Methods of modulating cd160 function in antigen-specific immune cells and uses thereof

Abstract

The present disclosure provides modified antigen-specific immune cells expressing an 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 disclosure also provides methods of modulating CD160 activity in antigen-specific immune cells. The present disclosure also provides methods and pharmaceutical compositions for treating cancer using the modified antigen-specific immune cells and modulators of CD160 activity described herein.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 812,897, filed March 1, 2019, the entire contents of each of which are incorporated herein by reference.

[0003] Submitting sequence lists as ASCII text files

[0004] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Computer-readable form of sequence listings (CRF) (filename: 756592000240SEQLIST.TXT, record date: February 24, 2020, size: 17KB). Technical Field

[0005] This invention relates to methods for regulating CD160 function in antigen-specific immune cells and their uses. Antigen-specific immune cells include 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. This invention also relates to antigen-specific immune cells expressing exogenous CD160 protein, and methods for regulating the function of antigen-specific immune cells in cancer treatment. Background Technology

[0006] T cells are natural medicines that protect our bodies from infections and cancer. Notably, tumor-targeting T cells can be effectively re-engaged through checkpoint blockade and chimeric antigen receptor (CAR) T-cell therapy to treat cancer patients. The success of these immunotherapies clearly demonstrates the effectiveness of T cells in cancer treatment, inspiring the exploration of multiple methods to activate and enable T cells to fight cancer. Over the past half-century, pioneering researchers and clinicians have explored the adoption of tumor-targeting T cells to treat human solid tumors. Various tumor-targeting T cells, including in vitro activated T cells, tumor-infiltrating lymphocytes (TILs), T cells carrying specific T-cell receptors (TCRs) that recognize tumor antigens or tumor-associated antigens (TCR-T), and T cells derived from dendritic cells loaded with tumor antigens (DC-T), have been tested and shown promising efficacy in preclinical models and human patients. Recently, T cells that recognize neoantigens from mutated cancer cells have been used in adoptive T-cell therapy for lung and breast cancer, and have shown efficacy in some cases. However, even with the help of other therapies, such as radiation, vaccination, chemotherapy and combined infusion of the anti-tumor cytokine IL-2, it is difficult to achieve sustained control and elimination of solid tumors through adoptive transfer of tumor-targeting T cells.

[0007] The relatively low response rate and eventual loss of tumor control via infused tumor-targeting T cells can be attributed to a number of intrinsic and extrinsic barriers to T cells. These barriers originate from both homeostatic immune tolerance mechanisms and tumor-induced immunosuppressive mechanisms. Notably, these tolerances create barriers that prevent T cells from effectively controlling tumors. For example, naturally occurring anti-tumor T cells, controlled by central tolerance mechanisms involved in positive and negative thymic selection, typically possess T cell receptors (TCRs) with low or intermediate affinity for unmutated tumor antigens or tumor-associated antigens. Furthermore, developing tumors can reduce the expression of major histocompatibility complex (MHC) molecules of classes I and II, effectively limiting the presentation of tumor antigens on tumor cells and thus limiting T cell recognition via homologous TCRs. Many efforts in this field have been dedicated to overcoming these barriers, with varying degrees of success. CARs can effectively enable T cells to engage with tumor cells and help overcome the inadequacy or loss of T cell tumor recognition. Remarkably, CAR-T cells targeting CD19 or BCMA antigens have eliminated B-cell leukemia / lymphoma or multiple myeloma, respectively. Other approaches focus on overcoming recognition barriers through in vitro evolution by increasing the affinity of tumor-specific TCRs or by selecting TCRs that recognize neoantigens, thereby moderately improving efficacy.

[0008] While these strategies show promise, additional barriers beyond antigen recognition must be overcome for tumor-targeting T cells to eliminate or sustain established tumor control. Notably, cold tumors—tumors lacking activated T cells—are associated with poor prognosis and low responsiveness to immunotherapy. It is speculated that checkpoint inhibition, the closure of T cell-specific chemical inducers, and T cell exhaustion due to adverse nutrition and hypoxia may contribute to the cold tumor phenomenon, in addition to many other unknown factors in the tumor microenvironment. Various genetic engineering strategies have been explored to enhance T cell transport and activation in tumors. Ectopic expression of chemokine receptors or VEGFR-recognizing CARs in tumor-specific T cells has been shown to increase tumor control via metastatic T cells. Furthermore, inactivation of negative T cell regulators in tumor-specific T cells, such as PD-1, CBLB, or adenosine 2A receptors, leads to enhanced anti-tumor function of T cells. Interestingly, ectopic expression of signals that enhance mitochondrial biogenesis and oxidative phosphorylation, such as PGC1α or OPA1, or CARs carrying the CD278 signaling domain, can also increase the anti-tumor function of metastatic T cells. While T cell function in tumor control can be enhanced through various molecular and cellular processes, these strategies are often insufficient to enable sustained tumor control or elimination of established tumors via T cells. Therefore, it is important to identify molecules that can be adapted to target tumor-specific T cells for sustained control and elimination of solid tumors.

[0009] CD160 is a 27 kDa glycoprotein that was initially identified on human natural killer cells using the monoclonal antibody BY55. (LeBouteiller et al., 1993, J Exp Med..178(3):1121-6). Later, the major form of CD160 was found to be a glycosyl-phosphatidylinositol (GPI)-anchored immunoglobulin (Ig)-like cell membrane receptor, which was found on a subset of major CD16+ NK cells, NK-T cells, γδ-T cells, some subsets of CD4 T cells, and CD8+ cytolytic T cells, as well as in activated endothelial cells (LeBouteiller et al., 2011, Immunol Lett., 138(2):93-6). The cDNA sequence of human CD160 encodes a 181-amino acid-rich, glycosyl-phosphatidylinositol-anchored protein with a single Ig-like domain. Subsequently, other isoforms with transmembrane domains and / or lacking extracellular Ig-like domains have been identified. CD160 is expressed on the cell surface as a tightly disulfide-linked multimer. CD160 is a ligand for HVEM, and the binding of CD160 to HVEM leads to T cell suppression and unresponsiveness (Cai et al., 2009, Nat Immunol., 9(2):176–185). CD160 is generally considered, along with anti-PD-1 antibodies, as an immune checkpoint inhibitor with anti-cancer activity (Stecher et al., 2017, Front Immunol., 8:572). It has been suggested that CD160 competes with BTLA (CD272) for binding to HVEM (Kojima et al., 2011, J Mol Biol., 413(4):762-72). Mouse and human CD160 on NK cells and some subsets of T cells have low affinity for MHC classes 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 novel target in cases of pathological ocular and tumor angiogenesis in humans that are unresponsive to or resistant to existing anti-angiogenic drugs (Chabot et al., 2011, J Exp Med., 208(5):973-86). Loss-of-function analysis of CD160 in mice showed that CD160 is essential for NK-mediated IFN-γ production, but not important for NK cell cytolytic activity, and 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 showing the role of CD160 in the control and elimination of established tumors by antigen-specific T cells.

[0010] All publications, patents, patent applications, and published patent applications mentioned herein are incorporated herein by reference in their entirety. Summary of the Invention

[0011] This application provides antigen-specific immune cells modified with exogenous CD160 protein on their surface, and methods for using them to treat cancer. The invention also provides methods for modulating CD160 activity in antigen-specific immune cells.

[0012] One aspect of this application provides antigen-specific immune cells modified with exogenous CD160 protein on their surface, wherein the exogenous CD160 protein leads to upregulation of the modified antigen-specific immune cells, wherein the immune cells are T cells, compared to precursor antigen-specific immune cells that do not contain exogenous CD160 protein. 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, antigen-presenting cell (APC)-activated anti-tumor T cells, 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 APC-activated anti-tumor T cells. In some embodiments, the APC-activated anti-tumor T cells are dendritic cell (DC)-activated anti-tumor T cells. 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, γδT cells, and macrophages. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NO:1-4, or a variant thereof having at least about 90% identity with any one of SEQ ID NO:1-4.

[0013] In some embodiments of antigen-specific immune cells according to any of the modifications described above, the exogenous CD160 protein is membrane-bound. In some embodiments, the exogenous CD160 protein binds to the membrane via a GPI linker. In some embodiments, the exogenous CD160 protein binds to the modified antigen-specific immune cell via an immune cell-binding portion. In some embodiments, the immune cell-binding portion binds to surface molecules of the immune cell. In some embodiments, the exogenous CD160 protein includes a transmembrane domain. In some embodiments, the exogenous CD160 protein further includes an intracellular domain. In some embodiments, the exogenous CD160 protein further includes an intracellular domain derived from a CD160 splice variant. In some embodiments, the intracellular domain includes an intracellular signal transduction domain derived from a signal transduction subunit of the TCR complex. In some embodiments, the signal transduction subunit of the TCR complex is selected from the group consisting of CD3γ, CD3δ, and CD3ε.

[0014] In some embodiments of antigen-specific immune cells according to any of the modifications described above, the exogenous CD160 protein is membrane-bound and includes an intracellular domain. In some embodiments, the intracellular domain includes a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, or both. In some embodiments, the exogenous CD160 protein, from its N-terminus to its C-terminus, includes: an extracellular CD160 domain, a transmembrane domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain. In some embodiments, the exogenous CD160 protein, from its N-terminus to its C-terminus, includes: an extracellular CD160 domain, a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD28 co-stimulatory domain. In some embodiments, the intracellular domain includes a primary signal transduction domain. In some embodiments, the primary signal transduction domain includes a CD3ζ domain. In some embodiments, the intracellular domain does not include a primary signal transduction domain.

[0015] In some embodiments of the antigen-specific immune cells according to any of the above-described modifications, the modified antigen-specific immune cells further include 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).

[0016] One aspect of this application provides a method for generating modified antigen-specific immune cells comprising a foreign CD160 protein on their surface, comprising: contacting a precursor antigen-specific immune cell with the foreign CD160 protein or a first nucleic acid encoding the foreign CD160 protein to generate the modified antigen-specific immune cell, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cell compared to the precursor antigen-specific immune cell, wherein the immune cell is a T cell. 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, APC-activated anti-tumor T cells, 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 APC-activated anti-tumor T cell. In some embodiments, the APC-activated anti-tumor T cell is a DC-activated anti-tumor T cell. 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, γδT cells, and macrophages.

[0017] In some embodiments of the generation method according to any of the above-described methods, the method includes contacting a precursor antigen-specific immune cell with an exogenous CD160 protein. In some embodiments, the exogenous CD160 protein includes an immune cell-binding portion of a surface molecule that binds to the immune cell. In some embodiments, the generation method includes introducing a nucleic acid encoding the exogenous CD160 protein into the precursor antigen-specific immune cell. 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 cell by transfection. In some embodiments, the nucleic acid is introduced into the precursor antigen-specific immune cell by transduction or electroporation. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NO: 1-4, or a variant thereof having at least about 90% identity with any one of SEQ ID NO: 1-4.

[0018] In some embodiments of any of the above-described methods of production, the exogenous CD160 protein is membrane-bound. In some embodiments, the exogenous CD160 protein binds to the membrane via a GPI linker. In some embodiments, the immune cell-binding portion binds to surface molecules of immune cells. In some embodiments, the exogenous CD160 protein binds to modified antigen-specific immune cells via the immune cell-binding portion. In some embodiments, the exogenous CD160 protein includes a transmembrane domain. In some embodiments, the exogenous CD160 protein further includes an intracellular domain. In some embodiments, the exogenous CD160 protein further includes an intracellular domain derived from a CD160 splice variant. In some embodiments, the intracellular domain includes an intracellular signal transduction domain derived from a signal transduction subunit of the TCR complex. In some embodiments, the signal transduction subunit of the TCR complex is selected from the group consisting of CD3γ, CD3δ, and CD3ε.

[0019] In some embodiments of any of the above-described generation methods, the exogenous CD160 protein is membrane-bound and includes an intracellular domain. In some embodiments, the intracellular domain includes a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, or both. In some embodiments, the exogenous CD160 protein, from its N-terminus to its C-terminus, includes: an extracellular CD160 domain, a transmembrane domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain. In some embodiments, the exogenous CD160 protein, from its N-terminus to its C-terminus, includes: an extracellular CD160 domain, a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD28 co-stimulatory domain. In some embodiments, the intracellular domain includes a primary signal transduction domain. In some embodiments, the primary signal transduction domain includes a CD3ζ domain. In some embodiments, the intracellular domain does not include a primary signal transduction domain.

[0020] In some embodiments of any of the above-described generation methods, the precursor antigen-specific immune cells include a second nucleic acid encoding a functional exogenous receptor. In some embodiments, the generation method further includes 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 and second nucleic acids are operatively linked to the same promoter. In some embodiments, the first and second nucleic acids are operatively linked to separate promoters. In some embodiments, the first and second nucleic acids are on the same vector. In some embodiments, the first and / or second nucleic acids 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: adenovirus vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, augmentative expression vectors, herpes simplex virus vectors, and derivatives thereof. In some embodiments, the vector is a non-viral vector.

[0021] In some embodiments of any of the above-described generation methods, the method further includes isolating or enriching immune cells comprising a first and / or a second nucleic acid. In some embodiments, the method further includes formulating antigen-specific immune cells expressing CD160 modified with at least one pharmaceutically acceptable carrier.

[0022] Modified antigen-specific immune cells generated by a method according to any of the above-described generation methods are also provided.

[0023] Further, a pharmaceutical composition is provided comprising an antigen-specific immune cell modified according to any of the above-described modifications of immune cells, and a pharmaceutically acceptable carrier.

[0024] Another aspect of this application provides a method for treating a disease in an individual, comprising administering to the individual an effective amount of modified antigen-specific immune cells according to any of the above-described modified antigen-specific immune cells or a pharmaceutical composition according to any of the above-described pharmaceutical compositions. In some embodiments, the modified antigen-specific immune cells are derived from the individual. Yet another aspect of this application provides a method for treating a disease in an individual, comprising administering to the individual an effective amount of exogenous CD160 protein or nucleic acid encoding exogenous CD160 protein, wherein the exogenous CD160 protein includes a binding portion that recognizes a surface molecule on an immune cell in the individual.

[0025] In some embodiments of any of the treatment methods described above, administration is intratumoral. In some embodiments, administration is into 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 person.

[0026] One aspect of the present invention provides a method for activating the immunostimulatory activity of CD160 in antigen-specific immune cells, 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 includes enhancing the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells, and wherein the agent enhances the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0027] One aspect of the invention provides a method for treating an immunological 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 immunological disease is an autoimmune disease or an inflammatory disease, and the agent inhibits the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0028] One aspect of the invention provides a method 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. Another aspect of the invention provides a method 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.

[0029] It also provides compositions, uses, kits, and products that include antigen-specific immune cells of any kind. Attached Figure Description

[0030] Figure 1A The ectopic expression of mouse CD160 in Pmel T cells compared to control Pmel T cells is shown, as analyzed by FACS. Figure 1B The median fluorescence intensity (MFI) of CD160 staining in Pmel T cells modified with exogenous CD160 is shown compared to the control.

[0031] Figure 2A The effects of exogenous CD160 expression on granzyme A and perforin levels in T cells were shown, as determined by intracellular staining and FACS analysis. Figure 2BThe effects of exogenous CD160 expression on INF-γ and TNFα levels in T cells were shown, as determined by intracellular staining and FACS analysis. Figure 2C The study presents an analysis of the cytolytic activity of Pmel T cells infected with either a control virus or a CD160 virus when co-cultured with B16F0 melanoma tumor cells.

[0032] Figure 3A The effects on tumor size in mice carrying B16F0 were shown by administration of either control Pmel T cells (T cells specific to the tumor antigen in B16F0) or 0.1, 0.2, 0.3, or 0.4 million CD160-modified Pmel T cells. Figure 3B The effects on tumor size in mice carrying B16F0 were shown by administration of control Pmel T cells, CD160-modified splenic T cells (non-specific to the B16F0 antigen), and CD160-modified Pmel T cells.

[0033] Figure 4A The relative changes in tumor size in mice carrying B16F0 after pretreatment with cyclophosphamide (CYP) and (a) control Pmel T cells; or (b) CD160-modified Pmel T cells are shown. Figure 4B Growth curves and selected tumor images of representative mice treated with CD160-modified Pmel T cells at different time points are shown. Figure 4C Kaplan-Meier survival analysis of mice carrying B16F0 melanoma, pretreated with CYP and administered (a) control Pmel T cells; or (b) CD160-modified Pmel T cells.

[0034] Figure 5A The relative changes in tumor size in mice carrying B16F0 were shown by treatment with CYP and administration of (a) PBS; (b) 0.15 million control Pmel T cells; or (c) 0.15 million control CD160-modified Pmel T cells. Figure 5B The relative changes in tumor size in mice carrying B16F0 were shown after treatment with CYP and administration of (a) PBS; (b) 0.3 million control Pmel T cells; or (c) 0.3 million control CD160-modified Pmel T cells. Figure 5C Kaplan-Meier survival analysis of mice carrying B16F0 melanoma, pretreated with CYP and administered with (a) PBS; (b) control Pmel T cells; (c) 0.15 million control CD160-modified Pmel T cells; or (d) 0.3 million control CD160-modified Pmel T cells.

[0035] Figure 6A The effects of treatment on mean tumor size in mice carrying metastatic B16F10, either untreated or pretreated with CYP and administered with (a) PBS; (b) control Pmel T cells; or (c) CD160-modified Pmel T cells, are shown. Figure 6B The effects of treatment on various tumors in mice carrying metastatic B16F10 were shown in either untreated or CYP-pretreated mice and (a) PBS; (b) control Pmel T cells; or (c) CD160-modified Pmel T cells.

[0036] Figure 7A The relative changes in tumor size in mice carrying B16F10 after treatment with CYP and administration of (a) control Pmel T cells; or (b) CD160-modified Pmel T cells are shown. Figure 7B Kaplan-Meier survival analysis of mice carrying B16F10 melanoma, pretreated with CYP and administered with (a) PBS; (b) control Pmel T cells; or (c) CD160-modified Pmel T cells, is shown.

[0037] Figure 8A -C shows the relative changes in tumor size in mice carrying B16F0 after treatment with CYP and PBS, mCD160-modified Pmel T cells, or Pmel T cells modified with one of the CD160 chimeras as elucidated. Figure 8A GEM 124, 125; Figure 8B GEM 126, 127; and Figure 8C (GEM 123, 128).

[0038] Figure 9A This is a schematic diagram showing, from left to right, GPI-anchored mCD160, GPI-anchored hCD160 isoforms, transmembrane hCD160 isoforms, and transmembrane hCD160 isoforms containing intracellular domains. Figure 9B The nucleotide sequences and degree of conservation between mouse and human CD160 isotypes were shown.

[0039] Figure 10A The relative changes in tumor size in mice carrying B16F0 were shown after treatment with CYP and (a) control Pmel T cells (VECTOR); or (b) Pmel T cells modified with exogenous GPI-anchored mCD160, GPI-anchored hCD160 isotype, transmembrane hCD160 isotype, or transmembrane hCD160 isotype containing intracellular domains. Figure 10BKaplan-Meier survival analysis is shown for mice carrying B16F0 melanoma treated with CYP and (a) control Pmel T cells (VECTOR); or (b) Pmel T cells modified with exogenous GPI-anchored hCD160 isotype, transmembrane hCD160 isotype, or transmembrane hCD160 isotype containing intracellular domains.

[0040] Figure 11A The study presents an analysis of the cytolytic activity of tumor-infiltrating T cells (TILs) extracted from Lewis lung cancer (LLC) infected with either a control virus or mCD160 virus when co-cultured with LLC cells. Figure 11B This diagram shows a representative in vivo experimental illustration of the ability of LLC TILs modified with exogenous CD160 or CD160 chimera (GEM 124) to control LLC tumors. Figure 11C Kaplan-Meier survival analysis is shown for mice carrying LLC that were left untreated or pretreated with CYP and (a) PBS; (b) control TIL; (c) mCD160-modified TIL; or (d) TIL modified with CD160 chimera GEM124.

[0041] Figure 12A A representative schematic diagram of CD19-CAR-T cells that overexpress human CD160 or its variants, such as CD160TC, is shown. Figure 12B The lentiviral vector configuration is shown, which is designed to overexpress human CD160 or a variant thereof together with a CAR that recognizes tumor-specific antigens, such as CD19.

[0042] Figure 13A The proliferation of CD19-CAR-T cells overexpressing huCD160TC and CD19-CAR-T cells not overexpressing CD160 (wild-type CD19-CAR-T) was shown. Figure 13B The study demonstrated the viability of CD19-CAR-T cells overexpressing huCD160TC compared to wild-type CD19-CAR-T cells after 2 weeks of culture. Figure 13C The study presents an analysis of the cytolytic activity of CD19-CAR-T cells overexpressing huCD160TC and wild-type CD19-CAR-T cells at various effector-to-target (E:T) ratios. Figure 13D The study showed that CD19-CAR-T cells overexpressing huCD160TC produced IFN-g in comparison to wild-type CD19-CAR-T cells. Figure 13EThe trend of tumor size in mice carrying Nalm6 tumors was shown by administration of: (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 Kaplan-Meier survival analysis of mice carrying Ramos tumors is shown, administered with (a) CAR-T cell-free (--); (b) wild-type CD19-CAR-T cells or (c) CD19-CAR-T cells (CD160TC) that overexpress huCD160TC.

[0043] Figure 14A A representative schematic diagram of NY-ESO-1 specific TCR-T cells that overexpress human CD160 or its variants, such as huCD160TC, is shown. Figure 14B The lentiviral vector configuration is shown, which is designed to overexpress human CD160 or a variant thereof together with a TCR that recognizes tumor-specific antigens, such as the NY-ESO-1-specific TCR.

[0044] Figure 15A FACS analysis showing NY-ESO-1-specific 1G4-TCR levels in wild-type 1G4-TCR-T cells and 1G4-TCR-T cells expressing huCD160TC, as determined by Tetramer analysis, is presented. Figure 15B The proliferation of 1G4-TCR-T cells overexpressing huCD160TC and 1G4-TCR-T cells not overexpressing CD160 (wild-type 1G4-TCR-T) was shown. Figure 15C The percentage of stem cells / memory T cells is shown in wild-type 1G4-TCR-T cells and 1G4-TCR-T cells expressing huCD160TC. Figure 15D The study presents an analysis of the cytolytic activity of 1G4-TCR-T cells overexpressing huCD160TC and wild-type 1G4-TCR-T cells at various effector-to-target (E:T) ratios. Figure 15E The study showed the production of IFN-g in 1G4-TCR-T cells that overexpress huCD160TC compared to wild-type 1G4-TCR-T cells. Figure 15F The tumor size in mice carrying A375 melanoma was shown when administered with: (a) no TCR-T cells; (b) control 1G4-TCR-T cells; or (c) 1G4-TCR-T cells overexpressing huCD160TC.

[0045] Figure 16This diagram illustrates an experiment using autologous tumor-infiltrating leukocytes (TILs) in a patient-derived xenograft (PDX) mouse tumor model. Tumor tissue from resected cancer patients with various cancers, such as lung, esophageal, colon, gastric, or pancreatic cancer, was implanted into NSG immunodeficient mice to generate PDX models with human tumors. Autologous tumor-infiltrating leukocytes (TILs) were extracted from the resected tumors for CD160 modification and subsequent functional testing of autologous human tumors in the PDX models.

[0046] Figure 17A This diagram shows a representative antitumor TIL with overexpression of human CD160 or its variants, such as huCD160TC. Figure 17B The lentiviral vector configuration for overexpressing human CD160 or its variants is shown, with GFP as a co-expressed reporter gene.

[0047] Figure 18A The cytolytic activity analysis of tumor-specific TILs (CD160TC) overexpressing huCD160TC in various effector-to-target (E:T) ratios is shown. Figure 18B The tumor size is shown in mice carrying autologous esophageal tumors treated with: (a) a control TIL that does not overexpress CD160 (none) or (c) a TIL that overexpresses huCD160TC. GEM indicates the genetically enhanced modifier expressed by the corresponding TIL. Invention Details

[0049] This application provides methods and compositions for modulating the immunostimulatory activity of CD160. This application is based on the surprising discovery that CD160, previously thought to primarily function as an inhibitory checkpoint molecule for T cells, is responsible for stimulating immune responses in antigen-specific immune cells, such as T cells.

[0050] We have demonstrated that tumor-specific T cells can be reprogrammed with CD160 to control and eliminate established solid tumors in immunocompetent mice. Moreover, CD160-programmed T cells have also shown remarkable efficacy in controlling metastatic melanoma and lung cancer in these highly difficult-to-treat mouse models. Ectopic expression of CD160 enhances the function of tumor-specific T cells carrying a TCR that specifically recognizes GP100 and polyclonal lung cancer tumor-infiltrating T cells (“TILs”) that recognize multiple antigens. CD160-modified tumor-specific T cells provide effective control of solid tumors from diverse tissue origins, regardless of their metastatic nature. Furthermore, by creating CD160 chimeras with TCRs and co-stimulatory signaling domains, we have shown that CD28 co-stimulatory signaling synergizes with CD160 and further enhances the function of antigen-specific T cells.

[0051] Importantly, human and mouse CD160 possesses a conserved function in enhancing the role of antigen-specific T cells in tumor control and elimination in vivo, suggesting that CD160 can control highly conserved pathways 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 potent agents for controlling and eliminating cancer, including established solid tumors. Furthermore, the findings strongly suggest that CD160-based reprogramming of immune cells (e.g., T cells) could be broadly applicable to all tumor-targeting immune cells (e.g., T cells) and tumors of diverse tissue origins.

[0052] The findings discussed above further suggest that CD160 can be an important target for the extrinsic regulation that promotes or inhibits antigen-specific T cell function. For example, endogenous CD160 in antigen-specific immune cells can be targeted to promote antigen-specific T cell activity and activate inflammatory responses, such as against viral and bacterial infections. Conversely, CD160 can be targeted to inhibit antigen-specific T cell activity during unintended immune responses, such as in inflammation and autoimmune diseases. Given the important function of CD160 in antigen-specific T cells, CD160 expression levels may be associated with the effective and functional status of antigen-specific T cells at inflammatory sites, including tumors or inflammatory tissues. Higher CD160 expression in those cells may indicate an activated state of antigen-specific T cells, while low levels or absence of CD160 expression may indicate an inactivated state. Therefore, CD160 can be used as a biomarker to predict the functional status of antigen-specific T cells and the efficacy of immunotherapies.

[0053] Therefore, in one aspect, an antigen-specific immune cell (e.g., a T cell) modified with an exogenous CD160 protein (e.g., on its surface) is provided, wherein the exogenous CD160 protein leads to upregulation of the modified antigen-specific immune cell compared to a precursor antigen-specific immune cell that does not contain the exogenous CD160 protein. In some embodiments, the exogenous CD160 protein binds to the cell membrane of the modified antigen-specific immune cell via a GPI linker. In some embodiments, the exogenous CD160 protein is a transmembrane protein including a transmembrane domain. In some embodiments, the exogenous CD160 protein includes a transmembrane domain derived from a co-stimulatory molecule and an intracellular signal transduction domain. In some embodiments, the exogenous CD160 protein binds to the modified antigen-specific immune cell via an immune cell-binding portion, such as an antibody that recognizes and activates T cell surface molecules. In some embodiments, the modified antigen-specific immune cell further includes a functional exogenous receptor, such as a modified T-cell receptor, an engineered T-cell receptor, or a chimeric antigen receptor (CAR). A method for generating the modified antigen-specific immune cell described above is also provided.

[0054] In another aspect, methods for modulating the immunostimulatory activity of CD160 in antigen-specific immune cells are provided, for example, for treating immunological diseases such as autoimmune diseases 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 them with an agent that activates (e.g., increases) CD160 activity (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 them with an agent that inhibits (e.g., downregulates) CD160 activity (e.g., an antagonist anti-CD160 antibody).

[0055] It also provides compositions (such as pharmaceutical compositions) including modified antigen-specific immune cells, kits and articles thereof, and methods for treating cancer using the modified antigen-specific immune cells described herein.

[0056] I. Definition

[0057] As used herein, “treatment” is a method for obtaining beneficial or desired results, including clinical outcomes. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease; reducing the severity of the disease; stabilizing the disease (e.g., preventing or delaying the worsening of the disease); preventing or delaying the spread of the disease (e.g., metastasis); preventing or delaying the recurrence of the disease; delaying or slowing the progression of the disease; alleviating the disease state; providing remission of the disease (partial or complete); reducing the dosage of one or more other medications required to treat the disease; delaying the progression of the disease; improving quality of life; and / or prolonging survival. “Treatment” also encompasses reducing the pathological outcomes of the disease. The methods of this invention are contemplated in relation to any one or more of these therapeutic aspects.

[0058] The term "prevention" and similar words such as "preventive" refer to methods used to prevent, suppress, or reduce the likelihood of a disease or condition, such as a recurrence of cancer. It also refers to delaying the recurrence of a disease or condition or the recurrence of its symptoms. As used herein, "prevention" and similar words also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition before it recurs.

[0059] As used in this article, “delaying” cancer development means postponing, hindering, slowing, delaying, stabilizing, and / or slowing the progression of the disease. This delay can have varying durations, depending on the history of the disease and / or the individual to be treated. Methods of “delaying” cancer development involve reducing the probability of disease development and / or reducing the severity of the disease within a given timeframe compared to not using these methods. Such comparisons are typically based on clinical studies using statistically significant numbers of individuals. Cancer development can be detectable using standard methods, including but not limited to computed tomography (CAT) scans, magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Development can also refer to cancer progression that may initially be undetectable, including occurrence, recurrence, and onset.

[0060] As used herein, the term "effective amount" refers to an amount of reagent or combination of reagents sufficient to treat a specific disorder, condition, or disease, such as alleviating, mitigating, reducing, and / or delaying one or more of its symptoms. In the context of cancer, an effective amount includes amounts sufficient to cause tumor shrinkage and / or reduce the rate of tumor growth (e.g., inhibit tumor growth) or prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay disease progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered once or multiple times. An effective amount of a drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) to some extent inhibit, delay, slow, and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., to some extent slow and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors and / or (vii) alleviate one or more cancer-related symptoms to some extent.

[0061] As used herein, "individual" or "subject" refers to a mammal, including but not limited to humans, bovines, horses, felines, canines, rodents, or primates. In some embodiments, the individual is a human.

[0062] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally present in cells containing nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0063] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell that has been introduced into it. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0064] As used herein, the terms “transfection,” “transformation,” or “transduction” refer to the process of transferring or introducing a heterologous nucleic acid into a host cell. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with a heterologous nucleic acid. These cells include primary subject cells and their progeny.

[0065] The "percentage (%) amino acid sequence identity" or "homology" relative to the polypeptide sequence identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the compared polypeptide, after taking into account any conserved substitutions as part of the sequence identity alignment sequence. For example, alignments for determining the percentage of amino acid sequence identity can be performed in various ways within the scope of the art using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine suitable parameters for measuring alignments, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this paper, the amino acid sequence identity value was 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).

[0066] As used herein, “antigen-specific immune cells” are immune cells that specifically recognize antigens on target cells via natural and / or engineered antigen recognition receptors. Immune cells include, but are not limited to, αβT cells, γδT cells, natural killer (NK) cells, natural killer T cells (NK-T cells), iNK-T cells, NK-T-like cells, and macrophages with or without engineered antigen recognition. Antigen-specific immune cells can be polyclonal or monoclonal. For example, in some embodiments, “antigen-specific immune cells” are tumor-infiltrating lymphocytes (TILs) that can be isolated from resected tumors; or neoantigen-specific T cells that can be isolated using their respective antigen-binding tetramers; or dendritic cell-activated T cells that are generated by co-culturing and activating peripheral blood T cells with dendritic cells loaded with tumor antigens; or other immune cells, such as γδT cells, NK cells, NK-T cells, iNK-T cells, NK-T-like cells, and macrophages expressing CAR or TCR-like antigen receptors.

[0067] As used in this article, “antigen-specific receptor” refers to a natural or engineered T-cell receptor (TCR) or an engineered antigen receptor, such as a chimeric antigen receptor (CAR).

[0068] As used herein, "T-cell receptor" or "TCR" refers to an endogenous or modified T-cell receptor that includes an extracellular antigen-binding domain that binds to a specific antigenic peptide bound to an MHC molecule. In some embodiments, a TCR includes a TCRα polypeptide chain and a TCRβ polypeptide chain. In some embodiments, a TCR specifically binds to a tumor antigen. "TCR-T" refers to a T cell expressing a recombinant TCR.

[0069] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that specifically transplants one or more antigens onto cells, such as αβT cells, γδT cells, NK cells, and macrophages. CAR is also known as "artificial T-cell receptor," "chimeric T-cell receptor," or "chimeric immune receptor." In some embodiments, CAR includes an extracellular variable domain of an antibody specific to a tumor antigen and an intracellular signaling domain of a T cell or other receptor, such as one or more co-stimulatory domains. "CAR-T" refers to T cells expressing CAR.

[0070] The term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. The term "antibody" includes, but is not limited to, fragments capable of binding antigens, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. The term "antibody" includes conventional tetrachain antibodies and single-domain antibodies, such as antibodies containing only the heavy chain or fragments thereof, such as V... H H.

[0071] As used herein, the terms “binding,” “specific binding,” or “specific to…” refer to a measurable and reproducible interaction, such as the binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous group 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 that target with greater affinity, affinity, ease, and / or longer duration than it binds to other targets. In one embodiment, the degree of antibody binding to an irrelevant target is less than about 10% of the antibody binding to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, antibodies that specifically bind to a target have a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In some embodiments, the 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 is not required to be, exclusive binding.

[0072] It should be understood that the embodiments of the present invention described herein include "consisting of embodiments" and / or "consisting substantially of embodiments".

[0073] The “about” values ​​or parameters mentioned in this article include (and describe) variations with respect to that value or parameter itself. For example, the description of “about X” includes a description of “X”.

[0074] As used in this article, mentioning "not" a value or parameter generally means and describes "other than" a value or parameter. For example, "this method is not used to treat type X cancer" means that this method is used to treat cancer types other than X.

[0075] The term “about XY” used in this article has the same meaning as “about X to about Y”.

[0076] As used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators.

[0077] II. Antigen-specific immune cells expressing exogenous CD160 protein

[0078] One aspect of the invention provides antigen-specific immune cells comprising (e.g., on their surface) a modified exogenous CD160 protein, wherein the exogenous CD160 protein leads to upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the exogenous CD160 protein. In some embodiments, upregulation includes increased cytolytic lymphocyte (CTL) activity. In some embodiments, upregulation includes enhanced tumor-killing activity in an immunocompetent host. In some embodiments, upregulation includes enhanced T cell- and / or NK cell-mediated killing. In some embodiments, upregulation includes increased expression of granzyme A and / or perforin. In some embodiments, upregulation includes enhanced inflammatory responses. In some embodiments, upregulation includes enhanced expression and / or secretion of inflammatory cytokines. In some embodiments, the inflammatory cytokines include IFN-γ and / or TNF-α. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NO: 1-4, or a variant thereof having at least about 80% identity with any one of SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90% identity with any of SEQ ID NO:1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO:1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (e.g., but not limited to dimers, trimers, tetramers, pentamers, or hexamers). 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, APC-activated anti-tumor T cells, 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 APC-activated anti-tumor T cells. In some embodiments, the APC-activated anti-tumor T cells are DC-activated anti-tumor T cells. 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, γδT cells, and macrophages. In some embodiments, the precursor antigen-specific immune cells are isolated from an individual tumor. 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 include 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 a further embodiment, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the modified immune cells are multiple immune cells specific to the same epitope. Non-limiting examples include multiple T cells, each including the same functional exogenous receptor (e.g., CAR). In some embodiments, the modified immune cells are multiple immune cells, each specific to one of multiple non-identical epitopes (e.g., locally overlapping or completely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0079] In some embodiments, antigen-specific immune cells modified with an exogenous CD160 protein on their surface are provided, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared with precursor antigen-specific immune cells that do not contain the exogenous CD160 protein, and wherein the exogenous CD160 protein is membrane-bound.

[0080] In some embodiments, antigen-specific immune cells modified with a foreign CD160 protein on their surface are provided, wherein the foreign CD160 protein leads to upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the foreign CD160 protein, wherein the foreign CD160 protein is membrane-bound, and wherein the foreign CD160 protein is bound to the membrane via a glycophosphatidylinositol (GPI) linker. In some embodiments, the foreign CD160 protein includes a GPI-anchored peptide sequence.

[0081] In some embodiments, antigen-specific immune cells modified with a foreign CD160 protein on their surface are provided, wherein the foreign CD160 protein leads to upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the foreign CD160 protein, wherein the foreign CD160 protein is membrane-bound, and wherein the foreign CD160 protein includes a transmembrane domain. In some embodiments, the transmembrane domain is derived from molecules 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) α subunit, TCRβ subunit, or TCRζ subunit. In some embodiments, the transmembrane domain is derived from molecules selected from the group consisting of: CD28, 4-1BB, CD80, CD152, and PD-1.

[0082] In some embodiments, antigen-specific immune cells modified with exogenous CD160 protein on their surface are provided, wherein the exogenous CD160 protein leads to upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain exogenous CD160 protein, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein includes a transmembrane domain and an intracellular domain. In some embodiments, the transmembrane domain is derived from molecules 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) α subunit, TCRβ subunit, or TCRζ subunit. In some embodiments, the transmembrane domain is derived from molecules 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 includes an intracellular signaling domain derived from a signaling subunit of the TCR complex. In some embodiments, the signaling subunit of the TCR complex is selected from the group consisting of CD3γ, CD3δ, and CD3ε. In some embodiments, the intracellular domain includes one or more signaling domains derived from T cell stimulating molecules. 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 includes a combination of two signaling domains selected from the group consisting of OX40, CD27, CD28, CD80, and CD258. [[CD160-TM+co-stim; covering 5-7+]]

[0083] In some embodiments, antigen-specific immune cells modified with exogenous CD160 protein on their surface are provided, wherein the exogenous CD160 protein leads to upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain exogenous CD160 protein, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein includes a transmembrane domain and an intracellular domain, and wherein the intracellular domain includes one or more co-stimulatory signaling domains. In some embodiments, the intracellular domain includes any 1, 2, 3, 4, 5, 6, 7, 8 or more co-stimulatory signaling domains. In some embodiments, the intracellular domain contains no more than 1, 2, 3, 4 or 5 co-stimulatory signaling domains. In some embodiments, the intracellular domain does not include a CD3ζ signaling domain or a combination of 4-1BB and CD3ζ domains. In some embodiments, the co-stimulatory signal transduction domain is derived from a co-stimulatory 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, CD83, and combinations thereof. In some embodiments, the intracellular domain includes a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, or both. In some embodiments, the exogenous CD160 protein, from its N-terminus to its C-terminus, includes: an extracellular CD160 domain, a transmembrane domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain. In some embodiments, the exogenous CD160 protein, from its N-terminus to its C-terminus, includes: an extracellular CD160 domain, a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain is adjacent to the transmembrane domain. In some embodiments, the CD28 co-stimulatory domain is adjacent to the C-terminus of the transmembrane domain. In some embodiments, the intracellular domain includes a primary signaling domain. In some embodiments, the primary signaling domain includes a CD3ζ domain. In other embodiments, the intracellular domain does not include a primary signaling domain. In other embodiments, the intracellular domain does not include a CD3ζ domain or a combination of 4-1BB and CD3ζ domains.

[0084] In some embodiments, an antigen-specific immune cell modified with a foreign CD160 protein on its surface is provided, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cell compared to a precursor antigen-specific immune cell that does not contain the foreign CD160 protein, wherein the foreign CD160 protein is membrane-bound, and wherein the foreign CD160 protein binds to the modified antigen-specific immune cell via an immune cell-binding portion. In some embodiments, the immune cell-binding portion binds to surface molecules of the immune cell. In some embodiments, the immune cell-binding portion includes an antibody that recognizes a T-cell surface molecule. In some embodiments, the antibody may be a full-length antibody or an antibody fragment, such as scFv, Fv, Fab, (Fab')2, a single-domain antibody (sdAb), or V. H H domain. Non-limiting examples include anti-CD3ε antibodies that recognize the TCR and / or activate TCR signaling. In some embodiments, the immune cell binding portion includes a ligand that binds to an associated T cell surface receptor. Non-limiting examples include tumor-specific peptide MHC complexes that recognize the TCR and IL-2.

[0085] In some embodiments of antigen-specific immune cells according to any modifications described herein, the exogenous CD160 protein comprises the amino acid sequence of any of SEQ ID NO: 1-4, or a variant thereof having at least about 80% identity with any of SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence having at least about 90% identity with any of SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence having about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (e.g., 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, APC-activated anti-tumor T cells, 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 APC-activated anti-tumor T cells. In some embodiments, the APC-activated anti-tumor T cells are DC-activated anti-tumor T cells. 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 an individual tumor. 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 include 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 a further embodiment, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the modified immune cells are multiple immune cells specific to the same epitope. Non-limiting examples include multiple T cells, each including the same functional exogenous receptor (e.g., CAR). In some embodiments, the modified immune cells are multiple immune cells, each specific to one of multiple non-identical epitopes (e.g., locally overlapping or completely different epitopes).Unrestricted examples include various polyclonal immune cells, such as polyclonal TILs.

[0086] 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 at least partially conferred by a functional exogenous receptor, such as, but not limited to, CAR and TCR. In some embodiments, the modified antigen-specific immune cells target tumor-associated antigens, mutated oncogenic antigens, and random somatic antigens, as well as 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 one or more modifications of TCR-T cells, CAR-T cells, TILs, or endogenous antigen-specific T cells. Examples of human and mouse TCR-T cells, CAR-T cells, TILs, or endogenous antigen-specific T cells have been 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 spectrum of antigens. In some embodiments, the modified antigen-specific immune cells target one or more of the antigens listed in Table 1.

[0087] Table 1: Exemplary List of Tumor Antigens and Related Cancer Symptoms

[0088]

[0089]

[0090]

[0091] In some embodiments, a pharmaceutical composition comprising any of the modified antigen-specific immune cells described herein is provided. In some embodiments, a method for generating any of the modified antigen-specific immune cells described herein is provided.

[0092] This includes libraries of antigen-specific immune cells modified with exogenous CD160 and methods for screening antigen-specific immune cells.

[0093] In some embodiments, libraries of antigen-specific immune cells (e.g., T cells) each having a functional exogenous receptor that recognizes different antigens (e.g., tumor antigens or tumor-associated antigens) are provided. In one aspect, a library of antigen-specific immune cells (e.g., T cells) each having a functional exogenous receptor that recognizes different tumors or tumor-associated antigens is provided, wherein each antigen-specific immune cell in the library further includes an exogenous CD160 protein on its surface, wherein the exogenous CD160 protein leads to upregulation of the modified immune cells compared to precursor immune cells that do not contain the exogenous CD160 protein.

[0094] In one aspect, a library of polyclonal immune cells (such as TILs) is provided, wherein each immune cell in the polyclonal composition is specific to one of a variety of non-identical epitopes (such as locally overlapping or completely different epitopes), wherein the exogenous CD160 protein leads to upregulation of the modified immune cells compared to precursor immune cells that do not contain the exogenous CD160 protein.

[0095] In some embodiments, a method is provided for screening immune cells that include functional exogenous receptors specific to a test antigen. This includes contacting a test antigen with a library of antigen-specific immune cells (e.g., T cells) each having a functional exogenous receptor that recognizes a different antigen (e.g., a tumor or tumor-associated antigen). Each antigen-specific immune cell in the library further includes an exogenous CD160 protein on its surface, wherein the exogenous CD160 protein leads to upregulation of the modified 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 (e.g., a desired functional exogenous receptor) can be identified by contacting the test antigen with a library of antigen-specific immune cells (e.g., T cells) each having a functional exogenous receptor that recognizes a different antigen, followed by analysis of 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, an ELISA analysis of any cytokine secretion (e.g., IFN-γ, TNF-α, and / or IL-2).

[0096] In some embodiments, a method for screening immune cells specific to a test antigen is provided, comprising contacting a test antigen with a library of polyclonal immune cells (e.g., TILs), wherein each immune cell in the polyclonal composition is specific for one of a plurality of non-identical epitopes (e.g., locally overlapping, or completely different epitopes), wherein the exogenous CD160 protein leads to upregulation of the modified 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 (e.g., TILs), wherein each cell is specific for one of a plurality of non-identical epitopes, followed by analysis of 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).

[0097] In some embodiments of the methods described above, the test antigen comprises one or more immunogenic epitopes. In some embodiments, the test antigen is derived from lysates, such as tumor lysates. In some embodiments, a library of antigen-specific immune cells modified with exogenous CD160 protein on its surface is generated by a process comprising contacting a plurality of precursor antigen-specific immune cells with exogenous CD160 protein or nucleic acid encoding exogenous CD160 protein to generate a library of modified antigen-specific immune cells. In some embodiments, the CD160 protein comprises any amino acid sequence of SEQ ID NO:1-4, or a variant thereof having at least about 80% identification identity with SEQ ID NO: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, APC-activated anti-tumor T cells, 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 APC-activated anti-tumor T cells. In some embodiments, the APC-activated anti-tumor T cells are DC-activated anti-tumor T cells. 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, γδT cells, and macrophages.

[0098] CD160 protein

[0099] The modified antigen-specific immune cells described herein express exogenous CD160 protein, wherein the exogenous CD160 protein leads to upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain exogenous CD160 protein. This application also provides exogenous CD160 proteins and compositions thereof. Table 2 shows the sequences of exemplary exogenous CD160 proteins.

[0100]

[0101] In some embodiments, an exogenous CD160 protein comprising a naturally occurring CD160 polypeptide or a fragment thereof is provided, wherein the exogenous CD160 protein leads to upregulation of modified 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 is substantially composed of a naturally occurring CD160 protein or a fragment thereof. In some embodiments, the exogenous CD160 protein includes an Ig-like V-type domain of a naturally occurring CD160 protein or a fragment thereof. In some embodiments, the exogenous CD160 protein includes a cysteine-enriched 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 NO: 1-3.

[0102] In some embodiments, the exogenous CD160 protein on the cell surface is in monomeric form. In some embodiments, the exogenous CD160 protein on the cell surface is in polymeric form. In some embodiments, the exogenous CD160 protein on the cell surface is in polymeric form, such as a dimer, trimer, tetramer, pentamer, or hexamer. In some embodiments, the polymeric form comprises one or more exogenous CD160 proteins and one or more naturally occurring CD160 proteins. In some embodiments, the polymeric form comprises one or more exogenous CD160 proteins and one or more endogenous CD160 proteins. In some embodiments, the CD160 protein polymeric forms on the cell surface are covalently linked. In some embodiments, the CD160 protein polymeric forms on the cell surface are disulfidated linked. 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 NO: 1-3, further comprising one or more mutations of 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 of cysteine ​​residues Cys29, Cys47, Cys64, Cys71, Cys115, Cys116, or any combination thereof. In some embodiments, the exogenous CD160 protein carrying one or more of the above mutations is unable to form multimers.

[0103] In some embodiments, the exogenous CD160 protein exhibits the same or substantially the same binding affinity for MHC-I as the naturally occurring CD160 protein. In some embodiments, the exogenous CD160 protein exhibits an increased binding affinity for MHC-I compared to the naturally occurring CD160 protein. In some embodiments, the MHC-I binding affinity of the exogenous CD160 protein is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of the naturally occurring CD160 protein. In some embodiments, the MHC-I binding affinity of the exogenous CD160 protein is about 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or 100 times higher than that of the naturally occurring CD160 protein. In some embodiments, the exogenous CD160 protein exhibits a reduced binding affinity for MHC-I compared to the naturally occurring CD160 protein. In some embodiments, the MHC-I binding affinity of the exogenous CD160 protein is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than that of the naturally occurring CD160 protein. In some embodiments, the MHC-I binding affinity of the exogenous CD160 protein is about 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or 100 times lower than that of the naturally occurring CD160 protein.

[0104] In some embodiments, the exogenous CD160 protein exhibits the same or substantially the same binding affinity for herpesvirus invasion mediators (HVEMs) as the naturally occurring CD160 protein. In some embodiments, the exogenous CD160 protein exhibits increased binding affinity for HVEMs compared to the naturally occurring CD160 protein. In some embodiments, the HVEM binding affinity of the exogenous CD160 protein is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 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, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or 100 times higher than that of the naturally occurring CD160 protein. In some embodiments, the exogenous CD160 protein exhibits a 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 approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than that of the naturally occurring CD160 protein. In some embodiments, the HVEM binding affinity of the exogenous CD160 protein is approximately 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or 100 times lower than that of the naturally occurring CD160 protein.

[0105] 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 similar binding affinity to HVEM as BTLA. In some embodiments, the exogenous CD160 protein exhibits a higher binding affinity to HVEM compared to BTLA. In some embodiments, the exogenous CD160 protein exhibits a lower binding affinity to HVEM compared to BTLA. In some embodiments, the exogenous CD160 protein exhibits the same or substantially the same binding affinity to HVEM as BTLA. In some embodiments, the exogenous CD160 protein exhibits a higher binding affinity to HVEM compared to BTLA. In some embodiments, the exogenous CD160 protein exhibits a lower binding affinity to HVEM compared to BTLA. In some embodiments, the exogenous CD160 protein exhibits the same or substantially the same dissociation rate from HVEM 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.

[0106] In some embodiments, the exogenous CD160 protein leads to upregulation of modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the exogenous CD160 protein, and wherein the CD160 protein comprises the amino acid sequence of any one of SEQ ID NO: 1-4, or a variant thereof having at least about 90% identity with any one of SEQ ID NO: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 80% sequence identity with any one of SEQ ID NO: 1-4, such as at least about 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 95% sequence identity with any one of SEQ ID NO: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having at least about 99% sequence identity with any one of SEQ ID NO: 1-4. In some embodiments, the CD160 protein comprises an amino acid sequence having about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO:1-4.

[0107] In some embodiments, the exogenous CD160 protein is derived from the CD160 protein of mammals. In some embodiments, the exogenous CD160 protein is derived from the CD160 protein of mice, dogs, cats, horses, rats, goats, or rabbits. In some embodiments, the exogenous CD160 protein is derived from the CD160 protein of humans.

[0108] In some embodiments, the exogenous CD160 protein comprises the full-length CD160 protein. In some embodiments, the exogenous CD160 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4. CD160 protein sequences are known in the art, including but not limited to sequences having UniProt (worldwide web.uniprot.org) accession numbers O95971 and O88875. The mRNA sequence encoding the CD160 protein is also known in the art, including but not limited to sequences having NCBI (worldwide web ncbi.nlm.nih.gov) accession numbers NM_007053.3, XM_005272929.3, and NM_001163497.1.

[0109] In some embodiments, the exogenous CD160 protein comprises any one of at least about 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more amino acids. In some embodiments, the exogenous CD160 protein comprises any one of no more than about 600, 550, 500, 450, 350, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50 or fewer amino acids. In some embodiments, the exogenous CD160 protein comprises any one of about 50-60, 50-75, 50-100, 50-150, 50-200, 50-250, 100-150, 100-200, 100-250, 150-250, 250-500, or 50-550 amino acids.

[0110] 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. The amino acid sequence variant of the CD160 protein can be prepared by introducing suitable modifications into the nucleotide sequence encoding the CD160 protein, or by peptide synthesis. Such modifications include, for example, deletions from the amino acid sequence of the CD160 protein, and / or insertions into the amino acid sequence of the CD160 protein and / or substitutions of residues within the amino acid sequence of the CD160 protein. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as pro-inflammatory activity.

[0111] In some embodiments, the exogenous CD160 protein comprises a naturally occurring CD160 protein or fragment thereof having one or more (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20 or more amino acid 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 fragment thereof having at least about 80% sequence identity with the sequence of a naturally occurring CD160 protein or fragment thereof, such as at least about 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0112] Conservative replacements are shown in Table 3 below.

[0113] Table 3: Conservative Replacement

[0114]

[0115]

[0116] Based on their typical side-chain properties, amino acids can be grouped into different categories:

[0117] a. Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0118] b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0119] c. Acids: Asp, Glu;

[0120] d. Alkaline: His, Lys, Arg;

[0121] e. Residues that affect chain orientation: Gly, Pro;

[0122] f. Aromatic tribes: Trp, Tyr, Phe.

[0123] Non-conservative permutations require swapping members of one of these categories with members of another category.

[0124] Those skilled in the art will recognize that any suitable method can be used to generate mutations in the gene of interest, including mutagenesis, polymerase chain reaction, homologous recombination, or any other genetic engineering technique known to those skilled in the art. Mutations may involve a single nucleotide (e.g., a point mutation, which involves the removal, addition, or substitution of a single nucleotide base within a DNA sequence), or may involve the insertion or deletion of a large number of nucleotides. Mutations can arise spontaneously from, for example, erroneous events in DNA replication fidelity, or be induced upon exposure to chemical or physical mutagens. Mutations can also be performed at specific sites using particular targeting methods well known to those skilled in the art.

[0125] As described by Cunningham and Wells (1989) Science, 244:1081-1085, a useful method for identifying residues or regions of peptides that can be targeted for mutagenesis is called "alanine scan mutagenesis." In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and substituted by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the upregulation of a peptide reagent (e.g., a CD160 variant) is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Optionally or additionally, the crystal structures of the CD160:MHC I complex or the CD160:HVEM complex can be determined to identify contact points between CD160 and MHC-1 or between CD160 and HVEM, respectively. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution to enhance or inhibit the function of CD160 in antigen-specific immune cells, depending on the disease indication. Variants can be filtered to determine whether they contain the desired properties.

[0126] Amino acid sequence insertions include amino and / or carboxyl terminus fusions of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues.

[0127] In some embodiments, the exogenous CD160 protein is secreted from modified antigen-specific immune cells. In some embodiments, the exogenous CD160 protein includes a signal peptide. The signal peptide (also known as a “lead sequence”) is typically inserted at the N-terminus of the protein immediately following the Met initiator. The signal peptide can be cleaved upon export of the exogenous CD160 protein from modified antigen-specific immune cells to form the mature protein. The signal peptide can be native or synthetic, and it can be heterologous or homologous to the protein to which it is linked. The selection of signal peptides is broad and available to those skilled in the art, including, for example, an 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. WO 2006 / 081430.

[0128] Functional exogenous receptors

[0129] Any of the antigen-specific immune cells modified as described above 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 includes an extracellular domain, a transmembrane domain, and an intracellular signaling domain that specifically bind to an antigen (e.g., a tumor antigen). In some embodiments, the intracellular signaling domain includes a primary intracellular signaling domain and / or a co-stimulatory domain. In some embodiments, the intracellular signaling domain includes the 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 operatively linked to a promoter (e.g., a constitutive or inducible promoter). In some embodiments, the protein is inserted into the cell membrane while the cell is traversed through a microfluidic system, such as a cell. Introducing a functional exogenous receptor into modified antigen-specific immune cells (see, for example, U.S. Patent Application Publication No. 20140287509). In some embodiments, the functional exogenous receptor is introduced into the modified immune cells via CRISPR-mediated gene editing. The functional exogenous receptor can enhance the function of the modified antigen-specific immune cells, for example, by targeting the modified antigen-specific immune cells, by signal transduction, and / or by enhancing the cytotoxicity of the modified antigen-specific immune cells. In some embodiments, the modified antigen-specific immune cells do not express the functional exogenous receptor, such as CAR or TCR.

[0130] In some implementations, the functional exogenous receptor includes 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 co-stimulatory domains.

[0131] In some embodiments, the functional exogenous receptor is a chimeric antigen receptor (CAR). Many chimeric antigen receptors are known in the art and are suitable for modifying antigen-specific immune cells according to the present invention. CARs specific to any cell surface marker can also be constructed by utilizing, for example, antigen-binding fragments of antibody molecules or variable domains of antibodies. Any method for generating CARs may be used herein. See, for example, US6,410,319, US7,446,191, US7,514,537, US9765342B2, WO 2002 / 077029, WO2015 / 142675, US2010 / 065818, US 2010 / 025177, US2007 / 059298, WO2017025038A1, and Berger C et al., J. Clinical Investigation 118:1 294-308 (2008), which are incorporated herein by reference. In some implementations, the modified antigen-specific immune cells are CAR-αβT cells, CAR-γδT cells, CAR-NK cells, or CAR-macrophages.

[0132] The CAR of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, including at least one targeting domain that specifically binds to at least one tumor antigen. In some embodiments, the intracellular signaling domain generates signals that promote the immune effector function of CAR-containing cells, such as CAR-T cells. "Immune effector function or immune effector response" refers, for example, the function or response of immune effector cells that enhances or promotes the immune attack of target cells. For example, an immune effector function or response may refer to the properties of T or NK cells that promote the killing of target cells or inhibit the growth or proliferation of target cells. Examples of immune effector functions in CAR-T cells include cytolytic activity (e.g., antibody-dependent cytotoxicity, or ADCC) and helper activities (e.g., cytokine secretion). In some embodiments, the CAR has an intracellular signaling domain with attenuated immune effector function. In some embodiments, compared to CARs having full-length and wild-type CD3ζ and optionally one or more co-stimulatory domains, the intracellular signaling domains of the CAR have no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of any immune effector function (such as cytolytic function against target cells). In some embodiments, the intracellular signaling domains generate signals that promote the proliferation and / or survival of CAR-containing cells. In some embodiments, the CAR includes one or more intracellular signaling domains selected from CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. The signaling domains of naturally occurring molecules may include the entire intracellular (i.e., cytoplasmic) portion of the molecule or a fragment or derivative thereof, or the entire natural intracellular signaling domain.

[0133] In some embodiments, the intracellular signaling domains of the CAR include primary intracellular signaling domains. A "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulatory manner to induce the function of an immune effector. In some embodiments, the primary intracellular signaling domain contains a signaling motif called an immune receptor tyrosine-based activation motif, or ITAM. In some embodiments, the primary intracellular signaling domain includes functional signaling domains of proteins selected from the group consisting of: CD3ζ, CD3γ, CD3δ, CD3ε, ordinary FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. In some embodiments, the primary intracellular signaling domain includes a nonfunctional or attenuated signaling domain of a protein selected from the group consisting of: CD3ζ, CD3γ, CD3δ, CD3ε, ordinary FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. The nonfunctional or attenuated signaling domain may be a mutant signaling domain having a point mutation, insertion, or deletion that attenuates or eliminates one or more immune effector functions (such as cytolytic or accessory activities, including antibody-dependent cytotoxicity (ADCC)). In some embodiments, the CAR includes a nonfunctional or attenuated CD3ζ (i.e., CD3ζ or CD3z) signaling domain. In some embodiments, the intracellular signaling domain does not include the primary intracellular signaling domain. Compared to CARs with the same construct but with wild-type primary intracellular signal transduction domains, attenuated primary intracellular signal transduction domains can induce no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of any of the immune effector functions (such as cytolytic function against target cells).

[0134] In some implementations, the intracellular signaling domains of a CAR include one or more (e.g., 1, 2, 3, or more) co-stimulatory domains. A “co-stimulatory domain” can be an intracellular portion of a co-stimulatory molecule. The term “co-stimulatory molecule” refers to a related binding partner on an immune cell (e.g., T cell) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response through the immune cell, such as, but not limited to, proliferation and survival. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response. Co-stimulatory molecules can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and NK cell activation receptors. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Other examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, 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.

[0135] In some embodiments, the CAR includes a single co-stimulatory domain. In some embodiments, the CAR includes two or more co-stimulatory domains. In some embodiments, the intracellular signaling domain includes a functional primary intracellular signaling domain and one or more co-stimulatory domains. In some embodiments, the CAR does not include a functional primary intracellular signaling domain (e.g., CD3ζ). In some embodiments, the CAR includes an intracellular signaling domain consisting of one or more co-stimulatory domains or substantially consisting of one or more co-stimulatory domains. In some embodiments, the CAR includes an intracellular signaling domain consisting of a non-functional or attenuated primary intracellular signaling domain (e.g., mutant CD3ζ) and one or more co-stimulatory domains, or substantially consisting of a non-functional or attenuated primary intracellular signaling domain (e.g., mutant CD3ζ) and one or more co-stimulatory domains. When the targeting domain binds to a tumor antigen, the CAR co-stimulatory domain can transduce signals that enhance the proliferation, survival, and differentiation of CAR-engineered immune cells (e.g., T cells) and inhibit activation-induced cell death. In some embodiments, one or more co-stimulatory 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 ligands that specifically bind to CD83.

[0136] In some embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain in the chimeric receptor of this application includes a co-stimulatory signaling domain derived from 4-1BB (i.e., CD137). In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of CD3ζ and a co-stimulatory signaling domain of 4-1BB.

[0137] In some embodiments, the intracellular signal transduction domain of the CAR includes a CD28 costimulatory signal transduction domain and a 4-1BB costimulatory signal transduction domain. In some embodiments, the intracellular signal transduction domain includes a CD3ζ cytoplasmic signal transduction domain, a CD28 costimulatory signal transduction domain, and a 4-1BB costimulatory signal transduction domain. In some embodiments, the intracellular signal transduction domain comprises a polypeptide that includes, from its N-terminus to its C-terminus, a CD28 costimulatory signal transduction domain, a 4-1BB costimulatory signal transduction domain, and a CD3ζ cytoplasmic signal transduction domain.

[0138] In some implementations, the target domain of the CAR is an antibody or antibody fragment, such as scFv, Fv, Fab, (Fab')2, a single-domain antibody (sdAb), or V. H 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 having 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 (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other clinically significant tumor antigens, and combinations thereof.

[0139] In some embodiments, the transmembrane domain of the CAR includes a transmembrane domain selected from the following transmembrane domains: α, β, or ζ chains of T-cell receptors, CD28, CD3ε, 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 (KLRF1), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7R a, 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 AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), 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 a CD4, CD3, CD8α, or CD28 transmembrane domain. In some embodiments, the transmembrane domain of the CAR includes a CD8α transmembrane domain.

[0140] In some embodiments, the extracellular domain is connected to the transmembrane domain via a hinge region. In one embodiment, the hinge region includes the hinge region of CD8α.

[0141] In some implementations, CAR includes a signal peptide, such as CD8αSP.

[0142] In some embodiments, the functional exogenous receptor is a modified T-cell receptor. In some embodiments, the engineered TCR is specific to the tumor antigen. 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 (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other clinically significant tumor antigens. In some embodiments, the tumor antigen is derived from intracellular proteins of tumor cells. Many TCRs specific to tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, p53 tumor suppressor antigen, TCRs for tumor antigens in melanoma (e.g., MARTI, gp 100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (HER2, NY-BR1, etc.). Any TCR known in the art may be used in this application. In some embodiments, the TCR has enhanced affinity for the tumor antigen. Exemplary TCRs and methods for introducing TCRs into immune cells have been described, for example, in US5830755 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.

[0143] The TCR receptor complex is an octameric complex formed by variable TCR receptor α and β chains (γ and δ chains in the case of γδT cells) with three dimer signal transduction modules: CD3δ / ε, CD3γ / ε, and CD247 (the CD3ζ chain of the T-cell surface glycoprotein) ζ / ζ or ζ / η. Ionizable residues in the transmembrane domains of each subunit form an interacting polar network that binds the complex together. The TCR complex has the function of activating the signal transduction cascade in T cells.

[0144] In some implementations, the modified antigen-specific immune cells express more than one functional exogenous receptor, such as any combination of CAR or TCR receptors.

[0145] In some embodiments, a functional exogenous receptor (such as a 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 elicit an immune response, particularly a T-cell-mediated immune response. The selection of the targeted antigen in this 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, muco-hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survival proteins and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelial protein.

[0146] In some implementations, tumor antigens include one or more cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can be used 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 prostate 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. Another group of target antigens is onco-fetal antigens, such as CEA. In B-cell lymphoma, tumor-specific individual genotype immunoglobulins constitute true tumor-specific immunoglobulin antigens, which are unique to the individual tumor. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates for target antigens in B-cell lymphoma.

[0147] In some implementations, tumor antigens are tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not appear on other cells in the body. TAA-associated antigens are not unique to tumor cells but are expressed on normal cells under conditions that do not induce an immunological tolerance state to the antigen. Expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0148] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER2 / neu; unique tumor antigens derived from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other major 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, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 242, 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, RCAS 1, SDCCAG16, TA-90 / Mac-2 binding proteins / cyclic protein C-related proteins, TAAL6, TAG72, TLP, and TPS.

[0149] Nucleic acid

[0150] In some embodiments, the modified antigen-specific immune cells described herein include one or more heterologous nucleic acid sequences encoding any of the exogenous CD160 proteins described herein and / or any of the functional exogenous receptors.

[0151] In some embodiments, an isolated nucleic acid is provided comprising a nucleic acid sequence encoding any of the exogenous CD160 proteins described herein. In some embodiments, an isolated nucleic acid is provided comprising a nucleic acid sequence encoding any 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.

[0152] Nucleic acid sequences encoding exogenous CD160 proteins and / or nucleic acids encoding functional exogenous receptors can be operatively linked to one or more regulatory sequences. Exemplary regulatory sequences controlling transcription and / or translation of the coding sequences are known in the art and may include, but are not limited to, promoters, appropriate initiation, regulation, and / or termination of transcription (e.g., polyA transcription termination sequences), mRNA delivery (e.g., nuclear localization signal sequences), processing (e.g., splicing signals), stabilization (e.g., introns and non-coding 5' and 3' sequences), translation (e.g., initiator Met, triplet leader sequences, IRES ribosome binding sites, signal peptides, etc.), and additional elements for introducing inserts into viral vectors. In some embodiments, the regulatory sequence is a promoter, transcription enhancer, and / or sequence that allows appropriate expression of exogenous CD160 proteins and / or functional exogenous receptors.

[0153] The term "regulatory sequence" or "control sequence" refers to a DNA sequence that influences the expression of a coding sequence to which it is operatively linked. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, regulatory sequences typically include promoters, ribosome binding sites, and terminators. In eukaryotes, regulatory sequences include promoters, terminators, and in some cases, enhancers, transactivators, or transcription factors.

[0154] The term "operably linked" refers to a parallel arrangement in which the components, as described, are in a relationship that allows them to function in their intended manner. "Operably linked" means a connection to a regulatory sequence of a coding sequence in such a way that the expression of the coding sequence is realized under conditions compatible with the regulatory sequence.

[0155] As used herein, a “promoter” or “promoter region” refers to a segment of DNA or RNA that controls transcription of DNA or RNA operatively linked to it. A promoter region includes specific sequences involved in RNA polymerase recognition, binding, and transcription initiation. Additionally, a promoter includes sequences that regulate the recognition, binding, and transcription initiation activities of RNA polymerases (i.e., the binding of one or more transcription factors). These sequences can be cis-acting or responsive to trans-acting factors. Depending on the nature of the regulation, a promoter can be constitutive or regulatory. Regulatory promoters can be inducible or environmentally responsive (e.g., responding to cues such as pH, anaerobic conditions, osmotic pressure, temperature, light, or cell density). Many such promoter sequences are known in the art. See, for example, 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, Second Edition, Cold Spring Harbor Press (1989).

[0156] In some embodiments, the nucleic acid sequence encoding the exogenous CD160 protein is operatively linked to a first promoter. In some embodiments, the nucleic acid sequence encoding a functional exogenous receptor is operatively 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 operatively 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 operatively linked to different promoters.

[0157] In some embodiments, the promoter is an endogenous promoter. For example, using any method known in the art, such as the CRISPR / Cas9 method, nucleic acid encoding a foreign CD160 protein and / or a functional foreign receptor is knocked into the genome of a modified antigen-specific immune cell downstream of an endogenous promoter. In some embodiments, the endogenous promoter is a protein-rich promoter, such as a promoter for β-actin. In some embodiments, the endogenous promoter is an inducible promoter, for example, induced by an endogenous activation signal from the modified antigen-specific immune cell. In some embodiments, the modified antigen-specific immune cell is a T cell, and the promoter is a T cell activation-dependent promoter (e.g., the IL-2 promoter, the NFAT promoter, or the NFκB promoter). In some embodiments, the promoter is a heterologous promoter.

[0158] Various promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in this invention. Promoters can be broadly classified as constitutive promoters or regulatory promoters, such as inducible promoters. In some embodiments, a heterologous nucleic acid sequence encoding a foreign CD160 protein and / or a functional foreign receptor is operatively linked to a constitutive promoter. In some embodiments, a heterologous nucleic acid sequence encoding a foreign CD160 protein and / or a functional foreign receptor is operatively linked to an inducible promoter. In some embodiments, a constitutive promoter is operatively linked to a nucleic acid sequence encoding a foreign CD160 protein, and an inducible promoter is operatively linked to a nucleic acid sequence encoding a functional foreign receptor. In some embodiments, a constitutive promoter is operatively linked to a nucleic acid sequence encoding a functional foreign receptor, and an inducible promoter is operatively linked to a nucleic acid sequence encoding a foreign CD160 protein. In some embodiments, a first inducible promoter is operatively linked to a nucleic acid sequence encoding a foreign CD160 protein, and a second inducible promoter is operatively linked to a nucleic acid sequence encoding a functional foreign receptor. In some embodiments, the first inducible promoter is induced by a first inducible condition, and the second inducible promoter is induced by a second inducible condition. In some embodiments, the first inducible condition and the second inducible condition are the same. In some embodiments, the first and second inducible promoters are induced simultaneously. In some embodiments, the first and second inducible promoters are induced sequentially, for example, the first inducible promoter is induced before or after the second inducible promoter.

[0159] Constitutive promoters allow the constitutive expression of heterologous genes (also known as transgenes) in host cells. Exemplary constitutive promoters considered herein include, but are not limited to, the cytomegalovirus (CMV) promoter, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), glycerol phosphokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter coupled to the CMV early enhancer (CAGG). The efficiency of such constitutive promoters in driving transgene expression has been extensively compared in numerous studies. In some embodiments, the promoter is the hEF1α promoter.

[0160] In some embodiments, the promoter is an inducible promoter. Inducible promoters belong to the category of regulatory promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the modified antigen-specific immune cells or the physiological state of the modified antigen-specific immune cells, an inducer (i.e., an inducing agent), or a combination thereof. In some embodiments, the inducing conditions do not induce the expression of endogenous genes in the modified antigen-specific immune cells and / or in the subject receiving the pharmaceutical composition. In some embodiments, the inducing conditions are selected from the group consisting of: an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and the activated state of the modified antigen-specific immune cells.

[0161] In some embodiments, the promoter is induced by an inducing agent. In some embodiments, the inducing agent 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, metal, or steroid. Chemically induced promoters have been the most extensively explored. Such promoters include those whose transcriptional activity is regulated by the presence or absence of small molecule chemicals, such as doxycycline, tetracycline, alcohol, steroid, metal, and other compounds. Doxycycline-induced systems with inverse tetracycline-controlled trans-activator (rtTA) and tetracycline-responsive element promoters (TRE) are currently the most mature systems. WO9429442 describes the tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters. WO9601313 discloses tetracycline-regulated transcription regulators. Additionally, Tet technologies, such as the Tet-on system, have been described on websites such as TetSystems.com. In this application, any known chemically regulated promoter can be used to drive the expression of therapeutic proteins.

[0162] In some embodiments, the inducer is a polypeptide, such as a growth factor, hormone, or ligand of a cell surface receptor, for example, a polypeptide that specifically binds to a tumor antigen. In some embodiments, the polypeptide is expressed by modified antigen-specific immune cells. In some embodiments, the polypeptide is encoded by nucleic acids in a heterologous nucleic acid. Many polypeptide inducers are also known in the art, and they may be suitable for use in this invention. For example, gene switches based on ecdysone receptors, progesterone receptors, and estrogen receptors all belong to gene switches employing steroid receptor-derived transactivators (WO9637609 and WO9738117, etc.).

[0163] In some embodiments, the inducer comprises both a small molecule component and one or more peptides. For example, inducible promoters that depend on peptide dimerization are known in the art and may be suitable for use in this invention. The first small molecule CID system developed in 1993 used FK1012, a derivative of the drug FK506, to induce homodimerization of FKBP. By employing a similar strategy, Wu et al. successfully made CAR-T cells titratable via an ON-switch mechanism using Rapalog / FKPB-FRB* and Gibberelline / GID1-GAI dimerization-dependent gene switches (C.-Y. Wu et al., Science 350, aab4077 (2015)). Other dimerization-dependent switch systems include coumarin / GyrB-GyrB (Nature 383(6596): 178-81) and HaXS / Snap-tag-HaloTag (Chemistry and Biology 20(4): 549-57).

[0164] In some implementations, the promoter is a photoinducible promoter, and the induction condition is light. Photoinducible promoters used to regulate 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 regulatory systems can be broadly classified into two categories based on their regulation of (1) DNA binding or (2) the recruitment of transcriptional activation domains to DNA-binding proteins. For example, a melanin-based synthetic mammalian blue light-controlled transcription system was developed and tested in mammalian cells, which responds to blue light (480 nm) to trigger an increase in intracellular calcium, leading to calcineurin-mediated NFAT mobilization. Recently, Motta-Mena et al. described a novel inducible gene expression system developed from the naturally occurring EL222 transcription factor, which conferred high levels of blue light-sensitive control over transcriptional initiation in human cell lines and zebrafish embryos (Nat. Chem. Biol. 10(3):196-202(2014)). Additionally, the red light-induced interaction photoreceptors Physiocin B (PhyB) and Physiocin Interacting Factor 6 (PIF6) in Arabidopsis thaliana were used for red light-triggered gene expression regulation. Furthermore, a UVB-inducible gene expression system has been developed and demonstrated to be effective in target gene transcription in mammalian cells (Chapter 25: Therapeutic Mechanisms and Strategies, Gene and Cell Therapy, 4th Edition, CRC Press, January 20, 2015). Any photoinducible promoter described herein can be used to drive the expression of the therapeutic proteins of this invention.

[0165] In some embodiments, the promoter is a photoinducible promoter induced by a combination of a photoinducible molecule and light. For example, a photocleavable photocage group on a chemical inducer renders the inducer inactive unless the photocage group is removed by radiation or other means. Such photoinducible molecules include small molecule compounds, oligonucleotides, and proteins. For example, cage-like ecdysone, cage-like IPTG for use with the lac operon, cage-like fulvamycin for ribozyme-mediated gene expression, cage-like doxycycline for use with the Tet-on system, and cage-like Rapalog for light-mediated FKBP / FRB dimerization have been developed (see, for example, Curr Opin Chem Biol. 16(3-4): 292-299(2012)).

[0166] In some embodiments, the promoter is a radiation-inducible promoter, and the induction condition is radiation, such as ionizing radiation. Radiation-inducible promoters are also known in the art for controlling transgene expression. Changes in gene expression occur after cell irradiation. For example, a group of genes known as “immediate early genes” can respond rapidly after ionizing radiation. Exemplary immediate early genes include, but are not limited to, Erg-1, p21 / WAF-1, GADD45α, t-PA, c-Fos, c-Jun, NF-κB, and AP1. Immediate early genes include radiation-responsive sequences in their promoter regions. A concordant sequence CC(A / T)6GG (SEQ ID NO:7) has been found in the Erg-1 promoter and is referred to as a serum-responsive element or CArG element. Combinations of radiation-inducible promoters and transgenes have been extensively studied and have demonstrated efficacy with therapeutic benefits. See, for example, CancerBiol Ther. 6(7):1005-12 (2007) and Chapter 25 of Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, 4th Edition, CRC Press, January 20, 2015.

[0167] In some implementations, the promoter is a heat-inducible promoter, and the induction condition is heat. Heat-inducible promoters driving transgene expression have been extensively studied in the art. Heat shock or stress proteins (HSPs), including Hsp90, Hsp70, Hsp60, Hsp40, and Hsp10, play important roles in protecting cells under heat or other physical and chemical stresses. Several heat-inducible promoters have been explored in preclinical studies, including the heat shock protein (HSP) promoter and the growth arrest and DNA damage (GADD) 153 promoter. The promoter of the human hsp70B gene, first described in 1985, appears to be one of the most highly effective heat-inducible promoters. Huang et al. reported that after introducing the coding sequences for hsp70B-EGFP, hsp70B-TNFα, and hsp70B-IL12, tumor cells expressed extremely high transgene expression upon heat treatment, while no transgene expression was detected without heat treatment. In mice treated with the IL12 transgene at heat, tumor growth was significantly delayed (Cancer Res. 60:3435 (2000)). Another group of scientists linked the HSV-tk suicide gene to the hsp70B promoter and tested the system in nude mice carrying mouse breast cancer. Mice whose tumors had been administered the hsp70B-HSVtk coding sequence and heat-treated showed tumor regression and significant survival compared to untreated controls (Hum. Gene Ther. 11:2453 (2000)). Other heat-inducible promoters known in the art can be found, for example, in Chapter 25 of Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, 4th Edition, CRC Press, January 20, 2015. Any heat-inducible promoters discussed herein can be used to drive the expression of the therapeutic proteins of the present invention.

[0168] In some implementations, the promoter is induced by a redox state. Exemplary promoters induced by a redox state include inducible promoters and hypoxia-inducible promoters. For example, Post DE et al. developed a hypoxia-inducible factor (HIF) response promoter that specifically and strongly induces transgene expression in HIF-active tumor cells (GeneTher.8:1801-1807 (2001); Cancer Res.67:6872-6881 (2007)).

[0169] In some embodiments, the promoter is induced by the physiological state of the modified antigen-specific immune cells, such as endogenous activation signals. In some embodiments, the modified antigen-specific immune cells are T cells, and the promoter is a T cell activation-dependent promoter induced by the endogenous activation signals of the modified T cells. In some embodiments, the modified T cells are activated by an inducer, such as phorbol myristate acetate (PMA), iomycin, or phytohemagglutinin. In some embodiments, the modified T cells are activated by recognizing tumor antigens on tumor cells via a functional exogenous receptor (such as CAR or TCR). In some embodiments, the T cell activation-dependent promoter is the IL-2 promoter. In some embodiments, the T cell activation-dependent promoter is the NFAT promoter. In some embodiments, the T cell activation-dependent promoter is the NFκB promoter.

[0170] The heterologous nucleic acid sequences described herein may be present in heterologous gene expression cassettes, which include one or more protein-coding sequences and optionally one or more promoters. In some embodiments, the heterologous gene expression cassette includes a single protein-coding sequence. In some embodiments, the heterologous gene expression cassette includes two or more protein-coding sequences driven by a single promoter (i.e., polycistronic). In some embodiments, the heterologous gene expression cassette further includes one or more regulatory sequences (e.g., 5'UTR, 3'UTR, enhancer sequences, IRES, transcription termination sequences), recombination sites, one or more selection markers (e.g., antibiotic resistance genes, reporter genes, etc.), signal sequences, or combinations thereof.

[0171] In some embodiments, a vector is provided comprising any one of the nucleic acids encoding the exogenous CD160 protein and / or a functional exogenous receptor 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.

[0172] A “vector” is a composition of matter comprising isolated nucleic acids and which can be used to deliver the isolated nucleic acids into cells. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids, and viruses. Generally, a suitable vector contains at least one origin of replication function in an organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers. The term “vector” should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc.

[0173] 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, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. 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.

[0174] Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vitro or ex vivo to modified antigen-specific immune cells. Many 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 methods known in the art. Using methods known in the art, the resulting lentiviral vectors can be used to transduce mammalian cells (e.g., human T cells).

[0175] In some implementations, the vector is a non-viral vector, such as a plasmid or an augmentation expression vector.

[0176] 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 provide expression of a coding sequence in the selected host. Expression vectors can be, for example, cloning vectors, binary vectors, or integrative vectors. Expression involves the transcription of a nucleic acid molecule, preferably, into a translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells, these typically include a promoter that ensures transcription initiation and optionally a poly-A signal that ensures transcription termination and transcript stability. Examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV- promoters (Rouse sarcoma virus), CMV-enhancers, SV40-enhancers, or globin introns in mammalian and other animal cells. Furthermore, depending on the expression system, a leader sequence capable of guiding the polypeptide into a cellular compartment or secreting it into a medium may be added to the coding sequence of the nucleic acid sequence and is well known in the art. The leader sequence assembles with the translation, initiation, and termination sequences at appropriate stages, preferably being a leader sequence capable of directing the translation of the protein or a portion thereof into the periplasmic space or extracellular medium. Optionally, the nucleic acid sequence may encode a fusion protein comprising an N-terminal recognition peptide conferring the desired characteristics, for example, for stabilization or simplified purification of the expression of the 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).

[0177] Methods for preparing antigen-specific immune cells modified with exogenous CD160

[0178] This application also provides methods for generating any of the antigen-specific immune cells modified herein.

[0179] In some aspects, a method is provided for generating antigen-specific immune cells whose surface includes a foreign CD160 protein, comprising: contacting a precursor antigen-specific immune cell with a foreign CD160 protein or a nucleic acid encoding the foreign CD160 protein to generate the modified antigen-specific immune cell, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cell compared to the precursor antigen-specific immune cell. In some embodiments, the method includes contacting the precursor antigen-specific immune cell with the foreign CD160 protein. In some embodiments, the foreign CD160 protein includes an immune cell-binding portion of a surface molecule that binds to the immune cell. In some embodiments, the method includes introducing a nucleic acid encoding the foreign CD160 protein into the precursor antigen-specific immune cell. 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 cell 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, such as using calcium phosphate, dendritic polymers, liposomes, or cationic polymers (e.g., DEAE-glucan or polyethyleneimine); non-chemical methods, such as electroporation, cell extrusion, acoustic perforation, optical transfection, puncture, protoplast fusion, hydrodynamic delivery, or transposons; particle-based methods, such as using gene guns, magnetic transfection or magnet-assisted transfection, particle bombardment; and hybrid methods, such as nuclear transfection. In some embodiments, nucleic acids are introduced into precursor antigen-specific immune cells via transfection. In some embodiments, nucleic acids are introduced into precursor antigen-specific immune cells via transduction or electroporation.

[0180] In some embodiments, a method is provided for generating antigen-specific immune cells modified with a foreign CD160 protein on their surface, comprising: contacting a precursor antigen-specific immune cell with a foreign CD160 protein or a nucleic acid encoding the foreign CD160 protein to generate the modified antigen-specific immune cell, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cell compared to the precursor antigen-specific immune cell. In some embodiments, the foreign CD160 protein comprises an amino acid sequence of any one of SEQ ID NO:1-4, or a variant thereof having at least about 80% identity with SEQ ID NO:1-4. In some embodiments, the foreign CD160 protein comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:1-4. In some embodiments, the foreign CD160 protein comprises an amino acid sequence having at least about 95% identity with SEQ ID NO:1-4. In some embodiments, the exogenous CD160 protein comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (e.g., but not limited to, dimers, trimers, tetramers, pentamers, or hexamers). 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, APC-activated anti-tumor T cells, 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 APC-activated anti-tumor T cells. In some embodiments, the APC-activated anti-tumor T cells are DC-activated anti-tumor T cells. 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, γδT 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 include 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 tumor antigens or tumor-associated antigens. 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 to the same epitope. Non-limiting examples include multiple T cells, each including the same functional exogenous receptor (e.g., CAR). In some embodiments, the modified antigen-specific immune cells are multiple immune cells that are specific to different epitopes (e.g., locally overlapping or completely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0181] In some embodiments, a method is provided for generating antigen-specific immune cells modified with an exogenous CD160 protein on their surface, comprising: contacting a precursor antigen-specific immune cell with an exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein to generate the modified antigen-specific immune cell, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cell compared to the precursor antigen-specific immune cell, and wherein the exogenous CD160 protein is membrane-bound.

[0182] In some embodiments, a method is provided for generating antigen-specific immune cells modified with an exogenous CD160 protein on their surface, comprising: contacting a precursor antigen-specific immune cell with the exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein to generate the modified antigen-specific immune cell, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cell compared to the precursor antigen-specific immune cell, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein is bound to the membrane via a glycophosphatidylinositol (GPI) linker. In some embodiments, the exogenous CD160 protein includes a GPI-anchored peptide sequence.

[0183] In some embodiments, a method is provided for generating antigen-specific immune cells modified with an exogenous CD160 protein on their surface, comprising: contacting a precursor antigen-specific immune cell with an exogenous CD160 protein or a nucleic acid encoding the exogenous CD160 protein to generate the modified antigen-specific immune cell, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cell compared to the precursor antigen-specific immune cell, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein includes a transmembrane domain. In some embodiments, the transmembrane domain is derived from molecules 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) α subunit, TCRβ subunit, or TCRζ subunit. In some implementations, the transmembrane domain is derived from molecules selected from the group consisting of CD28, 4-1BB, CD80, CD152, and PD1.

[0184] In some embodiments, a method is provided for generating antigen-specific immune cells whose surface includes a foreign CD160 protein, comprising: contacting a precursor antigen-specific immune cell with a foreign CD160 protein or a nucleic acid encoding the foreign CD160 protein to generate the modified antigen-specific immune cell, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cell compared to the precursor antigen-specific immune cell, wherein the foreign CD160 protein is membrane-bound, and wherein the foreign CD160 protein includes a transmembrane domain and an intracellular domain. In some embodiments, the transmembrane domain is derived from molecules 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) α subunit, TCRβ subunit, or TCRζ 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 includes an intracellular signaling domain derived from a signaling subunit of the TCR complex. In some embodiments, the signaling subunit of the TCR complex is selected from the group consisting of CD3γ, CD3δ, and CD3ε. In some embodiments, the intracellular domain includes one or more signaling domains derived from T cell stimulating molecules. 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 includes a combination of two signaling domains selected from the group consisting of OX40, CD27, CD28, CD80, and CD258.

[0185] In some embodiments, a method is provided for generating antigen-specific immune cells modified with a foreign CD160 protein on their surface, comprising: contacting a precursor antigen-specific immune cell with a foreign CD160 protein or a nucleic acid encoding the foreign CD160 protein to generate the modified antigen-specific immune cell, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cell compared to the precursor antigen-specific immune cell, wherein the foreign CD160 protein is membrane-bound, wherein the CD160 protein includes a transmembrane domain and an intracellular domain, and wherein the intracellular domain includes one or more co-stimulatory signaling domains. In some embodiments, the intracellular domain includes any 1, 2, 3, 4, 5, 6, 7, 8 or more co-stimulatory signaling domains. In some embodiments, the intracellular domain contains no more than 1, 2, 3, 4 or 5 co-stimulatory signaling domains. In some embodiments, the intracellular domain does not include a CD3ζ signaling domain or a combination of a 4-1BB and CD3ζ domain. In some embodiments, the co-stimulatory signal transduction domain is derived from a co-stimulatory 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, CD83, and combinations thereof. In some embodiments, the intracellular domain includes a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, or both. In some embodiments, the exogenous CD160 protein, from its N-terminus to its C-terminus, includes: an extracellular CD160 domain, a transmembrane domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain. In some embodiments, the exogenous CD160 protein, from its N-terminus to its C-terminus, includes: an extracellular CD160 domain, a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain is adjacent to the transmembrane domain. In some embodiments, the CD28 co-stimulatory domain is adjacent to the C-terminus of the transmembrane domain. In some embodiments, the intracellular domain includes a primary signaling domain. In some embodiments, the primary signaling domain includes a CD3ζ domain. In other embodiments, the intracellular domain does not include a primary signaling domain. In other embodiments, the intracellular domain does not include a CD3ζ domain or a combination of 4-1BB and CD3ζ domains.

[0186] In some embodiments, a method is provided for generating antigen-specific immune cells modified with a foreign CD160 protein on their surface, comprising: contacting a precursor antigen-specific immune cell with a foreign CD160 protein or a nucleic acid encoding the foreign CD160 protein to generate the modified antigen-specific immune cell, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cell compared to the precursor antigen-specific immune cell, wherein the foreign CD160 protein is membrane-bound, and wherein the foreign CD160 protein binds to the modified antigen-specific immune cell via an immune cell-binding portion. In some embodiments, the immune cell-binding portion binds to surface molecules of the immune cell. In some embodiments, the immune cell-binding portion includes an antibody that recognizes a T-cell surface molecule. In some embodiments, the antibody may be a full-length antibody or an antibody fragment, such as scFv, Fv, Fab, (Fab')2, a single-domain antibody (sdAb), or a VHH domain. Non-limiting examples include anti-CD3ε antibodies that recognize the TCR and / or activate TCR signaling. In some embodiments, the immune cell binding portion includes a ligand that binds to an associated T cell surface receptor. Non-limiting examples include a tumor-specific peptide MHC complex that recognizes TCR and IL-2.

[0187] In some embodiments of the methods described herein, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NO: 1-4, or a variant thereof having at least about 80% identity with SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence having at least about 90% identity with SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence having at least about 95% identity with SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (e.g., 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, APC-activated anti-tumor T cells, 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 APC-activated anti-tumor T cells. In some embodiments, the APC-activated anti-tumor T cells are DC-activated anti-tumor T cells. 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 include 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 to the same epitope. Non-limiting examples include multiple T cells, each including the same functional exogenous receptor (e.g., CAR).In some embodiments, the modified antigen-specific immune cells are multiple immune cells, each specific to a different epitope (e.g., locally overlapping or completely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0188] In some embodiments of any of the methods for producing modified antigen-specific immune cells, the surface of which comprises the exogenous CD160 protein as described herein, 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 a second nucleic acid encoding 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 modified T-cell receptor (TCR). In some embodiments, the engineered T-cell receptor (TCR) recognizes a tumor antigen or a tumor-associated antigen. In a further embodiment, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the first and second nucleic acids are operatively linked to the same promoter. In some embodiments, the first and second nucleic acid sequences are operatively linked to different promoters. In some embodiments, the first and second nucleic acids are on the same vector. In some embodiments, the first and second nucleic acids are on different vectors. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of adenovirus vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, herpes simplex virus vectors, and derivatives thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is an addendum gene expression vector. In some embodiments, the method further includes isolating or enriching immune cells comprising a first nucleic acid and / or a second nucleic acid. In some embodiments, the method further includes formulating modified antigen-specific immune cells using at least one pharmaceutically acceptable vector.

[0189] In some embodiments, modified antigen-specific immune cells obtained by any of the methods described herein are provided. In some embodiments, pharmaceutical compositions comprising any of the modified antigen-specific immune cells described herein and a pharmaceutically acceptable carrier are provided.

[0190] In some embodiments, an isolated host cell is provided, comprising any of the nucleic acids or vectors described herein. The host cell can be used to express or clone exogenous CD160 protein and / or a functional exogenous receptor, or a nucleic acid or vector encoding exogenous CD160 protein and / or a functional exogenous receptor. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as 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 containing an isolated nucleic acid 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.

[0191] Precursor antigen-specific immune cells can be prepared using a variety of methods known in the art. For example, primary immune cells, such as T cells, can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection sites, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, immune cells (such as T cells) can be prepared using any number of techniques known in the art, such as FICOLL. TM Cells are separated from blood units collected from an individual. In some embodiments, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected via apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, cells are washed with phosphate-buffered saline (PBS) or a wash solution lacking divalent cations, such as calcium and magnesium. As will be readily understood by those skilled in the art, the washing step can be performed by methods known to those skilled in the art, such as using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, BaxterCytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, cells may be resuspended in various biocompatible buffers, such as, for example, calcium-free buffers. 2+ Mg-free 2+ PBS, PlasmaLyte A, or other saline solutions with or without buffer. Optionally, unwanted components in apheresis samples can be removed and cells can be directly resuspended in the culture medium.

[0192] In some implementations, this is achieved by lysing red blood cells and consuming monocytes, for example, by PERCOLLTM Primary T cells are isolated from peripheral blood lymphocytes by gradient centrifugation or countercurrent centrifugation. Specific T cell subsets, such as CD3+, are isolated. + CD28 + CD4 + CD8 + CD45RA and CD45RO cells can be further separated using positive or negative selection techniques. For example, in one embodiment, this can be achieved by using beads conjugated with anti-CD3 / anti-CD28 (i.e., 3x28) beads, such as... M-450 CD3 / CD28 T incubation is sufficient for a period of time to isolate T cells for positive selection of the desired T cells.

[0193] In some implementations, the T cell population is further enriched by negative selection using a combination of antibodies targeting surface markers specific to the negatively selected cells. For example, one method involves cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For instance, to enrich CD4 by negative selection... + Cellular monoclonal antibody mixtures typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, enrichment or positive selection of cells that normally express CD4 may be desired. + CD25 + CD62L hi GITR + and FoxP3 + Regulatory T cells. Alternatively, in some embodiments, regulatory T cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0194] Methods for introducing vectors or nucleic acids into host cells (such as precursor antigen-specific immune cells) are known in the art. Vectors or nucleic acids can be transferred into host cells by physical, chemical, or biological methods.

[0195] Physical methods for introducing vectors or nucleic acids into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells comprising 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, the vector is introduced into the cells via electroporation.

[0196] 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 cells, such as human cells.

[0197] Chemical methods for introducing carriers or nucleic acids into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0198] In some embodiments, precursor antigen-specific immune cells are proliferated in vitro after the introduction of heterologous nucleic acids. In some embodiments, the transduced or transfected precursor antigen-specific immune cells are cultured to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 10, 12, or 14 days. In some embodiments, the transduced or transfected precursor antigen-specific immune cells are cultured for no more than about 1, 2, 3, 4, 5, 6, 7, 10, 12, or 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.

[0199] Reporter genes can be used to identify potentially transfected cells and to assess the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and encodes a polypeptide whose expression is demonstrated by easily detectable properties such as enzyme activity. Reporter gene expression is measured at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., FEBS Letters 479:79-82 (2000)).

[0200] Other methods for confirming the presence of heterologous nucleic acids in precursor antigen-specific immune cells include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; and biochemical assays, such as detecting the presence or absence of a specific peptide, for example by immunological methods (such as ELISA and Western blotting).

[0201] In some embodiments, antigen-specific immune cells modified to express one or more of the exogenous CD160 proteins described herein are provided. In some embodiments, antigen-specific immune cells modified to overexpress the CD160 protein are provided. In some embodiments, the CD160 protein is an endogenous protein. In some embodiments, the CD160 protein is an exogenous protein. In some embodiments, CD160-modified antigen-specific immune cells exhibit increased proliferation and / or increased viability compared to non-CD160-modified antigen-specific immune cells. In some embodiments, the yield and / or viability of CD160-modified antigen-specific immune cells are increased by at least about 0.5-fold, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 10000-fold or more compared to non-CD160-modified antigen-specific immune cells. In some embodiments, the yield and / or viability of CD160-modified antigen-specific immune cells are increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to non-CD160-modified antigen-specific immune cells. In some embodiments, the yield and / or viability of CD160-modified antigen-specific immune cells are increased by at least about 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 20 times, 20 to 50 times, 50 to 100 times, 100 to 500 times, 500 to 1000 times, or 1000 to 10000 times compared to non-CD160-modified antigen-specific immune cells. In some embodiments, the yield and / or viability of CD160-modified antigen-specific immune cells are increased by at least about 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% compared to non-CD160-modified antigen-specific immune cells.

[0202] In some aspects, methods for increasing the yield and / or viability of antigen-specific immune cells are provided, comprising introducing a nucleic acid encoding a foreign CD160 protein into immune cells. In some embodiments, the yield and / or viability of antigen-specific immune cells expressing the foreign CD160 protein are increased by at least about 0.5-fold, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 10000-fold or more compared to antigen-specific immune cells not expressing the foreign CD160 protein. In some embodiments, the yield and / or viability of antigen-specific immune cells expressing the foreign CD160 protein are increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to antigen-specific immune cells not expressing the foreign CD160 protein. In some embodiments, compared with antigen-specific immune cells that do not express exogenous CD160 protein, the yield and / or viability of antigen-specific immune cells expressing exogenous CD160 protein are increased by at least about 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 20 times, 20 to 50 times, 50 to 100 times, 100 to 500 times, 500 to 1000 times, or 1000 to 10000 times. In some embodiments, compared with antigen-specific immune cells that do not express exogenous CD160 protein, the yield and / or viability of antigen-specific immune cells expressing exogenous CD160 protein are increased by at least about 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, or 80% to 90% or 90% to 100%. In some implementations, the increase in the production of CD160-modified antigen-specific immune cells is caused by an increase in the proliferation rate of CD160-modified antigen-specific immune cells.

[0203] In some aspects, methods for increasing the yield and / or viability of antigen-specific immune cells are provided, including inducing overexpression of the 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 overexpressing the CD160 protein are increased by at least about 0.5-fold, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 10000-fold or more compared to antigen-specific immune cells not overexpressing the CD160 protein. In some embodiments, the yield and / or viability of antigen-specific immune cells overexpressing the CD160 protein are increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to antigen-specific immune cells not overexpressing the CD160 protein. In some embodiments, compared with antigen-specific immune cells that do not overexpress CD160 protein, the yield and / or viability of antigen-specific immune cells overexpressing CD160 protein are increased by at least about 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 20 times, 20 to 50 times, 50 to 100 times, 100 to 500 times, 500 to 1000 times, or 1000 to 10000 times. In some embodiments, compared with antigen-specific immune cells that do not overexpress CD160 protein, the yield and / or viability of antigen-specific immune cells overexpressing CD160 protein are increased by at least about 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, or 80% to 90% or 90% to 100%. In some implementations, the increase in the production of CD160-modified antigen-specific immune cells is caused by an increase in the proliferation rate of CD160-modified antigen-specific immune cells.

[0204] In some embodiments, a method for manufacturing therapeutic antigen-specific immune cells is provided, including a method for increasing the yield and / or viability of any selected antigen-specific immune cells according to the methods described herein. In some embodiments, the therapeutic antigen-specific immune cells include tumor-infiltrating lymphocytes (TILs). In some embodiments, the therapeutic antigen-specific immune cells include 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). Therapeutic antigen-specific immune cells manufactured according to any of the methods described herein are also provided. 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 cells 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 cells is provided.

[0205] In some embodiments, antigen-specific immune cells modified to express one or more of the exogenous CD160 proteins described herein are provided. In some embodiments, antigen-specific immune cells modified to overexpress the CD160 protein are provided. In some embodiments, the CD160 protein is an endogenous protein. In some embodiments, the CD160 protein is an exogenous protein. In some embodiments, CD160-modified antigen-specific immune cells exhibit increased in vitro and / or in vivo cytolytic activity compared to non-CD160-modified antigen-specific immune cells. In some embodiments, the in vitro and / or in vivo cytolytic activity of CD160-modified antigen-specific immune cells is increased by at least about 0.5-fold, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 10000-fold or more compared to non-CD160-modified antigen-specific immune cells. In some embodiments, compared with antigen-specific immune cells that are not CD160-modified, the in vitro and / or in vivo cytolytic activity of CD160-modified antigen-specific immune cells is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more. In some embodiments, compared with antigen-specific immune cells that are not CD160-modified, the in vitro and / or in vivo cytolytic activity of CD160-modified antigen-specific immune cells is increased by at least about 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 20 times, 20 to 50 times, 50 to 100 times, 100 to 500 times, 500 to 1000 times, or 1000 to 10000 times. In some embodiments, compared with antigen-specific immune cells that are not CD160-modified, the in vitro and / or in vivo cytolytic activity of CD160-modified antigen-specific immune cells is increased by at least about 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

[0206] In some aspects, methods for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells are provided, comprising introducing a nucleic acid encoding a foreign CD160 protein into the immune cells. In some embodiments, the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells expressing the foreign CD160 protein is increased by at least about 0.5-fold, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 10000-fold or more compared to antigen-specific immune cells not expressing the foreign CD160 protein. In some embodiments, the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells expressing the foreign CD160 protein is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more compared to antigen-specific immune cells not expressing the foreign CD160 protein. In some embodiments, compared with antigen-specific immune cells that do not express exogenous CD160 protein, antigen-specific immune cells expressing exogenous CD160 protein exhibit at least one to two-fold, two to five-fold, five to ten-fold, ten to twenty-fold, twenty to fifty-fold, fifty to one hundred-fold, one hundred to five hundred-fold, five hundred to one hundred-hundred-fold, or one hundred to one hundred-thousand-fold increase in in vitro and / or in vivo cytolytic activity. In some embodiments, antigen-specific immune cells expressing exogenous CD160 protein exhibit at least one increase in in vitro and / or in vivo cytolytic activity of at least about 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, or 80% to 90% or 90% to 100% compared to antigen-specific immune cells that do not express exogenous CD160 protein.

[0207] In some aspects, methods are provided for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells, including inducing overexpression of the CD160 protein in the 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 in vitro and / or in vivo cytolytic activity of antigen-specific immune cells overexpressing the CD160 protein is increased by at least about 0.5-fold, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 10000-fold or more compared to antigen-specific immune cells that do not overexpress the CD160 protein. In some embodiments, compared with antigen-specific immune cells that do not overexpress CD160 protein, antigen-specific immune cells that overexpress CD160 protein exhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more of increased in vitro and / or in vivo cytolytic activity. In some embodiments, compared with antigen-specific immune cells that do not overexpress CD160 protein, antigen-specific immune cells that overexpress CD160 protein exhibit at least about 1 to 2 times, 2 to 5 times, 5 to 10 times, 10 to 20 times, 20 to 50 times, 50 to 100 times, 100 to 500 times, 500 to 1000 times, or 1000 to 10000 times of increased in vitro and / or in vivo cytolytic activity. In some embodiments, antigen-specific immune cells overexpressing CD160 protein exhibit at least a 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, or 80% to 90% or 90% to 100% increase in in vitro and / or in vivo cytolytic activity compared to antigen-specific immune cells that do not overexpress CD160 protein. In some embodiments, a method for manufacturing therapeutic antigen-specific immune cells is provided, comprising a method for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells selected according to any 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). Therapeutic antigen-specific immune cells manufactured according to any of the methods described herein are also provided.In some embodiments, the use of CD160 overexpression is provided to increase the cytolytic activity of therapeutic antigen-specific T cells (including, but not limited to, TILs, TCR-T cells, and CAR-T cells) in vitro and / or in vivo. In some embodiments, the use of exogenous CD160 expression is provided to increase the cytolytic activity of therapeutic antigen-specific T cells (including, but not limited to, TILs, TCR-T cells, and CAR-T cells) in vitro and / or in vivo.

[0208] III. Therapeutic methods using exogenous CD160 protein or antigen-specific immune cells modified to express exogenous CD160 protein.

[0209] One aspect of this application relates to a method of treating an individual's disease, comprising administering to the individual an effective amount of any of the modified antigen-specific immune cells described herein or any of the pharmaceutical compositions described herein. This application contemplates modified antigen-specific immune cells that 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 combined with suitable pharmaceutical carriers and excipients well known in the art prior to administration. In some embodiments, the modified antigen-specific immune cells are derived from an individual.

[0210] Another aspect of this application relates to a method of treating an individual's disease, including administering to the individual an effective amount of exogenous CD160 protein or nucleic acid encoding exogenous CD160 protein, wherein the exogenous CD160 protein includes a binding portion that recognizes a surface molecule on an immune cell in the individual.

[0211] Therefore, in some embodiments, a method of treating a disease (e.g., cancer) in an individual (e.g., a person) is provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising (e.g., on their surface) a foreign CD160 protein, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the foreign CD160 protein. 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 (e.g., on their surface) a foreign CD160 protein, said modified antigen-specific immune cells being generated by a process comprising contacting precursor antigen-specific immune cells with a foreign CD160 protein or a first nucleic acid encoding the foreign CD160 protein to generate the modified antigen-specific immune cells, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells. In some embodiments, the modified antigen-specific immune cells are derived from the individual. In some embodiments, a method of treating an individual's disease includes administering to the individual an effective amount of a pharmaceutical composition comprising (a) a modified antigen-specific immune cell comprising (e.g., on its surface) a foreign CD160 protein, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cell compared to a precursor antigen-specific immune cell that does not contain the foreign CD160 protein; and (b) a pharmaceutically acceptable carrier. In some embodiments, the modified antigen-specific immune cell is derived from the individual. In some embodiments of any of the treatment methods described herein, the foreign CD160 protein comprises an amino acid sequence of any one of SEQ ID NO:1-4, or a variant thereof having at least about 80% identity with any one of SEQ ID NO:1-4. In some embodiments, the foreign CD160 protein comprises an amino acid sequence having at least about 90% identity with any one of SEQ ID NO:1-4. In some embodiments, the exogenous CD160 protein comprises any amino acid sequence having about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO:1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (e.g., but not limited to, dimers, trimers, tetramers, pentamers, or hexamers). 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, APC-activated anti-tumor T cells, 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 APC-activated anti-tumor T cells. In some embodiments, the APC-activated anti-tumor T cells are DC-activated anti-tumor T cells. 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, γδT 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 include 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 tumor antigens or tumor-associated antigens. In a further embodiment, 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 to the same epitope. Non-limiting examples include multiple T cells, each including the same functional exogenous receptor (e.g., CAR). In some embodiments, the modified antigen-specific immune cells are multiple immune cells that are specific to different epitopes (e.g., locally overlapping or completely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0212] In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein causes upregulation 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. In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, said modified antigen-specific immune cells being generated by a process comprising: contacting precursor antigen-specific immune cells with the exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein to generate the modified antigen-specific immune cells, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells, and wherein the exogenous CD160 protein is membrane-bound.

[0213] In some embodiments, a method of treating an individual's disease includes administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the exogenous CD160 protein, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein is bound to the membrane via a GPI linker. In some embodiments, a method of treating an individual's disease includes administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, said modified antigen-specific immune cells being generated by a process comprising: contacting a precursor antigen-specific immune cell with the exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein to generate the modified antigen-specific immune cells, wherein the exogenous CD160 protein causes upregulation 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, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein is bound to the membrane via a GPI linker. In some embodiments, the exogenous CD160 protein includes a GPI-anchored peptide sequence.

[0214] In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the exogenous CD160 protein, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein includes a transmembrane domain. In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, said modified antigen-specific immune cells being generated by a process comprising: contacting precursor antigen-specific immune cells with the exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein to generate the modified antigen-specific immune cells, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein includes a transmembrane domain. In some embodiments, the transmembrane domain is derived from molecules 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) α subunit, TCR β subunit, or TCR ζ subunit. In some embodiments, the transmembrane domain is derived from molecules selected from the group consisting of: CD28, 4-1BB, CD80, CD152, and PD-1.

[0215] In some embodiments, a method of treating an individual's disease includes administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the exogenous CD160 protein, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein includes a transmembrane domain and an intracellular domain. In some embodiments, a method of treating an individual's disease is provided, comprising administering to the individual an effective amount of antigen-specific immune cells that include a foreign CD160 protein on their surface, the modified antigen-specific immune cells being generated by a process comprising: contacting a precursor antigen-specific immune cell with a foreign CD160 protein or a first nucleic acid encoding the foreign CD160 protein to generate the modified antigen-specific immune cells, wherein the foreign CD160 protein causes upregulation 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, wherein the foreign CD160 protein is membrane-bound, and wherein the foreign CD160 protein includes a transmembrane domain and an intracellular domain. In some embodiments, the transmembrane domain is derived from molecules 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) α subunit, TCR β subunit, or TCR ζ subunit. In some embodiments, the transmembrane domain is derived from molecules 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 includes an intracellular signal transduction domain derived from a signal transduction subunit of the TCR complex. In some embodiments, the signal transduction subunit of the TCR complex is selected from the group consisting of: CD3γ, CD3δ, and CD3ε. In some embodiments, the intracellular domain includes one or more signal transduction domains derived from T cell stimulating molecules. In some embodiments, the signal transduction domain is one or more of 4-1BB, OX40, CD27, CD28, CD80, or CD258. In some embodiments, the intracellular domain includes a combination of two signal transduction domains selected from the group consisting of OX40, CD27, CD28, CD80, and CD258.

[0216] In some embodiments, a method of treating an individual's disease includes administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the exogenous CD160 protein, wherein the exogenous CD160 protein is membrane-bound, wherein the exogenous CD160 protein includes a transmembrane domain and an intracellular domain, and wherein the intracellular domain includes one or more co-stimulatory signal transduction domains. In some embodiments, a method of treating an individual's disease includes administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, said modified antigen-specific immune cells being generated by a process comprising: contacting a precursor antigen-specific immune cell with the exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein to generate the modified antigen-specific immune cells, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, wherein the exogenous CD160 protein is membrane-bound, wherein the exogenous CD160 protein includes a transmembrane domain and an intracellular domain, and wherein the intracellular domain includes one or more co-stimulatory signaling domains. In some embodiments, the intracellular domain includes any 1, 2, 3, 4, 5, 6, 7, 8 or more co-stimulatory signaling domains. In some embodiments, the intracellular domain contains no more than any one of 1, 2, 3, 4 or 5 co-stimulatory signaling domains. In some embodiments, the intracellular domain does not include a CD3ζ signaling domain or a combination of 4-1BB and CD3ζ domains. In some embodiments, the co-stimulatory signaling domain is derived from ligands 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, CD83, and combinations thereof. In some embodiments, the intracellular domain includes a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, or both. In some embodiments, the exogenous CD160 protein, from its N-terminus to its C-terminus, includes: an extracellular CD160 domain, a transmembrane domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain. In some embodiments, the exogenous CD160 protein includes, from the N-terminus to the C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain is adjacent to the transmembrane domain. In some embodiments, the CD28 co-stimulatory domain is adjacent to the C-terminus of the transmembrane domain.In some embodiments, the intracellular domain includes a primary signal transduction domain. In some embodiments, the primary signal transduction domain includes a CD3ζ domain. In other embodiments, the intracellular domain does not include a primary signal transduction domain. In other embodiments, the intracellular domain does not include a CD3ζ domain or a combination of 4-1BB and CD3ζ domains.

[0217] In some embodiments, a method of treating an individual's disease includes administering to the individual an effective amount of antigen-specific immune cells modified with exogenous CD160 protein on their surface, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain exogenous CD160 protein, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein binds to the modified antigen-specific immune cells via an immune cell-binding portion. In some embodiments, a method of treating an individual's disease includes administering to the individual an effective amount of antigen-specific immune cells modified with an exogenous CD160 protein on their surface, said modified antigen-specific immune cells being generated by a process comprising: contacting a precursor antigen-specific immune cell with the exogenous CD160 protein or a first nucleic acid encoding the exogenous CD160 protein to generate the modified antigen-specific immune cells, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cells compared to the precursor antigen-specific immune cells, wherein the exogenous CD160 protein is membrane-bound, and wherein the exogenous CD160 protein binds to the modified antigen-specific immune cells via an immune cell-binding portion. In some embodiments, the immune cell-binding portion binds to surface molecules of the immune cells. In some embodiments, the immune cell-binding portion includes an antibody that recognizes T-cell surface molecules. In some embodiments, the antibody may be a full-length antibody or an antibody fragment, such as scFv, Fv, Fab, (Fab')2, a single-domain antibody (sdAb), or AV. H H domain. Non-limiting examples include anti-CD3ε antibodies that recognize the TCR and / or activate TCR signaling. In some embodiments, the immune cell binding portion includes a ligand that binds to an associated T cell surface receptor. Non-limiting examples include tumor-specific peptide MHC complexes that recognize the TCR and IL-2.

[0218] In some embodiments of any of the treatment methods described herein, the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NO: 1-4, or a variant thereof having at least about 80% identity with any one of SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence having at least about 90% identity with any one of SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein comprises the amino acid sequence having about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 1-4. In some embodiments, the exogenous CD160 protein on the cell surface is in the form of a multimer (e.g., 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, APC-activated anti-tumor T cells, 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 APC-activated anti-tumor T cells. In some embodiments, the APC-activated anti-tumor T cells are DC-activated anti-tumor T cells. 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 include 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 a further embodiment, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the modified antigen-specific immune cells are multiple immune cells specific to the same epitope. Non-limiting examples include multiple T cells, each including the same functional exogenous receptor (e.g., CAR).In some embodiments, the modified antigen-specific immune cells are multiple immune cells, each specific to a different epitope (e.g., locally overlapping or completely different epitopes). Non-limiting examples include multiple polyclonal immune cells, such as polyclonal TILs.

[0219] In some embodiments of any of the treatment methods described herein, the method of generating modified antigen-specific immune cells includes contacting the precursor antigen-specific immune cells with an exogenous CD160 protein. In some embodiments, the exogenous CD160 protein includes an immune cell-binding portion of a surface molecule that binds to the immune cell. In some embodiments, the method includes 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 transfections such as those using calcium phosphate, dendritic polymers, liposomes, or cationic polymers (e.g., DEAE-glucan or polyethyleneimine); non-chemical methods such as electroporation, cell squeezing, acoustic perforation, optical transfection, puncture, protoplast fusion, hydrodynamic delivery, or transposons; particle-based methods such as those using a gene gun, magnetic transfection or magnet-assisted transfection, particle bombardment; and hybrid methods such as nuclear transfection. In some embodiments, nucleic acids are introduced into precursor antigen-specific immune cells via transfection. In some embodiments, nucleic acids are introduced into precursor antigen-specific immune cells via transduction or electroporation. In some embodiments, the CD160 protein comprises the amino acid sequence of any one of SEQ ID NO:1-4, or a variant thereof having at least about 80% identification identity with any one of SEQ ID NO:1-4. In some embodiments, the CD160 protein comprises the amino acid sequence having at least about 90% identity with any one of SEQ ID NO:1-4. In some embodiments, the CD160 protein comprises the amino acid sequence having at least about 95% identity with any one of SEQ ID NO:1-4. In some embodiments, the CD160 protein comprises the amino acid sequence having at least about 99% identity with any one of SEQ ID NO:1-4. In some embodiments, the CD160 protein comprises the amino acid sequence having about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:1-4.

[0220] In some embodiments, a method of treating an individual's disease is provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells, said modified antigen-specific immune cells being generated 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 to generate modified antigen-specific immune cells, wherein the exogenous CD160 protein causes upregulation 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 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 generating modified antigen-specific immune cells further comprises introducing a 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 a further embodiment, the functional exogenous receptor is a chimeric antigen receptor (CAR). In some embodiments, the first and second nucleic acids are operatively linked to the same promoter. In some embodiments, the first and second nucleic acid sequences are operatively linked to different promoters. In some embodiments, the first and second nucleic acids are on the same vector. In some embodiments, the first and second nucleic acids are on different vectors. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of adenovirus vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, herpes simplex virus vectors, and derivatives thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is an appendage gene expression vector. In some embodiments, the method further includes isolating or enriching immune cells comprising the first and / or second nucleic acids. In some embodiments of any of the treatment methods described herein, the method of generating modified antigen-specific immune cells further includes formulating the modified antigen-specific immune cells with at least one pharmaceutically acceptable vector.

[0221] In some embodiments, a method of treating an individual's cancer is provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells comprising (e.g., on their surface) a foreign CD160 protein, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the foreign CD160 protein. In some embodiments, a method of treating an individual's cancer is provided, comprising administering to the individual an effective amount of modified antigen-specific immune cells, said modified antigen-specific immune cells being generated by a process comprising contacting precursor antigen-specific immune cells with a foreign CD160 protein or a first nucleic acid encoding the foreign CD160 protein to generate modified antigen-specific immune cells, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cells compared to 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 virus-related cancer, such as HPV-related cancer or EBV-related cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting the proliferation of cancer cells; (3) inducing peripheral T cell redistribution; (4) inducing an immune response in the tumor; (5) reducing tumor size; (6) alleviating one or more symptoms in a patient with cancer; (7) inhibiting tumor metastasis; (8) prolonging survival; (9) prolonging the time of cancer progression; (10) preventing, inhibiting, or reducing the likelihood of cancer recurrence; (11) improving an individual's quality of life; (12) promoting T cell infiltration in the tumor; and (13) reducing the incidence or burden of pre-existing tumor metastases (e.g., metastasis to lymph nodes). In some embodiments, the method achieves a tumor cell mortality rate of at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method reduces tumor size by at least about 10% (including, for example, any one of at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method inhibits metastasis by at least about 10% (including, for example, any one of at least 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method prolongs individual survival by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or more months.In some implementations, the method prolongs the time to cancer progression by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 or more months.

[0222] In some embodiments of any of the treatment methods described herein, administration is intratumoral. In some embodiments, administration is into the lymph nodes. In some embodiments, administration is performed parenterally, percutaneously (into the dermis), intracavitarily, intra-arterially (into the artery), intramuscularly (into the muscle), intrathecally, or intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously (under the skin). In some embodiments, administration is intravenously.

[0223] The methods described herein are suitable for treating various cancers, including both solid and liquid cancers. The methods are applicable to all stages of cancer, including early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. The methods described herein can be used as a first-line, second-line, third-line therapy, or in combination with other types of cancer therapies known in the art, such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (e.g., T-cell therapy), bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, etc., in an adjunctive or neoadjunctive setting (i.e., the method can be performed prior to primary / final therapy). In some embodiments, the method is used to treat individuals who have previously been treated. In some embodiments, the cancer is refractory to previous treatments. In some embodiments, the method is used to treat individuals who have not previously received treatment.

[0224] In some embodiments of the methods described herein, antigen-specific immune cells modified with exogenous CD160 protein are used as short-term cytolytic agents for controlling and eliminating established solid tumors. In some embodiments, the modified antigen-specific immune cells comprise exogenous CD160 protein. In some embodiments, the method includes administering the modified antigen-specific immune cells or pharmaceutical composition approximately every 7, 10, 14, 21, or 30 days. In some embodiments, the method includes administering the modified antigen-specific immune cells or pharmaceutical composition approximately every 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, the modified antigen-specific immune cells or pharmaceutical composition are administered multiple times (e.g., any one of 2, 3, 4, 5, 6, or more times). In some embodiments, the method further includes administering one or more therapeutic agents. In some embodiments, the therapeutic agent is one or more of the following: radiotherapy, chemotherapy, or immunotherapy. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets any 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 cytokines. In some embodiments, the cytokines are 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 induces 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.

[0225] In some embodiments, the method further includes treatment or pretreatment. In some embodiments, treatment or pretreatment is used to reduce or eliminate underlying disease to create space for new bone marrow. In some embodiments, the method further includes chemotherapy. In some embodiments, chemotherapy is administered before the administration of modified antigen-specific immune cells or a modified pharmaceutical composition. In some embodiments, chemotherapy is administered after the administration of modified antigen-specific immune cells or a modified pharmaceutical composition. In some embodiments, chemotherapy is used to pretreat an individual carrying cancer. In some embodiments, the method does not include pretreatment. In some embodiments, the method does not further include chemotherapy or chemotherapy pretreatment. In some embodiments, the method does not further include radiation therapy or radiation pretreatment. In some embodiments, the method does not further include the use of vaccination. In some embodiments, the method does not further include the use of interleukins, such as, but not limited to, interleukin-2 (IL-2).

[0226] The effective amount of the modified antigen-specific immune cell or pharmaceutical composition administered in the methods described herein will depend on many factors, such as the specific type and stage of the cancer being treated, the route of administration, the activity of the exogenous CD160 protein and / or functional exogenous receptor, etc. An appropriate dosing regimen may be determined by a physician based on clinical factors, including patient size, body surface area, age, the specific compound to be administered, sex, time and route of administration, overall health status, and other concurrently administered medications. In some embodiments, the effective amount of the modified antigen-specific immune cell or pharmaceutical composition is below the level that induces a toxicological effect (i.e., an effect above a clinically acceptable level of toxicity) or at a level that is manageable or tolerable as a potential side effect when the pharmaceutical composition is administered to an individual. In some embodiments, the effective amount of the modified antigen-specific immune cell or pharmaceutical composition comprises about 10 5 To about 10 10 The effective amount of modified antigen-specific immune cells or pharmaceutical composition comprises any one of about 0.1, 0.2, 0.5, 0.75, 1, 2, 5, 10, 20, 50, 100, 200, or 500 million modified antigen-specific immune cells. In some embodiments, the effective amount of modified antigen-specific immune cells or pharmaceutical composition comprises any one of about 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 billion modified antigen-specific immune cells.

[0227] In some embodiments, the modified antigen-specific immune cells or pharmaceutical composition are administered in a single dose (e.g., by pellet injection). In other embodiments, the modified antigen-specific immune cells or pharmaceutical composition are administered multiple times (e.g., any one of 2, 3, 4, 5, 6, or more times). If administered multiple times, they can be carried out via the same or different routes and can occur at the same or alternative sites. The pharmaceutical composition can be administered at a suitable frequency, such as once daily to once a year. Those skilled in the medical field can easily determine the optimal dosage and treatment regimen for a particular patient by monitoring signs of the patient's disease and adjusting treatment accordingly.

[0228] In some embodiments, the method includes administering a modified antigen-specific immune cell or pharmaceutical composition approximately every 7, 10, 14, 21, or 30 days. In some embodiments, the method includes administering a modified antigen-specific immune cell or pharmaceutical composition approximately every 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, the method includes administering a modified antigen-specific immune cell or pharmaceutical composition every 1, 2, 3, 4, 5, 6, 7, or 8 months. In some embodiments, the individual to be 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, cattle, etc. In some embodiments, the individual is a human.

[0229] Pharmaceutical Composition

[0230] This application further provides pharmaceutical compositions comprising any of the modified antigen-specific immune cells described herein, and optionally a pharmaceutically acceptable carrier.

[0231] The applicant's pharmaceutical compositions may include any number of modified antigen-specific immune cells. In some embodiments, the pharmaceutical composition includes a single copy of a modified antigen-specific immune cell. In some embodiments, the pharmaceutical composition includes at least about 1, 10, 100, 1000, or 10 4 10 5 10 6 10 7 10 8 10 9Any of one or more copies of modified antigen-specific immune cells. In some embodiments, the pharmaceutical composition comprises at least about 0.1, 0.2, 0.5, 0.75, 1, 2, 5, 10, 20, 50, 100, 200, or 500 million modified antigen-specific immune cells. In some embodiments, the pharmaceutical composition comprises at least about 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 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 in their cell origin, cell type, expressed chimeric receptors, and / or promoters.

[0232] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cells or individuals exposed thereto at the doses and concentrations used. Typically, physiologically acceptable carriers are aqueous pH buffer solutions. Examples of suitable drug 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, etc. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used.

[0233] Pharmaceutical compositions including such carriers can be formulated using 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 sucrose solution, Hank's solution, and other physiologically balanced saline solutions (see, for example, the latest edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins).

[0234] The pharmaceutical compositions described herein can be administered via any suitable route. In some embodiments, the pharmaceutical compositions are administered parenterally, percutaneously (into the dermis), intracavitarily, intra-arterially (into the artery), intramuscularly (into the muscle), intrathecally, or intravenously. In some embodiments, the pharmaceutical compositions are administered subcutaneously (under the skin). In some embodiments, the pharmaceutical compositions are administered intravenously. In some embodiments, the pharmaceutical compositions are administered to an individual via infusion or injection. In some embodiments, the pharmaceutical compositions are administered directly to a target site, for example, by delivery via a biological projectile to an internal or external target site or via a catheter to a site in an artery. In some embodiments, the pharmaceutical compositions are administered locally, such as intratumorally. Administration may be performed using conventional syringes and needles or any compound or device available in the art capable of facilitating or improving the delivery of the active agent in the subject.

[0235] Parenteral formulations 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, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral media include sodium chloride solutions, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextran), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. Furthermore, the pharmaceutical compositions of this disclosure may include protein carriers, such as serum albumin or immunoglobulins, preferably of human origin. Various viral preparations are available in the art in frozen, liquid, or lyophilized forms (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 involving vacuum drying and freeze-drying (see, for example, WO2014 / 053571). It is envisioned that, in addition to the modified antigen-specific immune cells described herein, the pharmaceutical compositions of this disclosure may include other bioactive agents, depending on the intended use of the pharmaceutical composition.

[0236] In some embodiments, the pharmaceutical composition is appropriately buffered for human use. Suitable buffers include, but are not limited to, phosphate buffers (e.g., PBS), bicarbonate buffers, and / or Tris buffers capable of maintaining a physiological or slightly alkaline pH (e.g., from about pH 7 to about pH 9). In some embodiments, the pharmaceutical composition may also be isotonic with blood by adding a suitable tension modifier, such as glycerol.

[0237] 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 reusable vial. In some embodiments, the pharmaceutical composition is contained in a large quantity in a container.

[0238] In some embodiments, the pharmaceutical composition must meet certain standards for individual administration. For example, the U.S. Food and Drug Administration has issued regulatory guidance setting standards for cell-based immunotherapy products, including 21 CFR 610 and 21 CFR 610.13. Methods for assessing the appearance, identity, purity, safety, and / or efficacy of a pharmaceutical composition are known in the art. In some embodiments, the pharmaceutical composition is substantially free of foreign proteins capable of producing allergic effects, such as animal-derived proteins used for cell culture, rather than modified antigen-specific immune cells. In some embodiments, "substantially free" means less than about 10%, 5%, 1%, 0.1%, 0.01%, 0.001%, 1 ppm, or less of the total volume or weight of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is prepared in a GMP-level facility. In some embodiments, the pharmaceutical composition includes less than about 5 EU / kg body weight / hour 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 “growth-free” result when evaluated using the 14-day direct inoculation test method described in the United States Pharmacopeia (USP). In some embodiments, a sample comprising both modified antigen-specific immune cells and pharmaceutically acceptable excipients should be collected for sterility testing approximately 48-72 hours prior to final harvest (or consistent with the last refeeding of the culture) before administration of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is free from mycoplasma contamination. In some embodiments, the pharmaceutical composition is free from detectable microbial agents. In some embodiments, the pharmaceutical composition is free from agents of infectious diseases such as HIV type I, HIV type II, HBV, HCV, human T-lymphovirus type I, and human T-lymphovirus type II.

[0239] 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 at least partially conferred by a functional exogenous receptor, such as, but not limited to, CAR and TCR. In some embodiments, the modified antigen-specific immune cells target tumor-associated antigens, mutated oncogenic 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 one or more modifications of TCR-T cells, CAR-T cells, TILs, or endogenous antigen-specific T cells. Examples of human and mouse TCR-T cells, CAR-T cells, TILs, or endogenous antigen-specific T cells have been 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 spectrum of antigens. In some embodiments, the modified antigen-specific immune cells target one or more of the antigens listed in Table 1.

[0240] IV. Regulating the immunostimulatory activity of CD160 in antigen-specific immune cells

[0241] 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 for modulating the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0242] In some aspects, methods for identifying modulators of endogenous CD160 expression, function, or activity are also provided, including contacting CD160-expressing immune cells (e.g., NK cells) with a test reagent; and measuring the expression and / or function of effectors of cytolytic or inflammatory pathways in the tested immune cells. In one embodiment, the test reagent is identified as a modulator of CD160 expression, function, or activity if the reagent modulates the expression and / or function of effectors of cytolytic or inflammatory pathways 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.

[0243] In some aspects, methods are provided for identifying modulators of endogenous CD160 expression, function, or activity, including contacting antigen-specific immune cells (e.g., NK cells) expressing CD160 with a test reagent; and measuring in vivo immune function induced by the tested immune cells, such as cytokine secretion by the immune cells after antigen challenge. In one embodiment, the test reagent is identified as a modulator of CD160 expression, function, or activity if, compared to control immune cells, the reagent modulates the expression and / or function of effectors in cytolytic or inflammatory pathways in the tested immune cells. Preferably, the expression, function, or activity of CD160 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.

[0244] One aspect of the invention provides a method for treating an immunological 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 immunological disease is an autoimmune disease or an inflammatory disease, and wherein the agent inhibits the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0245] Inhibits the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0246] One aspect of the present invention provides a method for inhibiting the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells, comprising contacting the antigen-specific immune cells with an effective amount of an agent that inhibits the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0247] In some embodiments, a method for treating an individual's autoimmune disease is provided, 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, a method for treating an individual's inflammatory disease is provided, 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.

[0248] In some embodiments, the agent inhibiting the endogenous immunostimulatory activity of the CD160 protein includes an antagonistic antibody. In some embodiments, the agent inhibiting the endogenous immunostimulatory activity of the CD160 protein includes an antagonistic protein. In some embodiments, the agent inhibiting the endogenous immunostimulatory activity of the CD160 protein includes 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 the following: siRNA, shRNA, or miRNA. In some embodiments, the agent inhibiting the endogenous immunostimulatory activity of the CD160 protein includes a small molecule. In some embodiments, the agent includes a dominant-negative form of the CD160 protein.

[0249] In some embodiments, the reagent inhibits the endogenous immunostimulatory activity of CD160 by any one of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the reagent inhibits the endogenous immunostimulatory activity of CD160 by any one of about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 times.

[0250] In some embodiments, methods of treating an autoimmune response include suppressing the immune response and / or inducing tolerance. Reducing the autoimmune response may include, but is not limited to, reducing the immune response or inducing tolerance against antigens associated with type 1 diabetes, rheumatoid arthritis, psoriasis, multiple sclerosis, Alzheimer's disease, ALS, Huntington's disease, Parkinson's disease, systemic lupus erythematosus, Sjögren's disease, Crohn's disease, or ulcerative colitis. In some embodiments, suppressing the immune response and / or inducing tolerance includes reducing allergic reactions. For example, reducing allergic reactions may include reducing the immune response or inducing tolerance against 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 the immune response and / or inducing tolerance includes reducing the immune response or inducing tolerance against transplanted tissue. In some embodiments, the antigen is viral. In some embodiments, suppressing the immune response and / or inducing tolerance includes reducing the pathogenic immune response or inducing tolerance against a virus. For example, a pathogenic immune response may include a cytokine storm generated by certain viruses. A cytokine storm is a potentially lethal immune response consisting of a positive feedback loop between cytokines and leukocytes. Therefore, in some implementations, suppressing the immune response and / or inducing tolerance includes reducing or eliminating the cytokine storm.

[0251] In some embodiments, the antigen recognized by 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 peptide or a fragment of a therapeutic peptide. 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.

[0252] Activate the immunostimulatory activity of CD160 in immune cells

[0253] In some embodiments, a method for activating the immunostimulatory activity of CD160 in antigen-specific immune cells includes 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, and the agent enhances the endogenous immunostimulatory activity of CD160 in the antigen-specific immune cells. In some embodiments, the method includes contacting an exogenous CD160 protein with the antigen-specific immune cells. In some embodiments, the method includes contacting a nucleotide encoding an exogenous CD160 protein with the antigen-specific immune cells.

[0254] In some embodiments, a method for treating cancer in an individual is provided, 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, a method for treating an infection in an individual is provided, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0255] In some embodiments, the agent activating the immunostimulatory activity of the CD160 protein includes an agonist peptide or a protein. In some embodiments, the agent includes a small molecule. In some embodiments, the agent activating the endogenous immunostimulatory activity of the CD160 protein includes an agonist antibody.

[0256] In some embodiments, the reagent that activates the endogenous immunostimulatory activity of the CD160 protein includes one or more nucleic acids. In some embodiments, the reagent is DNA and / or mRNA.

[0257] In some embodiments, the reagent activates the immunostimulatory activity of CD160 in antigen-specific immune cells, wherein the antigen-specific immune cells do not exhibit detectable CD160 activity prior to contact with the reagent. In some embodiments, the reagent enhances the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells. In some embodiments, the reagent enhances the endogenous immunostimulatory activity of CD160 by any one of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the reagent enhances the endogenous immunostimulatory activity of CD160 by any one of about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 times.

[0258] In some embodiments, methods of treating cancer include increasing the immune response to tumor antigens or tumor-associated antigens. In some embodiments, the antigen recognized by antigen-specific immune cells is a protein. In some embodiments, the antigen-specific immune cells are specific to tumor antigens or tumor-associated antigens.In some embodiments, tumor-associated antigens are selected from the group consisting of: mesotheliin, EGFRvIII, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, GD2, GD3, BCMA, TnAg, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, and interleukin-11 receptor α (IL-1 lRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-β (PDGFR-β), SSEA-4, CD20, folate receptor α (FRa), ERBB2 (Her2 / neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, prostaglandin B2, PAP, ELF2M, liver glycoside B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, pod protein, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B l, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyesterase, 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 novel antibody, such as a novel cancer-related antibody. In some embodiments, the antigen includes a novel epitope, such as a novel cancer-related epitope.

[0259] In some embodiments, a method for treating an infection in an individual is provided, comprising administering to the individual a therapeutically effective amount of an agent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0260] In some embodiments, methods of treating an infection include increasing an immune response to an antigen associated with an 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.

[0261] V. Methods for treating cancer based on the level or activity of CD160 in the tumor environment.

[0262] One aspect of the invention relates to a method of treating cancer, wherein CD160 can be used as a biomarker to predict the functional state of antigen-specific T cells and thus the efficacy of immunotherapy. Higher CD160 expression in those cells indicates an activated state of antigen-specific T cells, while low levels or absence of CD160 expression indicates an inactivated state of antigen-specific T cells.

[0263] Therefore, in some embodiments, a method of treating an individual's cancer is provided, comprising administering to the individual a therapeutically effective amount of a composition comprising antigen-specific immune cells, wherein the individual's endogenous CD160 level or activity serves as the basis for selecting the individual for treatment. In some embodiments, an individual is selected for treatment if they have high CD160 levels or activity. In some embodiments, an individual is selected for treatment if they have low CD160 levels or activity. In some embodiments, CD160 levels or activity are determined by immunohistochemical methods. In some embodiments, CD160 levels or activity are based on CD160 protein expression levels. In some embodiments, CD160 levels or activity are based on CD160 mRNA levels. In some embodiments, CD160 levels or activity are measured in the individual's tumor. In some embodiments, CD160 levels or activity are measured in the individual's tumor-infiltrating T cells (TILs). In some embodiments, CD160 levels or activity are measured in the individual's peripheral T cells.

[0264] In some embodiments, a method of treating an individual's cancer is provided, comprising administering to the individual a therapeutically effective amount of a composition comprising modified antigen-specific immune cells containing (e.g., on their surface) a foreign CD160 protein, wherein the foreign CD160 protein causes upregulation of the modified antigen-specific immune cells compared to precursor antigen-specific immune cells that do not contain the foreign CD160 protein, wherein the immune cells are T cells, and wherein the individual's endogenous CD160 level or activity serves as a basis for selecting the individual for treatment. In some embodiments, the individual is selected for treatment if they have high CD160 levels or activity. In some embodiments, the individual is selected for treatment if they have low CD160 levels or activity. In some embodiments, CD160 levels are determined by immunohistochemical methods. In some embodiments, CD160 levels or activity are based on CD160 protein expression levels. In some embodiments, CD160 levels or activity are based on CD160 mRNA levels. In some embodiments, CD160 levels or activity are measured in the individual's tumor. In some embodiments, the level or activity of CD160 in an individual's tumor-infiltrating T cells (TILs) is measured. In some embodiments, the level or activity of CD160 in an individual's peripheral T cells is measured.

[0265] In some embodiments, a method of treating an individual's cancer includes administering to the individual a therapeutically effective amount of a composition comprising one or more immune checkpoint inhibitors, wherein the individual's endogenous CD160 level or activity serves as a basis for selecting the individual for treatment. In some embodiments, an individual is selected for treatment if they have high CD160 levels or activity. In some embodiments, an individual is selected for treatment if they have low CD160 levels or activity. In some embodiments, CD160 levels are determined by immunohistochemical methods. In some embodiments, CD160 levels or activity are based on CD160 protein expression levels. In some embodiments, CD160 levels or activity are based on CD160 mRNA levels. In some embodiments, CD160 levels or activity are measured in the individual's tumor. In some embodiments, CD160 levels or activity are measured in the individual's tumor-infiltrating T cells (TILs). In some embodiments, CD160 levels or activity are measured in the individual's peripheral T cells. In some implementations, immune checkpoint inhibitors target any one of PD-1, PD-L1, CTLA-4, TIM-3, LAG3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA.

[0266] In some embodiments, a method of treating an individual's cancer is provided, comprising administering to the individual a therapeutically effective amount of a composition comprising an immunostimulatory activity that activates CD160, wherein the individual's endogenous CD160 level or activity serves as the basis for selecting the individual for treatment. In some embodiments, an individual is selected for treatment if they have high CD160 levels or activity. In some embodiments, an individual is selected for treatment if they have low CD160 levels or activity. In some embodiments, CD160 levels are determined by immunohistochemical methods. In some embodiments, CD160 levels or activity are based on CD160 protein expression levels. In some embodiments, CD160 levels or activity are based on CD160 mRNA levels. In some embodiments, CD160 levels or activity are measured in an individual's tumor. In some embodiments, CD160 levels or activity are measured in an individual's tumor-infiltrating T cells (TILs). In some embodiments, CD160 levels or activity are measured in an individual's peripheral T cells. In some embodiments, the agent activating the immunostimulatory activity of the CD160 protein comprises an agonist peptide or a protein. In some embodiments, the agent comprises a small molecule. In some embodiments, the reagent that activates the endogenous immunostimulatory activity of the CD160 protein includes an agonist antibody. In some embodiments, the reagent that activates the endogenous immunostimulatory activity of the CD160 protein includes one or more nucleic acids. In some embodiments, the reagent is DNA and / or mRNA. In some embodiments, the reagent activates the immunostimulatory activity of CD160 in antigen-specific immune cells, wherein the antigen-specific immune cells do not exhibit detectable CD160 activity prior to contact with the reagent. In some embodiments, the reagent enhances the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells. In some embodiments, the reagent enhances the endogenous immunostimulatory activity of CD160 by any one of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the reagent enhances the endogenous immunostimulatory activity of CD160 by any one of approximately 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 times.

[0267] In other aspects, methods are provided for selecting (including identifying) individuals with cancer (such as melanoma or lung cancer) to treat with a composition comprising a therapeutic agent, wherein the method includes determining the individual's CD160 level or activity. In some embodiments, methods are provided for selecting (including identifying) individuals with cancer (such as melanoma or lung cancer) to treat with a composition comprising immunotherapy, wherein the method includes determining the individual's CD160 level or activity. 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, CD160 levels are determined based on protein expression levels. In some embodiments, CD160 levels are determined based on mRNA levels. In some embodiments, CD160 levels are determined by immunohistochemical assay.

[0268] In some embodiments, the level (e.g., high or low) is determined by comparison with a control (such as any of the controls described herein). In some embodiments, the method further includes comparing CD160 levels or activity with a control. In some embodiments, the level (e.g., high or low) is determined based on a scoring system, such as the H-scoring system described herein. Control samples can be obtained using the same sources and methods as non-control samples. In some embodiments, control samples are obtained from different individuals (e.g., individuals without cancer and / or individuals sharing similar racial, age, and sex characteristics). In some embodiments, when the sample is a tumor tissue sample, the control sample can be a non-cancerous sample from the same individual. In some embodiments, multiple control samples (e.g., from different individuals) are used to determine a range of CD160 activity levels in a particular tissue, organ, or cell population. In some embodiments, the control sample is a cultured tissue or cell that has been identified as a suitable control. In some embodiments, the control is a cell that does not express CD160. In some embodiments, the control is a cell that expresses high levels of CD160. In some embodiments, clinically accepted normal levels in standardized tests are used as control levels to determine CD160 activity in the relevant tissue. In some implementations, reference CD160 levels or activity in subjects are classified as high, intermediate, or low based on a scoring system, such as an immunohistochemical scoring system for CD160 staining, like the H-score discussed further herein. In some implementations, reference CD160 levels or activity in subjects are classified as low samples when the H-score is less than or equal to the total median H-score.

[0269] In some embodiments, a method of treating an individual with cancer is provided, comprising administering to the individual a therapeutically effective amount of a composition comprising modified antigen-specific immune cells containing functional exogenous receptors on their surface, wherein the immune cells are T cells, and wherein the individual's CD160 level or activity serves as the basis for selecting modified antigen-specific immune cells for cancer treatment. In some embodiments, modified antigen-specific immune cells are selected for treatment if the cells have high CD160 levels or activity. In some embodiments, modified antigen-specific immune cells are selected for treatment if the cells have low CD160 levels or activity. In some embodiments, CD160 levels are determined by immunohistochemical methods. In some embodiments, CD160 levels or activity are based on CD160 protein expression levels. In some embodiments, CD160 levels or activity are based on CD160 mRNA levels. In some embodiments, CD160 levels or activity are compared with precursor immune cells that do not include exogenous functional receptors. In some embodiments, the CD160 level or activity of antigen-specific immune cells modified with exogenous functional receptors on their surface is compared with that of antigen-specific immune cells modified with exogenous CD160 protein on their surface.

[0270] In some embodiments of any of the methods described herein, CD160 levels are determined based on CD160 protein expression levels. In some embodiments, CD160 levels are determined based on mRNA levels. In some embodiments, nucleoside transporter levels are determined by immunohistochemical assays. In some embodiments, levels (e.g., high or low) are determined by comparison with a control (such as any control described herein). In some embodiments, levels (e.g., high or low) are determined based on a scoring system, such as the H-score system described herein. In some embodiments, the scoring is based on the “H-score” as described in U.S. Patent Publication No. 2013 / 0005678. The H-score is obtained by the following formula: 3 × percentage of strongly stained cells + 2 × percentage of moderately stained cells + percentage of weakly stained cells, resulting in a range of 0 to 300.

[0271] VI. Reagent kits and products

[0272] Kits, unit doses, and articles thereof are also provided, comprising any of the modified antigen-specific immune cells or compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, kits containing any of the pharmaceutical compositions described herein and preferably instructions for use thereof are provided. In some embodiments, in addition to modified antigen-specific immune cells, the kit further comprises a secondary cancer therapy, such as chemotherapy, hormone therapy, and / or immunotherapy. The kit can be tailored to an individual's specific cancer and includes a corresponding secondary cancer therapy for that individual.

[0273] Kits, unit doses, and preparations are also provided that include any regulator (such as an inhibitor or activator) of CD160 expression, function, or activity, or any agent that modulates (such as inhibiting or activating) CD160 activity.

[0274] The kit may contain one or more additional components, such as containers, reagents, culture media, inducers, cytokines, buffers, antibodies, etc., to allow for the proliferation or induction of modified antigen-specific immune cells. The kit may also contain a device for the local application (e.g., intratumoral injection) of the drug composition to the tumor site.

[0275] In another embodiment, a kit is provided comprising 1) a composition including modified antigen-specific cells containing an exogenous functional receptor (e.g., CAR), a modulator of CD160 activity, and / or an immunotherapy (e.g., an immune checkpoint inhibitor) and 2) a reagent for determining CD160 levels or activity. In some embodiments, the reagent for determining CD160 expression levels is an antibody that recognizes the CD160 protein.

[0276] The kit of this application is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth flasks, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally include additional components, such as buffers and explanatory information. This application therefore also provides articles of manufacture, including vials (e.g., sealed vials), bottles, wide-mouth flasks, flexible packaging, etc. Some components of the kit may be packaged in aqueous media or lyophilized form.

[0277] Articles may include containers and labels or packaging inserts on or associated with the containers. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from various materials such as glass or plastic. Generally, containers contain compositions effective for treating the diseases or disorders described herein (such as cancer) and may have a sterile inlet (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). Labels or packaging inserts indicate that the composition is intended to treat the specific condition of an individual. Labels or packaging inserts will further include instructions for administering the composition to an individual. Labels may indicate instructions for reconstitution and / or use. Containers containing pharmaceutical compositions may be multi-purpose vials that allow for repeated administration of the reconstituted formulation (e.g., 2-6 administrations). Packaging inserts refer to instructions typically included in the commercial packaging of therapeutic products, containing information on the indications, usage, dosage, administration, contraindications, and / or warnings for using such therapeutic products. Furthermore, the article may further include a second container comprising pharmaceutically acceptable buffer solutions, such as antibacterial water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. From a commercial and user perspective, it may further include other desired materials, including additional buffers, diluents, filters, needles, and syringes.

[0278] The kit or product may include a pharmaceutical composition in multiple unit doses and instructions for use, packaged in quantities sufficient for storage and use in pharmacies such as hospital pharmacies and multi-purpose pharmacies.

[0279] Exemplary Implementation

[0280] The present invention provides the following listed embodiments.

[0281] Implementation 1. An antigen-specific immune cell having a modified exogenous CD160 protein on its surface, wherein the exogenous CD160 protein causes upregulation of the modified antigen-specific immune cell, wherein the immune cell is a T cell, compared to a precursor antigen-specific immune cell that does not contain the exogenous CD160 protein.

[0282] Implementation Method 2. The modified antigen-specific immune cells according to Implementation Method 1, 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, antigen-presenting cell (APC)-activated anti-tumor T cells, and natural killer T cells (NK-T cells).

[0283] Implementation Method 3. The modified antigen-specific immune cells according to Implementation Method 1, wherein the modified antigen-specific immune cells are cytotoxic T cells.

[0284] Implementation Method 4. The modified antigen-specific immune cells according to Implementation Method 2, wherein the modified antigen-specific immune cells are tumor-infiltrating T cells or APC-activated anti-tumor T cells.

[0285] Implementation Method 5. The modified antigen-specific immune cells according to Implementation Method 1, wherein 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, γδT cells and macrophages.

[0286] Embodiment 6. An antigen-specific immune cell modified according to any one of Embodiments 1-5, wherein the exogenous CD160 protein comprises the amino acid sequence of any one of SEQ ID NO: 1-4, or a variant thereof having at least about 90% identity with any one of SEQ ID NO: 1-4.

[0287] Implementation Method 7. An antigen-specific immune cell modified according to any one of Implementation Methods 1-5, wherein the exogenous CD160 protein is membrane-bound.

[0288] Implementation Method 8. The modified antigen-specific immune cell according to Implementation Method 7, wherein the exogenous CD160 protein is bound to the membrane via a GPI linker.

[0289] Implementation Method 9. The modified antigen-specific immune cell according to Implementation Method 7, wherein the exogenous CD160 protein includes a transmembrane domain.

[0290] Implementation 10. The modified antigen-specific immune cell according to Implementation 9, wherein the exogenous CD160 protein further includes an intracellular domain.

[0291] Implementation 11. Modified antigen-specific immune cells according to Implementation 9 or 10, wherein the exogenous CD160 protein further includes an intracellular domain derived from a CD160 splice variant.

[0292] Implementation Method 12. The modified antigen-specific immune cell according to Implementation Method 10, wherein the intracellular domain includes an intracellular signal transduction domain derived from the signal transduction subunit of the TCR complex.

[0293] Implementation Method 13. The modified antigen-specific immune cell according to Implementation Method 12, wherein the signal transduction subunit of the TCR complex is selected from the group consisting of CD3γ, CD3δ and CD3ε.

[0294] Implementation Method 14. The modified antigen-specific immune cell according to Implementation Method 10, wherein the intracellular domains include a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, or both.

[0295] Implementation Method 15. The modified antigen-specific immune cell according to Implementation Method 14, wherein the exogenous CD160 protein comprises, from the N-terminus to the C-terminus, an extracellular CD160 domain, a transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0296] Implementation Method 16. The modified antigen-specific immune cell according to Implementation Method 14, wherein the exogenous CD160 protein comprises, from the N-terminus to the C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain.

[0297] Implementation 17. An antigen-specific immune cell modified according to any one of Implementations 10-16, wherein the intracellular structural domains include primary signal transduction domains.

[0298] Implementation 18. The modified antigen-specific immune cell according to Implementation 17, wherein the primary signal transduction domain includes the CD3ζ domain.

[0299] Implementation 19. An antigen-specific immune cell modified according to any one of Implementations 10-16, wherein the intracellular structural domains do not include primary signal transduction domains.

[0300] Implementation Method 20. The modified antigen-specific immune cell according to Implementation Method 7, wherein the exogenous CD160 protein is bound to the modified antigen-specific immune cell via the immune cell binding portion.

[0301] Implementation Method 21. The modified antigen-specific immune cell according to Implementation Method 20, wherein the immune cell binding portion binds to the surface molecules of the immune cell.

[0302] Implementation Method 22. Modified antigen-specific immune cells according to any one of Implementation Methods 1-21, wherein the modified antigen-specific immune cells further include a functional exogenous receptor.

[0303] Implementation Method 23. The modified antigen-specific immune cell according to Implementation Method 22, wherein the functional exogenous receptor is an engineered T cell receptor (TCR).

[0304] Implementation Method 24. Modified antigen-specific immune cells according to Implementation Method 22, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR).

[0305] Implementation Method 25. A method for generating antigen-specific immune cells whose surface includes a modified exogenous CD160 protein, comprising:

[0306] Modified antigen-specific immune cells are generated by contacting precursor antigen-specific immune cells with exogenous CD160 protein or the first nucleic acid encoding exogenous CD160 protein.

[0307] Compared with precursor antigen-specific immune cells, exogenous CD160 protein leads to the upregulation of modified antigen-specific immune cells, of which T cells are immune cells.

[0308] Implementation Method 26. According to the method of Implementation Method 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, APC-activated anti-tumor T cells, and natural killer T cells (NK-T cells).

[0309] Implementation 27. The method according to Implementation 25, wherein the modified antigen-specific immune cell is a cytotoxic T cell.

[0310] Implementation 28. The method according to Implementation 26, wherein the modified antigen-specific immune cell is a tumor-infiltrating T cell or an APC-activated anti-tumor T cell.

[0311] Implementation 29. The method according to Implementation 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, γδT cells and macrophages.

[0312] Implementation 30. The method according to any one of Implementations 25-29, wherein the method includes contacting precursor antigen-specific immune cells with exogenous CD160 protein.

[0313] Implementation 31. The method according to Implementation 30, wherein the exogenous CD160 protein includes an immune cell binding portion of a surface molecule that binds to immune cells.

[0314] Implementation Method 32. The method according to any one of Implementation Methods 25-29, wherein the method includes introducing a nucleic acid encoding an exogenous CD160 protein into a precursor antigen-specific immune cell.

[0315] Implementation Method 33. The method according to Implementation Method 32, wherein the nucleic acid is mRNA.

[0316] Implementation Method 34. The method according to Implementation Method 32, wherein the nucleic acid is DNA.

[0317] Implementation Method 35. The method according to any one of Implementation Methods 32-34, wherein nucleic acid is introduced into precursor antigen-specific immune cells by transfection.

[0318] Implementation 36. The method according to any one of Implementations 32-34, wherein nucleic acids are introduced into precursor antigen-specific immune cells by transduction or electroporation.

[0319] Implementation 37. The method according to any one of Implementations 25-36, wherein the CD160 protein comprises the amino acid sequence of any one of SEQ ID NO:1-4, or a variant thereof having at least about 90% identity with any one of SEQ ID NO:1-4.

[0320] Implementation Method 38. The method according to any one of Implementation Methods 25-37, wherein the exogenous CD160 protein is membrane-bound.

[0321] Implementation Method 39. The method according to Implementation Method 38, wherein the exogenous CD160 protein is bound to the membrane via a GPI linker.

[0322] Implementation 40. The method according to Implementation 38, wherein the exogenous CD160 protein includes a transmembrane domain.

[0323] Implementation 41. The method according to Implementation 39, wherein the exogenous CD160 protein further includes an intracellular domain.

[0324] Implementation 42. The method according to implementation 40 or 41, wherein the exogenous CD160 protein further includes an intracellular domain from a CD160 splice variant.

[0325] Implementation 43. The method according to implementation 41, wherein the intracellular domain includes an intracellular signal transduction domain derived from the signal transduction subunit of the TCR complex.

[0326] Implementation 44. The modified antigen-specific immune cell according to Implementation 43, wherein the signal transduction subunit of the TCR complex is selected from the group consisting of CD3γ, CD3δ and CD3ε.

[0327] Implementation 45. The method according to implementation 41, wherein the intracellular domain includes a CD28 costimulatory domain, a 4-1BB costimulatory domain, or both.

[0328] Implementation 46. The method according to Implementation 45, wherein the exogenous CD160 protein comprises, from the N-terminus to the C-terminus, an extracellular CD160 domain, a transmembrane domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0329] Implementation 47. The method according to Implementation 45, wherein the exogenous CD160 protein comprises, from the N-terminus to the C-terminus, an extracellular CD160 domain, a transmembrane domain, a 4-1BB costimulatory domain, and a CD28 costimulatory domain.

[0330] Implementation 48. The method according to any one of Implementations 41-47, wherein the intracellular structural domain includes a primary signal transduction structural domain.

[0331] Implementation 49. The method according to implementation 48, wherein the primary signal transduction structure domain includes the CD3ζ structure domain.

[0332] Implementation 50. The method according to any one of Implementations 41-47, wherein the intracellular structural domains do not include primary signal transduction domains.

[0333] Implementation 51. The method according to Implementation 38, wherein the exogenous CD160 protein is bound to the modified antigen-specific immune cell via the immune cell binding portion.

[0334] Implementation 52. The method according to Implementation 51, wherein the immune cell binding portion binds to the surface molecules of the immune cell.

[0335] Implementation 53. The method according to any one of Implementations 25-52, wherein the precursor antigen-specific immune cells comprise a second nucleic acid encoding a functional exogenous receptor.

[0336] Implementation 54. The method according to any one of Implementations 25-52, further comprising contacting a precursor antigen-specific immune cell with a second nucleic acid encoding a functional exogenous receptor.

[0337] Implementation 55. The method according to implementation 53 or 54, wherein the functional exogenous receptor is an engineered T-cell receptor (TCR).

[0338] Implementation 56. The method according to implementation 53 or 54, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR).

[0339] Implementation 57. The method according to any one of Implementations 54-56, wherein the first nucleic acid and the second nucleic acid are operatively linked to the same promoter.

[0340] Implementation 58. The method according to any one of Implementations 54-56, wherein the first nucleic acid and the second nucleic acid are operatively linked to different promoters.

[0341] Implementation 59. The method according to any one of Implementations 54-58, wherein the first nucleic acid and the second nucleic acid are on the same vector.

[0342] Implementation 60. The method according to any one of Implementations 54-59, wherein the first nucleic acid and / or the second nucleic acid are on different vectors.

[0343] Implementation method 61. The method according to implementation method 59 or 60, wherein the vector is a viral vector.

[0344] Implementation Method 62. The method according to Implementation Method 61, wherein the viral vector is selected from the group consisting of: adenovirus vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, augmentative expression vectors, herpes simplex virus vectors and their derivatives.

[0345] Implementation method 63. The method according to implementation method 59 or 60, wherein the vector is a non-viral vector.

[0346] Implementation 64. The method according to any one of Implementations 25-63, further comprising isolating or enriching immune cells comprising a first and / or a second nucleic acid.

[0347] Implementation 65. The method according to any one of Implementations 25-64, further comprising formulating modified antigen-specific immune cells expressing CD160 with at least one pharmaceutically acceptable carrier.

[0348] Implementation method 66. Modified antigen-specific immune cells obtained by any one of implementation methods 25-65.

[0349] Embodiment 67. A pharmaceutical composition comprising a modified antigen-specific immune cell and a pharmaceutically acceptable carrier as described in any one of Embodiments 1-24 and 66.

[0350] Implementation method 68. A method for treating an individual's disease, comprising administering to the individual an effective amount of modified antigen-specific immune cells according to any one of Implementation methods 1-24 and 66 or a pharmaceutical composition according to Implementation method 67.

[0351] Implementation 69. The method according to Implementation 68, wherein the modified antigen-specific immune cells are derived from an individual.

[0352] Implementation 70. A method of treating a disease in an individual, comprising administering to the individual an effective amount of exogenous CD160 protein or nucleic acid encoding exogenous CD160 protein, wherein the exogenous CD160 protein includes a binding portion that recognizes a surface molecule on an immune cell in the individual.

[0353] Implementation Method 71. The method according to any one of Implementation Methods 68-70, wherein the application is intratumoral application.

[0354] Implementation method 72. The method according to any one of implementation methods 68-70, wherein the application is administered into the lymph node.

[0355] Implementation method 73. The method according to any one of implementation methods 68-72, wherein the disease is cancer.

[0356] Implementation 74. The method according to implementation 73, wherein the cancer is a solid tumor.

[0357] Implementation 75. The method according to implementation 73 or 74, wherein the cancer is metastatic cancer.

[0358] Implementation Method 76. The method according to any one of Implementation Methods 73-75, 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.

[0359] Implementation method 77. The method according to any one of implementation methods 68-76, wherein the individual is a person.

[0360] Implementation Method 78. A method for inhibiting the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells, comprising contacting antigen-specific immune cells with an effective amount of a reagent that inhibits the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0361] Implementation Method 79. A method for activating the immunostimulatory activity of CD160 in antigen-specific immune cells, comprising contacting antigen-specific immune cells with an effective amount of a reagent for activating the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0362] Implementation Method 80. The method according to Implementation Method 79, wherein the method enhances the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells, and wherein the reagent enhances the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0363] Implementation Method 81. A method for treating an immunological disease in an individual, comprising administering to the individual a therapeutically effective amount of a reagent that modulates the endogenous immunostimulatory activity of CD160 in antigen-specific immune cells.

[0364] Implementation Method 82. The method according to Implementation Method 81, wherein the immunological disease is an autoimmune disease or an inflammatory disease, and wherein the reagent inhibits the endogenous immune-stimulating activity of CD160 in antigen-specific immune cells.

[0365] Implementation Method 83. A method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of a reagent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0366] Implementation Method 84. A method for treating an infection in an individual, comprising administering to the individual a therapeutically effective amount of a reagent that activates the immunostimulatory activity of CD160 in antigen-specific immune cells.

[0367] Implementation Method 85. A method for increasing the yield and / or viability of antigen-specific immune cells, comprising introducing a nucleic acid encoding an exogenous CD160 protein into immune cells.

[0368] Implementation Method 86. A method for increasing the yield and / or viability of antigen-specific immune cells, comprising inducing overexpression of the CD160 protein in immune cells.

[0369] Implementation Method 87. The method according to Implementation Method 86, wherein the CD160 protein is an endogenous protein.

[0370] Implementation Method 88. The method according to Implementation Method 86, wherein the CD160 protein is an exogenous protein.

[0371] Implementation Method 89. The method according to Implementation Method 85, wherein the yield of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least about: 0.5 times, 1 time, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times, or 10000 times compared with antigen-specific immune cells that do not express exogenous CD160 protein.

[0372] Implementation 90. The method according to Implementation 85, wherein the viability of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least about: 0.5 times, 1 time, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times, or 10000 times compared with antigen-specific immune cells that do not express exogenous CD160 protein.

[0373] Implementation 91. The method according to any one of Implementations 85-88, wherein the yield of antigen-specific immune cells overexpressing CD160 protein is increased by at least about: 0.5 times, 1 time, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times, or 10000 times compared with antigen-specific immune cells that do not overexpress CD160 protein.

[0374] Implementation 92. The method according to any one of Implementations 85-88, wherein the viability of antigen-specific immune cells overexpressing CD160 protein is increased by at least about: 0.5 times, 1 time, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times, or 10000 times compared with antigen-specific immune cells that do not overexpress CD160 protein.

[0375] Embodiment 93. A method for manufacturing therapeutic antigen-specific immune cells, comprising a method selected from the method according to any one of Embodiments 85 to 92 for increasing the yield and / or viability of antigen-specific immune cells.

[0376] Implementation 94. The method according to Implementation 93, wherein the therapeutic antigen-specific immune cells include tumor-infiltrating lymphocytes (TILs).

[0377] Implementation 95. The method according to Implementation 93, wherein the therapeutic antigen-specific immune cells include functional exogenous receptors.

[0378] Implementation 96. The method according to Implementation 95, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR).

[0379] Implementation 97. The method according to Implementation 95, wherein the functional exogenous receptor is an engineered T-cell receptor (TCR).

[0380] Implementation 98. A method for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells, comprising introducing a nucleic acid encoding an exogenous CD160 protein into the immune cells.

[0381] Implementation 99. A method for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells, comprising inducing overexpression of the CD160 protein in immune cells.

[0382] Implementation 100. The method according to Implementation 99, wherein the CD160 protein is an endogenous protein.

[0383] Implementation 101. The method according to Implementation 99, wherein the CD160 protein is an exogenous protein.

[0384] Implementation 102. The method according to Implementation 98, wherein, compared with antigen-specific immune cells that do not express exogenous CD160 protein, the in vitro cytolytic activity of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least about: 0.5 times, 1 time, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times, or 10000 times.

[0385] Implementation 103. The method according to Implementation 98, wherein, compared with antigen-specific immune cells that do not express exogenous CD160 protein, the in vivo cytolytic activity of antigen-specific immune cells expressing exogenous CD160 protein is increased by at least about: 0.5 times, 1 time, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times, or 10000 times.

[0386] Implementation Method 104. The method according to any one of Implementation Methods 98-101, wherein the in vitro cytolytic activity of antigen-specific immune cells overexpressing CD160 protein is increased by at least about: 0.5 times, 1 time, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times, or 10000 times compared with antigen-specific immune cells that do not overexpress CD160 protein.

[0387] Implementation 105. The method according to any one of Implementations 98-101, wherein, compared with antigen-specific immune cells that do not overexpress CD160 protein, the in vivo cytolytic activity of antigen-specific immune cells that overexpress CD160 protein is increased by at least about: 0.5 times, 1 time, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times, or 10000 times.

[0388] Embodiment 106. A method for manufacturing therapeutic antigen-specific immune cells, comprising a method selected from the method according to any one of Embodiments 98 to 105 for increasing the in vitro and / or in vivo cytolytic activity of antigen-specific immune cells.

[0389] Implementation 107. The method according to implementation 106, wherein the therapeutic antigen-specific immune cells include tumor-infiltrating lymphocytes (TILs).

[0390] Implementation 108. The method according to implementation 106, wherein the therapeutic antigen-specific immune cells include functional exogenous receptors.

[0391] Implementation 109. The method according to Implementation 108, wherein the functional exogenous receptor is a chimeric antigen receptor (CAR).

[0392] Implementation 110. The method according to Implementation 108, wherein the functional exogenous receptor is an engineered T-cell receptor (TCR). Example

[0393] Example 1: Ectopic expression of mCD160 in Pmel T cells

[0394] To examine the function of CD160 in immune cells, the GPI-anchored form of mouse CD160 was ectopically expressed in anti-tumor T cells and quantified by FACS analysis.

[0395] Specifically, full-length mouse CD160 (mCD160) was cloned into an MSCV-based retroviral vector and fused with a GFP reporter gene via a P2A spacer, thereby allowing the independent synthesis of CD160 and GFP proteins. Figure 1A The mCD160 virus was then transduced into Pmel T cells, which carried a mouse homolog of the human melanoma antigen GP100.

[0396] As indicated by FACS analysis, ectopic expression of CD160 led to an approximately 2.5-fold increase in CD160 expression on Pmel T cells, which typically express low levels of endogenous CD160. Figure 1B 、1C).

[0397] Example 2: CD160 expression enhances the CTL function of Pmel T cells against cultured B16F0 melanoma cells.

[0398] To demonstrate the effect of ectopic CD160 expression on the cytolytic function of T cells, the expression of granzyme A and perforin, the expression of inflammatory cytokines, and the cytotoxic activity of CD160-modified Pmel T cells were measured. Simulated infection Pmel T cells were used as a control.

[0399] Specifically, as described in Example 1, mCD160 virus was transfected into Pmel T cells, 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, used for T cell and NK cell-mediated killing. Figure 2AThe results showed that, compared with control Pmel T cells, the expression of granzyme A and perforin was increased, indicating that exogenous mCD160 enhanced the intrinsic CTL function of tumor-specific T cells.

[0400] Using FACS, the expression profiles of the inflammatory cytokines IFN-γ and TNF-α were measured in CD160-modified Pmel T cells compared to control Pmel T cells. Figure 2B The results showed that, compared with control Pmel T cells, the expression of IFN-γ and TNF-α was increased, indicating that exogenous mCD160 enhanced the inflammatory activity of tumor-specific T cells.

[0401] The ability of CD160-modified Pmel T cells to kill tumor cells was also examined and compared with control Pmel T cells. In short, Pmel T cells were stimulated with anti-CD3 and anti-CD28 beads and cultured for 3 days in T cell expansion medium containing IL-2. On day 3, target tumor cells B16F0 were warmed at 37°C for 10 min and treated with 1 μM CellTrace. TM Violet (Invitrogen) was used to label cells at 37°C for 30 min, and then seeded into 96-well culture plates. Control or CD160-modified Pmel T cells were added to each well at a defined effector-to-target cell ratio, and then incubated for four to six hours. Target cells were subsequently harvested, labeled with 7-aminoactinomycin D (7-AAD, BD Pharmingen), and analyzed by FACS to determine any killing effector T cells. TM A population of Violet dye+ / 7-AAD+ cells represents target cells that have been killed, and CELLTRACE TM The Violet dye + / 7-AAD- population represents the remaining live target cells. (e.g.) Figure 2C As shown, CD160-modified Pmel T cells were more effective at killing B16F0 melanoma cells in co-culture compared to control T cells.

[0402] In summary, the results show that ectopic expression of CD160 enhances intrinsic cytolytic activity, promotes inflammatory function, and improves the tumor-killing activity of antigen-specific T cells.

[0403] Example 3: CD160 expression enhanced the control of B16F0 melanoma by Pmel T cells in mice.

[0404] To examine whether ectopic CD160 can enhance the tumor control activity of antigen-specific T cells in vivo, CD160-modified Pmel T cells were adoptively transferred into recipient mice carrying subcutaneous B16F0 melanoma tumors.

[0405] Specifically, 1x10 5 B16F0 cells were subcutaneously injected into 6- to 8-week-old female C57BL / 6 mice. Mice were randomly assigned prior to adoptive cell transfer to ensure no size bias at the start of the experiment. In one experiment, on day 8 post-implantation, a single dose of 0.1, 0.2, 0.3, or 0.4 million CD160-modified Pmel T cells or 0.3 million control Pmel T cells was adoptively transferred into tumor-bearing mice. Figure 3A Tumor formation in mice was examined twice weekly by palpation, and tumor area was measured using calipers. Tumor area represents the mean measurement (+ / - SEM, two-tailed t-test) from at least 5 mice in each group. Figure 3A As shown in the study, CD160-modified Pmel exhibits a dose-dependent effect on tumor control in response to CD160-Pmel T cell metastasis.

[0406] In separate experiments, the effects of adoptive transfer of CD160-modified antigen-specific T cells and non-antigen-specific T cells on the control of B16F0 melanoma tumors were analyzed. Figure 3B In short, on day 8 post-implantation, a single dose of 0.3 million of each of the following types of cells were adoptively transferred into tumor-bearing mice: (i) control Pmel T cells, (ii) CD160-modified antigen-specific Pmel T cells, or (iii) CD160-modified non-antigen-specific spleen T cells. Figure 3B The study showed that, although non-antigen-specific splenic T cells had only a modest and statistically insignificant effect on tumor control, ectopic expression of CD160 significantly enhanced the tumor control activity of tumor-specific Pmel T cells.

[0407] In summary, these results indicate that ectopic CD160 expression in tumor-specific T cells enhances tumor control in syngeneically immunocompetent mouse tumor models.

[0408] Example 4: CD160-modified T cells can control and eliminate established B16F0 melanoma tumors without IL-2 and vaccination.

[0409] To examine the ability of CD160-modified Pme1 T cells to eliminate established tumors, CD160-Pmel T cells were adoptively transferred into mice carrying established B16F0 melanoma tumors after chemotherapy pretreatment.

[0410] In short, 1x10 5 B16F0 cells were subcutaneously injected into female C57BL / 6 mice aged 6 to 8 weeks. Mice were observed daily after tumor implantation and sacrificed when signs of disease appeared. Tumor formation was examined twice weekly by palpation, and tumor area was measured using calipers. Mice were randomly assigned before adoptive cell transfer to ensure no size bias at the start of the experiment. Starting on day 7 post-tumor implantation, mice were infused with Pmel T cells at 14-day intervals, with each T cell infusion preceded by a cyclophosphamide (CYP) pretreatment regimen (100 mg / kg per treatment), but without any vaccination or IL-2 infusion. The mean tumor size peaked on day 35 post-implantation. A normalized spider plot was plotted accordingly, with the peak tumor size on day 35 normalized to "1" to elucidate the relative changes in tumor size in response to treatment with control Pmel T cells or CD160-modified Pmel T cells. Figure 4A The study showed that adoptive transfer of CD160-modified Pmel T cells in mice resulted in a near 100% response rate, with tumors shrinking by more than 90% or being completely eliminated in over 80% of the mice.

[0411] The improved survival rate of adoptive transfer of CD160-modified Pmel T cells was also measured. Figure 4C As shown, in mice treated with CYP and CD160-modified Pmel T cells every two weeks, no death was observed up to 110 days post-implantation. In contrast, mice treated with CYP alone or with CYP and control Pmel T cells died before 75 days post-implantation and exhibited median survival at 27 days or 60 days post-implantation, respectively.

[0412] In summary, the results indicate that adoptive transfer of CD160-modified Pmel T cells with CYP pretreatment can effectively control and eliminate established B16F0 melanoma tumors, and in particular, without requiring any IL-2 cytokine or vaccination regimen.

[0413] Example 5: CD160-modified Pmel T cells showed dose-dependent control of B16F0 melanoma.

[0414] To characterize the dose-dependent effect of CD160-modified Pmel T cells on the control of B16F0 melanoma, mice carrying B16F0 were infused with 0.15 million or 0.3 million CD160-modified Pmel T cells.

[0415] In short, 1x10 5B16F0 cells were subcutaneously injected into female C57BL / 6 mice aged 6 to 8 weeks. Mice were observed daily after tumor implantation and euthanized when signs of disease appeared. Tumor formation was examined twice weekly by palpation, and tumor area was measured using calipers. Mice were randomly assigned prior to adoptive cell transfer to ensure no size bias at the start of the experiment. Starting on day 7 post-tumor implantation, mice were infused with either (A) 0.15 million or (B) 0.3 million CD160-modified Pmel T cells at 14-day intervals (every two weeks), following a CYP treatment regimen (100 mg / kg per treatment). Normalization and spider plotting were performed similarly as described in Example 4.

[0416] A dosage regimen involving the transfer of 0.15 million CD160-modified Pmel T cells every two weeks resulted in a 100% response rate to tumor control in treated mice, although only 20-30% of mice showed a tumor size reduction of more than 90%. Figure 5A In contrast, a dosage regimen of transferring 0.3 million CD160-Pmel T cells every two weeks resulted in a 100% response rate of tumor shrinkage in treated mice, with approximately 40-50% of mice showing a tumor size reduction of over 90%. Figure 5B ).

[0417] The survival improvement achieved by adoptive transfer of CD160-modified Pmel T cells at two doses was also measured. Tumor-carrying mice treated with CD160-modified Pmel T cells showed significant survival improvements at both doses, with mice treated with 0.15 or 0.3 million CD160-modified Pmel T cells, respectively, achieving 80% or 100% survival at 120 days post-implantation. Figure 5C Conversely, mice treated with CYP alone or with CYP and control Pmel T cells died before 90 days, with median survival of 61.5 days or 56 days post-implantation, respectively.

[0418] In summary, these results indicate that adoptive transfer of CD160-modified Pmel T cells with CYP pretreatment can effectively control and eliminate established B16F0 melanoma tumors, and that the tumor control effect is dose-dependent.

[0419] Example 6: CD160-modified Pmel T cells effectively control the growth of metastatic B16F10 melanoma tumors.

[0420] To examine the ability of CD160-modified Pmel1 T cells to suppress the growth of metastatic tumors, CD160-Pmel T cells were adoptively transferred into mice carrying metastatic B16F10 melanoma tumors after chemotherapy pretreatment.

[0421] In short, 1x10 5 B16F10 cells were subcutaneously injected into female C57BL / 6 mice aged 6 to 8 weeks. Mice were observed daily after tumor implantation and sacrificed when signs of disease appeared. Tumor formation was examined twice weekly by palpation, and tumor area was measured using calipers. Mice were randomly assigned before adoptive cell transfer to ensure no size bias at the start of the experiment. Starting on day 7 post-tumor implantation, mice were infused with 0.3 million CD160-modified Pmel T cells at 14-day intervals (every two weeks), with each T cell infusion preceded by a CYP treatment regimen (100 mg / kg per treatment).

[0422] like Figure 6A The results showed that CD160-modified Pmel T cells, when combined with CYP pretreatment, significantly prevented the increase in mean tumor size compared to untreated mice, mice treated with CYP chemotherapy alone, or mice treated with control Pmel T cells and CYP pretreatment. Tumor area represents the mean measurement (+ / - SEM, two-tailed t-test) from at least 5 mice in each group. Figure 6B This study demonstrated the ability of CD160-modified Pmel T cells (with CYP pretreatment) to control individual tumor growth compared to untreated, CYP-only, or treatment with control Pmel T cells and CYP.

[0423] In summary, compared with untreated mice, mice treated with CYP chemotherapy alone, or mice treated with control Pmel T cells and CYP pretreatment, adoptive transfer of CD160-modified Pmel T cells, when combined with CYP pretreatment, showed effective control of subcutaneous tumor growth.

[0424] The long-term tumor control and survival improvement of CD160-modified Pmel T cells were also examined. Spider plots of normalized tumor size were used, similar to those in Example 4.

[0425] like Figure 7A As shown, subcutaneous tumors can be controlled or eliminated by adoptive transfer of CD160-Pmel T cells during treatment. Figure 7B The results showed that untreated mice, mice treated with CYP chemotherapy alone, and mice treated with control Pmel T cells and CYP pretreatment all died before 80 days post-implantation due to metastasis, with median survival of 39 days, 39 days, and 59 days, respectively. Conversely, the group of mice treated with CD160-Pmel T cells and CYP pretreatment had a median survival of 120 days post-implantation. Figure 7BThe survival rate remained at 80% throughout the duration of continuous infusion of CD160-modified Pmel T cells (data not shown).

[0426] In summary, these results indicate that CD160-modified Pmel T cells, in addition to controlling the growth of subcutaneous tumors, can also help control and inhibit tumor metastasis.

[0427] Example 7: Enhanced tumor suppressor activity of mouse CD160-activated chimeras

[0428] To investigate whether the tumor suppressor activity of CD160 can be modulated by other domains, the extracellular domains of mouse CD160 were fused with intracellular signaling domains from the TCR and their co-stimulatory pathways in various configurations, including CD3ξ, CD28, and 4-1BB, to drive CD160 activation chimeras, such as... Figure 8A As shown in -C. The ability of Pmel T cells expressing these CD160 chimeras in established B16F0 melanoma mice was measured and compared with that expressing GPI-anchored mouse CD160 (mCD160).

[0429] In short, 1x10 5 B16F10 cells were subcutaneously injected into female C57BL / 6 mice aged 6 to 8 weeks. Mice were observed daily after tumor implantation and sacrificed when signs of disease appeared. Tumor formation was examined twice weekly by palpation, and tumor area was measured using calipers. Mice were randomly assigned before adoptive cell transfer to ensure no size bias at the start of the experiment. Starting on day 7 post-tumor implantation, mice were infused with 0.3 million Pmel T cells ectopically expressing mCD160, GEM 123, GEM124, GEM 125, GEM 126, GEM 127, or GEM 128 at 14-day intervals (every two weeks), with each T cell infusion preceded by a CYP treatment regimen (100 mg / kg per treatment).

[0430] like Figure 8A In the study, Pmel T cells expressing GEM 125 (a chimera with a CD28 signaling domain located distant from the transmembrane domain) exhibited weaker tumor control compared to Pmel T cells expressing mCD160; while Pmel T cells expressing GEM 124 (a chimera with a CD28 signaling domain located adjacent to the transmembrane domain) exhibited stronger tumor control compared to Pmel T cells expressing mCD160. These results suggest that a CD28 signaling domain located adjacent to the transmembrane domain can further enhance the ability of CD160 chimeras to enhance the immune response of antigen-specific T cells.

[0431] like Figure 8B In our observations, Pmel T cells expressing GEM 127 (a chimera with a 4-1BB signaling domain adjacent to a transmembrane domain) exhibited weaker tumor control compared to Pmel T cells expressing mCD160; while Pmel T cells expressing GEM 126 (a chimera with a CD28 signaling domain adjacent to a transmembrane domain) exhibited stronger tumor control compared to Pmel T cells expressing mCD160. These results suggest that the CD28 signaling domain, rather than the 4-1BB domain, when located adjacent to a transmembrane domain, can further enhance the ability of CD160 chimeras to enhance antigen-specific T cell immune responses.

[0432] like Figure 8C In our observations, Pmel T cells expressing GEM 123 (a chimera with a CD3ξ signaling domain adjacent to the transmembrane domain) exhibited weaker tumor control compared to Pmel T cells expressing mCD160. Furthermore, GEM 128, a chimera with three signaling domains, including a CD3ξ domain distal to the transmembrane domain, showed significantly lower tumor control compared to mCD160, despite the presence of a CD28 signaling domain in the adjacent transmembrane domain.

[0433] In summary, these results indicate that the CD28 co-stimulatory domain, when adjacent to the transmembrane domain, can further enhance the antigen-specificity of CD160 chimeric T cells. Figure 8A The immune response capacity of GEM 124 and GEM 126 in B. However, the ability of CD160 to enhance tumor control may be related to the integration of CD3ξ ( Figure 8B , 8C GEM 123, GEM 127, and GEM128 are incompatible.

[0434] Example 8: Human CD160 and its variants have conserved function in established B16F0 melanomas in mice.

[0435] To compare the tumor suppressor activity of human CD160 variants with that of mouse CD160, the ability of Pmel T cells expressing these respective forms of CD160 to control established B16F0 melanoma mice was measured.

[0436] In short, 1x10 5B16F10 cells were subcutaneously injected into 6- to 8-week-old female C57BL / 6 mice. Mice were observed daily after tumor implantation and sacrificed when signs of disease appeared. Tumor formation was examined twice weekly by palpation, and tumor area was measured using calipers. Mice were randomly assigned prior to adoptive cell transfer to ensure no size bias at the start of the experiment. To achieve ectopic expression of CD160 entities, 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 intracellular domains. Starting on day 7 post-tumor implantation, mice were infused with 0.3 million ectopically expressed mouse or human CD160 variant Pmel T cells at 14-day intervals (every two weeks), with each T cell infusion preceded by a CYP treatment regimen (100 mg / kg per treatment).

[0437] like Figure 10A In our study, Pmel T cells expressing all human CD160 variants exhibited stronger activity in controlling and eliminating established B16F0 melanoma tumors compared to Pmel T cells expressing mCD160. Specifically, Pmel T cells expressing GPI-anchored human CD160 and Pmel T cells expressing human CD160 with intracellular domains demonstrated the strongest activity in tumor control.

[0438] The improved survival rate of Pmel T cells expressing various CD160 variants was also measured through adoptive transfer. Figure 10B As shown, Pmel T cells expressing GPI-anchored human CD160 and Pmel T cells expressing human CD160 with intracellular domains provided the strongest improvement in survival.

[0439] In summary, these results demonstrate that human CD160 variants exhibit the same conserved function as mouse CD160 in enhancing tumor-specific T cells to control and eliminate established B16F0 melanoma tumors in mice, suggesting that they may have similar functions in controlling established solid tumors in humans.

[0440] Example 9: CD160-modified LLC TILs inhibit the development of metastatic Lewis lung cancer in mice.

[0441] To examine the ability of CD160-modified antigen-specific immune cells to suppress the development of metastatic lung cancer, the ability of CD160-modified tumor-infiltrating lymphocytes (TILs) to kill Lewis lung cancer in vitro and improve in vivo survival in a Lewis lung cancer mouse model was investigated.

[0442] To obtain TILs, lung tumors were isolated from mice carrying Lewis lung cancer and carefully cut into small pieces, then digested with collagenase V at 37°C. Single-cell suspensions were obtained by passing the digested sample through a 70–100 μm cell filter. As needed, the digested tissue was gently squeezed through the cell filter using a syringe plunger. The single-cell suspensions were then stained with anti-TCRβ conjugated phycoerythrin (PE), further enriched with anti-PE magnetic beads, and finally sorted on a SONY SH800 FACS sorter. As determined by FACS analysis, the purity of the TILs sorted in the described experiments was greater than 85%. The TILs were then modified to express mCD160 or the CD160 chimera GEM124 (see [link to FACS]). Figure 8A ).

[0443] First, the ability of CD160-modified TILs to kill tumor cells was examined and compared with control TILs. In short, TILs were stimulated with anti-CD3 and anti-CD28 beads and cultured for 3 days in T cell expansion medium containing IL-2. On day 3, target tumor cells (LLCs) were warmed at 37°C for 10 min and then treated with 1 μM CellTrace at 37°C. TM Violet (Invitrogen) labeling for 30 min followed by seeding into 96-well plates. Control or CD160-modified TILs were added to each well at defined effector-to-target cell ratios, and incubation was performed for four to six hours. Target tumor cells were then harvested, labeled with 7-aminoactinomycin D (7-AAD, BDPharmingen), and analyzed by FACS to determine any TIL-induced killing. TM The population of Violet dye+ / 7-AAD+ cells represents the target cells that have been killed, and CELLTRACE TM Violet dye + / 7-AAD- populations represent the remaining live target cells.

[0444] like Figure 11A The results showed that mCD160-modified TILs were more effective than TILs in killing co-cultured Lewis lung cancer cells.

[0445] The ability of CD160-modified TILs to inhibit the development of metastatic lung cancer in vivo was also examined. To generate a metastatic lung cancer model, 2x10 TILs were administered via intratracheal infusion. 5 -2x10 6Lewis lung cancer cells (LLCs) were directly introduced into the lungs of 6- to 8-week-old female C57BL / 6 mice. Mice were randomly assigned prior to adoptive cell transfer to ensure no size bias at the start of the experiment. Starting on day 10 post-tumor implantation, mice were infused with 0.3 million control TILs or TILs ectopically expressing mCD160 or GEM124 at 12-day intervals, with each T-cell infusion preceded by a CYP treatment regimen (100 mg / kg per treatment). Primary subcutaneous tumors were generally controlled by the CYP treatment regimen, and mice subsequently died due to lung metastases. Mice were observed daily after tumor implantation and euthanized upon the appearance of signs of disease. Tumor formation in mice was examined twice weekly by palpation or caliper measurement. Mice were euthanized and the tumor harvested once the tumor reached a diameter of 1.2–1.5 cm or skin ulceration occurred. Figure 11B ).

[0446] Notably, at the end of the experiment, mice treated with TIL expressing GEM124 had a 90% survival rate at 75 days post-implantation, while mice infused with TIL expressing mCD160 had a median survival of 70 days. Conversely, untreated mice, mice treated with CYP only, and mice treated with control TIL and CYP showed median survivals of 42, 47, and 47 days, respectively. Figure 11C ).

[0447] In summary, these results indicate that CD160 can enhance the tumor control activity of lung cancer TILs both in vitro and in vivo. Furthermore, since TILs extracted from mouse lung cancer are inherently polyclonal, these results also suggest that CD160 and its activating chimeras can enhance tumor control through endogenous polyclonal anti-tumor T cells carrying TCRs that recognize multiple tumor antigens.

[0448] Example 10: CD160-modified human CAR-T cells exhibited improved proliferation, reduced apoptosis, and enhanced in vivo and in vitro tumor control.

[0449] To determine whether CD160 can enhance the function of human CAR-T cells, CD19-CAR-T cells were modified to overexpress CD160, and their ability to proliferate in culture, as well as their functional activity against tumors in vitro and in vivo, were examined.

[0450] In short, human T cells were engineered to co-express a human CD160 variant (called huCD160TC) possessing both a transmembrane domain and a cytosol domain, as well as a CD19-chimeric antigen receptor (CD19-CAR) for recognizing tumor-associated antigens on CD19-positive tumor cells. Figure 12AAn exemplary method for co-expressing CD19-CAR and huCD160TC linked by a 2A peptide using a lentiviral vector is also shown. Figure 12B CD160-modified CD19-CAR-T cells are produced by transducing human T cells with lentivirus. Figure 12A , 12B ), and then tested functional improvements in culture and tumor models.

[0451] To examine the ability of CD160 to improve the function of cultured human CAR-T cells, CD160-modified CD19-CAR-T cells were expanded and cultured, and their growth rate was recorded daily and compared with non-CD160-modified CD19-CAR-T cells. In short, T cells were isolated from human peripheral blood and modified using a described lentiviral vector to co-express CD19-CAR and huCD160TC. During the first week of culture, cells were analyzed using… Figure 12B Lentiviral transduction was used to modify corresponding T cells to express huCD160TC and CD19-CAR, or to modify them to express CD19-CAR only. T cells expressing only CD19-CAR or CD160-modified CD19-CAR-T cells were then cultured in T cell expansion medium with appropriate growth factors. Subsequently, to determine cell concentration and viability, cells were stained with 1 μg / ml propidium iodide, mixed with fluorescent counting beads (Spherotech), and analyzed on an SP6800 Sony Spectral flow cytometer. Data were analyzed on the instrument or directly using FCS Express to determine absolute cell counts and the percentage of live and dead cells. The extent of T cell expansion during a two-week culture period was calculated by combining cell counts and mitotic factors, and plotted using PRISM software.

[0452] like Figure 13A As shown, human CD19-CAR T cells with huCD160TC overexpression consistently expanded more efficiently than those without. In this culture system, T cells underwent activation and infection processes during the first week and generally expanded only to a limited extent. Consistent with this process, the difference in proliferation was less significant during the first week of culture but became more apparent during the second week. Notably, at days 14–16 after the start of culture, human CD19-CAR T cells with CD160TC overexpression had a significantly lower percentage of dead cells compared to control CAR-T cells without huCD160TC overexpression, as determined by trypan blue staining or FACS analysis using propidium iodide (PI) or 7AAD. Figure 13BThese results indicate that CD19-CAR T cells expressing CD160TC exhibit higher proliferative potential and are less prone to cell death in later stages of cell culture, thus suggesting that CD160 overexpression in CAR-T cells can be used to enhance CAR-T cell production. Importantly, CD19-CAR T cells expressing CD160TC and control CAR T cells essentially ceased proliferation after two weeks of culture (data not shown), indicating that CD160 overexpression in CAR-T cells does not lead to uncontrolled T cell proliferation.

[0453] To examine the effect of CD160TC overexpression on the enhanced functional activity of CD19-CAR-T cells against tumors in vitro, the cytolytic function of CD160-modified CD19-CAR T cells on CD19-positive tumor cultures was evaluated. In short, target tumor cells Nalm6, Ramos, or CD19+ / K562 were incubated at 37°C for 10 min and treated with 1 μM CellTrace. TM Violet (Invitrogen) was used to label the cells at 37°C for 30 min, and then seeded onto 96-well culture plates. Control or CD160-modified CAR-T cells were added to each well at a specified effector-to-target cell ratio, and then incubated for 24–48 hours. Target cells were subsequently harvested and treated with 7-aminoactinomycin D (7-AAD, BD PHARMINGEN). TM The cells were labeled and analyzed using FACS to determine any killing effector T cells. TM A population of Violet dye+ / 7-AAD+ cells represents target cells that have been killed, and CELLTRACE TM The Violet dye + / 7-AAD- population represents the remaining live target cells. (e.g.) ...

Claims

1. An antigen-specific immune cell comprising a modified exogenous membrane-binding CD160 protein on its surface, wherein the exogenous CD160 protein leads to an upregulation of the activity of the modified antigen-specific immune cell compared to the activity of a precursor antigen-specific immune cell that does not contain the exogenous CD160 protein. The exogenous CD160 protein is the amino acid sequence of any one of SEQ ID NO:1-4, and The immune cells are T cells.

2. The modified antigen-specific immune cell according to claim 1, wherein the T cell is a cytotoxic αβ T cell or a γδ T cell.

3. The modified antigen-specific immune cell according to claim 1, wherein the T cell is a cytotoxic T cell.

4. The modified antigen-specific immune cell according to claim 1, wherein the T cell is a tumor-infiltrating T cell or an APC-activated anti-tumor T cell.

5. The modified antigen-specific immune cell according to claim 1, wherein the T cell is a helper T cell.

6. The modified antigen-specific immune cell according to claim 1, wherein the exogenous CD160 protein: (a) is bound to the membrane via a GPI linker; or (b) includes a transmembrane domain.

7. The modified antigen-specific immune cell according to claim 6, wherein the exogenous CD160 protein further includes an intracellular domain.

8. The modified antigen-specific immune cell according to claim 7, wherein the intracellular domain includes an intracellular signal transduction domain derived from the signal transduction subunit of the TCR complex.

9. The modified antigen-specific immune cell according to claim 1, wherein the exogenous CD160 protein is bound to the modified antigen-specific immune cell via an immune cell binding portion.

10. The modified antigen-specific immune cell according to claim 9, wherein the immune cell binding portion binds to the surface molecules of the modified antigen-specific immune cell.

11. The modified antigen-specific immune cell according to any one of claims 1-10, wherein the modified antigen-specific immune cell further comprises a functional exogenous receptor.

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

13. A method for generating antigen-specific immune cells whose surface includes a modified exogenous membrane-binding CD160 protein, comprising: Modified antigen-specific immune cells are generated by contacting precursor antigen-specific immune cells with exogenous CD160 protein or the first nucleic acid encoding exogenous CD160 protein. The exogenous CD160 protein is any one of the amino acid sequences shown in SEQ ID NO:1-4; Among them, exogenous CD160 protein leads to upregulation of the activity of modified antigen-specific immune cells compared to the activity of precursor antigen-specific immune cells; and The immune cells are T cells.

14. The method of claim 13, wherein the T cell is a cytotoxic αβ T cell or a γδ T cell.

15. The method of claim 13, wherein the T cell is a cytotoxic T cell.

16. The method of claim 13, wherein the T cell is a tumor-infiltrating T cell or an APC-activated anti-tumor T cell.

17. The method of claim 13, wherein the T cell is a helper T cell.

18. The method of claim 13, wherein the method comprises contacting precursor antigen-specific immune cells with exogenous CD160 protein.

19. The method of claim 18, wherein the exogenous CD160 protein comprises an immune cell binding portion of a surface molecule that binds to a precursor antigen-specific immune cell.

20. The method of claim 13, wherein the method comprises introducing a nucleic acid encoding an exogenous CD160 protein into a precursor antigen-specific immune cell.

21. The method of claim 13, wherein the exogenous CD160 protein: (a) is bound to the membrane via a GPI linker; or (b) includes a transmembrane domain.

22. The method of claim 13, wherein the exogenous CD160 protein is bound to the modified antigen-specific immune cell via the immune cell binding portion.

23. The method of claim 22, wherein the immune cell binding portion binds to the surface molecules of the modified antigen-specific immune cell.

24. The method of claim 13, wherein the precursor antigen-specific immune cells comprise a second nucleic acid encoding a functional exogenous receptor.

25. The method of claim 13, further comprising contacting a precursor antigen-specific immune cell with a second nucleic acid encoding a functional exogenous receptor.

26. The method of claim 24 or 25, wherein the functional exogenous receptor is an engineered T-cell receptor (TCR) or a chimeric antigen receptor (CAR).

27. The method of claim 24 or 25, wherein the first nucleic acid and the second nucleic acid are operatively linked to the same promoter.

28. The method according to claim 24 or 25, wherein the first nucleic acid and the second nucleic acid are on the same vector.

29. The method according to claim 24 or 25, further comprising isolating or enriching immune cells comprising a first and / or a second nucleic acid.

30. The method according to any one of claims 13-25, further comprising formulating modified antigen-specific immune cells expressing CD160 with at least one pharmaceutically acceptable carrier.

31. Modified antigen-specific immune cells obtained by the method according to any one of claims 13-30.

32. A pharmaceutical composition comprising a modified antigen-specific immune cell according to any one of claims 1-12 and 31 and a pharmaceutically acceptable carrier.

33. Use of an effective amount of the modified antigen-specific immune cells according to any one of claims 1-12 and 31 in the preparation of a medicament for treating cancer in an individual.

34. The use according to claim 33, wherein the modified antigen-specific immune cells are derived from an individual.

35. Use of an effective amount of the pharmaceutical composition according to claim 32 in the preparation of a medicament for treating cancer in an individual.

36. The use according to claim 35, wherein the modified antigen-specific immune cells are derived from an individual.

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