Anti-CD94 antibodies and chimeric antigen receptors and methods of use thereof

Abstract

According to various aspects of the disclosure, the disclosure relates to an antibody or antigen-binding fragment thereof capable of binding to CD94, a chimeric antigen receptor capable of binding to CD94, and engineered T cells containing a chimeric antigen receptor capable of binding to CD94.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 298,423, filed January 11, 2022, which is incorporated by reference herein in its entirety.

[0002] Reference to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (Name: 4443_010PC01_Seqlisting_ST26.xml, Size: 73,637 bytes, and Creation Date: January 5, 2023) are submitted with this application and are incorporated by reference in their entirety herein.

[0003] The present disclosure relates generally to the fields of immunology and cancer therapy. For example, the present disclosure provides anti-CD94 antibodies, anti-CD94 chimeric antigen receptors, and anti-CD94 CAR-T cells, as well as methods of making and using them. [Background technology]

[0004] Chimeric antigen receptor (CAR) T-cell therapy has shown efficacy in B-cell leukemia and lymphoma. However, targeting natural killer (NK) cells and T-cell lymphoproliferative disorders has been challenging due to the difficulty in identifying suitable antigens for CAR-T-cell therapy.

[0005] CD94 is a C-type lectin receptor expressed on NK cells and a subset (<5%) of CD8+ T cells in normal tissues. CD94 can function as a stimulatory or inhibitory receptor when heterodimerizing with NKG2C or NKG2A, respectively. Studies in patients with primary immunodeficiencies in which NK cell deficiency is the major immunological defect indicate that they are primarily at risk for recurrent herpesvirus infections, which can be controlled by a combination of vaccination, prophylaxis, and / or treatment strategies. Collectively, these reports suggest that CD94 may be a safe and effective target for CAR T cell therapy in CD94-expressing NK / T cell lymphoproliferative disorders.

[0006] For example, there is a need to develop antibodies and CAR-T cells for the treatment of natural killer cell and T-cell lymphoproliferative disorders. Summary of the Invention

[0007] The present disclosure provides an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody). In some embodiments, the antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:19, a CDR H2 comprising the amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:20, a CDR H3 comprising the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:21, a CDR L1 comprising the amino acid sequence represented by SEQ ID NO:6 or SEQ ID NO:22, a CDR L2 comprising the amino acid sequence YTS, SEQ ID NO:23, or SEQ ID NO:24, and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO:8.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1, and the VL comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2.

[0009] In some aspects, the antibody or antigen-binding fragment thereof comprises i) CDR H1, CDR H2, and CDR H3 comprising the CDR H1, CDR H2, and CDR H3 amino acid sequences of SEQ ID NO:1, and ii) CDR L1, CDR L2, and CDR L3 comprising the CDR L1, CDR L2, and CDR L3 amino acid sequences of SEQ ID NO:2.

[0010] In some embodiments, the CDRs are those defined in Kabat, Chothia, AbM, or IMGT.

[0011] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 85% identical to the amino acid sequence SEQ ID NO:1.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence SEQ ID NO:1.

[0013] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 95% identical to the amino acid sequence SEQ ID NO:1.

[0014] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 98% identical to the amino acid sequence SEQ ID NO:1.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 99% identical to the amino acid sequence SEQ ID NO:1.

[0016] In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:2.

[0017] In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2.

[0018] In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:2.

[0019] In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:2.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:2.

[0021] In some aspects, the antibody or antigen-binding fragment thereof is human, humanized, or chimeric.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody.

[0023] In some embodiments, the IgG antibody is an IgG1 antibody or an IgG4 antibody.

[0024] In some embodiments, the antibody is an antigen-binding fragment of an antibody.

[0025] In some aspects, the fragment is selected from the group consisting of a Fab, F(ab')2, Fv, scFv, scFv-Fc, dsFv, and a single domain molecule.

[0026] In some embodiments, the fragment is an scFv.

[0027] In some embodiments, the fragment is a Fab.

[0028] In some embodiments, the fragment is an intrabody.

[0029] In some embodiments, the antigen-binding fragment lacks the Fc region.

[0030] In some embodiments, the antibody or antigen-binding fragment comprises a VH and a VL on the same polypeptide chain.

[0031] In some aspects, the VH and VL are connected by a linker.

[0032] In some aspects, the antibody or antigen-binding fragment thereof is conjugated to an agent selected from the group consisting of a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, and PEG.

[0033] In some aspects, the antibody or antigen-binding fragment thereof is a bispecific antibody.

[0034] In some aspects, disclosed herein is a chimeric antigen receptor (CAR), comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding domain comprising an antigen binding domain capable of binding to CD94, (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain. In some aspects, the antigen binding domain comprises CDR H1 comprising an amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:19, CDR H2 comprising an amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:20, CDR H3 comprising an amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:21, CDR L1 comprising an amino acid sequence represented by SEQ ID NO:6 or SEQ ID NO:22, CDR L2 comprising an amino acid sequence represented by SEQ ID NO:23 or SEQ ID NO:24, and CDR L3 comprising an amino acid sequence represented by SEQ ID NO:8.

[0035] In some aspects, the chimeric antigen receptor comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:2.

[0036] In some embodiments, the antigen binding domain is an scFv.

[0037] In some embodiments, the costimulatory domain comprises a 4-1BB costimulatory domain, a CD28 costimulatory domain, or an OX40 costimulatory domain.

[0038] In some embodiments, the hinge, the transmembrane domain, or both, are from a CD8α polypeptide.

[0039] In some embodiments, the signaling domain comprises a CD3zeta signaling domain.

[0040] In some aspects, disclosed herein is an engineered human T cell comprising a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding domain comprising an scFv domain capable of binding to CD94, the scFv domain comprising a VL and a VH, (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain. In some aspects, the VH comprises a CDR H1 comprising an amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:19, a CDR H2 comprising an amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:20, and a CDR H3 comprising an amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:21. In some aspects, the VL comprises a CDR L1 comprising an amino acid sequence represented by SEQ ID NO:6 or SEQ ID NO:22, a CDR L2 comprising an amino acid sequence YTS or an amino acid sequence represented by SEQ ID NO:23 or SEQ ID NO:24, and a CDR L3 comprising an amino acid sequence represented by SEQ ID NO:8.

[0041] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor (CAR) comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, 56, or 57.

[0042] In some aspects, disclosed herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the VH or heavy chain of any of the antibodies or antigen-binding fragments thereof disclosed herein.

[0043] In some embodiments, the isolated polynucleotide further comprises a nucleic acid molecule encoding either the VL or light chain of an antibody or antigen-binding fragment thereof disclosed herein.

[0044] In some aspects, disclosed herein is an isolated polynucleotide comprising a nucleic acid molecule encoding either the VL or light chain of an antibody or antigen-binding fragment thereof disclosed herein.

[0045] In some aspects, disclosed herein is an isolated polynucleotide comprising a nucleic acid molecule encoding any of the chimeric antigen receptors disclosed herein.

[0046] In some aspects, disclosed herein is an isolated vector comprising any of the polynucleotides disclosed herein.

[0047] In some aspects, disclosed herein is a host cell comprising any of the polynucleotides disclosed herein, or any of the vectors disclosed herein.

[0048] In some aspects, the host cell is selected from the group consisting of CHO, HEK-293T, HeLa, and BHK cells, and optionally, the CHO cell is a CHO-K1SP cell.

[0049] In some aspects, disclosed herein are methods for producing an antibody or antigen-binding fragment thereof capable of binding to CD94, the method comprising: (a) culturing in a cell culture any of the host cells disclosed herein under conditions allowing expression of the antibody or antigen-binding fragment thereof; and (b) recovering the antibody or antigen-binding fragment thereof from the cell culture.

[0050] In some aspects, disclosed herein are antibodies or antigen-binding fragments thereof, obtainable by any of the production methods disclosed herein.

[0051] In some aspects, disclosed herein is a pharmaceutical composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein, any of the chimeric antigen receptors disclosed herein, any of the engineered T cells disclosed herein, or any of the vectors disclosed herein. In some aspects, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0052] In some aspects, disclosed herein are methods of treating cancer in an individual, the methods comprising administering to the individual a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof disclosed herein, any of the chimeric antigen receptors disclosed herein, any of the engineered T cells disclosed herein, or any of the pharmaceutical compositions disclosed herein.

[0053] In some embodiments, the cancer is leukemia. In some embodiments, the cancer is a CD94-expressing cancer.

[0054] In some embodiments, the leukemia is T-cell leukemia, T-cell large granular leukemia, natural killer cell large granular leukemia, or natural killer cell leukemia.

[0055] In some aspects, the cancer is lymphoma.

[0056] In some embodiments, the lymphoma is T-cell lymphoma, extranodal natural killer / T-cell lymphoma, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell lymphoma, or anaplastic large cell lymphoma.

[0057] In some aspects, described herein are methods of treating or preventing graft rejection of a transplant in a patient, the methods comprising administering to the individual a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof described herein, any of the chimeric antigen receptors described herein, any of the engineered T cells described herein, or any of the pharmaceutical compositions described herein.

[0058] In some embodiments, the transplant is an allogeneic transplant.

[0059] In some embodiments, the transplant is an organ transplant.

[0060] In some aspects, the transplant is a hematopoietic cell transplant.

[0061] In some aspects, the transplant is an induced pluripotent cell therapy.

[0062] In some aspects, described herein are methods of modulating an immune response in a subject, the methods comprising administering to the individual a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof described herein, any of the chimeric antigen receptors described herein, any of the engineered T cells disclosed herein, or any of the pharmaceutical compositions disclosed herein.

[0063] In some aspects, the immune response is enhanced.

[0064] In some embodiments, the immune response is mediated by natural killer cells and / or T cells. In some embodiments, natural killer cells and / or T cells mediate the immune response of an autoimmune disease. In some embodiments, the autoimmune disease is a systemic autoimmune disease. In some embodiments, the systemic autoimmune disease is systemic lupus erythematosus (SLE), Sjogren's syndrome, systemic sclerosis, rheumatoid arthritis (RA), multiple sclerosis, type 1 diabetes mellitus (T1DM), or autoimmune liver disease (ALD). [Brief description of the drawings]

[0065] [Figure 1A] FIG. 1 shows the expression of CD94 mRNA relative to β-actin mRNA in the natural killer (NK) cell lymphoma cell line NK-92 and normal human tissues. [Figure 1B]FIG. 1 shows CD94 mRNA expression in 79 normal human tissues and lymphoma subtypes by high-density oligonucleotide arrays from BioGPS. [Diagram 2] Flow cytometry analysis showing the generation of CD94-L cells by measuring positive staining for CD94 (x-axis). [Figure 3A] Specificity of purified anti-CD94 monoclonal antibody ("UT-CD94") produced by hybridoma technology is shown. Staining of CD94-L cells (CD94-L) and L cells (L) with UT-CD94 monoclonal antibody conjugated to AF647 is shown. [Figure 3B] Specificity of purified anti-CD94 monoclonal antibody ("UT-CD94") produced by hybridoma technology is shown. Staining of CD56+ NK cells (Nk cells), CD3+ T cells (T cells), CD19+ B cells (B cells), and CD14+ monocytes (monocytes) from normal donor peripheral blood mononuclear cells by UT-CD94 monoclonal antibody conjugated to AF647 is shown. [Figure 3C] Specificity of purified anti-CD94 monoclonal antibody ("UT-CD94") produced by hybridoma technology is shown. Staining of NK lymphoma or leukemia lines (NK-92, NKL, and KHYG-1) and B cell lymphoma lines (SP53, Daudi, and Jeko-1) with UT-CD94 monoclonal antibody conjugated to AF647 is shown. [Figure 4A] 1 shows an exemplary design of an anti-CD94 chimeric antigen receptor ("UT-CD94 CAR"), which comprises (i) a CD94 scFv comprising a VL and VH domain, (ii) a CD8α hinge domain, (iii) a CD8α transmembrane domain, (iv) a 4-1BB costimulatory domain, and (v) a CD3zeta signaling domain. [Figure 4B]Figure 1 shows expression of UT-CD94 CAR 72 hours after lentiviral transduction as determined by staining with His-tagged CD94 extracellular domain (ECD) protein and AF647-conjugated anti-His antibody. Flow cytometry analysis shows positive staining for UT-CD94 CAR on the X-axis. [Figure 5A] Cytotoxicity of anti-CD94 CAR-T cells ("UT-CD94 CAR-T cells"). Figures 5A-5B show the percentage of lysis of the CD94-positive NK cell line NKL (Figure 5A) or NK-92 (Figure 5B) at effector:target ratios of 8:1, 4:1, and 1:1 after co-culture with UT-CD94 CAR-T cells (CD94 CAR-T), with non-transduced T cells (control T), or without co-culture (tumor only). [Figure 5B] Cytotoxicity of anti-CD94 CAR-T cells ("UT-CD94 CAR-T cells"). Figures 5A-5B show the percentage of lysis of the CD94-positive NK cell line NKL (Figure 5A) or NK-92 (Figure 5B) at effector:target ratios of 8:1, 4:1, and 1:1 after co-culture with UT-CD94 CAR-T cells (CD94 CAR-T), with non-transduced T cells (control T), or without co-culture (tumor only). [Figure 5C] Cytotoxicity of anti-CD94 CAR-T cells ("UT-CD94 CAR-T cells"). Figures 5C-E show a 4-day period with the corresponding lysis percentages of the CD94-positive NK cell line NKL (Figure 5C), the CD94-positive NK cell line NK-92 (Figure 5D), or the B-cell lymphoma cell line SP53 (Figure 5E) after co-culture with UT-CD94 CAR-T cells (CD94 CAR-T), co-culture with untransduced T cells (control T), or no co-culture (tumor only). [Figure 5D]Cytotoxicity of anti-CD94 CAR-T cells ("UT-CD94 CAR-T cells"). Figures 5C-E show a 4-day period with the corresponding lysis percentages of the CD94-positive NK cell line NKL (Figure 5C), the CD94-positive NK cell line NK-92 (Figure 5D), or the B-cell lymphoma cell line SP53 (Figure 5E) after co-culture with UT-CD94 CAR-T cells (CD94 CAR-T), co-culture with untransduced T cells (control T), or no co-culture (tumor only). [Figure 5E] Cytotoxicity of anti-CD94 CAR-T cells ("UT-CD94 CAR-T cells"). Figures 5C-E show a 4-day period with the corresponding lysis percentages of the CD94-positive NK cell line NKL (Figure 5C), the CD94-positive NK cell line NK-92 (Figure 5D), or the B-cell lymphoma cell line SP53 (Figure 5E) after co-culture with UT-CD94 CAR-T cells (CD94 CAR-T), co-culture with untransduced T cells (control T), or no co-culture (tumor only). [Figure 5F] Figure 1 shows the cytotoxicity of anti-CD94 CAR-T cells ("UT-CD94 CAR-T cells"), showing the percentage of killed target cells in L cells, human CD94-transfected L cells, the B cell lymphoma cell line SP53, and the NK92 cell line when cultured alone or in combination with non-transduced T cells, anti-CD94 CAR-T cells, or anti-CD19 CAR-T cells at an effector:target ratio of 6:1 for 24 hours. [Figure 6] Flow cytometry analysis measuring the effect of chimeric anti-CD94 antibody ("cUT-CD94") or isotype antibody on staining of HLA-E tetramers (measured by median fluorescence intensity) is shown. [Figure 7]Flow cytometry analysis measuring CD137+ NK cells after incubation with HLA-E expressing SP-53 mantle cell lymphoma cells and treatment with mouse anti-human CD94 antibodies (UT-CD94 / mCD94 and DX22 from Biolegend), cUT-CD94 antibody (cCD94), and relevant isotype controls (mIgG1 and hIgG1 from Biolegend). [Figure 8] Flow cytometry analysis measuring CD107a+ NK cells after incubation with HLA-E expressing SP-53 mantle cell lymphoma cells and treatment with mouse anti-human CD94 antibodies (UT-CD94 / mCD94 and DX22 from Biolegend), cUT-CD94 antibody (cCD94), and relevant isotype controls (mIgG1 and hIgG1 from Biolegend). [Figure 9A] Figure 9 shows scRNAseq analysis of CD94 (KLRD1) mRNA expression. CD94 mRNA expression overview based on scRNAseq analysis across 25 normal human tissues (Figure 9A) and in single cell types from all 25 tissues (Figure 9B), heart (Figure 9C), lung (Figure 9D), liver (Figure 9E), and kidney (Figure 9F). In addition, CD94 mRNA expression in 29 immune cell types from normal donor peripheral blood was analyzed (Figure 9G). nTPM - normalized transcripts per million. Source: Human Protein Atlas. [Figure 9B] Figure 9 shows scRNAseq analysis of CD94 (KLRD1) mRNA expression. CD94 mRNA expression overview based on scRNAseq analysis across 25 normal human tissues (Figure 9A) and in single cell types from all 25 tissues (Figure 9B), heart (Figure 9C), lung (Figure 9D), liver (Figure 9E), and kidney (Figure 9F). In addition, CD94 mRNA expression in 29 immune cell types from normal donor peripheral blood was analyzed (Figure 9G). nTPM - normalized transcripts per million. Source: Human Protein Atlas. [Figure 9C]Figure 9 shows scRNAseq analysis of CD94 (KLRD1) mRNA expression. CD94 mRNA expression overview based on scRNAseq analysis across 25 normal human tissues (Figure 9A) and in single cell types from all 25 tissues (Figure 9B), heart (Figure 9C), lung (Figure 9D), liver (Figure 9E), and kidney (Figure 9F). In addition, CD94 mRNA expression in 29 immune cell types from normal donor peripheral blood was analyzed (Figure 9G). nTPM - normalized transcripts per million. Source: Human Protein Atlas. [Figure 9D] Figure 9 shows scRNAseq analysis of CD94 (KLRD1) mRNA expression. CD94 mRNA expression overview based on scRNAseq analysis across 25 normal human tissues (Figure 9A) and in single cell types from all 25 tissues (Figure 9B), heart (Figure 9C), lung (Figure 9D), liver (Figure 9E), and kidney (Figure 9F). In addition, CD94 mRNA expression in 29 immune cell types from normal donor peripheral blood was analyzed (Figure 9G). nTPM - normalized transcripts per million. Source: Human Protein Atlas. [Figure 9E] Figure 9 shows scRNAseq analysis of CD94 (KLRD1) mRNA expression. CD94 mRNA expression overview based on scRNAseq analysis across 25 normal human tissues (Figure 9A) and in single cell types from all 25 tissues (Figure 9B), heart (Figure 9C), lung (Figure 9D), liver (Figure 9E), and kidney (Figure 9F). In addition, CD94 mRNA expression in 29 immune cell types from normal donor peripheral blood was analyzed (Figure 9G). nTPM - normalized transcripts per million. Source: Human Protein Atlas. [Figure 9F]Figure 9 shows scRNAseq analysis of CD94 (KLRD1) mRNA expression. CD94 mRNA expression overview based on scRNAseq analysis across 25 normal human tissues (Figure 9A) and in single cell types from all 25 tissues (Figure 9B), heart (Figure 9C), lung (Figure 9D), liver (Figure 9E), and kidney (Figure 9F). In addition, CD94 mRNA expression in 29 immune cell types from normal donor peripheral blood was analyzed (Figure 9G). nTPM - normalized transcripts per million. Source: Human Protein Atlas. [Figure 9G] Figure 9 shows scRNAseq analysis of CD94 (KLRD1) mRNA expression. CD94 mRNA expression overview based on scRNAseq analysis across 25 normal human tissues (Figure 9A) and in single cell types from all 25 tissues (Figure 9B), heart (Figure 9C), lung (Figure 9D), liver (Figure 9E), and kidney (Figure 9F). In addition, CD94 mRNA expression in 29 immune cell types from normal donor peripheral blood was analyzed (Figure 9G). nTPM - normalized transcripts per million. Source: Human Protein Atlas. [Figure 10A] Immunohistochemistry assays for expression of CD94 in normal donor tissue arrays (FIG. 10A), normal tonsils (FIG. 10B), and tumor biopsies from subjects with NK / T cell lymphoma (FIG. 10C) are shown. [Figure 10B] Immunohistochemistry assays for expression of CD94 in normal donor tissue arrays (FIG. 10A), normal tonsils (FIG. 10B), and tumor biopsies from subjects with NK / T cell lymphoma (FIG. 10C) are shown. [Figure 10C] Immunohistochemistry assays for expression of CD94 in normal donor tissue arrays (FIG. 10A), normal tonsils (FIG. 10B), and tumor biopsies from subjects with NK / T cell lymphoma (FIG. 10C) are shown. [Figure 11]1 shows the binding of anti-CD94 antibodies in wild-type and CD94 knockout NK cell leukemia cell lines. The binding specificity of anti-CD94 antibodies was assessed by flow cytometry on wild-type KHYG-1 and NK92 cell lines, as well as their isogenic cell lines with CD94 knockout (KO). [Figure 12A] 1 shows exemplary CD94-targeted CAR constructs and their cytotoxic activity in vitro. Three different CD94-targeted CAR designs are shown. [Figure 12B] 1 shows exemplary CD94-targeted CAR constructs and their cytotoxic activity in vitro.Flow cytometry evaluation of lentivirally transduced CD94-targeted CARs into normal donor T cells. [Figure 12C] Exemplary CD94-targeted CAR constructs and their cytotoxic activity in vitro. Figures 12C-12E show the percentage of cells undergoing lysis or growth monitored by continuous imaging using an Incucyte Live-Cell Analysis System. NK cell leukemia cell lines (NKL-Figure 12C, KHYG-1-Figure 12D, and NK92-Figure 12E) were stably transduced with red fluorescent protein (RFP). Tumor cells were then cultured in the presence or absence of CD94-targeted CAR T cells or untransduced T cells at an effector:target ratio of 0.5:1. [Figure 12D] Exemplary CD94-targeted CAR constructs and their cytotoxic activity in vitro. Figures 12C-12E show the percentage of cells undergoing lysis or growth monitored by continuous imaging using an Incucyte Live-Cell Analysis System. NK cell leukemia cell lines (NKL-Figure 12C, KHYG-1-Figure 12D, and NK92-Figure 12E) were stably transduced with red fluorescent protein (RFP). Tumor cells were then cultured in the presence or absence of CD94-targeted CAR T cells or untransduced T cells at an effector:target ratio of 0.5:1. [Figure 12E]Exemplary CD94-targeted CAR constructs and their cytotoxic activity in vitro. Figures 12C-12E show the percentage of cells undergoing lysis or growth monitored by continuous imaging using an Incucyte Live-Cell Analysis System. NK cell leukemia cell lines (NKL-Figure 12C, KHYG-1-Figure 12D, and NK92-Figure 12E) were stably transduced with red fluorescent protein (RFP). Tumor cells were then cultured in the presence or absence of CD94-targeted CAR T cells or untransduced T cells at an effector:target ratio of 0.5:1. [Figure 13A] Shows CD94 expression in hepatosplenic T-cell lymphoma and anti-CD94 cytotoxicity against PDX tumor cells. Shows images of liver and spleen at necropsy of NSG mice intravenously injected with hepatosplenic T-cell lymphoma (TCL) PDX cells (1x106 tumor cells / mouse). [Figure 13B] Shown is the expression of CD94 in hepatosplenic T cell lymphoma and the cytotoxicity of anti-CD94 against PDX tumor cells. Shown is the expression of CD3 (hCD3), CD94 (hCD94), and CD45 (hCD45) in liver, spleen, bone marrow, and blood as measured by flow cytometry among hCD3+hCD45+ vs. hCD3-hCD45- cells. NSG mice without tumor injection were used as controls. [Figure 13C] Shows CD94 expression in hepatosplenic T cell lymphoma and cytotoxicity of anti-CD94 against PDX tumor cells. Shows survival in five different mouse cohorts: 1) tumor only, 2) untransduced T cells, 3) mice administered 10x106 anti-CD94 targeted CAR+ T cells (CD94 / CD28), 4) mice administered 10x106 anti-CD94 targeted CAR+ T cells (CD94 / 4-1BB), 5) mice administered 10x106 anti-CD94 targeted CAR+ T cells (CD94 / OX40). Anti-tumor efficacy of CAR T cells was assessed by monitoring mice for survival. Log-rank test was used to assess differences in survival between treatment groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] Certain aspects of the disclosure are directed to anti-CD94 antibodies, anti-CD94 chimeric antigen receptors, and anti-CD94 CAR-T cells, as well as methods of making and using them. In some aspects, disclosed herein is an antibody or antigen-binding fragment thereof capable of binding to CD94. In some aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising a complementarity determining region (CDR) H1 comprising an amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:19, a CDR H2 comprising an amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:20, and a CDR H3 comprising an amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:21, and a light chain variable region (VL) comprising a CDR L1 comprising an amino acid sequence represented by SEQ ID NO:6 or SEQ ID NO:22, a CDR L2 comprising an amino acid sequence YTS or an amino acid sequence represented by SEQ ID NO:23 or SEQ ID NO:24, and a CDR L3 comprising an amino acid sequence represented by SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and a light chain variable region (VL) comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment thereof comprises an anti-CD94 scFv comprising a VL and a VH domain. In some aspects, disclosed herein are chimeric antigen receptors (CARs) or CAR T cells comprising an anti-CD94 antibody or antigen-binding fragment thereof (e.g., an anti-CD94 scFv) disclosed herein.

[0067] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated herein by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0068] Although methods and materials similar or equivalent to those disclosed herein can be used in the practice or testing of the present disclosure, suitable methods and materials are disclosed below. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description, and from the claims.

[0069] In order to further define this disclosure, the following terms and definitions are provided.

[0070] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. In certain aspects, the terms "a" or "an" mean "single." In other aspects, the terms "a" or "an" include "two or more" or "plural."

[0071] The term "about" is used herein to mean approximately, roughly, roughly, or almost. When the term "about" is used in conjunction with a numerical range, the term modifies that range by extending the boundaries to around the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10 percent above or below (higher or lower).

[0072] Throughout this disclosure, various aspects of the disclosure are presented in the form of ranges. Numerical ranges are intended to be inclusive of the numbers that define the range. When a range of values ​​is listed, it is understood that each intervening integer value between the upper and lower limit values ​​listed for that range, and each fractional part thereof, is also expressly disclosed, along with each subrange between such values. The upper and lower limits of any range may be independently included or excluded from the range, and each range that includes either the upper or lower limit, each range that does not include either, or each range that includes both, is also encompassed by this disclosure. Thus, ranges listed herein are understood to be shorthand for all of the values ​​within the range, including the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0073] When a value is explicitly stated, it should be understood that values ​​that are approximately the same number or amount as the stated value are also included within the scope of this disclosure.When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and included within the scope of this disclosure.Conversely, when different elements or groups of elements are disclosed individually, their combinations are also disclosed.When any element of a disclosure is disclosed as having multiple alternatives, the disclosed examples in which each alternative is excluded alone or in any combination with other alternatives are also disclosed herein, and two or more elements of a disclosure may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0074] As used herein, the term "and / or" should be considered as a specific disclosure of each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0075] As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, formulations, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the bounds of safe medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0076] The term "excipient" refers to any substance that is not itself a therapeutic agent, but may be used in a composition for delivering an active therapeutic agent to a subject, or in combination with an active therapeutic agent (e.g., to make a pharmaceutical composition), to improve its handling or storage characteristics, or to enable or facilitate the formation of a dosage unit of the composition (e.g., the formation of a hydrogel, which can then be optionally incorporated into a patch). Excipients include, but are not limited to, solvents, penetration enhancers, humectants, antioxidants, lubricants, emollients, substances added to improve the appearance or texture of the composition, and substances used to form hydrogels. Any such excipients may be used in any dosage form according to the present disclosure. The aforementioned classes of excipients are not meant to be exhaustive, but merely exemplary, so that one skilled in the art will recognize that additional types and combinations of excipients can be used to achieve the desired goal for the delivery of a drug. Excipients may be inert, non-active, and / or non-pharmaceutical active substances. Excipients may serve a variety of purposes. Those skilled in the art can select one or more excipients for specific desired properties without undue burden and through routine experimentation. The amount of each excipient used can vary within the range conventional in the art. The techniques and excipients that can be used to formulate dosage forms are described in Handbook of Pharmaceutical Excipients, 6th edition, Rowe et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2009), and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

[0077] The term "effective amount" or "pharmaceutical effective amount" or "therapeutically effective amount" as used herein refers to the amount or content of a drug or pharma- ceutical active substance that is sufficient to elicit a necessary or desired therapeutic response, or in other words, the amount is sufficient to elicit an appreciable biological response when administered to a patient.

[0078] The terms "treat" or "treatment" as used herein refer to the administration of a composition to a subject for therapeutic purposes.

[0079] The term "antibody" refers to an immunoglobulin molecule that recognizes and binds to a target, e.g., a protein, a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, or a combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antibody, and any other modified immunoglobulin molecule, provided that the antibody exhibits the desired biological activity. Antibodies can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different well-known subunit structures and three-dimensional structures. Antibodies can be naked or can be conjugated to other molecules, such as, for example, toxins, radioisotopes, etc.

[0080] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can contain an antigen recognition site of an intact antibody (e.g., a sufficient complementarity determining region (CDR) to bind to the antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially produced.

[0081] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of a light or heavy chain, typically about the amino terminal 110-120 or 110-125 amino acids in a mature heavy chain and about 90-115 amino acids in a mature light chain, which differ in sequence between antibodies and are used in the binding and specificity of a particular antibody to its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions within the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen.

[0082] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or antigen-binding fragment thereof.

[0083] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or antigen-binding fragment thereof.

[0084] The "hypervariable regions" in each chain are held together in close proximity by FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, 1992). As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally include amino acid residues from the "complementarity determining regions" or "CDRs", the latter of which are most highly variable in sequence and / or involved in antigen recognition. Numerous CDR definitions are in use and are encompassed herein. The Kabat definition is based on sequence variation and is the most commonly used (Kabat EA et al. (Kabat supra)). Chothia instead refers to the location of the structural loops (Chothia C & Lesk AM (1987) J. Mol. Biol. 196:901-917). The AbM definition is a compromise between the Kabat and Chothia definitions and is used by Oxford Molecular's AbM antibody modeling software (Martin AC R et al., (1989) Proc. Natl Acad. Sci. USA, 86: 9268-72; Martin AC R et al., (1991) Methods Enzymol. 203: 121-153; Pedersen JT et al., (1992) Immunomethods, 1: 126-136; Rees AR et al., (1996) In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172). The contact definition has been recently introduced (Maccallum RM et al., (1996) J. Mol. Biol. 262:732-745) and is based on the analysis of complex structures available in the Protein Data Bank.The definition of CDRs according to IMGT®, the international ImMunoGeneTics information System® (http: / / www.imgt.org), is based on the IMGT numbering for all immunoglobulin and T cell receptor V-REGIONS of all species (IMGT®, the international ImMunoGeneTics information System®, Lefranc MP et al., (1991) Nucleic Acids Res. 27(1): 209-12; Ruiz M et al., (2000) Nucleic Acids Res. 28(1): 219-21; Lefranc MP (2001) Nucleic Acids Res. 29(1): 207-9; Lefranc MP (2003) Nucleic Acids Res. 31(1): 307-10; Lefranc MP et al. al.,(2005)Dev.Comp.Immunol.29(3):185-203, Kaas Q et al.,(2007)Briefings in Functional Genomics & Proteomics,6(4):253-64).

[0085] In some embodiments, the complementarity determining regions (CDRs) disclosed herein are defined according to IMGT®. In some embodiments, the CDRs are defined according to Chothia. In some embodiments, the CDRs are defined according to Kabat. For example, for the light chain, the variable domain residues for each of the CDRs can be (numbering according to Kabat EA, et al., supra): LCDR1: 27-32, LCDR2: 50-52, LCDR3: 89-97. As used herein, the "non-CDR regions" of the VL region include the amino acid sequences: 1-26 (FRI), 33-49 (FR2), 53-88 (FR3), and 98-approximately 107 (FR4). For the heavy chain, the variable domain residues for each of the three CDRs can be HCDR1: 26-35, HCDR2: 51-57, and HCDR3: 93-102. As used herein, the "non-CDR regions" of the VH region may include the amino acid sequences: 1-25 (FR1), 36-50 (FR2), 58-92 (FR3), and 103 to approximately 113 (FR4).

[0086] The CDRs of the present disclosure are based on the above definitions and can include "extended CDRs" which can have variable domain residues as follows: LCDR1: 24-36, LCDR2: 46-56, LCDR3: 89-97, HCDR1: 26-36, HCDR2: 47-65, HCDR3: 93-102. These extended CDRs are also numbered according to Kabat et al. supra.

[0087] In some embodiments, alternative CDR sequences are provided for the same framework amino acid sequence of the variable region. In some embodiments, the alternative CDR sequences are generated using a software program. In some embodiments, different software can be used to generate alternative CDR sequences for the framework sequence of the variable region with different CDR sequences resulting from the use of different software programs. In some embodiments, the use of alternative CDR sequences can improve the binding affinity of the bispecific antibody to at least one antigen. In some embodiments, the alternative CDR sequences are used for affinity optimization of one or both antigen binding sites of the bispecific antibody according to the invention. In some embodiments, the alternative CDRs are defined according to the Kabat, Chothia, Paratome, AbM, Contact, and / or IMGT annotations. In some embodiments, the CDRs are defined according to two or more annotations.

[0088] The "non-extended CDR region" of the VL region can include the amino acid sequences: 1-23 (FRI), 37-45 (FR2), 57-88 (FR3), and 98-approximately 107 (FR4). The "non-extended CDR region" of the VH region can include the amino acid sequences: 1-25 (FRI), 37-46 (FR2), 66-92 (FR3), and 103-approximately 113 (FR4).

[0089] As used herein, the term "Fab region" refers to the VH and CHI domains of an immunoglobulin heavy chain (a "Fab heavy chain"), or the VL and CL domains of the light chain (a "Fab light chain").

[0090] As used herein, the term "scFv" or "single-chain antibody fragment" refers to a single chain consisting of an antibody heavy chain variable region and a light chain variable region linearly linked together by a linker (e.g., a short peptide of 10 to 25 amino acids) that exhibits specific binding to an antigen.

[0091] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to the immunoglobulin variable domain.

[0092] As used herein, the term "heavy chain" when used in reference to an antibody can refer to any of the different types, e.g., alpha (a), delta (d), epsilon (e), gamma (g), and mu (m), based on the amino acid sequence of the constant domain, which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0093] As used herein, the term "light chain" when used in reference to an antibody can refer to any of the different types, such as kappa (K) or lambda (1), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some embodiments, the light chain is a human light chain.

[0094] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequences are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and function, while the constant regions are homologous to sequences in an antibody or antigen-binding fragment thereof from another species (usually human) to avoid eliciting an immune response in that species.

[0095] The term "humanized" antibody or antigen-binding fragment thereof refers to a form of a non-human (e.g., murine) antibody or antigen-binding fragment that is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof that contains minimal non-human (e.g., murine) sequence. Typically, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues from its complementarity determining regions (CDRs) are replaced with residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) that has the desired specificity, affinity, and function ("CDR grafting") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, the Fv framework regions (FR) of a human immunoglobulin are replaced with corresponding residues in an antibody or fragment from a non-human species having the desired specificity, affinity, and function. The humanized antibody or antigen-binding fragment thereof can be further modified by substituting additional residues either within the Fv framework regions and / or within the non-human CDR residues to refine and optimize the specificity, affinity, and / or function of the antibody or antigen-binding fragment thereof. Generally, a humanized antibody or antigen-binding fragment thereof will contain substantially all of at least one, and typically two or three, variable domains, including all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof can also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some embodiments, a "humanized antibody" is a resurfaced antibody.

[0096] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin gene locus, and such an antibody or antigen-binding fragment is made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0097] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof, and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, the equilibrium dissociation constant (KD) and the equilibrium association constant (KA). KD is defined as the k off / k on KA is calculated from the quotient of k on / k off It is calculated from the quotient of k on k refers to, for example, the association rate constant of an antibody or an antigen-binding fragment thereof to an antigen, off refers, for example, to the dissociation of an antibody or antigen-binding fragment thereof from an antigen. on and k off can be determined by techniques known to those of skill in the art, such as BIAcore® or KinExA.

[0098] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (linear or consecutive epitope), or an epitope can be, for example, taken together from a polypeptide or two or more discontinuous regions of a polypeptide (conformational, non-linear, discontinuous, or discontinuous epitope). In certain aspects, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al, (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303).Antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc., see e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al., US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW, Roversi P et al, (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. See, e.g., Champe M et al, (1995) J Biol Chem 270:1388-1394, and Cunningham BC & Wells JA (1989) Science 244:1081-1085, for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0099] An antibody is said to "competitively inhibit" the binding of a reference antibody to a given epitope if it preferentially binds to that epitope, or to an overlapping epitope, to such an extent that it blocks the reference antibody from binding to the epitope to some extent. Competitive inhibition can be determined by any method known in the art, for example, by competitive ELISA assay. An antibody can be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0100] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition refers to a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0101] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified, naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), and other modifications known in the art, are also included within this definition. Because the polypeptides of this disclosure are based on antibodies, it is understood that in certain aspects, the polypeptides can occur as single chains or associated chains.

[0102] "Percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Percent identity can be determined by aligning two sequences and introducing gaps to maximize the identity between sequences. Alignment can be generated using programs known in the art. For the purposes of this specification, alignment of nucleotide sequences can be performed with the blastn program set with default parameters, and alignment of amino acid sequences can be performed with the blastp program set with default parameters (see National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov).

[0103] As used herein, the term "host cell" can be any type of cell, for example, a primary cell, a cell in culture, or a cell from a cell line. In certain aspects, the term "host cell" refers to a cell that has been transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due, for example, to mutations or environmental influences that may occur upon subsequent generation or integration of the nucleic acid molecule into the host cell genome.

[0104] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient is in a form such that it is effective and does not contain additional components that are unacceptably toxic to the subject to which the composition is administered. The composition may be sterile.

[0105] As used herein, the terms "administer," "administering," "administration," and the like refer to methods that can be used to enable delivery of a drug, e.g., an anti-CD94 antibody or antigen-binding fragment thereof, to a desired site(s) of biological action (e.g., intravenous administration). Administration techniques that can be used with the agents and methods described herein are found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon, and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0106] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be an animal. In some aspects, a subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate, etc.). In some aspects, a subject is a human.

[0107] As used herein, the term "nucleic acid" or "polynucleotide" refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by either ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have changes in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Additionally, the entire sugar moiety can be replaced by sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications at a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids can be either single-stranded or double-stranded.

[0108] The term "chimeric antigen receptor" (CAR) refers to a chimeric molecule that includes a binding domain (e.g., the binding domain can include an antibody or antigen-binding fragment thereof specific for a desired antigen (e.g., CD94)) that binds to a component (e.g., a ligand) present on a target cell, and a T cell receptor activation intracellular domain. In some aspects, the CAR exhibits specific anti-target cell immune activity. In certain aspects, the CAR can include an extracellular ligand binding domain (e.g., a single-chain antibody binding domain (scFv)) fused to an intracellular signaling domain of the T cell antigen receptor complex zeta chain. In some aspects, the CAR can include an extracellular ligand binding domain, a hinge, a transmembrane domain, and a cytoplasmic domain. In some aspects, the cytoplasmic domain includes a costimulatory domain (e.g., 4-1BB, CD28, or OX40) and a signaling domain (e.g., CD3zeta). In some aspects, the CAR, when expressed in a T cell, can redirect antigen recognition based on the specificity of the antibody.

[0109] The term "vector" as used herein includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that may include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acids. In some cases, vectors are capable of autonomous replication (episomal vectors) and / or expression of nucleic acids to which they are linked (expression vectors). Many suitable vectors are known to those of skill in the art and are commercially available. Viral vectors include negative-stranded RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), positive-stranded RNA viruses such as rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), picornaviruses and alphaviruses, as well as double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian C, B, and D viruses, HTLV-BLV group, and lentivirus.

[0110] "Costimulatory domain" or "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (e.g., a proliferative response) by the cell. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. In some embodiments, a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that promotes an efficient immune response.

[0111] "Costimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory molecule on a T cell. In some embodiments, the costimulatory ligand provides a signal that mediates a T cell response in addition to the primary signal provided, for example, by binding of the TCR / CD3 complex with a peptide-loaded MHC molecule. In some embodiments, the T cell response includes, but is not limited to, proliferation activation, differentiation, and the like. Costimulatory ligands can include, but are not limited to, agonists or antibodies that bind to a ligand that specifically binds CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, Toll ligand receptor, and B7-H3.

[0112] A "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of molecules.

[0113] As used herein, the term "extracellular ligand-binding domain" refers to an oligo- or polypeptide capable of binding to a ligand, e.g., a cell surface molecule. For example, the extracellular ligand-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state (e.g., cancer). Examples of cell surface markers that can act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0114] A "signal transducing domain" or "signaling domain" as used herein with respect to CARs is responsible for intracellular signal transduction following binding of the extracellular ligand binding domain to a target. In some embodiments, the signaling domain used in CARs results in activation of immune cells and immune responses. In other words, the signaling domain can be responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. Thus, a signaling domain can refer to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Examples of signaling domains for use in CARs can be the cytoplasmic sequences of T cell receptors and co-receptors that act together to initiate signal transduction following antigen receptor binding, as well as any derivative or variant of these sequences, and any synthetic sequence with the same function. In some cases, the signaling domain includes two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act in an antigen-independent manner to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).

[0115] "ITAM" refers to a signaling motif found in the cytoplasmic tails of various receptors that function as binding sites for the syk / zap70 class of tyrosine kinases. Exemplary ITAMs include those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD3zeta, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the signaling domain of the CAR can comprise a CD3zeta signaling domain.

[0116] As used herein, a "transmembrane region" or "transmembrane domain" is a portion of a CAR that anchors the extracellular binding moiety to the plasma membrane of an immune effector cell and facilitates binding of the binding domain to a target antigen. In some embodiments, the transmembrane domain can be a CD3zeta transmembrane domain, although other transmembrane domains that can be used include those obtained from CD8α, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, or CD154.

[0117] The binding domain of a CAR can be followed by a "spacer" or "hinge," which refers to a region that separates or moves the antigen binding domain away from the effector cell surface. In some embodiments, the hinge allows for cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6:412-419). The hinge region in a CAR can be between the transmembrane (TM) domain and the binding domain. In some embodiments, the hinge region is an immunoglobulin hinge region, which can be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs disclosed herein can include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, and CD7, which can be wild-type hinge regions from these molecules or can be altered. In some embodiments, the hinge region comprises a CD8α hinge.

[0118] Whenever an embodiment is disclosed herein with the language "comprising," it is understood that similar embodiments separately disclosed in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0119] The following examples are illustrative and do not limit the scope of the claimed embodiments.

[0120] II. Anti-CD94 Antibodies and Antigen-Binding Fragments Thereof In some aspects, provided herein are antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to CD94 (e.g., human CD94). In some aspects, the amino acid sequence of human CD94 comprises SEQ ID NO: 31 (see RefSeq Accession No. NP_001107868.2).

[0121] In some aspects, the antibodies or antigen-binding fragments thereof provided herein bind to CD94 and contain six CDRs (e.g., CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3). The CDRs can be determined by a number of algorithms in the art, such as IMGT (Table 1), Abyss (Table 2), and Kabat (Table 3).

[0122] For example, the international ImMunoGeneTics information System® (http: / / www.imgt.org) is based on the IMGT numbering for all immunoglobulin and T cell receptor V-REGIONS of all species (IMGT®, international ImMunoGeneTics information System®, Lefranc MP et al., (1991) Nucleic Acids Res. 27(1): 209-12; Ruiz M et al., (2000) Nucleic Acids Res. 28(1): 219-21; Lefranc MP (2001) Nucleic Acids Res. 29(1): 207-9; Lefranc MP (2003) Nucleic Acids Res. 31(1): 307-10; Lefranc MP et al., (2005) Dev. Comp. Immunol. 29(3): 185-203; Kaas Q et al., (2006) Nucleic Acids Res. 29(3): 185-203; al., (2007) Briefings in Functional Genomics & Proteomics, 6(4):253-64).

[0123] The ABYSIS algorithm compiles antibody protein sequences from EMBLIG, which contains antibody information extracted from the EMBL-ENA databank, the Kabat collection, and the Protein Databank (Swindells MB et al., (2017) J Mol Biol. 2017 Feb 3; 429(3): 356-364).

[0124] The Kabat definition is based on sequence variation and is commonly used (Kabat EA et al., supra). [Table 1] [Table 2] [Table 3]

[0125] In some aspects, provided herein is an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody), the antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) H1 comprising the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:19, a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:20, a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:21, a CDR L1 comprising the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:22, a CDR L2 comprising the amino acid sequence YTS or the amino acid sequence set forth in SEQ ID NO:23 or SEQ ID NO:24, and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO:8.

[0126] In some embodiments, an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody) comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence set forth in SEQ ID NO:3, a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO:4, a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO:5, a CDR L1 comprising the amino acid sequence set forth in SEQ ID NO:6, a CDR L2 comprising the amino acid sequence YTS, and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO:8.

[0127] In some embodiments, an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody) comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO: 21, a CDR L1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR L2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0128] In some embodiments, an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody) comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO: 20, a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO: 21, a CDR L1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR L2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a CDR L3 comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0129] In some embodiments, an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody) comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the VL comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2.

[0130] In some embodiments, an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody) comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1. In some embodiments, the VL comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:2.

[0131] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 85% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 95% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 98% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence at least 99% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:1.

[0132] In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:2.

[0133] In some embodiments, the antibody or antigen-binding fragment comprises a VH that comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment comprises a VH that comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment comprises a VH that comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment comprises a VH that comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the antibody or antigen-binding fragment comprises a VH that comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:1.

[0134] In some embodiments, the antibody or antigen-binding fragment comprises a VL that comprises an amino acid sequence about 85% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment comprises a VL that comprises an amino acid sequence about 90% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment comprises a VL that comprises an amino acid sequence about 95% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment comprises a VL that comprises an amino acid sequence about 98% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment comprises a VL that comprises an amino acid sequence about 99% identical to the amino acid sequence of SEQ ID NO:2.

[0135] In some aspects, provided herein is an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody), the antibody or antigen-binding fragment thereof comprising i) CDR H1, CDR H2, and CDR H3 comprising the CDR H1, CDR H2, and CDR H3 amino acid sequences of SEQ ID NO:1, and ii) CDR L1, CDR L2, and CDR L3 comprising the CDR L1, CDR L2, and CDR L3 amino acid sequences of SEQ ID NO:2.

[0136] In some embodiments, the CDRs are those defined in Kabat, Chothia, AbM, or IMGT.

[0137] In some aspects, the antibody or antigen-binding fragment thereof is human, humanized, or chimeric.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody.

[0139] In some embodiments, the IgG antibody is an IgG1 antibody or an IgG4 antibody.

[0140] In some aspects, the antibody is an antigen-binding fragment of an antibody.

[0141] In some aspects, the fragment is selected from the group consisting of a Fab, F(ab')2, Fv, scFv, scFv-Fc, dsFv, and a single domain molecule.

[0142] In some embodiments, the fragment is an scFv.

[0143] In some embodiments, the fragment is a Fab.

[0144] In some embodiments, the fragment is an intrabody.

[0145] In some embodiments, the antigen-binding fragment lacks the Fc region.

[0146] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH and a VL on the same polypeptide chain.

[0147] In some aspects, the VH and VL are connected by a linker.

[0148] In some aspects, the antibody or antigen-binding fragment thereof is conjugated to an agent selected from the group consisting of a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, and PEG.

[0149] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody. The antibody may be in the form of an scFv, Fab, F(ab)2, or IgG conjugated to an anti-CD3 antibody, an anti-CD16 antibody, or other molecule to mediate an effector response via T, NK, or macrophage immune cells against CD94-expressing target cells.

[0150] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein can be described by its VL domain alone or its VH domain alone.See, for example, Rader C et al., (1998) PNAS 95:8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of a mouse anti-αvβ3 antibody, resulting in a humanized antibody variant with affinity similar to or higher than that of the original antibody by identifying a complementary light or heavy chain from a human light or heavy chain library, respectively.See also Clackson T et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, describing a method for producing an antibody that binds to a specific antigen by using a specific VL domain (or VH domain) and screening a library for a complementary variable domain. See also Kim SJ & Hong HJ, (2007) J Microbiol 45:572-577, which is incorporated herein by reference in its entirety, describing methods of using a specific VH domain to generate antibodies that bind to a specific antigen by screening a library (e.g., a human VL library) for complementary VL domains. The selected VL domain can in turn be used to guide the selection of additional complementary (e.g., human) VH domains.

[0151] In some embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme, which refers to the location of the structural loops of an immunoglobulin (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226). Typically, when using the Kabat numbering convention, a Chothia CDR-H1 loop is located at heavy chain amino acids 26-32, 33, or 34, a Chothia CDR-H2 loop is located at heavy chain amino acids 52-56, a Chothia CDR-H3 loop is located at heavy chain amino acids 95-102, a Chothia CDR-L1 loop is located at light chain amino acids 24-34, a Chothia CDR-L2 loop is located at light chain amino acids 50-56, and a Chothia CDR-L3 loop is located at light chain amino acids 89-97. When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34).

[0152] In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to CD94 (e.g., human CD94) and comprise the Chothia VH and VL CDRs of the antibody. In some aspects, an antibody or antigen-binding fragment thereof that specifically binds to CD94 (e.g., human CD94) comprises one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to CD94 (e.g., human CD94) and comprise a combination of Kabat and Chothia CDRs.

[0153] In some embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7:132-136, and Lefranc MP et al., (1999) Nucleic Acids Res 27:209-212. According to the IMGT numbering scheme, CDR H1 is at position 26-35, CDR H2 is at position 51-57, CDR H3 is at position 93-102, CDR L1 is at position 27-32, CDR L2 is at position 50-52, and CDR L3 is at position 89-97. In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to CD94 (e.g., human CD94) and comprise the IMGT VH and VL CDRs of the antibodies listed in Tables 3 and 4, e.g., as described in Lefranc MP (1999) supra and Lefranc MP et al., (1999) supra.

[0154] In some embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745. Also see, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001). In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD94 (e.g., human CD94) as determined by the method in MacCallum RM et al.

[0155] In some embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions that represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CD94 (e.g., human CD94) as used herein is determined by the AbM numbering scheme.

[0156] In some aspects, the antibody or antigen-binding fragment thereof disclosed herein is an isolated antibody or antigen-binding fragment thereof. In some aspects, the antibody or antigen-binding fragment thereof disclosed herein is a monoclonal antibody or antigen-binding fragment thereof. In some aspects, the antibody or antigen-binding fragment thereof disclosed herein is not a polyclonal antibody or antigen-binding fragment thereof.

[0157] In some embodiments, the antigen-binding fragment disclosed herein that immunospecifically binds to CD94 (e.g., human CD94) is selected from the group consisting of Fab, Fab', F(ab')2, and scFv, where the Fab, Fab', F(ab')2, or scFv comprises the heavy chain variable region sequence and the light chain variable region sequence of an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein. The Fab, Fab', F(ab')2, or scFv can be produced by any technique known to one of skill in the art. In some embodiments, the Fab, Fab', F(ab')2, or scFv further comprises a moiety that extends the half-life of the antibody in vivo. This moiety is also referred to as a "half-life extending moiety." Any moiety known to one of skill in the art for extending the half-life of a Fab, Fab', F(ab')2, or scFv in vivo can be used. For example, the half-life extending moiety can comprise an Fc region, a polymer, albumin, or an albumin binding protein or compound. The polymer may comprise a natural or synthetic, optionally substituted, linear or branched polyalkylene, polyalkenylene, polyoxylalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen, or derivatives thereof. The substituent may comprise one or more hydroxy, methyl, or methoxy groups. In some embodiments, the Fab, Fab', F(ab')2, or scFv may be modified by the addition of one or more C-terminal amino acids for attachment of a half-life extending moiety. In some embodiments, the half-life extending moiety is polyethylene glycol or human serum albumin. In some embodiments, the Fab, Fab', F(ab')2, or scFv is fused to an Fc region.

[0158] In some embodiments, the anti-CD94 antibodies or antigen-binding fragments thereof disclosed herein are bispecific antibodies or antigen-binding fragments thereof.

[0159] The anti-CD94 antibody or antigen-binding fragment thereof may be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance. Examples of detectable labels or substances include enzyme labels, such as glucose oxidase, iodine ( 125 I, 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 These include radioisotopes such as Tc, luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and biotin. Such labeled antibodies or antigen-binding fragments thereof can be used to detect CD94 (e.g., human CD94) protein.

[0160] III. Anti-CD94 Chimeric Antigen Receptor Chimeric antigen receptors (CARs) redirect T cell specificity against antibody-recognized antigens expressed on the surface of cells (e.g., cancer cells).

[0161] In some embodiments, the present disclosure includes a chimeric antigen receptor (CAR) that is specific for CD94. In some embodiments, the CAR disclosed herein includes an extracellular target-specific binding domain, a transmembrane domain, an intracellular signaling domain (such as a signaling domain derived from CD3zeta or FcRgamma), and / or one or more costimulatory signaling domains derived from a costimulatory molecule, such as, but not limited to, 4-1BB, CD28, or OX40. In some embodiments, the CAR includes a hinge or spacer region between the extracellular binding domain and the transmembrane domain, such as a CD8α hinge.

[0162] In some aspects, provided herein is a chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding domain comprising an antigen binding domain capable of binding to CD94, (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain. In some aspects, the antigen binding domain comprises CDR H1 comprising the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:19, CDR H2 comprising the amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:20, CDR H3 comprising the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:21, CDR L1 comprising the amino acid sequence represented by SEQ ID NO:6 or SEQ ID NO:22, CDR L2 comprising the amino acid sequence YTS or the amino acid sequence represented by SEQ ID NO:23 or SEQ ID NO:24, and CDR L3 comprising the amino acid sequence represented by SEQ ID NO:8.

[0163] In some embodiments, the antigen-binding domain comprises a CDR H1 comprising the amino acid sequence represented by SEQ ID NO:3, a CDR H2 comprising the amino acid sequence represented by SEQ ID NO:4, a CDR H3 comprising the amino acid sequence represented by SEQ ID NO:5, a CDR L1 comprising the amino acid sequence represented by SEQ ID NO:6, a CDR L2 comprising the amino acid sequence YTS, and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO:8.

[0164] In some embodiments, the antigen-binding domain comprises a CDR H1 comprising the amino acid sequence represented by SEQ ID NO: 19, a CDR H2 comprising the amino acid sequence represented by SEQ ID NO: 20, a CDR H3 comprising the amino acid sequence represented by SEQ ID NO: 21, a CDR L1 comprising the amino acid sequence represented by SEQ ID NO: 22, a CDR L2 comprising the amino acid sequence represented by SEQ ID NO: 23, and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO: 8.

[0165] In some embodiments, the antigen-binding domain comprises a CDR H1 comprising the amino acid sequence represented by SEQ ID NO: 19, a CDR H2 comprising the amino acid sequence represented by SEQ ID NO: 20, a CDR H3 comprising the amino acid sequence represented by SEQ ID NO: 21, a CDR L1 comprising the amino acid sequence represented by SEQ ID NO: 22, a CDR L2 comprising the amino acid sequence represented by SEQ ID NO: 24, and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO: 8.

[0166] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9.

[0167] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:9.

[0168] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:56.

[0169] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:56.

[0170] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:57.

[0171] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:57.

[0172] In some aspects, the chimeric antigen receptor comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:2.

[0173] In some embodiments, the VH comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the VL comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2.

[0174] In some embodiments, the VH comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1. In some embodiments, the VL comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:2.

[0175] In some embodiments, the VH comprises an amino acid sequence at least 85% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the VH comprises an amino acid sequence at least 90% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the VH comprises an amino acid sequence at least 95% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the VH comprises an amino acid sequence at least 98% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the VH comprises an amino acid sequence at least 99% identical to the amino acid sequence SEQ ID NO:1. In some embodiments, the VH comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:1.

[0176] In some embodiments, the VL comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the VL comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the VL comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the VL comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the VL comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the VL comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:2.

[0177] In some embodiments, the VH comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO: 1. In some embodiments, the VH comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO: 1. In some embodiments, the VH comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO: 1. In some embodiments, the VH comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO: 1. In some embodiments, the VH comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO: 1.

[0178] In some embodiments, the VL comprises an amino acid sequence about 85% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VL comprises an amino acid sequence about 90% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VL comprises an amino acid sequence about 95% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VL comprises an amino acid sequence about 98% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VL comprises an amino acid sequence about 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0179] In some embodiments, the antigen binding domain is an scFv. In some embodiments, the scFv comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the scFv comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the scFv comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:33.

[0180] In some embodiments, the scFv comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:33. In some embodiments, the scFv comprises an amino acid sequence that is about 99% identical to the amino acid sequence SEQ ID NO:33.

[0181] In some embodiments, the costimulatory domain comprises a 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:37.

[0182] In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence that is about 95% identical to the amino acid sequence SEQ ID NO: 37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence that is about 98% identical to the amino acid sequence SEQ ID NO: 37. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence that is about 99% identical to the amino acid sequence SEQ ID NO: 37.

[0183] In some embodiments, the costimulatory domain comprises a CD28 costimulatory domain. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:52.

[0184] In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence that is about 98% identical to the amino acid sequence SEQ ID NO: 52. In some embodiments, the CD28 costimulatory domain comprises an amino acid sequence that is about 99% identical to the amino acid sequence SEQ ID NO: 52.

[0185] In some embodiments, the costimulatory domain comprises an OX40 costimulatory domain. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:53.

[0186] In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence that is about 98% identical to the amino acid sequence SEQ ID NO: 53. In some embodiments, the OX40 costimulatory domain comprises an amino acid sequence that is about 99% identical to the amino acid sequence SEQ ID NO:53.

[0187] In some embodiments, the hinge, the transmembrane domain, or both, are from a CD8α polypeptide.

[0188] In some embodiments, the CD8 alpha hinge domain comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35. In some embodiments, the CD8 alpha hinge domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:35. In some embodiments, the CD8α hinge domain comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:35.

[0189] In some embodiments, the CD8α hinge domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the CD8α hinge domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the CD8α hinge domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the CD8α hinge domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the CD8α hinge domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:35. In some embodiments, the CD8α hinge domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:35. In some embodiments, the CD8α hinge domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:35. In some embodiments, the CD8α hinge domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:35. In some embodiments, the CD8 alpha hinge domain comprises an amino acid sequence that is about 95% identical to the amino acid sequence SEQ ID NO: 35. In some embodiments, the CD8 alpha hinge domain comprises an amino acid sequence that is about 98% identical to the amino acid sequence SEQ ID NO: 35. In some embodiments, the CD8 alpha hinge domain comprises an amino acid sequence that is about 99% identical to the amino acid sequence SEQ ID NO: 35.

[0190] In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:36.

[0191] In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:36. In some embodiments, the CD8α transmembrane domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:36. In some embodiments, the CD8 α transmembrane domain comprises an amino acid sequence that is about 98% identical to the amino acid sequence SEQ ID NO: 36. In some embodiments, the CD8 α transmembrane domain comprises an amino acid sequence that is about 99% identical to the amino acid sequence SEQ ID NO:36.

[0192] In some embodiments, the signaling domain comprises a CD3zeta signaling domain. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:38.

[0193] In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence that is about 98% identical to the amino acid sequence SEQ ID NO: 38. In some embodiments, the CD3zeta signaling domain comprises an amino acid sequence that is about 99% identical to the amino acid sequence SEQ ID NO: 38.

[0194] In some embodiments, the chimeric antigen receptor comprises any of the amino acid sequences shown in Table 4, or a combination thereof. [Table 4-1] [Table 4-2]

[0195] In some embodiments, the chimeric antigen receptor comprises any of the nucleic acid sequences shown in Table 5, or a combination thereof. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0196] In some embodiments, the binding domain or extracellular domain of the CAR provides the CAR with the ability to bind to a target antigen of interest. The binding domain (e.g., ligand binding domain or antigen binding domain) can be any protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof). The binding domain can include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for the biological molecule of interest. For example, as further disclosed herein, the binding domain can be an antibody light and heavy chain variable region, or the light and heavy chain variable regions can be linked together in a single chain and in either orientation (e.g., VL-VH or VH-VL). Various assays are known to identify the binding domain of the present disclosure that specifically binds to a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis). The target can be an antigen of clinical interest where it is desired to elicit an effector immune response that results in tumor killing. In some embodiments, the target antigen of the binding domain of the chimeric antigen receptor is the CD94 protein.

[0197] Exemplary ligand binding domains include antigen binding proteins, such as antigen binding fragments of antibodies, such as scFvs, extracellular domains of receptors, ligands for cell surface molecules / receptors or receptor binding domains thereof, and tumor binding proteins. In some aspects, the antigen binding domains included in the CARs of the present disclosure can be variable regions (Fvs), CDRs, Fabs, scFvs, VHs, VLs, domain antibody variants (dAbs), camelid antibodies (VHHs), and other antigen-specific binding domains derived from other protein scaffolds.

[0198] In some embodiments, the binding domain of the CAR is an anti-CD94 single chain antibody (scFv), which can be a murine, chimeric, human, or humanized scFv. Single chain antibodies can be cloned from the V region genes of hybridomas specific for the desired target. Techniques that can be used to clone the variable heavy chain (VH) and variable light chain (VL) are disclosed, for example, in Orlandi et al., PNAS, 1989;86:3833-3837. Thus, in some embodiments, the binding domain comprises an antibody-derived binding domain, but can be a non-antibody-derived binding domain. An antibody-derived binding domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody, which fragment is involved in binding to an antigen.

[0199] In some embodiments, the CAR of the present disclosure can include a linker between one or more domains, e.g., provided for proper spacing and conformation of the molecule. For example, in some embodiments, a linker may be present between the VH or VL of the binding domain. In some embodiments, the linker may be 1-10 amino acids in length. In some embodiments, the linker between any of the domains of the chimeric antigen receptor may be 1-20 amino acids in length or 20 amino acids in length. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. Ranges inclusive of the numbers described herein, e.g., linkers 10-30 amino acids in length, are also included herein.

[0200] In some embodiments, a linker suitable for use in the CARs disclosed herein is a flexible linker. Suitable linkers can be easily selected and can be of any of different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0201] Exemplary flexible linkers may include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art, where n is at least an integer. Glycine and glycine-serine polymers are relatively unstructured and may potentially function as neutral tethers between domains of fusion proteins such as the CARs disclosed herein. Glycine has significant access to the phi-psi space even more than alanine and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that CAR designs may include linkers that are all or partially flexible, such that the linker may include one or more moieties that confer flexible linkers and less flexible structures to provide the desired CAR structure. Particular linkers include (G4S)n linkers, where n=1-3. In some embodiments, the linker comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the linker comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the linker comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:34.

[0202] In some embodiments, the linker comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:34. In some embodiments, the linker comprises an amino acid sequence that is about 99% identical to the amino acid sequence SEQ ID NO:34.

[0203] In some embodiments, the transmembrane region or domain is the part of the CAR that anchors the extracellular binding moiety to the plasma membrane of the immune effector cell and facilitates binding of the binding domain to the target antigen. The transmembrane domain can be the CD3zeta transmembrane domain, but other transmembrane domains that can be used include those obtained from CD8α, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain is the transmembrane domain of CD8α. In some embodiments, the transmembrane domain is synthetic, in which case it will contain primarily hydrophobic residues such as leucine and valine.

[0204] Signals generated through the T cell receptor alone are insufficient for full activation of T cells: a costimulatory signal or signaling domain must also be present to activate the T cell.

[0205] Examples of signaling domains for use in CARs can be the cytoplasmic sequences of T cell receptors and co-receptors that act together to initiate signaling after antigen receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same function. In some cases, the signaling domains include two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act in an antigen-independent manner to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can include signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs can be found in the cytoplasmic tails of various receptors that function as binding sites for tyrosine kinases of the syk / zap70 class.

[0206] Exemplary ITAMs can include those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD3zeta, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the signaling domain of the CAR can comprise a CD3zeta signaling domain.

[0207] In some aspects, the CARs disclosed herein can include a costimulatory domain, for example, derived from a costimulatory molecule. Costimulatory molecules can include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include, for example, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. A costimulatory molecule can be a cell surface molecule other than an antigen receptor or its ligand that can contribute to an efficient immune response. Thus, although the present disclosure provides an exemplary costimulatory domain derived from 4-1BB, other costimulatory domains are contemplated for use with the CARs disclosed herein. The inclusion of one or more costimulatory signaling domains can enhance the efficacy and expansion of T cells expressing a CAR receptor disclosed herein. The intracellular signaling and costimulatory signaling domains can be linked in tandem in any order to the carboxyl terminus of the transmembrane domain.

[0208] In some embodiments, the CAR disclosed herein can include a signal peptide. In some embodiments, the signal peptide is a CD8α signal peptide. In some embodiments, the CD8α signal peptide includes an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32. In some embodiments, the CD8α signal peptide includes an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:32.

[0209] In some embodiments, the CD8α signal peptide comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence that is about 98% identical to the amino acid sequence SEQ ID NO: 32. In some embodiments, the CD8α signal peptide comprises an amino acid sequence that is about 99% identical to the amino acid sequence SEQ ID NO: 32.

[0210] Although scFv-based CARs engineered to contain signaling domains from CD3 or FcRgamma have been shown to deliver potent signals for T cell activation and effector function, they are not sufficient to induce signals that promote T cell survival and expansion in the absence of concomitant costimulatory signals. Other CARs that contain binding domains, hinges, transmembrane, and signaling domains derived from CD3zeta or FcRgamma, along with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134, and CD278), can more effectively direct anti-tumor activity, as well as increased cytokine secretion, lytic activity, survival, and proliferation in CAR-expressing T cells in vitro, as well as in animal models and cancer patients (Milone et al., Molecular Therapy, 2009; 17:1453-1464; Zhong et al., Molecular Therapy, 2010; 18:413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0211] In some aspects, a CD94 binding CAR of the present disclosure comprises (a) an anti-CD94 scFv (e.g., an scFv having a binding region (e.g., CDR or variable domain) from an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein) as a binding domain, (b) a hinge region (e.g., derived from human CD8α), (c) a transmembrane domain (e.g., human CD8α transmembrane domain), and (d) a signaling domain (e.g., human T cell receptor CD3zeta chain signaling domain), and optionally one or more costimulatory signaling domains, e.g., a 4-1BB costimulatory domain, a CD28 costimulatory domain, or an OX40 costimulatory domain. In some aspects, the different protein domains are arranged in the following order from amino to carboxyl terminus: binding domain, hinge, and transmembrane domain. The intracellular signaling domain and optional costimulatory signaling domain are linked in tandem in any order to the transmembrane carboxy terminus to form a single-chain chimeric polypeptide.

[0212] In some embodiments, the nucleic acid construct encoding the CD94 binding CAR is a chimeric nucleic acid molecule that comprises different coding sequences, for example (5' to 3') coding sequences for a human anti-CD94 scFv, a human CD8α hinge region, a human CD8α transmembrane domain, and a CD3zeta signaling domain. In some embodiments, the nucleic acid construct encoding the CD94 binding CAR is a chimeric nucleic acid molecule that comprises different coding sequences, for example (5' to 3') coding sequences for a human anti-CD94 scFv, a human CD8α hinge, a human CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3zeta signaling domain.

[0213] In some aspects, the polynucleotide encoding the CAR disclosed herein is inserted into a vector. In some aspects, a vector as used herein is a vehicle into which a polynucleotide encoding a protein can be covalently inserted to result in expression of the protein and / or cloning of the polynucleotide. An isolated polynucleotide can be inserted into a vector using any suitable method known in the art, for example, but not limited to, a vector can be digested using an appropriate restriction enzyme and then ligated with an isolated polynucleotide having a matching restriction end. In some aspects, an expression vector has the ability to incorporate and express a heterologous or modified nucleic acid sequence that encodes at least a portion of a gene product that can be transcribed in a cell. In most cases, the RNA molecule is then translated into a protein. An expression vector can contain a variety of control sequences, which refer to nucleic acid sequences for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that also perform other functions, which are discussed below. An expression vector can contain additional elements, for example, an expression vector can have two replication systems, thus allowing it to be maintained in two organisms, for example, human cells for expression and a prokaryotic host for cloning and amplification.

[0214] The expression vector can have 5' upstream and 3' downstream regulatory elements such as promoter sequences (e.g., CMV, PGK, or EF1 alpha promoter), ribosome recognition and binding TATA boxes, and 3'UTR AAUAAA transcriptional sequences for efficient gene transcription and translation in their respective host cells. Other suitable promoters can include constitutive promoters of Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukosis virus promoter, EBV immediate early promoter, and Rous sarcoma virus promoter. Human gene promoters can also be used, including but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In some aspects, an inducible promoter is also contemplated as part of a vector expressing a chimeric antigen receptor. This provides a molecular switch that can turn on or off the expression of a polynucleotide sequence of interest. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.

[0215] Expression vectors can have additional sequences such as 6x-histidine, c-Myc, and FLAG tags that are incorporated into the expressed CAR. Thus, expression vectors can be engineered to contain 5' and 3' untranslated regulatory sequences, which can sometimes function as enhancer sequences, promoter regions, and / or terminator sequences that can facilitate or enhance efficient transcription of the nucleic acid(s) of interest carried on the expression vector. Expression vectors can also be engineered for replication and / or expression functionality (e.g., transcription and translation) in a particular cell type, cell location, or tissue type. Expression vectors can include selectable markers for maintenance of the vector in the host or recipient cell.

[0216] In some embodiments, the vector is a plasmid, an autonomously replicating sequence, and a transposable element. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Examples of expression vectors are Lenti-X™ Bicistronic Expression System (Neo) vector (Clontrch), pClneo vector (Promega) for expression in mammalian cells, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. The coding sequence of the CAR disclosed herein can be ligated into such an expression vector for expression of the chimeric protein in mammalian cells.

[0217] In some aspects, for the expression of CD94-binding CAR, a vector can be introduced into a host cell to allow expression of the polypeptide in the host cell. An expression vector can contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements can be appropriately selected by those skilled in the art as disclosed herein. For example, a promoter sequence can be selected to promote transcription of a polynucleotide in the vector. Suitable promoter sequences include but are not limited to T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. An enhancer sequence can be selected to enhance transcription of a polynucleotide. A selectable marker can be selected to allow selection of a host cell into which the vector has been inserted from a host cell that has not, for example, a selectable marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to allow transport of the expressed polypeptide out of the host cell.

[0218] For cloning of polynucleotides, a vector can be introduced into a host cell (isolated host cell) to allow the vector to replicate itself, thereby amplifying copies of the polynucleotides contained therein. A cloning vector can contain sequence components, which generally include, but are not limited to, a replication origin, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements can be appropriately selected by those skilled in the art. For example, a replication origin can be selected to promote the autonomous replication of the vector in the host cell.

[0219] In some aspects, the present disclosure provides an isolated host cell containing the vector provided herein. The host cell containing the vector can be useful in expressing or cloning the polynucleotide contained in the vector. Suitable host cells can include, but are not limited to, higher eukaryotic cells, such as prokaryotic cells, fungal cells, yeast cells, or mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa and Streptomyces.

[0220] The CAR of the present disclosure can be introduced into a host cell using transfection and / or transduction techniques known in the art. As used herein, the terms "transfection" and "transduction" refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid can be integrated into the host cell DNA or maintained extrachromosomally. The nucleic acid can be maintained transiently or can be stably introduced. Transfection can be achieved by various means known in the art, including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by means of viral infection rather than transfection. In some aspects, retroviral vectors are transduced by packaging the vector into virions prior to contacting the cell. For example, a nucleic acid encoding a CD94 CAR carried by a retroviral vector can be transduced into cells through infection and proviral integration.

[0221] In some aspects, the CARs of the present disclosure are introduced into and expressed in immune effector cells to redirect their specificity to a target antigen of interest.

[0222] The present disclosure provides a method for producing immune effector cells expressing a CAR disclosed herein. In some embodiments, immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. Such cells can then be directly re-administered to the individual. In some embodiments, immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a CAR. In this regard, immune effector cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express a CAR disclosed herein).

[0223] Prior to in vitro manipulation or genetic modification of immune effector cells disclosed herein, a cell source can be obtained from a subject. In particular, immune effector cells for use with the CARs disclosed herein include T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL separation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction and the cells placed in a buffer or medium appropriate for subsequent processing. In some embodiments, the cells are washed with PBS. In some aspects, the wash solution lacks calcium and may lack magnesium, or may lack many, but not all, divalent cations. As will be appreciated by those of skill in the art, the wash step may be accomplished by methods known to those of skill in the art, for example, by using a semi-automated flow-through centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers, or other saline solutions with or without buffers. In some aspects, undesirable components of the apheresis sample may be removed in the culture medium in which the cells are directly resuspended.

[0224] In some embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CD1b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present disclosure.

[0225] PBMCs can be used directly for genetic modification with CARs using the methods disclosed herein. In some embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in some embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion. CD8+ cells can be obtained by using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of those types of CD8+ cells. In some embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. After staining with anti-CD8 and anti-CD62L antibodies, PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions. In some embodiments, expression of phenotypic markers of central memory TCMs includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and are positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.

[0226] In some embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods.

[0227] Immune effector cells such as T cells can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of precursor cells) in vitro before being genetically modified. In another embodiment, immune effector cells such as T cells are genetically modified with a chimeric antigen receptor disclosed herein (e.g., transduced with a viral vector containing a nucleic acid encoding a CAR) and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are disclosed, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, 6,797,514, and WO2012079000. In general, such methods include contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, e.g., anti-CD3 and anti-CD28 antibodies, typically bound to beads or other surfaces, in a medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies bound to the same bead function as "surrogate" antigen presenting cells (APCs). In some embodiments, T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those disclosed in U.S. Patent Nos. 6,040,177, 5,827,642, and WO2012 / 129514.

[0228] CAR-expressing immune effector cells prepared as disclosed herein can be utilized in methods and compositions for adoptive immunotherapy according to known techniques or variations thereof that will be apparent to those of skill in the art based on this disclosure. See, e.g., U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al., and U.S. Patent No. 4,690,915 by Rosenberg.

[0229] In some embodiments, the cells are formulated by first harvesting them from their culture medium, then washing the cells and concentrating them in a therapeutically effective amount in a medium and container system suitable for administration (a "pharmaceutical acceptable" carrier). A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0230] The cells may be autologous or xenogeneic to the patient receiving the therapy. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) disclosed herein to enhance induction of an immune response.

[0231] The CAR-expressing immune effector cell population of the present disclosure can be administered either alone or as a pharmaceutical composition in combination with other components such as diluents and / or IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical composition of the present disclosure can include the CAR-expressing immune effector cell population, such as T cells, disclosed herein, in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions include, for example, a buffer, such as neutral buffered saline, phosphate buffered saline; a carbohydrate, such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The composition of the present disclosure is preferably formulated for intravenous administration.

[0232] Humoral immune response may also be induced, which is mainly mediated by helper T cells, which can activate B cells and thereby lead to antibody production.Various techniques may be used to analyze the type of immune response induced by the composition of the present disclosure, and are fully disclosed in the art, for example, Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0233] In some aspects, provided herein are methods of treating cancer in an individual, the methods comprising administering to the individual a therapeutically effective amount of any of the chimeric antigen receptors provided herein.

[0234] In some embodiments, the cancer is leukemia. In some embodiments, the cancer is a CD94-expressing cancer.

[0235] In some embodiments, the leukemia is T-cell leukemia, T-cell large granular leukemia, natural killer cell large granular leukemia, or natural killer cell leukemia.

[0236] In some aspects, the cancer is lymphoma.

[0237] In some embodiments, the lymphoma is T-cell lymphoma, extranodal natural killer / T-cell lymphoma, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell lymphoma, or anaplastic large cell lymphoma.

[0238] In some aspects, the cancer is lung cancer, bladder cancer, or melanoma.

[0239] In some aspects, provided herein are methods of treating or preventing graft rejection of a transplant in a patient, the methods comprising administering to the individual a therapeutically effective amount of any of the chimeric antigen receptors provided herein.

[0240] In some embodiments, the transplant is an allogeneic transplant.

[0241] In some embodiments, the transplant is an organ transplant.

[0242] In some aspects, the transplant is a hematopoietic cell transplant.

[0243] In some aspects, the transplant is an induced pluripotent cell therapy.

[0244] In some aspects, provided herein are methods of modulating an immune response in a subject, the methods comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment of any of the chimeric antigen receptors provided herein.

[0245] In some aspects, the immune response is enhanced.

[0246] In some embodiments, the immune response is mediated by natural killer cells and / or T cells.

[0247] Methods for administering the cell compositions disclosed herein include any method that is effective to result in the reintroduction in a subject of ex vivo genetically modified immune effector cells that directly express a CAR of the present disclosure, or of genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells expressing a CAR upon introduction into a subject. One method includes transducing peripheral blood T cells ex vivo with a nucleic acid construct according to the present disclosure and returning the transduced cells to the subject.

[0248] The present disclosure encompasses methods of preparing immune cells for immunotherapy comprising introducing into such immune cells ex vivo a polynucleotide or vector encoding one of the CD94-specific chimeric antigen receptors disclosed herein.

[0249] The present disclosure also encompasses immune cells comprising a polynucleotide or lentiviral vector encoding one of the CD94-specific chimeric antigen receptors disclosed herein. In some embodiments, these immune cells are used for immunotherapy (e.g., treating cancer).

[0250] The present disclosure also encompasses the method of genetically modifying immune cells to make them more suitable for allogeneic transplantation. According to the first aspect, immune cells can be made allogeneic by inactivating at least one gene expressing one or more components of T cell receptor (TCR), as disclosed in WO2013 / 176915, which can be combined with the inactivation of genes encoding or regulating the expression of HLA or β2m protein. Thus, the risk of graft-versus-host syndrome and graft rejection is significantly reduced.

[0251] In some embodiments, provided herein is an immune cell comprising the chimeric antigen receptor of the present disclosure. In some embodiments, the immune cell is an immune effector cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T lymphocyte selected from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. In some cases, the immune cell is a CD8+ cytotoxic T lymphocyte.

[0252] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor, the chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding domain comprising an scFv domain capable of binding to CD94, (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain. In some embodiments, the VH comprises a CDR H1 comprising an amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:19, a CDR H2 comprising an amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:20, and a CDR H3 comprising an amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:21. In some embodiments, the VL comprises a CDR L1 comprising an amino acid sequence represented by SEQ ID NO:6 or SEQ ID NO:22, a CDR L2 comprising an amino acid sequence YTS or an amino acid sequence represented by SEQ ID NO:23 or SEQ ID NO:24, and a CDR L3 comprising an amino acid sequence represented by SEQ ID NO:8. In some embodiments, the scFv domain comprises a VH and a VL.

[0253] In some embodiments, the VH comprises a CDR H1 comprising the amino acid sequence represented by SEQ ID NO: 3, a CDR H2 comprising the amino acid sequence represented by SEQ ID NO: 4, and a CDR H3 comprising the amino acid sequence represented by SEQ ID NO: 5. In some embodiments, the VL comprises a CDR L1 comprising the amino acid sequence represented by SEQ ID NO: 6, a CDR L2 comprising the amino acid sequence YTS, and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO: 8.

[0254] In some embodiments, the VH comprises a CDR H1 comprising the amino acid sequence represented by SEQ ID NO: 19, a CDR H2 comprising the amino acid sequence represented by SEQ ID NO: 20, and a CDR H3 comprising the amino acid sequence represented by SEQ ID NO: 21. In some embodiments, the VL comprises a CDR L1 comprising the amino acid sequence represented by SEQ ID NO: 22, a CDR L2 comprising the amino acid sequence represented by SEQ ID NO: 23, and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO: 8. In some embodiments, the VL comprises a CDR L1 comprising the amino acid sequence represented by SEQ ID NO: 22, a CDR L2 comprising the amino acid sequence represented by SEQ ID NO: 24, and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO: 8.

[0255] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9.

[0256] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:9.

[0257] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:56.

[0258] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:56.

[0259] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:57.

[0260] In some embodiments, the engineered human T cells comprise a chimeric antigen receptor comprising an amino acid sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:57.

[0261] Whether or not engineered cells (e.g., T cells) are genetically modified before or after genetic modification, the immune cells of the present disclosure, particularly T cells, may generally be activated and expanded independently of antigen binding mechanisms, as described, for example, in U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,460, 6,905,680, 6,692,964, 5,858,358, 6,887,460, 6,905,680, 6,692,964, 6,887,460, 6,905,68 ... T cells may be further activated and expanded using methods disclosed in U.S. Patent Application Publication Nos. 6, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 2006 / 0121005. T cells may be expanded in vitro or in vivo.

[0262] In some embodiments, the T cells of the present disclosure are expanded by contact with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of the T cell to generate an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate an activation signal for the T cell.

[0263] As non-limiting examples, T cell populations can be stimulated in vitro, for example, by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, T cell populations can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 5 (Lonza)), which can contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp, and TNF-α, or any other additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokine(s) in sufficient amounts for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells injected into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% O2). T cells exposed to various stimulation times can exhibit different characteristics.

[0264] In some embodiments, the cells can be expanded by co-culture with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0265] IV. Antibody Production Antibodies and antigen-binding fragments thereof that immunospecifically bind to CD94 (e.g., human CD94) can be produced by any method known in the art for the synthesis of antibodies and antigen-binding fragments thereof, e.g., by chemical synthesis or by recombinant expression techniques. The methods disclosed herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, synthesis and modification of oligonucleotides, nucleic acid hybridization, and related fields, within the skill of the art. These techniques are disclosed, for example, in the references cited herein and are fully described therein. For example, Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates), Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates). See updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press, Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0266] In some aspects, provided herein is a method of making an antibody or antigen-binding fragment that immunospecifically binds to CD94 (e.g., human CD94), comprising culturing a cell or host cell disclosed herein. In some aspects, provided herein is a method of making an antibody or antigen-binding fragment that immunospecifically binds to CD94 (e.g., human CD94), comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment using a cell or host cell disclosed herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody or antigen-binding fragment disclosed herein). In some aspects, the cell is an isolated cell. In some aspects, an exogenous polynucleotide has been introduced into the cell. In some aspects, the method further comprises purifying the antibody or antigen-binding fragment obtained from the cell or host cell.

[0267] Methods for producing polyclonal antibodies are known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0268] Monoclonal antibodies or antigen-binding fragments thereof can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technology, yeast-based display technology, or a combination thereof.For example, monoclonal antibodies or antigen-binding fragments thereof can be produced using hybridoma techniques known in the art, including those taught in, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988), Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981), or those disclosed in Kohler G & Milstein C (1975) Nature 256:495. Examples of yeast-based display methods that can be used to select and generate the antibodies disclosed herein include, for example, those disclosed in WO2009 / 036379A2, WO2010 / 105256, and WO2012 / 009568, each of which is incorporated by reference in its entirety.

[0269] In certain embodiments, monoclonal antibodies or antigen-binding fragments thereof may be produced using the hybridoma method first disclosed by Kohler et al., Nature, 256:495 (1975), as described above. In the hybridoma method, a mouse or another suitable host animal is immunized as disclosed above to elicit lymphocytes that produce or can produce antibodies that specifically bind to the protein used for immunization, e.g., a mixture of variants of CD94. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1986).

[0270] In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment produced by a clonal cell (e.g., a hybridoma or host cell producing a recombinant antibody or antigen-binding fragment), and the antibody or antigen-binding fragment immunospecifically binds to CD94 (e.g., human CD94) as determined, for example, by ELISA or other antigen-binding assays known in the art or in the Examples provided herein. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof may be a rodent or murine antibody or antigen-binding fragment thereof. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof may be a chimeric or humanized antibody or antigen-binding fragment thereof. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof may be a Fab fragment or a F(ab')2 fragment. The monoclonal antibodies or antigen-binding fragments disclosed herein may be made by hybridoma methods, e.g., as disclosed in Kohler G & Milstein C (1975) Nature 256:495, or may be isolated, for example, from a phage library, using, for example, the techniques disclosed herein. Other methods for preparation of clonal cell lines and the monoclonal antibodies and antigen-binding fragments thereof expressed thereby are well known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al.).

[0271] Antigen-binding fragments of the antibodies disclosed herein can be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments disclosed herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). The Fab fragment corresponds to one of the two identical arms of a tetrameric antibody molecule and contains an intact light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains the two antigen-binding arms of the tetrameric antibody molecule linked by disulfide bonds in the hinge region.

[0272] Additionally, the antibodies or antigen-binding fragments thereof disclosed herein can also be generated using various phage display and / or yeast-based display methods known in the art. In phage display methods, proteins are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of diseased tissue). The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, which is infected with helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are usually recombinantly fused to either phage gene III or gene VIII. Phage expressing antibodies or antigen-binding fragments thereof that bind to a particular antigen can be selected or identified by antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to generate the antibodies or fragments disclosed herein include those described in Brinkman U et al., (1995) J Immunol Methods 182:41-50, Ames RS et al., (1995) J Immunol Methods 184:177-186, Kettleborough CA et al., (1994) Eur J Immunol 24:952-958, Persic L et al., (1997) Gene 187:9-18, Burton DR & Barbas CF (1994) Advan Immunol 57:191-280, PCT Application No. PCT / GB91 / 001134, International Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844, and U.S. Pat. Nos. 5,698,426, 5,223, 5,423, ... ,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0273] The antibody or antigen-binding fragment thereof can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.

[0274] A polynucleotide In some aspects, provided herein are polynucleotides comprising nucleotide sequences encoding the antibodies or antigen-binding fragments thereof disclosed herein, or domains thereof (e.g., variable light chain regions and / or variable heavy chain regions) that immunospecifically bind to the CD94 (e.g., human CD94) antigen, as well as vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).

[0275] In some aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) disclosed herein.

[0276] In some aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that immunospecifically binds to a CD94 polypeptide (e.g., human CD94) and comprises an amino acid sequence disclosed herein.

[0277] In some aspects, the antibodies or antigen-binding fragments thereof provided herein bind to CD94 and comprise six CDRs (e.g., CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3). The CDRs may be determined by a number of algorithms in the art, such as IMGT (Table 6), Abyss (Table 7), and Kabat (Table 8).

[0278] For example, the international ImMunoGeneTics information System® (http: / / www.imgt.org) is based on the IMGT numbering for all immunoglobulin and T cell receptor V-REGIONS of all species (IMGT®, international ImMunoGeneTics information System®, Lefranc MP et al., (1991) Nucleic Acids Res. 27(1): 209-12; Ruiz M et al., (2000) Nucleic Acids Res. 28(1): 219-21; Lefranc MP (2001) Nucleic Acids Res. 29(1): 207-9; Lefranc MP (2003) Nucleic Acids Res. 31(1): 307-10; Lefranc MP et al., (2005) Dev. Comp. Immunol. 29(3): 185-203; Kaas Q et al., (2006) Nucleic Acids Res. 29(3): 185-203; al., (2007) Briefings in Functional Genomics & Proteomics, 6(4):253-64).

[0279] The ABYSIS algorithm compiles antibody protein sequences from EMBLIG, which contains antibody information extracted from the EMBL-ENA databank, the Kabat collection, and the Protein Databank (Swindells MB et al., (2017) J Mol Biol. 2017 Feb 3; 429(3): 356-364).

[0280] The Kabat definition is based on sequence variation and is the most commonly used (Kabat EA et al., supra). [Table 6] [Table 7] [Table 8]

[0281] In some aspects, isolated polynucleotides are disclosed herein, including nucleic acid molecules encoding either the VH or heavy chain of an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the VH is encoded by a polynucleotide sequence that comprises a sequence corresponding to the polynucleotide sequence of SEQ ID NO:10.

[0282] In some aspects, disclosed herein is an isolated polynucleotide comprising a nucleic acid molecule encoding either the VL or light chain of an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the VL is encoded by a polynucleotide sequence comprising a sequence corresponding to the polynucleotide sequence of SEQ ID NO:11.

[0283] In some aspects, provided herein is an antibody or antigen-binding fragment thereof capable of binding to CD94, the antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) H1 comprising the polynucleotide sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 25, a CDR H2 comprising the polynucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 26, a CDR H3 comprising the polynucleotide sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 27, a CDR L1 comprising the polynucleotide sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 28, a CDR L2 comprising the polynucleotide sequence TACACCAGC or the polynucleotide sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 30, and a CDR L3 comprising the polynucleotide sequence set forth in SEQ ID NO: 17.

[0284] In some aspects, provided herein is an antibody or antigen-binding fragment thereof capable of binding to CD94, the antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) H1 comprising the polynucleotide sequence set forth in SEQ ID NO: 12, a CDR H2 comprising the polynucleotide sequence set forth in SEQ ID NO: 13, a CDR H3 comprising the polynucleotide sequence set forth in SEQ ID NO: 14, a CDR L1 comprising the polynucleotide sequence set forth in SEQ ID NO: 15, a CDR L2 comprising the polynucleotide sequence TACACCAGC, and a CDR L3 comprising the polynucleotide sequence set forth in SEQ ID NO: 17.

[0285] In some aspects, provided herein is an antibody or antigen-binding fragment thereof capable of binding to CD94, the antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) H1 comprising the polynucleotide sequence set forth in SEQ ID NO:25, a CDR H2 comprising the polynucleotide sequence set forth in SEQ ID NO:26, a CDR H3 comprising the polynucleotide sequence set forth in SEQ ID NO:27, a CDR L1 comprising the polynucleotide sequence set forth in SEQ ID NO:28, a CDR L2 comprising the polynucleotide sequence set forth in SEQ ID NO:29, and a CDR L3 comprising the polynucleotide sequence set forth in SEQ ID NO:17.

[0286] In some aspects, provided herein is an antibody or antigen-binding fragment thereof capable of binding to CD94, the antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) H1 comprising the polynucleotide sequence set forth in SEQ ID NO:25, a CDR H2 comprising the polynucleotide sequence set forth in SEQ ID NO:26, a CDR H3 comprising the polynucleotide sequence set forth in SEQ ID NO:27, a CDR L1 comprising the polynucleotide sequence set forth in SEQ ID NO:28, a CDR L2 comprising the polynucleotide sequence set forth in SEQ ID NO:30, and a CDR L3 comprising the polynucleotide sequence set forth in SEQ ID NO:17.

[0287] In some embodiments, an antibody or antigen-binding fragment thereof capable of binding to CD94 comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH comprises a polynucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the VL comprises a polynucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0288] In some embodiments, an antibody or antigen-binding fragment thereof capable of binding to CD94 comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 10. In some embodiments, the VL comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 11.

[0289] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence at least 85% identical to the polynucleotide sequence SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence at least 90% identical to the polynucleotide sequence SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence at least 95% identical to the polynucleotide sequence SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence at least 98% identical to the polynucleotide sequence SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence at least 99% identical to the polynucleotide sequence SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 10.

[0290] In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 11.

[0291] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO: 10.

[0292] In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence about 85% identical to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence about 90% identical to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence about 95% identical to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence about 98% identical to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence about 99% identical to the polynucleotide sequence of SEQ ID NO: 11.

[0293] In some embodiments, disclosed herein is an isolated polynucleotide comprising a nucleic acid molecule encoding any of the chimeric antigen receptors (CARs) disclosed herein. In some embodiments, the chimeric antigen receptor comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the sequence corresponding to the polynucleotide sequence of SEQ ID NO: 18. In some embodiments, the chimeric antigen receptor comprises a polynucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence corresponding to the polynucleotide sequence of SEQ ID NO: 18. In some embodiments, the chimeric antigen receptor comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 18.

[0294] In some embodiments, a chimeric antigen receptor comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 58. In some embodiments, a chimeric antigen receptor comprises a polynucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 58. In some embodiments, a chimeric antigen receptor comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:58.

[0295] In some embodiments, a chimeric antigen receptor comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 59. In some embodiments, a chimeric antigen receptor comprises a polynucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 59. In some embodiments, a chimeric antigen receptor comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:59.

[0296] In some embodiments, the chimeric antigen receptor comprises, from N-terminus to C-terminus, (a) an extracellular ligand binding domain comprising an scFv domain capable of binding CD94, (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain. In some embodiments, the signal peptide comprises a CD8α signal peptide. In some embodiments, the scFv comprises a linker. In some embodiments, the hinge comprises a CD8α hinge domain. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD8α. In some embodiments, the costimulatory domain comprises a 4-1BB costimulatory domain. In some embodiments, the signaling domain comprises a CD3zeta signaling domain.

[0297] In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 39.

[0298] In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO:39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO:39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:39.

[0299] In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence that is about 85% identical to the polynucleotide sequence SEQ ID NO: 39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence that is about 90% identical to the polynucleotide sequence SEQ ID NO: 39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence that is about 95% identical to the polynucleotide sequence SEQ ID NO: 39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence that is about 98% identical to the polynucleotide sequence SEQ ID NO: 39. In some embodiments, the CD8α signal peptide comprises a polynucleotide sequence that is about 99% identical to the polynucleotide sequence SEQ ID NO: 39.

[0300] In some embodiments, the scFv comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 40.

[0301] In some embodiments, the scFv comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 40.

[0302] In some embodiments, the scFv comprises a polynucleotide sequence that is about 85% identical to the polynucleotide sequence SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence that is about 90% identical to the polynucleotide sequence SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence that is about 95% identical to the polynucleotide sequence SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence that is about 98% identical to the polynucleotide sequence SEQ ID NO: 40. In some embodiments, the scFv comprises a polynucleotide sequence that is about 99% identical to the polynucleotide sequence SEQ ID NO: 40.

[0303] In some embodiments, the linker comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 41. In some embodiments, the linker comprises a polynucleotide sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 41.

[0304] In some embodiments, the linker comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO:41. In some embodiments, the linker comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:41. In some embodiments, the linker comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:41. In some embodiments, the linker comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO:41. In some embodiments, the linker comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:41. In some embodiments, the linker comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:41.

[0305] In some embodiments, the linker comprises a polynucleotide sequence that is about 85% identical to the polynucleotide sequence SEQ ID NO: 41. In some embodiments, the linker comprises a polynucleotide sequence that is about 90% identical to the polynucleotide sequence SEQ ID NO: 41. In some embodiments, the linker comprises a polynucleotide sequence that is about 95% identical to the polynucleotide sequence SEQ ID NO: 41. In some embodiments, the linker comprises a polynucleotide sequence that is about 98% identical to the polynucleotide sequence SEQ ID NO: 41. In some embodiments, the linker comprises a polynucleotide sequence that is about 99% identical to the polynucleotide sequence SEQ ID NO: 41.

[0306] In some embodiments, the CD8 alpha hinge domain comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 42. In some embodiments, the CD8 alpha hinge domain comprises a polynucleotide sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 42.

[0307] In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO: 42. In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO: 42. In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO: 42. In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO: 42. In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO: 42. In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 42.

[0308] In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO: 42. In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO: 42. In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO: 42. In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO: 42. In some embodiments, the CD8 α hinge domain comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO: 42.

[0309] In some embodiments, the CD8 alpha transmembrane domain comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 43. In some embodiments, the CD8 alpha transmembrane domain comprises a polynucleotide sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 43.

[0310] In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO: 43. In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO: 43. In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO: 43. In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO: 43. In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO: 43. In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 43.

[0311] In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO: 43. In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO: 43. In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO: 43. In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO: 43. In some embodiments, the CD8α transmembrane domain comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO: 43.

[0312] In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 44.

[0313] In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 44.

[0314] In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO: 44. In some embodiments, the 4-1BB costimulatory domain comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO: 44.

[0315] In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence having about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 45.

[0316] In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 45.

[0317] In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence that is about 85% identical to the polynucleotide sequence SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence that is about 90% identical to the polynucleotide sequence SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence that is about 95% identical to the polynucleotide sequence SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence that is about 98% identical to the polynucleotide sequence SEQ ID NO: 45. In some embodiments, the CD3zeta signaling domain comprises a polynucleotide sequence that is about 99% identical to the polynucleotide sequence SEQ ID NO: 45.

[0318] The nucleic acid encoding the heavy chain variable domain or heavy chain and the nucleic acid encoding the light chain variable domain or light chain can be in the same polynucleotide or in different polynucleotides. The nucleic acid encoding the heavy chain variable domain or heavy chain and the nucleic acid encoding the light chain variable domain or light chain can be in the same vector or in different vectors.

[0319] Also provided herein are polynucleotides encoding the anti-CD94 antibodies or antigen-binding fragments thereof, or domains thereof disclosed herein, that are optimized, for example, by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods of generating optimized nucleic acids encoding anti-CD94 antibodies or antigen-binding fragments thereof, or domains thereof (e.g., heavy chains, light chains, VH domains, or VL domains) for recombinant expression by introducing codon changes (e.g., codon changes that encode the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in the mRNA can be performed, for example, by adapting the optimization methods disclosed in U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498.

[0320] Polynucleotides encoding the antibodies or antigen-binding fragments thereof, or domains thereof disclosed herein, can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from a hybridoma cell producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acid comprising sequences encoding the light and / or heavy chains of an antibody or antigen-binding fragment thereof. Such PCR amplification methods can be used to obtain nucleic acid comprising sequences encoding the variable light and / or variable heavy chain regions of an antibody or antigen-binding fragment thereof. Amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning, e.g., to generate chimeric and humanized antibodies or antigen-binding fragments thereof.

[0321] The polynucleotides provided herein may be, for example, in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and the DNA may be double-stranded or single-stranded. If single-stranded, the DNA may be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide is a cDNA or DNA lacking one or more endogenous introns. In some embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In some embodiments, the polynucleotide is recombinantly produced. In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure. In some embodiments, the polynucleotide is purified from natural components.

[0322] b. Cells and Vectors In some aspects, provided herein is a vector (e.g., an expression vector) comprising a polynucleotide comprising a nucleotide sequence encoding an anti-CD94 antibody, or antigen-binding fragment thereof, or a domain thereof, for recombinant expression in a host cell, preferably a mammalian cell. Also provided herein is a cell, e.g., a host cell, comprising such a vector for recombinantly expressing an anti-CD94 antibody, or antigen-binding fragment thereof, disclosed herein. In some aspects, provided herein is a method for producing an antibody, or antigen-binding fragment thereof, disclosed herein, comprising expressing such an antibody, or antigen-binding fragment thereof, in a host cell.

[0323] In some aspects, recombinant expression of an antibody or antigen-binding fragment thereof disclosed herein that specifically binds to CD94 (e.g., human CD94), or a domain thereof (e.g., a heavy or light chain disclosed herein), involves the construction of an expression vector containing a polynucleotide encoding the antibody or antigen-binding fragment thereof, or a domain thereof. Once a polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein, or a domain thereof (e.g., a heavy or light chain variable domain) is obtained, a vector for the production of the antibody or antigen-binding fragment thereof can be produced by recombinant DNA technology using techniques well known in the art. Thus, disclosed herein are methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, or a domain thereof (e.g., a light or heavy chain). Methods well known to those skilled in the art can be used to construct expression vectors containing an antibody or antigen-binding fragment thereof, or a domain thereof (e.g., a light or heavy chain) coding sequence, and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein, a heavy or light chain, a heavy or light chain variable domain, or a heavy or light chain CDR, operably linked to a promoter. Such vectors may, for example, comprise a nucleotide sequence encoding the constant region of an antibody or antigen-binding fragment thereof (see, for example, International Publication Nos. WO86 / 05807 and WO89 / 01036, and U.S. Patent No. 5,122,464), and the variable domain of an antibody or antigen-binding fragment thereof may be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0324] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody or antigen-binding fragment thereof disclosed herein, e.g., an antibody or antigen-binding fragment thereof, including any of the anti-CD94 antibodies disclosed herein.

[0325] In some aspects, disclosed herein is an isolated vector comprising any of the polynucleotides disclosed herein.

[0326] In some aspects, provided herein is a host cell comprising any of the polynucleotides disclosed herein.

[0327] In some embodiments, the host cell is selected from the group consisting of a CHO, HEK-293T, HeLa, and BHK cell. In some embodiments, the CHO cell is a CHO-K1SP cell.

[0328] In some aspects, disclosed herein are methods for producing an antibody or antigen-binding fragment thereof capable of binding to CD94, the methods comprising (a) culturing any of the host cells disclosed herein in a cell culture under conditions that allow expression of the antibody or antigen-binding fragment thereof, and (b) recovering the antibody or antigen-binding fragment thereof from the cell culture. In some aspects, disclosed herein are antibodies or antigen-binding fragments thereof obtainable by any of the production methods disclosed herein.

[0329] In some embodiments, for expression of a double-chain antibody or antigen-binding fragment thereof, vectors encoding both the heavy and light chains individually may be co-expressed in a host cell for expression of the entire immunoglobulin, as detailed below. In some embodiments, the host cell contains a vector comprising polynucleotides encoding both the heavy and light chains of an antibody or domain thereof disclosed herein. In some embodiments, the host cell contains two different vectors, a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof disclosed herein, and a second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody or domain thereof disclosed herein. In some embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof disclosed herein, and the second host cell comprises a second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein. In some aspects, provided herein is a population of host cells comprising such a first host cell and such a second host cell.

[0330] In some aspects, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein, and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein. Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides can be used.

[0331] A variety of host-expression vector systems can be utilized to express the antibodies and antigen-binding fragments thereof disclosed herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, can express the antibodies or antigen-binding fragments thereof disclosed herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK) harboring recombinant expression constructs containing promoters derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells). In some embodiments, the cells for expressing the antibodies and antigen-binding fragments thereof disclosed herein are CHO cells, e.g., CHO cells from CHO GS System™ (Lonza).In some embodiments, the cells for expressing the antibodies disclosed herein are human cells, e.g., human cell lines. In some embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some embodiments, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used for the expression of recombinant antibody molecules, particularly for expressing whole recombinant antibody molecules. For example, mammalian cells, such as Chinese hamster ovary (CHO) cells, are effective expression systems for antibodies, in conjunction with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus (Foecking MK & Hofstetter H (1986) Gene 45:101-105, and Cockett MI et al., (1990) Biotechnology 8:662-667). In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are produced by CHO cells or NS0 cells.

[0332] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can contribute to the function of the protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In some embodiments, the anti-CD94 antibodies disclosed herein are produced in mammalian cells, e.g., CHO cells, e.g., CHO-K1 cells. In some embodiments, the anti-CD94 antibodies disclosed herein are produced in mammalian cells, such as HEK-293 cells.

[0333] In some embodiments, signal peptides are used in constructing vectors containing the VH and / or VL, or heavy and / or light chains, of an antibody or antigen-binding fragment thereof provided herein.

[0334] Once an antibody or antigen-binding fragment thereof disclosed herein is produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen following Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Additionally, the antibodies or antigen-binding fragments thereof disclosed herein can be fused to heterologous polypeptide sequences disclosed herein or otherwise known in the art to facilitate purification.

[0335] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are isolated or purified. Generally, an isolated antibody or antigen-binding fragment thereof is substantially free of other antibodies or antigen-binding fragments thereof that have an antigen specificity different from that of the isolated antibody or antigen-binding fragment thereof. For example, in some embodiments, preparations of the antibodies or antigen-binding fragments thereof disclosed herein are substantially free of cellular material and / or chemical precursors.

[0336] In some embodiments, the nucleic acid encoding the antibody or antigen-binding fragment thereof disclosed herein, or the CAR disclosed herein, is provided in a viral vector. In some embodiments, the viral vector is a retrovirus, a lentivirus, or a foamy virus.

[0337] In some embodiments, the viral vector containing the coding sequence for the antibody or antigen-binding fragment thereof disclosed herein or the CAR disclosed herein is a retroviral vector or a lentiviral vector.The term "retroviral vector" refers to a vector that contains structural and functional genetic elements that are mainly derived from retroviruses.The term "lentiviral vector" refers to a vector that contains structural and functional genetic elements outside the LTR that are mainly derived from lentiviruses.

[0338] Retroviral vectors for use herein may be derived from any known retrovirus (e.g., C-type retroviruses, such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)). "Retrovirus" of the present disclosure also includes the lentivirus family of retroviruses, such as human T-cell leukemia viruses, HTLV-1 and HTLV-2, as well as human immunodeficiency viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), as well as other classes of retroviruses.

[0339] Lentiviral vector as used herein refers to vectors derived from lentiviruses, a group (or genus) of retroviruses that cause slowly developing diseases. Viruses included within this group include HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2), visna-maedi, caprine arthritis encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). Preparation of recombinant lentiviruses can be achieved using methods according to Dull et al. and Zufferey et al. (Dull et al., J.Virol., 1998;72:8463-8471 and Zufferey et al., J.Virol.1998;72:9873-9880).

[0340] Retroviral vectors (i.e., both lentiviral and non-lentiviral) for use in the present disclosure can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation disclosed herein (Current Protocols in Molecular Biology, Ausubel, FM et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals, Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 85:3014-3018). 87:6141-6145, Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043, Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381, Chowdhury et al. (1991) Science 254:1802-1805, van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644, Kay et al. (1992) Human Gene Therapy 3:641-647, Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895, Hwu et. al. (1993) J. Immunol 150:4104-4115, U.S. Patent Nos. 4,868,116, 4,980,286, PCT Application No. WO89 / 07136, PCT Application No. WO89 / 02468, PCT Application No. WO89 / 05345, and PCT Application No. WO92 / 07573).

[0341] Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in forming vectors include, for example, genomic RNA and cDNA available from commercial sources including Type Culture Collection (ATCC), Rockville, Md. Sequences can also be chemically synthesized.

[0342] V. Pharmaceutical Compositions Provided herein are compositions comprising an antibody or antigen-binding fragment thereof disclosed herein, a chimeric antigen receptor (CAR) disclosed herein, or an engineered T cell disclosed herein having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.

[0343] In various embodiments, compositions comprising anti-CD94 antibodies or antigen-binding fragments thereof, anti-CD94 chimeric antigen receptors, or engineered T cells targeting CD94 are provided in a composition with a pharma- ceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)).

[0344] The pharmaceutical compositions disclosed herein, in one embodiment, are for use as medicaments. The pharmaceutical compositions disclosed herein, in one embodiment, are for use as diagnostics, for example, for detecting the presence of CD94 in a sample obtained from a patient (e.g., a human patient).

[0345] Compositions to be used for in vivo administration can be sterilized, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0346] In some aspects, a pharmaceutical composition is provided, the pharmaceutical composition comprising an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein, a chimeric antigen receptor disclosed herein, or an engineered T cell disclosed herein and a pharma- ceutical acceptable carrier.

[0347] In some aspects, disclosed herein are pharmaceutical compositions comprising any of the antibodies or antigen-binding fragments thereof disclosed herein.

[0348] In some aspects, disclosed herein are pharmaceutical compositions comprising any of the chimeric antigen receptors disclosed herein.

[0349] In some embodiments, disclosed herein is a pharmaceutical composition comprising any of the engineered T cells disclosed herein. In some embodiments, the engineered T cells are CAR-T cells.

[0350] In some aspects, disclosed herein are pharmaceutical compositions comprising any of the vectors disclosed herein.

[0351] In some aspects, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0352] VI. How to use In some aspects, provided herein are methods of treating cancer in an individual, the methods comprising administering to the individual a therapeutically effective amount of any of an anti-CD94 antibody or antigen-binding portion thereof, a chimeric antigen receptor (CAR), an engineered T cell, or a composition comprising the same as provided herein.

[0353] In some aspects, provided herein are methods of treating cancer in an individual, the methods comprising administering to the individual a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof disclosed herein.

[0354] In some embodiments, provided herein are methods of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of any of the engineered T cells disclosed herein. In some embodiments, the methods comprise administering to the individual a therapeutically effective amount of a CAR natural killer cell, a CAR macrophage, or a CAR natural killer T cell. In some embodiments, the methods comprise administering to the individual a therapeutically effective amount of an engineered immune cell. In some embodiments, the engineered immune cell comprises any of the chimeric antigen receptors disclosed herein. In some embodiments, the engineered immune cell is a macrophage. In some embodiments, the engineered immune cell is a monocyte.

[0355] In some aspects, provided herein are methods of treating cancer in an individual, the methods comprising administering to the individual a therapeutically effective amount of any of the vectors disclosed herein.

[0356] In some aspects, provided herein are methods of treating cancer in an individual, the methods comprising administering to the individual a therapeutically effective amount of any of the pharmaceutical compositions provided herein.

[0357] In some aspects, the cancer is leukemia.

[0358] In some embodiments, the leukemia is T-cell leukemia, T-cell large granular leukemia, natural killer cell large granular leukemia, or natural killer cell leukemia.

[0359] In some aspects, the cancer is lymphoma.

[0360] In some embodiments, the lymphoma is T-cell lymphoma, extranodal natural killer / T-cell lymphoma, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell, or anaplastic large cell lymphoma.

[0361] In some embodiments, the cancer is a CD94-expressing cancer.

[0362] In some aspects, the cancer is lung cancer, bladder cancer, or melanoma.

[0363] In some aspects, provided herein is a method of treating or preventing graft rejection of a transplant in a patient, the method comprising administering to the individual a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof disclosed herein.

[0364] In some aspects, provided herein are methods of treating or preventing graft rejection of a transplant in a patient, the methods comprising administering to the individual a therapeutically effective amount of any of the chimeric antigen receptors provided herein.

[0365] In some aspects, provided herein is a method of treating or preventing graft rejection of a transplant in a patient, the method comprising administering to the individual a therapeutically effective amount of any of the engineered T cells disclosed herein.

[0366] In some aspects, provided herein are methods of treating or preventing transplant graft rejection in a patient, the methods comprising administering to the individual a therapeutically effective amount of any of the vectors disclosed herein.

[0367] In some aspects, provided herein are methods of treating or preventing graft rejection of a transplant in a patient, the methods comprising administering to the individual a therapeutically effective amount of any of the pharmaceutical compositions provided herein.

[0368] In some embodiments, the transplant is an allogeneic transplant.

[0369] In some embodiments, the transplant is an organ transplant.

[0370] In some aspects, the transplant is a hematopoietic cell transplant.

[0371] In some aspects, the transplant is an induced pluripotent cell therapy.

[0372] In some aspects, provided herein is a method of modulating an immune response in a subject, the method comprising administering to the individual a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof disclosed herein.

[0373] In some aspects, provided herein are methods of modulating an immune response in a subject, the methods comprising administering to the individual a therapeutically effective amount of any of the chimeric antigen receptors provided herein.

[0374] In some aspects, provided herein is a method of modulating an immune response in a subject, the method comprising administering to the individual a therapeutically effective amount of any of the engineered T cells disclosed herein.

[0375] In some aspects, provided herein is a method of modulating an immune response in a subject, the method comprising administering to the individual a therapeutically effective amount of any of the vectors disclosed herein.

[0376] In some aspects, provided herein are methods of modulating an immune response in a subject, the methods comprising administering to an individual a therapeutically effective amount of any of the pharmaceutical compositions provided herein.

[0377] In some aspects, the immune response is enhanced.

[0378] In some embodiments, the immune response is mediated by natural killer cells and / or T cells. In some embodiments, the antibody or antigen-binding fragment thereof or composition disclosed herein can be delivered to a subject by various routes, such as parenteral, subcutaneous, intravenous, intradermal, transdermal, intrathecal, and intranasal. In some embodiments, the antibody or antigen-binding fragment thereof or composition is administered by intraperitoneal route.

[0379] The amount of the antibody or antigen-binding fragment thereof or composition that will be effective in treating or preventing a condition will depend on the nature of the disease. The precise dose to be employed in the composition will also depend on the route of administration, and the severity of the disease.

[0380] In some aspects, described herein are methods of modulating an immune response in a subject, the methods comprising administering to the individual a therapeutically effective amount of any of the antibodies or antigen-binding fragments thereof described herein, any of the chimeric antigen receptors described herein, any of the engineered T cells disclosed herein, or any of the pharmaceutical compositions disclosed herein.

[0381] In some aspects, the immune response is enhanced.

[0382] In some embodiments, the immune response is mediated by natural killer cells and / or T cells. In some embodiments, natural killer cells and / or T cells mediate the immune response of an autoimmune disease. In some embodiments, the autoimmune disease is a systemic autoimmune disease. In some embodiments, the systemic autoimmune disease is systemic lupus erythematosus (SLE), Sjogren's syndrome, systemic sclerosis, rheumatoid arthritis (RA), multiple sclerosis, type 1 diabetes mellitus (T1DM), or autoimmune liver disease (ALD).

[0383] The anti-CD94 antibodies or antigen-binding fragments thereof disclosed herein can be used to assay CD94 protein levels or CD94+ cells in biological samples using classical methods known to those of skill in the art, including immunoassays, for example, enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, iodine ( 125 I, 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 Labels include radioisotopes such as Tc), luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and biotin. Such labels can be used to label the antibodies or antigen-binding fragments thereof disclosed herein. Alternatively, a second antibody or antigen-binding fragment thereof that recognizes the anti-CD94 antibody or antigen-binding fragment thereof disclosed herein can be labeled and used in combination with the anti-CD94 antibody or antigen-binding fragment thereof to detect CD94 protein levels (e.g., soluble CD94 protein levels).

[0384] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other cell source that potentially expresses CD94. Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans) are well known in the art. A biological sample can also be a blood sample.

[0385] The anti-CD94 antibodies and antigen-binding fragments thereof disclosed herein can carry a detectable or functional label.

[0386] Examples of detectable moieties that can be used herein include, but are not limited to, radioisotopes, phosphorescent chemicals, chemiluminescent chemicals, fluorescent chemicals, enzymes, fluorescent polypeptides, and epitope tags. Detectable moieties can be members of a binding pair that are identifiable through their interaction with an additional member of the binding pair, as well as labels that are directly visualized.

[0387] When using fluorescent labels, currently available microscopy and fluorescence activated cell sorter analysis (FACS), or a combination of both method procedures known in the art, can be utilized to identify and quantify specific binding members. The anti-CD94 antibodies or antigen-binding fragments thereof disclosed herein can carry a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes, such as aminocoumarins, fluorescein and Texas Red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. Anti-CD94 antibodies can be radiolabeled, e.g., isotope-based. 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I,131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186 The label may carry Re. When a radioactive label is used, currently available counting procedures known in the art may be utilized to identify and quantify specific binding of the anti-CD94 antibody or antigen-binding fragment to CD94 (e.g., human CD94). When the label is an enzyme, detection may be accomplished by any of the currently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques known in the art. This may be accomplished by contacting the sample or control sample with the anti-CD94 antibody or antigen-binding fragment thereof under conditions that allow for the formation of a complex between the antibody or antigen-binding fragment thereof and CD94. Any complexes formed between the antibody or antigen-binding fragment thereof and CD94 are detected and compared in the sample and the control.

[0388] In some aspects, provided herein is a method for detecting CD94 in a sample in vitro, comprising contacting the sample with an antibody or antigen-binding fragment thereof, and optionally detecting binding of the antibody or antigen-binding fragment thereof to CD94. In some aspects, provided herein is a use of an antibody or antigen-binding fragment thereof provided herein for detecting CD94 in a sample in vitro. In one aspect, provided herein is an antibody or antigen-binding fragment thereof or a pharmaceutical composition provided herein for use in detecting CD94 (e.g., soluble CD94) in a subject or a sample obtained from a subject.

[0389] In some embodiments, the subject is a mammal. In some embodiments, the mammal is selected from the group consisting of a human, a mouse, a hamster, a rabbit, a non-human primate, a guinea pig, a rat, a zebrafish, a pig, a sheep, a cat, or a dog. In some embodiments, the subject is a human.

[0390] VII. Kits Provided herein are kits that include one or more of the antibodies or antigen-binding fragments thereof disclosed herein, or conjugates thereof (eg, detection conjugates).

[0391] In some embodiments, the kit comprises one or more of the chimeric antigen receptors provided herein.

[0392] In some embodiments, the kit comprises one or more of any of the engineered T cells disclosed herein.

[0393] In some embodiments, the kit comprises one or more of any of the vectors disclosed herein.

[0394] In some embodiments, the kit comprises one or more of any of the pharmaceutical compositions provided herein.

[0395] In some embodiments, the kit further comprises instructions for use.

[0396] In some embodiments, the kit is used to treat a subject in need of treatment.

[0397] Thus, in some embodiments, the kits will contain, in suitable container means, the cells, proteins, or nucleic acid constructs of the invention, or associated reagents. In some embodiments, means for obtaining a sample from an individual and / or means for assaying the sample may be provided in the kit. In some embodiments, the kits will include, for example, cells, buffers, cell culture media, vectors, primers, restriction enzymes, salts, and the like.

[0398] The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, preferably, suitably aliquoted. If there are two or more components in the kit, the kit generally also includes a second, third, or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in the vial. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are held.

[0399] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The composition may also be formulated into an injectable composition. In that case, the container means itself may be a syringe, pipette, and / or other similar device from which the formulation may be applied to an infected area of ​​the body, injected into an animal, and / or further applied to and / or mixed with other components of the kit. However, the components of the kit may also be provided as a dry powder(s). When the reagents and / or components are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. EXAMPLES

[0400] Example 1. Analysis of the specificity of anti-CD94 monoclonal antibodies Anti-CD94 monoclonal antibodies were designed to treat NK cell or T cell lymphoproliferative disorders.

[0401] First, the expression of CD94 transcripts was evaluated in normal tissues. The Human Total RNA Survey Panel, containing RNA from 21 normal human tissues, was obtained from Applied Biosystems, Foster City, CA. Approximately 10 μg of total RNA from each source was reverse transcribed into cDNA using the Superscript III kit (Invitrogen). Quantitative PCR was performed with CD94 and β-actin primers using the following conditions: 50° C. for 2 min, 94° C. for 10 min, followed by 94° C. for 15 s, and 60° C. for 60 s, for 40 cycles on an Applied Biosystems StepOne™ / StepOnePlus™ Real-Time PCR System. The expression of CD94 mRNA relative to β-actin mRNA was calculated in each sample (FIG. 1A).

[0402] High levels of CD94 mRNA were detected in lymphoid organs, lymph nodes, spleen, and NK cells (Figure 1A). Low levels of CD94 mRNA were detected in the lung and small intestine, which are known to contain small amounts of lymphoid tissue.

[0403] Using the publicly available gene expression profiling database, BioGPS (http: / / biogps.org), CD94 mRNA expression was analyzed in 79 normal human tissues and lymphoid tissue subtypes by high-density oligonucleotide arrays. The analysis determined that CD94 transcripts were highly expressed in peripheral blood NK cells, whereas low levels were observed in CD8 T cells. CD94 mRNA was not expressed in other normal tissues (Figure 1B).

[0404] Next, we generated an anti-human CD94 monoclonal antibody ("UT-CD94"). Human CD94 was stably transfected into L cells (a mouse fibroblast cell line), and CD94-expressing L cells were used to immunize BALB / c mice. Splenocytes were isolated from the immunized mice and hybridized with mouse myeloma cells to induce monoclonal antibodies targeting human CD94 by hybridoma technology (Figure 2).

[0405] The specificity of the UT-CD94 monoclonal antibody was then tested by flow cytometry. The UT-CD94 monoclonal antibody was fluorochrome conjugated with AF647 and used to stain NK cell lymphoma and leukemia cell lines and normal donor peripheral blood mononuclear cells (PBMCs). Antibodies against CD3, CD20, CD14, and CD56 were also used to stain the cells. The UT-CD94 monoclonal antibody specifically stained human CD94-expressing L cells and human peripheral blood NK cells (Figure 3A), but did not stain parental L cells, human B cells, T cells, or monocytes (Figure 3B). The UT-CD94 monoclonal antibody also bound to NK lymphoma and leukemia cell lines (NK-92, NKL, KHYG-1), but not to B cell lymphoma cell lines (SP53, Daudi, and Jeko-1) (Figure 3C). Flow cytometry analysis demonstrated that the UT-CD94 monoclonal antibody had specificity for CD94-expressing cells.

[0406] Example 2. Analysis of cytotoxicity of CD94-specific CAR-T cells The VH and VL sequences of the UT-CD94 monoclonal antibody from Example 1 were used to generate an anti-CD94 chimeric antigen receptor (CAR). The VH and VL sequences of the UT-CD94 monoclonal antibody were converted into a single chain variable fragment (scFv), and the CD8α hinge, CD8α transmembrane domain, 4-1BB signaling domain, and CD3zeta signaling domain were used to generate an anti-CD94 CAR ("UT-CD94-CAR") (Figure 4A). The entire CAR sequence was incorporated into a lentiviral vector (pLVMG). The CDRs of the CAR amino acid and nucleotide sequences are shown above in Tables 1-3 (amino acid sequences) and Tables 4-6 (nucleotide sequences).

[0407] Normal donor T cells were purified from buffy coats, activated with anti-CD3, anti-CD28, and anti-CD2 antibodies in the presence of IL-2 for 2–3 days, and then transduced with lentivirus containing UT-CD94-CAR. Transduction efficiency was assessed by staining with His-tagged CD94 extracellular domain protein (ACROBiosystems, USA) followed by flow cytometry analysis with anti-His AF647-conjugated antibody. Flow cytometry analysis for surface expression of UT-CD94-CAR using CD94 extracellular domain protein staining revealed high transduction efficiency after 72 h (Figure 4B).

[0408] Next, the cytotoxicity of UT-CD94-CAR T cells was analyzed against NK-92 (NK cell lymphoma cell line) and NKL (NK cell leukemia cell line). NK-92 and NKL were labeled with CellTrace™ Far Red cell staining kit (Thermofisher), while the B cell lymphoma cell line SP53 (Mantle cell lymphoma cell line) was stained with CFSE (Thermofisher). NK-92, NKL, and SP53 cell lines were then co-cultured with UT-CD94-CAR transduced T cells at a 1:1:1 effector:tumor 1:tumor 2 (E:T1:T2) ratio. The percentage of dead cells was determined by flow cytometry on days 1, 2, 3, and 4 after dead cell staining (Thermofisher). The cytotoxicity of UT-CD94-CAR T cells was also analyzed against L cells (a murine fibroblast cell line) and human CD94-expressing L cells. Non-transduced T cells or tumor alone were used as negative controls.

[0409] UT-CD94-CAR T cells induced significant lysis of CD94-positive NK lymphoma and leukemia cell lines (NK-92 and NKL), as well as L cells transfected with human CD94, but not CD94-negative B cell lymphoma cell line (SP-53) or parental L cells (Figures 5A-5F). No significant lysis was observed in non-transduced T cells.

[0410] This example demonstrates that the UT-CD94-CAR T cells disclosed herein exhibit high specificity and high cytotoxic activity against CD94-expressing cells.

[0411] Example 3. Chimeric anti-CD94 antibodies block HLA-E interactions. A chimeric anti-human CD94 antibody ("cUT-CD94") was synthesized by replacing the mouse Fc with human IgG1 Fc in the mouse UT-CD94 monoclonal antibody produced using hybridoma technology in Example 1. The mouse and chimeric anti-human CD94 antibodies were then used in various assays in vitro.

[0412] Because CD94 can function as a stimulatory or inhibitory receptor when heterodimerizing with NKG2C or NKG2A, respectively, we determined whether cUT-CD94 antibodies could block its interaction with its ligand, HLA-E. After incubating the NK leukemia cell line, KHYG-1, with different concentrations of cUT-CD94 antibodies or an isotype control antibody for 1 h, the cells were stained with HLA-E tetramer and analyzed by flow cytometry. The results showed that cUT-CD94 antibodies blocked the interaction with HLA-E (Figure 6).

[0413] Since the interaction of the NKG2A / CD94 heterodimer with its ligand, HLA-E, can result in inhibition of NK cell function, we determined whether blocking CD94 could result in enhanced activation of NK cells. NK cells were isolated from normal donor peripheral blood mononuclear cells by magnetic cell separation and incubated with HLA-E expressing SP-53 mantle cell lymphoma cell line at an effector:target (E:T) ratio of 2:1 in duplicate wells in the presence or absence of UT-CD94 antibody (mCD94 Ab) and cUT-CD94 antibody (cCD94 Ab), or a commercially available mouse anti-CD94 antibody (clone DX22 from Biolegend catalog no. 305502) and their respective isotype controls (mouse IgG1 isotype control antibody from Biolegend catalog no. 401402 and human IgG1 isotype control antibody from Biocell catalog no. BE0297). All antibodies were used at a concentration of 10 μg / mL. After overnight incubation, CD137 expression was assessed by flow cytometry (Figure 7).

[0414] For CD107a expression analysis, NK cells were isolated from normal donor peripheral blood mononuclear cells by magnetic cell separation, activated overnight with IL-2 (1200 IU / mL) and incubated with HLA-E expressing SP-53 mantle cell lymphoma cell line at an effector:target (E:T) ratio of 2:1 in duplicate wells in the presence or absence of UT-CD94 antibody (mCD94 Ab) and cUT-CD94 antibody (cCD94 Ab), or a commercial mouse anti-CD94 antibody (clone DX22 from Biolegend catalog no. 305502) and their respective isotype controls (mouse IgG1 isotype control antibody from Biolegend catalog no. 401402 and human IgG1 isotype control antibody from Biocell catalog no. BE0297). All antibodies were used at a concentration of 10 μg / mL. After 16 h of incubation, CD107a expression was evaluated by flow cytometry (Figure 8).

[0415] It was observed that blocking CD94 with both UT-CD94 and cUT-CD94 antibodies resulted in increased activation of NK cells as determined by expression of CD137 (Figure 7) and degranulation of CD107a (Figure 8). This increased expression of CD137 and CD107a was significantly higher with cUT-CD94 antibody compared to a commercial mouse anti-CD94 antibody (clone DX22 from Biolegend). Specifically, expression of CD137 was found in 44-49% of cells treated with cUT-CD94 antibody, which was significantly higher than the 22-23% of cells with expression of CD137 after treatment with either UT-CD94 antibody (mCD94 Ab) or commercial mouse anti-CD94 antibody (DX22 Ab). Furthermore, expression of CD107a was found in 33-34% of cells treated with the cUT-CD94 antibody, which was significantly higher than the 16% of cells with CD107a expression after treatment with a commercially available mouse anti-CD94 antibody (DX22) and the 14-16% of cells with CD107a expression after treatment with the UT-CD94 antibody (mCD94 Ab) generated in Example 1. Taken together, these results indicate that the cUT-CD94 antibody generated herein is more effective at driving expression of CD107a and CD137 than current commercially available embodiments, as demonstrated by the DX22 mouse anti-CD94 antibody.

[0416] Example 4. Anti-CD94 antibody drug conjugates (prophetic) The UT-CD94 antibody of Example 1 or the chimeric cUT-CD94 antibody of Example 3 is used to generate an anti-CD94 antibody drug conjugate (AdC). The anti-CD94 antibody is conjugated to a cytotoxic compound via a linker known in the art, such as those disclosed in Kostoba et al., Pharmaceuticals, 2021; 14(5): 442, Tong et al., Molecules, 2021 Sep 27; 26(19): 5847, and Drago et al., Nat Rev Clin Oncol, 2021 Jun; 18(6): 327-344. doi: 10.1038 / s41571-021-00470-8. Epub 2021 Feb 8. The cytotoxicity of the anti-CD94 AdC is analyzed against NK cell lymphoma or leukemia lines NK-92 and NKL. NK-92 and NKL are labeled with CellTrace™ Far Red cell staining kit (Thermofisher), and B cell lymphoma cell line SP53 (mantle cell lymphoma cell line) is stained with CFSE (Thermofisher). NK-92, NKL, and SP53 cell lines are co-cultured with anti-CD94 AdC. The percentage of dead cells is determined by flow cytometry on days 1, 2, 3, and 4 after staining for dead cells (Thermofisher). Tumor cells alone are used as a negative control.

[0417] Example 5. Expression of CD94 in normal and tumor tissues Expression of CD94 transcripts is restricted in normal tissues. Analysis of single-cell RNAseq (scRNASeq) data from the Human Protein Atlas project revealed that CD94 mRNA transcripts were present only in infiltrating immune cells with highest expression in NK cells across 25 normal tissues, including vital organs such as heart, lung, liver, and kidney. Importantly, CD94 transcripts were not detected in epithelial cells, endothelial cells, muscle, adipocytes, cardiomyocytes, hepatocytes, neurons, renal tubule cells, etc. (Figure 9A-9F). Analysis of 29 immune cell types from normal donor peripheral blood mononuclear cells (PBMCs) showed that CD94 transcripts were predominantly present in NK cells, CD8+ terminal effector T cells, and gd T cells, and at low levels in CD8+ effector T cells, but absent in dendritic cells, B cells, and monocytes (Figure 9G).

[0418] A tissue array containing 32 different organs from three different healthy donors was tested with a commercially available anti-CD94 monoclonal antibody (clone EPR21003, Abcam) at a titration of 1:400. Standard immunohistochemistry protocols were followed and signals were detected with a Leica refined polymer detection kit. Positive signals (brown) were detected in a diffuse manner in reactive lymphocytes in different organs (lymph nodes, spleen, thymus, tonsils, breast, small intestine, colon, stomach, uterus, ovaries, larynx, and esophagus). Cerebrum, cerebellum, adrenal gland, liver, thyroid, lung, pancreas, bone marrow, trachea, testis, heart, skeletal muscle, skin, nerve, salivary gland, kidney, pericardium, eye, prostate, and cervical organs did not show a positive signal (Figure 10A). Tonsil from a normal donor (FIG. 10B) and NK / T cell lymphoma tumor tissue from a patient (FIG. 10C) were used as positive controls, and strong signals for CD94 were detected in both tissue slides.

[0419] CD94 protein expression was restricted in normal tissues. Consistent with the scRNAseq data, immunohistochemical analysis of normal donor tissue arrays showed only scattered expression of CD94 protein in reactive lymphocytes in different organs (Figure 10A). Strong staining for CD94 was detected in normal tonsils (Figure 10B) and NK / T cell lymphoma tissues (Figure 10C), as expected.

[0420] Example 6. Anti-CD94 antibody binding in NK cell leukemia cell lines To evaluate the specificity of the CD94 monoclonal antibody, CD94 was stably knocked out in two NK cell leukemia cell lines (KHYG-1 and NK92) using CRISPR-Cas9 technology. The binding specificity of the anti-CD94 antibody was evaluated by flow cytometry on wild-type KHYG-1 and NK92 cell lines, as well as their isogenic cell lines with CD94 knockout (KO) (Figure 11). The anti-CD94 monoclonal antibody bound to wild-type KHYG-1 and NK92, but not to their isogenic cell lines with CD94 knockout, confirming its specificity.

[0421] Example 7. Cytotoxic activity of anti-CD94 targeted CAR in vitro Three different CAR molecules were synthesized using VH and VL from anti-CD94 monoclonal antibodies (Figure 12A). The anti-CD94 scFv is linked to the hinge / transmembrane domain of CD8a and the costimulatory domain from CD28, 4-1BB, or OX-40. The CD3z signaling domain is present in all three constructs. Anti-CD94 CAR molecules were lentivirally transduced into normal donor T cells, and CAR transduction efficiency was assessed by flow cytometry 72 hours after staining the cells with recombinant human CD94 protein (Figure 12B). Good transduction efficiency was observed with all three constructs. NK cell leukemia cell lines (NKL-Figure 12C, KHYG-1-Figure 12D, and NK92-Figure 12E) were stably transduced with red fluorescent protein (RFP). Tumor cells were then cultured in the presence or absence of CD94-targeted CAR T cells or untransduced T cells at an effector:target ratio of 0.5:1. Tumor cell lysis or growth was monitored by continuous imaging using the Live-Cell Analysis System. Robust cytotoxic activity was observed with anti-CD94 CAR T cells containing CD28, 4-1BB, or OX-40 costimulatory domains against three different NK cell leukemia cell lines (NKL, KHYG-1, and NK92) at a very low effector:target ratio of 0.5:1 (P<0.05) (Figures 12C-12E). In contrast, no significant lysis was observed with untransduced T cells.

[0422] Example 8. Cytotoxic activity of anti-CD94 targeted CAR in vivo To evaluate the antitumor activity of CD94-targeted CAR T cells in vivo, we established a patient-derived xenograft model of hepatosplenic T-cell lymphoma that was CD94 positive. Tumor cells (1 × 10 6NSG mice were intravenously injected with 10 ... Tumor cells (1.5 × 10 6 tumor cells / mouse) were injected intravenously via the tail vein into NSG mice (5 mice / group), and 2 weeks later, 10 × 10 6 Anti-CD94-targeted CAR+ T cells or non-transduced T cells were administered by intravenous injection. The anti-tumor efficacy of CAR T cells was evaluated by monitoring mice for survival. A log-rank test was used to evaluate differences in survival between treatment groups. Treatment of NSG mice with CD94-targeted CAR T cells resulted in a significant extension of survival compared to untreated mice or mice treated with non-transduced T cells (P<0.05). Survival at day 56 was 100% for mice treated with each of the three different CAR constructs bearing CD28, 4-1BB, or OX-40 costimulatory domains. * * *

[0423] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of the art, and such techniques are fully described in the literature.

[0424] All of the references cited above, and all references cited herein, are hereby incorporated by reference in their entirety.

[0425] Any examples provided herein are offered by way of illustration and not by way of limitation.

Claims

1. 1. An antibody or antigen-binding fragment thereof capable of binding to CD94, comprising: a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:19; a CDR H2 comprising the amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:20; a CDR H3 comprising the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:21; a CDR L1 comprising the amino acid sequence represented by SEQ ID NO:6 or SEQ ID NO:22; a CDR L2 comprising the amino acid sequence YTS, or the amino acid sequence represented by SEQ ID NO:23 or SEQ ID NO:24; and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO:

8.

2. 2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1, and the VL comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

2.

3. An antibody or antigen-binding fragment thereof capable of binding to CD94, i) CDR H1, CDR H2, and CDR H3 comprising the CDR H1, CDR H2, and CDR H3 amino acid sequences of SEQ ID NO:1; ii) CDR L1, CDR L2, and CDR L3 comprising the CDR L1, CDR L2, and CDR L3 amino acid sequences of SEQ ID NO:

2.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the CDR is a Kabat-defined CDR, a Chothia-defined CDR, an AbM-defined CDR, or an IMGT-defined CDR.

5. 10. The antibody or antigen-binding fragment of any of the preceding claims, wherein the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence that is at least 85% identical to the amino acid sequence SEQ ID NO:

1.

6. 10. The antibody or antigen-binding fragment of any of the preceding claims, wherein the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO:

1.

7. 2. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence SEQ ID NO:

1.

8. 2. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence that is at least 98% identical to the amino acid sequence SEQ ID NO:

1.

9. 2. The antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence that is at least 99% identical to the amino acid sequence SEQ ID NO:

1.

10. 2. An antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

2.

11. 2. An antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

2.

12. 2. An antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

2.

13. 2. An antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO:

2.

14. 2. An antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

2.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof is human, humanized, or chimeric.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof is an IgG antibody.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the IgG antibody is an IgG1 antibody or an IgG4 antibody.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody is an antigen-binding fragment of the antibody.

19. 20. The antigen-binding fragment of the antibody of claim 18, wherein the fragment is selected from the group consisting of Fab, F(ab')2, Fv, scFv, scFv-Fc, dsFv, and single domain molecules.

20. 20. The antigen-binding fragment of the antibody of claim 19, wherein the fragment is an scFv.

21. 20. The antigen-binding fragment of the antibody of claim 19, wherein the fragment is a Fab.

22. 20. The antigen-binding fragment of the antibody of claim 18, wherein the fragment is an intrabody.

23. The antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or 18 to 22, wherein the antigen-binding fragment lacks the Fc region.

24. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or 18 to 23, comprising VH and VL on the same polypeptide chain.

25. The antibody or antigen-binding fragment thereof of claim 24 , wherein the VH and the VL are connected by a linker.

26. 26. The antibody or antigen-binding fragment thereof of any one of claims 1 to 25, wherein the antibody or antigen-binding fragment thereof is conjugated to an agent selected from the group consisting of a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, and PEG.

27. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody.

28. 20. A chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand-binding domain comprising an antigen-binding domain capable of binding to CD94, (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain, wherein the antigen-binding domain comprises: a CDR H1 comprising the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:19; a CDR H2 comprising the amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:20; a CDR H3 comprising the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:21; a CDR L1 comprising the amino acid sequence represented by SEQ ID NO:6 or SEQ ID NO:22; a CDR L2 comprising the amino acid sequence YTS, SEQ ID NO:23, or SEQ ID NO:24; and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO:

8.

29. 29. The chimeric antigen receptor of claim 28, comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

2.

30. The chimeric antigen receptor of claim 28 or 29, wherein the antigen binding domain is an scFv.

31. The chimeric antigen receptor of any one of claims 28 to 30, wherein the costimulatory domain is selected from the group consisting of a 4-1BB costimulatory domain, a CD28 costimulatory domain, or an OX40 costimulatory domain.

32. The chimeric antigen receptor of any one of claims 28 to 31, wherein the hinge, the transmembrane domain, or both, are from a CD8α polypeptide.

33. The chimeric antigen receptor of any one of claims 28 to 32, wherein the signaling domain comprises a CD3zeta signaling domain.

34. 1. An engineered human T cell comprising a chimeric antigen receptor, the chimeric antigen receptor comprising, from N-terminus to C-terminus, (a) an extracellular ligand binding domain comprising an scFv domain capable of binding to CD94, the scFv domain comprising a VL and a VH, (b) a hinge, (c) a transmembrane domain, and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain, wherein the VH comprises a CDR H1 comprising the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:19, a CDR H2 comprising the amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:20, and a CDR H3 comprising the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:21, and the VL comprises a CDR L1 comprising the amino acid sequence represented by SEQ ID NO:6 or SEQ ID NO:22, a CDR L2 comprising the amino acid sequence represented by SEQ ID NO:23 or SEQ ID NO:24, and a CDR L3 comprising the amino acid sequence represented by SEQ ID NO:

8.

35. 35. The engineered human T cell of claim 34, comprising a chimeric antigen receptor comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9, 56, or 57.

36. An isolated polynucleotide comprising a nucleic acid molecule encoding the VH or heavy chain of the antibody or antigen-binding fragment thereof of any one of claims 1 to 26.

37. 37. The isolated polynucleotide of claim 36, further comprising a nucleic acid molecule encoding the VL or light chain of the antibody or antigen-binding fragment thereof of any one of claims 1 to 26.

38. 27. An isolated polynucleotide comprising a nucleic acid molecule encoding the VL or light chain of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 26.

39. An isolated polynucleotide comprising a nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 28 to 33.

40. An isolated vector comprising the polynucleotide of any one of claims 36 to 39.

41. A host cell comprising the polynucleotide according to any one of claims 33 to 39 or the vector according to claim 40.

42. 42. The host cell of claim 41, selected from the group consisting of CHO, HEK-293T, HeLa, and BHK cells, optionally wherein the CHO cell is a CHO-K1SP cell.

43. 1. A method for producing an antibody or antigen-binding fragment thereof capable of binding to CD94, comprising the steps of: (a) culturing in a cell culture the cell of claim 41 or 42 under conditions allowing expression of the antibody or the antigen-binding fragment thereof; (b) recovering the antibody or antigen-binding fragment thereof from the cell culture.

44. 44. An antibody or antigen-binding fragment thereof obtainable by the method of claim 43.

45. 43. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 26 or 44, the chimeric antigen receptor of any one of claims 28 to 33, the engineered T cell of claim 34 or 35, or the vector of claim 40, and a pharma- ceutical acceptable excipient.

46. 11. A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-26 or 44, the chimeric antigen receptor of any one of claims 28-33, the engineered T cell of claim 34 or 35, or the pharmaceutical composition of claim 45.

47. 47. The method of claim 46, wherein the cancer is leukemia.

48. 48. The method of claim 47, wherein the leukemia is T-cell leukemia, T-cell large granular leukemia, natural killer cell large granular leukemia, or natural killer cell leukemia.

49. 47. The method of claim 46, wherein the cancer is lymphoma.

50. 50. The method of claim 49, wherein the lymphoma is T-cell lymphoma, extranodal natural killer / T-cell lymphoma, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell lymphoma, or anaplastic large cell lymphoma.

51. 47. The method of claim 46, wherein the cancer is a CD94-expressing cancer.

52. 11. A method of treating or preventing graft rejection of a transplant in a patient, comprising administering to an individual a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 26 or 44, the chimeric antigen receptor of any one of claims 28 to 33, the engineered T cell of claim 34 or 35, or the pharmaceutical composition of claim 45.

53. 53. The method of claim 52, wherein the transplant is an allogeneic transplant.

54. 53. The method of claim 52, wherein the transplant is an organ transplant.

55. 53. The method of claim 52, wherein the transplant is a hematopoietic cell transplant.

56. 53. The method of claim 52, wherein the transplant is an induced pluripotent cell therapy.

57. 11. A method of modulating an immune response in a subject, comprising administering to an individual a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 26 or 44, the chimeric antigen receptor of any one of claims 28 to 33, the engineered T cell of claim 34 or 35, or the pharmaceutical composition of claim 45.

58. 58. The method of claim 57, wherein the immune response is enhanced.

59. 58. The method of claim 57, wherein the immune response is mediated by natural killer cells and / or T cells.

60. 60. The method of claim 59, wherein the natural killer cells and / or the T cells mediate the immune response in an autoimmune disease.

61. 61. The method of claim 60, wherein the autoimmune disease is a systemic autoimmune disease.

62. 62. The method of claim 61, wherein the systemic autoimmune disease is systemic lupus erythematosus (SLE), Sjogren's syndrome, systemic sclerosis, rheumatoid arthritis (RA), multiple sclerosis, type 1 diabetes mellitus (T1DM), or autoimmune liver disease (ALD).

63. 34. A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of immune cells engineered to express the chimeric antigen receptor of any one of claims 28-33.

64. 64. The method of claim 63, wherein the immune cell is a natural killer cell, a natural killer T cell, a macrophage, or a monocyte.

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