Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2851—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/31—Chimeric antigen receptors [CAR]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70517—CD8
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70521—CD28, CD152
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/53—Hinge
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2510/00—Genetically modified cells

Definitions

• the disclosure generally relates to the fields of immunology and cancer therapy.
• the present disclosure provides anti-CD94 antibodies, anti-CD94 chimeric antigen receptors and anti-CD94 CAR-T cells, and methods of making and using the same.
• Chimeric antigen receptor (CAR) T-cell therapy has shown efficacy in B cell leukemias and lymphomas.
• CAR Natural Killer
• NK Natural Killer
• T cell lymphoproliferative disorders has been challenging due to difficulty in identifying appropriate antigens for CAR-T cell therapy.
• CD94 is a C-type lectin receptor expressed on NK cells and a subset of CD8+ T cells ( ⁇ 5%) amongst normal tissues. It can function as a stimulatory or inhibitory receptor when it heterodimerizes with NKG2C or NKG2A, respectively. Studies in patients with primary immunodeficiency where NK-cell deficiency is the major immunological defect indicate that they are primarily at risk of recurrent herpes virus infections, which can be controlled with a combination of vaccination, prophylaxis, and/or therapeutic strategies. Collectively, these reports suggest that CD94 could be a safe and effective target for CAR T- cell therapy in CD94-expressing NK/T-cell lymphoproliferative disorders.
• the present disclosure provides 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 comprises a complementary determining region (CDR) Hl 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 NON 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 LI 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, 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.
• CDR complementary determining region
• the antibody or antigen-binding fragment thereof of comprises a heavy chain variable region (VH) and a light chain variable region (VL).
• 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
• 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.
• the antibody or antigen-binding fragment thereof comprises: i) a CDR Hl, CDR H2, and CDR H3 comprising the CDR Hl, CDR H2, and CDR H3 amino acid sequences of SEQ ID NO: 1; and ii) a CDR LI, CDR L2, and CDR L3 comprising the CDR LI, CDR L2, and CDR L3 amino acid sequences of SEQ ID NO:2.
• the CDRs are the Kabat-defined CDRs, the Chothia-defined CDRs, the AbM-defined CDRs, or the IMGT-defined CDRs.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the antibody or antigen-binding fragment thereof is human, humanized, or chimeric.
• the antibody or antigen-binding fragment thereof is an IgG antibody.
• the IgG antibody is an IgGl antibody or an IgG4 antibody.
• said antibody is an antigen-binding fragment of an antibody.
• said fragment is selected from the group consisting of Fab, F(ab’)2, Fv, scFv, scFv-Fc, dsFv and a single domain molecule.
• said fragment is a scFv.
• said fragment is a Fab.
• said fragment is an intrabody.
• the antigen-binding fragment is devoid of an Fc region.
• the antibody or antigen-binding fragment comprises a VH and a
• VL on the same polypeptide chain.
• VH and VL are connected by a linker.
• 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.
• the antibody or antigen-binding fragment thereof is a bispecific antibody.
• a chimeric antigen receptor which comprises, 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.
• the antigen-binding domain comprises a CDR Hl 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 LI 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.
• 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.
• the antigen-binding domain is a scFv.
• the costimulatory domain comprises a 4- IBB costimulatory domain, a CD28 costimulatory domain, or an 0X40 costimulatory domain.
• the hinge, the transmembrane domain, or both, are from a CD8a polypeptide.
• the signaling domain comprises a CD3zeta signaling domain.
• an engineered human T cell comprising a chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus: (a) an extracellular ligand-binding domain comprising a scFv domain capable of binding to CD94, wherein the scFv domain comprises 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.
• CAR chimeric antigen receptor
• the VH comprises a CDR Hl 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.
• the VL comprises a CDR LI 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 NOR
• the engineered human T cell comprises 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 NOs:9, 56, or 57.
• CAR chimeric antigen receptor
• an isolated polynucleotide comprising a nucleic acid molecule encoding the VH or heavy chain of any of the antibody or antigenbinding fragment thereof disclosed herein.
• the isolated polynucleotide further comprises a nucleic acid molecule encoding any of the VL or light chain of the antibody or antigen-binding fragment thereof disclosed herein.
• an isolated polynucleotide comprising a nucleic acid molecule encoding any of the VL or light chain of the antibody or antigenbinding fragment thereof disclosed herein.
• an isolated polynucleotide comprising a nucleic acid molecule encoding any of the chimeric antigen receptor disclosed herein.
• disclosed herein is an isolated vector comprising the 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.
• the host cell is selected form the group consisting of CHO, HEK- 293T, HeLa and BHK cells, optionally wherein the CHO cell is a CHO-K1 SP cell.
• a method of producing an antibody or antigenbinding fragment thereof capable of binding to CD94 comprising: (a) culturing any of the host cells disclosed herein in a cell culture under conditions which allow expression of the antibody or antigen-binding fragment thereof; and (b) recovering the antibody or antigen-binding fragment thereof from said cell culture.
• an antibody or antigen-binding fragment thereof obtainable by any of the methods of producing disclosed herein.
• a pharmaceutical composition comprising any of the antibody 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.
• the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
• disclosed herein is a method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of any of the antibody 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.
• the cancer is a leukemia. In some aspects, the cancer is a CD94 expressing cancer.
• the leukemia is T cell leukemia, T cell large granular leukemia, Natural Killer cell large granular leukemia, or Natural Killer cell leukemia.
• the cancer is a lymphoma.
• 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.
• described herein is a method of treating or preventing graftrejection of a transplant in a patient, the method comprising administering to the individual therapeutically effective amount of any of the antibody 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.
• the transplant is an allogenic transplant.
• the transplant is an organ transplant.
• the transplant is a hematopoietic cell transplant.
• the transplant is an induced pluripotent cell therapy.
• described herein is a method of modulating an immune response in a subject, the method comprising administering to the individual therapeutically effective amount of any of the antibody 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.
• the immune response is enhanced.
• the immune response is mediated by Natural Killer cells and/or T cells.
• the Natural Killer cells and/or T cells mediate the immune response of an autoimmune disease.
• the autoimmune disease is a Systemic Autoimmune Disease.
• 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).
• FIG. 1 A shows expression of CD94 mRNA relative to P-actin mRNA in the Natural Killer (NK) cell lymphoma cell line NK-92 and normal human tissues.
• NK Natural Killer
• FIG. IB shows expression of CD94 mRNA in 79 normal human tissues and lymphoma subtypes by high-density oligonucleotide arrays from BioGPS.
• FIG. 2 shows a flow cytometry analysis indicating generation of CD94-L cells by measuring positive staining for CD94 (x-axis).
• FIG. 3A-3C show the specificity of a purified anti-CD94 monoclonal antibody generated by hybridoma technology ("UT-CD94").
• FIG. 3 A shows staining of CD94-L cells (CD94-L) and L cells (L) by the UT-CD94 monoclonal antibody conjugated to AF647.
• FIG. 3B shows staining of CD56+ NK cells (Nk cells), CD3 + T cells (T cells), CD 19+ B cells (B cells), and CD14+ monocytes (Monocytes) from normal donor peripheral blood mononuclear cells by the UT-CD94 monoclonal antibody conjugated to AF647.
• FIG. 3 A shows staining of CD94-L cells (CD94-L) and L cells (L) by the UT-CD94 monoclonal antibody conjugated to AF647.
• FIG. 3B shows staining of CD56+ NK cells (Nk cells), CD3 + T cells (T cells), CD 19+ B cells (
• 3C shows staining of NK lymphoma or leukemia lines (NK-92, NKL, and KHYG-1) and B cell lymphoma lines (SP53, Daudi, and Jeko-1) by the UT-CD94 monoclonal antibody conjugated to AF647.
• FIG. 4A illustrates an exemplary design of an anti-CD94 Chimeric Antigen Receptor ("UT-CD94 CAR").
• the UT-CD94 CAR includes (i) a CD94 scFv including a VL and VH domain, (ii) a CD8a hinge domain, (iii) a CD8a transmembrane domain, (iv) a 4- 1BB costimulatory domain, and (v) a CD3zeta signaling domain.
• FIG. 4B shows the expression of the UT-CD94 CAR 72 hours after lentiviral transduction as determined by staining with a His-tagged CD94 extracellular domain (ECD) protein and an AF647-conjugated anti-His antibody.
• ECD extracellular domain
• FIG. 5 A-5F show the cytotoxicity of anti-CD94 CAR-T cells ("UT-CD94 CAR-T cells").
• FIGs. 5A-5B show the lysis percentage of CD94 positive NK cell lines NKL (FIG. 5A) or NK-92 (FIG. 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), co-culture with untransduced T cells (Control T), or no co-culture (Tumor Only).
• FIGs. 5C-E show a 4 day period with the corresponding lysis percentage of CD94 positive NK cell line NKL (FIG.
• FIG. 5C shows percentage of dead target cells in L cells, human CD94-transfected L cells, B cell lymphoma cell line SP53, and NK92 cell line when cultured alone or in combination with untransduced T cells, anti-CD94 CAR-T cells or anti-CD19 CAR-T cells at an effector : target ratio of 6: 1 for 24 hours.
• FIG. 6 shows a flow cytometry analysis measuring the effect of a chimeric anti- CD94 antibody ("cUT-CD94") or isotype antibody on the staining of HLA-E tetramers (as measured by Median Fluorescence Intensity).
• FIG. 7 shows flow cytometry analysis measuring CD137+ NK cells after incubation with HLA-E expressing SP-53 mantle cell lymphoma cells and treatment with murine anti-human CD94 antibodies (UT-CD94/mCD94 and DX22 from Biolegend), cUT- CD94 antibody (cCD94), and relevant isotype controls (mlgGl and hlgGl from Biolegend).
• FIG. 8 shows flow cytometry analysis measuring CD107a+ NK cells after incubation with HLA-E expressing SP-53 mantle cell lymphoma cells and treatment with murine anti-human CD94 antibodies (UT-CD94/mCD94 and DX22 from Biolegend), cUT- CD94 antibody (cCD94), and relevant isotype controls (mlgGl and hlgGl from Biolegend).
• FIGs. 9A-9G show scRNAseq analysis of CD94 (KLRD1) mRNA expression. Overview of CD94 mRNA expression was based on scRNAseq analysis across 25 normal human tissues (FIG. 9A), in single cell types from all 25 tissues (FIG.
• FIG. 9B heart (FIG. 9C), lung (FIG. 9D), liver (FIG. 9E), and kidney (FIG. 9F). Additionally, CD94 mRNA expression in 29 immune cell types from normal donor peripheral blood was analyzed (FIG. 9G). nTPM - normalized transcripts per million. Source: Human Protein Atlas.
• FIGs. 10A-10C show an immunohistochemistry assay for CD94 expression in normal donor tissue array (FIG. 10A), normal tonsil (FIG. 10B), and tumor biopsy from a subject with NK/T-cell lymphoma (FIG. 10C).
• FIG. 11 shows binding of anti-CD94 antibody in wild-type and CD94 knock-out NK-cell leukemia cell lines.
• the binding specificity of anti-CD94 antibody was assessed by flow cytometry against wild-type KHYG-1 and NK92 cell lines and their isogenic cell lines with CD94 knock-out (KO).
• FIGs. 12A-12E show exemplary CD94-targeting CAR constructs and their cytotoxic activity in vitro.
• FIG. 12A shows the design of three different CD94-targeting CARs.
• FIG. 12B shows flow cytometry assessment of CD94-targeting CARs were lentivirally transduced into normal donor T cells.
• FIGs. 12C-12E shows the percentage of cells that underwent lysis or growth as monitored by serial imaging using the Incucyte Live- Cell Analysis System.
• NK-cell leukemia cell lines (NKL - FIG. 12C, KHYG-1 - FIG. 12D, and NK92 - FIG. 12E) were stably transduced with red fluorescent protein (RFP).
• the tumor cells were then cultured in the presence or absence of CD94-targeting CAR T cells or untransduced T cells at an Effector: Target ratio of 0.5:1.
• FIGs 13A-13C show the expression of CD94 in hepatosplenic T-cell lymphoma and cytotoxicity of anti-CD94 against PDX tumor cells.
• FIG. 13 A shows a picture of liver and spleen at necropsy of NSG mice that were injected intravenously with hepatosplenic T- cell lymphoma (TCL) PDX cells (IxlO 6 tumor cells/mouse).
• FIG. 13B shows CD3 (hCD3), CD94 (hCD94), and CD45 (hCD45) expression in liver, spleen, bone marrow, and blood as measured by flow cytometry between hCD3+hCD45+ cells vs. hCD3-hCD45- cells.
• FIG. 13C shows survival in 5 different mouse cohorts: 1) tumor only, 2) untransduced T cells, 3) mice administered with 10xl0 6 anti-CD94-targeting CAR+ T cells (CD94/CD28), 4) mice administered with 10xl0 6 anti-CD94-targeting CAR+ T cells (CD94/4-1BB), 5) mice administered with 10xl0 6 anti- CD94-targeting CAR+ T cells (CD94/OX40).
• Antitumor effects of CAR T cells was assessed by monitoring the mice for survival. Log-rank test was used to evaluate the difference in survival between treatment groups.
• Certain aspects of the disclosure are directed to anti-CD94 antibodies, anti-CD94 chimeric antigen receptors and anti-CD94 CAR-T cells, and methods of making and using the same.
• disclosed herein is an antibody or antigen-binding fragment thereof capable of binding to CD94.
• the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising complementary determining region (CDR) Hl 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, and a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:21 and a light chain variable region (VL) comprising a CDR LI 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.
• VH heavy chain variable region
• CDR complementary determining region
• 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.
• VH heavy chain variable region
• VL light chain variable region
• the antibody or antigen-binding fragment thereof comprises an anti-CD94 scFv comprising the VL and VH domains.
• a chimeric antigen receptors (CAR) or CAR T-cell comprising an anti-CD94 antibody or antigen-binding fragment thereof (e.g., an anti-CD94 scFv) disclosed herein.
• pharmaceutically acceptable refers to those compounds, materials, compositions, formulations, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• excipient refers to any substance, not itself a therapeutic agent, which can be used in a composition for delivery of an active therapeutic agent to a subject or combined with an active therapeutic agent (e.g., to create a pharmaceutical composition) to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition (e.g., formation of a hydrogel which can then be optionally incorporated into a patch).
• Excipients include, but are not limited to, solvents, penetration enhancers, wetting agents, antioxidants, lubricants, emollients, substances added to improve appearance or texture of the composition and substances used to form hydrogels. Any such excipients can be used in any dosage forms according to the present disclosure.
• excipients are not meant to be exhaustive but merely illustrative as a person of ordinary skill in the art would recognize that additional types and combinations of excipients could be used to achieve the desired goals for delivery of a drug.
• the excipient can be an inert substance, an inactive substance, and/or a not medicinally active substance.
• the excipient can serve various purposes. A person skilled in the art can select one or more excipients with respect to the particular desired properties by routine experimentation and without any undue burden. The amount of each excipient used can vary within ranges conventional in the art.
• an effective amount or “pharmaceutically effective amount” or “therapeutically effective amount” as used herein refers to the amount or quantity of a drug or pharmaceutically active substance which is sufficient to elicit the required or desired therapeutic response, or in other words, the amount which is sufficient to elicit an appreciable biological response when administered to a patient.
• treating refers to the administration of a composition to a subject for therapeutic purposes.
• antibody means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
• a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
• the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity.
• An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
• the different classes of immunoglobulins have different and well known subunit structures and three- dimensional configurations.
• Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.
• 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., complementarity determining regions (CDRs) sufficient to bind antigen).
• CDRs complementarity determining regions
• 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.
• An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.
• variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the aminoterminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
• the variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR).
• CDRs complementarity determining regions
• FR framework regions
• VH and "VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody or antigen-binding fragment thereof.
• VL and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody or antigen-binding fragment thereof.
• hypervariable regions in each chain are held together in close proximity by FRs, and with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest, 1992).
• the term "hypervariable region” as used herein refers to the amino acid residues of an antibody, which are responsible for antigen binding.
• the hypervariable region generally comprises amino acid residues from a "complementary determining region" or "CDR", the latter being of highest sequence variability and/or involved in antigen recognition. A number of CDR definitions are in use and are encompassed herein.
• the Kabat definition is based on sequence variability and is the most commonly used (Kabat EA et al., supra). Chothia refers instead to the location of the structural loops (Chothia C & LeskAM (1987) J. Mol. Biol. 196: 901-917).
• the AbM definition is a compromise between the Kabat and the Chothia definitions and is used by Oxford Molecular's AbM antibody modelling software (Martin AC R et al., (1989) Proc. Natl Acad. Sci. USA, 86: 9268-72; Martin AC R et al., (1991) Methods Enzymol.
• 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 M P (2001) Nucleic Acids Res. 29(1): 207-9; Lefranc M P (2003) Nucleic Acids Res.
• the Complementarity Determining Regions (CDRs) disclosed herein are defined according to IMGT®.
• the CDRs are defined according to Chothia.
• the CDRs are defined according to Kabat.
• the variable domain residues for each of the CDRs can be (numbering according to Kabat E A, et al., supra): LCDR1 : 27-32, LCDR2: 50-52, LCDR3: 89-97.
• the "non-CDR region" of the VL region as used herein comprise the amino acid sequences: 1-26 (FRI), 33- 49 (FR2), 53-88 (FR3), and 98-approximately 107 (FR4).
• the variable domain residues for each of the three CDRs can be HCDR1 : 26-35, HCDR2: 51-57 and HCDR3: 93-102.
• the "non-CDR region” of the VH region as used herein can comprise the amino acid sequences: 1-25 (FRI), 36-50 (FR2), 58-92 (FR3), and 103 to approximately 113 (FR4).
• the CDRs of the present disclosure can comprise "extended CDRs" which are based on the aforementioned definitions and 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 numbered as well according to Kabat et al., supra.
• alternate CDR sequences are provided for the same framework amino acid sequences of a variable region.
• the alternate CDR sequences are generated using software programs.
• different software can be used to generate alternate CDR sequences for the framework sequences of a variable region with different CDR sequences resulting from the use of the different software programs.
• the use of alternate CDR sequences can improve binding affinities of a bispecific antibody to at least one antigen.
• alternate CDR sequences are used for affinity optimization of one or both antigen binding sites of a bispecific antibody according to the present invention.
• the alternate CDRs are defined according to Kabat, Chothia, Paratome, AbM, Contact and/or IMGT annotations. In some aspects, the CDRs are defined according to more than one annotation.
• the "non-extended CDR region" of the VL region can comprise the amino acid sequences: 1-23 (FRI), 37-45 (FR2), 57-88 (FR3), and 98 to approximately 107 (FR4).
• the "non-extended CDR region” of the VH region can comprise the amino acid sequences: 1-25 (FRI), 37-46 (FR2), 66-92 (FR3), and 103 to approximately 113 (FR4).
• Fab region refers to VH and CHI domains of a heavy chain (“Fab heavy chain”), or VL and CL domains of a light chain (“Fab light chain”) of an immunoglobulin.
• scFv single chain antibody fragment
• a linker e.g., a short peptide of 10-25 amino acids
• constant region and “constant domain” are interchangeable and have their meaning common in the art.
• the constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor.
• the constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.
• the term "heavy chain” when used in reference to an antibody can refer to any distinct type, 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 IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g. , IgGl, 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.
• the term "light chain” when used in reference to an antibody can refer to any distinct type, e.g. , kappa (K) or lambda (1) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.
• the term "chimeric" antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof derived from one species of mammals (e.g.
• humanized antibody or antigen-binding fragment thereof refers to forms of non-human (e.g. murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences.
• humanized antibodies or antigenbinding fragments thereof are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non- human species (e.g.
• CDR grafted mouse, rat, rabbit, hamster
• Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody or fragment from a non-human species that has the desired specificity, affinity, and capability.
• the humanized antibody or antigenbinding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and/or within the non-human CDR residues to refine and optimize the specificity, affinity, and/or capability of the antibody or antigen-binding fragment thereof.
• the humanized antibody or antigen-binding fragment thereof will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
• a "humanized antibody” is a resurfaced antibody.
• 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, where such antibody or antigen-binding fragment is made using any technique known in the art. This definition of a human antibody or antigenbinding fragment thereof includes intact or full-length antibodies and fragments thereof.
• Binding affinity generally refers to the strength of the sum total 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 indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., antibody or antigen binding fragment thereof and antigen).
• the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD).
• Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA).
• KD is calculated from the quotient of koff/kon
• KA is calculated from the quotient of kon/koff.
• k on refers to the association rate constant of, e.g., an antibody or antigen binding fragment thereof to an antigen
• koft refer to the dissociation of, e.g., an antibody or antigen-binding fragment thereof from an antigen.
• the kon and k O ff can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA.
• an "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, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more noncontiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope).
• the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, e.g., 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).
• crystallization can be accomplished using any of the known methods 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 can be 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.,; U.S.
• An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope.
• Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays.
• An antibody can be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
• a polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated” refers to a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. As used herein, “substantially pure” refers to material which 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.
• polypeptide polypeptide
• peptide protein
• the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
• the polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids.
• the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
• polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
• the polypeptides of this disclosure are based upon antibodies, in certain aspects, the polypeptides can occur as single chains or associated chains.
• Percent identity refers to the extent of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Percent identity can be determined by aligning two sequences, introducing gaps to maximize identity between the sequences. Alignments can be generated using programs known in the art. For purposes herein, alignment of nucleotide sequences can be performed with the blastn program set at default parameters, and alignment of amino acid sequences can be performed with the blastp program set at default parameters (see National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).
• NCBI National Center for Biotechnology Information
• the term "host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line.
• the term “host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
• composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.
• the composition can be sterile.
• administer refers to methods that can be used to enable delivery of a drug, e.g., an anti-CD94 antibody or antigen-binding fragment thereof, to the desired site(s) of biological action (e.g., intravenous administration).
• Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.
• the terms "subject” and “patient” are used interchangeably.
• the subject can be an animal.
• the subject is a mammal such as a non-human animal (e.g., cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.).
• the subject is a human.
• nucleic acid or “polynucleotides” refers to nucleotides and/or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• PCR polymerase chain reaction
• 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 a combination of both.
• Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
• Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
• sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
• modifications in 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.
• chimeric antigen receptor refers to a chimeric molecule that includes a binding domain which binds to a component (e.g., a ligand) present on a target cell (for example, the binding domain can include an antibody or antigen-binding fragment thereof specific for a desired antigen (e.g., CD94)) and a T cell receptor-activating intracellular domain.
• the CAR exhibits a specific anti-target cellular immune activity.
• CARs can include an extracellular ligand-binding domain (e.g., a single chain antibody -binding domain (scFv)) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain.
• the CAR can include an extracellular ligand-binding domain, a hinge, a transmembrane domain, and a cytoplasmic domain.
• the cytoplasmic domain comprises a costimulatory domain (e.g., 4- 1BB, CD28, or 0X40) and a signaling domain (e.g., CD3zeta).
• the CAR when expressed in T cells, can redirect antigen recognition based on the antibody's specificity.
• vector includes, but is not limited to, a viral vector, a plasmid, an RNA vector or a linear or circular DNA or RNA molecule which can include chromosomal, non-chromosomal, semi -synthetic or synthetic nucleic acids.
• the vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and are commercially available.
• Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
• RNA viruses such as picomavirus and alphavirus
• double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
• herpesvirus e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus
• poxvirus e.g., vaccinia, fowlpox and canarypox
• Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
• retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, and lentivirus.
• costimulatory domain or “costimulatory molecule” refers to the cognate binding partner on a T-cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the cell (e.g., a proliferation response).
• Costimulatory molecules include, but are not limited to, an MHC class I molecule, BTLA and Toll ligand receptor.
• costimulatory molecules examples include CD27, CD28, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like.
• the costimulatory molecule is a cell surface molecule, other than an antigen receptor or their ligands, that is promotes an efficient immune response.
• costimulatory ligand refers to a molecule on an antigen presenting cell that specifically binds a cognate costimulatory molecule on a T-cell.
• the costimulatory ligand provides a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response.
• the T cell response includes, but is not limited to, proliferation activation, differentiation and the like.
• a costimulatory ligand can include but is not limited to 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, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3.
• a "costimulatory signal” refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of a molecule.
• extracellular ligand-binding domain refers to an oligo- or polypeptide that is capable of binding a ligand, e.g., a cell surface molecule.
• the extracellular ligand-binding domain can be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state (e.g., cancer).
• a particular disease state e.g., cancer
• cell surface markers that can act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.
• a “signal transducing domain” or “signaling domain”, as used herein with respect to CARs, is responsible for intracellular signaling following the binding of an extracellular ligand binding domain to a target.
• the signaling domain as used in a CAR results in the activation of the immune cell and immune response.
• the signal transducing domain can responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed.
• the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines.
• the signal transducing domain can refer to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function.
• Examples of signal transducing domains for use in a CAR can be the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivate or variant of these sequences and any synthetic sequence that has the same functional capability.
• signaling domains comprise two distinct classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
• Primary cytoplasmic signaling sequences can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (IT AMs).
• ITAMs refer to signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases.
• ITAMs include those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD3zeta, CD5, CD22, CD79a, CD79b and CD66d.
• the signaling domain of the CAR can comprise the CD3zeta signaling domain.
• transmembrane region or “transmembrane domain” as used herein is a portion of a CAR that anchors the extracellular binding portion to the plasma membrane of an immune effector cell, and facilitates binding of the binding domain to the target antigen.
• the transmembrane domain can be a CD3zeta transmembrane domain, however other transmembrane domains that can be employed include those obtained from CD8a, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, or CD154.
• the binding domain of a CAR can be followed by a "spacer,” or, “hinge,” which refers to the region that separates or moves the antigen binding domain away from the effector cell surface.
• the hinge enables 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) and the binding domain.
• a hinge region is an immunoglobulin hinge region and can be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region.
• hinge regions used in the CARs disclosed herein can include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a, CD4, CD28 and CD7, which may be wild-type hinge regions from these molecules or may be altered.
• the hinge region comprises a CD8a hinge.
• antibodies e.g. monoclonal antibodies
• antigen-binding fragments thereof which specifically bind to CD94
• CD94 e.g., human CD94
• the amino acid sequence for human CD94 comprises SEQ ID NO: 31 (see RefSeq Accession Number NP_001107868.2).
• an antibody or antigen-binding fragment thereof provided herein binds to CD94 and comprises six CDRs (e.g., a CDR Hl, a CDR H2, a CDR H3, a CDR LI, a CDR L2, and a CDR L3).
• the CDRs can be determined by a number of algorithms in the art, such as IMGT (Table 1), abYsis (Table 2), and Kabat (Table 3).
• 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 M P (2001) Nucleic Acids Res. 29(1): 207-9; Lefranc M P (2003) Nucleic Acids Res. 31(1): 307-10; Lefranc M P et al., (2005) Dev. Comp. Immunol. 29(3): 185-203; Kaas Q et al., (2007) Briefings in Functional Genomics & Proteomics, 6(4): 253-64).
• 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).
• an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody), wherein the antibody or antigen-binding fragment thereof comprises a complementary determining region (CDR) Hl 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 LI 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.
• CDR complementary determining region
• the antibody or antigen-binding fragment thereof capable of binding CD94 comprises a complementary determining region (CDR) Hl 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 LI 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.
• CDR complementary determining region
• the antibody or antigen-binding fragment thereof capable of binding CD94 comprises a complementary determining region (CDR) Hl 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 LI 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.
• CDR complementary determining region
• the antibody or antigen-binding fragment thereof capable of binding CD94 comprises a complementary determining region (CDR) Hl 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 LI 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.
• CDR complementary determining region
• the antibody or antigen-binding fragment thereof capable of binding CD94 comprises a heavy chain variable region (VH) and a light chain variable region (VL).
• 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.
• 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.
• the antibody or antigen-binding fragment thereof capable of binding CD94 comprises a heavy chain variable region (VH) and a light chain variable region (VL).
• 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.
• 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.
• 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 aspects, 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 aspects, 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 aspects, 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.
• 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 aspects, 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.
• 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 aspects, 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 aspects, 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 aspects, 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.
• 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 aspects, 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.
• the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:1. In some aspects, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO: 1.
• the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence about 85% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence about 90% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence about 95% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence about 98% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the antibody or antigen-binding fragment comprises a VL comprising an amino acid sequence about 99% identical to the amino acid sequence of SEQ ID NO:2.
• an antibody or antigen-binding fragment thereof capable of binding to CD94 (e.g., an anti-CD94 specific antibody), wherein the antibody or antigen-binding fragment thereof comprises: i) a CDR Hl, CDR H2, and CDR H3 comprising the CDR Hl, CDR H2, and CDR H3 amino acid sequences of SEQ ID NO: 1; and ii) a CDR LI, CDR L2, and CDR L3 comprising the CDR LI, CDR L2, and CDR L3 amino acid sequences of SEQ ID NO:2.
• the CDRs are the Kabat-defined CDRs, the Chothia-defined CDRs, the AbM-defined CDRs, or the IMGT-defined CDRs.
• the antibody or antigen-binding fragment thereof is human, humanized, or chimeric.
• the antibody or antigen-binding fragment thereof is an IgG antibody.
• the IgG antibody is an IgGl antibody or an IgG4 antibody.
• the antibody is an antigen-binding fragment of an antibody.
• said fragment is selected from the group consisting of Fab, F(ab’)2, Fv, scFv, scFv-Fc, dsFv and a single domain molecule.
• said fragment is a scFv.
• said fragment is a Fab.
• said fragment is an intrabody.
• the antigen-binding fragment is devoid of an Fc region.
• the antibody or antigen-binding fragment thereof comprises a VH and a VL on the same polypeptide chain.
• VH and VL are connected by a linker.
• 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.
• the antibody or antigen-binding fragment thereof is a bispecific antibody.
• the antibody can be in the form of scFv, Fab, F(ab)2, or IgG conjugated with anti- CD3 antibody, anti-CD16 antibody, or other molecules, to mediate effector response via T, NK, or macrophage immune cells against CD94-expressing target cells.
• an 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 the mouse anti-avP3 antibody by identifying a complementing light chain or heavy chain, respectively, from a human light chain or heavy chain library, resulting in humanized antibody variants having affinities as high or higher than the affinity of the original antibody.
• 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 immunoglobulin structural loops (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; Tramontane A etal., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226).
• Chothia numbering scheme refers to the location of immunoglobulin structural loops
• the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34
• the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56
• the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102
• the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34
• the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56
• the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97.
• the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
• antibodies and antigen-binding fragments thereof that specifically bind to CD94 e.g., human CD94
• CD94 e.g., human CD94
• antibodies or antigen-binding fragments thereof that specifically bind to CD94 comprise one or more CDRs, in which the Chothia and Kabat CDRs have the same amino acid sequence.
• antibodies and antigen-binding fragments thereof that specifically bind to CD94 e.g., human CD94
• combinations of Kabat CDRs and Chothia CDRs are antibodies and antigen-binding fragments thereof that specifically bind to CD94 and comprise combinations of Kabat CDRs and Chothia CDRs.
• the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212.
• CDR Hl is at positions 26 to 35
• CDR H2 is at positions 51 to 57
• CDR H3 is at positions 93 to 102
• CDR LI is at positions 27 to 32
• CDR L2 is at positions 50 to 52
• CDR L3 is at positions 89 to 97.
• antibodies and antigen-binding fragments thereof that specifically bind to CD94 (e.g., human CD94) and comprise the IMGT VH and VL CDRs of an antibody listed in Tables 3 and 4, for example, as described in Lefranc M-P (1999) supra and Lefranc M-P et al., (1999) supra).
• CD94 e.g., human CD94
• 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. See also, e.g., Martin A.
• antibodies or antigen-binding fragments thereof that specifically bind to CD94 are determined by the method in MacCallum RM et al.
• the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers AbM hypervariable regions which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).
• AbM numbering scheme refers AbM hypervariable regions which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).
• CD94 e.g., human CD94
• an antibody or antigen-binding fragment thereof disclosed herein is an isolated antibody or antigen-binding fragment thereof. In some aspects, an antibody or antigen-binding fragment thereof disclosed herein is a monoclonal antibody or antigenbinding fragment thereof. In some aspects, an antibody or antigen-binding fragment thereof disclosed herein is not a polyclonal antibody or antigen-binding fragment thereof.
• an antigen-binding fragment as disclosed herein which immunospecifically binds to CD94 (e.g., human CD94), is selected from the group consisting of a Fab, Fab', F(ab')2, and scFv, wherein the Fab, Fab', F(ab')2, or scFv comprises a heavy chain variable region sequence and a light chain variable region sequence of an anti-CD94 antibody or antigen-binding fragment thereof as disclosed herein.
• a Fab, Fab', F(ab')2, or scFv can be produced by any technique known to those of skill in the art.
• the Fab, Fab', F(ab')2, or scFv further comprises a moiety that extends the half-life of the antibody in vivo.
• the moiety is also termed a "half-life extending moiety.” Any moiety known to those of skill in the art for extending the half-life of a Fab, Fab', F(ab')2, or scFv in vivo can be used.
• the half-life extending moiety can include a Fc region, a polymer, an albumin, or an albumin binding protein or compound.
• the polymer can include a natural or synthetic, optionally substituted straight or branched chain polyalkylene, polyalkenylene, polyoxylalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen, or derivative thereof.
• Substituents can include one or more hydroxy, methyl, or methoxy groups.
• the Fab, Fab', F(ab')2, or scFv can be modified by the addition of one or more C-terminal amino acids for attachment of the half-life extending moiety.
• the half-life extending moiety is polyethylene glycol or human serum albumin.
• the Fab, Fab', F(ab')2, or scFv is fused to an Fc region.
• an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein is a bispecific antibody or antigen-binding fragment thereof.
• An anti-CD94 antibody or antigen-binding fragment thereof can be fused or conjugated (e.g., covalently or noncovalently linked) to a detectable label or substance.
• detectable labels or substances include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium (“Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• detectable labels or substances include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium (“Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluoresc
• Chimeric antigen receptors redirect T cell specificity toward antibody- recognized antigens expressed on the surface of cells (e.g., cancer cells).
• the present disclosure includes a chimeric antigen receptor (CAR) which is specific for CD94.
• a CAR as disclosed herein comprises 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 co-stimulatory signaling domains derived from a co-stimulatory molecule, such as, but not limited to, 4- IBB, CD28, or 0X40.
• the CAR includes a hinge or spacer region between the extracellular binding domain and the transmembrane domain, such as a CD8a hinge.
• a chimeric antigen receptor which comprises, 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.
• the antigen-binding domain comprises a CDR Hl 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 LI 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.
• the antigen-binding domain comprises a CDR Hl 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 LI 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.
• the antigen-binding domain comprises a CDR Hl 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 LI 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.
• the antigen-binding domain comprises a CDR Hl 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 LI 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the VH comprises an amino acid sequence at least 85% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the VH comprises an amino acid sequence at least 90% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the VH comprises an amino acid sequence at least 95% identical to the amino acid sequence SEQ ID NO:1. In some aspects, the VH comprises an amino acid sequence at least 98% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the VH comprises an amino acid sequence at least 99% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the VH comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1.
• the VL comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the VL comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the VL comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the VL comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the VL comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the VL comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:2.
• the VH comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the VH comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the VH comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:1. In some aspects, the VH comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO: 1. In some aspects, the VH comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO: 1.
• the VL comprises an amino acid sequence about 85% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the VL comprises an amino acid sequence about 90% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the VL comprises an amino acid sequence about 95% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the VL comprises an amino acid sequence about 98% identical to the amino acid sequence of SEQ ID NO:2. In some aspects, the VL comprises an amino acid sequence about 99% identical to the amino acid sequence of SEQ ID NO:2.
• the antigen-binding domain is a scFv.
• 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.
• 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.
• 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.
• the scFv comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:33. In some aspects, the scFv comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:33. In some aspects, the scFv comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:33. In some aspects, the scFv comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:33. In some aspects, the scFv comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:33.
• the scFv comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:33. In some aspects, the scFv comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:33. In some aspects, the scFv comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:33. In some aspects, the scFv comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:33. In some aspects, the scFv comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:33. In some aspects, the scFv comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:33.
• the costimulatory domain comprises a 4- IBB costimulatory domain.
• 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.
• 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.
• 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.
• the 4- IBB costimulatory domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:37. In some aspects, the 4- IBB costimulatory domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:37. In some aspects, 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 aspects, 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 aspects, the 4-1BB costimulatory domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:37.
• the 4- IBB costimulatory domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:37. In some aspects, the 4-1BB costimulatory domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:37. In some aspects, the 4-1BB costimulatory domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:37. In some aspects, the 4-1BB costimulatory domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:37. In some aspects, the 4-1BB costimulatory domain comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:37. In some aspects, the 4-1BB costimulatory domain comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:37.
• the costimulatory domain comprises a CD28 costimulatory domain.
• 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.
• 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.
• 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.
• 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 aspects, 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 aspects, 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 aspects, 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 aspects, the CD28 costimulatory domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:52.
• the CD28 costimulatory domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:52. In some aspects, the CD28 costimulatory domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:52. In some aspects, the CD28 costimulatory domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:52. In some aspects, the CD28 costimulatory domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:52. In some aspects, the CD28 costimulatory domain comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:52. In some aspects, the CD28 costimulatory domain comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:52.
• the costimulatory domain comprises an 0X40 costimulatory domain.
• the 0X40 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.
• the 0X40 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.
• the 0X40 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.
• the 0X40 costimulatory domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:53. In some aspects, the 0X40 costimulatory domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:53. In some aspects, the 0X40 costimulatory domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:53. In some aspects, the 0X40 costimulatory domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:53. In some aspects, the 0X40 costimulatory domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:53.
• the 0X40 costimulatory domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:53. In some aspects, the 0X40 costimulatory domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:53. In some aspects, the 0X40 costimulatory domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:53. In some aspects, the 0X40 costimulatory domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:53. In some aspects, the 0X40 costimulatory domain comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:53. In some aspects, the 0X40 costimulatory domain comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:53.
• the hinge, the transmembrane domain, or both are from a CD8a polypeptide.
• the CD8a 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.
• the CD8a 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.
• the CD8a 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.
• the CD8a hinge domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:35. In some aspects, the CD8a hinge domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:35. In some aspects, the CD8a hinge domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:35. In some aspects, the CD8a hinge domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:35. In some aspects, the CD8a hinge domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:35.
• the CD8a hinge domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:35. In some aspects, the CD8a hinge domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:35. In some aspects, the CD8a hinge domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:35. In some aspects, the CD8a hinge domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:35. In some aspects, the CD8a hinge domain comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:35. In some aspects, the CD8a hinge domain comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:35.
• the CD8a 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.
• the CD8a 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.
• the CD8a 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.
• the CD8a transmembrane domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:36. In some aspects, the CD8a transmembrane domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:36. In some aspects, the CD8a transmembrane domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:36. In some aspects, the CD8a transmembrane domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:36. In some aspects, the CD8a transmembrane domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:36.
• the CD8a transmembrane domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:36. In some aspects, the CD8a transmembrane domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:36. In some aspects, the CD8a transmembrane domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:36. In some aspects, the CD8a transmembrane domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:36. In some aspects, the CD8a transmembrane domain comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:36. In some aspects, the CD8a transmembrane domain comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:36.
• the signaling domain comprises a CD3zeta signaling domain.
• 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.
• 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.
• 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.
• 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 aspects, 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 aspects, 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 aspects, 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 aspects, the CD3zeta signaling domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:38.
• the CD3zeta signaling domain comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:38. In some aspects, the CD3zeta signaling domain comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:38. In some aspects, the CD3zeta signaling domain comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:38. In some aspects, the CD3zeta signaling domain comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:38. In some aspects, the CD3zeta signaling domain comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:38. In some aspects, the CD3zeta signaling domain comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:38.
• the chimeric antigen receptor comprises any of the amino acid sequences shown in Table 4 or combinations thereof.
• the chimeric antigen receptor comprises any of the nucleic acid sequences shown in Table 5 or combinations thereof.
• the binding domain or the extracellular domain of the CAR provides the CAR with the ability to bind to the target antigen of interest.
• a binding domain e.g., a ligand-binding domain or antigen-binding domain
• a 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).
• a binding domain can include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest.
• a binding domain can be antibody light chain and heavy chain variable regions, or the light and heavy chain variable regions can be joined together in a single chain and in either orientation (e.g., VL-VH or VH-VL).
• assays are known for identifying binding domains of the present disclosure that specifically bind with 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 against which it would be desirable to trigger an effector immune response that results in tumor killing.
• the target antigen of the binding domain of the chimeric antigen receptor is a CD94 protein.
• Illustrative ligand-binding domains include antigen binding proteins, such as antigen binding fragments of an antibody, such as scFv, extracellular domains of receptors, ligands for cell surface molecules/receptors, or receptor binding domains thereof, and tumor binding proteins.
• the antigen binding domains included in a CAR of the disclosure can be a variable region (Fv), a CDR, a Fab, an scFv, a VH, a VL, a domain antibody variant (dAb), a camelid antibody (VHH) and other antigen-specific binding domain derived from other protein scaffolds.
• the binding domain of the CAR is an anti-CD94 single chain antibody (scFv), and can be a murine, chimeric, human or humanized scFv.
• Single chain antibodies can be cloned from the V region genes of a hybridoma specific for a desired target.
• VH variable region heavy chain
• VL variable region light chain
• a 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 the antibody, which fragment is involved in binding with the antigen.
• the CARs of the present disclosure can comprise a linker between one or more domains, e.g., added for appropriate spacing and conformation of the molecule.
• the linker can be between 1-10 amino acids long.
• the linker between any of the domains of the chimeric antigen receptor can be between 1-20 or 20 amino acids long.
• the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long.
• the linker can be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. Ranges including the numbers described herein are also included herein, e.g., a linker 10-30 amino acids long.
• linkers suitable for use in the CAR disclosed herein are flexible linkers.
• Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 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.
• Exemplary flexible linkers can include glycine polymers (G)n, glycine-serine polymers, where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
• Glycine and glycine-serine polymers are relatively unstructured, and therefore can be able to serve as a neutral tether between domains of fusion proteins such as the CARs disclosed herein. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).
• linker that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired CAR structure.
• 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.
• 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.
• 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.
• the linker comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:34. In some aspects, the linker comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:34. In some aspects, the linker comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:34. In some aspects, the linker comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:34. In some aspects, the linker comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:34. In some aspects, the linker comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:34.
• the linker comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:34. In some aspects, the linker comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:34. In some aspects, the linker comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:34. In some aspects, the linker comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:34. In some aspects, the linker comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:34.
• the transmembrane region or domain is the portion of the CAR that anchors the extracellular binding portion 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 a CD3zeta transmembrane domain, however other transmembrane domains that can be employed include those obtained from CD8a, CD4, CD28, CD45, CD9, CD 16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154.
• the transmembrane domain is the transmembrane domain of CD8a.
• the transmembrane domain is synthetic in which case it would comprise predominantly hydrophobic residues such as leucine and valine.
• Examples of signal transducing domains for use in a CAR can be the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivate or variant of these sequences and any synthetic sequence that has the same functional capability.
• signaling domains comprise two distinct classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation, and those that act in an antigenindependent manner to provide a secondary or co- stimulatory signal.
• Primary cytoplasmic signaling sequences can comprise signaling motifs that are known as immunoreceptor tyrosine-based activation motifs (IT AMs). IT AMs can be found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases.
• Exemplary ITAMs can include those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD3zeta, CD5, CD22, CD79a, CD79b and CD66d.
• the signal transducing domain of the CAR can comprise the CD3zeta signaling domain.
• the CAR disclosed herein can comprise a costimulatory domain, e.g., derived from a costimulatory molecule.
• Costimulatory molecules can include, but are not limited to, an MHC class I molecule, BTLA and Toll ligand receptor.
• Examples of costimulatory molecules include, e.g., CD27, CD28, CD8, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like.
• a costimulatory molecule can be a cell surface molecule other than an antigen receptor or their ligands that can contribute to an efficient immune response. Accordingly, while the present disclosure provides exemplary costimulatory domains derived from 4- IBB, 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 CAR receptors disclosed herein. The intracellular signaling and costimulatory signaling domains can be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.
• the CAR disclosed herein can comprises a signal peptide.
• the signal peptide is a CD8a signal peptide.
• the CD8a signal peptide 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:32.
• the CD8a signal peptide 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:32.
• the CD8a 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.
• the CD8a signal peptide comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:32. In some aspects, the CD8a signal peptide comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:32. In some aspects, the CD8a signal peptide comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:32. In some aspects, the CD8a signal peptide comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:32. In some aspects, the CD8a signal peptide comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:32.
• the CD8a signal peptide comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:32. In some aspects, the CD8a signal peptide comprises an amino acid sequence about 85% identical to the amino acid sequence SEQ ID NO:32. In some aspects, the CD8a signal peptide comprises an amino acid sequence about 90% identical to the amino acid sequence SEQ ID NO:32. In some aspects, the CD8a signal peptide comprises an amino acid sequence about 95% identical to the amino acid sequence SEQ ID NO:32. In some aspects, the CD8a signal peptide comprises an amino acid sequence about 98% identical to the amino acid sequence SEQ ID NO:32. In some aspects, the CD8a signal peptide comprises an amino acid sequence about 99% identical to the amino acid sequence SEQ ID NO:32.
• scFv-based CARs engineered to contain a signaling domain from CD3 or FcRgamma have been shown to deliver a potent signal for T cell activation and effector function, they are not sufficient to elicit signals that promote T cell survival and expansion in the absence of a concomitant costimulatory signal.
• CARs containing a binding domain, a hinge, a transmembrane and the signaling domain derived from CD3zeta or FcRgamma together with one or more costimulatory signaling domains can more effectively direct antitumor activity as well as increased cytokine secretion, lytic activity, survival and proliferation in CAR expressing T cells in vitro, and 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).
• costimulatory signaling domains e.g., intracellular costimulatory domains derived from CD28, CD137, CD134 and CD278
• the CD94-binding CARs of the disclosure comprise (a) an anti- CD94 scFv as a binding domain (e.g., an scFv having binding regions (e.g., CDRs or variable domains) from an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein); (b) a hinge region (e.g., derived from human CD8a); (c) a transmembrane domain (e.g., a human CD8a transmembrane domain); and (d) a signaling domain (e.g., a human T cell receptor CD3zeta chain signaling domain), and optionally one or more costimulatory signaling domains, e.g., a 4- IBB co-stimulatory domain, a CD28 costimulatory domain, or an 0X40 costimulatory domain.
• a binding domain e.g., an scFv having binding regions (e.g., CDRs or variable domains) from an anti
• the different protein domains are arranged from amino to carboxyl terminus in the following order: binding domain, hinge and transmembrane domain.
• the intracellular signaling domain and optional co-stimulatory signaling domains are linked to the transmembrane carboxy terminus in any order in tandem to form a single chain chimeric polypeptide.
• a nucleic acid construct encoding a CD94-binding CAR is a chimeric nucleic acid molecule comprising different coding sequences, for example, (5' to 3') the coding sequences of a human anti-CD94 scFv, a human CD8a-hinge region, a human CD8a transmembrane domain and a CD3zeta signaling domain.
• a nucleic acid construct encoding a CD94-binding CAR is a chimeric nucleic acid molecule comprising different coding sequences, for example, (5' to 3') the coding sequences of a human anti- CD94 scFv, a human CD8a-hinge, a human CD8a transmembrane domain, a 4-1BB co- stimulatory domain, and a CD3zeta signaling domain.
• the polynucleotide encoding the CAR disclosed herein is inserted into a vector.
• the vector as used herein is a vehicle into which a polynucleotide encoding a protein can be covalently inserted so as to bring about the expression of that protein and/or the cloning of the polynucleotide.
• the isolated polynucleotide can be inserted into a vector using any suitable methods known in the art, for example, without limitation, the vector can be digested using appropriate restriction enzymes and then can be ligated with the isolated polynucleotide having matching restriction ends.
• expression vectors have the ability to incorporate and express heterologous or modified nucleic acid sequences coding for at least part of a gene product capable of being transcribed in a cell.
• Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism.
• control sequences which refer to nucleic acid sequences for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism.
• vectors and expression vectors can contain nucleic acid sequences that serve other functions as well and are discussed infra.
• An expression vector can comprise additional elements, for example, the expression vector can have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
• the expression vector can have 5' upstream and 3' downstream regulatory elements such as promoter sequences (e.g., CMV, PGK or EFl alpha promoters), ribosome recognition and binding TATA box, and 3' UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell.
• promoter sequences e.g., CMV, PGK or EFl alpha promoters
• ribosome recognition and binding TATA box e.g., ribosome recognition and binding TATA box
• 3' UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell.
• Other suitable promoters can include the constitutive promoter of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and rous sarcoma virus promoter.
• Human gene promoters can also be used, including, but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
• inducible promoters are also contemplated as part of the vectors expressing chimeric antigen receptor. This provides a molecular switch capable of turning on expression of the polynucleotide sequence of interest or turning off expression. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.
• the expression vector can have additional sequence such as 6x-histidine, c-Myc, and FLAG tags that are incorporated into the expressed CARs.
• the expression vector can be engineered to contain 5' and 3' untranslated regulatory sequences that sometimes can 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.
• An expression vector 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 a selectable marker for maintenance of the vector in the host or recipient cell.
• the vectors are plasmid, autonomously replicating sequences, and transposable elements.
• Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
• animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
• retrovirus including lentivirus
• adenovirus e.g., adeno-associated virus
• herpesvirus e.g., herpes simplex virus
• poxvirus baculovirus
• papillomavirus papillomavirus
• papovavirus e.g., SV40
• expression vectors are Lenti-XTM Bicistronic Expression System (Neo) vectors (Clontrch), pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DESTTM, pLenti6/V5-DESTTM, and pLenti6.2N5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
• the coding sequences of the CARs disclosed herein can be ligated into such expression vectors for the expression of the chimeric protein in mammalian cells.
• the vector for expression of a CD94-binding CAR, can be introduced into a host cell to allow expression of the polypeptide within the host cell.
• the expression vectors can contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements can be selected as appropriate by a person of ordinary skill in the art, as disclosed herein.
• the promoter sequences can be selected to promote the transcription of the polynucleotide in the vector. Suitable promoter sequences include, without limitation, T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EFla promoter, CMV promoter, and SV40 promoter.
• Enhancer sequences can be selected to enhance the transcription of the polynucleotide.
• Selectable markers can be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers can be genes that confer antibiotic resistance.
• Signal sequences can be selected to allow the expressed polypeptide to be transported outside of the host cell.
• the vector can be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein.
• the cloning vectors can contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements can be selected as appropriate by a person of ordinary skill in the art.
• the origin of replication can be selected to promote autonomous replication of the vector in the host cell.
• the present disclosure provides isolated host cells containing the vectors provided herein.
• the host cells containing the vector can be useful in expression or cloning of the polynucleotide contained in the vector.
• Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.
• Suitable prokaryotic cells for this purpose include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E.
• the CARs of the present disclosure can be introduced into a host cell using transfection and/or transduction techniques known in the art.
• transfection and/or transduction,” refer to the processes 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 can be maintained extrachromosomally.
• the nucleic acid can be maintained transiently or can be a stable introduction.
• Transfection can be accomplished by a variety of 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 by transfection.
• retroviral vectors are transduced by packaging the vectors into virions prior to contact with a cell.
• a nucleic acid encoding a CD94 CAR carried by a retroviral vector can be transduced into a cell through infection and pro virus integration.
• the CAR of the present disclosure is introduced and expressed in immune effector cells so as to redirect their specificity to a target antigen of interest.
• the present disclosure provides methods for making the immune effector cells that express the CAR as disclosed herein.
• the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual.
• the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a CAR.
• the immune effector cells can be cultured before or after being genetically modified (i.e., transduced or transfected to express a CAR as disclosed herein).
• the source of cells can be obtained from a subject.
• the immune effector cells for use with the CARs as disclosed herein comprise T cells.
• T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
• T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL separation.
• cells from the circulating blood of an individual are obtained by apheresis.
• the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets.
• the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing.
• the cells are washed with PBS.
• the washed solution lacks calcium, and can lack magnesium or can lack many, if not all, divalent cations.
• a washing step can be accomplished by methods known to those in the art, such as by using a semiautomated flowthrough centrifuge.
• T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
• PBMCs peripheral blood mononuclear cells
• a specific subpopulation of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
• enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
• One method for use herein is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
• a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDlb, 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.
• PBMCs can be used directly for genetic modification with the CARs using methods as disclosed herein.
• T lymphocytes after isolation of PBMC, T lymphocytes are further isolated and in some aspects, 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.
• CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of those types of CD8+ cells.
• memory T cells are present in both CD62L+ and CD62L-subsets of CD8+ peripheral blood lymphocytes.
• PBMC are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies.
• the expression of phenotypic markers of central memory TCM include CD45RO, CD62L, CCR7, CD28, CD3, and CD 127 and are negative for granzyme B.
• central memory T cells are CD45RO+, CD62L+, CD8+ T cells.
• effector T cells are negative for CD62L, CCR7, CD28, and CD 127, and positive for granzyme B and perforin.
• naive CD8+T lymphocytes are characterized by the expression of phenotypic markers of naive T cells including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.
• CD4+ T cells are further sorted into subpopulations.
• CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.
• CD4+ lymphocytes can be obtained by standard methods.
• the immune effector cells, such as T cells can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified.
• the immune effector cells such as T cells
• 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; W02012079000.
• such methods include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2.
• a stimulatory agent and costimulatory agent such as anti-CD3 and anti-CD28 antibodies
• cytokines such as IL-2.
• Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC).
• the 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. Pat. Nos. 6,040,177; 5,827,642; and WO2012129514.
• CAR-expressing immune effector cells prepared as disclosed herein can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure. See, e.g., US Patent Application Publication No. 2003/0170238 to Gruenberg et al; see also U.S. Pat. No. 4,690,915 to Rosenberg.
• the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatmenteffective amount.
• a “pharmaceutically acceptable” carrier can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized.
• the infusion medium can be supplemented with human serum albumin.
• the cells can be autologous or heterologous to the patient undergoing therapy.
• the treatment can also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g., IFN-y, IL-2, IL-12, TNF-a, IL-18, and TNF-p, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIPla, etc.) as disclosed herein to enhance induction of the immune response.
• mitogens e.g., PHA
• lymphokines e.g., lymphokines, cytokines, and/or chemokines (e.g., IFN-y, IL-2, IL-12, TNF-a, IL-18, and TNF-p, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIPla, etc.) as disclosed herein to enhance induction of the immune response.
• compositions of the present disclosure can be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
• pharmaceutical compositions of the present disclosure can comprise a CAR- expressing immune effector cell population, such as T cells, as disclosed herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
• compositions comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
• buffers such as neutral buffered saline, phosphate buffered saline and the like
• carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
• proteins polypeptides or amino acids
• antioxidants such as glycine
• chelating agents such as EDTA or glutathione
• adjuvants e.g., aluminum hydroxide
• preservatives e.g., aluminum hydroxide
• Humoral immune responses mediated primarily by helper T cells capable of activating B cells thus leading to antibody production, may be induced.
• a variety of techniques may be used for analyzing the type of immune responses induced by the compositions of the present disclosure, which are well disclosed in the art; e.g., Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, N.Y., N.Y.
• provided herein is a method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of any of the chimeric antigen receptors provided herein.
• the cancer is a leukemia. In some aspects, the cancer is a CD94 expressing cancer.
• the leukemia is T cell leukemia, T cell large granular leukemia, Natural Killer cell large granular leukemia, or Natural Killer cell leukemia.
• the cancer is a lymphoma.
• 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.
• the cancer is lung cancer, bladder cancer, or melanoma.
• provided herein is a method of treating or preventing graftrejection of a transplant in a patient, the method comprising administering to the individual a therapeutically effective amount of any of the chimeric antigen receptors provided herein.
• the transplant is an allogenic transplant.
• the transplant is an organ transplant.
• the transplant is a hematopoietic cell transplant.
• the transplant is an induced pluripotent cell therapy.
• provided herein is a method of modulating an immune response in a subject, the method comprising administering to the individual therapeutically effective amount of the antibody or antigen-binding fragment thereof of any of the chimeric antigen receptors provided herein.
• the immune response is enhanced.
• the immune response is mediated by Natural Killer cells and/or T cells.
• the methods for administering the cell compositions disclosed herein includes any method which is effective to result in reintroduction of ex vivo genetically modified immune effector cells that either directly express a CAR of the disclosure in the subject or on reintroduction of the genetically modified progenitors of immune effector cells that on introduction into a subject differentiate into mature immune effector cells that express the CAR.
• One method comprises transducing peripheral blood T cells ex vivo with a nucleic acid construct in accordance with the disclosure and returning the transduced cells into the subject.
• the present disclosure encompasses methods of preparing immune cells for immunotherapy comprising introducing, ex vivo, into such immune cells the polynucleotides or vectors encoding one of the CD94-specific chimeric antigen receptors disclosed herein.
• 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 aspects, these immune cells are used for immunotherapy (e.g., treatment of cancer).
• the present disclosure also encompasses methods of genetically modifying immune cells to make them more suitable for allogeneic transplantation.
• the immune cell can be made allogeneic, for instance, by inactivating at least one gene expressing one or more component of T-cell receptor (TCR) as disclosed in WO 2013/176915, which can be combined with the inactivation of a gene encoding or regulating HLA or P2m protein expression. Accordingly, the risk of graft versus host syndrome and graft rejection is significantly reduced.
• TCR T-cell receptor
• 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.
• the engineered human T cell comprises a chimeric antigen receptor comprising, from N-terminus to C-terminus: (a) an extracellular ligand-binding domain comprising a 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.
• the VH comprises a CDR Hl 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 NON 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.
• the VL comprises a CDR LI 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.
• the scFv domain comprises a VH and a VL.
• the VH comprises a CDR Hl comprising the amino acid sequence set forth in SEQ ID NON, a CDR H2 comprising the amino acid sequence set forth in SEQ ID NON, and a CDR H3 comprising the amino acid sequence set forth in SEQ ID NON.
• the VL comprises a CDR LI comprising the amino acid sequence set forth in SEQ ID NON, 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.
• the VH comprises a CDR Hl 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, and a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO:21.
• the VL comprises a CDR LI 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 NON.
• the VL comprises a CDR LI 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.
• the engineered human T cell comprises 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.
• the engineered human T cell comprises 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.
• the engineered human T cell comprises 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.
• the engineered human T cell comprises 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.
• the engineered human T cell comprises 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.
• the engineered human T cell comprises 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.
• the immune cells particularly T-cells of the present disclosure can be further activated and expanded generally using methods as disclosed, for example, in U.S. Pat. Nos.
• T cells can be expanded in vitro or in vivo.
• the T cells of the disclosure are expanded by contact with an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of the T cells to create an activation signal for the T-cell.
• an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of the T cells to create an activation signal for the T-cell.
• chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T-cell.
• T cell populations can be stimulated in vitro such as by contact with an anti-CD3 antibody, or 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.
• a protein kinase C activator e.g., bryostatin
• a ligand that binds the accessory molecule is used.
• a population of T cells can be contacted with an anti-CD3 antibody and an anti- CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
• Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza)) that can contain factors necessary for proliferation and viability, 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-a or any other additives for the growth of cells known to the skilled artisan.
• Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
• Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added 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 an amount of cytokine(s) sufficient for the growth and expansion of T cells.
• Antibiotics e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject.
• the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% 02). T cells that have been exposed to varied stimulation times can exhibit different characteristics.
• the cells can be expanded by co-culturing with tissue or cells.
• the cells can also be expanded in vivo, for example in the subject's blood after administrating said cell into the subject. IV. Antibody Production
• Antibodies and antigen-binding fragments thereof that immunospecifically bind to CD94 can be produced by any method known in the art for the synthesis of antibodies and antigen-binding fragments thereof, for example, 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, oligonucleotide synthesis and modification, 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 explained in the literature.
• provided herein is a method of making an antibody or antigenbinding fragment which immunospecifically binds to CD94 (e.g., human CD94) comprising culturing a cell or host cell disclosed herein.
• a method of making an antibody or antigen-binding fragment thereof which immunospecifically binds to CD94 comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using a cell or host cell disclosed herein (e.g., a cell or a host cell comprising polynucleotides encoding an antibody or antigen-binding fragment thereof disclosed herein).
• the cell is an isolated cell.
• the exogenous polynucleotides have been introduced into the cell.
• the method further comprises the step of purifying the antibody or antigen-binding fragment obtained from the cell or host cell.
• 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 technologies, yeast-based presentation technologies, or a combination thereof.
• monoclonal antibodies or antigen-binding fragments thereof can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
• a monoclonal antibody or antigen-binding fragment thereof can be produced using the hybridoma method first disclosed by Kohler et al., Nature, 256:495 (1975), as mentioned above.
• a mouse or another appropriate host animal is immunized as above disclosed to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization, for example, variant mixtures of CD94.
• Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1986).
• a suitable fusing agent such as polyethylene glycol
• a monoclonal antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment produced by a clonal cell (e.g., hybridoma or host cell producing a recombinant antibody or antigen-binding fragment), wherein the antibody or antigen-binding fragment immunospecifically binds to CD94 e.g., human CD94) as determined, e.g., by ELISA or other antigen-binding assays known in the art or in the Examples provided herein.
• a monoclonal antibody or antigen-binding fragment thereof can be a rodent or murine antibody or antigen-binding fragment thereof.
• a monoclonal antibody or antigen-binding fragment thereof can be a chimeric or a humanized antibody or antigen-binding fragment thereof. In some aspects, a monoclonal antibody or antigen-binding fragment thereof can be a Fab fragment or an F(ab')2 fragment. Monoclonal antibodies or antigen-binding fragments thereof disclosed herein can, for example, be made by the hybridoma method as disclosed in Kohler G & Milstein C (1975) Nature 256: 495 or can, e.g., be isolated from phage libraries using the techniques as disclosed herein, for example.
• Antigen-binding fragments of antibodies disclosed herein can be generated by any technique known to those of skill in the art.
• 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).
• a Fab fragment corresponds to one of the two identical arms of a tetrameric antibody molecule and contains the complete light chain paired with the VH and CHI domains of the heavy chain.
• An F(ab')2 fragment contains the two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds in the hinge region.
• the antibodies or antigen-binding fragments thereof disclosed herein can also be generated using various phage display and/or yeast-based presentation methods known in the art.
• phage display methods proteins are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
• DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of affected tissues).
• the DNA encoding the VH and VL domains are recombined together with a scFv linker by PCR and cloned into a phagemid vector.
• the vector is electroporated in E. coli and the E.
• Phage used in these methods are typically filamentous phage including fd and Ml 3, and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII.
• Phage expressing an antibody or antigen-binding fragment thereof that binds to a particular antigen can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
• phage display methods that can be used to make the antibodies or fragments disclosed herein include those disclosed 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.
• An 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 IgGl, IgG2, IgG3 and IgG4. a. Polynucleotides
• polynucleotides comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein or a domain thereof (e.g., a variable light chain region and/or variable heavy chain region) that immunospecifically binds to a CD94 (e.g., human CD94) antigen
• vectors e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells).
• polynucleotides comprising a nucleotide sequence encoding the chimeric antigen receptors (CARs) disclosed herein.
• polynucleotides comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof, which immunospecifically bind to a CD94 polypeptide (e.g., human CD94) and comprise an amino acid sequence as disclosed herein.
• a CD94 polypeptide e.g., human CD94
• an antibody or antigen-binding fragment thereof provided herein binds to CD94 and comprises six CDRs (e.g., a CDR Hl, a CDR H2, a CDR H3, a CDR LI, a CDR L2, and a CDR L3).
• the CDRs may be determined by a number of algorithms in the art, such as IMGT (Table 6), abYsis (Table 7), and Kabat (Table 8).
• 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 M P (2001) Nucleic Acids Res. 29(1): 207-9; Lefranc M P (2003) Nucleic Acids Res. 31(1): 307-10; Lefranc M P et al., (2005) Dev. Comp. Immunol. 29(3): 185-203; Kaas Q et al., (2007) Briefings in Functional Genomics & Proteomics, 6(4): 253-64).
• 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).
• the Kabat definition is based on sequence variability and is the most commonly used (Kabat EA et al., supra).
• an isolated polynucleotide comprises a nucleic acid molecule encoding any of the VH or heavy chain of the anti-CD94 antibody or antigenbinding fragment thereof disclosed herein.
• the VH is encoded by a polynucleotide sequence comprising a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 10.
• an isolated polynucleotide comprising a nucleic acid molecule encoding any of the VL or light chain of the anti-CD94 antibody or antigen-binding fragment thereof disclosed herein.
• the VL is encoded by a polynucleotide sequence comprising a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 11.
• an antibody or antigen-binding fragment thereof capable of binding to CD94, wherein the antibody or antigen-binding fragment thereof comprises a complementary determining region (CDR) Hl 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 LI 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
• an antibody or antigen-binding fragment thereof capable of binding to CD94, wherein the antibody or antigen-binding fragment thereof comprises a complementary determining region (CDR) Hl 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 LI 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.
• CDR complementary determining region
• an antibody or antigen-binding fragment thereof capable of binding to CD94, wherein the antibody or antigen-binding fragment thereof comprises a complementary determining region (CDR) Hl 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 LI 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.
• CDR complementary determining region
• an antibody or antigen-binding fragment thereof capable of binding to CD94, wherein the antibody or antigen-binding fragment thereof comprises a complementary determining region (CDR) Hl 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 LI 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.
• CDR complementary determining region
• the antibody or antigen-binding fragment thereof capable of binding CD94 comprises a heavy chain variable region (VH) and a light chain variable region (VL).
• 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.
• 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.
• the antibody or antigen-binding fragment thereof capable of binding CD94 comprises a heavy chain variable region (VH) and a light chain variable region (VL).
• 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.
• 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.
• 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 aspects, 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 aspects, 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.
• 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 aspects, 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 aspects, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 10.
• 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 aspects, 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 aspects, 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.
• 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 aspects, 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 aspects, the antibody or antigen-binding fragment comprises a VL comprising a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 11.
• 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 aspects, 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 aspects, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO: 10.
• 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 aspects, the antibody or antigen-binding fragment comprises a VH comprising a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO: 10.
• 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 aspects, 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 aspects, 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.
• 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 aspects, 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.
• 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 a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 18.
• 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 a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 18.
• the chimeric antigen receptor comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 18.
• 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 a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 58.
• 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 a sequence corresponding to the polynucleotide sequence of SEQ ID NO: 58.
• the chimeric antigen receptor comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO: 58.
• 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 a sequence corresponding to the polynucleotide sequence of SEQ ID NO:59.
• 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 a sequence corresponding to the polynucleotide sequence of SEQ ID NO:59.
• the chimeric antigen receptor comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:59.
• the chimeric antigen receptor comprises from N-terminus to C- terminus: (a) an extracellular ligand-binding domain comprising a 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.
• the signal peptide comprises a CD8a signal peptide.
• the scFv comprises a linker.
• the hinge comprises a CD8a hinge domain.
• the transmembrane domain comprises a CD8a transmembrane domain.
• the costimulatory domain comprises a 4- IBB costimulatory domain.
• the signaling domain comprises a CD3zeta signaling domain.
• the CD8a 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.
• the CD8a 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.
• the CD8a signal peptide comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO:39. In some aspects, the CD8a signal peptide comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:39. In some aspects, the CD8a signal peptide comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:39. In some aspects, the CD8a signal peptide comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO:39.
• the CD8a signal peptide comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:39. In some aspects, the CD8a signal peptide comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:39.
• the CD8a signal peptide comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO:39. In some aspects, the CD8a signal peptide comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO:39. In some aspects, the CD8a signal peptide comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO:39. In some aspects, the CD8a signal peptide comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO:39. In some aspects, the CD8a signal peptide comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO:39.
• 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.
• 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.
• the scFv comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO:40. In some aspects, the scFv comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:40. In some aspects, the scFv comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:40. In some aspects, the scFv comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO:40.
• the scFv comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:40. In some aspects, the scFv comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:40.
• the scFv comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO:40. In some aspects, the scFv comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO:40. In some aspects, the scFv comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO:40. In some aspects, the scFv comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO:40. In some aspects, the scFv comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO:40.
• 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.
• 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.
• the linker comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO:41. In some aspects, the linker comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:41. In some aspects, the linker comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:41. In some aspects, the linker comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO:41.
• the linker comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:41. In some aspects, the linker comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID N0:41.
• the linker comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO:41. In some aspects, the linker comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO:41. In some aspects, the linker comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO:41. In some aspects, the linker comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO:41. In some aspects, the linker comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO:41.
• the CD8a 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.
• the CD8a 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.
• the CD8a hinge domain comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO:42. In some aspects, the CD8a hinge domain comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:42. In some aspects, the CD8a hinge domain comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:42. In some aspects, the CD8a hinge domain comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO:42.
• the CD8a hinge domain comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:42. In some aspects, the CD8a hinge domain comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:42.
• the CD8a hinge domain comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO:42. In some aspects, the CD8a hinge domain comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO:42. In some aspects, the CD8a hinge domain comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO:42. In some aspects, the CD8a hinge domain comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO:42. In some aspects, the CD8a hinge domain comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO:42.
• the CD8a 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.
• the CD8a 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.
• the CD8a transmembrane domain comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO:43. In some aspects, the CD8a transmembrane domain comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:43. In some aspects, the CD8a transmembrane domain comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:43.
• the CD8a transmembrane domain comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO:43. In some aspects, the CD8a transmembrane domain comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:43. In some aspects, the CD8a transmembrane domain comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:43.
• the CD8a transmembrane domain comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO:43. In some aspects, the CD8a transmembrane domain comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO:43. In some aspects, the CD8a transmembrane domain comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO:43. In some aspects, the CD8a transmembrane domain comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO:43. In some aspects, the CD8a transmembrane domain comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO:43.
• the 4- IBB 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.
• 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.
• the 4- IBB costimulatory domain comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO:44. In some aspects, the 4- IBB costimulatory domain comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:44. In some aspects, the 4-1BB costimulatory domain comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:44. In some aspects, the 4-1BB costimulatory domain comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO:44.
• the 4-1BB costimulatory domain comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:44. In some aspects, the 4-1BB costimulatory domain comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:44.
• the 4- IBB costimulatory domain comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO:44. In some aspects, the 4- IBB costimulatory domain comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO:44. In some aspects, the 4-1BB costimulatory domain comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO:44. In some aspects, the 4-1BB costimulatory domain comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO:44. In some aspects, the 4-1BB costimulatory domain comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO:44.
• 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.
• 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.
• the CD3zeta signaling domain comprises a polynucleotide sequence at least 85% identical to the polynucleotide sequence of SEQ ID NO:45. In some aspects, the CD3zeta signaling domain comprises a polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:45. In some aspects, the CD3zeta signaling domain comprises a polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:45. In some aspects, the CD3zeta signaling domain comprises a polynucleotide sequence at least 98% identical to the polynucleotide sequence of SEQ ID NO:45.
• the CD3zeta signaling domain comprises a polynucleotide sequence at least 99% identical to the polynucleotide sequence of SEQ ID NO:45. In some aspects, the CD3zeta signaling domain comprises a polynucleotide sequence corresponding to the polynucleotide sequence of SEQ ID NO:45.
• the CD3zeta signaling domain comprises a polynucleotide sequence about 85% identical to the polynucleotide sequence SEQ ID NO:45. In some aspects, the CD3zeta signaling domain comprises a polynucleotide sequence about 90% identical to the polynucleotide sequence SEQ ID NO:45. In some aspects, the CD3zeta signaling domain comprises a polynucleotide sequence about 95% identical to the polynucleotide sequence SEQ ID NO:45. In some aspects, the CD3zeta signaling domain comprises a polynucleotide sequence about 98% identical to the polynucleotide sequence SEQ ID NO:45. In some aspects, the CD3zeta signaling domain comprises a polynucleotide sequence about 99% identical to the polynucleotide sequence SEQ ID NO:45.
• a nucleic acid encoding a heavy chain variable domain or heavy chain and a nucleic acid encoding a light chain variable domain or light chain can be in the same polynucleotide or in different polynucleotides.
• a nucleic acid encoding a heavy chain variable domain or heavy chain and a nucleic acid encoding a light chain variable domain or light chain can be in the same vector or in different vectors.
• polynucleotides encoding an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein or a domain thereof that are optimized, e.g., by codon/RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements.
• Methods to generate optimized nucleic acids encoding an anti-CD94 antibody or antigen-binding fragment thereof or a domain thereof (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., a codon change that encodes the same amino acid due to the degeneracy of the genetic code) and/or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods disclosed in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.
• a polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein or a domain thereof 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 hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the light chain and/or heavy chain of an antibody or antigen-binding fragment thereof.
• Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the variable light chain region and/or the variable heavy chain region of an antibody or antigen-binding fragment thereof.
• the amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies or antigen-binding fragments thereof.
• Polynucleotides provided herein can be, e.g., in the form of RNA or in the form of DNA.
• DNA includes cDNA, genomic DNA, and synthetic DNA, and DNA can be doublestranded or single-stranded. If single stranded, DNA can be the coding strand or non-coding (anti-sense) strand.
• the polynucleotide is a cDNA or a DNA lacking one more endogenous introns.
• a polynucleotide is a non-naturally occurring polynucleotide.
• a polynucleotide is recombinantly produced.
• the polynucleotides are isolated.
• the polynucleotides are substantially pure.
• a polynucleotide is purified from natural components.
• vectors comprising polynucleotides comprising nucleotide sequences encoding anti-CD94 antibodies and antigen-binding fragments thereof or a domain thereof for recombinant expression in host cells, preferably in mammalian cells.
• cells e.g., host cells, comprising such vectors for recombinantly expressing anti-CD94 antibodies or antigenbinding fragments thereof disclosed herein.
• recombinant expression of an antibody or antigen-binding fragment thereof or domain thereof disclosed herein involves construction of an expression vector containing a polynucleotide that encodes the antibody or antigen-binding fragment thereof or domain thereof.
• a polynucleotide encoding an antibody or antigenbinding fragment thereof or domain thereof e.g., heavy or light chain variable domain
• the vector for the production of the antibody or antigenbinding fragment thereof can be produced by recombinant DNA technology using techniques well known in the art.
• a polynucleotide containing an antibody or antigen-binding fragment thereof or domain thereof e.g., light chain or heavy chain
• methods for preparing a protein by expressing a polynucleotide containing an antibody or antigen-binding fragment thereof or domain thereof (e.g., light chain or heavy chain) encoding nucleotide sequence are disclosed herein.
• Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody or antigen-binding fragment thereof or domain thereof (e.g., light chain or heavy chain) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
• 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 can, for example, include the nucleotide sequence encoding the constant region of the antibody or antigen-binding fragment thereof (see, e.g., International Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Patent No. 5,122,464), and variable domains of the antibody or antigen-binding fragment thereof can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
• An expression vector can be transferred to a cell (e.g., 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 comprising any of the anti-CD94 antibodies disclosed herein.
• disclosed herein is an isolated vector comprising any of the polynucleotides disclosed herein.
• a host cell comprising any of the polynucleotide disclosed herein.
• the host cell is selected form the group consisting of CHO, HEK- 293 T, HeLa and BHK cells.
• the CHO cell is a CHO-K1SP cell.
• a method of producing an antibody or antigenbinding fragment thereof capable of binding to CD94 comprising (a) culturing any of the host cells disclosed herein in a cell culture under conditions which allow expression of the antibody or antigen-binding fragment thereof; and (b) recovering the antibody or antigen-binding fragment thereof from said cell culture.
• an antibody or antigen-binding fragment thereof obtainable by any of the methods of production disclosed herein.
• vectors encoding both the heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin, as detailed below.
• a host cell contains a vector comprising a polynucleotide encoding both the heavy chain and light chain of an antibody disclosed herein or a domain thereof.
• a host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof disclosed herein, and a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody disclosed herein or a domain thereof.
• a first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof disclosed herein
• a second host cell comprises a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody or antigen-binding fragment thereof disclosed herein.
• a heavy chain/heavy chain variable region expressed by a first cell associated with a light chain/light chain variable region of a second cell to form an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein.
• a population of host cells comprising such first host cell and such second host cell.
• a population of vectors comprising a first vector comprising a polynucleotide encoding a 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 a heavy chain/heavy chain variable region of an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein.
• a single vector can be used which encodes, and is capable of expressing, both heavy and light chain polypeptides.
• host-expression vector systems can be utilized to express antibodies and antigen-binding fragments thereof disclosed herein (see, e.g., U.S. Patent No. 5,807,715).
• host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or antigen-binding fragment thereof disclosed herein in situ. These include but are not limited to microorganisms such as bacteria (e.g. ,E. coli and B.
• yeast e.g., Saccharomyces Pichid
• insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences
• plant cell systems e.g., green algae such as Chlamydomonas reinhardtii
• plant cell systems e.g., green algae such as Chlamydomonas reinhardtii
• plant cell systems e.g., green algae such as Chlamydomonas reinhardtii
• plant cell systems e.g., green algae such as Chlamydomonas reinhardtii
• plant cell systems e.g., green algae such as Chlamydomonas reinhardtii
• plant cell systems e.g., green algae such as Chlamydomonas reinhardtii
• plant cell systems e.g., green algae such as Chlamydomonas reinhardtii
• plant cell systems e.g., green algae such
• cells for expressing antibodies and antigen-binding fragments thereof disclosed herein are CHO cells, for example CHO cells from the CHO GS SystemTM (Lonza).
• cells for expressing antibodies disclosed herein are human cells, e.g., human cell lines.
• a mammalian expression vector is pOptiVECTM or pcDNA3.3.
• bacterial cells such as Escherichia coli, or eukaryotic cells e.g., mammalian cells), especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
• mammalian cells such as Chinese hamster ovary (CHO) cells in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667).
• CHO Chinese hamster ovary
• antibodies or antigen-binding fragments thereof disclosed herein are produced by CHO cells or NSO cells.
• a host cell strain can 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.
• eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can 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 murine 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, Rl.l, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells.
• COS e.g., COS1 or COS
• PER.C6 VERO
• HsS78Bst HEK-293T
• HepG2 SP210
• Rl.l B-W
• L-M low-M
• anti-CD94 antibodies disclosed herein are produced in mammalian cells, such as CHO cells, e.g., CHO-K1 cells. In some aspects, anti-CD94 antibodies disclosed herein are produced in mammalian cells, such as HEK-293 cells.
• a signal peptide is used in constructing a vector containing the VH and/ or VL or the heavy and/or light chain of an antibody or antigen-binding fragment thereof provided herein.
• an antibody or antigen-binding fragment thereof disclosed herein can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, 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. [0349] In some aspects, an antibody or antigen-binding fragment thereof disclosed herein is isolated or purified.
• an isolated antibody or antigen-binding fragment thereof is one that is substantially free of other antibodies or antigen-binding fragments thereof with different antigenic specificities than the isolated antibody or antigen-binding fragment thereof.
• a preparation of an antibody or antigen-binding fragment thereof disclosed herein is substantially free of cellular material and/or chemical precursors.
• the nucleic acids encoding the antibody or antigen-binding fragment thereof disclosed herein or the CAR disclosed herein are provided in a viral vector.
• the viral vector is a retrovirus, a lentivirus, or a foamy virus.
• 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.
• retroviral vector refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus.
• lentiviral vector refers to a vector containing structural and functional genetic elements outside the LTRs that are primarily derived from a lentivirus.
• the retroviral vectors for use herein can be derived from any known retrovirus (e.g., type c retroviruses, such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine 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)).
• type c retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine 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)).
• Retroviruses also include human T cell leukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family of retroviruses, such as Human Immunodeficiency Viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immnodeficiency virus (EIV), and other classes of retroviruses.
• retroviruses such as Human Immunodeficiency Viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immnodeficiency virus (EIV), and other classes of retroviruses.
• a lentiviral vector for use herein refers to a vector derived from a lentivirus, a group (or genus) of retroviruses that give rise to slowly developing disease.
• Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi; a caprine arthritis-encephalitis virus; equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
• HIV human immunodeficiency virus
• HIV type 1 HIV type 1
• visna-maedi a caprine arthritis-encephalitis virus
• equine infectious anemia virus feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
• Preparation of the recombinant lentivirus can be achieved
• Retroviral vectors 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, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10- 9.14 and other standard laboratory manuals; Eglitis, et al.
• Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in forming the vectors include, for example, genomic RNA and cDNAs available from commercially available sources, including the Type Culture Collection (ATCC), Rockville, Md. The sequences also can be synthesized chemically.
• 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 the 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.
• CAR chimeric antigen receptor
• compositions comprising an anti-CD94 antibody or antigenbinding fragment thereof, an anti-CD94 chimeric antigen receptor, or an engineered T cell targeting CD94 are provided in compositions with a pharmaceutically 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)).
• the pharmaceutical compositions disclosed herein are in one aspect for use as a medicament.
• the pharmaceutical compositions disclosed herein are in one aspect for use as a diagnostic, e.g., to detect the presence of CD94 in a sample obtained from a patient (e.g., a human patient).
• compositions to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.
• compositions wherein the pharmaceutical composition comprises anti-CD94 antibodies or antigen-binding fragments thereof disclosed herein, a chimeric antigen receptor disclosed herein, or an engineered T cell disclosed herein and a pharmaceutically acceptable carrier.
• composition comprising any of the antibody or antigen-binding fragments thereof disclosed herein.
• composition comprising any of the chimeric antigen receptors disclosed herein.
• a pharmaceutical composition comprising any of the engineered T cells disclosed herein.
• the engineered T cells are CAR-T cells.
• composition comprising any of the vectors disclosed herein.
• the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
• provided herein is a method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of any of the anti-CD94 antibodies or antigen-binding portions thereof, chimeric antigen receptors (CARs), engineered T cells, or compositions comprising the same as provided herein.
• CARs chimeric antigen receptors
• provided herein is a method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of any of the antibody or antigen-binding fragments thereof disclosed herein.
• a method of treating cancer in an individual comprising administering to the individual a therapeutically effective amount of any of the engineered T cells disclosed herein.
• the method comprises administering to the individual a therapeutically effective amount of CAR Natural Killer cells, CAR macrophages, or CAR Natural Killer T Cells.
• the method comprises administering to the individual a therapeutically effective amount of an engineered immune cell.
• the engineered immune cell comprises any of the chimeric antigen receptors disclosed herein.
• the engineered immune cell is a macrophage.
• the engineered immune cell is a monocyte.
• provided herein is a method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of any of the vectors disclosed herein.
• provided herein is a method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of any of the pharmaceutical compositions provided herein.
• the cancer is a leukemia.
• the leukemia is T cell leukemia, T cell large granular leukemia,
• Natural Killer cell large granular leukemia or Natural Killer cell leukemia.
• the cancer is a lymphoma.
• the lymphoma is T cell lymphoma, extranodal Natural Killer/T cell lymphoma, hepatosplenic T cell lymphoma, angioimmunoblastic T cell, or anaplastic large cell lymphoma.
• the cancer is a CD94 expressing cancer.
• the cancer is lung cancer, bladder cancer, or melanoma.
• provided herein is a method of treating or preventing graftrejection of a transplant in a patient, the method comprising administering to the individual a therapeutically effective amount of any of the antibody or antigen-binding fragments thereof disclosed herein.
• provided herein is a method of treating or preventing graftrejection of a transplant in a patient, the method comprising administering to the individual a therapeutically effective amount of any of the chimeric antigen receptors provided herein.
• provided herein is a method of treating or preventing graftrejection 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.
• a method of treating or preventing graftrejection of a transplant in a patient the method comprising administering to the individual a therapeutically effective amount of any of the vectors disclosed herein.
• provided herein is a method of treating or preventing graftrejection of a transplant in a patient, the method comprising administering to the individual a therapeutically effective amount of any of the pharmaceutical compositions provided herein.
• the transplant is an allogenic transplant.
• the transplant is an organ transplant.
• the transplant is a hematopoietic cell transplant.
• the transplant is an induced pluripotent cell therapy.
• provided herein is a method of modulating an immune response in a subject, the method comprising administering to the individual therapeutically effective amount of any of the antibody or antigen-binding fragments thereof disclosed herein.
• provided herein is a method of modulating an immune response in a subject, the method comprising administering to the individual therapeutically effective amount of any of the chimeric antigen receptors provided herein.
• provided herein is a method of modulating an immune response in a subject, the method comprising administering to the individual therapeutically effective amount of any of the engineered T cells disclosed herein.
• provided herein is a method of modulating an immune response in a subject, the method comprising administering to the individual therapeutically effective amount of any of the vectors disclosed herein.
• provided herein is a method of modulating an immune response in a subject, the method comprising administering to the individual therapeutically effective amount of any of the pharmaceutical compositions provided herein.
• the immune response is enhanced.
• the immune response is mediated by Natural Killer cells and/or T cells.
• the antibody or antigen-binding fragment thereof or composition disclosed herein can be delivered to a subject by a variety of routes, such as parenteral, subcutaneous, intravenous, intradermal, transdermal, intrathecal, and intranasal.
• the antibody or antigen-binding fragment thereof or composition is administered by an intraperitoneal route.
• the amount of an antibody or antigen-binding fragment thereof or composition which will be effective in the treatment or prevention of a condition will depend on the nature of the disease. The precise dose to be employed in a composition will also depend on the route of administration, and the seriousness of the disease.
• described herein is a method of modulating an immune response in a subject, the method comprising administering to the individual therapeutically effective amount of any of the antibody 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.
• the immune response is enhanced.
• the immune response is mediated by Natural Killer cells and/or T cells.
• the Natural Killer cells and/or T cells mediate the immune response of an autoimmune disease.
• the autoimmune disease is a Systemic Autoimmune Disease.
• 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).
• An anti-CD94 antibody or antigen-binding fragment thereof disclosed herein can be used to assay CD94 protein levels or CD94+ cells in a biological sample using classical methods known to those of skill in the art, including immunoassays, such as the enzyme linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting.
• immunoassays such as the enzyme linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting.
• Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, glucose oxidase
• radioisotopes such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium ( 99 Tc)
• luminescent labels such as luminol
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• Such labels can be used to label an antibody or antigen-binding fragment thereof disclosed herein.
• a second antibody or antigen-binding fragment thereof that recognizes an anti-CD94 antibody or antigen-binding fragment thereof disclosed herein can be labeled and used in combination with an anti-CD94 antibody or antigen-binding fragment thereof to detect CD94 protein levels (e.g., soluble CD94 protein levels).
• biological sample refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing 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.
• Anti-CD94 antibodies and antigen-binding fragments thereof disclosed herein can carry a detectable or functional label.
• detectable moieties examples include but are not limited to radioactive isotopes, phosphorescent chemicals, chemiluminescent chemicals, fluorescent chemicals, enzymes, fluorescent polypeptides, and epitope tags.
• the detectable moiety can be a member of a binding pair, which is identifiable via its interaction with an additional member of the binding pair, and a label which is directly visualized.
• fluorescence labels When fluorescence labels are used, currently available microscopy and fluorescence-activated cell sorter analysis (FACS) or combination of both methods procedures known in the art can be utilized to identify and to quantitate the specific binding members.
• FACS fluorescence-activated cell sorter analysis
• Anti-CD94 antibodies or antigen-binding fragments thereof disclosed herein can carry a fluorescence label.
• fluorescence labels include, for example, reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes.
• An anti-CD94 antibody can carry a radioactive label, such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, "Tc, m In, 117 Lu, 121 I, 124 I, 1 25 I, 131 I, 198 AU, 211 At, 213 Bi, 225 Ac and 186 Re.
• radioactive labels are used, currently available counting procedures known in the art can be utilized to identify and quantitate the specific binding of anti-CD94 antibody or antigen-binding fragment to CD94 (e.g., human CD94).
• detection can be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques as known in the art. This can be achieved by contacting a sample or a control sample with an 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.
• methods for in vitro detecting CD94 in a sample comprising contacting said sample with an antibody or antigen-binding fragment thereof, are provided herein, and optionally detecting binding of the antibody or antigen-binding fragment thereof to CD94.
• provided herein is the use of an antibody or antigen-binding fragment thereof provided herein, for in vitro detecting CD94 in a sample.
• the subject is a mammal.
• the mammal is selected from the group consisting of a human, a mouse, a hamster, a rabbit, a nonhuman primate, a guinea pig, a rat, a zebrafish, a pig, a sheep, a cat, or a dog.
• the subject is a human.
• kits comprising one or more antibodies or antigen-binding fragments thereof disclosed herein or conjugates (e.g., detection conjugates) thereof.
• the kit comprises one or more of the chimeric antigen receptors provided herein.
• the kit comprises one or more of any of the engineered T cells disclosed herein.
• the kit comprises one or more of any of the vectors disclosed herein.
• the kit comprises one or more of any of the pharmaceutical compositions provided herein.
• the kit further comprises instructions for use.
• the kit is used for treating a subject in need thereof.
• kits will thus include, in suitable container means, cells, proteins or nucleic acid constructs or related reagents of the present invention.
• means of taking a sample from an individual and/or of assaying the sample may be provided in the kit.
• the kit includes cells, buffers, cell media, vectors, primers, restriction enzymes, salts, and so forth, for example.
• kits may be packaged either in aqueous media or in lyophilized form.
• the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed.
• various combinations of components may be comprised in a vial.
• Such containers may include injection or blow- molded plastic containers into which the desired vials are retained.
• the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
• the compositions may also be formulated into a syringeable composition.
• the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
• the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
• Anti-CD94 monoclonal antibodies were designed to for treating NK cell or T cell lymphoproliferative disorders.
• CD94 transcript expression was assessed in normal tissues.
• Human Total RNA Survey Panel containing RNA from 21 normal human tissues was obtained from Applied Biosystems, Foster City, CA. About 10 pg of total RNA from each source was reverse transcribed into cDNA with Superscript III kit (Invitrogen). Quantitative PCR was performed with CD94 and P-actin primers using the following conditions: 50 °C for 2 minutes, 94 °C for 10 minutes, followed by 94 °C for 15 seconds, and 60 °C for 60 seconds for 40 cycles on Applied Biosystems StepOneTM/StepOnePlusTM Real-Time PCR System. The expression of CD94 mRNA relative to the P-actin mRNA was calculated in each sample (FIG. 1A).
• CD94 mRNA High levels of CD94 mRNA were detected in lymphoid organs, lymph node, spleen, and NK cells (FIG. 1 A). Lower levels of CD94 mRNA were detected in lung and small intestine, which are known to contain small amounts of lymphoid tissue.
• a publicly available gene expression profiling database BioGPS (http://biogps.org), was used to analyze CD94 mRNA expression in 79 normal human tissues and lymphoid tissue subtypes by high-density oligonucleotide arrays. The analysis determined that CD94 transcript was highly expressed in peripheral blood NK cells, while low levels were observed in CD8 T cells. CD94 mRNA was not expressed in other normal tissues (FIG. IB).
• UT-CD94 an anti-human CD94 monoclonal antibody was generated ("UT-CD94").
• Human CD94 was stably transfected into L cells (a mouse fibroblast cell line) and the CD94- expressing L cells were used to immunize BALB/c mice.
• Splenocytes from immunized mice were isolated and hybridized with mouse myeloma cells to derive a monoclonal antibody targeting human CD94 by hybridoma technology (FIG. 2).
• the UT-CD94 monoclonal antibody was fluorochrome conjugated with AF647 and used to stain NK-cell lymphoma and leukemia cell lines as well as normal donor peripheral blood mononuclear cells (PBMC). 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 (FIG. 3 A), but did not stain parental L cells, human B cells, T cells, or monocytes (FIG. 3B).
• the UT-CD94 monoclonal antibody also bound to NK lymphoma and leukemia cell lines (NK-92, NKL, KHYG-1), but did not bind to the B cell lymphoma cell lines (SP53, Daudi, and Jeko-1) (FIG. 3C).
• the flow cytometry analysis demonstrated that the UT-CD94 monoclonal antibody had specificity for CD94 expressing cells.
• the VH and VL sequence of the UT-CD94 monoclonal antibody of Example 1 were used to generate an anti-CD94 chimeric antigen receptor (CAR).
• the VH and VL sequence of the UT-CD94 monoclonal antibody were converted to a single chain variable fragment (scFv) and an anti-CD94 CAR ("UT-CD94-CAR") was generated using a CD8a hinge, a CD8a transmembrane domain, a 4-1BB signaling domain, and a CD3zeta signaling domain (FIG. 4A).
• the entire CAR sequence was incorporated into a lentivirus 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).
• Normal donor T cells were purified from buffy coat, activated for 2-3 days with anti-CD3, anti-CD28, and anti-CD2 antibodies in the presence of IL-2, and then transduced with a lentivirus containing the UT-CD94-CAR.
• the transduction efficiency was assessed by staining with His-tagged CD94 extra cellular domain protein (ACROBiosystems, USA) followed by flow cytometry analysis with the anti-His AF647-conjugated antibody.
• the flow cytometry analysis for surface expression of the UT-CD94-CAR using CD94 extracellular domain protein staining revealed high transduction efficiency after 72 hours (FIG. 4B).
• NK-92 and NKL were labeled with CellTraceTM Far Red cell staining kit (Thermofisher), while the B cell lymphoma cell line SP53 (mantle cell lymphoma cell line) was stained with CFSE (Thermofisher).
• the NK-92, NKL, and SP53 cell lines were then co-cultured with UT-CD94- CAR -transduced T cells at 1 : 1 : 1 Effector: Tumor ETumor 2 (E:T1 :T2) ratio.
• the percentage of dead cells were determined by flow cytometry at days 1, 2, 3 and 4 after staining for dead cells (Thermofisher).
• the cytotoxicity of UT-CD94-CAR T cells was also analyzed against L cells (a mouse fibroblast cell line) and the human CD94-expressing L cells. Untransduced T cells or tumor alone were used as negative controls.
• UT-CD94-CAR T cells induced significant lysis of CD94-positive NK lymphoma and leukemia cell lines (NK-92 and NKL) and L cells transfected with human CD94 but not CD94-negative B-cell lymphoma cell line (SP-53) or parental L cells (FIG. 5A-5F). There was no significant lysis observed with untransduced T cells.
• a chimeric anti-human CD94 antibody (“cUT-CD94") was synthesized by replacing the mouse Fc with human IgGl Fc in the murine UT-CD94 monoclonal antibody that was generated in example 1 using hybridoma technology. The murine and chimeric antihuman CD94 antibodies were then used in various in vitro assays.
• CD94 can function as a stimulatory or inhibitory receptor when it heterodimerizes with NKG2C or NKG2A, respectively. It was determined whether the cUT- CD94 antibody can block interaction with its ligand, HLA-E.
• the NK leukemia cell line, KHYG-1 was incubated with different concentrations of the cUT-CD94 antibody or isotype control antibody for 1 hour and then the cells were stained with HLA-E tetramer and analyzed by flow cytometry. The results showed that the cUT-CD94 antibody blocked interaction with HLA-E (FIG. 6).
• 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 Effector: Target (E:T) ratio of 2: 1 in duplicate wells in the presence or absence of the UT-CD94 antibody (mCD94 Ab) and cUT-CD94 antibody (cCD94 Ab) or commercial murine anti-CD94 antibody (clone DX22 from Biolegend Catalog # 305502) and their respective isotype controls (mouse IgGl isotype control antibody from Biolegend Catalog # 401402 and human IgGl isotype control antibody from Biocell Catalog # BE0297). All antibodies were used at a concentration of 10 pg/mL. CD137 expression was assessed by flow cytometry
• NK cells were isolated from normal donor peripheral blood mononuclear cells by magnetic cell separation, activated with IL-2 (1200 lU/mL) overnight, and incubated with HLA-E-expressing SP-53 mantle cell lymphoma cell line at Effector: Target (E:T) ratio of 2: 1 in duplicate wells in the presence or absence of the UT-CD94 antibody (mCD94 Ab) and cUT-CD94 antibody (cCD94 Ab) or commercial murine anti-CD94 antibody (clone DX22 from Biolegend Catalog # 305502) and their respective isotype controls (mouse IgGl isotype control antibody from Biolegend Catalog # 401402 and human IgGl isotype control antibody from Biocell Catalog # BE0297). All antibodies were used at a concentration of 10 pg/mL. CD 107a expression was assessed by flow cytometry after 16 hours of incubation (FIG. 8).
• CD137 expression was significantly higher with cUT-CD94 antibody compared to a commercial murine anti-CD94 antibody (clone DX22 from Biolegend). Specifically, CD137 expression was found in 44-49% of cells treated with the cUT-CD94 antibody, which was significantly higher than the 22-23% of cells with CD137 expression after treatment with either the UT- CD94 antibody (mCD94 Ab) or the commercial murine anti-CD94 antibody (DX22 Ab).
• CD107a expression was found in 33-34% of cells treated with the cUT-CD94 antibody, which was significantly higher than the 16% of cells with CD 107a expression after treatment with the commercial murine anti-CD94 antibody (DX22) and the 14-16% of cells with CD107a expression after treatment with the UT-CD94 antibody generated in example 1 (mCD94 Ab).
• DX22 commercial murine anti-CD94 antibody
• mCD94 Ab 14-16% of cells with CD107a expression after treatment with the UT-CD94 antibody generated in example 1
• an anti-CD94 antibody drug conjugate (AdC) is generated.
• 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.
• NK-cell lymphoma or leukemia lines NK-92 and NKL The cytotoxicity of the anti-CD94 AdC is analyzed against NK-cell lymphoma or leukemia lines NK-92 and NKL.
• NK-92 and NKL is labeled with CellTraceTM Far Red cell staining kit (Thermofisher), and the B-cell lymphoma cell line SP53 (mantle cell lymphoma cell line) is stained with CFSE (Thermofisher).
• the NK-92, NKL and SP53 cell lines are co-cultured with the anti-CD94 AdC.
• the percentage of dead cells is determined by flow cytometry at days 1, 2, 3 and 4 after staining for dead cells (Thermofisher). Tumor cells alone are used as negative controls.
• CD94 transcript expression is restricted in normal tissues. Analysis of single cell RNAseq (scRNASeq) data from the Human Protein Atlas project revealed that CD94 mRNA transcript was only present 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, neuronal cells, renal tubular cells, etc. (FIGs. 9A- 9F).
• PBMC peripheral blood mononuclear cells
• Cerebrum, cerebellum, adrenal, liver, thyroid, lung, pancreas, bone marrow, trachea, testicle, heart, skeletal muscle, skin, nerve, salivary gland, kidney, pericardium, eye, prostate, and cervix organs did not show positive signals (FIG. 10A).
• Tonsil derived from a normal donor (FIG. 10B) and NK/T-cell lymphoma tumor tissue derived from a patient (FIG. 10C) were used as positive controls and strong signals of CD94 were detected in both tissue slides.
• CD94 protein expression was restricted in normal tissues. Consistent with scRNAseq data, immunohistochemical analysis of normal donor tissue array showed CD94 protein expression only in scattered in reactive lymphocytes in different organs (FIG. 10A). Strong staining for CD94 was detected in normal tonsil (FIG. 10B) and NK/T-cell lymphoma tissue (FIG. 10C) as expected.
• CRISPR-Cas9 technology was used to stably knock-out CD94 in two NK-cell leukemia cell lines (KHYG-1 and NK92).
• the binding specificity of anti-CD94 antibody was assessed by flow cytometry against wild-type KHYG-1 and NK92 cell lines and their isogenic cell lines with CD94 knock-out (KO) (FIG. 11).
• the anti-CD94 monoclonal antibody bound to the wild-type KHYG-1 and NK92 but not their isogeneic cell lines with CD94 knock-out confirming its specificity.
• Anti-CD94 scFv is linked to hinge/transmembrane domains of CD8a and costimulatory domains derived from CD28, 4- 1BB, or OX-40. 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 after 72 hours after staining cells with recombinant human CD94 protein (FIG. 12B). Good transduction efficiency was noted with all three constructs.
• NK-cell leukemia cell lines (NKL - FIG.
• FIG. 12C KHYG-1 - FIG. 12D, and NK92 - FIG. 12E were stably transduced with red fluorescent protein (RFP).
• the tumor cells were then cultured in the presence or absence of CD94-targeting CAR T cells or untransduced T cells at an Effector: Target ratio of 0.5:1. Lysis or growth of tumor cells was monitored by serial imaging using the Incucyte Live-Cell Analysis System.
• mice Intravenous injection of these tumor cells into NSG mice resulted in massive hepatosplenomegaly in mice and growth of tumor predominantly in liver, spleen, and bone marrow with minimal involvement of peripheral blood very similar to the clinical presentation in humans. These mice died of progressive tumor growth by about 50 days (data not shown).
• NSG mice that were not injected with tumor were used as controls.
• Tumor cells 1.5 x lO 6 tumor cells/mouse
• TCL hepatosplenic T-cell lymphoma
• 10xl0 6 anti-CD94-targeting CAR+ T cells or untransduced T cells were administrated by intravenous injection.
• Antitumor effects of CAR T cells was assessed by monitoring the mice for survival. Log-rank test was used to evaluate the difference in survival between treatment groups.

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