treating cancer using the modified antigen-specific immune cells described herein. I. Definition

[0053] As used herein, "treatment" or "treating" refers to an approach for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms resulting from the disease, reduction in the extent of the disease, stabilization of the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improvement of the disease state, remission of the disease (partial or total), reduction in the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, increasing quality of life, and / or prolonging survival. Also encompassed by "treatment" is reduction in the pathological consequences of the disease. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0054] The term "prevent" and similar words such as "prevented," "preventing," etc., refer to an approach for preventing, inhibiting, or reducing the likelihood of recurrence of a disease or condition, such as cancer. It also refers to delaying the recurrence of a disease or condition, or delaying the recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the recurrence of the disease or condition.

[0055] As used herein, "delaying" the onset of cancer means postponing, preventing, slowing, retarding, stabilizing, and / or postponing the onset of the disease. This delay may be for a varying length of time, depending on the history of the disease and / or individual being treated. A method for "delaying" the onset of cancer is one that reduces the probability of disease onset in a given time frame and / or reduces the extent of disease in a given time frame compared to not using the method. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. Cancer onset may be detectable using standard methods, such as, but not limited to, computed tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset may also refer to cancer progression, which may be undetectable initially, and includes onset, recurrence, and onset.

[0056] As used herein, the term "effective amount" refers to an amount of an agent or combination of agents sufficient to treat a particular disorder, condition, or disease, e.g., ameliorate, alleviate, reduce, and / or delay one or more symptoms thereof. With reference to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or a decrease in the rate of tumor growth (e.g., tumor growth inhibition) or to prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay disease onset. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent, and preferably stop cancer cell invasion of peripheral organs; (iv) inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0057] As used herein, "individual" or "subject" refers to a mammal, such as, but not limited to, a human, cow, horse, cat, dog, rodent, or primate. In some embodiments, the individual is a human.

[0058] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in cells that ordinarily contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0059] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0060] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which heterologous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with heterologous nucleic acid. The cell includes the primary subject cell and its progeny.

[0061] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after aligning the sequences and taking into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0062] As used herein, "antigen-specific immune cells" refer to immune cells that specifically recognize antigens on target cells via natural and / or engineered antigen-recognition receptors. Examples of immune cells include, but are not limited to, αβ T cells, γδ T cells, natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, and macrophages, which may or may not exhibit engineered antigen recognition. Antigen-specific immune cells may be polyclonal or monoclonal. For example, in some embodiments, "antigen-specific immune cells" refer to tumor-infiltrating lymphocytes (TILs) that can be isolated from resected tumors; neoantigen-specific T cells that can be isolated using the respective antigen-binding tetramers; dendritic cell-activated T cells generated by co-culture and activation of peripheral blood T cells with tumor antigen-loaded dendritic cells; or other immune cells, such as γδ T cells, NK cells, NK-T cells, iNK-T cells, NK-T-like cells, and macrophages, that express CAR or TCR-like antigen receptors.

[0063] As used herein, an "antigen-specific receptor" is a natural or engineered T cell receptor (TCR), or an engineered antigen receptor such as a chimeric antigen receptor (CAR).

[0064] As used herein, "T cell receptor" or "TCR" refers to an endogenous or modified T cell receptor that comprises an extracellular antigen-binding domain that binds to a specific antigenic peptide bound to an MHC molecule. In some embodiments, the TCR comprises a TCR alpha polypeptide chain and a TCR beta polypeptide chain. In some embodiments, the TCR specifically binds to a tumor antigen. "TCR-T" refers to a T cell that expresses a recombinant TCR.

[0065] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that transfers one or more antigen specificities into cells such as αβ T cells, γδ T cells, NK cells, macrophages, etc. CARs are also known as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, a CAR comprises the extracellular variable domain of an antibody specific for a tumor antigen and the intracellular signaling domain of a T cell or other receptor, such as one or more costimulatory domains. "CAR-T" refers to a T cell that expresses a CAR.

[0066] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. The term antibody includes fragments capable of binding to antigen, such as, but not limited to, Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody includes traditional four-chain antibodies, as well as heavy-chain-only antibodies or fragments thereof, such as V H This includes single domain antibodies such as H.

[0067] As used herein, the terms "bind," "specifically bind," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

[0068] Embodiments of the invention described herein are understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0069] Reference herein to a value or parameter with "about" includes (and describes) the variation that the value or parameter itself is subject to. For example, a statement referring to "about X" includes the statement of "X."

[0070] As used herein, a reference to "not being" a value or parameter generally means and describes "other than" a value or parameter. For example, a method that is not used to treat cancer type X means that the method is used to treat cancer types other than X.

[0071] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0072] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. II. Modified antigen-specific immune cells expressing exogenous CD160 protein

[0073] One aspect of the present invention provides modified antigen-specific immune cells comprising exogenous CD160 protein (e.g., on the surface), wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein. In some embodiments, the upmodulation comprises increased cytolytic lymphocyte (CTL) activity. In some embodiments, the upmodulation comprises enhanced tumor killing activity in an immunocompetent host. In some embodiments, the upmodulation comprises enhanced T cell and / or NK cell-mediated killing. In some embodiments, the upmodulation comprises enhanced expression of granzyme A and / or perforin. In some embodiments, the upmodulation comprises enhanced inflammatory response. In some embodiments, the upmodulation comprises enhanced expression and / or secretion of pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines comprise IFN-γ and / or TNF-α. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, or a variant thereof having at least about 80% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having any one of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (such as, but not limited to, a dimer, trimer, tetramer, pentamer, or hexamer). In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells). In some embodiments, the modified antigen-specific immune cells are cytotoxic αβ T cells.In some embodiments, the modified antigen-specific immune cells are tumor-infiltrating T cells or anti-tumor T cells activated by APCs. In some embodiments, the anti-tumor T cells activated by APCs are anti-tumor T cells activated by DCs. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, gamma delta T cells, and macrophages. In some embodiments, the precursor antigen-specific immune cells are isolated from a tumor of an individual. In some embodiments, the precursor antigen-specific immune cells are monoclonal. In some embodiments, the precursor antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells are monoclonal. In some embodiments, the modified antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells further comprise a functional exogenous receptor. In some embodiments, the functional exogenous receptor is a modified T cell receptor (TCR). In some embodiments, the engineered T cell receptor (TCR) recognizes a tumor antigen or a tumor-associated antigen. In further embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the engineered immune cells are multiple immune cells specific for the same epitope. Non-limiting examples include multiple T cells each comprising the same functional exogenous receptor (such as a CAR). In some embodiments, the engineered immune cells are multiple immune cells each specific for one of multiple non-identical epitopes (such as partially overlapping or entirely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0074] In some embodiments, modified antigen-specific immune cells are provided that comprise an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound.

[0075] In some embodiments, modified antigen-specific immune cells are provided that comprise an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound and the exogenous CD160 protein is attached to the membrane by a glycophosphatidylinositol (GPI) linker. In some embodiments, the exogenous CD160 protein comprises a GPI-anchor peptide sequence.

[0076] In some embodiments, modified antigen-specific immune cells are provided that comprise an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD160, CD4, CD8, CD5, CD6, CD16, CD22, CD33, CD37, CD80, CD86, CD134, CD137, CD154, CD244, T cell receptor (TCR) alpha subunit, TCR beta subunit, or TCR zeta subunit. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, CD80, CD152, and PD-1.

[0077] In some embodiments, modified antigen-specific immune cells are provided that comprise an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain and an intracellular domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD160, CD4, CD8, CD5, CD6, CD16, CD22, CD33, CD37, CD80, CD86, CD134, CD137, CD154, CD244, T cell receptor (TCR) alpha subunit, TCR beta subunit, or TCR zeta subunit. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, CD80, CD152, and PD-1. In some embodiments, the intracellular domain is derived from a CD160 splice variant. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from a signaling subunit of a TCR complex. In some embodiments, the signaling subunit of the TCR complex is selected from the group consisting of CD3 gamma, CD3 delta, and CD3 epsilon. In some embodiments, the intracellular domain comprises one or more signaling domains derived from a T cell stimulatory molecule. In some embodiments, the signaling domain is one or more of 4-1BB, OX40, CD27, CD28, CD80, or CD258. In some embodiments, the intracellular domain comprises a combination of two signaling domains selected from the group consisting of OX40, CD27, CD28, CD80, and CD258. [[CD160-TM+Co-Stimulation; Covers 5-7+]]

[0078] In some embodiments, modified antigen-specific immune cells are provided that comprise an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, and the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain and an intracellular domain, and the intracellular domain comprises one or more costimulatory signaling domains. In some embodiments, the intracellular domain comprises one, two, three, four, five, six, seven, eight, or more costimulatory signaling domains. In some embodiments, the intracellular domain contains no more than one, two, three, four, or five costimulatory signaling domains. In some embodiments, the intracellular domain does not comprise a CD3ζ signaling domain or a combination of 4-1BB and a CD3ζ domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, DAP10, CD30, CD40, CD3, CD80, CD258, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, or both. In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain is adjacent to the transmembrane domain. In some embodiments, the CD28 costimulatory domain is adjacent to the C-terminus of the transmembrane domain. In some embodiments, the intracellular domain comprises a primary signaling domain. In some embodiments, the primary signaling domain comprises a CD3ζ domain. In other embodiments, the intracellular domain does not comprise a primary signaling domain.In other embodiments, the intracellular domain does not include a CD3 zeta domain or a combination of 4-1BB and a CD3 zeta domain.

[0079] In some embodiments, modified antigen-specific immune cells are provided that comprise an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cell compared to a precursor antigen-specific immune cell that does not contain the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound and the exogenous CD160 protein is bound to the modified antigen-specific immune cell by an immune cell binding moiety. In some embodiments, the immune cell binding moiety binds to a surface molecule of the immune cell. In some embodiments, the immune cell binding moiety comprises an antibody that recognizes a T cell surface molecule. In some embodiments, the antibody is a full-length antibody or an scFv, Fv, Fab, (Fab')2, single domain antibody (sdAb), or V H The immune cell binding moiety may be an antibody fragment such as an H domain. Non-limiting examples include anti-CD3ε antibodies that recognize TCRs and / or activate TCR signaling. In some embodiments, the immune cell binding moiety comprises a ligand that binds to a cognate T cell surface receptor. Non-limiting examples include tumor-specific peptide-MHC complexes that recognize TCRs and IL-2.

[0080] In some embodiments of any of the modified antigen-specific immune cells described herein, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, or a variant thereof having at least about 80% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (such as, but not limited to, a dimer, trimer, tetramer, pentamer, or hexamer). In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells). In some embodiments, the modified antigen-specific immune cells are cytotoxic T cells. In some embodiments, the modified antigen-specific immune cells are tumor-infiltrating T cells or anti-tumor T cells activated by APCs. In some embodiments, anti-tumor T cells activated by APCs are anti-tumor T cells activated by DCs. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, γδ T cells, and macrophages. In some embodiments, the precursor antigen-specific immune cells are isolated from a tumor of an individual. In some embodiments, the precursor antigen-specific immune cells are monoclonal. In some embodiments, the precursor antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells are monoclonal. In some embodiments, the modified antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells further comprise a functional exogenous receptor.In some embodiments, the functional exogenous receptor is an engineered T cell receptor (TCR). In some embodiments, the engineered T cell receptor (TCR) recognizes a tumor antigen or a tumor-associated antigen. In further embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the engineered immune cells are multiple immune cells specific for the same epitope. Non-limiting examples include multiple T cells each comprising the same functional exogenous receptor (such as a CAR). In some embodiments, the engineered immune cells are multiple immune cells each specific for one of multiple non-identical epitopes (such as partially overlapping or entirely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0081] In some embodiments, the modified antigen-specific immune cells exhibit natural antigen recognition. In some embodiments, the modified antigen-specific immune cells exhibit engineered antigen recognition. In some embodiments, the antigen recognition of the modified antigen-specific immune cells is conferred at least in part by a functional exogenous receptor, such as, but not limited to, a CAR and a TCR. In some embodiments, the modified antigen-specific immune cells target tumor-associated antigens, mutated oncogenic antigens and random somatic antigens, and other neoantigens. In some embodiments, the modified antigen-specific immune cells are human immune cells. In some embodiments, the modified antigen-specific immune cells are mouse immune cells. In some embodiments, the modified antigen-specific immune cells are modified from one or more of TCR-T cells, CAR-T cells, TILs, or endogenous antigen-specific T cells. Some examples of human and mouse TCR-T cells, CAR-T cells, TILs, or endogenous antigen-specific T cells are reported in Tran et al., Nat Immunol. 2017;18(3):255-62, MacKay et al., Nat Biotechnol. 2020;38(2):233-44, and Schumacher et al., Cancer Neoantigens. Annu Rev Immunol. 2019;37:173-200, which are incorporated herein by reference. In some embodiments, the modified antigen-specific immune cells target a broad range of antigens. In some embodiments, the modified antigen-specific immune cells target one or more of the antigens listed in Table 1. Table 1: Exemplary list of tumor antigens and associated cancer indications [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0082] In some embodiments, a pharmaceutical composition is provided comprising any of the modified antigen-specific immune cells described herein. In some embodiments, a method is provided for producing any one of the modified antigen-specific immune cells described herein. Libraries of modified antigen-specific immune cells containing exogenous CD160 and methods for screening antigen-specific immune cells

[0083] In some embodiments, a library of antigen-specific immune cells (such as T cells) is provided, each having a functional exogenous receptor that recognizes a different antigen (such as a tumor antigen or a tumor-associated antigen). In one aspect, a library of antigen-specific immune cells (such as T cells) is provided, each having a functional exogenous receptor that recognizes a different tumor or tumor-associated antigen, wherein each antigen-specific immune cell in the library further comprises exogenous CD160 protein on its surface, and wherein the exogenous CD160 protein results in upmodulation of the engineered immune cell compared to a precursor immune cell that does not contain the exogenous CD160 protein.

[0084] In one aspect, a library of polyclonal immune cells (such as TILs) is provided, wherein each immune cell in the polyclonal composition is specific for one of multiple non-identical epitopes (such as partially overlapping or completely different epitopes), and wherein exogenous CD160 protein results in upmodulation of the engineered immune cells compared to precursor immune cells that do not contain the exogenous CD160 protein.

[0085] In some embodiments, a method of screening for immune cells comprising a functional exogenous receptor specific for a test antigen is provided, comprising contacting the test antigen with a library of antigen-specific immune cells (such as T cells), each having a functional exogenous receptor that recognizes a different antigen (such as a tumor or tumor-associated antigen), wherein each antigen-specific immune cell in the library further comprises an exogenous CD160 protein on its surface, and the exogenous CD160 protein results in up-modulation of the engineered immune cells compared to precursor immune cells that do not contain the exogenous CD160 protein. Desired antigen-specific immune cells expressing a functional exogenous receptor (such as a desired functional exogenous receptor) can be identified by contacting the test antigen with the library of antigen-specific immune cells (such as T cells), each having a functional exogenous receptor that recognizes a different antigen, and then analyzing the binding activity of the cells in the library to the test antigen, or by measuring the antigen-specific immune activity of the cells in the library, such as, but not limited to, ELISA analysis of any cytokine secretion (e.g., IFN-γ, TNF-α, and / or IL-2).

[0086] In some embodiments, a method for screening immune cells specific to a test antigen is provided, comprising contacting the test antigen with a library of polyclonal immune cells (such as TILs), wherein each immune cell in the polyclonal composition is specific for one of a plurality of non-identical epitopes (such as partially overlapping or completely different epitopes), and wherein the exogenous CD160 protein results in up-modulation of the engineered immune cells compared to precursor immune cells that do not contain the exogenous CD160 protein. Desired antigen-specific immune cells within the polyclonal composition can be identified by contacting the test antigen with a library of polyclonal immune cells (such as TILs), each cell of which is specific for one of a plurality of non-identical epitopes, and then analyzing the binding activity of the cells in the library to the test antigen, or by measuring the antigen-specific immune activity of the cells in the library, such as, but not limited to, ELISA analysis of any cytokine secretion (e.g., IFN-γ, TNF-α, and / or IL-2).

[0087] In some embodiments of any one of the above methods, the test antigen comprises one or more immunogenic epitopes. In some embodiments, the test antigen is derived from a lysate, such as a tumor lysate. In some embodiments, the library of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface is produced by a process comprising contacting a plurality of precursor antigen-specific immune cells with an exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein, thereby producing the library of modified antigen-specific immune cells. In some embodiments, the CD160 protein comprises the amino acid sequence of any of SEQ ID NOs: 1-4, or a variant thereof having at least about 80% identity to SEQ ID NOs: 1-4. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells). In some embodiments, the modified antigen-specific immune cells are cytotoxic T cells. In some embodiments, the modified antigen-specific immune cells are tumor-infiltrating T cells or anti-tumor T cells activated by APCs. In some embodiments, the anti-tumor T cells activated by APCs are anti-tumor T cells activated by DCs. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, γδ T cells, and macrophages. CD160 protein

[0088] The modified antigen-specific immune cells described herein express exogenous CD160 protein, which results in up-modulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain exogenous CD160 protein. The present application also provides exogenous CD160 proteins and compositions thereof. Table 2 shows the sequences of exemplary exogenous CD160 proteins. [Table 2]

[0089] In some embodiments, an exogenous CD160 protein or a fragment thereof is provided, comprising a naturally occurring CD160 polypeptide, wherein the exogenous CD160 protein results in up-modulation of engineered antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the exogenous CD160 protein. In some embodiments, the exogenous CD160 protein consists of, or consists essentially of, a naturally occurring CD160 protein or a fragment thereof. In some embodiments, the exogenous CD160 protein comprises an Ig-like V-type domain of a naturally occurring CD160 protein, or a fragment thereof. In some embodiments, the exogenous CD160 protein comprises a cysteine-rich domain of a naturally occurring CD160 protein, or a fragment thereof. In some embodiments, the exogenous CD160 protein comprises amino acids 25-133 of a naturally occurring CD160 protein, wherein the amino acid sequence numbering is based on any one of SEQ ID NOs: 1-3.

[0090] In some embodiments, the exogenous CD160 protein on the cell surface is in a monomeric form. In some embodiments, the exogenous CD160 protein on the cell surface is in a multimer. In some embodiments, the exogenous CD160 protein on the cell surface is in a multimer, such as a dimer, trimer, tetramer, pentamer, or hexamer. In some embodiments, the multimer comprises one or more exogenous CD160 proteins and one or more naturally occurring CD160 proteins. In some embodiments, the multimer comprises one or more exogenous CD160 proteins and one or more endogenous CD160 proteins. In some embodiments, the CD160 protein multimer on the cell surface is covalently linked. In some embodiments, the CD160 protein multimer on the cell surface is disulfide-bonded. In some embodiments, at least one, two, three, or four cysteine ​​residues in the exogenous CD160 protein are mutated. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-3, further comprising one or more mutations in cysteine ​​residues Cys26, Cys44, Cys61, Cys68, Cys112, Cys113, or any combination thereof. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence of SEQ ID NO: 4, further comprising one or more mutations in cysteine ​​residues Cys29, Cys47, Cys64, Cys71, Cys115, Cys116, or any combination thereof. In some embodiments, the exogenous CD160 protein having one or more of the above mutations is unable to form multimers.

[0091] In some embodiments, the exogenous CD160 protein exhibits the same or essentially the same binding affinity for MHC-I as a naturally occurring CD160 protein. In some embodiments, the exogenous CD160 protein exhibits increased binding affinity for MHC-I compared to a naturally occurring CD160 protein. In some embodiments, the MHC-I binding affinity of the exogenous CD160 protein is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% higher than that of a naturally occurring CD160 protein. In some embodiments, the MHC-I binding affinity of the exogenous CD160 protein is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, or about 100-fold higher than that of a naturally occurring CD160 protein. In some embodiments, the exogenous CD160 protein exhibits reduced binding affinity to MHC-I compared to a naturally occurring CD160 protein. In some embodiments, the MHC-I binding affinity of the exogenous CD160 protein is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% lower than that of a naturally occurring CD160 protein. In some embodiments, the MHC-I binding affinity of the exogenous CD160 protein is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, or about 100-fold lower than that of a naturally occurring CD160 protein.

[0092] In some embodiments, the exogenous CD160 protein exhibits the same or essentially the same binding affinity for herpesvirus entry mediator (HVEM) as the naturally occurring CD160 protein. In some embodiments, the exogenous CD160 protein exhibits increased binding affinity for HVEM compared to the naturally occurring CD160 protein. In some embodiments, the HVEM binding affinity of the exogenous CD160 protein is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% higher than that of the naturally occurring CD160 protein. In some embodiments, the HVEM-binding affinity of the exogenous CD160 protein is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, or about 100-fold higher than that of the naturally occurring CD160 protein. In some embodiments, the exogenous CD160 protein exhibits reduced binding affinity for HVEM compared to the naturally occurring CD160 protein. In some embodiments, the HVEM-binding affinity of the exogenous CD160 protein is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% lower than that of the naturally occurring CD160 protein. In some embodiments, the HVEM binding affinity of the exogenous CD160 protein is any one of about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, or about 100-fold lower than that of a naturally occurring CD160 protein.

[0093] In some embodiments, the exogenous CD160 protein competes with BTLA (also known as CD272) for binding to HVEM. In some embodiments, the exogenous CD160 protein does not compete with BTLA for binding to HVEM. In some embodiments, the exogenous CD160 protein exhibits a binding affinity for HVEM similar to that of BTLA. In some embodiments, the exogenous CD160 protein exhibits a higher binding affinity for HVEM compared to BTLA. In some embodiments, the exogenous CD160 protein exhibits a lower binding affinity for HVEM compared to BTLA. In some embodiments, the exogenous CD160 protein exhibits the same or essentially the same binding affinity for HVEM as BTLA. In some embodiments, the exogenous CD160 protein exhibits a higher binding affinity for HVEM compared to BTLA. In some embodiments, the exogenous CD160 protein exhibits a lower binding affinity for HVEM compared to BTLA. In some embodiments, the exogenous CD160 protein exhibits the same or essentially the same dissociation rate from HVEM binding as BTLA. In some embodiments, the exogenous CD160 protein exhibits a higher dissociation rate from HVEM binding compared to BTLA. In some embodiments, the exogenous CD160 protein exhibits a lower dissociation rate from HVEM binding compared to BTLA.

[0094] In some embodiments, the exogenous CD160 protein results in upmodulation of engineered antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the exogenous CD160 protein, and the CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, or a variant thereof having at least about 90% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 80% sequence identity, such as at least about 85%, about 87%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to any one of SEQ ID NOs: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 99% sequence identity to any one of SEQ ID NOs: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having any one of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any one of SEQ ID NOs: 1-4.

[0095] In some embodiments, the exogenous CD160 protein is derived from a mammalian CD160 protein. In some embodiments, the exogenous CD160 protein is derived from a mouse, dog, cat, horse, rat, goat, or rabbit CD160 protein. In some embodiments, the exogenous CD160 protein is derived from a human CD160 protein.

[0096] In some embodiments, the exogenous CD160 protein comprises a full-length CD160 protein. In some embodiments, the exogenous CD160 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4. CD160 protein sequences are known in the art, including, but not limited to, sequences having UniProt (World Wide Web at uniprot.org) accession numbers O95971 and O88875. Sequences of mRNA encoding CD160 proteins are also known in the art, including, but not limited to, sequences having NCBI (World Wide Web at ncbi.nlm.nih.gov) accession numbers NM_007053.3, XM_005272929.3, and NM_001163497.1.

[0097] In some embodiments, the exogenous CD160 protein comprises any one or more of at least about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600 amino acids. In some embodiments, the exogenous CD160 protein comprises any one or fewer of about 600, about 550, about 500, about 450, about 350, about 300, about 250, about 200, about 175, about 150, about 125, about 100, about 90, about 80, about 70, about 60, about 50 amino acids or less. In some embodiments, the exogenous CD160 protein comprises any one of about 50-60, about 50-75, about 50-100, about 50-150, about 50-200, about 50-250, about 100-150, about 100-200, about 100-250, about 150-250, about 250-500, or about 50-550 amino acids.

[0098] In some embodiments, the exogenous CD160 protein comprises an amino acid sequence variant of a naturally occurring CD160 protein or a fragment thereof. For example, it may be desirable to improve the binding affinity and / or other biological properties of the CD160 protein. Amino acid sequence variants of the CD160 protein can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the CD160 protein or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the CD160 protein. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, for example, pro-inflammatory activity.

[0099] In some embodiments, the exogenous CD160 protein comprises a naturally occurring CD160 protein or a fragment thereof having one or more (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, or more amino acids) conservative substitutions compared to the sequence of a naturally occurring CD160 protein or fragment thereof. In some embodiments, the exogenous CD160 protein comprises a naturally occurring CD160 protein or a fragment thereof having at least about 80% sequence identity to the sequence of a naturally occurring CD160 protein or fragment thereof, such as at least any one of about 85%, about 87%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity.

[0100] Conservative substitutions are shown in Table 3 below. Table 3: Conservative substitutions [Table 3]

[0101] Amino acids can be divided into different classes according to common side chain properties: a. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues that influence chain orientation: Gly, Pro; f. Aromatics: Trp, Tyr, Phe.

[0102] Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0103] Those skilled in the art will recognize that any suitable method can be used to generate mutations in a gene of interest, including mutagenesis, polymerase chain reaction, homologous recombination, or any other genetic engineering technique known to those skilled in the art. Mutations can involve a single nucleotide (such as point mutations, which involve the removal, addition, or substitution of a single nucleotide base in a DNA sequence), or they can involve the insertion or deletion of multiple nucleotides. Mutations can occur spontaneously as a result of events such as errors in the fidelity of DNA replication, or can be induced after exposure to chemical or physical mutagens. Mutations can also be site-specific by using specific targeting methods well known to those skilled in the art.

[0104] A useful method for identifying residues or regions of a polypeptide that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether immune upmodulation by a polypeptide agent (e.g., a CD160 variant) is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the CD160:MHC I complex or the CD160:HVEM complex can be determined to identify contact points between CD160 and MHC-I or between CD160 and HVEM, respectively. Such contact and neighboring residues can be targeted or removed as candidates for substitution to enhance or suppress CD160 function in antigen-specific immune cells depending on the disease indication. Variants can be screened to determine whether they contain the desired properties.

[0105] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0106] In some embodiments, the exogenous CD160 protein is secreted from the modified antigen-specific immune cells. In some embodiments, the exogenous CD160 protein comprises a signal peptide. The signal peptide (also known as a "leader sequence") is typically inserted at the N-terminus of the protein immediately after the Met initiator. The signal peptide can be cleaved to form the mature protein upon transport of the exogenous CD160 protein from the modified antigen-specific immune cells. Signal peptides can be natural or synthetic, and they can be heterologous or homologous to the protein to which they bind. The selection of signal peptides is wide and accessible to those skilled in the art, for example, in the online Leader sequence Database maintained by the Department of Biochemistry, National University of Singapore. See Choo et al., BMC Bioinformatics, 6: 249 (2005); and PCT Publication No. WO2006 / 081430. Functional exogenous receptors

[0107] Any of the above-described modified antigen-specific immune cells may further express a functional exogenous receptor. In some embodiments, the functional exogenous receptor is an engineered receptor. Exemplary functional exogenous receptors include, but are not limited to, CARs and engineered TCRs. In some embodiments, the functional exogenous receptor comprises an extracellular domain that specifically binds to an antigen (e.g., a tumor antigen), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain and / or a costimulatory domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of a TCR co-receptor. In some embodiments, the functional exogenous receptor is encoded by a heterologous nucleic acid sequence encoding an exogenous CD160 protein. In some embodiments, the functional exogenous receptor is encoded by a second heterologous nucleic acid operably linked to a promoter (such as a constitutive promoter or an inducible promoter). In some embodiments, a functional exogenous receptor is introduced into the modified antigen-specific immune cells by inserting a protein into the cell membrane while passing the cells through a microfluidic system, such as CELL SQUEEZE® (see, e.g., U.S. Patent Application Publication No. 20140287509). In some embodiments, the functional exogenous receptor is introduced into the modified immune cells by CRISPR-mediated gene editing. The functional exogenous receptor can enhance the function of the modified antigen-specific immune cells, such as by targeting the modified antigen-specific immune cells, transducing a signal, and / or enhancing the cytotoxicity of the modified antigen-specific immune cells. In some embodiments, the modified antigen-specific immune cells do not express a functional exogenous receptor, such as a CAR or TCR.

[0108] In some embodiments, a functional exogenous receptor comprises one or more specific binding domains that target at least one tumor antigen and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and / or costimulatory domains.

[0109] In some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). Many chimeric antigen receptors are known in the art and may be suitable for the modified antigen-specific immune cells of the present invention. For example, by utilizing an antigen-binding fragment of an antibody molecule or an antibody variable domain, a CAR with specificity for any cell surface marker can also be constructed. Any method for producing a CAR can be used herein. See, e.g., U.S. Patent No. 6,410,319, U.S. Patent No. 7,446,191, U.S. Patent No. 7,514,537, U.S. Patent No. 9,765,342B2, WO2002 / 077029, WO2015 / 142675, US2010 / 065818, US2010 / 025177, US2007 / 059298, WO2017025038A1, and Berger C. et al., J. Clinical Investigation 118: 1 294-308 (2008), which are incorporated by reference herein. In some embodiments, the engineered antigen-specific immune cells are CAR-αβ T cells, CAR-γδ T cells, CAR-NK cells, or CAR-macrophages.

[0110] The CAR of the present invention comprises an extracellular domain including at least one targeting domain that specifically binds to at least one tumor antigen, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain generates a signal that promotes the immune effector function of a CAR-containing cell, e.g., a CAR-T cell. "Immune effector function or immune effector response" refers to, for example, a function or response of an immune effector cell that enhances or promotes immune attack of a target cell. For example, an immune effector function or response may refer to a property of a T or NK cell that promotes killing of a target cell or inhibiting its growth or proliferation. For example, examples of immune effector functions in CAR-T cells include cytolytic activity (such as antibody-dependent cellular cytotoxicity, or ADCC) and helper activity (such as cytokine secretion). In some embodiments, the CAR has an intracellular signaling domain with attenuated immune effector function. In some embodiments, the CAR has an intracellular signaling domain that has about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or less of an immune effector function (such as cytolytic function against target cells) compared to a CAR having full-length and wild-type CD3ζ and, optionally, one or more costimulatory domains. In some embodiments, the intracellular signaling domain generates a signal that promotes the proliferation and / or survival of the CAR-containing cell. In some embodiments, the CAR comprises one or more intracellular signaling domains selected from the signaling domains of CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. The signaling domain of a naturally occurring molecule may comprise the entire intracellular (i.e., cytoplasmic) portion of the molecule, or the entire native intracellular signaling domain, or a fragment or derivative thereof.

[0111] In some embodiments, the intracellular signaling domain of the CAR comprises a primary intracellular signaling domain. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector function. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. In some embodiments, the primary intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12. In some embodiments, the primary intracellular signaling domain comprises a non-functional or attenuated signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12. The non-functional or attenuated signaling domain may be a mutant signaling domain having a point mutation, insertion, or deletion that attenuates or abolishes one or more immune effector functions, such as cytolytic activity or helper activity, including antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the CAR comprises a non-functional or attenuated CD3 zeta (i.e., CD3ζ or CD3z) signaling domain. In some embodiments, the intracellular signaling domain does not comprise a primary intracellular signaling domain. An attenuated primary intracellular signaling domain may induce about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% or less of an immune effector function (such as cytolytic function against target cells) compared to a CAR having the same construct but with a wild-type primary intracellular signaling domain.

[0112] In some embodiments, the intracellular signaling domain of the CAR comprises one or more (e.g., one, two, three, or more) costimulatory domains. A "costimulatory domain" may be the intracellular portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand to mediate a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that contributes to an efficient immune response. Costimulatory molecules may be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, and CD18 , LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0113] In some embodiments, the CAR comprises a single costimulatory domain. In some embodiments, the CAR comprises two or more costimulatory domains. In some embodiments, the intracellular signaling domain comprises a functional primary intracellular signaling domain and one or more costimulatory domains. In some embodiments, the CAR does not comprise a functional primary intracellular signaling domain (such as CD3ζ). In some embodiments, the CAR comprises an intracellular signaling domain consisting of, or consisting essentially of, one or more costimulatory domains. In some embodiments, the CAR comprises an intracellular signaling domain consisting of, or consisting essentially of, a non-functional or attenuated primary intracellular signaling domain (such as a mutant CD3ζ) and one or more costimulatory domains. Upon binding of the targeting domain to a tumor antigen, the costimulatory domain of the CAR can transduce signals for enhanced proliferation, survival, and differentiation of engineered immune cells (such as T cells) bearing the CAR, and inhibit activation-induced cell death. In some embodiments, the one or more costimulatory signaling domains are derived from one or more molecules selected from the group consisting of CD27, CD28, 4-1BB (i.e., CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0114] In some embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain in the chimeric receptor of the present application comprises a costimulatory signaling domain derived from 4-1BB (i.e., CD137). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of 4-1BB.

[0115] In some embodiments, the intracellular signaling domain of the CAR comprises the costimulatory signaling domain of CD28 and the costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ, the costimulatory signaling domain of CD28, and the costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a polypeptide comprising, from N-terminus to C-terminus, the costimulatory signaling domain of CD28, the costimulatory signaling domain of 4-1BB, and the cytoplasmic signaling domain of CD3ζ.

[0116] In some embodiments, the targeting domain of the CAR is selected from the group consisting of scFv, Fv, Fab, (Fab'), single domain antibody (sdAb), or V H The targeting domain of the CAR is an antibody or antibody fragment, such as an H domain. In some embodiments, the targeting domain of the CAR is a ligand or extracellular portion of a receptor that specifically binds to a tumor antigen. In some embodiments, one or more targeting domains of the CAR specifically bind to a single tumor antigen. In some embodiments, the CAR is a bispecific or multispecific CAR that includes targeting domains that bind to two or more tumor antigens. In some embodiments, the tumor antigen is selected from the group consisting of CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (such as EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens with clinical significance, and combinations thereof.

[0117] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD1 60, CD19, IL-2R beta, IL-2R gamma, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1 , ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT In some embodiments, the CAR comprises a transmembrane domain selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain of the CAR is the transmembrane domain of CD4, CD3, CD8α, or CD28. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of CD8α.

[0118] In some embodiments, the extracellular domain is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises the hinge region of CD8α.

[0119] In some embodiments, the CAR comprises a signal peptide, such as CD8αSP.

[0120] In some embodiments, the functional exogenous receptor is a modified T cell receptor. In some embodiments, the engineered TCR is specific for a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (such as EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens with clinical significance. In some embodiments, the tumor antigen is derived from an intracellular protein of a tumor cell. Many TCRs specific for tumor antigens (including tumor-associated antigens) have been described, including, for example, TCRs for the NY-ESO-1 cancer-testis antigen, p53 tumor suppressor antigen, melanoma (e.g., MARTI, gp100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1). Any TCR known in the art can be used in the present application. In some embodiments, the TCR has enhanced affinity for the tumor antigen. Exemplary TCRs and methods for introducing TCRs into immune cells are described, for example, in U.S. Patent No. 5,830,755 and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001). In some embodiments, the modified antigen-specific immune cells are TCR-T cells.

[0121] The TCR receptor complex is an octameric complex formed by the variable TCR receptor α and β chains (γ and δ chains in the case of γδ T cells) containing three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247 (T cell surface glycoprotein CD3 zeta chain) ζ / ζ or ζ / η. Ionizable residues in the transmembrane domains of each subunit form a polarized network of interactions that hold the complex together. The TCR complex functions to activate signaling cascades in T cells.

[0122] In some embodiments, the modified antigen-specific immune cells express more than one functional exogenous receptor, such as any combination of CAR or TCR receptors.

[0123] In some embodiments, the functional exogenous receptor (such as CAR or TCR) expressed by modified antigen-specific immune cells targets one or more tumor antigens.Tumor antigens are proteins produced by tumor cells that can induce immune responses, particularly T cell-mediated immune responses.The selection of the antigen targeted by the present invention will depend on the specific type of cancer to be treated. Exemplary tumor antigens include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0124] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute true tumor-specific immunoglobulin antigens that are unique to each individual tumor. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates for target antigens in B-cell lymphomas.

[0125] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSA is unique to tumor cells and does not exist on other cells in the body. TAA-associated antigens are not unique to tumor cells, but are instead expressed on normal cells under conditions that cannot induce immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAA may be antigens that are expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they may be antigens that are normally present at extremely low levels on normal cells but are expressed at much higher levels on tumor cells.

[0126] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as the Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, and BT. These include AA, CA125, CA15-3, CA27.29, BCAA, CA195, CA242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS. nucleic acid

[0127] In some embodiments, the modified antigen-specific immune cells described herein comprise one or more heterologous nucleic acid sequences encoding any one of the exogenous CD160 proteins and / or any one of the functional exogenous receptors described herein.

[0128] In some embodiments, an isolated nucleic acid is provided comprising a nucleic acid sequence encoding any one of the exogenous CD160 proteins described herein. In some embodiments, an isolated nucleic acid is provided comprising a nucleic acid sequence encoding any one of the functional exogenous receptors described herein. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is linear. In some embodiments, the nucleic acid is circular.

[0129] The nucleic acid sequence encoding the exogenous CD160 protein and / or the nucleic acid encoding the functional exogenous receptor can be operably linked to one or more regulatory sequences. Exemplary regulatory sequences that control the transcription and / or translation of the coding sequence are known in the art and may include, but are not limited to, a promoter, proper initiation, regulation, and / or termination of transcription (e.g., a polyA transcription termination sequence), mRNA transport (e.g., a nuclear localization signal sequence), processing (e.g., a splicing signal), stability (e.g., introns and non-coding 5' and 3' sequences), translation (e.g., an initiation Met, a tripartite leader sequence, an IRES ribosome binding site, a signal peptide, etc.), and additional elements for introducing the insert into a viral vector. In some embodiments, the regulatory sequence is a promoter, a transcription enhancer, and / or a sequence that allows proper expression of the exogenous CD160 protein and / or the functional exogenous receptor.

[0130] The term "regulatory sequence" or "control sequence" refers to a DNA sequence that influences the expression of an operably linked coding sequence. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, regulatory sequences generally include promoters, ribosome binding sites, and terminators. In eukaryotes, regulatory sequences include promoters, terminators, and in some cases, enhancers, transactivators, or transcription factors.

[0131] The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences.

[0132] As used herein, a "promoter" or "promoter region" refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operably linked. A promoter region contains specific sequences involved in RNA polymerase recognition, binding, and transcription initiation. In addition, a promoter contains sequences that modulate RNA polymerase recognition, binding, and transcription initiation activity (i.e., binding of one or more transcription factors). These sequences may be cis-acting or responsive to trans-acting factors. Depending on the nature of regulation, a promoter may be constitutive or regulatable. Regulatable promoters may be inducible or environmentally responsive (e.g., responsive to cues such as pH, anaerobic conditions, osmolytes, temperature, light, or cell density). Many such promoter sequences are known in the art. See, e.g., U.S. Patent Nos. 4,980,285; 5,631,150; 5,707,928; 5,759,828; 5,888,783; 5,919,670, and Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).

[0133] In some embodiments, the nucleic acid sequence encoding the exogenous CD160 protein is operably linked to a first promoter. In some embodiments, the nucleic acid sequence encoding the functional exogenous receptor is operably linked to a second promoter. In some embodiments, the nucleic acid sequence encoding the exogenous CD160 protein and the nucleic acid sequence encoding the functional exogenous receptor are operably linked to the same promoter. In some embodiments, the nucleic acid sequence encoding the exogenous CD160 protein and the nucleic acid sequence encoding the functional exogenous receptor are operably linked to separate promoters.

[0134] In some embodiments, the promoter is an endogenous promoter. For example, a nucleic acid encoding an exogenous CD160 protein and / or a functional exogenous receptor can be knocked into the genome of the modified antigen-specific immune cell downstream of the endogenous promoter using any method known in the art, such as CRISPR / Cas9 technology. In some embodiments, the endogenous promoter is a promoter for an abundant protein such as beta-actin. In some embodiments, the endogenous promoter is an inducible promoter, for example, inducible by an endogenous activation signal of the modified antigen-specific immune cell. In some embodiments, when the modified antigen-specific immune cell is a T cell, the promoter is a T cell activation-dependent promoter (such as an IL-2 promoter, an NFAT promoter, or an NFκB promoter). In some embodiments, the promoter is a heterologous promoter.

[0135] Various promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in the present invention. Promoters can be broadly classified as constitutive promoters or regulatable promoters, such as inducible promoters. In some embodiments, a heterologous nucleic acid sequence encoding an exogenous CD160 protein and / or a functional exogenous receptor is operably linked to a constitutive promoter. In some embodiments, a heterologous nucleic acid sequence encoding an exogenous CD160 protein and / or a functional exogenous receptor is operably linked to an inducible promoter. In some embodiments, a constitutive promoter is operably linked to a nucleic acid sequence encoding an exogenous CD160 protein, and an inducible promoter is operably linked to a nucleic acid sequence encoding a functional exogenous receptor. In some embodiments, a constitutive promoter is operably linked to a nucleic acid sequence encoding a functional exogenous receptor, and an inducible promoter is operably linked to a nucleic acid sequence encoding an exogenous CD160 protein. In some embodiments, the first inducible promoter is operably linked to a nucleic acid sequence encoding an exogenous CD160 protein, and the second inducible promoter is operably linked to a nucleic acid sequence encoding a functional exogenous receptor. In some embodiments, the first inducible promoter is induced by a first induction condition, and the second inducible promoter is induced by a second induction condition. In some embodiments, the first induction condition is the same as the second induction condition. In some embodiments, the first inducible promoter and the second inducible promoter are induced simultaneously. In some embodiments, the first inducible promoter and the second inducible promoter are induced sequentially, for example, the first inducible promoter is induced before the second inducible promoter, or the first inducible promoter is induced after the second inducible promoter.

[0136] A constitutive promoter can constitutively express a heterologous gene (also called a transgene) in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, the cytomegalovirus (CMV) promoter, human elongation factor-1 alpha (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter (CAGG) linked to the CMV early enhancer. The efficiency of such constitutive promoters for driving transgene expression has been widely compared in numerous studies. In some embodiments, the promoter is the hEF1α promoter.

[0137] In some embodiments, the promoter is an inducible promoter. Inducible promoters belong to the category of regulatable promoters. Inducible promoters can be induced by one or more conditions, such as a physical condition, the microenvironment of the modified antigen-specific immune cells, or the physiological state of the modified antigen-specific immune cells, an inducer (e.g., an inducer), or a combination thereof. In some embodiments, the inducing condition does not induce expression of an endogenous gene in the modified antigen-specific immune cells and / or in a subject receiving the pharmaceutical composition. In some embodiments, the inducing condition is selected from the group consisting of an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and activation state of the modified antigen-specific immune cells.

[0138] In some embodiments, the promoter is induced by an inducer. In some embodiments, the inducer is a small molecule such as a chemical compound. In some embodiments, the small molecule is selected from the group consisting of doxycycline, tetracycline, alcohol, metals, or steroids. Chemically inducible promoters are the most widely explored. Such promoters include promoters whose transcriptional activity is regulated by the presence or absence of small chemical molecules such as doxycycline, tetracycline, alcohol, steroids, metals, and other compounds. The doxycycline-inducible system using a reverse tetracycline-controlled transactivator (rtTA) and a tetracycline-responsive element promoter (TRE) is currently the most established system. WO9429442 describes the tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters. WO9601313 discloses tetracycline-regulated transcriptional modulators. Furthermore, Tet technologies, such as the Tet-on system, are described, for example, on the TetSystems.com website. Any known chemically regulated promoter can be used to drive expression of the therapeutic protein in the present application.

[0139] In some embodiments, the inducer is a polypeptide such as a growth factor, a hormone, or a ligand for a cell surface receptor, for example, a polypeptide that specifically binds to a tumor antigen. In some embodiments, the polypeptide is expressed by a modified antigen-specific immune cell. In some embodiments, the polypeptide is encoded by a nucleic acid in a heterologous nucleic acid. Many polypeptide inducers are also known in the art and may be suitable for use in the present invention. For example, ecdysone receptor-based gene switches, progesterone receptor-based gene switches, and estrogen receptor-based gene switches belong to gene switches that use steroid receptor-derived transactivators (e.g., WO9637609 and WO9738117).

[0140] In some embodiments, the inducer comprises both a small molecule component and one or more polypeptides. For example, inducible promoters that depend on the dimerization of polypeptides are known in the art and may be suitable for use in the present invention. The first small molecule CID system, developed in 1993, used FK1012, a derivative of the drug FK506, to induce the homodimerization of FKBP. Using a similar strategy, Wu et al. * and successfully generated titratable CAR-T cells via an ON switch mode by using a gibberellin / GID1-GAI dimerization-dependent gene switch (C.-Y. Wu et al., Science 350, aab4077 (2015)). Other dimerization-dependent switch systems include coumermycin / GyrB-GyrB (Nature 383 (6596): 178-81) and HaXS / Snap-tag-HaloTag (Chemistry and Biology 20 (4): 549-57).

[0141] In some embodiments, the promoter is a light-inducible promoter, and the inducing condition is light.Light-inducible promoters for regulating gene expression in mammalian cells are also well known in the art (see, for example, Science 332, 1565-1568 (2011); Nat. Methods 9, 266-269 (2012); Nature 500: 472-476 (2013); Nature Neuroscience 18: 1202-1212 (2015)).Such gene regulation systems can be roughly divided into two categories based on their regulation of (1) DNA binding or (2) the recruitment of transcription activation domains to DNA-bound proteins.For example, a synthetic mammalian blue light-controlled transcription system based on melanopsin has been developed and tested in mammalian cells, which responds to blue light (480 nm) and induces intracellular calcium increase, which leads to calcineurin-mediated translocation of NFAT. More recently, Motta-Mena et al. described a new inducible gene expression system developed from the naturally occurring EL222 transcription factor that confers high-level blue-light-sensitive control of transcription initiation in human cell lines and zebrafish embryos (Nat. Chem. Biol. 10(3):196-202 (2014)). Furthermore, the red-light-induced interaction between the Arabidopsis thaliana photoreceptor phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) was utilized to regulate red-light-induced gene expression. Furthermore, an ultraviolet B (UVB)-inducible gene expression system was also developed and proved effective in target gene transcription in mammalian cells (Chapter 25 of Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth Edition, CRC Press, Jan. 2014). th , 2015). Any of the light-inducible promoters described herein can be used to drive expression of a therapeutic protein in the present invention.

[0142] In some embodiments, the promoter is a light-inducible promoter that is induced by the combination of a light-inducible molecule and light.For example, a photocleavable photocage group on a chemical inducer keeps the inducer inactive unless the photocage group is removed by irradiation or other means.Such light-inducible molecules include small molecules, oligonucleotides, and proteins.For example, caged ecdysone, caged IPTG for use with the lac operon, caged toyocamycin for ribozyme-mediated gene expression, caged doxycycline for use with the Tet-on system, and caged rapalogs for light-mediated FKBP / FRB dimerization have been developed (see, for example, Curr Opin Chem Biol. 16(3-4): 292-299 (2012)).

[0143] In some embodiments, the promoter is a radiation-inducible promoter, and the inducing condition is radiation, such as ionizing radiation. Radiation-inducible promoters are also known in the art to control transgene expression. Alterations in gene expression occur upon irradiation of cells. For example, a group of genes known as "immediate early genes" can respond immediately to ionizing radiation. Exemplary immediate early genes include, but are not limited to, Erg-1, p21 / WAF-1, GADD45 alpha, t-PA, c-Fos, c-Jun, NF-kappa B, and AP1. Immediate early genes contain radiation-responsive elements in their promoter regions. The consensus sequence CC(A / T)6GG (SEQ ID NO: 7) is found in the Erg-1 promoter and is called the serum response element, or known as the CArG element. The combination of radiation-inducible promoters with transgenes has been intensively studied and has proven effective in terms of therapeutic benefit. See, for example, Cancer Biol Ther. 6(7):1005-12 (2007) and Chapter 25 of Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth Edition, CRC Press, Jan. 20th , 2015.

[0144] In some embodiments, the promoter is a heat-inducible promoter, and the inducing condition is heat. Heat-inducible promoters for driving transgene expression have also been widely studied in the art. Heat shock or stress proteins (HSPs), including Hsp90, Hsp70, Hsp60, Hsp40, Hsp10, etc., play an important role in protecting cells under heat or other physical and chemical stresses. Several heat-inducible promoters, including heat shock protein (HSP) promoters and growth arrest and DNA damage (GADD)153 promoters, have been tested in preclinical trials. The promoter of the human hsp70B gene, first described in 1985, is considered to be one of the most efficient heat-inducible promoters. Huang et al. reported that after introducing hsp70B-EGFP, hsp70B-TNFalpha, and hsp70B-IL12 coding sequences, tumor cells expressed significantly higher transgene expression upon heat treatment, but no transgene expression was detected in the absence of heat treatment. And tumor growth was significantly delayed in vivo in the IL12 transgene + heat-treated mice (Cancer Res. 60:3435 (2000)). Another group of scientists linked the HSV-tk suicide gene with the hsp70B promoter and tested the system in nude mice bearing mouse breast cancer. Mice injected with the hsp70B-HSVtk coding sequence into their tumors and heat-treated showed tumor regression and significant survival rates compared with non-heat-treated controls (Hum. Gene Ther. 11:2453 (2000)). Additional heat-inducible promoters known in the art can be found, for example, in Chapter 25 of Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth Edition, CRC Press, Jan. 20 th, 2015. Any of the heat-inducible promoters discussed herein can be used to drive expression of the therapeutic proteins of the invention.

[0145] In some embodiments, promoter is induced by redox condition.The exemplary promoter that is induced by redox condition includes inducible promoter and hypoxia-induced promoter.For example, Post DE et al. have developed hypoxia-inducible factor (HIF) responsive promoter, which specifically and strongly induces transgene expression in HIF-active tumor cells (Gene Ther.8:1801-1807(2001);Cancer Res.67:6872-6881(2007)).

[0146] In some embodiments, the promoter is induced by a physiological state, such as an endogenous activation signal of the modified antigen-specific immune cell. In some embodiments, when the modified antigen-specific immune cell is a T cell, the promoter is a T cell activation-dependent promoter that is induced by an endogenous activation signal of the modified T cell. In some embodiments, the modified T cell is activated by an inducer such as phorbol myristate acetate (PMA), ionomycin, or phytohemagglutinin. In some embodiments, the modified T cell is activated by recognition of a tumor antigen on a tumor cell via a functional exogenous receptor (such as a CAR or TCR). In some embodiments, the T cell activation-dependent promoter is an IL-2 promoter. In some embodiments, the T cell activation-dependent promoter is an NFAT promoter. In some embodiments, the T cell activation-dependent promoter is an NFκB promoter.

[0147] The heterologous nucleic acid sequences described herein may be present in a heterologous gene expression cassette comprising one or more protein-coding sequences and, optionally, one or more promoters. In some embodiments, the heterologous gene expression cassette comprises a single protein-coding sequence. In some embodiments, the heterologous gene expression cassette comprises two or more protein-coding sequences driven by a single promoter (i.e., polycistronic). In some embodiments, the heterologous gene expression cassette further comprises one or more regulatory sequences (such as a 5'UTR, a 3'UTR, an enhancer sequence, an IRES, a transcription termination sequence), a recombination site, one or more selectable markers (such as antibiotic resistance genes, reporter genes), a signal sequence, or a combination thereof.

[0148] In some embodiments, a vector is provided comprising any one of the nucleic acids encoding the exogenous CD160 proteins and / or functional exogenous receptors described herein. In some embodiments, a vector is provided comprising a first nucleic acid sequence encoding any one of the exogenous CD160 proteins described herein and a second nucleic acid sequence encoding any one of the functional exogenous receptors described herein. In some embodiments, a composition is provided comprising a first vector comprising a first nucleic acid sequence encoding any one of the exogenous CD160 proteins described herein and a second vector comprising a second nucleic acid sequence encoding any one of the functional exogenous receptors described herein.

[0149] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Several vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Generally, a suitable vector contains a replication origin functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers. The term "vector" should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, etc.

[0150] In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, vaccinia vectors, herpes simplex virus vectors, and their derivatives.Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0151] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using techniques known in the art, heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles. The recombinant viruses can then be isolated and delivered to engineered antigen-specific immune cells in vitro or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors can be packaged using protocols known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells (such as human T cells) using methods known in the art.

[0152] In some embodiments, the vector is a non-viral vector, such as a plasmid, or an episomal expression vector.

[0153] In some embodiments, the vector is an expression vector. An "expression vector" is a construct that can be used to transform a selected host and provides for expression of a coding sequence in the selected host. Expression vectors may be, for example, cloning vectors, binary vectors, or integrating vectors. Expression involves transcription of a nucleic acid molecule, preferably into translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells are well known to those skilled in the art. In eukaryotic cells, they usually include a promoter to ensure initiation of transcription and, optionally, a polyA signal to ensure termination of transcription and stabilization of the transcript. Examples of regulatory elements enabling expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the CMV, SV40, or RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells. Furthermore, depending on the expression system used, leader sequences capable of directing a polypeptide to a cellular compartment or secreting it into the medium can be added to the coding sequence of the described nucleic acid sequence, and are well known in the art. The leader sequence is assembled in an appropriate step with translation, initiation, and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein, or a portion thereof, into the periplasmic space or extracellular medium. If desired, the nucleic acid sequence may encode a fusion protein containing an N-terminal identification peptide that provides desirable properties, such as stabilization or simplified purification of the expressed recombinant product. Suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pEF-DHFR, and pEF-ADA (Raum et al., Cancer Immunol Immunother (2001) 50(3), 141-150) or pSPORT1 (GIBCO BRL). Method for preparing modified antigen-specific immune cells containing exogenous CD160

[0154] The present application also provides methods of producing any one of the modified antigen-specific immune cells described herein.

[0155] In certain aspects, methods are provided for producing modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, comprising producing the modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells. In some embodiments, the method comprises contacting precursor antigen-specific immune cells with exogenous CD160 protein. In some embodiments, the exogenous CD160 protein comprises an immune cell binding moiety that binds to a surface molecule of the immune cell. In some embodiments, the method comprises introducing a nucleic acid encoding the exogenous CD160 protein into the precursor antigen-specific immune cells. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is DNA. The nucleic acid can be introduced into the modified antigen-specific immune cells using any transfection or transduction method known in the art, including viral or non-viral methods. Exemplary non-viral transfection methods include, but are not limited to, chemical-based transfection using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods such as electroporation, cell squeezing, sonoporation, optical transfection, impale infection, protoplast fusion, hydrodynamic delivery, or transposon; particle-based methods using gene guns, magnetofection or magnet-assisted transfection, particle bombardment, and the like; and hybrid methods such as nucleofection. In some embodiments, nucleic acids are introduced into precursor antigen-specific immune cells by transfection. In some embodiments, nucleic acids are introduced into precursor antigen-specific immune cells by transduction or electroporation.

[0156] In some embodiments, methods are provided for producing modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, the method comprising producing the modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells relative to the precursor antigen-specific immune cells. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence of any of SEQ ID NOs: 1-4, or a variant thereof having at least about 80% identity to SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (such as, but not limited to, a dimer, a trimer, a tetramer, a pentamer, or a hexamer). In some embodiments, the modified antigen-specific immune cell is selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells). In some embodiments, the modified antigen-specific immune cell is a cytotoxic T cell. In some embodiments, the modified antigen-specific immune cell is a tumor-infiltrating T cell or an anti-tumor T cell activated by APCs. In some embodiments, the anti-tumor T cells activated by APCs are anti-tumor T cells activated by DCs. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, γδ T cells, and macrophages.In some embodiments, the precursor antigen-specific immune cells are isolated from a tumor in an individual. In some embodiments, the precursor antigen-specific immune cells are monoclonal. In some embodiments, the precursor antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells are monoclonal. In some embodiments, the modified antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells further comprise a functional exogenous receptor. In some embodiments, the functional exogenous receptor is a modified T cell receptor (TCR). In some embodiments, the engineered T cell receptor (TCR) recognizes a tumor antigen or a tumor-associated antigen. In further embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the modified antigen-specific immune cells are multiple immune cells specific for the same epitope. Non-limiting examples include multiple T cells each comprising the same functional exogenous receptor (such as a CAR). In some embodiments, the modified antigen-specific immune cells are multiple immune cells each specific for a non-identical epitope (such as partially overlapping or entirely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0157] In some embodiments, methods are provided for producing modified antigen-specific immune cells comprising exogenous CD160 protein on the surface thereof, the method comprising producing the modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with exogenous CD160 protein or a nucleic acid encoding an exogenous CD160 protein, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, and wherein the exogenous CD160 protein is membrane-bound.

[0158] In some embodiments, methods are provided for producing modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, the method comprising producing the modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, and wherein the exogenous CD160 protein is membrane-bound and the exogenous CD160 protein is bound to the membrane via a glycophosphatidylinositol (GPI) linker. In some embodiments, the exogenous CD160 protein comprises a GPI-anchor peptide sequence.

[0159] In some embodiments, methods are provided for producing modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, the method comprising contacting precursor antigen-specific immune cells with exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein to produce the modified antigen-specific immune cells, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, and wherein the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD160, CD4, CD8, CD5, CD6, CD16, CD22, CD33, CD37, CD80, CD86, CD134, CD137, CD154, CD244, T cell receptor (TCR) alpha subunit, TCR beta subunit, or TCR zeta subunit. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, CD80, CD152 and PD1.

[0160] In some embodiments, methods are provided for producing modified antigen-specific immune cells comprising an exogenous CD160 protein on the surface thereof, the method comprising producing the modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with an exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, and wherein the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain and an intracellular domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD160, CD4, CD8, CD5, CD6, CD16, CD22, CD33, CD37, CD80, CD86, CD134, CD137, CD154, CD244, T cell receptor (TCR) alpha subunit, TCR beta subunit, or TCR zeta subunit. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, CD80, CD152, and PD-1. In some embodiments, the intracellular domain is derived from a CD160 splice variant. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from a signaling subunit of a TCR complex. In some embodiments, the signaling subunit of a TCR complex is selected from the group consisting of CD3 gamma, CD3 delta, and CD3 epsilon. In some embodiments, the intracellular domain comprises one or more signaling domains derived from a T cell stimulatory molecule. In some embodiments, the signaling domain is one or more of 4-1BB, OX40, CD27, CD28, CD80, or CD258. In some embodiments, the intracellular domain comprises a combination of two signaling domains selected from the group consisting of OX40, CD27, CD28, CD80, and CD258.

[0161] In some embodiments, methods are provided for producing modified antigen-specific immune cells comprising an exogenous CD160 protein on the surface thereof, the method comprising contacting precursor antigen-specific immune cells with an exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein to produce the modified antigen-specific immune cells, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, the exogenous CD160 protein being membrane-bound, the CD160 protein comprising a transmembrane domain and an intracellular domain, and the intracellular domain comprising one or more costimulatory signaling domains. In some embodiments, the intracellular domain comprises one, two, three, four, five, six, seven, eight, or more costimulatory signaling domains. In some embodiments, the intracellular domain contains no more than one, two, three, four, or five costimulatory signaling domains. In some embodiments, the intracellular domain does not comprise a CD3ζ signaling domain or a combination of 4-1BB and a CD3ζ domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, DAP10, CD30, CD40, CD3, CD80, CD258, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, or both. In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain is adjacent to the transmembrane domain. In some embodiments, the CD28 costimulatory domain is adjacent to the C-terminus of the transmembrane domain. In some embodiments, the intracellular domain comprises a primary signaling domain.In some embodiments, the primary signaling domain comprises a CD3ζ domain. In other embodiments, the intracellular domain does not comprise a primary signaling domain. In other embodiments, the intracellular domain does not comprise a CD3ζ domain or a combination of 4-1BB and CD3ζ domains.

[0162] In some embodiments, methods are provided for producing modified antigen-specific immune cells comprising an exogenous CD160 protein on their surface, the method comprising producing the modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with an exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, the exogenous CD160 protein being membrane-bound, and the exogenous CD160 protein is bound to the modified antigen-specific immune cells via an immune cell binding moiety. In some embodiments, the immune cell binding moiety binds to a surface molecule of an immune cell. In some embodiments, the immune cell binding moiety comprises an antibody that recognizes a T cell surface molecule. In some embodiments, the antibody is a full-length antibody or an scFv, Fv, Fab, (Fab')2, single domain antibody (sdAb), or V H The immune cell binding moiety may be an antibody fragment such as an H domain. Non-limiting examples include anti-CD3ε antibodies that recognize TCRs and / or activate TCR signaling. In some embodiments, the immune cell binding moiety comprises a ligand that binds to a cognate T cell surface receptor. Non-limiting examples include tumor-specific peptide-MHC complexes that recognize TCRs and IL-2.

[0163] In some embodiments of any one of the methods described herein, the exogenous CD160 protein comprises the amino acid sequence of any of SEQ ID NOs: 1-4, or a variant thereof having at least about 80% identity to SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (such as, but not limited to, a dimer, trimer, tetramer, pentamer, or hexamer). In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells). In some embodiments, the modified antigen-specific immune cells are cytotoxic T cells. In some embodiments, the modified antigen-specific immune cells are tumor-infiltrating T cells or anti-tumor T cells activated by APCs. In some embodiments, anti-tumor T cells activated by APCs are anti-tumor T cells activated by DCs. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, γδ T cells, and macrophages. In some embodiments, the precursor antigen-specific immune cells are isolated from a tumor in an individual. In some embodiments, the precursor antigen-specific immune cells are monoclonal. In some embodiments, the precursor antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells are monoclonal. In some embodiments, the modified antigen-specific immune cells are derived from a polyclonal population.In some embodiments, the modified antigen-specific immune cells further comprise a functional exogenous receptor. In some embodiments, the functional exogenous receptor is a modified T cell receptor (TCR). In some embodiments, the engineered T cell receptor (TCR) recognizes a tumor antigen or a tumor-associated antigen. In further embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the modified antigen-specific immune cells are multiple immune cells specific for the same epitope. Non-limiting examples include multiple T cells each comprising the same functional exogenous receptor (such as a CAR). In some embodiments, the modified antigen-specific immune cells are multiple immune cells each specific for non-identical epitopes (such as partially overlapping or entirely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0164] In some embodiments of any one of the methods for producing modified antigen-specific immune cells comprising an exogenous CD160 protein described herein on the surface thereof, the precursor antigen-specific immune cells further comprise a second nucleic acid encoding a functional exogenous receptor. In some embodiments, the method further comprises contacting the precursor antigen-specific immune cells with the second nucleic acid encoding the functional exogenous receptor. In some embodiments, the functional exogenous receptor is an engineered T cell receptor (TCR). In some embodiments, the functional exogenous receptor is an engineered T cell receptor (TCR). In some embodiments, the engineered T cell receptor (TCR) recognizes a tumor antigen or a tumor-associated antigen. In further embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to the same promoter. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to separate promoters. In some embodiments, the first nucleic acid and the second nucleic acid are on the same vector. In some embodiments, the first nucleic acid and the second nucleic acid are on separate vectors. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, and derivatives thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is an episomal expression vector. In some embodiments, the method further comprises isolating or enriching immune cells comprising the first nucleic acid and / or the second nucleic acid. In some embodiments, the method further comprises formulating the modified antigen-specific immune cells with at least one pharmaceutically acceptable carrier.

[0165] In some embodiments, provided are modified antigen-specific immune cells obtained by any of the methods described herein. In some embodiments, provided are pharmaceutical compositions comprising any of the modified antigen-specific immune cells described herein and a pharmaceutically acceptable carrier.

[0166] In some embodiments, an isolated host cell is provided comprising any one of the nucleic acids or vectors described herein. The host cell may be useful for expressing or cloning exogenous CD160 protein and / or a functional exogenous receptor, or a nucleic acid or vector encoding the exogenous CD160 protein and / or a functional exogenous receptor. Suitable host cells may include higher eukaryotic cells, such as, but not limited to, prokaryotic cells, fungal cells, yeast cells, or mammalian cells. In some embodiments, the host cell comprises a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In some embodiments, the host cell comprises a single vector comprising isolated nucleic acids encoding the first and second polypeptides. In some embodiments, the first polypeptide is an exogenous CD160 protein. In some embodiments, the second polypeptide is a functional exogenous receptor.

[0167] Precursor antigen-specific immune cells can be prepared using various methods known in the art. For example, primary immune cells, such as T cells, can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, immune cells (such as T cells) can be obtained from a unit of blood collected from an individual using any number of techniques known in the art, such as FICOLL™ separation. In some embodiments, cells derived from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS) or a wash solution lacking divalent cations, such as calcium and magnesium. As one of ordinary skill in the art would readily appreciate, the washing step can be accomplished by methods known in the art, for example, by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be centrifuged, for example, using Ca 2+ Contains no Mg 2+ The cells can be resuspended in a variety of biocompatible buffers, such as free PBS, PlasmaLyte A, or other aqueous salt solutions with or without buffering agents. Alternatively, undesirable components of the apheresis sample can be removed and the cells can be resuspended directly in culture medium.

[0168] In some embodiments, primary T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. + , CD28 + , CD4 + , CD8+ Specific subpopulations of T cells, such as CD45RA, and CD45RO cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient for positive selection of the desired T cells.

[0169] In some embodiments, the T cell population can be further enriched by negative selection using a combination of antibodies against surface markers unique to the negatively selected cells. For example, one method involves cell sorting and / or selection by negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can be used to enrich the T cell population for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, the cocktail typically includes antibodies against CD4 + , CD25 + , CD62L hi , G.I.T.R. + , and FoxP3 + It may be desirable to enrich for or positively select for regulatory T cells expressing T-cell receptor 2 (TCR2). Alternatively, in certain embodiments, regulatory T cells are depleted by anti-CD25 conjugated beads or other similar selection methods.

[0170] Methods for introducing vectors or nucleic acids into host cells (such as precursor antigen-specific immune cells) are known in the art. The vectors or nucleic acids can be transferred into host cells by physical, chemical, or biological methods.

[0171] Physical methods for introducing vectors or nucleic acids into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, vectors are introduced into cells by electroporation.

[0172] Biological methods for introducing vectors or nucleic acids into host cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human, cells.

[0173] Chemical means for introducing vectors or nucleic acids into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro is a liposome (e.g., an artificial membrane vesicle).

[0174] In some embodiments, the transduced or transfected precursor antigen-specific immune cells are expanded ex vivo after introduction of the heterologous nucleic acid. In some embodiments, the transduced or transfected precursor antigen-specific immune cells are cultured and expanded for at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 12 days, or about 14 days. In some embodiments, the transduced or transfected precursor antigen-specific immune cells are cultured for no more than about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 12 days, or about 14 days. In some embodiments, the transduced or transfected precursor antigen-specific immune cells are further evaluated or screened to select modified antigen-specific immune cells.

[0175] Reporter genes can be used to identify potentially transfected cells and evaluate the function of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues, and encode polypeptides whose expression is indicated by some easily detectable characteristic, such as enzymatic activity. The expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al. FEBS Letters 479: 79-82 (2000)).

[0176] Other methods for confirming the presence of heterologous nucleic acids in precursor antigen-specific immune cells include molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; biochemical assays, such as detecting the presence or absence of specific peptides by immunological methods (such as ELISA and Western blot).

[0177] In some embodiments, modified antigen-specific immune cells are provided that express one or more exogenous CD160 proteins as described herein. In some embodiments, modified antigen-specific immune cells are provided that overexpress a CD160 protein. In some embodiments, the CD160 protein is an endogenous protein. In some embodiments, the CD160 protein is an exogenous protein. In some embodiments, the CD160-modified antigen-specific immune cells exhibit increased proliferation and / or increased viability compared to antigen-specific immune cells that are not CD160-modified. In some embodiments, the yield and / or viability of the CD160-modified antigen-specific immune cells is increased by at least one of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10,000-fold, or greater, compared to antigen-specific immune cells that are not CD160-modified. In some embodiments, the yield and / or viability of CD160-modified antigen-specific immune cells is increased by at least one of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to antigen-specific immune cells that are not CD160-modified. In some embodiments, the yield and / or viability of CD160-modified antigen-specific immune cells is increased by at least one of about 1-fold to about 2-fold, about 2-fold to about 5-fold, about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 50-fold, about 50-fold to about 100-fold, about 100-fold to about 500-fold, about 500-fold to about 1000-fold, or about 1000-fold to about 10000-fold, compared to antigen-specific immune cells that are not CD160-modified. In some embodiments, the yield and / or viability of CD160-modified antigen-specific immune cells is increased by at least any one of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% compared to antigen-specific immune cells that are not CD160-modified.

[0178]

[0010] In some aspects, methods of increasing the yield and / or viability of antigen-specific immune cells are provided, comprising introducing into immune cells a nucleic acid encoding an exogenous CD160 protein. In some embodiments, the yield and / or viability of antigen-specific immune cells expressing the exogenous CD160 protein is increased by at least one of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold, or greater, compared to antigen-specific immune cells that do not express the exogenous CD160 protein. In some embodiments, the yield and / or viability of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least any one of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to antigen-specific immune cells that do not express exogenous CD160 protein. In some embodiments, the yield and / or viability of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least one of the following fold changes: about 1-fold to about 2-fold, about 2-fold to about 5-fold, about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 50-fold, about 50-fold to about 100-fold, about 100-fold to about 500-fold, about 500-fold to about 1000-fold, or about 1000-fold to about 10000-fold, compared to antigen-specific immune cells not expressing exogenous CD160 protein. In some embodiments, the yield and / or viability of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% compared to antigen-specific immune cells that do not express exogenous CD160 protein. In some embodiments, the increased yield of CD160-modified antigen-specific immune cells is caused by an increased proliferation rate of the CD160-modified antigen-specific immune cells.

[0179] In some aspects, a method for increasing the yield and / or viability of antigen-specific immune cells is provided, comprising causing overexpression of CD160 protein in immune cells. In some embodiments, the CD160 protein is an endogenous protein. In some embodiments, the CD160 protein is an exogenous protein. In some embodiments, the yield and / or viability of antigen-specific immune cells that overexpress CD160 protein is increased by at least one of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold, or a greater fold change, compared to antigen-specific immune cells that do not overexpress CD160 protein. In some embodiments, the yield and / or viability of antigen-specific immune cells that overexpress CD160 protein is increased by at least any one of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to antigen-specific immune cells that do not overexpress CD160 protein. In some embodiments, the yield and / or viability of antigen-specific immune cells that overexpress CD160 protein is increased by at least one of the following fold changes, compared to antigen-specific immune cells that do not overexpress CD160 protein: about 1-fold to about 2-fold, about 2-fold to about 5-fold, about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 50-fold, about 50-fold to about 100-fold, about 100-fold to about 500-fold, about 500-fold to about 1000-fold, or about 1000-fold to about 10000-fold. In some embodiments, the yield and / or viability of antigen-specific immune cells overexpressing CD160 protein is increased by at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% compared to antigen-specific immune cells that do not overexpress CD160 protein. In some embodiments, the increased yield of CD160-modified antigen-specific immune cells is caused by an increased proliferation rate of the CD160-modified antigen-specific immune cells.

[0180] In some embodiments, methods are provided for producing therapeutic antigen-specific immune cells, including methods for increasing the yield and / or viability of antigen-specific immune cells selected according to any one of the methods described herein. In some embodiments, the therapeutic antigen-specific immune cells comprise tumor-infiltrating lymphocytes (TILs). In some embodiments, the therapeutic antigen-specific immune cells comprise a functional exogenous receptor. In some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the functional exogenous receptor is an engineered T cell receptor (TCR). Also provided are therapeutic antigen-specific immune cells produced according to any one of the methods described herein. In some embodiments, the use of CD160 overexpression to increase the yield and / or viability of therapeutic TILs, TCR-T cells, and / or CAR-T cell production is provided. In some embodiments, the use of exogenous CD160 expression to increase the yield and / or viability of therapeutic TILs, TCR-T cells, and / or CAR-T cell production is provided.

[0181] In some embodiments, modified antigen-specific immune cells are provided that express one or more exogenous CD160 proteins described herein. In some embodiments, modified antigen-specific immune cells are provided that overexpress a CD160 protein. In some embodiments, the CD160 protein is an endogenous protein. In some embodiments, the CD160 protein is an exogenous protein. In some embodiments, the CD160-modified antigen-specific immune cells exhibit increased in vitro and / or in vivo cytolytic activity compared to antigen-specific immune cells that are not CD160-modified. In some embodiments, the in vitro and / or in vivo cytolytic activity of the CD160-modified antigen-specific immune cells is increased by any one of at least about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10,000-fold, or greater, compared to antigen-specific immune cells that are not CD160-modified. In some embodiments, the in vitro and / or in vivo cytolytic activity of the CD160-modified antigen-specific immune cells is increased by at least any one of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to antigen-specific immune cells that are not CD160-modified. In some embodiments, the in vitro and / or in vivo cytolytic activity of CD160-modified antigen-specific immune cells is increased by at least one of the following fold changes compared to non-CD160-modified antigen-specific immune cells: about 1-fold to about 2-fold, about 2-fold to about 5-fold, about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 50-fold, about 50-fold to about 100-fold, about 100-fold to about 500-fold, about 500-fold to about 1000-fold, or about 1000-fold to about 10000-fold.In some embodiments, the in vitro and / or in vivo cytolytic activity of the CD160-modified antigen-specific immune cells is increased by at least any one of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% compared to antigen-specific immune cells that are not CD160-modified.

[0182]

[0010] In some aspects, methods are provided for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells, comprising introducing into the immune cells a nucleic acid encoding an exogenous CD160 protein. In some embodiments, the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least one of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold, or greater, compared to antigen-specific immune cells that do not express exogenous CD160 protein. In some embodiments, the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least any one of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to antigen-specific immune cells that do not express exogenous CD160 protein. In some embodiments, the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least one of the following fold changes: about 1-fold to about 2-fold, about 2-fold to about 5-fold, about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 50-fold, about 50-fold to about 100-fold, about 100-fold to about 500-fold, about 500-fold to about 1000-fold, or about 1000-fold to about 10000-fold, compared to antigen-specific immune cells not expressing exogenous CD160 protein. In some embodiments, the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least any one of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%, compared to antigen-specific immune cells that do not express exogenous CD160 protein.

[0183] In some aspects, a method for increasing the in vitro and / or in vivo cytolytic activity of an antigen-specific immune cell is provided, comprising causing overexpression of CD160 protein in the immune cell. In some embodiments, the CD160 protein is an endogenous protein. In some embodiments, the CD160 protein is an exogenous protein. In some embodiments, the in vitro and / or in vivo cytolytic activity of an antigen-specific immune cell that overexpresses CD160 protein is increased by at least one of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold, or a greater fold change, compared to an antigen-specific immune cell that does not overexpress CD160 protein. In some embodiments, the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells that overexpress CD160 protein is increased by at least any one of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to antigen-specific immune cells that do not overexpress CD160 protein. In some embodiments, the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells that overexpress CD160 protein is increased by at least one of the following fold changes: about 1-fold to about 2-fold, about 2-fold to about 5-fold, about 5-fold to about 10-fold, about 10-fold to about 20-fold, about 20-fold to about 50-fold, about 50-fold to about 100-fold, about 100-fold to about 500-fold, about 500-fold to about 1000-fold, or about 1000-fold to about 10000-fold, compared to antigen-specific immune cells that do not overexpress CD160 protein.In some embodiments, the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells overexpressing CD160 protein is increased by at least about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% compared to antigen-specific immune cells that do not overexpress CD160 protein. In some embodiments, methods are provided for producing therapeutic antigen-specific immune cells, including methods for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells selected according to any one of the methods described herein. In some embodiments, the therapeutic antigen-specific immune cells comprise tumor-infiltrating lymphocytes (TILs). In some embodiments, the therapeutic antigen-specific immune cells comprise a functional exogenous receptor. In some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the functional exogenous receptor is an engineered T cell receptor (TCR). Also provided are therapeutic antigen-specific immune cells produced according to any one of the methods described herein. In some embodiments, provided is the use of CD160 overexpression to increase the cytolytic activity of therapeutic antigen-specific T cells (including but not limited to TIL, TCR-T cells, and CAR-T cells) in vitro and / or in vivo. In some embodiments, provided is the use of exogenous CD160 expression to increase the cytolytic activity of therapeutic antigen-specific T cells (including but not limited to TIL, TCR-T cells, and CAR-T cells) in vitro and / or in vivo. III. Treatment Methods Using Exogenous CD160 Protein or Modified Antigen-Specific Immune Cells Expressing Exogenous CD160 Protein

[0184] One aspect of the present application relates to a method of treating a disease in an individual, the method comprising administering to the individual an effective amount of any one of the modified antigen-specific immune cells described herein or any one of the pharmaceutical compositions described herein. The present application contemplates the modified antigen-specific immune cells, which can be administered alone or in any combination with another therapy, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient. In some embodiments, the modified antigen-specific immune cells may be mixed with suitable pharmaceutical carriers and excipients known in the art prior to administration. In some embodiments, the modified antigen-specific immune cells are derived from an individual.

[0185] Another aspect of the present application relates to a method of treating a disease in an individual, comprising administering to the individual an effective amount of exogenous CD160 protein or a nucleic acid encoding an exogenous CD160 protein, wherein the exogenous CD160 protein comprises a binding moiety that recognizes a surface molecule on an immune cell in the individual.

[0186] Thus, in some embodiments, methods are provided for treating a disease (e.g., cancer) in an individual (e.g., a human), comprising administering to the individual an effective amount of modified antigen-specific immune cells that comprise (e.g., on their surface) exogenous CD160 protein, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein. In some embodiments, methods are provided for treating a disease in an individual, comprising administering to the individual an effective amount of modified antigen-specific immune cells that comprise (e.g., on their surface) exogenous CD160 protein, produced by a process that includes contacting precursor antigen-specific immune cells with exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein, thereby producing the modified antigen-specific immune cells, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells. In some embodiments, the modified antigen-specific immune cells are derived from the individual. In some embodiments, methods of treating a disease in an individual are provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (a) modified antigen-specific immune cells comprising (e.g., on their surface) exogenous CD160 protein, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein; and (b) a pharmaceutically acceptable carrier. In some embodiments, the modified antigen-specific immune cells are derived from the individual. In some embodiments of any one of the treatment methods described herein, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, or a variant thereof having at least about 80% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 1-4.In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (such as, but not limited to, a dimer, a trimer, a tetramer, a pentamer, or a hexamer). In some embodiments, the modified antigen-specific immune cell is selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells). In some embodiments, the modified antigen-specific immune cell is a cytotoxic T cell. In some embodiments, the modified antigen-specific immune cell is a tumor-infiltrating T cell or an anti-tumor T cell activated by APCs. In some embodiments, the anti-tumor T cells activated by APCs are anti-tumor T cells activated by DCs. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, γδ T cells, and macrophages. In some embodiments, the precursor antigen-specific immune cells are isolated from a tumor in an individual. In some embodiments, the precursor antigen-specific immune cells are monoclonal. In some embodiments, the precursor antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells are monoclonal. In some embodiments, the modified antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells further comprise a functional exogenous receptor. In some embodiments, the functional exogenous receptor is a modified T cell receptor (TCR). In some embodiments, the engineered T cell receptor (TCR) recognizes a tumor antigen or a tumor-associated antigen. In further embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the modified antigen-specific immune cells are multiple immune cells that are specific for the same epitope. A non-limiting example includes multiple T cells that each contain the same functional exogenous receptor (such as a CAR).In some embodiments, the modified antigen-specific immune cells are multiple immune cells each specific for a non-identical epitope (such as partially overlapping or entirely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0187] In some embodiments, methods of treating disease in an individual are provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound. In some embodiments, methods of treating disease in an individual are provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, wherein the modified antigen-specific immune cells are produced by a process comprising contacting precursor antigen-specific immune cells with exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein, thereby producing the modified antigen-specific immune cells, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells, and wherein the exogenous CD160 protein is membrane-bound.

[0188] In some embodiments, methods of treating a disease in an individual are provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein is membrane-bound via a GPI linker. In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, the modified antigen-specific immune cells being produced by a process comprising contacting precursor antigen-specific immune cells with an exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein, thereby producing the modified antigen-specific immune cells, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, the modified antigen-specific immune cells are derived from the individual, the exogenous CD160 protein is membrane-bound, and the exogenous CD160 protein is membrane-bound via a GPI linker. In some embodiments, the exogenous CD160 protein comprises a GPI-anchor peptide sequence.

[0189] In some embodiments, methods of treating disease in an individual are provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain. In some embodiments, methods of treating disease in an individual are provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, produced by a process comprising producing the modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells, and wherein the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD160, CD4, CD8, CD5, CD6, CD16, CD22, CD33, CD37, CD80, CD86, CD134, CD137, CD154, CD244, a T cell receptor (TCR) alpha subunit, a TCR beta subunit, or a TCR zeta subunit. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, CD80, CD152, and PD-1.

[0190] In some embodiments, methods of treating a disease in an individual are provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain and an intracellular domain. In some embodiments, methods of treating a disease in an individual are provided comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, produced by a process comprising producing the modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, wherein the modified antigen-specific immune cells are derived from the individual, and the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain and an intracellular domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD160, CD4, CD8, CD5, CD6, CD16, CD22, CD33, CD37, CD80, CD86, CD134, CD137, CD154, CD244, T cell receptor (TCR) alpha subunit, TCR beta subunit, or TCR zeta subunit. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD28, 4-1BB, CD80, CD152, and PD-1. In some embodiments, the intracellular domain is derived from a CD160 splice variant. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from a signaling subunit of a TCR complex. In some embodiments, the signaling subunit of a TCR complex is selected from the group consisting of CD3 gamma, CD3 delta, and CD3 epsilon.In some embodiments, the intracellular domain comprises one or more signaling domains derived from a T cell stimulatory molecule. In some embodiments, the signaling domain is one or more of 4-1BB, OX40, CD27, CD28, CD80, or CD258. In some embodiments, the intracellular domain comprises a combination of two signaling domains selected from the group consisting of OX40, CD27, CD28, CD80, and CD258.

[0191] In some embodiments, methods of treating a disease in an individual are provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein comprises a transmembrane domain and an intracellular domain, and wherein the intracellular domain comprises one or more costimulatory signaling domains. In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising an exogenous CD160 protein on their surface, the modified antigen-specific immune cells being produced by a process comprising contacting precursor antigen-specific immune cells with an exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein, thereby producing the modified antigen-specific immune cells; the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells; the exogenous CD160 protein is membrane-bound and comprises a transmembrane domain and an intracellular domain, the intracellular domain comprising one or more costimulatory signaling domains. In some embodiments, the intracellular domain comprises one, two, three, four, five, six, seven, eight, or more costimulatory signaling domains. In some embodiments, the intracellular domain contains no more than one, two, three, four, or five costimulatory signaling domains. In some embodiments, the intracellular domain does not comprise a CD3ζ signaling domain or a combination of 4-1BB and a CD3ζ domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, DAP10, CD30, CD40, CD3, CD80, CD258, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, or both.In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain. In some embodiments, the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain is adjacent to the transmembrane domain. In some embodiments, the CD28 costimulatory domain is adjacent to the C-terminus of the transmembrane domain. In some embodiments, the intracellular domain comprises a primary signaling domain. In some embodiments, the primary signaling domain comprises a CD3 zeta domain. In other embodiments, the intracellular domain does not comprise a primary signaling domain. In other embodiments, the intracellular domain does not comprise a CD3 zeta domain or a combination of 4-1BB and CD3 zeta domains.

[0192] In some embodiments, methods of treating a disease in an individual are provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein is bound to the modified antigen-specific immune cells via an immune cell binding moiety. In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising exogenous CD160 protein on their surface, the modified antigen-specific immune cells being produced by a process comprising contacting precursor antigen-specific immune cells with an exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein, thereby producing the modified antigen-specific immune cells, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, and the exogenous CD160 protein is membrane-bound, and the exogenous CD160 protein is bound to the modified antigen-specific immune cells via an immune cell binding moiety. In some embodiments, the immune cell binding moiety binds to a surface molecule of an immune cell. In some embodiments, the immune cell binding moiety comprises an antibody that recognizes a T cell surface molecule. In some embodiments, the antibody is a full-length antibody or an scFv, Fv, Fab, (Fab')2, single domain antibody (sdAb), or V H The immune cell binding moiety may be an antibody fragment such as an H domain. Non-limiting examples include anti-CD3ε antibodies that recognize TCRs and / or activate TCR signaling. In some embodiments, the immune cell binding moiety comprises a ligand that binds to a cognate T cell surface receptor. Non-limiting examples include tumor-specific peptide-MHC complexes that recognize TCRs and IL-2.

[0193] In some embodiments of any one of the treatment methods described herein, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, or a variant thereof having at least about 80% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having any one of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (such as, but not limited to, a dimer, trimer, tetramer, pentamer, or hexamer). In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells). In some embodiments, the modified antigen-specific immune cells are cytotoxic T cells. In some embodiments, the modified antigen-specific immune cells are tumor-infiltrating T cells or anti-tumor T cells activated by APCs. In some embodiments, anti-tumor T cells activated by APCs are anti-tumor T cells activated by DCs. In some embodiments, the modified antigen-specific immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, γδ T cells, and macrophages. In some embodiments, the precursor antigen-specific immune cells are isolated from a tumor in an individual. In some embodiments, the precursor antigen-specific immune cells are monoclonal. In some embodiments, the precursor antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells are monoclonal. In some embodiments, the modified antigen-specific immune cells are derived from a polyclonal population. In some embodiments, the modified antigen-specific immune cells further comprise a functional exogenous receptor.In some embodiments, the functional exogenous receptor is an engineered T cell receptor (TCR). In some embodiments, the engineered T cell receptor (TCR) recognizes a tumor antigen or a tumor-associated antigen. In further embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the engineered antigen-specific immune cells are multiple immune cells specific for the same epitope. Non-limiting examples include multiple T cells each comprising the same functional exogenous receptor (such as a CAR). In some embodiments, the engineered antigen-specific immune cells are multiple immune cells each specific for non-identical epitopes (such as partially overlapping or entirely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0194] In some embodiments of any of the treatment methods described herein, the method of producing modified antigen-specific immune cells comprises contacting precursor antigen-specific immune cells with exogenous CD160 protein. In some embodiments, the exogenous CD160 protein comprises an immune cell binding moiety that binds to a surface molecule of the immune cell. In some embodiments, the method comprises introducing a nucleic acid encoding the exogenous CD160 protein into the precursor antigen-specific immune cells. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is DNA. The nucleic acid can be introduced into the modified antigen-specific immune cells using any transfection or transduction method known in the art, including viral or non-viral methods. Exemplary non-viral transfection methods include, but are not limited to, chemical-based transfection using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods such as electroporation, cell squeezing, sonoporation, optical transfection, impale infection, protoplast fusion, hydrodynamic delivery, or transposons; particle-based methods using gene guns, magnetofection or magnet-assisted transfection, particle bombardment, and the like; and hybrid methods such as nucleofection. In some embodiments, the nucleic acid is introduced into precursor antigen-specific immune cells by transfection. In some embodiments, the nucleic acid is introduced into precursor antigen-specific immune cells by transduction or electroporation. In some embodiments, the CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, or a variant thereof having at least about 80% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 95% identity to any one of SEQ ID NOs: 1-4.In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 99% identity to any one of SEQ ID NOs: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to any one of SEQ ID NOs: 1-4.

[0195] In some embodiments, methods of treating a disease in an individual are provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells produced by a process comprising producing modified antigen-specific immune cells by contacting precursor antigen-specific immune cells with exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells relative to the precursor antigen-specific immune cells. In some embodiments, the modified antigen-specific immune cells are modified T cells. In some embodiments, the precursor antigen-specific immune cells are precursor T cells. In some embodiments, the precursor antigen-specific immune cells further comprise a second nucleic acid encoding a functional exogenous receptor. In some embodiments of any of the treatment methods described herein, the method of producing modified antigen-specific immune cells further comprises introducing the second nucleic acid encoding a functional exogenous receptor into the precursor antigen-specific immune cells. In some embodiments, the functional exogenous receptor is an engineered T cell receptor (TCR). In some embodiments, the functional exogenous receptor is a modified T cell receptor (TCR). In some embodiments, the engineered T cell receptor (TCR) recognizes a tumor antigen or a tumor-associated antigen. In further embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to the same promoter. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to separate promoters. In some embodiments, the first nucleic acid and the second nucleic acid are on the same vector. In some embodiments, the first nucleic acid and the second nucleic acid are on separate vectors. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, and derivatives thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is an episomal expression vector.In some embodiments, the method further comprises isolating or enriching immune cells comprising the first nucleic acid and / or the second nucleic acid. In some embodiments of any of the methods of treatment described herein, the method of producing the modified antigen-specific immune cells further comprises formulating the modified antigen-specific immune cells with at least one pharmaceutically acceptable carrier.

[0196] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising (e.g., on the surface) exogenous CD160 protein, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein. In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells produced by a process comprising contacting precursor antigen-specific immune cells with exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein to produce the modified antigen-specific immune cells, wherein the exogenous CD160 protein results in up-modulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the cancer is selected from the group consisting of melanoma, lung cancer, esophageal cancer, pancreatic cancer, breast cancer, liver cancer, brain cancer, and ovarian cancer. In some embodiments, the cancer is a virus-associated cancer, such as HPV-associated cancer or EBV-associated cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the method for treating cancer has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting cancer cell proliferation; (3) inducing peripheral T cell redistribution; (4) inducing an immune response in tumors; (5) reducing tumor size; (6) alleviating one or more symptoms in individuals with cancer; (7) inhibiting tumor metastasis; (8) prolonging survival; (9) extending the time to cancer progression; (10) preventing, inhibiting, or reducing the likelihood of cancer recurrence; (11) improving the quality of life of individuals; (12) facilitating T cell infiltration in tumors, and (13) reducing the occurrence or burden of pre-existing tumor metastasis (such as metastasis to lymph nodes). In some embodiments, the method achieves a tumor cell death rate of at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more.In some embodiments, the method reduces tumor size by at least about 10% (including, for example, at least about 20%, about 30%, about 40%, about 60%, about 70%, about 80%, about 90%, or about 100%). In some embodiments, the method inhibits metastasis by at least about 10% (including, for example, at least about 20%, about 30%, about 40%, about 60%, about 70%, about 80%, about 90%, or about 100%). In some embodiments, the method extends survival of an individual by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months or more. In some embodiments, the method extends time to cancer progression by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months or more.

[0197] In some embodiments of any of the treatment methods described herein, the administration is intratumoral administration. In some embodiments, the administration is to a lymph node. In some embodiments, the administration is parenteral, percutaneous (into the dermis), intraluminal, intraarterial (into an artery), intramuscular (into a muscle), intrathecal, or intravenous. In some embodiments, the pharmaceutical composition is administered subcutaneously (under the skin). In some embodiments, the administration is intravenous.

[0198] The methods described herein are suitable for treating a variety of cancers, including both solid and liquid cancers. The methods are applicable to cancers at any stage, including early-stage, non-metastatic, primary, advanced, locally advanced, metastatic, or in remission. The methods described herein can be used in adjuvant or neoadjuvant settings as a first, second, or third therapy, or in combination with other types of cancer therapy known in the art, such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (such as T-cell therapy), bone marrow transplant, stem cell transplant, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, and radiofrequency ablation (i.e., the method can be performed before primary / definitive therapy). In some embodiments, the method is used to treat individuals who have been previously treated. In some embodiments, the cancer was refractory to previous therapy. In some embodiments, the method is used to treat individuals who have not been previously treated.

[0199] In some embodiments of any of the methods described herein, the modified antigen-specific immune cells comprising exogenous CD160 protein are used as a short-term cytolytic agent to control and eliminate established solid tumors. In some embodiments, the modified antigen-specific immune cells comprise exogenous CD160 protein. In some embodiments, the method comprises administering the modified antigen-specific immune cells or pharmaceutical composition about every 7 days, about 10 days, about 14 days, about 21 days, or about 30 days. In some embodiments, the method comprises administering the modified antigen-specific immune cells or pharmaceutical composition about every 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks. In some embodiments, the modified antigen-specific immune cells or pharmaceutical composition are administered multiple times (e.g., 2, 3, 4, 5, 6 or more times). In some embodiments, the method further comprises administering one or more therapeutic agents. In some embodiments, the therapeutic agent is one or more of radiation therapy, chemotherapy, or immunotherapy. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets one of PD-1, PD-L1, CTLA-4, TIM-3, LAG3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor targets PD-1 and / or PD-L1. In some embodiments, the therapeutic agent comprises a cytokine. In some embodiments, the cytokine is IL-2, IL-7, IL-12a, IL-12b, or IL-15. In some embodiments, the therapeutic agent is a substance that further modulates and / or generates an immune response. In some embodiments, the therapeutic agent comprises a TLR agonist. In some embodiments, the therapeutic agent comprises a TLR3 agonist, a TLR4 agonist, a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist.

[0200] In some embodiments, the method further comprises a conditioning or preconditioning treatment. In some embodiments, the conditioning or preconditioning treatment is used to reduce or eliminate underlying disease to create space for new bone marrow. In some embodiments, the method further comprises chemotherapy. In some embodiments, the chemotherapy is administered before administration of the modified antigen-specific immune cells or modified pharmaceutical composition. In some embodiments, the chemotherapy is administered after administration of the modified antigen-specific immune cells or modified pharmaceutical composition. In some embodiments, the chemotherapy is used to precondition the individual bearing cancer. In some embodiments, the method does not comprise preconditioning. In some embodiments, the method does not further comprise chemotherapy or chemotherapy preconditioning. In some embodiments, the method does not further comprise radiation therapy or radiation preconditioning. In some embodiments, the method does not further comprise the use of vaccination. In some embodiments, the method does not further comprise the use of an interleukin, such as, but not limited to, interleukin-2 (IL-2).

[0201] The effective amount of modified antigen-specific immune cells or pharmaceutical composition administered in the methods described herein will depend on several factors, such as the specific type and stage of cancer being treated, the route of administration, the activity of the exogenous CD160 protein and / or functional exogenous receptor, etc. A physician can determine the appropriate dosage regimen based on clinical factors, such as the patient's size, body surface area, age, the specific compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly. In some embodiments, the effective amount of modified antigen-specific immune cells or pharmaceutical composition is below a level that induces toxic effects (i.e., effects beyond clinically acceptable levels of toxicity) or is at a level that allows potential side effects to be controlled or tolerated when the pharmaceutical composition is administered to an individual. In some embodiments, the effective amount of modified antigen-specific immune cells or pharmaceutical composition is about 10 5 ~about 10 10In some embodiments, an effective amount of modified antigen-specific immune cells or pharmaceutical composition comprises about 100,000, about 200,000, about 500,000, about 750,000, about 1 million, about 2 million, about 5 million, about 10 million, about 20 million, about 50 million, about 100 million, about 200 million, or about 500 million modified antigen-specific immune cells. In some embodiments, an effective amount of modified antigen-specific immune cells or pharmaceutical composition comprises about 100 million, about 200 million, about 500 million, about 750 million, about 1 billion, about 2 billion, about 3 billion, about 4 billion, about 5 billion, about 6 billion, about 7 billion, about 8 billion, about 9 billion, about 10 billion, about 11 billion, about 12 billion, about 13 billion, about 14 billion, or about 15 billion modified antigen-specific immune cells.

[0202] In some embodiments, the modified antigen-specific immune cells or pharmaceutical composition are administered once (e.g., bolus injection). In some embodiments, the modified antigen-specific immune cells or pharmaceutical composition are administered multiple times (e.g., 2, 3, 4, 5, 6, or more times). Multiple administrations may be performed by the same or different routes and may be administered at the same or different sites. The pharmaceutical composition may be administered at a suitable frequency, such as daily to once a year. One skilled in the art of medicine can readily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0203] In some embodiments, the methods involve administering the modified antigen-specific immune cells or pharmaceutical composition about every 7 days, about 10 days, about 14 days, about 21 days, or about 30 days. In some embodiments, the methods involve administering the modified antigen-specific immune cells or pharmaceutical composition about every 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks. In some embodiments, the methods involve administering the modified antigen-specific immune cells or pharmaceutical composition about every 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, or about 8 months. In some embodiments, the individual being treated is a mammal. Examples of mammals include, but are not limited to, humans, monkeys, rats, mice, hamsters, guinea pigs, dogs, cats, rabbits, pigs, sheep, goats, horses, cows, etc. In some embodiments, the individual is human. Pharmaceutical Composition

[0204] Further provided by the present application is a pharmaceutical composition comprising any one of the modified antigen-specific immune cells described herein and, optionally, a pharmaceutically acceptable carrier.

[0205] The pharmaceutical compositions of the present application may comprise any number of modified antigen-specific immune cells. In some embodiments, the pharmaceutical composition comprises a single copy of the modified antigen-specific immune cells. In some embodiments, the pharmaceutical composition comprises at least about 1, about 10, about 100, about 1000, about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9In some embodiments, the pharmaceutical composition comprises at least about 100,000, about 200,000, about 500,000, about 750,000, about 1 million, about 2 million, about 5 million, about 10 million, about 20 million, about 50 million, about 100 million, about 200 million, or about 500 million modified antigen-specific immune cells. In some embodiments, the pharmaceutical composition comprises at least about 100 million, about 200 million, about 500 million, about 750 million, about 1 billion, about 2 billion, about 3 billion, about 4 billion, about 5 billion, about 6 billion, about 7 billion, about 8 billion, about 9 billion, about 10 billion, about 11 billion, about 12 billion, about 13 billion, about 14 billion, or about 15 billion modified antigen-specific immune cells. In some embodiments, the pharmaceutical composition comprises a single type of modified antigen-specific immune cell. In some embodiments, the pharmaceutical composition comprises at least two types of modified antigen-specific immune cells, wherein the different types of modified antigen-specific immune cells differ by their cell source, cell type, expressed chimeric receptor, and / or promoter, etc.

[0206] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or individuals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers are often aqueous pH-buffered solutions. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed.

[0207] Pharmaceutical compositions containing such carriers can be formulated by well-known conventional methods.The solvent or diluent is preferably isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength.Representative examples include sterile water, physiological saline (e.g., sodium chloride), Ringer's solution, glucose, trehalose or saccharose solution, Hank's solution, and other aqueous physiologically balanced salt solutions (see, for example, Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins, latest edition).

[0208] The pharmaceutical compositions described herein can be administered by any suitable route. In some embodiments, the pharmaceutical compositions are administered parenterally, transdermally (into the dermis), intraluminally, intraarterially (into an artery), intramuscularly (into a muscle), intrathecally, or intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously (under the skin). In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered to an individual by infusion or injection. In some embodiments, the pharmaceutical composition is administered directly to the target site, for example, by biolistic delivery to an internal or external target site, or by a catheter to a site in an artery. In some embodiments, the pharmaceutical composition is administered locally, for example, intratumorally. Administration may use a conventional syringe and needle or any compound or device available in the artery that can facilitate or improve delivery of an active agent in a subject.

[0209] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present. In addition, the pharmaceutical compositions of the present disclosure may include a proteinaceous carrier, such as serum albumin or immunoglobulin, preferably of human origin. Various virus formulations in frozen, liquid, or lyophilized form are available in the art (e.g., WO98 / 02522, WO01 / 66137, WO03 / 053463, WO2007 / 056847, and WO2008 / 114021, etc.). Solid (e.g., dry powder or lyophilized) compositions can be obtained by processes including vacuum drying and freeze-drying (see, e.g., WO2014 / 053571). It is envisioned that the pharmaceutical compositions of the present disclosure may contain, in addition to the modified antigen-specific immune cells described herein, additional bioactive agents depending on the intended use of the pharmaceutical composition.

[0210] In some embodiments, the pharmaceutical composition is suitably buffered for human use. Suitable buffers include, but are not limited to, phosphate buffers (e.g., PBS), bicarbonate buffers, and / or Tris buffers, which can maintain a physiological or slightly basic pH (e.g., about pH 7 to about pH 9). In some embodiments, the pharmaceutical composition can be made isotonic with blood by the addition of a suitable tonicity-modifying agent, such as glycerol.

[0211] In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container.

[0212] In some embodiments, pharmaceutical compositions must meet certain criteria for administration to an individual. For example, the U.S. Food and Drug Administration has issued regulatory guidelines setting standards for cell-based immunotherapy products, including 21 CFR 610 and 21 CFR 610.13. Methods for evaluating the appearance, identity, purity, safety, and / or efficacy of pharmaceutical compositions are known in the art. In some embodiments, pharmaceutical compositions are substantially free of foreign proteins that may cause allergic effects, such as proteins of animal origin used in cell culture, other than the modified antigen-specific immune cells. In some embodiments, "substantially free" means less than about 10%, about 5%, about 1%, about 0.1%, about 0.01%, about 0.001%, or 1 ppm of the total volume or weight of the pharmaceutical composition, or less. In some embodiments, pharmaceutical compositions are prepared in a GMP-level facility. In some embodiments, pharmaceutical compositions contain less than about 5 EU / kg body weight / hr of endotoxin for parenteral administration. In some embodiments, at least about 70% of the modified antigen-specific immune cells in the pharmaceutical composition are viable for intravenous administration. In some embodiments, the pharmaceutical composition has a "no growth" result when evaluated using the 14-day direct inoculation test method described in the United States Pharmacopeia (USP). In some embodiments, prior to administration of the pharmaceutical composition, a sample containing both the modified antigen-specific immune cells and the pharmaceutically acceptable excipients should be collected for sterility testing approximately 48-72 hours prior to the last harvest (or simultaneously with the last refeed of the culture). In some embodiments, the pharmaceutical composition is free of mycoplasma contaminants. In some embodiments, the pharmaceutical composition is free of detectable microbial agents. In some embodiments, the pharmaceutical composition is free of infectious disease agents, such as HIV type I, HIV type II, HBV, HCV, human T-lymphotropic virus, type I; and human T-lymphotropic virus, type II.

[0213] In some embodiments, the modified antigen-specific immune cells exhibit natural antigen recognition. In some embodiments, the modified antigen-specific immune cells exhibit engineered antigen recognition. In some embodiments, the antigen recognition of the modified antigen-specific immune cells is conferred at least in part by a functional exogenous receptor, such as, but not limited to, a CAR and a TCR. In some embodiments, the modified antigen-specific immune cells target tumor-associated antigens, mutated oncogenic antigens and random somatic antigens, and other neoantigens. In some embodiments, the modified antigen-specific immune cells are human immune cells. In some embodiments, the modified antigen-specific immune cells are mouse immune cells. In some embodiments, the modified antigen-specific immune cells are modified from one or more of TCR-T cells, CAR-T cells, TILs, or endogenous antigen-specific T cells. Some examples of human and mouse TCR-T cells, CAR-T cells, TILs, or endogenous antigen-specific T cells are reported in Tran et al., Nat Immunol. 2017;18(3):255-62, MacKay et al., Nat Biotechnol. 2020;38(2):233-44, and Schumacher et al., Cancer Neoantigens. Annu Rev Immunol. 2019;37:173-200, which are incorporated herein by reference. In some embodiments, the modified antigen-specific immune cells target a broad range of antigens. In some embodiments, the modified antigen-specific immune cells target one or more of the antigens listed in Table 1. IV. Modulation of the immunostimulatory activity of CD160 in antigen-specific immune cells

[0214] One aspect of the present invention provides a method for modulating the immunostimulatory activity of CD160 protein in antigen-specific immune cells, comprising administering a therapeutically effective amount of an agent that modulates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0215] In some aspects, methods are also provided for identifying modulators of endogenous CD160 expression, function, or activity, comprising contacting CD160-expressing immune cells (such as NK cells) with a test agent; and measuring effector expression and / or function of a cytolytic or inflammatory pathway in the tested immune cells. In one embodiment, a test agent is identified as a modulator of CD160 expression, function, or activity if the agent modulates effector expression and / or function of a cytolytic or inflammatory pathway in the tested immune cells compared to control immune cells. In some embodiments, CD160 expression, function, or activity is substantially modulated compared to a normal baseline control. In some embodiments, the modulator is an inhibitor of CD160 expression, function, or activity. In some embodiments, the modulator is an activator of CD160 expression, function, or activity.

[0216] In some aspects, a method for identifying a modulator of endogenous CD160 expression, function, or activity is provided, comprising contacting antigen-specific immune cells (such as NK cells) expressing CD160 with a test agent; and measuring an in vivo immune function elicited by the tested immune cells, such as cytokine secretion by the immune cells upon antigen challenge. In one embodiment, a test agent is identified as a modulator of CD160 expression, function, or activity if the agent modulates effector expression and / or function of cytolytic or inflammatory pathways in the tested immune cells compared to control immune cells. Preferably, CD160 expression, function, or activity is substantially modulated compared to a normal baseline control. In some embodiments, the modulator is an inhibitor of CD160 expression, function, or activity. In some embodiments, the modulator is an activator of CD160 expression, function, or activity.

[0217] One aspect of the present invention provides a method of treating an immune disease in an individual, comprising administering to the individual a therapeutically effective amount of an agent that modulates the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells. In some embodiments, the immune disease is an autoimmune disease or an inflammatory disease, and the agent inhibits the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells. Inhibition of the immunostimulatory activity of CD160 in antigen-specific immune cells

[0218] One aspect of the present invention provides a method for inhibiting the endogenous immunostimulatory activity of CD160 in an antigen-specific immune cell, the method comprising contacting the antigen-specific immune cell with an effective amount of an agent that inhibits the immunostimulatory activity of CD160 in the antigen-specific immune cell.

[0219] In some embodiments, methods are provided for treating an autoimmune disease in an individual, comprising administering to the individual a therapeutically effective amount of an agent that inhibits the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells. In some embodiments, methods are provided for treating an inflammatory disease in an individual, comprising administering to the individual a therapeutically effective amount of an agent that inhibits the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0220] In some embodiments, the agent that inhibits the endogenous immunostimulatory activity of CD160 protein comprises an antagonist antibody. In some embodiments, the agent that inhibits the endogenous immunostimulatory activity of CD160 protein comprises an antagonist protein. In some embodiments, the agent that inhibits the endogenous immunostimulatory activity of CD160 protein comprises one or more nucleic acids. In some embodiments, the agent is in the form of RNA interference (RNAi). In some embodiments, the agent is one or more of siRNA, shRNA, or miRNA. In some embodiments, the agent that inhibits the endogenous immunostimulatory activity of CD160 protein comprises a small molecule. In some embodiments, the agent comprises a dominant-negative form of CD160 protein.

[0221] In some embodiments, the agent inhibits the endogenous immunostimulatory activity of CD160 by any one of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In some embodiments, the agent inhibits the endogenous immunostimulatory activity of CD160 by any one of about one-fold, about one-half, about one-third, about one-quarter, about one-fifth, about one-tenth, about one-twentieth, about one-thirtieth, about one-fortieth, about one-fifth, about one-sixtieth, about one-seventieth, about one-eighth, about one-ninth, about one-hundredth, about one-fifth, or about one-thousandth.

[0222] In some embodiments, the method of treating an autoimmune response includes suppressing an immune response and / or inducing tolerance. Reducing an autoimmune response includes, but is not limited to, reducing an immune response or inducing tolerance to antigens associated with type 1 diabetes, rheumatoid arthritis, psoriasis, multiple sclerosis, Alzheimer's disease, ALS, Huntington's disease, Parkinson's disease, systemic lupus erythematosus, Sjogren's disease, Crohn's disease, or ulcerative colitis. In some embodiments, suppressing an immune response and / or inducing tolerance includes reducing an allergic response. For example, reducing an allergic response may include reducing an immune response or inducing tolerance to antigens associated with allergic asthma, atopic dermatitis, allergic rhinitis (hay fever), food allergies, and gluten allergies. In some embodiments, the antigen is an antigen associated with transplanted tissue. In some embodiments, suppressing an immune response and / or inducing tolerance includes reducing an immune response or inducing tolerance to transplanted tissue. In some embodiments, the antigen is associated with a virus. In some embodiments, suppressing an immune response and / or inducing tolerance includes reducing or inducing tolerance to a pathogenic immune response to a virus. For example, the pathogenic immune response may include a cytokine storm generated by certain viral infections. A cytokine storm is a potentially lethal immune response consisting of a positive feedback loop between cytokines and leukocytes. Thus, in some embodiments, suppressing an immune response and / or inducing tolerance includes reducing or eliminating a cytokine storm.

[0223] In some embodiments, the antigen recognized by the antigen-specific immune cells is a protein. In some embodiments, the antigen is an autoantigen. In some embodiments, the autoantigen is associated with type 1 diabetes or rheumatoid arthritis. In some embodiments, the antigen is associated with a therapeutic agent. In some embodiments, the antigen is a therapeutic polypeptide or a fragment of a therapeutic polypeptide. In some embodiments, the therapeutic agent is a clotting factor, such as, but not limited to, Factor VIII and Factor IX. In some embodiments, the therapeutic agent is an antibody. In some embodiments, the therapeutic agent is a hormone. In some embodiments, the therapeutic agent is insulin. In some embodiments, the therapeutic agent is a recombinant cytokine. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor. Activation of the immunostimulatory activity of CD160 in immune cells

[0224] In some embodiments, methods are provided for activating the immunostimulatory activity of CD160 in antigen-specific immune cells, the methods comprising contacting the antigen-specific immune cells with an effective amount of an agent that activates the immunostimulatory activity of CD160 in the antigen-specific immune cells. In some embodiments, the method enhances the endogenous immunostimulatory activity of CD160 in the antigen-specific immune cells, wherein the agent enhances the endogenous immunostimulatory activity of CD160 in the antigen-specific immune cells. In some embodiments, the method comprises contacting the antigen-specific immune cells with exogenous CD160 protein. In some embodiments, the method comprises contacting the antigen-specific immune cells with nucleotides encoding the exogenous CD160 protein.

[0225] In some embodiments, methods are provided for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells. In some embodiments, methods are provided for treating infection in an individual, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0226] In some embodiments, the agent that activates the immunostimulatory activity of the CD160 protein comprises an agonist peptide or protein. In some embodiments, the agent comprises a small molecule. In some embodiments, the agent that activates the intrinsic immunostimulatory activity of the CD160 protein comprises an agonist antibody.

[0227] In some embodiments, the agent that activates the intrinsic immunostimulatory activity of the CD160 protein comprises one or more nucleic acids, hi some embodiments, the agent is DNA and / or mRNA.

[0228] In some embodiments, the agent activates the immunostimulatory activity of CD160 in antigen-specific immune cells, and the antigen-specific immune cells do not exhibit detectable CD160 activity prior to contact with the agent. In some embodiments, the agent enhances the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells. In some embodiments, the agent enhances the endogenous immunostimulatory activity of CD160 by any one of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In some embodiments, the agent enhances the intrinsic immunostimulatory activity of CD160 by any one of about 1, about 2, about 3, about 4, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 500, or about 1000 fold.

[0229] In some embodiments, the method of treating cancer comprises increasing an immune response to a tumor antigen or a tumor-associated antigen. In some embodiments, the antigen recognized by the antigen-specific immune cells is a protein. In some embodiments, the antigen-specific immune cells are specific for a tumor antigen or a tumor-associated antigen. In some embodiments, the tumor-associated antigen is mesothelin, EGFRvIII, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, GD2, GD3, BCMA, Tn Ag, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin-11 receptor (IL-11Ra), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, folate receptor alpha (FRa), ERBB2 (Her2 / neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, prostase, PAP, ELF2M, ephrin B2, IGF -I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, Melan-A / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG(TMPRSS2)ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1. In some embodiments, the antigen is derived from a neoantigen, e.g., a cancer-associated neoantigen. In some embodiments, the antigen comprises a neoepitope, e.g., a cancer-associated neoepitope.

[0230] In some embodiments, methods are provided for treating an infection in an individual, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells. In some embodiments, methods are provided for treating an infection in an individual, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0231] In some embodiments, the method of treating an infection comprises increasing an immune response to an antigen associated with the infectious agent. In some embodiments, the antigen is a non-self antigen. In some embodiments, the antigen is a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen. V. Methods for Treating Cancer Based on the Level or Activity of CD160 in the Tumor Environment

[0232] One aspect of the present invention relates to a method of treating cancer, in which CD160 can be used as a biomarker for predicting the functional status of antigen-specific T cells and therefore the effectiveness of immunotherapy. Higher CD160 expression in these cells suggests an activated state of antigen-specific T cells, while low levels or the absence of CD160 expression can suggest an inactive state of antigen-specific T cells.

[0233] Thus, in some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a therapeutically effective amount of a composition comprising antigen-specific immune cells, wherein endogenous CD160 levels or activity in the individual is used as a basis for selecting the individual for treatment. In some embodiments, the individual is selected for treatment if the individual has a high CD160 level or activity. In some embodiments, the individual is selected for treatment if the individual has a low CD160 level or activity. In some embodiments, the CD160 level or activity is determined by immunohistochemistry. In some embodiments, the CD160 level or activity is based on CD160 protein expression levels. In some embodiments, the CD160 level or activity is based on CD160 mRNA levels. In some embodiments, the CD160 level or activity is measured in the individual's tumor. In some embodiments, the CD160 level or activity is measured in the individual's tumor-infiltrating T cells (TILs). In some embodiments, the CD160 level or activity is measured in the individual's peripheral T cells.

[0234] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a therapeutically effective amount of a composition comprising modified antigen-specific immune cells comprising (e.g., on the surface) exogenous CD160 protein, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not comprise the exogenous CD160 protein, the immune cells being T cells, and wherein endogenous CD160 levels or activity in the individual are used as a basis for selecting the individual for treatment. In some embodiments, if the individual has a high CD160 level or activity, the individual is selected for treatment. In some embodiments, if the individual has a low CD160 level or activity, the individual is selected for treatment. In some embodiments, the CD160 level is determined by immunohistochemistry. In some embodiments, the CD160 level or activity is based on CD160 protein expression levels. In some embodiments, the CD160 level or activity is based on CD160 mRNA levels. In some embodiments, the CD160 level or activity in the individual's tumor is measured. In some embodiments, CD160 levels or activity in the individual's tumor-infiltrating T cells (TILs) is measured. In some embodiments, CD160 levels or activity in the individual's peripheral T cells is measured.

[0235] In some embodiments, methods are provided for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising one or more immune checkpoint inhibitors, wherein endogenous CD160 levels or activity in the individual are used as a basis for selecting the individual for treatment. In some embodiments, the individual is selected for treatment if the individual has a high CD160 level or activity. In some embodiments, the individual is selected for treatment if the individual has a low CD160 level or activity. In some embodiments, the CD160 level is determined by immunohistochemistry. In some embodiments, the CD160 level or activity is based on CD160 protein expression levels. In some embodiments, the CD160 level or activity is based on CD160 mRNA levels. In some embodiments, the CD160 level or activity is measured in the individual's tumor. In some embodiments, the CD160 level or activity is measured in the individual's tumor-infiltrating T cells (TILs). In some embodiments, the CD160 level or activity is measured in the individual's peripheral T cells. In some embodiments, the immune checkpoint inhibitor targets one of PD-1, PD-L1, CTLA-4, TIM-3, LAG3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA.

[0236] In some embodiments, methods are provided for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising an agent that activates the immunostimulatory activity of CD160, wherein endogenous CD160 levels or activity in the individual are used as a basis for selecting the individual for treatment. In some embodiments, the individual is selected for treatment if the individual has a high CD160 level or activity. In some embodiments, the individual is selected for treatment if the individual has a low CD160 level or activity. In some embodiments, the CD160 level is determined by immunohistochemistry. In some embodiments, the CD160 level or activity is based on CD160 protein expression levels. In some embodiments, the CD160 level or activity is based on CD160 mRNA levels. In some embodiments, the CD160 level or activity is measured in the individual's tumor. In some embodiments, the CD160 level or activity is measured in the individual's tumor-infiltrating T cells (TILs). In some embodiments, the CD160 level or activity is measured in the individual's peripheral T cells. In some embodiments, the agent that activates the immunostimulatory activity of the CD160 protein comprises an agonist peptide or protein. In some embodiments, the agent comprises a small molecule. In some embodiments, the agent that activates the endogenous immunostimulatory activity of the CD160 protein comprises an agonist antibody. In some embodiments, the agent that activates the endogenous immunostimulatory activity of the CD160 protein comprises one or more nucleic acids. In some embodiments, the agent is DNA and / or mRNA. In some embodiments, the agent activates the immunostimulatory activity of CD160 in antigen-specific immune cells, and the antigen-specific immune cells do not exhibit detectable CD160 activity prior to contact with the agent. In some embodiments, the agent enhances the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells. In some embodiments, the agent enhances the endogenous immunostimulatory activity of CD160 by any one of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99%.In some embodiments, the agent enhances the intrinsic immunostimulatory activity of CD160 by any one of about 1, about 2, about 3, about 4, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 500, or about 1000 fold.

[0237] In other aspects, methods are provided for selecting (including identifying) an individual having cancer (such as melanoma or lung cancer) for treatment using a composition comprising a therapeutic agent, the method comprising determining CD160 levels or activity in the individual. In some embodiments, methods are provided for selecting (including identifying) an individual having cancer (such as melanoma or lung cancer) for treatment using a composition comprising an immunotherapy, the method comprising determining CD160 levels or activity in the individual. In some embodiments, individuals with high levels of CD160 are selected for treatment. In some embodiments, individuals with low levels of CD160 are selected for treatment. In some embodiments, the level of CD160 is determined based on protein expression levels. In some embodiments, the level of CD160 is determined based on mRNA levels. In some embodiments, the level of CD160 is determined by immunohistochemistry.

[0238] In some embodiments, the level is determined (e.g., high or low) by comparison with a control (such as any of the controls described herein). In some embodiments, the method further includes comparing the CD160 level or activity to a control. In some embodiments, the level is determined (e.g., high or low) based on a scoring system, such as the H-score system described herein. The control sample can be obtained using the same source and method as the non-control sample. In some embodiments, the control sample is obtained from a different individual (e.g., an individual without cancer and / or an individual sharing a similar ethnic, age, and gender identity). In some embodiments, where the sample is a tumor tissue sample, the control sample may be a non-cancerous sample from the same individual. In some embodiments, multiple control samples (e.g., from different individuals) are used to determine the range of levels of CD160 activity in a particular tissue, organ, or cell population. In some embodiments, the control sample is cultured tissue or cells determined to be an appropriate control. In some embodiments, the control is cells that do not express CD160. In some embodiments, the control is cells that express high levels of CD160. In some embodiments, clinically accepted normal level in standardized test is used as control level for determining CD160 activity in relevant tissue.In some embodiments, the reference CD160 level or activity in subject is classified as high, medium or low according to a scoring system based on immunohistochemistry for CD160 staining, for example, H score as further discussed herein.In some embodiments, the reference CD160 level or activity in subject is classified as low sample if H score is lower than or equal to the overall median H score.

[0239] In some embodiments, methods are provided for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising modified antigen-specific immune cells comprising a functional exogenous receptor on their surface, wherein the immune cells are T cells, and wherein CD160 levels or activity in the individual is used as a basis for selecting the modified antigen-specific immune cells for use in treating the cancer. In some embodiments, the modified antigen-specific immune cells are selected for treatment if the cells have high CD160 levels or activity. In some embodiments, the modified antigen-specific immune cells are selected for treatment if the cells have low CD160 levels or activity. In some embodiments, the CD160 levels are determined by immunohistochemistry. In some embodiments, the CD160 levels or activity are based on CD160 protein expression levels. In some embodiments, the CD160 levels or activity are based on CD160 mRNA levels. In some embodiments, the CD160 levels or activity are compared to precursor immune cells that do not comprise the exogenous functional receptor. In some embodiments, the CD160 levels or activity of modified antigen-specific immune cells comprising an exogenous functional receptor on their surface are compared to modified antigen-specific immune cells comprising an exogenous CD160 protein on their surface. In some embodiments, the CD160 levels or activity of modified antigen-specific immune cells comprising an exogenous functional receptor on their surface are compared to modified antigen-specific immune cells comprising an exogenous dominant-negative form of CD160 protein on their surface.

[0240] In some embodiments of any of the methods described herein, the level of CD160 is determined based on CD160 protein expression levels. In some embodiments, the level of CD160 is determined based on mRNA levels. In some embodiments, the level of a nucleoside transporter is determined by immunohistochemistry. In some embodiments, the level is determined (e.g., high or low) by comparison with a control (such as any of the controls described herein). In some embodiments, the level is determined (e.g., high or low) based on a scoring system, such as the H-score system described herein. In some embodiments, scoring is based on the "H-score" described in U.S. Patent Application Publication No. 2013 / 0005678. The H-score is calculated by the formula: 3 x percentage of strongly staining cells + 2 x percentage of moderately staining cells + percentage of weakly staining cells, and ranges from 0 to 300. VI. Kits and Products

[0241] Also provided are kits, unit dosage forms, and articles of manufacture comprising any one of the modified antigen-specific immune cells or compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, kits are provided that contain any one of the pharmaceutical compositions described herein, preferably providing instructions for its use. In some embodiments, the kits further comprise, in addition to the modified antigen-specific immune cells, a second cancer therapy, such as chemotherapy, hormonal therapy, and / or immunotherapy. The kits may be tailored for a particular cancer for an individual, and include the corresponding second cancer therapy for the individual.

[0242] Also provided are kits, unit dosage forms, and articles of manufacture comprising any one of the modulators (such as inhibitors or activators) of CD160 expression, function, or activity, or any one of the agents that modulate (such as inhibit or activate) CD160 activity.

[0243] The kit may contain one or more additional components that allow for the expansion or induction of the modified antigen-specific immune cells, such as containers, reagents, culture media, inducers, cytokines, buffers, antibodies, etc. The kit may also contain a device for local administration of the pharmaceutical composition to the tumor site (such as intratumoral injection).

[0244] In another aspect, kits are provided that include 1) a composition comprising modified antigen-specific cells comprising an exogenous functional receptor (such as a CAR), a modulator of CD160 activity, and / or an immunotherapy (such as an immune checkpoint inhibitor), and 2) an agent for determining CD160 levels or activity. In some embodiments, the agent for determining CD160 expression levels is an antibody that recognizes CD160 protein.

[0245] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar bags or plastic pouches), and the like. The kits can provide additional components, such as buffers and interpretive information, as needed. Thus, the present application also provides articles of manufacture that include vials (such as sealed vials), bottles, jars, flexible packaging, and the like. Some components of the kits can be packaged in aqueous media or in lyophilized form.

[0246] The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials, such as glass or plastic. Generally, the container holds a composition effective for treating a disease or disorder (e.g., cancer) described herein and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). The label or package insert will indicate that the composition is used for treating a specific condition in an individual. The label or package insert will further include instructions for administering the composition to an individual. The label may indicate directions for reconstitution and / or use. The container holding the pharmaceutical composition may be a multi-dose vial that allows for repeated administration of the reconstituted formulation (e.g., 2-6 administrations). Package insert refers to instructions customarily included in commercial packaging of therapeutic products containing information regarding the indications, use, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. Moreover, the article of manufacture may further include a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fillers, needles, and syringes.

[0247] The kit or article of manufacture may include multiple unit doses of the pharmaceutical composition packaged in an amount sufficient for storage and use in a pharmacy, e.g., a hospital pharmacy or compounding pharmacy, and instructions for use. Exemplary Embodiments

[0248] The present invention provides the following enumerated embodiments.

[0249] Embodiment 1: A modified antigen-specific immune cell comprising an exogenous CD160 protein on its surface, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cell compared to a precursor antigen-specific immune cell that does not comprise the exogenous CD160 protein, and wherein the immune cell is a T cell.

[0250] Embodiment 2: The modified antigen-specific immune cell of embodiment 1, wherein the modified antigen-specific immune cell is selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by antigen-presenting cells (APCs), and natural killer T cells (NK-T cells).

[0251] Embodiment 3: The modified antigen-specific immune cell of embodiment 1, which is a cytotoxic T cell.

[0252] Embodiment 4: The modified antigen-specific immune cell of embodiment 2, which is a tumor-infiltrating T cell or an anti-tumor T cell activated by an APC.

[0253] Embodiment 5: The modified antigen-specific immune cell of embodiment 1, wherein the modified antigen-specific immune cell is selected from the group consisting of a natural killer (NK) cell, a natural killer T cell (NK-T cell), an iNK-T cell, an NK-T-like cell, a gamma delta T cell, and a macrophage.

[0254] Embodiment 6: The modified antigen-specific immune cell of any one of embodiments 1 to 5, wherein the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, or a variant thereof having at least about 90% identity to any one of SEQ ID NOs: 1-4.

[0255] Embodiment 7: The modified antigen-specific immune cell of any one of embodiments 1 to 5, wherein the exogenous CD160 protein is membrane-bound.

[0256] Embodiment 8: The modified antigen-specific immune cell of embodiment 7, wherein the exogenous CD160 protein is membrane-bound via a GPI linker.

[0257] Embodiment 9: The modified antigen-specific immune cell of embodiment 7, wherein the exogenous CD160 protein comprises a transmembrane domain.

[0258] Embodiment 10: The modified antigen-specific immune cell of embodiment 9, wherein the exogenous CD160 protein further comprises an intracellular domain.

[0259] Embodiment 11: The modified antigen-specific immune cell of embodiment 9 or 10, wherein the exogenous CD160 protein further comprises an intracellular domain derived from a CD160 splice variant.

[0260] Embodiment 12: The modified antigen-specific immune cell of embodiment 10, wherein the intracellular domain comprises an intracellular signaling domain derived from a signaling subunit of a TCR complex.

[0261] Embodiment 13: The modified antigen-specific immune cell of embodiment 12, wherein the signaling subunit of the TCR complex is selected from the group consisting of CD3 gamma, CD3 delta, and CD3 epsilon.

[0262] Embodiment 14: The modified antigen-specific immune cell of embodiment 10, wherein the intracellular domain comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, or both.

[0263] Embodiment 15: The modified antigen-specific immune cell of embodiment 14, wherein the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0264] Embodiment 16: The modified antigen-specific immune cell of embodiment 14, wherein the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain.

[0265] Embodiment 17: The modified antigen-specific immune cell of any one of embodiments 10 to 16, wherein the intracellular domain comprises a primary signaling domain.

[0266] Embodiment 18: The modified antigen-specific immune cell of embodiment 17, wherein the primary signaling domain comprises a CD3 zeta domain.

[0267] Embodiment 19: The modified antigen-specific immune cell of any one of embodiments 10 to 16, wherein the intracellular domain does not comprise a primary signaling domain.

[0268] Embodiment 20: The modified antigen-specific immune cell of embodiment 7, wherein the exogenous CD160 protein is bound to the modified antigen-specific immune cell via an immune cell binding moiety.

[0269] Embodiment 21: The modified antigen-specific immune cell of embodiment 20, wherein the immune cell binding moiety binds to a surface molecule of the immune cell.

[0270] Embodiment 22: The modified antigen-specific immune cell of any one of embodiments 1 to 21, further comprising a functional exogenous receptor.

[0271] Embodiment 23: The modified antigen-specific immune cell of embodiment 22, wherein the functional exogenous receptor is an engineered T cell receptor (TCR).

[0272] Embodiment 24: The modified antigen-specific immune cell of embodiment 22, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR).

[0273] Embodiment 25: A method of producing modified antigen-specific immune cells comprising exogenous CD160 protein on the surface thereof, comprising: producing the modified antigen-specific immune cells by contacting the precursor antigen-specific immune cells with an exogenous CD160 protein or a first nucleic acid encoding an exogenous CD160 protein; The method, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells relative to the precursor antigen-specific immune cells, and the immune cells are T cells.

[0274] Embodiment 26: The method of embodiment 25, wherein the modified antigen-specific immune cells are selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells).

[0275] Embodiment 27: The method of embodiment 25, wherein the modified antigen-specific immune cells are cytotoxic T cells.

[0276] Embodiment 28: The method of embodiment 26, wherein the modified antigen-specific immune cells are tumor-infiltrating T cells or anti-tumor T cells activated by APCs.

[0277] Embodiment 29: The method of embodiment 26, wherein the immune cells are selected from the group consisting of natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, gamma delta T cells, and macrophages.

[0278] Embodiment 30: The method of any one of embodiments 25 to 29, comprising contacting the precursor antigen-specific immune cells with exogenous CD160 protein.

[0279] Embodiment 31: The method of embodiment 30, wherein the exogenous CD160 protein comprises an immune cell binding moiety that binds to a surface molecule of an immune cell.

[0280] Embodiment 32: The method of any one of embodiments 25 to 29, comprising introducing into the precursor antigen-specific immune cells a nucleic acid encoding an exogenous CD160 protein.

[0281] Embodiment 33: The method of embodiment 32, wherein the nucleic acid is mRNA.

[0282] Embodiment 34: The method of embodiment 32, wherein the nucleic acid is DNA.

[0283] Embodiment 35: The method of any one of embodiments 32 to 34, wherein the nucleic acid is introduced into the precursor antigen-specific immune cells by transfection.

[0284] Embodiment 36: The method of any one of embodiments 32 to 34, wherein the nucleic acid is introduced into the precursor antigen-specific immune cells by transduction or electroporation.

[0285] Embodiment 37: The method of any one of embodiments 25 to 36, wherein the CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4, or a variant thereof having at least about 90% identity to any one of SEQ ID NOs: 1 to 4.

[0286] Embodiment 38: The method of any one of embodiments 25 to 37, wherein the exogenous CD160 protein is membrane-bound.

[0287] Embodiment 39: The method of embodiment 38, wherein the exogenous CD160 protein is membrane-bound via a GPI linker.

[0288] Embodiment 40: The method of embodiment 38, wherein the exogenous CD160 protein comprises a transmembrane domain.

[0289] Embodiment 41: The method of embodiment 39, wherein the exogenous CD160 protein further comprises an intracellular domain.

[0290] Embodiment 42: The method of embodiment 40 or 41, wherein the exogenous CD160 protein further comprises an intracellular domain derived from a CD160 splice variant.

[0291] Embodiment 43: The method of embodiment 41, wherein the intracellular domain comprises an intracellular signaling domain derived from a signaling subunit of a TCR complex.

[0292] Embodiment 44: The modified antigen-specific immune cell of embodiment 43, wherein the signaling subunit of the TCR complex is selected from the group consisting of CD3 gamma, CD3 delta, and CD3 epsilon.

[0293] Embodiment 45: The method of embodiment 41, wherein the intracellular domain comprises a CD28 costimulatory domain, a 4-1BB costimulatory domain, or both.

[0294] Embodiment 46: The method of embodiment 45, wherein the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0295] Embodiment 47: The method of embodiment 45, wherein the exogenous CD160 protein comprises, from N-terminus to C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain.

[0296] Embodiment 48: The method of any one of embodiments 41 to 47, wherein the intracellular domain comprises a primary signaling domain.

[0297] Embodiment 49: The method of embodiment 48, wherein the primary signaling domain comprises a CD3 zeta domain.

[0298] Embodiment 50: The method of any one of embodiments 41 to 47, wherein the intracellular domain does not comprise a primary signaling domain.

[0299] Embodiment 51: The method of embodiment 38, wherein the exogenous CD160 protein is bound to the modified antigen-specific immune cell via an immune cell binding moiety.

[0300] Embodiment 52: The method of embodiment 51, wherein the immune cell binding moiety binds to a surface molecule of an immune cell.

[0301] Embodiment 53: The method of any one of embodiments 25 to 52, wherein the precursor antigen-specific immune cells comprise a second nucleic acid encoding a functional exogenous receptor.

[0302] Embodiment 54: The method of any one of embodiments 25 to 52, further comprising contacting the precursor antigen-specific immune cells with a second nucleic acid encoding a functional exogenous receptor.

[0303] Embodiment 55: The method of embodiment 53 or 54, wherein the functional exogenous receptor is an engineered T cell receptor (TCR).

[0304] Embodiment 56: The method of embodiment 53 or 54, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR).

[0305] Embodiment 57: The method of any one of embodiments 54 to 56, wherein the first nucleic acid and the second nucleic acid are operably linked to the same promoter.

[0306] Embodiment 58: The method of any one of embodiments 54 to 56, wherein the first nucleic acid and the second nucleic acid are operably linked to separate promoters.

[0307] Embodiment 59: The method of any one of embodiments 54 to 58, wherein the first nucleic acid and the second nucleic acid are on the same vector.

[0308] Embodiment 60: The method of any one of embodiments 54 to 59, wherein the first nucleic acid and / or the second nucleic acid are on separate vectors.

[0309] Embodiment 61: The method of embodiment 59 or 60, wherein the vector is a viral vector.

[0310] Embodiment 62: The method of embodiment 61, wherein the viral vector is selected from the group consisting of adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, episomal vector expression vectors, herpes simplex viral vectors, and derivatives thereof.

[0311] Embodiment 63: The method of embodiment 59 or 60, wherein the vector is a non-viral vector.

[0312] Embodiment 64: The method of any one of embodiments 25 to 63, further comprising isolating or enriching immune cells comprising the first and / or second nucleic acid.

[0313] Embodiment 65: The method of any one of embodiments 25 to 64, further comprising formulating the modified antigen-specific immune cells expressing CD160 with at least one pharmaceutically acceptable carrier.

[0314] Embodiment 66: A modified antigen-specific immune cell obtained by the method according to any one of embodiments 25 to 65.

[0315] Embodiment 67: A pharmaceutical composition comprising the modified antigen-specific immune cells of any one of embodiments 1 to 24 and 66 and a pharmaceutically acceptable carrier.

[0316] Embodiment 68: A method of treating a disease in an individual, comprising administering to the individual an effective amount of the modified antigen-specific immune cells of any one of embodiments 1 to 24 and 66 or the pharmaceutical composition of embodiment 67.

[0317] Embodiment 69: The method of embodiment 68, wherein the modified antigen-specific immune cells are derived from an individual.

[0318] Embodiment 70: A method of treating a disease in an individual, comprising administering to the individual an effective amount of exogenous CD160 protein or a nucleic acid encoding an exogenous CD160 protein, wherein the exogenous CD160 protein comprises a binding moiety that recognizes a surface molecule on an immune cell in the individual.

[0319] Embodiment 71: The method of any one of embodiments 68 to 70, wherein the administration is intratumoral administration.

[0320] Embodiment 72: The method of any one of embodiments 68 to 70, wherein the administration is to a lymph node.

[0321] Embodiment 73: The method of any one of embodiments 68 to 72, wherein the disease is cancer.

[0322] Embodiment 74: The method of embodiment 73, wherein the cancer is a solid tumor.

[0323] Embodiment 75: The method of embodiment 73 or 74, wherein the cancer is metastatic cancer.

[0324] Embodiment 76: The method of any one of embodiments 73 to 75, wherein the cancer is selected from the group consisting of melanoma, lung cancer, esophageal cancer, pancreatic cancer, breast cancer, liver cancer, brain cancer, and ovarian cancer.

[0325] Embodiment 77: The method of any one of embodiments 68 to 76, wherein the individual is a human.

[0326] Embodiment 78: A method of inhibiting the endogenous immunostimulatory activity of CD160 in an antigen-specific immune cell, comprising contacting the antigen-specific immune cell with an effective amount of an agent that inhibits the immunostimulatory activity of CD160 in the antigen-specific immune cell.

[0327] Embodiment 79: A method of activating the immunostimulatory activity of CD160 in an antigen-specific immune cell, comprising contacting the antigen-specific immune cell with an effective amount of an agent that activates the immunostimulatory activity of CD160 in the antigen-specific immune cell.

[0328] Embodiment 80: The method of embodiment 79, wherein the method enhances the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells, wherein the agent enhances the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0329] Embodiment 81: A method of treating an immune disease in an individual, comprising administering to the individual a therapeutically effective amount of an agent that modulates the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0330] Embodiment 82: The method of embodiment 81, wherein the immune disease is an autoimmune disease or an inflammatory disease, and the agent inhibits the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0331] Embodiment 83: A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0332] Embodiment 84: A method of treating an infection in an individual, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0333] Embodiment 85: A method for increasing the yield and / or viability of antigen-specific immune cells, comprising introducing into the immune cells a nucleic acid encoding an exogenous CD160 protein.

[0334] Embodiment 86: A method for increasing the yield and / or viability of antigen-specific immune cells, comprising causing overexpression of CD160 protein in the immune cells.

[0335] Embodiment 87: The method of embodiment 86, wherein the CD160 protein is an endogenous protein.

[0336] Embodiment 88: The method of embodiment 86, wherein the CD160 protein is an exogenous protein.

[0337] Embodiment 89: The method of embodiment 85, wherein the yield of antigen-specific immune cells expressing exogenous CD160 protein is increased by any one of at least about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold compared to antigen-specific immune cells not expressing exogenous CD160 protein.

[0338] Embodiment 90: The method of embodiment 85, wherein the viability of antigen-specific immune cells expressing exogenous CD160 protein is increased by any one of at least about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold compared to antigen-specific immune cells not expressing exogenous CD160 protein.

[0339] Embodiment 91: The method of any one of embodiments 85 to 88, wherein the yield of antigen-specific immune cells that overexpress CD160 protein is increased by at least one of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold compared to antigen-specific immune cells that do not overexpress CD160 protein.

[0340] Embodiment 92: The method of any one of embodiments 85 to 88, wherein the viability of antigen-specific immune cells that overexpress CD160 protein is increased by at least one of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold compared to antigen-specific immune cells that do not overexpress CD160 protein.

[0341] Embodiment 93: A method for producing therapeutic antigen-specific immune cells, comprising a method for increasing the yield and / or viability of antigen-specific immune cells selected from the methods described in any one of embodiments 85 to 92.

[0342] Embodiment 94: The method of embodiment 93, wherein the therapeutic antigen-specific immune cells comprise tumor-infiltrating lymphocytes (TILs).

[0343] Embodiment 95: The method of embodiment 93, wherein the therapeutic antigen-specific immune cells comprise a functional exogenous receptor.

[0344] Embodiment 96: The method of embodiment 95, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR).

[0345] Embodiment 97: The method of embodiment 95, wherein the functional exogenous receptor is an engineered T cell receptor (TCR).

[0346] Embodiment 98: A method for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells, comprising introducing into the immune cells a nucleic acid encoding an exogenous CD160 protein.

[0347] Embodiment 99: A method for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells, comprising causing overexpression of CD160 protein in the immune cells.

[0348] Embodiment 100: The method of embodiment 99, wherein the CD160 protein is an endogenous protein.

[0349] Embodiment 101: The method of embodiment 99, wherein the CD160 protein is an exogenous protein.

[0350] Embodiment 102: The method of embodiment 98, wherein the in vitro cytolytic activity of antigen-specific immune cells expressing exogenous CD160 protein is increased by any one of at least about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold compared to antigen-specific immune cells not expressing exogenous CD160 protein.

[0351] Embodiment 103: The method of embodiment 98, wherein the in vivo cytolytic activity of antigen-specific immune cells expressing exogenous CD160 protein is increased by any one of at least about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold compared to antigen-specific immune cells not expressing exogenous CD160 protein.

[0352] Embodiment 104: The method of any one of embodiments 98 to 101, wherein the in vitro cytolytic activity of antigen-specific immune cells overexpressing CD160 protein is increased by at least one of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold compared to antigen-specific immune cells not overexpressing CD160 protein.

[0353] Embodiment 105: The method of any one of embodiments 98 to 101, wherein the in vivo cytolytic activity of antigen-specific immune cells that overexpress CD160 protein is increased by at least one of about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, about 1000-fold, or about 10000-fold compared to antigen-specific immune cells that do not overexpress CD160 protein.

[0354] Embodiment 106: A method for producing therapeutic antigen-specific immune cells, comprising a method for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells selected from the methods described in any one of embodiments 98 to 105.

[0355] Embodiment 107: The method of embodiment 106, wherein the therapeutic antigen-specific immune cells comprise tumor-infiltrating lymphocytes (TILs).

[0356] Embodiment 108: The method of embodiment 106, wherein the therapeutic antigen-specific immune cells comprise a functional exogenous receptor.

[0357] Embodiment 109: The method of embodiment 108, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR).

[0358] Embodiment 110: The method of embodiment 108, wherein the functional exogenous receptor is an engineered T cell receptor (TCR). [Example]

[0359] Example 1 Ectopic expression of mCD160 in Pmel T cells To examine the function of CD160 in immune cells, GPI-anchored murine CD160 was ectopically expressed in anti-tumor T cells and quantified by FACS analysis.

[0360] Specifically, mouse full-length CD160 (mCD160) was cloned into an MSCV-based retroviral vector and fused to a GFP reporter via a P2A spacer, allowing independent synthesis of CD160 and GFP proteins (Fig. 1A). The mCD160 virus was then transduced into Pmel T cells bearing a TCR that recognizes the murine homolog of the human melanoma antigen GP100.

[0361] As shown by FACS analysis, ectopic expression of CD160 resulted in an approximately 2.5-fold increase in CD160 expression on Pmel T cells, which normally express low levels of endogenous CD160 (Figures 1B, 1C). Example 2 CD160 expression enhances CTL function of Pmel T cells against B16F0 melanoma cells in culture

[0362] To demonstrate the effect of ectopic CD160 expression on the cytolytic function of T cells, we measured the expression of granzyme A and perforin, the expression of inflammatory cytokines, and the killing activity of CD160-modified Pmel T cells. Mock-infected Pmel T cells were used as a control.

[0363] Specifically, mCD160 virus was transduced into Pmel T cells as described in Example 1, and the expression of granzyme A and perforin was measured by FACS. Granzyme A and perforin are two essential proteins in the granule exocytosis pathway for killing mediated by T cells and NK cells. As shown in Figure 2A, the expression of granzyme A and perforin was increased compared to control Pmel T cells, indicating that exogenous mCD160 enhanced the endogenous CTL function of tumor-specific T cells.

[0364] The expression profiles of the inflammatory cytokines IFN-γ and TNF-α were measured in CD160-modified Pmel T cells compared with control Pmel T cells using FACS. As shown in Figure 2B, the expression of IFN-γ and TNF-α was increased compared with control Pmel T cells, indicating that exogenous mCD160 enhanced the inflammatory activity of tumor-specific T cells.

[0365] The ability of CD160-modified Pmel T cells to kill tumor cells was also examined and compared with control Pmel T cells. Briefly, Pmel T cells were stimulated with anti-CD3 and anti-CD28 beads and cultured for 3 days in T cell expansion medium with IL-2. On day 3, target tumor cells B16F0 were warmed at 37°C for 10 minutes, labeled with 1 μM CELLTRACE™ Violet (Invitrogen) for 30 minutes at 37°C, and then seeded onto a 96-well culture plate. Control or CD160-modified Pmel T cells were added to each well at a defined effector-to-target ratio and incubated for 4–6 hours. Subsequently, target cells were harvested, labeled with 7-aminoactinomycin D (7-AAD, BD Pharmingen), and analyzed by FACS to determine killing by effector T cells. The CELLTRACE™ Violet dye+ / 7-AAD+ cell population represented killed target cells, and the CELLTRACE™ Violet dye+ / 7-AAD- population represented remaining viable target cells. As shown in Figure 2C, CD160-modified Pmel T cells were more potent at killing B16F0 melanoma cells in coculture compared with control T cells.

[0366] Taken together, these results indicated that ectopic expression of CD160 enhanced endogenous cytolytic activity, boosted inflammatory function, and enhanced the tumor-killing activity of antigen-specific T cells. Example 3 CD160 expression enhances the control of B16F0 melanoma in mice by Pmel T cells

[0367] To test whether ectopic CD160 can enhance the tumor-regulatory activity of antigen-specific T cells in vivo, CD160-modified Pmel T cells were adoptively transferred into recipient mice bearing subcutaneous B16F0 melanoma tumors.

[0368] Specifically, 1×10 5 B16F0 cells were subcutaneously injected into 6- to 8-week-old female C57BL / 6 mice. Prior to adoptive cell transfer, mice were randomized to ensure there was no size bias at the start of the experiment. In one experiment, a single dose of 100,000, 200,000, 300,000, or 400,000 CD160-modified Pmel T cells or 300,000 control Pmel T cells was adoptively transferred into tumor-bearing mice 8 days after implantation (Figure 3A). Mice were checked twice weekly for tumor formation by palpation, and tumor area was measured by caliper measurement. Tumor area is the mean measurement of at least five mice per group (+ / - SEM, two-tailed t-test). As shown in Figure 3A, CD160-modified Pmel T cells demonstrated a dose-dependent effect on tumor control in response to CD160-Pmel T cell transfer.

[0369] In a separate experiment, we analyzed the effect of adoptive transfer of CD160-modified antigen-specific T cells and non-antigen-specific T cells on the control of B16F0 melanoma tumors (Figure 3B). Briefly, a single dose of 300,000 each of (i) control Pmel T cells, (ii) CD160-modified antigen-specific Pmel T cells, or (iii) CD160-modified, non-antigen-specific splenic T cells was adoptively transferred into tumor-bearing mice 8 days after implantation. As shown in Figure 3B, ectopic expression of CD160 significantly enhanced the tumor-controlling activity of tumor-specific Pmel T cells, whereas it had only a small, statistically insignificant effect on tumor control by non-antigen-specific splenic T cells.

[0370] Taken together, these results demonstrated that ectopic CD160 expression in tumor-specific T cells can enhance tumor control in a synergistic immunocompetent mouse tumor model. Example 4 CD160-modified T cells can control and eliminate established B16F0 melanoma tumors without IL-2 or vaccination

[0371] To examine the ability of CD160-modified Pmel T cells in eliminating established tumors, CD160-Pmel T cells were adoptively transferred into mice bearing established B16F0 melanoma tumors after chemotherapy preconditioning.

[0372] Simply put, 1 x 10 5 B16F0 cells were subcutaneously injected into 6-8 week-old female C57BL / 6 mice. After tumor implantation, mice were observed daily and sacrificed if signs of death were observed. Mice were checked twice weekly for tumor formation by palpation, and tumor area was measured by caliper measurement. Prior to adoptive cell transfer, mice were randomized to ensure size uniformity at the start of the experiment. Starting on day 7 after tumor implantation, mice were infused with Pmel T cells at 14-day intervals. Each T cell infusion was preceded by a cyclophosphamide (CYP) conditioning regimen (100 mg / kg per treatment), but no vaccination or IL-2 infusion was administered. The mean tumor size peaked on day 35 after implantation. Normalized spider plots were plotted appropriately, with the peak tumor size at day 35 normalized as "1" to show the relative change in tumor size in response to treatment with control Pmel T cells or CD160-modified Pmel T cells. As shown in Figure 4A, adoptive transfer of CD160-modified Pmel T cells resulted in a nearly 100% response rate in mice, with tumors shrinking in size by more than 90% or being completely eliminated in over 80% of mice.

[0373] We also measured the improvement in survival with adoptive transfer of CD160-modified Pmel T cells. As shown in Figure 4C, no deaths were observed in mice receiving 2-weekly treatments of CYP and CD160-modified Pmel T cells up to 110 days after implantation. In contrast, mice treated with CYP alone or CYP and control Pmel T cells died before 75 days after implantation, with median survival times of 27 and 60 days, respectively.

[0374] Taken together, these results indicated that adoptive transfer of CD160-modified Pmel T cells with CYP preconditioning could efficiently control and eliminate established B16F0 melanoma tumors, particularly without the need for IL-2 cytokines or vaccination regimens. Example 5 Regulation of CD160-modified Pmel T cells on B16F0 melanoma is dose-dependent

[0375] To characterize the dose-dependent effect of CD160-modified Pmel T cells on the control of B16F0 melanoma, B16F0-bearing mice were infused with 150,000 or 300,000 CD160-modified Pmel T cells.

[0376] Simply put, 1 x 10 5 B16F0 cells were subcutaneously injected into 6-8 week-old female C57BL / 6 mice. After tumor implantation, mice were observed daily and sacrificed if signs of death were observed. Mice were checked twice weekly for tumor formation by palpation, and tumor area was measured by caliper measurement. Prior to adoptive cell transfer, mice were randomized to ensure size uniformity at the start of the experiment. Starting 7 days after tumor implantation, mice were infused with either (A) 150,000 or (B) 300,000 CD160-modified Pmel T cells at 14-day intervals (every 2 weeks). Each T cell infusion was preceded by a CYP conditioning regimen (100 mg / kg per treatment). Spider plots were normalized and plotted as described in Example 4.

[0377] At a dose regimen of 150,000 CD160-modified Pmel T cells per 2-week transfer, a 100% response rate in tumor control was observed among treated mice, with only 20-30% of mice showing a greater than 90% reduction in tumor size (Figure 5A). In comparison, at a dose regimen of 300,000 CD160-Pmel T cells per 2-week transfer, a 100% response rate in tumor shrinkage was observed among treated mice, with approximately 40-50% of mice showing a greater than 90% reduction in tumor size (Figure 5B).

[0378] We also measured the improvement in survival following adoptive transfer of CD160-modified Pmel T cells at two doses. Tumor-bearing mice treated with CD160-modified Pmel T cells at both doses showed significant survival improvements, with 80% and 100% survival rates at 120 days post-implantation for mice treated with 150,000 or 300,000 CD160-modified Pmel T cells, respectively (Figure 5C). In contrast, mice treated with CYP alone or CYP and control Pmel T cells died before 90 days, with median survival times of 61.5 and 56 days post-implantation, respectively.

[0379] Taken together, these results indicated that adoptive transfer of CD160-modified Pmel T cells with CYP preconditioning could efficiently control and eliminate established B16F0 melanoma tumors, and the tumor-control effect was dose-dependent. Example 6 CD160-modified Pmel T cells efficiently controlled the growth of metastatic B16F10 melanoma tumors

[0380] To examine the ability of CD160-modified Pmel T cells in inhibiting metastatic tumor growth, CD160-Pmel T cells were adoptively transferred into mice bearing metastatic B16F10 melanoma tumors after chemotherapy preconditioning.

[0381] Simply put, 1 x 10 5B16F10 cells were subcutaneously injected into 6-8 week-old female C57BL / 6 mice. After tumor implantation, mice were observed daily and sacrificed if signs of death were observed. Mice were checked twice weekly for tumor formation by palpation, and tumor area was measured by caliper measurement. Prior to adoptive cell transfer, mice were randomized to ensure size uniformity at the start of the experiment. Starting 7 days after tumor implantation, mice were infused with 300,000 CD160-modified Pmel T cells at 14-day intervals (every 2 weeks). Each T cell infusion was preceded by a CYP conditioning regimen (100 mg / kg per treatment).

[0382] As shown in Figure 6A, CD160-modified Pmel T cells, when combined with CYP preconditioning, significantly halted the increase in mean tumor size compared with untreated mice, mice treated with CYP chemotherapy alone, or mice treated with control Pmel T cells and CYP preconditioning. Tumor area is the mean measurement of at least five mice per group (+ / - SEM, two-tailed t-test). Figure 6B shows the ability of CD160-modified Pmel T cells (with CYP preconditioning) to control individual tumor growth compared with no treatment, CYP treatment alone, or treatment with control Pmel T cells and CYP.

[0383] Taken together, adoptively transferred CD160-modified Pmel T cells, when combined with CYP preconditioning, were shown to efficiently control subcutaneous tumor growth when compared with untreated mice, mice treated with CYP chemotherapy alone, or mice treated with control Pmel T cells and CYP preconditioning.

[0384] Longer-term tumor control and improved survival with CD160-modified Pmel T cells was also examined. Spider plots for tumor size were normalized as in Example 4.

[0385] As shown in Figure 7A, adoptive transfer of CD160-Pmel T cells was able to control or eliminate subcutaneous tumors over the course of treatment. As shown in Figure 7B, untreated mice, mice treated with CYP chemotherapy alone, and mice treated with control Pmel T cells and CYP preconditioning all died before 80 days post-implantation as a result of metastasis, with median survival times of 39, 39, and 59 days post-implantation, respectively. In contrast, the group of mice treated with CD160-Pmel T cells and CYP preconditioning maintained an 80% survival rate at 120 days post-implantation (Figure 7B) and throughout the duration of continuous infusion of CD160-modified Pmel T cells (data not shown).

[0386] Taken together, these results indicated that CD160-modified Pmel T cells, in addition to controlling subcutaneous tumor growth, could also establish control and inhibition against tumor metastasis. Example 7 Enhanced tumor suppressor activity of mouse CD160 activating chimeras

[0387] To investigate whether the tumor-suppressive activity of CD160 can be modulated using additional domains, the extracellular domain of murine CD160 was fused in various configurations to intracellular signaling domains derived from the TCR and its costimulatory pathway, including CD3ζ, CD28, and 4-1BB, to derive CD160 activating chimeras, as shown in Figure 8A-C. The ability of PM1 T cells expressing these CD160 chimeras to control established B16F0 melanoma in mice was measured and compared with those expressing GPI-anchored murine CD160 (mCD160).

[0388] Simply put, 1 x 10 5B16F10 cells were subcutaneously injected into 6-8 week-old female C57BL / 6 mice. After tumor implantation, mice were observed daily and sacrificed if signs of death were observed. Mice were checked twice weekly for tumor formation by palpation, and tumor area was measured by caliper measurement. Prior to adoptive cell transfer, mice were randomized to ensure size uniformity at the start of the experiment. Starting 7 days after tumor implantation, mice were infused at 14-day intervals (every 2 weeks) with 300,000 Pmel T cells ectopically expressing mCD160, GEM123, GEM124, GEM125, GEM126, GEM127, or GEM128, respectively. Prior to each T cell infusion, a CYP conditioning regimen (100 mg / kg per treatment) was administered.

[0389] As seen in Figure 8A, Pmel T cells expressing GEM125, a chimera with a CD28 signaling domain distal to the transmembrane domain, showed weaker tumor suppression than Pmel T cells expressing mCD160, whereas Pmel T cells expressing GEM124, a chimera with a CD28 signaling domain adjacent to the transmembrane domain, showed stronger tumor suppression than Pmel T cells expressing mCD160. These results indicated that the CD28 signaling domain located adjacent to the transmembrane domain can further enhance the ability of CD160 chimeras to enhance antigen-specific T cell immune responses.

[0390] As seen in Figure 8B, Pmel T cells expressing GEM127, a chimera with a 4-1BB signaling domain adjacent to the transmembrane domain, showed weaker tumor suppression compared with Pmel T cells expressing mCD160, whereas Pmel T cells expressing GEM126, a chimera with a CD28 signaling domain adjacent to the transmembrane domain, showed stronger tumor suppression compared with Pmel T cells expressing mCD160. These results indicated that the CD28 signaling domain, but not the 4-1BB domain, when located adjacent to the transmembrane domain, can further enhance the ability of CD160 chimeras to enhance antigen-specific T cell immune responses.

[0391] As seen in Figure 8C, Pmel T cells expressing GEM123, a chimera with a CD3ζ signaling domain adjacent to the transmembrane domain, showed weaker tumor suppression than Pmel T cells expressing mCD160. Furthermore, GEM128, a chimera with three signaling domains, including the CD3ζ domain distal to the transmembrane domain, showed significantly lower tumor suppression than mCD160, despite having a CD28 signaling domain adjacent to the transmembrane domain.

[0392] Taken together, these results indicated that the ability of CD160 chimeras to enhance antigen-specific T cell immune responses could be further enhanced when the CD28 costimulatory domain was located adjacent to the transmembrane domain (GEM124, GEM126 in Figures 8A and 8B). However, the ability of CD160 to enhance tumor control may not be compatible with the integrated CD3ζ (GEM123, GEM127, GEM128 in Figures 8B and 8C). Example 8 Human CD160 and its variants have conserved functions in the control of established B16F0 melanoma in mice

[0393] To compare the tumor suppressor activity of human CD160 variants with murine CD160, we measured the ability of Pmel T cells expressing each of these forms of CD160 in controlling established B16F0 melanoma in mice.

[0394] Simply put, 1 x 10 5 B16F10 cells were subcutaneously injected into 6-8 week-old female C57BL / 6 mice. After tumor implantation, mice were observed daily and sacrificed if signs of death were observed. Mice were checked twice weekly for tumor formation by palpation, and tumor area was measured by caliper measurement. Prior to adoptive cell transfer, mice were randomized to ensure there was no size bias at the start of the experiment. To achieve ectopic expression of CD160, Pmel T cells were infected with viruses carrying GPI-anchored mouse CD160, GPI-anchored human CD160 variants, transmembrane human CD160 variants, or transmembrane human CD160 with an intracellular domain. Starting 7 days after tumor implantation, mice were infused at 14-day intervals (every 2 weeks) with 300,000 Pmel T cells ectopically expressing the indicated murine or human CD160 variants, with each T cell infusion preceded by a CYP conditioning regimen (100 mg / kg per treatment).

[0395] As seen in Figure 10A, all Pmel T cells expressing human CD160 variants showed stronger activity in controlling and eliminating established B16F0 melanoma tumors than Pmel T cells expressing mCD160. In particular, Pmel T cells expressing GPI-anchored human CD160 and human CD160 with the intracellular domain showed the most potent activity in tumor control.

[0396] We also measured the survival improvement by adoptive transfer of Pmel T cells expressing various CD160 variants. As shown in Figure 10B, Pmel T cells expressing GPI-anchored human CD160 and Pmel T cells expressing human CD160 with the intracellular domain provided the strongest survival improvement.

[0397] Taken together, these results indicate that human CD160 variants, like murine CD160, exhibit a conserved function in enhancing tumor-specific T cells for the control and elimination of established B16F0 melanoma tumors in mice, suggesting that they likely have a similar function in the control of established solid tumors in humans. Example 9 CD160-modified LLC TILs inhibit the development of metastatic Lewis lung carcinoma in mice

[0398] To examine the ability of CD160-modified antigen-specific immune cells to inhibit the development of metastatic lung cancer, CD160-modified tumor-infiltrating lymphocytes (TILs) were tested for their ability to kill Lewis lung carcinoma in vitro and for their ability to improve survival in vivo in a Lewis lung carcinoma mouse model.

[0399] To induce TILs, lung tumors were isolated from mice bearing Lewis lung carcinoma, carefully minced into small pieces, and then digested with collagenase V at 37°C. Single-cell suspensions were obtained by passing the digested samples through a 70-100 μm cell strainer. A syringe plunger was used to gently squeeze the digested tissue through the cell strainer mesh as needed. The single-cell suspension was then stained with anti-TCRβ conjugated to phycoerythrin (PE), further enriched using anti-PE magnetic beads, and finally sorted on a SONY SH800 FACS sorter. The purity of the sorted TILs used in the described experiments was greater than 85%, as determined by FACS analysis. TILs were then engineered to express mCD160 or the CD160 chimeric GEM124 (see Figure 8A).

[0400] The ability of CD160-modified TILs to kill tumor cells was first examined and compared with control TILs. Briefly, TILs were stimulated with anti-CD3 and anti-CD28 beads and cultured in T cell expansion medium with IL-2 for 3 days. On day 3, target tumor cells, LLC, were warmed to 37°C for 10 minutes, labeled with 1 μM CELLTRACE™ Violet (Invitrogen) for 30 minutes at 37°C, and then seeded onto 96-well culture plates. C...

Claims

1. 1. A modified antigen-specific immune cell comprising a heterologous nucleic acid sequence encoding an exogenous CD160 protein expressed on the surface of the modified antigen-specific immune cell, The modified antigen-specific immune cell, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cell compared to a precursor antigen-specific immune cell that does not contain the exogenous CD160 protein, and the immune cell is a T cell.

2. 2. The modified antigen-specific immune cell of claim 1, wherein the modified antigen-specific immune cell is selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by antigen-presenting cells (APCs), and natural killer T cells (NK-T cells).

3. (a) a cytotoxic T cell, or (b) selected from the group consisting of natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, and γδ T cells; The modified antigen-specific immune cell of claim 1 .

4. The modified antigen-specific immune cell of claim 2, which is a tumor-infiltrating T cell or an anti-tumor T cell activated by an APC.

5. 5. The modified antigen-specific immune cell of any one of claims 1 to 4, wherein the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4, or a variant thereof having at least 90% identity to any one of SEQ ID NOs: 1 to 4.

6. 6. The modified antigen-specific immune cell of claim 1, wherein the exogenous CD160 protein is membrane-bound.

7. the exogenous CD160 protein is (a) membrane-bound by a GPI linker; (b) comprising a transmembrane domain; The modified antigen-specific immune cell of claim 6 .

8. The modified antigen-specific immune cell of claim 7, wherein the exogenous CD160 protein further comprises an intracellular domain.

9. 9. The modified antigen-specific immune cell of claim 8, wherein the intracellular domain comprises an intracellular signaling domain derived from a signaling subunit of a TCR complex.

10. 10. The modified antigen-specific immune cell of any one of claims 1 to 9, further comprising a functional exogenous receptor.

11. 11. The modified antigen-specific immune cell of claim 10, wherein the functional exogenous receptor is an engineered T cell receptor (TCR) or a chimeric antigen receptor (CAR).

12. 1. A composition comprising a first nucleic acid encoding an exogenous CD160 protein for use in a method for producing modified antigen-specific immune cells comprising said exogenous CD160 protein on the surface thereof, the composition comprising: the method includes producing the modified antigen-specific immune cells by introducing into precursor antigen-specific immune cells a first nucleic acid encoding the exogenous CD160 protein; The composition, wherein the exogenous CD160 protein results in upmodulation of the modified antigen-specific immune cells relative to the precursor antigen-specific immune cells, and the immune cells are T cells.

13. 13. The composition of claim 12, wherein the modified antigen-specific immune cells are selected from the group consisting of cytotoxic αβ T cells, γδ T cells, helper T cells, tumor-infiltrating T cells, anti-tumor T cells activated by APCs, and natural killer T cells (NK-T cells).

14. the modified antigen-specific immune cells (a) a cytotoxic T cell; (b) selected from the group consisting of natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, and γδ T cells; The composition of claim 12.

15. 14. The composition of claim 13, wherein the modified antigen-specific immune cells are tumor-infiltrating T cells or anti-tumor T cells activated by APCs.

16. The composition of any one of claims 12 to 15, wherein the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4, or a variant thereof having at least 90% identity to any one of SEQ ID NOs: 1 to 4.

17. The composition of any one of claims 12 to 16, wherein the exogenous CD160 protein is membrane-bound.

18. the exogenous CD160 protein is (a) membrane-bound via a GPI linker; or (b) comprising a transmembrane domain; 18. The composition of claim 17.

19. (a) the precursor antigen-specific immune cells comprise a second nucleic acid encoding a functional exogenous receptor; and / or (b) the method further comprises contacting the precursor antigen-specific immune cells with a second nucleic acid encoding a functional exogenous receptor.

19. The composition of any one of claims 12 to 18.

20. (a) the functional exogenous receptor is an engineered T cell receptor (TCR) or a chimeric antigen receptor (CAR); (b) the first nucleic acid and the second nucleic acid are operably linked to the same promoter; and / or (c) the first nucleic acid and the second nucleic acid are on the same vector; 20. The composition of claim 19.

21. The method comprises: (a) isolating or enriching immune cells containing said first and / or said second nucleic acid; and / or (b) formulating said modified antigen-specific immune cells expressing CD160 with at least one pharmaceutically acceptable carrier.

21. The composition of any one of claims 12 to 20, further comprising:

22. 1. A modified antigen-specific immune cell obtained by a method for producing a modified antigen-specific immune cell comprising an exogenous CD160 protein on its surface, the method comprising: The method comprises contacting precursor antigen-specific immune cells with a composition comprising a heterologous nucleic acid sequence encoding an exogenous CD160 protein; wherein said exogenous CD160 protein results in upmodulation of said modified antigen-specific immune cell relative to said precursor antigen-specific immune cell, and said immune cell is a T cell.

23. 23. A pharmaceutical composition comprising the modified antigen-specific immune cells of any one of claims 1 to 11 and 22 and a pharmaceutically acceptable carrier.

24. 24. A composition comprising the modified antigen-specific immune cells of any one of claims 1 to 11 and 22, or the pharmaceutical composition of claim 23, for treating a disease in an individual.

25. 25. The composition of claim 24, wherein the modified antigen-specific immune cells are derived from the individual.

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