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WO2025049995A1 - Anticorps anti-ly6 et leurs utilisations - Google Patents

Anticorps anti-ly6 et leurs utilisations Download PDF

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WO2025049995A1
WO2025049995A1 PCT/US2024/044799 US2024044799W WO2025049995A1 WO 2025049995 A1 WO2025049995 A1 WO 2025049995A1 US 2024044799 W US2024044799 W US 2024044799W WO 2025049995 A1 WO2025049995 A1 WO 2025049995A1
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seq
amino acid
acid sequence
antibody
antigen
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Geeta UPADHYAY
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Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • 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
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/49—Breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • Ly6 belongs to the superfamily of lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) proteins.
  • Ly6 proteins are cysteine-rich proteins characterized by a distinct disulfide bridge pattern that creates the three-finger Ly6/uPAR (LU) domain.
  • Ly6D, Ly6E, Ly6K and Ly6H are high compared to adjacent normal tissues in a multitude of tumors including ovarian, colorectal, gastric, breast, lung, bladder, brain and CNS, cervical, esophageal, head and neck, and pancreatic cancer.
  • the increased expression of Ly6D, Ly6E, Ly6K and Ly6H was associated with poor survival in ovarian, colorectal, gastric, breast, and lung cancer. It is reported that Ly6 proteins play important role in TGF-P signaling, AKT pathways and immune regulation. A cumulative effect of Ly6 downstream pathways may lead to increased aggressiveness of cancer cells and leading to the poor survival outcome.
  • Ly6 The tumor- specific expression of Ly6 proteins indicates that the Ly6 is a potential therapeutic target for cancer immunotherapy.
  • the present disclosure provides antibodies or fragments thereof having binding specificity to the human Ly6 proteins, in particular, human Ly6D, Ly6E, Ly6K, or Ly6H proteins, respectively.
  • Blockage of Ly6 using the antibodies or antigen-binding fragment thereof, or the Ly6 (for example, Ly6D, Ly6E, Ly6K, or Ly6H) CAR-expressing immune cell can induce T cell activation and stimulate cellular immunity in the body.
  • Ly6 CAR-expressing immune cells are capable of killing cancer cells expressing Ly6 without the need for HL A matching.
  • the present disclosure provides an antibody or antigen-binding fragment thereof having specificity to a cell-surface Lymphocyte Antigen 6 Family Member K protein (Ly6K), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprise the sequences of: (a) HCDR1: SYNIH (SEQ ID NO: 1), HCDR2: AIYPGNGDTSYNQKFKD (SEQ ID NO: 2), HCDR3: GGYPFIY (SEQ ID NO: 3), LCDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 4), LCDR2: KVSNRFS (SEQ ID NO: 5), and LCDR3
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15 and 23, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15 and 23.
  • the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16 and 24 or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16 and 24.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8;
  • the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16; or
  • the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24.
  • the present disclosure provides an antibody or antigen-binding fragment thereof having specificity to a cell-surface Lymphocyte Antigen 6 Family Member D protein (Ly6D), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprise the sequences of:
  • HCDR1 DYIMH (SEQ ID NO: 25), HCDR2: YIQPNNGDNTYNQKFKG (SEQ ID NO: 26), HCDR3: TNWDGAMDY (SEQ ID NO: 27), LCDR1: RASQNIYDYLH (SEQ ID NO: 28), LCDR2: YASQSIS (SEQ ID NO: 29), and LCDR3: QSGHSFPYT (SEQ ID NO: 30);
  • HCDR1 EYPIH (SEQ ID NO: 33), HCDR2: MIYTDTGESTYAEEFKG (SEQ ID NO:34), HCDR3: DYYYSCPLAY (SEQ ID NO: 35), LCDR1: RASQDISNYLN (SEQ ID NO: 39) or RASDNIHNFLT (SEQ ID NO: 48), LCDR2: YTSRLYS (SEQ ID NO: 40) or NAKTLAD (SEQ ID NO: 49), and LCDR3: QQCNTLPWT (SEQ ID NO: 41) or QHFWSIPWT (SEQ ID NO: 50); (c) HCDR1: EYPIH (SEQ ID NO: 45), HCDR2: MIYTDTGESTYAEEFKG (SEQ ID NO:46), HCDR3: DYYYSCPLAY (SEQ ID NO: 47), LCDR1 : RASDNIHNFLT (SEQ ID NO: 48), LCDR2: NAKTLAD (SEQ ID NO:
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 42, 51 and 59, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 42, 51 and 59.
  • the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 44, 52 and 60, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 44, 52 and 60.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, the first light chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and a second light chain variable region comprising the amino acid sequence of SEQ ID NO: 44;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 52; or
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60.
  • the present disclosure provides an antibody or antigen-binding fragment thereof having specificity to a cell-surface Lymphocyte Antigen 6 Family Member E protein (Ly6E), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprise the sequences of: (a) HCDR1: TFWMH (SEQ ID NO: 61), HCDR2: NINPNNGGTNYNEKFKK (SEQ ID NO: 62), HCDR3: TAY (SEQ ID NO: 63), LCDR1: SGSSSGSYMH (SEQ ID NO: 64), LCDR2: ETSKLAS (SEQ ID NO: 65), and LCDR3: QQWSRNPPTLT (Ly6E), wherein
  • RIYPGDGDTNYNGKFKG (SEQ ID NO:78), HCDR3: EGYYGSNSYYTMDY (SEQ ID NO: 79), LCDR1: SASQGIRNYLN (SEQ ID NO: 80), LCDR2: YTSSLHS (SEQ ID NO: 81), and LCDR3: QQYSKVPWT (SEQ ID NO: 82).
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 75 and 83, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 75 and 83.
  • the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 76 and 84 or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 76 and 84.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 67, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68; (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 75, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 76; or (c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 83, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 84.
  • the antibody or antigen-binding fragment thereof further comprises
  • any antibody or antigen-binding fragment thereof of the preceding paragraphs may further comprises a heavy chain constant region, a light chain constant region, an Fc region, or the combination thereof.
  • the light chain constant region may be a kappa or lambda chain constant region.
  • any antibody or antigen-binding fragment thereof of the preceding paragraphs may be of an isotype of IgG, IgM, IgA, IgE or IgD.
  • the isotype may be IgGl, IgG2, IgG3 or IgG4.
  • the present disclosure provides a chimeric antigen receptor, comprising the antibody, or antigen binding fragment thereof of any of the preceding paragraphs.
  • the chimeric antigen receptor comprises one or more costimulatory domains, and an activation domain.
  • the one or more costimulatory domains may comprise one or more costimulatory signaling regions derived from the group consisting of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, 2B4, CD3 gamma, CD3 delta, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD27, CD28, CD28T, OX-40, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD 154, NKG2D, inducible T cell costimulator (1COS), CD247, 1g alpha (CD79a), Fc gamma receptor or a zeta chain of a T cell receptor.
  • 1COS inducible T cell costimulator
  • the one or more costimulatory domains may comprise one or more intracellular signaling regions of CD28, OX-40 and/or 4- IBB.
  • the costimulatory domain further comprises a transmembrane domain and optionally, a spacer domain.
  • the transmembrane domain may comprises a transmembrane domain selected from the group consisting of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, 2B4, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, NKG2D, or a zeta chain of a T cell receptor, or any combination thereof.
  • 4-1BB/CD137 an alpha chain of a T cell receptor
  • a beta chain of a T cell receptor 2B4, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, NKG2D, or a zeta
  • the spacer domain may comprise a hinge region selected from the group consisting of the hinge region of IgGl, lgG2, lgG3, lgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha.
  • the activation domain comprises a cytoplasmic region selected from the group consisting of CD3 zeta, CD3 epsilon, CD3 delta, and CD3 gamma. The activation domain may be mutated to prevent cell apoptosis.
  • the present disclosure provides an antibody or antigen-binding fragment thereof competing with the antibody or antigen-binding fragment thereof or the chimeric antigen receptor of preceding paragraphs.
  • the present disclosure provides a bifunctional molecule, comprising a first antigen-binding portion and a second portion having specificity to a second protein, wherein the first antigen-binding portion comprises an antibody or antigen-binding fragment thereof of preceding paragraphs.
  • the present disclosure provides an antibody conjugate comprising the antibody or antigen-binding fragment thereof, or the bifunctional molecule of preceding paragraphs, and a conjugate.
  • the present disclosure provides a composition comprising the antibody or antigen-binding fragment thereof of the preceding paragraphs, the bifunctional molecule of the preceding paragraphs, or any combination thereof, and a pharmaceutically acceptable carrier.
  • the present disclosure provides an isolated cell comprising one or more polynucleotide encoding the antibody or antigen-binding fragment thereof of the preceding paragraphs or the bifunctional molecule of the preceding paragraphs.
  • the present disclosure provides an engineered immune cell expressing at the cell surface membrane a chimeric antigen receptor according to the preceding paragraphs.
  • the present disclosure provides a polynucleotide encoding one or more chains of the antibody or antigen-binding fragment thereof of the preceding paragraphs, the chimeric antigen receptor of any one of the preceding paragraphs or the bifunctional molecule of the preceding paragraphs.
  • the present disclosure provides a method of engineering an immune cell comprising: (a) providing an immune cell, and (b) expressing at the surface of said cell at least one chimeric antigen receptor of the preceding paragraphs.
  • the present disclosure provides a method of engineering an immune cell, comprising: (a) providing an immune cell, (b) introducing into said cell at least one polynucleotide encoding said chimeric antigen receptor of the preceding paragraphs, and (c) expressing said polynucleotide into said immune cell.
  • the present disclosure provides a method of treating a cancer in a patient in need thereof, comprising administering to the patient the antibody or antigenbinding fragment thereof of any one of the preceding paragraphs or any combination thereof, the bifunctional molecule of the preceding paragraphs, or the engineered immune cell of the preceding paragraphs.
  • the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.
  • the breast cancer may be triple negative breast cancer.
  • the pancreatic cancer may be pancreatic ductal adenocarcinoma.
  • the method may further comprise administering to the patient a therapy for treating said cancer.
  • Said therapy may be selected from the group consisting of immunotherapy, chemotherapy and radiotherapy.
  • the present disclosure provides a method of detecting expression of Ly6 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-16 or the bifunctional molecule of claim 28 under conditions for the antibody or antigen-binding fragment thereof to bind to the Ly6, and detecting the binding which indicates expression of Ly6 in the sample.
  • the present disclosure provides a method of treating a breast cancer comprising administering to the patient the antibody or antigen-binding fragment thereof of the preceding paragraphs or an engineered immune cell expressing at the cell surface membrane a chimeric antigen receptor comprising the antibody, or antigen binding fragment thereof of the preceding paragraphs.
  • the breast cancer is triple negative breast cancer.
  • the present disclosure provides a method of treating a pancreatic cancer comprising administering to the patient the antibody or antigen-binding fragment thereof of the preceding paragraphs or an engineered immune cell expressing at the cell surface membrane a chimeric antigen receptor comprising the antibody, or antigen binding fragment thereof of the preceding paragraphs.
  • the pancreatic cancer is pancreatic ductal adenocarcinoma.
  • FIG. 1A illustrates Ly6K expression in normal breast and breast cancer tissues.
  • FIG. IB illustrates Ly6K expression in triple negative breast cancer (TNBC) and non- TNBC tissues.
  • FIG. 1C is a graph showing survival rate for TNBC with high and low Ly6K expression.
  • FIG. ID is a graph showing Ly6K mRNA expression rate in TNBC subtypes.
  • FIG. IE is a graph showing quantitative protein expression of Ly6K in normal tissues.
  • FIG. IF is a diagram showing expression of Ly6D, Ly6E, Ly6K in immune cells.
  • FIG. 2A illustrates Ly6K expression in 4T1 cells.
  • FIG. 3B illustrates purification of the extracellular portion of the human Ly6K protein.
  • FIG. 4B shows binding of the anti-human Ly6K antibody to paraffin fixed TNBC tissues in IHC studies.
  • FIG. 4C is graphs showing flow cytometry results showing recognition of Ly6K protein in various cancer cell lines by a Ly6K antibody.
  • FIG. 5 illustrates transduction of Ly6K CAR compared to CD 19 CAR.
  • FIG. 6B is a graph showing anti-tumor activity of Ly6K CAR T-cells and untransduced T cell against HeLa tumor cell line.
  • FIG. 7A is a graph showing cell counts of MDA-MB-231 cells and pancreatic cancer cells, after cultured with no T-cell.
  • FIG. 7B is a graph showing cell counts of MDA-MB-231 cells and pancreatic cancer cells, after cultured with untransduced T-cells.
  • FIG. 8C is a photograph of excised tumor of HS378t TNBC cell line xenograft model over time with in vivo administration with Ly6K CAR T-cells and untransduced T-cells.
  • FIG. 8D is a graph showing final open tumor volume of HS378t TNBC cell line xenograft model treated with in vivo administration with Ly6K CAR T-cells and untransduced T-cells.
  • FIGS. 11D-F are graphs showing immune cell specific cell death upon contacting with PBMC and an anti-Ly6D antibody.
  • a “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins.
  • the regions are connected with a short linker peptide of ten to about 25 amino acids.
  • the linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
  • ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.
  • immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
  • IgG a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000.
  • the four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
  • Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • Light chains are classified as either kappa or lambda (K, X). Each heavy chain class may be bound with either a kappa or lambda light chain.
  • the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • both the light and heavy chains are divided into regions of structural and functional homology.
  • the terms “constant” and “variable” are used functionally.
  • the variable domains of both the light (VK) and heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (CK) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CK domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
  • variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen-binding site.
  • This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VK chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3).
  • a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g. , Hamers-Casterman et al., Nature 363:446-448 (1993).
  • each antigen-binding domain is short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the antigen-binding domains referred to as “framework” regions, show less inter-molecular variability.
  • the framework regions largely adopt a p-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the [3 -sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigenbinding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non- covalent binding of the antibody to its cognate epitope.
  • the amino acids comprising the CDRs and the framework regions, respectively can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
  • CDR complementarity determining region
  • Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
  • One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself.
  • “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).
  • CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue.
  • CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue.
  • CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR- H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid.
  • CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue.
  • CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues.
  • CDR-L3 begins at approximately the thirty third residue after the end of CDR-L2 (i.e., following a cysteine residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid.
  • Antibodies disclosed herein may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.
  • the variable region may be condricthoid in origin (e.g., from sharks).
  • heavy chain constant region includes amino acid sequences derived from an immunoglobulin heavy chain.
  • a polypeptide comprising a heavy chain constant region comprises at least one of: a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • an antigen-binding polypeptide for use in the disclosure may comprise a polypeptide chain comprising a CHI domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHI domain and a CH3 domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain.
  • a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain.
  • an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g. , all or part of a CH2 domain).
  • a CH2 domain e.g. , all or part of a CH2 domain.
  • the heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules.
  • a heavy chain constant region of a polypeptide may comprise a CHI domain derived from an IgGi molecule and a hinge region derived from an IgGi molecule.
  • a heavy chain constant region can comprise a hinge region derived, in part, from an IgGi molecule and, in part, from an IgGs molecule.
  • a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGi molecule and, in part, from an IgG4 molecule.
  • the term “light chain constant region” includes amino acid sequences derived from antibody light chain.
  • the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.
  • a “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CHI domain of the heavy chain.
  • VH domain includes the amino terminal variable domain of an immunoglobulin heavy chain
  • CHI domain includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain.
  • the CHI domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
  • CH2 domain includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983).
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.
  • the term “hinge region” includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux el al., J. Immunol 161:4083 (1998)).
  • the term “disulfide bond” includes the covalent bond formed between two sulfur atoms.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
  • the CHI and CK regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
  • chimeric antibody will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species.
  • the target binding region or site will be from a non -human source (e.g., mouse or primate) and the constant region is human.
  • an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope.
  • an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
  • the term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
  • antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”
  • the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
  • phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.
  • the present disclosure provides antibodies, including antibodies or antigen-binding fragments thereof, that have binding specificity to the human Ly6 protein, in particular, human Ly6D, Ly6K, Ly6E, Ly6H proteins, respectively.
  • human Ly6D, Ly6K, Ly6E, Ly6H proteins are highly binding specificity to the human Ly6 protein, in particular, human Ly6D, Ly6K, Ly6E, Ly6H proteins, respectively.
  • numerous anti-human Ly6D, Ly6K, Ly6E, or Ly6H antibodies were separately obtained, having high binding affinity to the human Ly6D, Ly6K, Ly6E, or Ly6H proteins, respectively.
  • the anti-Ly6 antibodies or antigen-binding fragments thereof provided herein refer to any one of the anti-Ly6D antibodies or antigen-binding fragments thereof, any one of the anti-Ly6K antibodies or antigen-binding fragments thereof, any one of the anti-Ly6E antibodies or antigen-binding fragments thereof, or any one of the anti-Ly6H antibodies or antigen-binding fragments thereof provided herein.
  • antibodies or antigen-binding fragments thereof that include the heavy chain and light chain variable domains with the CDR regions of the antibodies prepared in the experimental examples.
  • the CDRs are summarized in Table 1 below (Kabat numbering).
  • VH CDR1, CDR2, and CDR3 are selected from any set of
  • VH CDR1, CDR2, and CDR3 shown in Table 1 and the VL CDR1, CDR2, and CDR3 are selected from any set of VL CDR1, CDR2, and CDR3 shown in Table 1.
  • the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 are selected from those derived from the same antibody in the examples.
  • At least one, or two, or three, or four, or five, or six of the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 of the above are modified by one, two or three amino acid additions, deletions, substitutions, or the combinations thereof.
  • the antibody is a humanized antibody.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Administration can be systemic or local.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the antigen-binding polypeptides or compositions of the disclosure may be administered locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g. , in conjunction, with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • care must be taken to use materials to which the protein does not absorb.
  • kits for detecting expression of a human Ly6K protein in a sample are provided.
  • Methods of detecting expression of a human Ly6D protein in a sample comprising contacting the sample with the antibody or fragment thereof, and detecting the binding which indicates expression of Ly6D in the sample.
  • methods of detecting expression of a human Ly6D protein in a sample comprising contacting the sample with the antibody or fragment thereof, and detecting the binding which indicates expression of Ly6D in the sample.
  • methods of detecting expression of a human Ly6D protein in a sample comprising contacting the sample with the antibody or fragment thereof, and detecting the binding which indicates expression of Ly6D in the sample.
  • Methods of inhibiting a human Ly6K protein are also provided, in some embodiments, comprising administering any of the anti-Ly6K antibody or fragment thereof described herein.
  • Methods of inhibiting a human Ly6K protein are also provided, in some embodiments, comprising administering any of the modified/engineered immune cells including anti-Ly6K CARs or anti-Ly6D CARs described herein (e.g., CAR T-cells).
  • compositions comprising the antibodies
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions comprising the antibodies or the antigen-binding fragment thereof is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the method in one embodiment, entails administering to the patient an effective amount of anti-Ly6 (for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR- expressing T cell of the present disclosure.
  • anti-Ly6 for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H
  • anti-Ly6 for example, anti-Ly6K, anti- Ly6D, anti-Ly6E, or anti-Ly6H
  • anti-Ly6 for example, anti-Ly6K, anti- Ly6D, anti-Ly6E, or anti-Ly6H
  • the anti-Ly6 for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H
  • the anti-Ly6 for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H
  • said T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time.
  • Said treatment can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment.
  • autologous it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor.
  • HLA Human Leucocyte Antigen
  • allogeneic is meant that the cells or population of cells used for treating patients are not originating from said patient but from a donor.
  • Said treatment can be used to treat patients diagnosed wherein a pre-malignant or malignant cancer condition characterized by anti-Ly6 (for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing cells, especially by an overabundance of anti-Ly6 (for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing cells.
  • the treatment with the engineered immune cells according to the invention may be in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
  • the treatment with the engineered immune cells according to the invention may be administered in combination (e.g., before, simultaneously or following) with one or more therapies against cancer selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine, chlorambucil, and combination thereof.
  • one or more therapies against cancer selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine, chloram
  • said treatment can be administrated into patients undergoing an immunosuppressive treatment.
  • the present disclosure preferably relies on cells or population of cells, which have been made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent.
  • the immunosuppressive treatment should help the selection and expansion of the T-cells according to the invention within the patient.
  • the administration of the cells or population of cells according to the present disclosure may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the compositions described herein may be administered to a patient subcutaneously, intradermaly, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally.
  • the cell compositions of the present disclosure are preferably administered by intravenous injection.
  • the administration of the cells or population of cells can consist of the administration of 10 4 -10 9 cells per kg body weight, preferably 10 5 to 10 6 cells/kg body weight including all integer values of cell numbers within those ranges.
  • the cells or population of cells can be administrated in one or more doses.
  • said effective amount of cells are administrated as a single dose.
  • said effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient.
  • the cells or population of cells may be obtained from any source, such as a blood bank or a donor.
  • An effective amount means an amount which provides a therapeutic or prophylactic benefit.
  • the dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.
  • said effective amount of cells or composition comprising those cells are administrated parenterally.
  • Said administration can be an intravenous administration.
  • Said administration can be directly done by injection within a tumor.
  • the treatment with the engineered immune cells according to the invention may be in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
  • the treatment with the engineered immune cells according to the invention may be administered in combination (e.g., before, simultaneously or following) with one or more therapies against cancer selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine ,Novantrone, Mitoxantrone, Vepeside, Etoposide (VP 16), arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine, chlorambucil, and combination thereof.
  • Example 1 Ly6K is a valid CAR T-cell therapeutic target in triple negative breast cancer [0180] Ly6K expression was measured in normal breast tissue, breast cancer tissue (FIG. 1A), and TNBC and non-TNBC tissue (FIG. IB). As shown in FIG. 1, Oncomine data analysis showed (A) increased mRNA expression of Ly6K in breast cancer than normal breast tissue using the cancer genome atlas data set, (B) increased mRNA expression in TNBC vs non-TNBC cases using the Curtis dataset. Two sample T-test, p ⁇ 0.05 considered significant.
  • Absolute Ly6K mRNA expression were quantified in TNBC subtypes.
  • Relevant GEO datasets HGU133 Plus 2.0 arrays
  • TNBC patients were normalized using Robust multi averaging RMA method and batch corrected using combat.
  • the analysis was done in R, using packages oligo, sva and limma.
  • n number of tissue samples.
  • the Welch two sample T-test was applied to determine p values, p ⁇ 0.05 considered significant (FIG. ID).
  • the publicly available data at “The human protein atlas” shows quantification of IHC using a NMR validated, affinity purified using the PrEST-antigen as affinity ligand HPA017770 (Sigma) antibody for Ly6K, in a panel of human normal tissues.
  • the intensity of IHC labeling is shown on the Y-axis, X-axis shows name of organs tested.
  • IHC images are shown from normal testis and breast using Ly6K antibody (FIG. IE). The results demonstrated that Ly6K was expressed in testis, but not in normal breast cells.
  • FIG. IF shows mRNA expression of Ly6D and Ly6E genes in purified immune cells. The absence of Ly6K mRNA expression is indicated by a sign and the presence is indicated by a ‘+’ sign.
  • FIG. 1G shows high over-expression of Ly6K in different types of cancer, such as, cervical, breast, head and neck, bladder, esophageal, lung, and colorectal cancer.
  • Example 2 Ly6K is required for in vivo tumor growth in syngeneic mammary tumor models.
  • Ly6K mAbs were generated against an extracellular portion of human Ly6K (hLy6K) using the NCI reagent development project.
  • the sequences associated with extracellular form of the protein (shown in FIG. 3A which shows the structure of Ly6K, SEQ ID NOs:86-89 below) were cloned in pET24.1 HIS tagged vectors respectively. Proteins were expressed in E.coli (BL21DE3) cells and purified using a His tag purification (Ni-NTA beads resulting) kit, Lane 10, resulting in a single product of the expected size of 17kDa (FIG. 3B). The protein was confirmed to be Ly6K by M ALDI/TOF analysis.
  • This protein was used for immunization generating several hybridoma cell lines.
  • the supernatants from these clones were screened with ELISA and Western Blottss to identify antibody producing clones.
  • This process yielded three high affinity Ly6K mAbs, as validated by an ELISA assay, as shown in FIG. 3C.
  • ELISA microtiter plates were coated with lOOul recombinant Ly6K protein for overnight. Indicated dilutions of the antibodies were added for 30 min. Bound antibodies were detected using TMB substrate.
  • Ly6D-2 (11A3) has two different VLs that pair with the same VH, respectively.
  • Ly6K mAbs recognize endogenously expressed Ly6K
  • Ly6K-3 anti-human Ly6K antibody 6E1 (Ly6K-3) was used in IHC studies in paraffin fixed section of human testis tissues. Slides were subjected to serial hydration, permeabilization and acidic antigen retrieval methods. Primary mAb (1:100 overnight 4°C). secondary goat anti-mouse Alexa 568 1:500, 1 h RT. The confocal images were acquired on 40X oil objective on Zeiss LSM700 normal human testis (FIG. 4A). As shown in FIG. 4A, the antibody recognized Ly6K on the basal sperm cell in human testis.
  • T cells were enriched from the PBMCs via negative selection and activated using the CTS Dynabeads CD3/CD28.
  • MSGV retrovirus vector was used to clone the scFv of Ly6K mAh sequences of 6E1 (CPCT-Ly6K-3) from polypeptide sequence of SEQ ID NO. 94.
  • pCL- Ampho Retrovirus Packaging Vector from Novus was used to generate retrovirus. The retrovirus was then transduced to the enriched and activated T cells.
  • the Ly6K CAR T cells were then cultured using CTS Optimizer Pro basal+Supplement-i-IL2 +Glutamax+P/S-i-No additional Serum.
  • Transduced CAR T-cells expressed scFV of Ly6K antibody.
  • the CAR expression was detected using protein L recognized by flow cytometry.
  • T-cells were isolated from PBMC and transduced with retrovirus particle CAR construct. The cells were subjected to protein L staining. Protein L can recognize the kappa chain of scFV which is the in the CD19 scFV and LY6K scFV. More than 30 million CAR T-cells was cryo-preserved. Ly6K CART cells have very good transduction comparable to CD 19 CAR T(more than 40% transduction) as shown in FIG. 5.
  • the CAR T-cells were tested for their cytotoxicity against triple negative breast cancer cells (MDA-MB-231).
  • the MDA-MB-231 cells were stained with cell titer blue (CTV) dye and designated with “CTV+”.
  • Ly6K negative pancreatic cancer cells were left unstained and designated with “CTV”
  • CTV+ and CTV- FIG. 7A
  • both cell population could be observed in the histogram as two peaks (FIG. 7B).
  • Example 7 In vivo antitumor activity of Ly6K CAR T-cells against HS378t Ti ’BC cell line xenograft model
  • ADCC antibody-dependent cell-mediated cytotoxicity

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Abstract

L'invention concerne des anticorps ou des fragments de ceux-ci ayant une spécificité de liaison à la protéine Ly6. Dans divers exemples, les anticorps ou fragments de ceux-ci comprennent des CDR de VH et VL tels que présentement divulgués, ou des variants de ceux-ci. L'invention concerne également un récepteur antigénique chimérique comprenant les anticorps ou des fragments de ceux-ci se liant à la protéine Ly6. L'invention concerne également des méthodes d'utilisation des anticorps ou des fragments de ceux-ci, ou de la cellule immunitaire exprimant CAR Ly6 pour le traitement du cancer.
PCT/US2024/044799 2023-09-01 2024-08-30 Anticorps anti-ly6 et leurs utilisations Pending WO2025049995A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140004536A1 (en) * 2012-05-10 2014-01-02 Perseus Proteomics Inc. Method for detecting cellular dna damage and antibody against cell surface antigen responsive to dna strand breaks
EP2893939A1 (fr) * 2014-01-10 2015-07-15 Netris Pharma Anticorps anti-nétrine-1
US20160199508A1 (en) * 2012-05-21 2016-07-14 Genentech, Inc. ANTI-Ly6E ANTIBODIES AND IMMUNOCONJUGATES AND METHODS OF USE
WO2018081642A1 (fr) * 2016-10-28 2018-05-03 Washington University Anticorps anti-apoe
WO2023136779A2 (fr) * 2022-01-14 2023-07-20 National University Of Singapore Séquences d'anticorps de trois anticorps candidats ciblant le membre k de la famille 6 des antigènes des lymphocytes (ly6k) humains

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140004536A1 (en) * 2012-05-10 2014-01-02 Perseus Proteomics Inc. Method for detecting cellular dna damage and antibody against cell surface antigen responsive to dna strand breaks
US20160199508A1 (en) * 2012-05-21 2016-07-14 Genentech, Inc. ANTI-Ly6E ANTIBODIES AND IMMUNOCONJUGATES AND METHODS OF USE
EP2893939A1 (fr) * 2014-01-10 2015-07-15 Netris Pharma Anticorps anti-nétrine-1
WO2018081642A1 (fr) * 2016-10-28 2018-05-03 Washington University Anticorps anti-apoe
WO2023136779A2 (fr) * 2022-01-14 2023-07-20 National University Of Singapore Séquences d'anticorps de trois anticorps candidats ciblant le membre k de la famille 6 des antigènes des lymphocytes (ly6k) humains

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