Anti-LY6 antibody and its use

Abstract

Provided are antibodies or fragments thereof having binding specificity to the Ly6 protein. In various examples, the antibodies or fragments include VH and VL CDR, or variants thereof, as disclosed herein. Also provided herein are chimeric antigen receptors comprising antibodies or fragments thereof that bind to the Ly6 protein. Methods using the above antibodies or fragments thereof or Ly6 CAR-expressing immune cells to treat cancer are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application asserts the interests under § 119(e) of U.S. Provisional Application No. 63 / 536,341 filed 1 September 2023, the contents of which Provisional Application are incorporated herein by reference in their entirety.

[0002] Description of research and development funded by the federal government. This invention was made with government support under grant numbers 1R01CA227694 and R21CA256424, both awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background Cell surface lymphocyte antigen 6 (Ly6) belongs to the superfamily of lymphocyte antigen-6 (Ly6) / urokinase-type plasminogen-activator receptor (uPAR) proteins. Members of the Ly6 protein family are cysteine-rich proteins characterized by a distinct disulfide crosslinking pattern that creates a three-finger Ly6 / uPAR(LU) domain. When the coding gene is located on human chromosome 8, RNA levels of Ly6 family proteins Ly6D, Ly6E, Ly6K, and Ly6H are elevated compared to adjacent normal tissue in numerous tumors, including ovarian cancer, colorectal cancer, gastric cancer, breast cancer, lung cancer, bladder cancer, brain cancer and CNS cancers, cervical cancer, esophageal cancer, head and neck cancer, and pancreatic cancer. Increased expression of Ly6D, Ly6E, Ly6K, and Ly6H has been associated with lower survival rates in ovarian cancer, colorectal cancer, gastric cancer, breast cancer, and lung cancer. The Ly6 protein has been reported to play a crucial role in TGF-β signaling, the AKT pathway, and immunomodulation. The cumulative effect of the Ly6 downstream pathway may lead to increased aggressiveness of cancer cells and a deterioration of survival outcomes.

[0004] Tumor-specific expression of the Ly6 protein indicates that Ly6 is a potential therapeutic target for cancer immunotherapy. [Overview of the project] [Means for solving the problem]

[0005] overview This disclosure provides antibodies or fragments thereof having binding specificity to human Ly6 proteins, particularly human Ly6D, Ly6E, Ly6K, or Ly6H proteins. Blocking Ly6 using antibodies or their antigen-binding fragments, or Ly6 (e.g., Ly6D, Ly6E, Ly6K, or Ly6H) CAR-expressing immune cells, can induce T cell activation and stimulate cellular immunity in the body. Since these antibodies and fragments bind to Ly6 proteins on the cell surface and neutralize their function, they are useful in treating diseases and symptoms such as cancer. Ly6 CAR-expressing immune cells can kill Ly6-expressing cancer cells without requiring HLA compatibility.

[0006] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment having specificity for cell surface lymphocyte antigen 6 family member K protein (Ly6K), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively: (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 LCDR The present invention provides an antibody or an antigen-binding fragment comprising the sequences 3:FQGSHVPYT (SEQ ID NO: 6); (b)HCDR1:DYNMH (SEQ ID NO: 9), HCDR2:YINPNNGGTRYNQKFKG (SEQ ID NO: 10), HCDR3:DDFY (SEQ ID NO: 11), LCDR1:SASSSVSYMY (SEQ ID NO: 12), LCDR2:LTSNLAS (SEQ ID NO: 13), and LCDR3:QQWSSNPLT (SEQ ID NO: 14); or (c)HCDR1:TNPIN (SEQ ID NO: 17), HCDR2:YSNSGSGKIYYASWAKG (SEQ ID NO: 18), HCDR3:GGIYFGDGLNL (SEQ ID NO: 19), LCDR1:QASQIINNYLA (SEQ ID NO: 20), LCDR2:DASNLAS (SEQ ID NO: 21), and LCDR3:QSYYGILSDGFA (SEQ ID NO: 22).

[0007] In some embodiments, the heavy chain variable region includes 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15, and 23. In some embodiments, the light chain variable region includes 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, and 24. In some embodiments, (a) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7 and the light chain variable region includes the amino acid sequence of SEQ ID NO: 8; (b) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 15 and the light chain variable region includes the amino acid sequence of SEQ ID NO: 16; or (c) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 23 and the light chain variable region includes the amino acid sequence of SEQ ID NO: 24.

[0008] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment having specificity for cell surface lymphocyte antigen 6 family member D protein (Ly6D), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, respectively: (a) HCDR1: DYI (b) MH (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); (b) HCDR1:EYPIH (SEQ ID NO: 33), HCDR2:MIYTDTGESTYAEEFKG (SEQ ID NO: 34), HCDR3:DYYYSCPLAY (SEQ ID NO: 35), LCDR1:RASQD ISNYLN (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), LCD The present invention provides an antibody or an antigen-binding fragment comprising the sequences R2:NAKTLAD (SEQ ID NO: 49) and LCDR3:QHFWSIPWT (SEQ ID NO: 50); or (d)HCDR1:SYAMG (SEQ ID NO: 53), HCDR2:TIDRSASTYYASWAKG (SEQ ID NO: 54), HCDR3:YGNEGVYDL (SEQ ID NO: 55), LCDR1:QASQSVYRNNYLT (SEQ ID NO: 56), LCDR2:FASTLAS (SEQ ID NO: 57), and LCDR3:QGEFSCDSSDCNA (SEQ ID NO: 58).

[0009] In some embodiments, the heavy chain variable region includes 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 42, 51, and 59. In some embodiments, the light chain variable region includes 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 44, 52, and 60. In some embodiments, (a) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 31 and the light chain variable region includes the amino acid sequence of SEQ ID NO: 32; (b) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 42, the first light chain variable region includes the amino acid sequence of SEQ ID NO: 52, and the second light chain variable region includes the amino acid sequence of SEQ ID NO: 44; (c) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 51 and the light chain variable region includes the amino acid sequence of SEQ ID NO: 52; or (d) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 59 and the light chain variable region includes the amino acid sequence of SEQ ID NO: 60.

[0010] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment having specificity for cell surface lymphocyte antigen 6 family member E protein (Ly6E), wherein the antibody or antigen-binding fragment comprises a heavy chain variable region including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are, respectively: (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: QQWSRNPPTL The present invention provides an antibody or an antigen-binding fragment comprising the sequences T (SEQ ID NO: 66); (b) HCDR1:SFGIC (SEQ ID NO: 69), HCDR2:EIYPRSGNTYYNEKFKG (SEQ ID NO: 70), HCDR3:RGDGYYVRYWYFDV (SEQ ID NO: 71), LCDR1:RSSQSIVHSNGNTYLE (SEQ ID NO: 72), LCDR2:KVSNRFY (SEQ ID NO: 73), and LCDR3:FQGSHVPWT (SEQ ID NO: 74); or (c) HCDR1:SSWMN (SEQ ID NO: 77), HCDR2: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).

[0011] In some embodiments, the heavy chain variable region includes 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 75, and 83. In some embodiments, the light chain variable region includes 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 76, and 84. In some embodiments, (a) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 67 and the light chain variable region includes the amino acid sequence of SEQ ID NO: 68; (b) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 75 and the light chain variable region includes the amino acid sequence of SEQ ID NO: 76; or (c) the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 83 and the light chain variable region includes the amino acid sequence of SEQ ID NO: 84.

[0012] In some embodiments, any antibody or antigen-binding fragment described in the preceding paragraph may further include a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. The light chain constant region may be a kappa or lambda chain constant region.

[0013] In some embodiments, any antibody or antigen-binding fragment described in the preceding paragraph may be an isotype of IgG, IgM, IgA, IgE, or IgD. The isotype may be IgG1, IgG2, IgG3, or IgG4.

[0014] In one embodiment, the present disclosure provides a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof as described in any of the preceding paragraphs.

[0015] In some embodiments, the chimeric antigen receptor comprises one or more costimulatory domains and an activation domain. The one or more costimulatory domains may include one or more costimulatory signaling regions derived from the group consisting of 4-1BB / CD137, the alpha chain of the T cell receptor, the beta chain of the 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, CD154, NKG2D, inducible T cell costimulator (ICOS), CD247, Ig alpha (CD79a), Fc gamma receptor, or the zeta chain of the T cell receptor. The one or more costimulatory domains may include one or more intracellular signaling regions of CD28, OX-40, and / or 4-1BB. In some embodiments, the costimulatory domains may further include a transmembrane domain and, optionally, a spacer domain. The transmembrane domains may include a transmembrane domain selected from the group consisting of 4-1BB / CD137, the alpha chain of the T cell receptor, the beta chain of the 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 the zeta chain of the T cell receptor, or any combination thereof. The spacer domain may include a hinge region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha hinge regions. In some embodiments, the activation domain includes 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 inhibit cell apoptosis.

[0016] In one embodiment, the present disclosure provides an antibody or its antigen-binding fragment that competes with the antibody or its antigen-binding fragment described in the preceding paragraph, or with a chimeric antigen receptor.

[0017] In one aspect, the present disclosure provides a bifunctional molecule comprising a first antigen-binding portion and a second portion having specificity for a second protein, wherein said first antigen-binding portion comprises the antibody or antigen-binding fragment thereof described in the preceding paragraph.

[0018] In one aspect, the present disclosure provides an antibody conjugate comprising the antibody or antigen-binding fragment thereof, or the bifunctional molecule described in the preceding paragraph, and a conjugate.

[0019] In one aspect, the present disclosure provides a composition comprising the antibody or antigen-binding fragment thereof described in the preceding paragraph, the bifunctional molecule described in the preceding paragraph, or any combination thereof, and a pharmaceutically acceptable carrier.

[0020] In one aspect, the present disclosure provides an isolated cell comprising one or more polynucleotides encoding the antibody or antigen-binding fragment thereof described in the preceding paragraph or the bifunctional molecule described in the preceding paragraph.

[0021] In one aspect, the present disclosure provides an engineered immune cell that expresses the chimeric antigen receptor described in the preceding paragraph on a cell surface membrane.

[0022] In one aspect, the present disclosure provides a polynucleotide encoding one or more chains of the antibody or antigen-binding fragment thereof described in the preceding paragraph, the chimeric antigen receptor described in any one of the preceding paragraphs, or the bifunctional molecule described in the preceding paragraph.

[0023] In one aspect, the present disclosure provides a method of engineering an immune cell, the method comprising: (a) providing an immune cell; and (b) expressing at least one chimeric antigen receptor described in the preceding paragraph on the surface of said cell.

[0024] In one aspect, the present disclosure provides a method for manipulating immune cells, comprising: (a) preparing immune cells; (b) introducing at least one polynucleotide encoding the chimeric antigen receptor described in the preceding paragraph into the cells; and (c) expressing the polynucleotide in the immune cells.

[0025] In one aspect, the present disclosure provides a method for treating cancer in a patient requiring treatment for cancer, comprising administering to the patient an antibody or antigen-binding fragment thereof as described in any one of the preceding paragraphs, or any combination thereof, a bifunctional molecule as described in the preceding paragraph, or engineered immune cells as described in the preceding paragraph.

[0026] In some embodiments, 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. In some embodiments, the method may further include administering a therapy to the patient to treat the cancer. The therapy may be selected from the group consisting of immunotherapy, chemotherapy, and radiotherapy.

[0027] In one embodiment, the present disclosure provides a method for detecting Ly6 expression in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 16, or a bifunctional molecule as described in claim 28, under conditions in which the antibody or antigen-binding fragment binds to Ly6, and detecting a binding indicating Ly6 expression in the sample.

[0028] In one aspect, the present disclosure provides a method for treating breast cancer, comprising administering to the patient engineered immune cells expressing an antibody or antigen-binding fragment thereof as described in the preceding paragraph, or a chimeric antigen receptor containing the antibody or antigen-binding fragment thereof as described in the preceding paragraph, on the cell surface membrane.

[0029] In some embodiments, the breast cancer is triple-negative breast cancer.

[0030] In one embodiment, the present disclosure provides a method for treating pancreatic cancer, comprising administering to the patient engineered immune cells expressing an antibody or antigen-binding fragment thereof as described in the preceding paragraph, or a chimeric antigen receptor containing the antibody or antigen-binding fragment thereof as described in the preceding paragraph, on the cell surface membrane.

[0031] In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma. [Brief explanation of the drawing]

[0032] [Figure 1A] Figure 1A illustrates Ly6K expression in normal breast tissue and breast cancer tissue.

[0033] [Figure 1B] Figure 1B illustrates Ly6K expression in triple-negative breast cancer (TNBC) and non-TNBC tissues.

[0034] [Figure 1C] Figure 1C is a graph showing the survival rates for TNBCs with high Ly6K expression and TNBCs with low Ly6K expression.

[0035] [Figure 1D] Figure 1D is a graph showing the Ly6K mRNA expression rate in TNBC subtypes.

[0036] [Figure 1E] Figure 1E is a graph showing the quantitative protein expression of Ly6K in normal tissue.

[0037] [Figure 1F] Figure 1F is a diagram showing the expression of Ly6D, Ly6E, and Ly6K in immune cells.

[0038] [Figure 1G] Figure 1G is a plot showing the overexpression of Ly6K in cancer cells.

[0039] [Figure 2A] Figure 2A illustrates Ly6K expression in 4T1 cells.

[0040] [Figure 2B] Figure 2B is a graph showing tumor volume growth of 4T1 cells with and without Ly6K.

[0041] [Figure 3A] Figure 3A is a diagram showing the structure of the Ly6K protein.

[0042] [Figure 3B] Figure 3B illustrates the purification of the extracellular portion of the human Ly6K protein.

[0043] [Figure 3C] Figure 3C is a graph showing the binding of three anti-Ly6K antibodies to the human Ly6K protein.

[0044] [Figure 4A] Figure 4A shows the binding of anti-human Ly6K antibody to paraffin-fixed testicular tissue in the IHC study.

[0045] [Figure 4B] Figure 4B shows the binding of anti-human Ly6K antibody to paraffin-fixed TNBC tissue in IHC studies.

[0046] [Figure 4C] Figure 4C is a graph showing flow cytometry results illustrating the recognition of the Ly6K protein in various cancer cell lines by the Ly6K antibody.

[0047] [Figure 5] Figure 5 illustrates the transduction of Ly6K CAR compared to CD19 CAR.

[0048] [Figure 6A] Figure 6A is a graph showing HeLa tumor cells stained with cell trace violet and co-cultured with untransduced T cells or Ly6K CAR T cells in various ratios.

[0049] [Figure 6B] Figure 6B is a graph showing the antitumor activity of Ly6K CAR T cells and untransduced T cells against HeLa tumor cell lines.

[0050] [Figure 7A-C] Figure 7A is a graph showing the cell counts of MDA-MB-231 cells and pancreatic cancer cells after culturing without T cells.

[0051] Figure 7B is a graph showing the cell counts of untransduced T cells, MDA-MB-231 cells after culture, and pancreatic cancer cells.

[0052] Figure 7C is a graph showing the cell counts of Ly6K CAR T cells, MDA-MB-231 cells after culture, and pancreatic cancer cells.

[0053] [Figure 7D] Figure 7D is a graph showing the cytotoxicity of Ly6K CAR T cells and untransduced T cells against the MDA-MB-231A cell line at various effector:target (E:T) ratios.

[0054] [Figure 7E] Figure 7E is a graph showing the cytotoxicity of Ly6K CAR T cells and untransduced T cells against the BT549 cell line at various effector:target (E:T) ratios.

[0055] [Figure 8A-B]Figures 8A and 8B are graphs showing the tumor volume over time in a xenograft model of the HS378t TNBC cell line, induced by in vivo administration of Ly6K CAR T cells and untransduced T cells.

[0056] [Figure 8C] Figure 8C is a photograph of resected tumors in a xenograft model of the HS378t TNBC cell line, induced by in vivo administration of Ly6K CAR T cells and untransduced T cells over time.

[0057] [Figure 8D] Figure 8D is a graph showing the final open tumor volume of the HS378t TNBC cell line xenograft model treated in vivo with Ly6K CAR T cells and untransduced T cells.

[0058] [Figure 8E] Figure 8E is a graph showing the final tumor weight of HS378t TNBC cell line xenograft models treated with in vivo administration of Ly6K CAR T cells and untransduced T cells.

[0059] [Figure 9A] Figure 9A is a graph showing the percentage of specific cancer cell lysis for untransduced T cells and anti-Ly6K CAR T cells at various effector:target (E:T) ratios.

[0060] [Figure 9B] Figure 9B is a graph showing the percentage of specific cancer cell lysis for untransduced T cells and anti-Ly6K CAR T cells at various effector:target (E:T) ratios.

[0061] [Figure 10A-D] Figures 10A-D are graphs showing the binding of anti-hLy6D antibodies to the human Ly6D protein.

[0062] [Figure 11A-C] Figures 11A-C are graphs showing the cell survival rate of pancreatic cancer cells when exposed to anti-Ly6D antibody.

[0063] [Figure 11D-F] Figures 11D-F are graphs showing immune cell-specific cell death when PBMCs are exposed to anti-Ly6D antibodies. [Modes for carrying out the invention]

[0064] Detailed explanation definition It should be noted that the terms “a” or “an” refer to one or more such entities. For example, “an antibody” is understood to represent one or more antibodies. Thus, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably in this specification.

[0065] As used herein, “antibody” or “antigen-binding polypeptide” refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be the entire antibody, any antigen-binding fragment, or a single chain thereof. Thus, the term “antibody” includes any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule having biological activity to bind to an antigen. Examples of such include, but are not limited to, the complementarity-determining region (CDR) or its ligand-binding portion of a heavy or light chain, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0066] As used herein, the terms “antibody fragment” or “antigen-binding fragment” refer to parts of antibodies such as F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv. Regardless of their structure, antibody fragments bind to the same antigens recognized by intact antibodies. The term “antibody fragment” includes aptamers, spygelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0067] "Single-stranded variable fragment" or "scFv" refers to the heavy chain (V) of immunoglobulins. H ) and light chain (V L This refers to a fusion protein of the variable region of ). In some embodiments, the region is linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and rich in serine or threonine for solubility. H The N-terminus of V L It can be linked to the C-terminus of and vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. The ScFv molecule is known in the art and is described, for example, in U.S. Patent No. 5,892,019.

[0068] The term antibody encompasses a broad range of polypeptide classes that can be biochemically distinguished. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses (e.g., γ1-γ4). It is the properties of the chain that determine the “class” of an antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and known to confer functional specialization. Each of these classes and isotypes, and its modified versions, are readily identifiable to those skilled in the art in light of this disclosure and are therefore within the scope of this disclosure. All immunoglobulin classes are clearly within the scope of this disclosure, and the following discussion will generally focus on the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule consists of two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy-chain polypeptides with a molecular weight of 53,000–70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, where the light chains begin at a "Y" mouth and the heavy chains follow through a variable region.

[0069] The antibodies, antigen-binding polypeptides, variants, or derivatives of the antigen-binding polypeptides, variants, or derivatives of the antibodies, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, or derivatives of the antibodies, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides, variants, antigen-binding polypeptides,

[0070] Light chains are classified as either kappa or lambda (Κ, λ). Each heavy chain class can bind to either a kappa or lambda light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the branched ends of the Y configuration to the C-terminus at the bottom of each chain.

[0071] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be understood that the variable domains of both the light chain (VK) and heavy chain (VH) determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases distally from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region. The CH3 and CK domains actually constitute the carboxyl terms of the heavy and light chains, respectively.

[0072] As described above, the variable region allows the antibody to selectively recognize and specifically bind to epitopes on the antigen. That is, a combination of the antibody's VK and VH domains, or subsets of the complementarity-determining regions (CDRs), forms a variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites present at the ends 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). In some examples, for instance, in certain immunoglobulin molecules derived from or engineered based on camelid animal immunoglobulins, the complete immunoglobulin molecule may consist only of heavy chains, without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0073] In naturally occurring antibodies, the six “complementarity-determining regions” or “CDRs” within each antigen-binding domain are short, discontinuous sequences of amino acids that are specifically positioned to form the antigen-binding domain when the antibody takes its three-dimensional configuration in an aqueous environment. The remaining amino acids within the antigen-binding domain, referred to as the “framework” region, exhibit less intermolecular variability. The framework region primarily takes the form of a β-sheet higher-order structure, and the CDRs form loops that link the β-sheet structure, and in some cases form part of the β-sheet structure. Thus, the framework region acts to form a scaffold that positions the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognitive epitope. The amino acids constituting the CDRs and framework regions can each be readily identified by those skilled in the art for any given heavy-chain or light-chain variable region. This is because they are precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E. et al., USD Department of Health and Human Services (1983); and Chothia and Lesk, J.MoI. Biol., 196:901-917 (1987)).

[0074] Where there are two or more definitions of a term used and / or permitted within the art, the definitions of terms used herein are intended to include all such meanings unless expressly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe discontinuous antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. This particular region is described by Kabat et al., USDept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J.MoI.Biol.196:901-917 (1987) (these in their entirety are incorporated herein by reference). The definitions of CDR according to Kabat and Chothia include duplication or subsets of amino acid residues when compared to one another. Nevertheless, the application of any definition to refer to the CDR of an antibody or its variant is intended to be within the scope of the terms defined and used herein. The following table shows, for comparison, the appropriate amino acid residues containing CDRs as defined by the references cited above. The exact residue numbers containing a particular CDR vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular CDR by considering the variable region amino acid sequence of the antibody. [Table 4]

[0075] Furthermore, Kabat et al. defined a numbering system for variable domain sequences applicable to any antibody. Those skilled in the art can clearly assign this system of “Kabat numbering” to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system presented by Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0076] In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), contains approximately 5-7 amino acids, and ends with the following tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, contains approximately 16-19 amino acids, and ends with the following arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2; contains 3-25 amino acids; and ends with the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., after the cysteine ​​residue); contains approximately 10-17 residues; and ends with the following tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1 and contains approximately 7 residues. CDR-L3 begins at approximately the 33rd residue after the end of CDR-L2 (i.e., after the cysteine ​​residue); contains approximately 7-11 residues and terminates with the sequence F or WGXG, where X is any amino acid.

[0077] The antibodies disclosed herein may be of any animal origin, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be of condricthoid origin (e.g., shark origin).

[0078] As used herein, the term “heavy chain constant region” includes an amino acid sequence derived from an immunoglobulin heavy chain. Polypeptides containing a heavy chain constant region include at least one of the CH1 domain, hinge (e.g., upper, middle, and / or lower hinge regions) domains, CH2 domains, CH3 domains, or variants or fragments thereof. For example, antigen-binding polypeptides used in this disclosure may include polypeptide chains containing a CH1 domain; polypeptide chains containing a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; polypeptide chains containing a CH1 domain and a CH3 domain; polypeptide chains containing a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or polypeptide chains containing a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptides of this disclosure include polypeptide chains containing a CH3 domain. Furthermore, antibodies used in this disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As previously described, it will be understood by those skilled in the art that the heavy chain constant region can be modified to have a different amino acid sequence from naturally occurring immunoglobulin molecules.

[0079] The heavy chain constant regions of antibodies disclosed herein may originate from various immunoglobulin molecules. For example, the heavy chain constant region of polypeptides may originate from IgG l It may include a CH1 domain derived from the molecule and a hinge region derived from the IgG3 molecule. In another example, the heavy chain constant region is partially IgG l The molecule may contain a hinge region derived, and partially, from the IgG3 molecule. In another example, the heavy chain portion is partially IgGl The molecule may contain a chimeric hinge derived in part from the IgG4 molecule.

[0080] As used herein, the term "light chain constant region" includes an amino acid sequence derived from the antibody light chain. Preferably, the light chain constant region includes at least one constant kappa domain or constant lambda domain.

[0081] A "light-heavy chain pair" refers to an aggregate of light and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0082] As previously shown, the subunit structures and three-dimensional arrangements of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" refers to the amino-terminal variable domain of the immunoglobulin heavy chain, and the term "CH1 domain" refers to the first (most amino-terminal) constant region domain of the immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and lies on the amino-terminal side of the hinge region of the immunoglobulin heavy chain molecule.

[0083] As used herein, the term “CH2 domain” refers to the portion of a heavy chain molecule extending from approximately residues 244 to 360 of an antibody (residues 244–360, Kabat numbering system; and residues 231–340, EU numbering system) using conventional numbering schemes (see Kabat et al., USDept. 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 sandwiched between the two CH2 domains of an intact, natural IgG molecule. It has also been well demonstrated that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

[0084] As used herein, the term "hinge region" includes a portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J.Immunol 161:4083 (1998)).

[0085] As used herein, the term “disulfide bond” includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 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 in the Kabat numbering system (positions 226 or 229, in the EU numbering system).

[0086] As used herein, the term “chimeric antibody” means any antibody in which the immunoreactive region or site is obtained from or derived from a first species, and the constant region (which may be intact, partial, or modified in accordance with this disclosure) is obtained from a second species. In certain embodiments, the target-binding region or site is derived from a non-human source (e.g., mouse or primate), and the constant region is human.

[0087] "Specifically binding" or "specific to" generally means that an antibody binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope more easily than it would to a random, unrelated epitope if it binds to that epitope via its antigen-binding domain. The term "specificity" is used herein to modify (qualify) the relative affinity of a particular antibody to a particular epitope. For example, antibody "A" may be considered to have higher specificity to a given epitope than antibody "B", or antibody "A" may be said to bind to epitope "C" with higher specificity than it has to related epitope "D".

[0088] As used herein, the terms “to treat” or “treatment” refer to both therapeutic and prophylactic or preventative measures, the purpose of which is to prevent or slow (reduce) undesirable physiological changes or impairments, such as cancer progression. Beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, reduction of disease severity, stabilization (i.e., no worsening) of the disease, delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” may also mean extending survival compared to the survival expected without treatment. Those who require treatment include those who already have symptoms or impairments, those who are prone to developing symptoms or impairments, or those for whom symptoms or impairments should be prevented.

[0089] "Subject," "individual," "animal," "patient," or "mammal" means any subject, especially a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Examples of mammalian subjects include humans, livestock, farm animals, and zoo animals, sporting animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cows.

[0090] As used herein, phrases such as “to patients requiring treatment” or “subjects requiring treatment” include, for example, subjects that would benefit from the administration of antibodies or compositions of this disclosure used for detection, diagnostic procedures, and / or treatment, such as mammalian subjects.

[0091] Anti-Ly6 antibody This disclosure provides antibodies comprising antibodies or antigen-binding fragments thereof having binding specificity to human Ly6 proteins, particularly human Ly6D, human Ly6K, human Ly6E, and human Ly6H proteins. As demonstrated in experimental examples, numerous anti-human Ly6D antibodies, anti-human Ly6K antibodies, anti-human Ly6E antibodies, or anti-human Ly6H antibodies, each having high binding affinity to human Ly6D, human Ly6K, human Ly6E, or human Ly6H proteins, were obtained separately.

[0092] The anti-Ly6 antibodies or their antigen-binding fragments provided herein refer to any one of the anti-Ly6D antibodies or their antigen-binding fragments, any one of the anti-Ly6K antibodies or their antigen-binding fragments, any one of the anti-Ly6E antibodies or their antigen-binding fragments, or any one of the anti-Ly6H antibodies or their antigen-binding fragments provided herein.

[0093] Additional functional studies have shown that these antibodies or their antigen-binding fragments efficiently bind to a wide range of cancer cells with potent activity. These antibodies or their derivatives significantly inhibit tumor growth.

[0094] According to one embodiment of the present disclosure, provided is an antibody or its antigen-binding fragment comprising a heavy chain variable domain and a light chain variable domain having a CDR region of the antibody prepared in the experimental example. The CDRs are summarized in Table 1 below (Kabat numbering). [Table 1-1] [Table 1-2]

[0095] In some embodiments, VH CDR1, CDR2, and CDR3 are selected from any set of VH CDR1, CDR2, and CDR3 shown in Table 1, and VL CDR1, CDR2, and CDR3 are selected from any set of VL CDR1, CDR2, and CDR3 shown in Table 1. In some embodiments, VH CDR1, CDR2, and CDR3, as well as VL CDR1, CDR2, and CDR3, are selected from those derived from the same antibody in the example.

[0096] In some embodiments, at least one, two, three, four, five, or six of the aforementioned VH CDR1, CDR2, and CDR3, as well as VL CDR1, CDR2, and CDR3, are modified by the addition, deletion, substitution, or combination thereof of one, two, or three amino acids.

[0097] In certain embodiments, the antibody is a humanized antibody. A humanized form of a non-human (e.g., mouse) antibody is a chimeric molecule of an immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing minimal sequences derived from the non-human immunoglobulin. Examples of humanized antibodies include human immunoglobulin (recipient antibody) in which residues forming the recipient's complementarity-determining region (CDR) are replaced by residues derived from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, possessing desired specificity, affinity, and capability. In some examples, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in either the recipient antibody or the imported CDR or framework sequence. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR region corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the FR region is the FR region of the human immunoglobulin consensus sequence. Furthermore, humanized antibodies will optimally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0098] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as import residues, which are typically taken from import variable domains. Humanization can essentially be carried out by using a rodent CDR or CDR sequence in place of its corresponding sequence in a human antibody, following the method of Winter and collaborators (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). Thus, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which a significantly smaller number of the intact human variable domains are replaced by corresponding sequences derived from non-human species. In practice, humanized antibodies are typically human antibodies in which several CDR residues and possibly several FR residues are substituted with residues derived from similar sites in rodent antibodies.

[0099] Antibodies can be produced by affinity maturation processes, which result in modified antibodies with improved affinity for an antigen compared to unmodified parent antibodies. Affinity-matured antibodies can be produced by procedures known in the art, such as Marks et al., Rio / Technology 10:779-783 (1992); Barbas et al., Proc Nat.Acad.Sci.USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J.Immunol.155:1994-2004 (1995); Jackson et al., J.Immunol.154(7):331 0-15 9 (1995); and Hawkins et al., J.Mol.Biol.226:889-896 (1992).

[0100] Furthermore, antibodies can be produced in animals using immunogenic peptides. In certain embodiments, Ly6K-4 antibodies were produced using peptides with sequences ERPKKPEEKRFLLEEP (SEQ ID NO: 90) and SMGESCGGLWAILLL (SEQ ID NO: 91). In certain embodiments, Ly6D-4 antibodies were produced using peptides with sequences QVSSGTSSTQCCQED (SEQ ID NO: 92) or APTRTALAHSALSLG (SEQ ID NO: 93).

[0101] In various embodiments, the present disclosure provides an antibody or antigen-binding fragment having specificity for human Ly6K protein, human Ly6D protein, human Ly6E protein, or human Ly6H protein, comprising a heavy chain variable region including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3.

[0102] The CDR, heavy chain variable region, and light chain variable region of this disclosure can be further modified. In some embodiments, the modified heavy chain variable region or light chain variable region retains at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity and can still bind to Ly6K, Ly6D, Ly6E, or Ly6H, respectively.

[0103] In certain embodiments, the antibody or its antigen-binding fragment comprises two pairs of identical VH / VL, for example, those listed in Table 2. In certain embodiments, the antibody or its antigen-binding fragment comprises two different light chains and two identical heavy chains, so as to form one pair of VH / VL1 and one pair of VH / VL2. For example, the antibody or its antigen-binding fragment provided herein may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof having at least 90%, at least 95%, or at least 98% identity; a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 52 or a variant thereof having at least 90%, at least 95%, or at least 98% identity; and a second light chain variable region comprising the amino acid sequence of SEQ ID NO: 44 or a variant thereof having at least 90%, at least 95%, or at least 98% identity, for example, the anti-Ly6D antibody Ly6D-2(11A3) in Table 2.

[0104] In some embodiments, the modification is the substitution of one or fewer hotspot locations from each CDR. In some embodiments, the modification is the substitution of one, two, or three such hotspot locations. In one embodiment, the modification is the substitution of one of the hotspot locations. Such substitutions are, in some embodiments, conservative substitutions.

[0105] A "conservative amino acid substitution" is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. In the art, families of amino acid residues having similar side chains are defined, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin polypeptides are preferably replaced by other amino acid residues from the same side chain family. In another embodiment, the amino acid string can be replaced with structurally similar strings that differ in the order and / or composition of their side-chain family members.

[0106] Non-restrictive examples of conserved amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conserved substitution between two amino acids. [Table 5] [Table 6]

[0107] It will also be understood by those skilled in the art that antibodies disclosed herein may be modified to have different amino acid sequences from the naturally occurring conjugated polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a given protein may be similar and may have a certain percentage of identity with respect to the starting sequence, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with respect to the starting sequence.

[0108] In certain embodiments, the antibodies provided herein further include a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0109] In certain embodiments, the heavy chain constant region includes a peptide having at least 80% (at least 85%, at least 90%, at least 95%, or at least 98%) the amino acid sequence of SEQ ID NO: 85, 36, or 37. In certain embodiments, the light chain constant region includes a peptide having at least 80% (at least 85%, at least 90%, at least 95%, or at least 98%) the amino acid sequence of SEQ ID NO: 38 or 43. [Table 2-1] [Table 2-2]

[0110] In certain embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 85, and the light chain constant region containing the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 37, and the light chain constant region containing the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 36, and the light chain constant region containing the amino acid sequence of SEQ ID NO: 43.

[0111] The Fc region can be manipulated to enhance or eliminate effector function. IgG antibodies can induce direct antitumor effects through indirect antitumor effects via Fc-mediated effector function involved in other immune cells or killer mechanisms. As used herein, “effector function” or “antibody effector function” refers to the biological activity that may result from the binding of the Fc region of an antibody to its effectors, such as the C1 complex and Fc receptors (FcγRIIa or FcγRIIIa). Exemplary effector functions include: complement-dependent cell-mediated cytotoxicity (CDC) induced by the interaction of an antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the binding of the Fc region of an antibody to an Fc receptor on an effector cell; and antibody-dependent cell-mediated phagocytosis (ADCP) in which nonspecific cytotoxic cells expressing the Fcγ receptor (FcγR) recognize the bound antibody on a target cell and then trigger phagocytosis of the target cell.

[0112] Of the four IgG subclasses, IgG1 and IgG3 induce the strongest Fc effector function. However, because IgG1 has the longest half-life and is more stable than IgG3, most therapeutic antibodies with Fc-mediated function are of the IgG1 isotype.

[0113] IgG2 and IgG4 isotypes exhibit significantly lower binding affinity to FcγR. Recent evidence suggests that while the IgG2 isotype does not completely lack effector function, the IgG4 isotype can undergo in vivo Fab-arm exchange to yield bispecific antibodies and off-target effects.

[0114] In one embodiment, the Disclosure provides a multispecific protein such as an antibody or a fragment thereof. In a particular embodiment, the Disclosure provides a bifunctional molecule comprising a first antigen-binding moiety specific to human Ly6K protein, human Ly6D protein, human Ly6E protein, or human Ly6H protein, and a second moiety specific to a second protein, wherein the first antigen-binding moiety includes an anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H antibody or a fragment thereof, as provided herein.

[0115] In certain embodiments, the second portion is an antibody or an antigen-binding fragment thereof. In certain embodiments, the second portion has specificity for an immune checkpoint. In certain embodiments, the second portion has specificity for other tumor antigens.

[0116] In certain embodiments, the antibody includes an amino acid sequence or one or more substructures not typically associated with the antibody. Exemplary modifications are described in more detail below. For example, the antibodies of this disclosure may include a flexible linker sequence or may be modified to include functional substructures (e.g., PEG, drugs, toxins, or labels).

[0117] Examples of antibodies, variants, or derivatives of the antibodies described herein include derivatives modified by covalent bonding of any type of molecule to the antibody, such that the covalent bond does not interfere with the antibody's binding to an epitope. For example, but not limited to, antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cell ligands or other proteins, etc. Any of the many chemical modifications can be carried out by known techniques, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, antibodies may contain one or more non-classical amino acids.

[0118] In some embodiments, the antibody may be conjugated with a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical, or PEG.

[0119] Antibodies may be conjugated or fused to therapeutic agents, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents which may include detectable labels such as radioactive labels, cytotoxic agents which may be drugs or toxins, ultrasound enhancers, non-radioactive labels, combinations thereof, and other such agents known in the art.

[0120] Furthermore, antibodies can be developed into bispecific T-cell engagers (BiTEs) or bispecific antibodies based on the Fv sequences of their heavy and light chains.

[0121] Chimeric antigen receptor (CAR) Furthermore, this disclosure provides chimeric antigen receptors (CARs) that target Ly6 proteins, particularly Ly6K proteins, Ly6K proteins, Ly6K proteins, or Ly6H proteins, respectively. CARs are generally fusion proteins consisting of an extracellular antibody-type antigen-binding domain fused to an intracellular T cell signaling protein. CARs have successfully redirected T cells to antigens expressed on the surface of tumor cells derived from various malignant tumors, including lymphomas and solid tumors.

[0122] A chimeric antigen receptor (CAR) targeting the Ly6 protein, as provided herein, refers to any one of the CARs targeting the Ly6K protein, any one of the CARs targeting the Ly6D protein, any one of the CARs targeting the Ly6E protein, or any one of the CARs targeting the Ly6H protein, as provided herein. In one embodiment, the CAR comprises a Ly6K-specific antigen-binding fragment as described herein. In an embodiment, the CAR comprises (ii) a Ly6K-specific antigen-binding fragment, (ii) an extracellular (possibly hinge-containing) domain, (iii) one or more costimulatory domains, and (iv) one or more activation domains. Since each domain may be heterogeneous, it will be understood that they may consist of sequences derived from (or corresponding to) different protein chains. In some embodiments, the CAR comprises an antibody or antigen-binding fragment having a CDR sequence as described in Table 1. For example, CAR is (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:FQGSHVPYT (SEQ ID NO: 6); (b) HCDR1:DYNMH (SEQ ID NO: 9), HCDR2:YINPNNGGTRYNQKFKG (SEQ ID NO: 10), HCDR3:DDFY (SEQ ID NO: 11), LCDR1:SASSS The Ly6K-specific antigen-binding fragments include VSYMY (SEQ ID NO: 12), LCDR2:LTSNLAS (SEQ ID NO: 13), and LCDR3:QQWSSNPLT (SEQ ID NO: 14); or (c)HCDR1:TNPIN (SEQ ID NO: 17), HCDR2:YSNSGSGKIYYASWAKG (SEQ ID NO: 18), HCDR3:GGIYFGDGLNL (SEQ ID NO: 19), LCDR1:QASQIINNYLA (SEQ ID NO: 20), LCDR2:DASNLAS (SEQ ID NO: 21), and LCDR3:QSYYGILSDGFA (SEQ ID NO: 22).

[0123] Similarly, Ly6 CAR-expressing immune cells provided herein refer to any one of the Ly6K CAR-expressing immune cells, any one of the Ly6D CAR-expressing immune cells, any one of the Ly6E CAR-expressing immune cells, or any one of the Ly6H CAR-expressing immune cells provided herein.

[0124] Typically, the external domain, which includes an antigen recognition region, comprises a signal peptide and an antigen recognition unit. According to this disclosure, the external domain comprises an anti-Ly6 (e.g., anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) single-chain domain. The single-chain domain is preferably an scFv comprising a heavy-chain variable region and a light-chain variable region as provided herein.

[0125] The external domain can be separated from the transmembrane domain by the presence of a spacer domain. The spacer domain, if necessary, links the antigen-binding domain to the transmembrane domain. The transmembrane domain is preferably flexible enough to allow the antigen-binding domain to be oriented in various directions to facilitate antigen recognition.

[0126] The transmembrane domain is typically a hydrophobic alpha-helix spanning the membrane. Other transmembrane domains may also be used. Immune cells such as T cells, NK cells, or BiTEs are recognized, containing a CAR as described, i.e., with the following composition: an anti-Ly6 (e.g., anti-Ly6K, anti-Ly6D, or anti-Ly6E) single-chain antigen-binding domain, optionally a spacer domain, one or more costimulatory domains, and an activating domain, containing a CAR from the N-terminus to the C-terminus. Therefore, the T cells of this disclosure exhibit effective antitumor effects.

[0127] In certain embodiments, the spacer domain includes a hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha. In other embodiments, the spacer domain of the CAR molecule is an IgG1 hinge region or a variant thereof.

[0128] The co-stimulatory domain may include a transmembrane domain and one or more signaling regions / signaling domains.

[0129] In some embodiments, a linker may be located between the spacer domain and the transmembrane domain.

[0130] Furthermore, in another embodiment relating to a T cell having a chimeric antigen receptor, the transmembrane domain may be derived from 4-1BB / CD137, the alpha chain of the T cell receptor, the beta chain of the 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 the zeta chain of the T cell receptor, or any combination thereof. In a particular embodiment, the transmembrane domain is the transmembrane domain of the CD28 molecule.

[0131] The activating domain contains CD3 zeta, CD3 epsilon, CD3 delta, and CD3 gamma or Fc epsilon receptor (IgE receptor) gamma chain signaling chains. In certain embodiments, the activating domain is mutated to inhibit cell apoptosis. Possible activating domains include the activating domain of CD3 zeta with inactive first and third immunoreceptor tyrosine activation motifs (ITAMs). For example, the activating domain is the CD3 zeta cytoplasmic region. The activating domain is responsible for cytotoxic activity in T cells or activation of interferon-gamma secretion by T cells.

[0132] CAR molecules may be so-called "second-generation" CAR molecules. Second-generation CAR molecules have enhanced intracellular signaling by additionally containing a costimulatory domain derived from, for example, CD28, OX40, or 4-1 BB. "Third-generation" CAR molecules contain a combined costimulatory domain, for example, CD28 combined with OX40 or 4-1 BB.

[0133] Polynucleotides encoding antibodies / CARs and methods for their preparation Furthermore, the Disclosure provides isolated polynucleotides or nucleic acid molecules encoding the antibodies / CARs of the Disclosure, their variants, or derivatives. For example, an isolated polynucleotide or nucleic acid molecule may encode the anti-Ly6K, Ly6D, or Ly6E antibodies / CARs of the Disclosure, their variants, or derivatives. The polynucleotides of the Disclosure may encode the entire heavy chain variable region and light chain variable region of an antigen-binding polypeptide, its variant, or derivative on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the Disclosure may encode portions of the heavy chain variable region and light chain variable region of an antigen-binding polypeptide, its variant, or derivative on the same polynucleotide molecule or on separate polynucleotide molecules.

[0134] Methods for producing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of this disclosure are entirely human. Fully human antibodies can be produced using techniques described in the art and are described herein. For example, a fully human antibody against a particular antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such an antibody in response to an antigen challenge, but whose endogenous locus has been deactivated. Exemplary techniques that can be used to produce such antibodies are described in U.S. Patents 6,150,584, 6,458,592 and 6,420,140 (all incorporated by reference).

[0135] In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of this disclosure are humanized. Humanized antibodies may be designed to minimize undesirable immunological responses toward rodent anti-human antibodies that limit the duration and effectiveness of the therapeutic use of their substructures in human recipients. Humanized antibodies may have one or more amino acid residues introduced from non-human sources. These non-human residues are often referred to as “import” residues, which are typically taken from the variable domain. Humanization may be performed by using a hypervariable region sequence in place of its corresponding sequence in a human antibody. Thus, such a “humanized” antibody is substantially a chimeric antibody in which less than an intact human variable domain is replaced by a corresponding sequence derived from a non-human species. See, for example, U.S. Patent No. 4,816,567, the contents of which are incorporated herein by reference. A humanized antibody may be a human antibody in which several hypervariable region residues and possibly several FR residues are replaced by residues derived from similar sites in the rodent antibody. Humanization or manipulation of the antibodies of this disclosure can be carried out using any known method, for example, the methods described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

[0136] For example, the polypeptide encoding the CAR of the ScFV Ly6K antibody may contain the following amino acid sequence: [ka] [ka]

[0137] As a non-limiting example, the protein of interest may be expressed in cells by introduction as a transgene, preferably encoded by at least one plasmid vector. The polypeptide may be expressed intracellularly as a result of introducing the polynucleotide encoding the polypeptide into the cell. Alternatively, the polypeptide may be produced extracellularly and then introduced into the cell.

[0138] Methods for introducing polynucleotide constructs into cells are known in the art and include, but are not limited to, stable transformation methods in which the polynucleotide construct is integrated into the cell's genome, transient transformation methods in which the polynucleotide construct is not integrated into the cell's genome, and virus-mediated methods. The polynucleotide can be introduced into cells by, for example, recombinant viral vectors (e.g., retroviruses, adenoviruses) and liposomes. For example, transient transformation methods include, for example, microinjection, electroporation or particle bombardment, and cell fusion. The polynucleotide can be contained in a vector, more specifically in a plasmid or virus, in terms of being expressed in the cell. The plasmid vector may contain a selection marker that enables the identification and / or selection of cells that have received the vector.

[0139] Various transgenes can be included in a single vector. The vector may contain nucleic acid sequences that encode ribosome skipping sequences, such as sequences encoding 2A peptides. The 2A peptides identified in the aftvirus subgroup of picornaviruses induce ribosome "skipping" from one codon to the next without forming a peptide bond between the two amino acids encoded by the codon (see Donnelly et al., J. of General Virology, 82:1013-1025 (2001); Donnelly et al., J. of Gen. Virology, 78:13-21 (1997); Doronina et al., Mol. And. Cell. Biology 28(13):4227-4239 (2008); Atkins et al., RNA 13:803-810 (2007)).

[0140] In a more preferred embodiment of the present invention, the polynucleotide encoding the polypeptide according to this disclosure may be mRNA that is directly introduced into a cell, for example by electroporation.

[0141] Various methods used herein include introducing CARs into cells. In non-limiting examples, the CAR may be introduced as a transgene encoded by a plasmid vector. The plasmid vector may also contain a selection marker to enable the identification and / or selection of cells receiving the vector. Polypeptides may be synthesized in situ within cells as a result of introducing the polynucleotide encoding the polypeptide into the cell. Alternatively, the polypeptide may be produced extracellularly and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art and, in non-limiting examples, include stable transformation methods in which the polynucleotide construct is integrated into the cell's genome, transient transformation methods in which the polynucleotide construct is not integrated into the cell's genome, and virus-mediated methods. The polynucleotide may be introduced into cells, for example, by recombinant viral vectors (e.g., retroviruses, adenoviruses) and liposomes. For example, transient transformation methods include, for example, microinjection, electroporation, or particle bombardment. The polynucleotide may be contained in a vector, more specifically a plasmid, or a virus, in terms of intracellular expression.

[0142] Manipulated immune cells The disclosure also relates to isolated cells or cell lines that can be obtained by the aforementioned methods for manipulating cells. In particular, the isolated cells include at least one CAR as described above. In another embodiment, the isolated cells include a population of CARs, each containing a different extracellular ligand-binding domain. In particular, the isolated cells include an exogenous polynucleotide sequence encoding a CAR. The genetically modified immune cells of the disclosure are activated and proliferate independently of antigen-binding mechanisms. The scope of the disclosure also includes isolated immune cells, preferably T cells, obtained according to any one of the previously described methods. The immune cells refer to hematopoietic-derived cells functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. The immune cells according to the disclosure may be derived from stem cells. Stem cells may be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. A typical human cell is the CD34+ cell. Furthermore, the isolated cells may be dendritic cells, killer dendritic cells, mast cells, NK cells, B cells, or T cells selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In another embodiment, the cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. Prior to the expansion and genetic modification of the cells of the present invention, the cell source can be obtained from the subject through a variety of non-limiting methods. Cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available and known to those skilled in the art may be used. In another embodiment, the cells may be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infection. In another embodiment, the cells are part of a mixed population of cells exhibiting various phenotypic characteristics. The scope of this disclosure also includes cell lines obtained from transformed T cells according to the methods described previously.Modified cells that are resistant to immunosuppressive treatment and can be obtained by prior methods are included within the scope of the present invention.

[0143] T cell activation and expansion Whether before or after the genetic modification of T cells, even if the genetically modified immune cells of this disclosure are activated and proliferate independently of the antigen-binding mechanism, the immune cells of this disclosure, particularly T cells, are further generally, for example, U.S. Patent Nos. 6,352,694; U.S. Patent Nos. 6,534,055; U.S. Patent Nos. 6,905,680; U.S. Patent Nos. 6,692,964; U.S. Patent Nos. 5,858,358; U.S. Patent No. 6,887, T cells can be activated and enlarged using the methods described in U.S. Patent No. 466; U.S. Patent No. 6,905,681; U.S. Patent No. 7,144,575; U.S. Patent No. 7,067,318; U.S. Patent No. 7,172,869; U.S. Patent No. 7,232,566; U.S. Patent No. 7,175,843; U.S. Patent No. 5,883,223; U.S. Patent No. 6,905,874; U.S. Patent No. 6,797,514; and U.S. Patent No. 6,867,041. T cells can be enlarged in vitro or in vivo. Generally, T cells are enlarged by contact with agents that stimulate the CD3 TCR complex and costimulatory molecules on the surface of the T cell to produce activation signals for the T cell. For example, activation signals for T cells can be generated using chemicals such as the calcium ionophore A23187, phorbol 12 myristate 13 acetate (PMA), or mitotic lectins such as phytohemagglutinin (PHA). As a non-limiting example, T cell populations can be stimulated in vitro, for example, by contact with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) combined with a calcium ionophore. Ligands that bind to accessory molecules are used for co-stimulation of accessory molecules on the surface of T cells. For example, a population of T cells can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation.Suitable conditions for T cell culture include a suitable medium (e.g., minimal essential medium or RPMI Media 1640 or X-vivo 5 (Lonza)) that may contain factors essential for proliferation and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, -10, -2, 1L-15, TGFp, and TNF-, or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may contain RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and is either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or sufficient amounts of cytokines for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture and not in the culture of cells intended to be injected into the target. Target cells are maintained under conditions essential to support growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). T cells exposed to various stimulation times may exhibit a variety of characteristics.

[0144] Antibody-based treatment and diagnostic methods The antibodies, variants, or derivatives described herein may be used in certain treatment and diagnostic methods.

[0145] This disclosure further covers antibody-based therapies, which involve administering the therapeutic compounds of this disclosure to patients, e.g., animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. The therapeutic compounds of this disclosure include, but are not limited to, the antibodies of this disclosure (including the variants and derivatives thereof described herein) and nucleic acids or polynucleotides encoding the antibodies of this disclosure (including the variants and derivatives thereof described herein).

[0146] In some embodiments, what is provided is a method for treating cancer in a patient requiring treatment for cancer. In one embodiment, the method involves administering an effective amount of the antibody or antigen-binding fragment of the present disclosure to the patient. In one embodiment, the method involves administering an effective amount of engineered immune cells, including anti-Ly6K CARs (e.g., CAR T cells), to the patient.

[0147] In some embodiments, what is provided is the use of the antibody or antigen-binding fragment of the present disclosure in the manufacture of a pharmaceutical for treating cancer in patients requiring cancer treatment. In some embodiments, what is provided is the use of engineered immune cells (e.g., CAR T cells) containing the anti-Ly6K CAR of the present disclosure in the manufacture of a pharmaceutical for treating cancer in patients requiring cancer treatment.

[0148] In some embodiments, what is provided is an antibody or antigen-binding fragment of the present disclosure used for the treatment of cancer in a patient requiring treatment for cancer.

[0149] Non-specific examples of cancer include 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.

[0150] In certain embodiments, the breast cancer is triple-negative breast cancer (TNBC).

[0151] In certain embodiments, pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0152] In certain embodiments, cancer cells may express or overexpress Ly6K, Ly6D, and / or Ly6E.

[0153] Additional diseases or conditions associated with increased cell viability that can be treated, prevented, diagnosed, and / or prognosticated by the antibodies or variants or derivatives of the present disclosure include, but are not limited to, the progression and / or metastasis of malignant tumors, and related disorders, such as leukemia (acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors (sarcomas and carcinomas, e.g., fibrosarcoma, Myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangioendosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial cancer Examples of cancers include, but are not limited to, tumors, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0154] The specific dosage and treatment regimen for any particular patient depends on various factors, including the specific antibody used, its variant or derivative, the patient's age, weight, overall health, sex, and diet, as well as the timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. Determining such factors by healthcare professionals is within the scope of the art. The dosage also depends on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0155] Methods of administering antibodies and variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding polypeptides or compositions may be administered by any convenient route, for example by injection or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of this disclosure may be administered orally, rectally, parenterally, intracapsularly, vaginally, intraperitoneally, topically (by powder, ointment, drops, or transdermal patch), orally, or as oral or nasal sprays.

[0156] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0157] Administration may be systemic or local. In addition, it may be desirable to introduce the antibodies of this disclosure into the central nervous system by any suitable route, including intracerebroventricular and intrathecal injection; intracerebroventricular injection may be facilitated, for example, by an intracerebroventricular catheter attached to a reservoir such as an Ommaya reservoir. Pulmonary administration may also be used, for example, by the use of an inhaler or nebulizer, and by formulation with an aerosolizing agent.

[0158] It may be desirable to administer the antigen-binding polypeptide or composition of this disclosure topically to an area requiring treatment; this can be achieved, for example, by topical injection during surgery, topical application together with postoperative wound dressings, injection, catheter, suppository, or implant, the implant being a membrane such as a sialastic membrane, or a porous, non-porous, or gelatinous material containing fibers. Preferably, when administering proteins such as antibodies of this disclosure, care should be taken to use a material that does not absorb proteins.

[0159] Furthermore, in some embodiments, a method is provided for detecting the expression of human Ly6K protein in a sample, which includes contacting the sample with an antibody or a fragment thereof and detecting binding indicating Ly6K expression in the sample.

[0160] In a particular embodiment, what is provided is the use of the antibody or its antigen-binding fragment in the preparation of a kit for detecting the expression of human Ly6K protein in a sample.

[0161] Furthermore, in some embodiments, a method is provided for detecting the expression of human Ly6D protein in a sample, which includes contacting the sample with an antibody or a fragment thereof and detecting binding that indicates Ly6D expression in the sample.

[0162] In a particular embodiment, what is provided is the use of the antibody or its antigen-binding fragment in the preparation of a kit for detecting the expression of human Ly6D protein in a sample.

[0163] Furthermore, in some embodiments, methods for inhibiting the human Ly6K protein are also provided, comprising administering any of the anti-Ly6K antibodies or fragments thereof described herein. Furthermore, in some embodiments, methods for inhibiting the human Ly6K protein are also provided, comprising administering any of the modified / manipulated immune cells (e.g., CAR T cells) containing anti-Ly6K CAR or anti-Ly6D CAR as described herein.

[0164] composition containing antibodies Furthermore, this disclosure provides pharmaceutical compositions comprising an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0165] In certain embodiments, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory agency, or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, more specifically in humans. Furthermore, “pharmaceutically acceptable carrier” generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0166] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with a therapeutic agent. Such pharmaceutical carriers may include sterile liquids, such as water, and oils, such as those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when a pharmaceutical composition is administered intravenously. Physiological saline, as well as aqueous solutions of dextrose and glycerol, can also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. The composition may also optionally contain small amounts of wetting or emulsifying agents, or pH buffers such as acetate, citrate, or phosphate. Antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and isotonic modifiers such as sodium chloride or dextrose are also conceivable. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, and sustained-release formulations. The compositions can be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, incorporated herein by reference. Such compositions preferably contain a therapeutically effective amount of antigen-binding polypeptide in a purified form, along with an appropriate amount of carrier, to provide a form for appropriate administration to a patient. The formulation should be suitable for the mode of administration.Parental preparations can be encapsulated in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0167] In one embodiment, a composition comprising an antibody or its antigen-binding fragment is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizer and a local anesthetic, such as lignocaine, to alleviate pain at the injection site. Generally, the components are supplied separately in unit dosage forms, for example, as lyophilized powder or anhydrous concentrate in sealed containers such as ampoules or sachets indicating the amount of the activator, or mixed together. If the composition is to be administered by injection, the composition can be dispensed in an injection bottle containing sterile pharmaceutical-grade water or saline. If the composition is to be administered by injection, ampoules of sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0168] Compositions comprising the antibodies or antigen-binding fragments of the present disclosure can be formulated in neutral or salt form. Examples of pharmaceutically acceptable salts include those formed by anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed by cations derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0169] Treatment methods using CAR immune cells In some embodiments, what is provided is a method for treating cancer in a patient requiring treatment for cancer. In one embodiment, the method involves administering an effective amount of anti-Ly6 (e.g., anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H)CAR-expressing T cells of the present disclosure to the patient.

[0170] In some embodiments, what is provided is the use of anti-Ly6 (e.g., anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing T cells of the present disclosure in the manufacture of a pharmaceutical for treating cancer in patients requiring cancer treatment.

[0171] In some embodiments, what is provided are anti-Ly6 (e.g., anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing T cells of the present disclosure, used for cancer treatment in patients requiring cancer treatment.

[0172] In another embodiment, the present disclosure relies on a method for treating a patient in need of treatment, the method comprising at least one of the following steps: (a) preparing engineered immune cells which can be obtained by any one of the methods described previously; (b) administering the transformed immune cells to the patient.

[0173] In one embodiment, the T cells of the present invention can undergo robust in vivo T cell expansion and can persist for a long period of time. The treatment may be mitigate, curative, or prophylactic. The treatment may be part of either autoimmunotherapy or allogeneic immunotherapy. Autologous means that the cells, cell lines, or cell populations used to treat the patient originate from the patient or a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cells or cell populations used to treat the patient originate from a donor but not from the patient.

[0174] The aforementioned treatment can be used to treat patients diagnosed with pre-malignant or malignant cancer symptoms characterized by excessive anti-Ly6 (e.g., anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing cells, particularly anti-Ly6 (e.g., anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing cells.

[0175] Treatment with engineered immune cells according to the present invention may be combined with one or more therapies against cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, and radiation therapy. Preferably, treatment with engineered immune cells according to the present invention may be administered in combination (e.g., before, concurrently with, or after) one or more therapies against cancer selected from the group consisting of aracytine, cytosine arabinoside, amsacrine, daunorubicin, idarubicin, novantrone, mitoxantrone, vepeside, etoposide (VP16), arsenic trioxide, tretinoin, a combination of arsenic trioxide and tretinoin, mechlorethamine, procarbazine, chlorambucil, and combinations thereof.

[0176] According to a preferred embodiment of the present invention, said treatment can be administered into a patient undergoing immunosuppressive treatment. Indeed, the present disclosure preferably relies on cells or a population of cells that are rendered resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. In this aspect, the immunosuppressive treatment should assist the selection and expansion of T cells according to the present invention within the patient.

[0177] Administration of the cells or cell population according to the present disclosure can be performed in any convenient manner including aerosol inhalation, injection, ingestion, blood transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient via subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular administration, intravenous or intralymphatic injection, or can be administered intraperitoneally. In one embodiment, the cell composition of the present disclosure is preferably administered by intravenous injection.

[0178] Administration of the cells or cell population is 10 4 ~10 9 cells / kg of body weight, preferably 10 5 ~10 6The administration may consist of a number of cells per kg of body weight (including all integer values ​​of cell count within that range). Cells or cell populations may be administered in doses of 1 or more. In another embodiment, the effective amount of cells is administered as a single dose. In another embodiment, the effective amount of cells is administered as multiple doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the patient's clinical condition. Cells or cell populations can be obtained from any source, such as a blood bank or donor. While individual needs vary, determining the optimal range of an effective amount of a given cell type for a particular disease or symptom is within the technical scope of the art. An effective amount means an amount that provides a therapeutic or preventive benefit. The dose administered depends on the recipient's age, health condition, and weight, the type and frequency of any concomitant treatments, and the nature of the desired effect. In another embodiment, the effective amount of cells or a composition containing such cells is administered parenterally. The administration may be intravenous. The administration may be carried out directly by injection into the tumor.

[0179] Treatment with engineered immune cells according to the present invention may be combined with one or more treatments for cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiotherapy. Preferably, treatment with engineered immune cells according to the present invention may be administered (for example, before, simultaneously, or after) one or more treatments for cancer selected from aracin, cytosine arabinoside, amsacrin, daunorubicin, idarubicin, novantrone, mitoxantrone, vepeside, etoposide (VP16), arsenic trioxide, trans retinoic acid, a combination of arsenic trioxide and trans retinoic acid, mechloretamine, procarbazine, chlorambucil, and combinations thereof. [Examples]

[0180] Example 1. Ly6K is a suitable CAR-T cell therapeutic target in triple-negative breast cancer. Ly6K expression was measured in normal breast tissue, breast cancer tissue (Figure 1A), and TNBC and non-TNBC tissue (Figure 1B). As shown in Figure 1, Oncomine data analysis showed (A) increased Ly6K mRNA expression in breast cancer compared to normal breast tissue using the cancer genome atlas dataset, and (B) increased mRNA expression in TNBC vs. non-TNBC cases using the Curtis dataset. In two-sample t-tests, p<0.05 was considered statistical significance.

[0181] Samples were divided into high and low Ly6K mRNA expression groups by using median gene expression levels as a branching point. Survival analysis was performed using survival data and continuous expression variables by fitting a Cox proportional hazards model using the library survival function "coxph". Hazard ratios (HR) and log-rank p-values ​​were obtained from the fitted model. To create prognostic plots, high and low expression categorical variables were used along with survival data. Plots were created using the same R library function "survfit". p<0.05 was considered statistically significant. Figure 1C shows that increased Ly6K expression was significantly associated with poor survival in TNBC (dataset ID: GSE19783), as observed by the prognostic software ProGgene V2.

[0182] Absolute Ly6K mRNA expression was quantified in TNBC subtypes. Relevant GEO datasets from TNBC patients (HGU133 Plus 2.0 array) were normalized using Robust multi-averaging RMA and batch-corrected using combat. Analysis was performed in R using the oligo, sva, and limma packages. n = number of tissue samples. Welch's two-sample t-test was applied to determine p-values, with p<0.05 considered significant (Figure 1D).

[0183] Publicly available data from "The Human Protein Atlas" show the quantification of IHC using affinity-purified NMR in a panel of normal human tissue, validated using the PrEST antigen as the affinity ligand HPA017770(Sigma) antibody for Ly6K. The intensity of the IHC label is shown on the Y axis, and the X axis indicates the name of the organ tested. The inset shows IHC images from normal testes and mammary glands using the Ly6K antibody (Figure 1E). The results demonstrate that Ly6K was expressed in the testes but not in normal mammary gland cells.

[0184] Figure 1F shows the mRNA expression of Ly6D and Ly6E genes in purified immune cells. The absence of Ly6K mRNA expression is indicated by a "-" symbol, and its presence is indicated by a "+" symbol.

[0185] Figure 1G shows high overexpression of Ly6K in various types of cancer, including cervical, breast, head and neck, bladder, esophageal, lung, and colorectal cancers.

[0186] Example 2. Ly6K is required for in vivo tumor growth in a syngeneic breast tumor model. 4T1 cells in the presence or absence of Ly6K (Figures 2A-2B) were transplanted into the submammary fat pad of BALB / c mice by subcutaneous injection (10 mice per group). Tumor measurements were performed using a Vernier caliper. Tumor volume was calculated using the formula 1 / 2 × length × width 2 ) was used for the calculation. ** This indicates p<0.001. Two-tailed Student's t-test. (Figure 2C).

[0187] Example 3. Preparation of Ly6K mouse monoclonal antibody (mAb) Ly6K mAbs were synthesized from the extracellular fraction of human Ly6K (hLy6K) using the NCI Reagent Development Project. Sequences associated with the extracellular morphology of the protein (shown in Figure 3A illustrating the structure of Ly6K; sequence numbers 86-89 below) were cloned into the pET24.1 HIS-tagged vector. The protein was expressed in E. coli (BL21DE3) cells and purified using a His-tagged purification kit (producing Ni-NTA beads) to obtain a single product of expected size (17 kDa) in lane 10 (Figure 3B). The protein was confirmed to be Ly6K by MALDI / TOF analysis. This protein was used for immunization to generate several hybridoma cell lines. Supernatants from these clones were screened by ELISA and Western blotting to identify antibody-producing clones. This process yielded three high-affinity Ly6K mAbs, as validated by the ELISA assay as shown in Figure 3C. For the ELISA, a microtiter plate was coated overnight with 100 μl of recombinant Ly6K protein. Dilutions of the antibodies shown were added for 30 minutes. The bound antibodies were detected using TMB substrate.

[0188] hLy6K-MF-C-HIS-noGPI-pcDNA3.1(Q17RY6)(Sequence ID 86) [ka]

[0189] hLy6E-MF-C-HIS-noGPI-pcDNA3.1(Q16553)(Sequence ID 87) [ka]

[0190] hLy6K-MF-C-HIS-noGPI-pcDNA3.1(Q17RY6)(Sequence ID 88) [ka]

[0191] hLy6H-MF-C-HIS-noGPI-pcDNA3.1(O94772)(Sequence ID 89) [ka]

[0192] Ten clones of Ly6 mAbs targeting the aforementioned Ly6K, Ly6D, or Ly6E proteins were obtained as Ly6K-2 / 1(5G1 / 2H5), Ly6K-3(6E1), Ly6K-4(3A12), Ly6D-1(10E7), Ly6D-2(11A3), Ly6D-3(11F4), Ly6D-4(1E1), Ly6E-1(1C11), Ly6E-2(6E2), and Ly6E-3(8H8). Of these, Ly6D-2(11A3) each possesses two different VLs paired with the same VH. [Table 3-1] [Table 3-2]

[0193] Example 4. Ly6K mAb recognizes endogenously expressed Ly6K. To test whether antibodies produced from bacterial-expressed peptides recognize endogenously expressed Ly6K protein on the cell surface, anti-human Ly6K antibody 6E1 (Ly6K-3) was used in IHC studies on paraffin-fixed sections of human testicular tissue. Slides were subjected to serial hydration, permeabilization, and acid antigen retrieval. Primary mAb (1:100, overnight, 4°C). Secondary goat anti-mouse Alexa 568 1:500, 1 hour, RT. Confocal images were obtained for normal human testes using a Zeiss LSM700 with a 40x oil immersion objective lens (Figure 4A). As shown in Figure 4A, the antibody recognized Ly6K on basal spermatids in human testes. TNBC clinical sample slides were scanned at 40x magnification for Ly6K staining and corresponding H&E staining using Axio Scan (Figure 4B). As shown in Figure 4B, clinical TNBC samples (n=3) were stained with CPTC-Ly6K-3 to show membrane / cytoplasmic staining. Similar results were achieved for testicular and clinical TNBC samples with the two other anti-Ly6K mAbs mentioned earlier.

[0194] To investigate whether Ly6K mAbs recognize cell surface Ly6K, unfixed living TNBC cells were stained with mAb Ly6K in flow cytometry analysis. Flow of living unfixed cells was measured, and cell surface Ly6K was analyzed with novel mAbs and secondary goat anti-mouse Alexa 488 (Figure 4C). As shown in Figure 4C, the antibodies recognized the cell surface Ly6K protein in the following human and mouse cancer cell lines: TNBC cell lines BT549, MDA-MB-231, and cervical cancer cell line Caski, as well as mouse syngeneic tumor lines 4T1, B16, and E0771.

[0195] Example 5. Production of Ly6K mouse chimeric antigen receptor (CAR) T cells were enriched from PBMCs via negative selection and activated using CTS Dynabeads CD3 / CD28. Using the MSGV retroviral vector, the scFv of the 6E1 Ly6K mAb sequence (CPCT-Ly6K-3) was cloned from the polypeptide sequence of SEQ ID NO: 94. The retrovirus was generated using Novus' pCL-Ampho Retrovirus Packaging Vector. The retrovirus was then transduced into enriched and activated T cells. Ly6K CAR T cells were then cultured using CTS Optimizer Pro basal + Supplement + IL2 + Glutamax + P / S + No additional Serum. Transduced CAR T cells expressed the scFV of the Ly6K antibody. CAR expression was detected using protein L, which is recognized by flow cytometry. T cells were isolated from PBMCs and transduced with the retroviral particle CAR construct. Cells were subjected to protein L staining. Protein L can recognize the kappa chain of scFVs in CD19 scFVs and LY6K scFVs. Over 30 million CAR T cells were cryopreserved. Ly6K CAR T cells exhibited very good transduction (over 40% transduction), comparable to CD19 CAR T cells, as shown in Figure 5.

[0196] Example 6. In vitro CTV-based cytotoxicity assay 5 x 10 4 HeLa tumor cell lines were stained with 5 μM cell trace violet and co-cultured in 96-well plates with UT (untransduced T cells) or Ly6K CAR T cells in 1:1 and 1:3 ratios (Figure 6A). CTV-positive residual viable cells were quantified by flow cytometry, and here, the results are represented by an overlay histogram of the CTV dilution quantification of Ly6K CAR T cells that induced cytotoxicity in HeLa tumor cells (Figure 6B). The p-value was determined by Bonferroni test using two-way ANOVA.

[0197] CAR T cells were tested for cytotoxicity against triple-negative breast cancer cells (MDA-MB-231). MDA-MB-231 cells were stained with cell titer blue (CTV) and named "CTV+". Ly6K-negative pancreatic cancer cells were left unstained and named "CTV-". These cells were mixed in a 1:1 ratio, and both cell populations were detected as two peaks marked as CTV+ and CTV- in a flow cytometry histogram (Figure 7A). When the cells were cultured together with T cells that do not express the CAR T protein (untransduced T cells), both cell populations could be observed as two peaks in the histogram (Figure 7B). When the cells were cultured in the presence of anti-LY6K-CAR-T cells, the LY6K-positive cancer cell population (CTV+) disappeared, and only the Ly6K-negative cancer cell population (CTV-) was observed (Figure 7C). T cells were tested in multiple cell lines, and non-transduced versus transduced CAR T cells were compared at various effector cell-to-target cell (E:T) ratios to demonstrate their efficacy in triple-negative breast cancer cells MDA-MB-231 (Figure 7D) and BT549 (Figure 7E).

[0198] Example 7. In vivo antitumor activity of Ly6K CAR T cells in a xenograft model of the HS378t TNBC cell line. The in vivo antitumor activity of Ly6K CAR T cells was tested against an HS378t triple-negative breast cancer (TNBC) tumor xenograft model. 0.5 × 10⁻⁶ 6 Individual HS378t tumor cells were subcutaneously (SC) injected near the memory fat pad, and tumor volume was measured using a Vernier caliper. Tumor volume was measured up to day 42 and day 48, respectively (Figures 8A-8B). The arrow marks indicate CAR T or UT (1×10). 6Individual cell injection (IV) was shown. P-values ​​were determined by Tukey's test using two-way repeated measures ANOVA. Figure 8C shows images of resected tumors from the UT (top) and CAR T (bottom) groups. Figures 8D–8E show measured tumor volume and tumor weight of the resected tumors. P-values ​​were determined by the Mann-Whitney unpaired Student's t-test. n=3 or 4.

[0199] Example 8. Activity of CAR Various effector cell-to-target cell ratios were tested using various Ly6K CAR T cells. The cell lysis percentage increased with increasing E:T ratio (Figures 9A-9B).

[0200] Example 9. Ly6D-specific antibody Four monoclonal anti-Ly6D antibodies (Ly6D-1, Ly6D-2, Ly6D-3, Ly6D-4) were prepared as shown in Table 1. For the SPR assay, the Ly6D monoclonal antibodies were captured on the surface of a CM5 chip by protein A / G immobilization. Purified human Ly6D protein was dose-dependently tested for binding to the human Ly6D monoclonal antibodies. As shown in Figures 10A-D, the SPR assay demonstrated that all four antibodies recognized recombinant human Ly6D protein with high affinity.

[0201] Of the four monoclonal anti-Ly6D antibodies in Example 9, three (Ly6D-1, Ly6D-2, and Ly6D-3) showed inhibition of pancreatic cancer cell growth. These monoclonal anti-Ly6D antibodies directly inhibited tumor cell growth in Capan-1 cells with IC50 ranging from 0.6 mg / ml to 1.0 mg / ml (Figure 11A-C).

[0202] Antibody-dependent cell-mediated cytotoxicity (ADCC) assays were performed on LY6D-1, LY6D-2, and LY6D-3. Target cancer cells (T) were incubated with effector PBMC cells (E) at various E:T ratios of 40:1, 20:1, 1:1, and 0:1. Co-cultured cells were labeled with IncuCyte® caspase 3 / 7 dye and imaged using the IncuCyte® S3 live cell analysis system to distinguish real-time caspase activation in cancer cells and PBMCs. Cancer cell-specific caspase activation was calculated using an automated image analysis program, and the data showed that the anti-LY6D antibody effectively produced ADCC in the presence of PBMC cells. For the ADCC assay, 0.25 μg / ml of antibody was used. While this dose may not be efficient in inducing cell death in cancer cells alone, in the presence of PBMCs, it effectively eliminated cancer cells as shown in Figures 11D-F. This demonstrates the ADCC activity of the anti-LY6D antibody. * * *

[0203] This disclosure should not be limited in scope by any specific embodiment described, which is intended as a single example of any aspect of this disclosure, and any functionally equivalent composition or method falls within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations of this disclosure insofar as they fall within the scope of the appended claims and their equivalents.

[0204] All publications and patent applications referenced herein are incorporated by reference as if each individual publication or patent application were specifically and individually incorporated by reference. [Table 7-1] [Table 7-2] Table 7-3 Table 7-4

Claims

1. An antibody or its antigen-binding fragment having specificity for cell surface lymphocyte antigen 6 family member K protein (Ly6K), wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, HCDR3 containing the amino acid sequence of SEQ ID NO: 3, LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6; (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 9, HCDR2 containing the amino acid sequence of SEQ ID NO: 10, HCDR3 containing the amino acid sequence of SEQ ID NO: 11, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence of SEQ ID NO: 13, and LCDR3 containing the amino acid sequence of SEQ ID NO: 14; or (c) HCDR1 containing the amino acid sequence of SEQ ID NO: 17, HCDR2 containing the amino acid sequence of SEQ ID NO: 18, HCDR3 containing the amino acid sequence of SEQ ID NO: 19, LCDR1 containing the amino acid sequence of SEQ ID NO: 20, LCDR2 containing the amino acid sequence of SEQ ID NO: 21, and LCDR3 containing the amino acid sequence of SEQ ID NO: 22 An antibody or its antigen-binding fragment containing the sequence of an antibody.

2. The antibody or antigen-binding fragment according to claim 1, wherein 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 with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15, and 23.

3. The antibody or antigen-binding fragment according to claim 1 or 2, wherein 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 with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, and 24.

4. (a) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 8; (b) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 15, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 16; or (c) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 23, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

24. The antibody or antigen-binding fragment according to any one of claims 1 to 3.

5. An antibody or its antigen-binding fragment having specificity for cell surface lymphocyte antigen 6 family member D protein (Ly6D), wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 25, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 27, LCDR1 containing the amino acid sequence of SEQ ID NO: 28, LCDR2 containing the amino acid sequence of SEQ ID NO: 29, and LCDR3 containing the amino acid sequence of SEQ ID NO: 30; (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 33, HCDR2 containing the amino acid sequence of SEQ ID NO: 34, HCDR3 containing the amino acid sequence of SEQ ID NO: 35, LCDR1 containing the amino acid sequence of SEQ ID NO: 39 or 48, LCDR2 containing the amino acid sequence of SEQ ID NO: 40 or 49, and LCDR3 containing the amino acid sequence of SEQ ID NO: 41 or 50; (c) HCDR1 containing the amino acid sequence of SEQ ID NO: 45, HCDR2 containing the amino acid sequence of SEQ ID NO: 46, HCDR3 containing the amino acid sequence of SEQ ID NO: 47, LCDR1 containing the amino acid sequence of SEQ ID NO: 48, LCDR2 containing the amino acid sequence of SEQ ID NO: 49, and LCDR3 containing the amino acid sequence of SEQ ID NO: 50; or (d) HCDR1 containing the amino acid sequence of SEQ ID NO: 53, HCDR2 containing the amino acid sequence of SEQ ID NO: 54, HCDR3 containing the amino acid sequence of SEQ ID NO: 55, LCDR1 containing the amino acid sequence of SEQ ID NO: 56, LCDR2 containing the amino acid sequence of SEQ ID NO: 57, and LCDR3 containing the amino acid sequence of SEQ ID NO: 58 An antibody or its antigen-binding fragment containing the sequence of an antibody.

6. The antibody or antigen-binding fragment according to claim 5, wherein 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 with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 42, 51, and 59.

7. The antibody or antigen-binding fragment according to claim 5 or 6, wherein 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 with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 44, 52, and 60.

8. (a) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 31, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 32; (b) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 42, the first light chain variable region includes the amino acid sequence of SEQ ID NO: 52, and the second light chain variable region includes the amino acid sequence of SEQ ID NO: 44; (c) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 51, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 52; or (d) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 59, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

60. The antibody or antigen-binding fragment according to any one of claims 5 to 7.

9. An antibody or its antigen-binding fragment having specificity for cell surface lymphocyte antigen 6 family member E protein (Ly6E), wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region including heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region including light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 61, HCDR2 containing the amino acid sequence of SEQ ID NO: 62, HCDR3 containing the amino acid sequence of SEQ ID NO: 63, LCDR1 containing the amino acid sequence of SEQ ID NO: 64, LCDR2 containing the amino acid sequence of SEQ ID NO: 65, and LCDR3 containing the amino acid sequence of SEQ ID NO: 66; (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 69, HCDR2 containing the amino acid sequence of SEQ ID NO: 70, HCDR3 containing the amino acid sequence of SEQ ID NO: 71, LCDR1 containing the amino acid sequence of SEQ ID NO: 72, LCDR2 containing the amino acid sequence of SEQ ID NO: 73, and LCDR3 containing the amino acid sequence of SEQ ID NO: 74; or (c) HCDR1 containing the amino acid sequence of SEQ ID NO: 77, HCDR2 containing the amino acid sequence of SEQ ID NO: 78, HCDR3 containing the amino acid sequence of SEQ ID NO: 79, LCDR1 containing the amino acid sequence of SEQ ID NO: 80, LCDR2 containing the amino acid sequence of SEQ ID NO: 81, and LCDR3 containing the amino acid sequence of SEQ ID NO: 82 An antibody or its antigen-binding fragment containing the sequence of an antibody.

10. The antibody or antigen-binding fragment according to claim 9, wherein 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 with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 75, and 83.

11. The antibody or antigen-binding fragment according to claim 9 or 10, wherein 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 with respect to an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 76, and 84.

12. (a) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 67, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 68; (b) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 75, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 76; or (c) The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 83, and the light chain variable region includes the amino acid sequence of SEQ ID NO:

84. The antibody or antigen-binding fragment according to any one of claims 9 to 11.

13. An antibody or antigen-binding fragment according to any one of claims 1 to 12, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

14. The antibody or antigen-binding fragment according to claim 13, wherein the light chain constant region is a kappa or lambda chain constant region.

15. The antibody or antigen-binding fragment according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is an isotype of IgG, IgM, IgA, IgE, or IgD.

16. The antibody or antigen-binding fragment according to claim 15, wherein the isotype is IgG1, IgG2, IgG3, or IgG4.

17. A chimeric antigen receptor comprising an antibody or antigen-binding fragment according to any one of claims 1 to 12.

18. The chimeric antigen receptor according to claim 17, further comprising one or more co-stimulatory domains and an activation domain.

19. The chimeric antigen receptor according to claim 18, wherein the one or more costimulatory domains include one or more costimulatory signaling regions derived from the group consisting of 4-1BB / CD137, the alpha chain of the T cell receptor, the beta chain of the 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, CD154, NKG2D, inducible T cell costimulator (ICOS), CD247, Ig alpha (CD79a), Fc gamma receptor, or the zeta chain of the T cell receptor.

20. The chimeric antigen receptor according to claim 18, wherein the one or more co-stimulatory domains include one or more intracellular signaling regions of CD28, OX-40, and / or 4-1BB.

21. The chimeric antigen receptor according to claim 19 or 20, wherein the costimulatory domain further comprises a transmembrane domain and optionally a spacer domain.

22. The chimeric antigen receptor according to claim 21, wherein the transmembrane domain comprises a transmembrane domain selected from the group consisting of 4-1BB / CD137, the alpha chain of the T cell receptor, the beta chain of the 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 the zeta chain of the T cell receptor, or any combination thereof.

23. The chimeric antigen receptor according to claim 22, wherein the transmembrane domain comprises the transmembrane domain of CD28.

24. The chimeric antigen receptor according to claim 21, wherein the spacer domain comprises a hinge region selected from the group consisting of hinge regions of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha.

25. The chimeric antigen receptor according to claim 18, wherein the activating domain comprises a cytoplasmic region selected from the group consisting of CD3 zeta, CD3 epsilon, CD3 delta, and CD3 gamma.

26. The chimeric antigen receptor according to claim 25, wherein the activating domain is mutated to inhibit cell apoptosis.

27. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, or an antibody or antigen-binding fragment thereof that competes with a chimeric antigen receptor according to any one of claims 17 to 26.

28. A bifunctional molecule comprising a first antigen-binding portion and a second portion having specificity for a second protein, wherein the first antigen-binding portion comprises an antibody or antigen-binding fragment according to any one of claims 1 to 16.

29. An antibody conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 and 27, or a bifunctional molecule according to claim 28, and a conjugate.

30. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, a bifunctional molecule according to claim 28, or any combination thereof, and a pharmaceutically acceptable carrier.

31. An isolated cell comprising one or more polynucleotides encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, or a bifunctional molecule according to claim 28.

32. Engineered immune cells expressing the chimeric antigen receptor described in any one of claims 17 to 26 on their cell surface membrane.

33. A polynucleotide encoding one or more chains of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, a chimeric antigen receptor according to any one of claims 17 to 26, or a bifunctional molecule according to claim 28.

34. A method of manipulating immune cells: (a) preparing immune cells, (b) expressing at least one chimeric antigen receptor according to any one of claims 17 to 26 on the surface of the cell Methods that include...

35. A method of manipulating immune cells: (a) preparing immune cells, (b) Introducing into the cells at least one polynucleotide encoding a chimeric antigen receptor according to any one of claims 17 to 26, (c) Expressing the polynucleotide in the immune cells Methods that include...

36. A method for treating cancer in a patient requiring treatment for cancer, comprising administering to the patient an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, or any combination thereof, a bifunctional molecule according to claim 28, or manipulated immune cells according to claim 32.

37. The method according to claim 36, wherein 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.

38. The method according to claim 37, wherein the breast cancer is triple-negative breast cancer.

39. The method according to claim 37, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

39. The method according to any one of claims 36 to 38, further comprising administering to the patient a therapy for treating the cancer.

40. The method according to claim 39, wherein the therapy is selected from the group consisting of immunotherapy, chemotherapy, and radiotherapy.

41. A method for detecting the expression of Ly6 in a sample, comprising: contacting the sample with an antibody or antigen-binding fragment according to any one of claims 1 to 16, or a bifunctional molecule according to claim 28, under conditions in which the antibody or antigen-binding fragment binds to Ly6; and detecting a binding that indicates the expression of Ly6 in the sample.

42. A method for treating breast cancer, comprising administering to the patient engineered immune cells that express an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a chimeric antigen receptor containing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, on their cell surface membrane.

43. The method according to claim 42, wherein the breast cancer is triple-negative breast cancer.

44. A method for treating pancreatic cancer, comprising administering to the patient engineered immune cells that express an antibody or antigen-binding fragment thereof according to any one of claims 5 to 8, or a chimeric antigen receptor containing the antibody or antigen-binding fragment thereof according to any one of claims 5 to 8, on their cell surface membrane.

45. The method according to claim 44, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

Images