WO2025049995A1 - Anti-ly6 antibodies and uses thereof - Google Patents

Abstract

Provided are antibodies or fragments thereof having binding specificity to the Ly6 protein. In various examples, the antibodies or fragments thereof include a VH and VL CDRs as disclosed herein, or variants thereof. Also provided herein is a chimeric antigen receptor comprising the antibodies or fragments thereof binding to the Ly6 protein. Methods of using the antibodies or fragments thereof, or the Ly6 CAR-expressing immune cell for treating cancer are also provided.

Description

ANTI-LY6 ANTIBODIES AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63/536,341, filed September 1, 2023, the content of which is hereby incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] This invention was made with government support under grant number 1 R01CA227694 awarded by the National Institutes of Health and under grant number R21CA256424 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] 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 proteins are cysteine-rich proteins characterized by a distinct disulfide bridge pattern that creates the three-finger Ly6/uPAR (LU) domain. With the encoding gene located on human chromosomes 8, the RNA levels of the Ly6 family proteins Ly6D, Ly6E, Ly6K and Ly6H are high compared to adjacent normal tissues in a multitude of tumors including ovarian, colorectal, gastric, breast, lung, bladder, brain and CNS, cervical, esophageal, head and neck, and pancreatic cancer. The increased expression of Ly6D, Ly6E, Ly6K and Ly6H was associated with poor survival in ovarian, colorectal, gastric, breast, and lung cancer. It is reported that Ly6 proteins play important role in TGF-P signaling, AKT pathways and immune regulation. A cumulative effect of Ly6 downstream pathways may lead to increased aggressiveness of cancer cells and leading to the poor survival outcome.

[0004] The tumor- specific expression of Ly6 proteins indicates that the Ly6 is a potential therapeutic target for cancer immunotherapy. SUMMARY

[0005] The present disclosure provides antibodies or fragments thereof having binding specificity to the human Ly6 proteins, in particular, human Ly6D, Ly6E, Ly6K, or Ly6H proteins, respectively. Blockage of Ly6 using the antibodies or antigen-binding fragment thereof, or the Ly6 (for example, Ly6D, Ly6E, Ly6K, or Ly6H) CAR-expressing immune cell can induce T cell activation and stimulate cellular immunity in the body. These antibodies and fragments thereof bind the Ly6 proteins on the cell surface and neutralize their function, therefore are useful in the treatment of diseases and conditions such as cancers. The Ly6 CAR-expressing immune cells are capable of killing cancer cells expressing Ly6 without the need for HL A matching.

[0006] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof having specificity to a cell-surface Lymphocyte Antigen 6 Family Member K protein (Ly6K), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprise the sequences of: (a) HCDR1: SYNIH (SEQ ID NO: 1), HCDR2: AIYPGNGDTSYNQKFKD (SEQ ID NO: 2), HCDR3: GGYPFIY (SEQ ID NO: 3), LCDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 4), LCDR2: KVSNRFS (SEQ ID NO: 5), and LCDR3: 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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15 and 23, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 15 and 23. In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16 and 24 or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16 and 24. In some embodiments, (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; (b) the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16; or (c) the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24.

[0008] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof having specificity to a cell-surface Lymphocyte Antigen 6 Family Member D protein (Ly6D), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprise the sequences of:

(a) HCDR1: DYIMH (SEQ ID NO: 25), HCDR2: YIQPNNGDNTYNQKFKG (SEQ ID NO: 26), HCDR3: TNWDGAMDY (SEQ ID NO: 27), LCDR1: RASQNIYDYLH (SEQ ID NO: 28), LCDR2: YASQSIS (SEQ ID NO: 29), and LCDR3: QSGHSFPYT (SEQ ID NO: 30);

(b) HCDR1: EYPIH (SEQ ID NO: 33), HCDR2: MIYTDTGESTYAEEFKG (SEQ ID NO:34), HCDR3: DYYYSCPLAY (SEQ ID NO: 35), LCDR1: RASQDISNYLN (SEQ ID NO: 39) or RASDNIHNFLT (SEQ ID NO: 48), LCDR2: YTSRLYS (SEQ ID NO: 40) or NAKTLAD (SEQ ID NO: 49), and LCDR3: QQCNTLPWT (SEQ ID NO: 41) or QHFWSIPWT (SEQ ID NO: 50); (c) HCDR1: EYPIH (SEQ ID NO: 45), HCDR2: MIYTDTGESTYAEEFKG (SEQ ID NO:46), HCDR3: DYYYSCPLAY (SEQ ID NO: 47), LCDR1 : RASDNIHNFLT (SEQ ID NO: 48), LCDR2: NAKTLAD (SEQ ID NO: 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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 42, 51 and 59, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 42, 51 and 59. In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 44, 52 and 60, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 44, 52 and 60. In some embodiments, (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32; (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, the first light chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and a second light chain variable region comprising the amino acid sequence of SEQ ID NO: 44; (c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 52; or (d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60.

[0010] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof having specificity to a cell-surface Lymphocyte Antigen 6 Family Member E protein (Ly6E), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprise the sequences of: (a) HCDR1: TFWMH (SEQ ID NO: 61), HCDR2: NINPNNGGTNYNEKFKK (SEQ ID NO: 62), HCDR3: TAY (SEQ ID NO: 63), LCDR1: SGSSSGSYMH (SEQ ID NO: 64), LCDR2: ETSKLAS (SEQ ID NO: 65), and LCDR3: QQWSRNPPTLT (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 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 75 and 83, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 75 and 83. In some embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 76 and 84 or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 76 and 84. In some embodiments, (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 67, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68; (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 75, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 76; or (c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 83, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the antibody or antigen-binding fragment thereof further comprises

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

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

[0014] In one aspect, the present disclosure provides a chimeric antigen receptor, comprising the antibody, or antigen binding fragment thereof of 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 comprise one or more costimulatory signaling regions derived from the group consisting of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, 2B4, CD3 gamma, CD3 delta, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD27, CD28, CD28T, OX-40, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD 154, NKG2D, inducible T cell costimulator (1COS), CD247, 1g alpha (CD79a), Fc gamma receptor or a zeta chain of a T cell receptor. The one or more costimulatory domains may comprise one or more intracellular signaling regions of CD28, OX-40 and/or 4- IBB. In some embodiments, the costimulatory domain further comprises a transmembrane domain and optionally, a spacer domain. The transmembrane domain may comprises a transmembrane domain selected from the group consisting of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, 2B4, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, NKG2D, or a zeta chain of a T cell receptor, or any combination thereof. The spacer domain may comprise a hinge region selected from the group consisting of the hinge region of IgGl, lgG2, lgG3, lgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha. In some embodiments, the activation domain comprises a cytoplasmic region selected from the group consisting of CD3 zeta, CD3 epsilon, CD3 delta, and CD3 gamma. The activation domain may be mutated to prevent cell apoptosis.

[0016] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof competing with the antibody or antigen-binding fragment thereof or the chimeric antigen receptor of preceding paragraphs.

[0017] In one aspect, the present disclosure provides a bifunctional molecule, comprising a first antigen-binding portion and a second portion having specificity to a second protein, wherein the first antigen-binding portion comprises an antibody or antigen-binding fragment thereof of preceding paragraphs.

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

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

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

[0021] In one aspect, the present disclosure provides an engineered immune cell expressing at the cell surface membrane a chimeric antigen receptor according to the preceding paragraphs. [0022] In one aspect, the present disclosure provides a polynucleotide encoding one or more chains of the antibody or antigen-binding fragment thereof of the preceding paragraphs, the chimeric antigen receptor of any one of the preceding paragraphs or the bifunctional molecule of the preceding paragraphs.

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

[0024] In one aspect, the present disclosure provides a method of engineering an immune cell, comprising: (a) providing an immune cell, (b) introducing into said cell at least one polynucleotide encoding said chimeric antigen receptor of the preceding paragraphs, and (c) expressing said polynucleotide into said immune cell.

[0025] In one aspect, the present disclosure provides a method of treating a cancer in a patient in need thereof, comprising administering to the patient the antibody or antigenbinding fragment thereof of any one of the preceding paragraphs or any combination thereof, the bifunctional molecule of the preceding paragraphs, or the engineered immune cell of the preceding paragraphs.

[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 comprise administering to the patient a therapy for treating said cancer. Said therapy may be selected from the group consisting of immunotherapy, chemotherapy and radiotherapy.

[0027] In one aspect, the present disclosure provides a method of detecting expression of Ly6 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-16 or the bifunctional molecule of claim 28 under conditions for the antibody or antigen-binding fragment thereof to bind to the Ly6, and detecting the binding which indicates expression of Ly6 in the sample. [0028] In one aspect, the present disclosure provides a method of treating a breast cancer comprising administering to the patient the antibody or antigen-binding fragment thereof of the preceding paragraphs or an engineered immune cell expressing at the cell surface membrane a chimeric antigen receptor comprising the antibody, or antigen binding fragment thereof of the preceding paragraphs.

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

[0030] In one aspect, the present disclosure provides a method of treating a pancreatic cancer comprising administering to the patient the antibody or antigen-binding fragment thereof of the preceding paragraphs or an engineered immune cell expressing at the cell surface membrane a chimeric antigen receptor comprising the antibody, or antigen binding fragment thereof of the preceding paragraphs.

[0031] In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1A illustrates Ly6K expression in normal breast and breast cancer tissues.

[0033] FIG. IB illustrates Ly6K expression in triple negative breast cancer (TNBC) and non- TNBC tissues.

[0034] FIG. 1C is a graph showing survival rate for TNBC with high and low Ly6K expression.

[0035] FIG. ID is a graph showing Ly6K mRNA expression rate in TNBC subtypes.

[0036] FIG. IE is a graph showing quantitative protein expression of Ly6K in normal tissues.

[0037] FIG. IF is a diagram showing expression of Ly6D, Ly6E, Ly6K in immune cells.

[0038] FIG. 1G is a plot showing over-expression of Ly6K in cancer cells.

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

[0040] FIG. 2B is a graph showing tumor volume growth of 4T 1 cells with or without Ly6K. [0041] FIG. 3A is a diagram showing the structure of the Ly6K protein.

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

[0043] FIG. 3C is graphs showing binding of three anti-Ly6K antibodies to the human Ly6K protein.

[0044] FIG. 4A shows binding of the anti-human Ly6K antibody to paraffin fixed testis tissues in IHC studies.

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

[0046] FIG. 4C is graphs showing flow cytometry results showing recognition of Ly6K protein in various cancer cell lines by a Ly6K antibody.

[0047] FIG. 5 illustrates transduction of Ly6K CAR compared to CD 19 CAR.

[0048] FIG. 6A is a graph showing staining of HeLa tumor cell with cell trace violet and coculturing with untransduced T cell or Ly6K CAR T-cells in various ratios.

[0049] FIG. 6B is a graph showing anti-tumor activity of Ly6K CAR T-cells and untransduced T cell against HeLa tumor cell line.

[0050] FIG. 7A is a graph showing cell counts of MDA-MB-231 cells and pancreatic cancer cells, after cultured with no T-cell.

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

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

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

[0054] FIG. 7E is a graph showing cytotoxicity of Ly6K CAR T-cells and untransduced T- cell against BT549 cell line at different effectortarget (E:T) ratios. [0055] FIGS. 8A-B are graphs showing tumor volume of HS378t TNBC cell line xenograft model over time with in vivo administration with Ly6K CAR T-cells and untransduced T- cells.

[0056] FIG. 8C is a photograph of excised tumor of HS378t TNBC cell line xenograft model over time with in vivo administration with Ly6K CAR T-cells and untransduced T-cells.

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

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

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

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

[0061] FIGS. 10A-D are graphs showing binding of an anti-hLy6D antibody to human Ly6D protein.

[0062] FIGS. 11A-C are graphs showing cell viability of pancreatic cancer cells upon contacting anti-Ly6D antibodies.

[0063] FIGS. 11D-F are graphs showing immune cell specific cell death upon contacting with PBMC and an anti-Ly6D antibody.

DETAILED DESCRIPTION

Definitions

[0064] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. [0065] As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus, the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

[0066] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, 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] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.

[0068] The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (y, p, a, S, e) with some subclasses among them (e.g. , y 1- y4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g. , IgGi, IgG₂, IgGs, IgG₄, IgGs, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.

[0069] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab’ and F(ab’)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0070] Light chains are classified as either kappa or lambda (K, X). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[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 appreciated that the variable domains of both the light (VK) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino- terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CK domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

[0072] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VK chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g. , Hamers-Casterman et al., Nature 363:446-448 (1993).

[0073] In naturally occurring antibodies, the six “complementarity determining regions” or “CDRs” present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, referred to as “framework” regions, show less inter-molecular variability. The framework regions largely adopt a p-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the [3 -sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non- covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)). [0074] In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia etal., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0075] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0076] In addition to table above, the Kabat number 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), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR- H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty third residue after the end of CDR-L2 (i.e., following a cysteine residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid.

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

[0078] As used herein, the term “heavy chain constant region” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of: a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the disclosure may comprise a polypeptide chain comprising a CHI domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHI domain and a CH3 domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain. Further, an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g. , all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.

[0079] The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain constant region of a polypeptide may comprise a CHI domain derived from an IgGi molecule and a hinge region derived from an IgGi molecule. In another example, a heavy chain constant region can comprise a hinge region derived, in part, from an IgGi molecule and, in part, from an IgGs molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGi molecule and, in part, from an IgG4 molecule.

[0080] As used herein, the term “light chain constant region” includes amino acid sequences derived from antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.

[0081] A “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CHI domain of the heavy chain.

[0082] As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CHI domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CHI domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0083] As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.

[0084] As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux el al., J. Immunol 161:4083 (1998)). [0085] As used herein the term “disulfide bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CHI and CK regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

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

[0087] By “specifically binds” or “has specificity to,” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”

[0088] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0089] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

[0090] As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.

Anti-Ly6 Antibodies

[0091] The present disclosure provides antibodies, including antibodies or antigen-binding fragments thereof, that have binding specificity to the human Ly6 protein, in particular, human Ly6D, Ly6K, Ly6E, Ly6H proteins, respectively. As demonstrated in the experimental examples, numerous anti-human Ly6D, Ly6K, Ly6E, or Ly6H antibodies were separately obtained, having high binding affinity to the human Ly6D, Ly6K, Ly6E, or Ly6H proteins, respectively.

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

[0093] Additional functional studies showed that these antibodies or antigen-binding fragments thereof efficiently bind to broad cancer cells with potent activity. These antibodies or their derivatives significantly suppress tumor growth.

[0094] In accordance with one embodiment of the present disclosure, provided are antibodies or antigen-binding fragments thereof that include the heavy chain and light chain variable domains with the CDR regions of the antibodies prepared in the experimental examples. The CDRs are summarized in Table 1 below (Kabat numbering).

Table 1. CDR sequences of the anti-Ly6 antibody clones

[0095] In some embodiments, the VH CDR1, CDR2, and CDR3 are selected from any set of

VH CDR1, CDR2, and CDR3 shown in Table 1, and the VL CDR1, CDR2, and CDR3 are selected from any set of VL CDR1, CDR2, and CDR3 shown in Table 1. In some embodiments, the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 are selected from those derived from the same antibody in the examples.

[0096] In some embodiments, at least one, or two, or three, or four, or five, or six of the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 of the above are modified by one, two or three amino acid additions, deletions, substitutions, or the combinations thereof.

[0097] According to specific embodiments, the antibody is a humanized antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a 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, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0099] Antibodies may be produced by a process of affinity maturation in which a modified antibody is generated that has an improvement in the affinity of the antibody for antigen, compared to an unmodified parent antibody. Affinity-matured antibodies may be produced by procedures known in the art, e.g., 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] Antibodies may also be produced using immunogenic peptides in animals. In certain embodiments, Ly6K-4 antibody was produced using the peptides of the sequence ERPKPEEKRFLLEEP (SEQ ID NO: 90) and SMGESCGGLWAILLL (SEQ ID NO: 91). In certain embodiments, Ly6D-4 antibody was produced using the peptides of the sequence QVSSGTSSTQCCQED (SEQ ID NO: 92) or APTRTALAHSALSLG (SEQ ID NO: 93).

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

[0102] The CDRs, heavy chain variable regions and light chain variable regions of the present 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 is still capable of binding to Ly6K, Ly6D, Ly6E or Ly6H, respectively.

[0103] In certain embodiments, the antibody or antigen-binding fragment thereof comprises two pairs of identical VH/VL, such as those listed in Table 2. In certain embodiments, the antibody or antigen-binding fragment thereof comprises two different light chains and two identical heavy chains, thus forming a pair of VH/VL 1 and a pair of VH/VL2. For example, the antibody or antigen-binding fragment thereof provided herein may comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42, or a variant having at least 90%, at least 95% or at least 98% identity thereof, a first light chain variable region comprising the amino acid sequence of SEQ ID NO: 52 or a variant having at least 90%, at least 95% or at least 98% identity thereof, and a second light chain variable region comprising the amino acid sequence of SEQ ID NO: 44 or a variant having at least 90%, at least 95% or at least 98% identity thereof, such as the anti Ly6D antibody Ly6D-2 (11 A3) in Table 2.

[0104] In some embodiments, the modification is substitution at no more than one hot spot position from each of the CDRs. In some embodiments, the modification is substitution at one, two or three such hot spot positions. In one embodiment, the modification is substitution at one of the hot spot positions. Such substitutions, in some embodiments, are conservative substitutions.

[0105] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar 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). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.

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

Amino Acid Similarity Matrix

Conservative Amino Acid Substitutions

[0107] It will also be understood by one of ordinary skill in the art that antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

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

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

Table 2

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

[0111] The Fc region can be engineered to enhance or eliminate effector function. IgG antibodies can induce direct anti-tumor effects by way of indirect anti-tumor effects via the Fc-mediated effector functions that engage other immune cells or killer mechanisms. “Effector functions” or “antibody effector functions” as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as Cl complex and Fc receptor(FcyRIIa or FcyRIIIa). Exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and Clq on the Cl complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and antibody dependent cell mediated phagocytosis (ADCP) , where nonspecific cytotoxic cells that express Fey receptors (FcyRs) recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell. [0112] Among the four IgG subclasses, IgGl and IgG3 induce the strongest Fc-effector functions. However, since IgGl has the longest half-life and is more stable than IgG3, most therapeutic antibodies with Fc-mediated functions are of IgGl isotype.

[0113] IgG2 and IgG4 isotypes have significantly lower binding affinity to FcyRs. Recent evidence suggests that the IgG2 isotype is not completely devoid of effector function, whereas the IgG4 isotype can undergo in vivo Fab arm exchange leading to bispecific antibody and off-target effects.

[0114] In one aspect, the present disclosure provides a multispecific proteins, such as antibodies or fragments thereof. In certain embodiments, the present disclosure provides a bifunctional molecule, comprising a first antigen-binding portion having specificity to a human Ly6K protein, human Ly6D protein, human Ly6E protein, or human Ly6H protein and a second portion having specificity to a second protein, wherein the first antigen-binding portion comprises an anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H antibody or 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 to immune checkpoints. In certain embodiments, the second portion has specificity to other tumor antigens.

[0116] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, an antibody of the disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0117] Antibodies, variants, or derivatives thereof of the disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope. For example, but not by way of limitation, the antibodies can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.

[0118] In some embodiments, the antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

[0119] The antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.

[0120] The antibodies may also be developed into bispecific T cell engagers (BiTEs) or bispecific antibodies based on heavy and light chain Fv sequences.

Chimeric Antigen Receptors (CARs)

[0121] The present disclosure also provides a chimeric antigen receptor (CAR) that targeting the Ly6 proteins, in particular, the Ly6K protein, the Ly6K protein, the Ly6K protein, or the Ly6H protein, respectively. The CAR are generally fusion proteins, consisting of an extracellular antibody type antigen-binding domain fused to intracellular T-cell signaling proteins. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors.

[0122] The chimeric antigen receptors (CARs) targeting the Ly6 protein provided herein refers to any one of the CARs targeting Ly6K protein, any one of the CARs targeting Ly6D protein, any one of the CARs targeting Ly6E protein, or any one of the CARs targeting Ly6H protein provided herein. In one embodiment, the CAR comprises a Ly6K specific antigen binding fragment as described herein. In embodiment, the CAR comprises: 1) Ly6K specific antigen binding fragment, (ii) an extracellular (which may comprise a hinge) domain, (iii) one or more costimulatory domain, and (iv) one or more activating domain. It will be appreciated that each domain may be heterogeneous, thus comprised of sequences derived from (or corresponding to) different protein chains. In some embodiments, the CAR comprises the antibody, or antigen binding fragment having CDR sequences described in Table 1. For example, the CAR comprises a Ly6K specific antigen binding fragment comprising: (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: 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).

[0123] Similarly, the Ly6 CAR-expressing immune cells provided herein refers 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 ectodomain containing the antigen recognition region comprises a signal peptide and an antigen recognition unit. According to the present disclosure, the ectodomain comprises an anti-Ly6 (for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti- Ly6H) single chain domain. It is preferred, that said single chain domain is a scFv comprising the heavy chain variable region and light chain variable region provided herein.

[0125] The ectodomain may be spaced apart from the transmembrane domain by the presence of a spacer domain. Said optional spacer domain links the antigen-binding domain to the transmembrane domain and it is preferred that said transmembrane domain is flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.

[0126] The transmembrane domain is typically a hydrophobic alpha helix that spans the membrane. Other transmembrane domains can also be used. It has been recognized that an immune cell including T-cell, NK cells or BiTEs containing the CAR as described, namely, containing a CAR starting from the N-terminus to the C-terminus having the following composition: an anti-Ly6 (for example, 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. Thus, the T-cells of the present disclosure show effective antitumor effect.

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

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

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

[0130] Further, another embodiment relates to a T-cell with a chimeric antigen receptor wherein the transmembrane domain can be a transmembrane domain derived from any of the following: 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, 2B4, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, NKG2D, or a zeta chain of a T cell receptor, or any combination thereof. In certain embodiments, the transmembrane domain is the transmembrane domain of the CD28 molecule .

[0131] The activation domain contains a CD3 zeta, CD3 epsilon, CD3 delta, and CD3 gamma or Fc Epsilon receptor (IgE receptor) gamma chain signaling chain. In certain embodiments, the activation domain is mutated to prevent cell apoptosis. The activation domain can include an activation domain of CD3 zeta with first and third Immune-receptor- Tyrosine-based- Activation- Motifs (IT AMs) inactive. For example, the activation domain is a CD3 zeta cytoplasmic region. The activation domain is responsible for the activation of the cytotoxic activity in T-cells or interferon-gamma secretion by T-cells.

[0132] The CAR molecule may be a so-called "second generation" CAR molecule. Second generation CAR molecules have improved intracellular signaling by additionally containing a costimulatory domain, e.g., derived from CD28, 0X40) or 4-1 BB. "Third generation" CAR molecules contain a combined co-stimulatory domain, e.g., CD28 combined with 0X40 or 4- 1BB. Polynucleotides encoding the antibodies/CARs and methods of preparing the same

[0133] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies/CARs, variants or derivatives thereof of the disclosure. For example, the isolated polynucleotides or nucleic acid molecules may encode the anti- Ly6K, Ly6D, or Ly6E antibodies/CARs, variants or derivatives thereof of the disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0134] Methods of making antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. patents: 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties.

[0135] In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are humanized. The humanized antibody may be designed to minimize unwanted immunological response toward rodent anti -human antibodies, which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. The humanized antibody may have one or more amino acid residues introduced into it from a source that is non-human. These non-human residues are often referred to as "import" residues, which are typically taken from a variable domain. Humanization may be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. For example, see U.S. Patent No. 4,816,567, the contents of which are herein incorporated by reference. The humanized antibody may be a human antibody in which some hypervariable region residues, and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanization or engineering of antibodies of the present disclosure can be performed using any known method, such as but not limited to those described in U.S. Pat. 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] According to one example, polypeptide encoding CAR of ScFV Ly6K antibody may comprise the amino acid sequence of: CTCGAGTTCGAACACCATGGCACTGCCAGTTACAGCTCTGCTGCTGCCACTCGCT CTCCTGCTCCATGCAGCTAGGCCACAAATCGTTCTGACACAGTCTCCAGCCCTCA TGTCTGCTTCTCCTGGCGAGAAGGTCACCATGACTTGTTCCGCTTCTTCCTCCGTT TCCTACATGTACTGGTATCAGCAGAAGCCTCGCTCTAGCCCAAAGCTCTGGATCT ACCTGACATCTAACCTGGCTTCCGGAGTTCCTGCAAGATTCTCCGGTTCCGGAAG CGGTACATCCTATTCCCTCACTATCTCTAGCATGGAGGCTGAGGACGCAGCCACC TACTACTGCCAACAGTGGAGCAGCAATCCTCTGACCTTCGGAGCAGGTACTAAGC TGGAGCTGAAAGGCAGCACCTCCGGATCTGGAAAGCCTGGATCTGGTGAGGGTA GCACCAAGGGCTCCGAGGTGCAGCTCCAGCAGTCCGGACCAGAACTCGTCAAAC CTGGTGCTTCCGTCAAGATGTCTTGCAAAGCAAGCGGTTACACCTTCACCGACTA CAACATGCACTGGGTGAAACAAAGCCACGGCAAGAGCCTCGAATGGATCGGTTA CATCAACCCTAACAACGGAGGCACACGCTACAACCAGAAGTTCAAGGGTAAGGC AACCCTGACTGTGAACAAATCTAGCTCTACTGCATACATGGAACTCCGCTCTCTG ACCTCTGAAGACTCTGCTGTTTACTACTGCGCAAGAGACGATTTCTATTGGGGAC AAGGTACAACCCTGACAGTTAGCTCCTTCGTTCCAGTCTTCCTCCCAGCTAAACC AACAACCACTCCTGCACCAAGACCTCCAACACCTGCTCCAACCATTGCAAGCCAG CCACTGTCCCTCAGACCTGAGGCTTGCAGACCTGCTGCAGGTGGTGCTGTGCACA CAAGAGGACTCGATTTCGCCTGCGATATCTACATTTGGGCTCCTCTGGCTGGAAC CTGTGGCGTTCTGCTCCTCAGCCTGGTCATTACTCTCTATTGCAACCATAGAAACA GGTCTAAGAGAAGCAGACTGCTGCATAGCGACTACATGAACATGACTCCAAGAA GACCAGGTCCTACCAGGAAGCACTACCAACCTTACGCTCCTCCTCGCGATTTCGC TGCTTACCGCTCCAGAGTCAAGTTCAGCAGGAGCGCTGACGCACCTGCTTACCAG CAGGGACAGAACCAGCTCTACAACGAACTGAACCTCGGCAGAAGGGAAGAGTAT GATGTGCTGGATAAGAGAAGAGGTCGCGACCCAGAGATGGGTGGCAAACCCAG AAGAAAGAACCCACAAGAAGGTCTGTACAACGAGCTGCAGAAAGATAAGATGG CAGAAGCCTATTCCGAGATCGGAATGAAAGGCGAGAGACGCAGAGGCAAAGGC CACGACGGTCTGTACCAAGGACTCTCCACAGCAACTAAGGACACATACGATGCC CTGCACATGCAAGCTCTCCCACCTAGATGATAATCTAGAGTCGAC (SEQ ID NO: 94)

[0137] As non-limiting example, said protein of interest can be expressed in the cell by its introduction as a transgene preferably encoded by at least one plasmid vector. Polypeptides may be expressed in the cell as a result of the introduction of polynucleotides encoding said polypeptides into the cell. Alternatively, said polypeptides could be produced outside the cell and then introduced thereto.

[0138] Methods for introducing a polynucleotide construct into cells are known in the art and include as non-limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell and virus mediated methods. Said polynucleotides may be introduced into a cell by for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposome and the like. For example, transient transformation methods include for example microinjection, electroporation or particle bombardment, cell fusion. Said polynucleotides may be included in vectors, more particularly plasmids or virus, in view of being expressed in cells. Said plasmid vector can comprise a selection marker which provides for identification and/or selection of cells which received said vector.

[0139] Different transgenes can be included in one vector. Said vector can comprise a nucleic acid sequence encoding ribosomal skip sequence such as a sequence encoding a 2A peptide. 2 A peptides, which were identified in the Aphtho virus subgroup of picornaviruses, causes a ribosomal "skip" from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons (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 invention, polynucleotides encoding polypeptides according to the present disclosure can be mRNA which is introduced directly into the cells, for example by electroporation. [0141] Different methods used herein involve introducing CAR into a cell. As non-limiting example, said CAR can be introduced as transgenes encoded by one plasmid vector. Said plasmid vector can also contain a selection marker which provides for identification and/or selection of cells which received said vector. Polypeptides may be synthesized in situ in the cell as a result of the introduction of polynucleotides encoding said polypeptides into the cell. Alternatively, said polypeptides could be produced outside the cell and then introduced thereto. Methods for introducing a polynucleotide construct into cells are known in the art and including as non-limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell and virus mediated methods. Said polynucleotides may be introduced into a cell by for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposome and the like. For example, transient transformation methods include for example microinjection, electroporation or particle bombardment. Said polynucleotides may be included in vectors, more particularly plasmids or virus, in view of being expressed in cells.

Engineered immune cells

[0142] The present disclosure also relates to isolated cells or cell lines susceptible to be obtained by said method to engineer cells. In particular said isolated cell comprises at least one CAR as described above. In another embodiment, said isolated cell comprises a population of CARs each one comprising different extracellular ligand binding domains. In particular, said isolated cell comprises exogenous polynucleotide sequence encoding CAR. Genetically modified immune cells of the present disclosure are activated and proliferate independently of antigen binding mechanisms. In the scope of the present disclosure is also encompassed an isolated immune cell, preferably a T-cell obtained according to any one of the methods previously described. Said immune cell refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptative immune response. Said immune cell according to the present disclosure can be derived from a stem cell. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+ cells. Said isolated cell can also be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T- lymphocytes or helper T-lymphocytes. In another embodiment, said cell can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. Prior to expansion and genetic modification of the cells of the invention, a source of cells can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen 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, said cell can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In another embodiment, said cell is part of a mixed population of cells which present different phenotypic characteristics. In the scope of the present disclosure is also encompassed a cell line obtained from a transformed T- cell according to the method previously described. Modified cells resistant to an immunosuppressive treatment and susceptible to be obtained by the previous method are encompassed in the scope of the present invention.

Activation and expansion of T cells

[0143] Whether prior to or after genetic modification of the T cells, even if the genetically modified immune cells of the present disclosure are activated and proliferate independently of antigen binding mechanisms, the immune cells, particularly T-cells of the present disclosure can be further activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041. T cells can be expanded in vitro or in vivo. Generally, the T cells are expanded by contact with an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of the T cells to create an activation signal for the T-cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PH A) can be used to create an activation signal for the T-cell. As non-limiting examples, T cell populations may be stimulated in vitro such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co- stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti- CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g , IL-4, IL-7, GM-CSF, -10, - 2, IL-15, TGFp, and TNF- or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanoi. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2). T cells that have been exposed to varied stimulation times may exhibit different characteristics.

Treatment and diagnostic methods using the antibodies

[0144] As described herein, the antibodies, variants or derivatives of the present disclosure may be used in certain treatment and diagnostic methods.

[0145] The present disclosure is further directed to antibody -based therapies which involve administering the therapeutic compounds of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein. Therapeutic compounds of the disclosure include, but are not limited to, antibodies of the disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and derivatives thereof as described herein).

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

[0147] In some embodiments, provided are uses of the antibodies or antigen-binding fragments thereof of the present disclosure in the manufacture of a medicament for treating a cancer in a patient in need thereof. In some embodiments, provided are uses of the engineered immune cells including anti-Ly6K CARs of the present disclosure (e.g., CAR T-cells) in the manufacture of a medicament for treating a cancer in a patient in need thereof.

[0148] In some embodiments, provided are the antibodies or antigen- binding fragments thereof of the present disclosure for use in the treatment of a cancer in a patient in need thereof.

[0149] Non-limiting examples of cancers 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, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

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

[0153] Additional diseases or conditions associated with increased cell survival, that may be treated, prevented, diagnosed and/or prognosed with the antibodies or variants, or derivatives thereof of the disclosure include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g. , chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin’s disease and non-Hodgkin’s disease), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyo sarcoma, 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 adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

[0154] A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient’s age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend 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 administration of the antibodies, variants or include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptides or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelia] or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), buccally, or as an oral or nasal spray. [0156] The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra- articular injection and infusion.

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

[0158] It may be desirable to administer the antigen-binding polypeptides or compositions of the disclosure locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g. , in conjunction, with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the disclosure, care must be taken to use materials to which the protein does not absorb.

[0159] Methods of detecting expression of a human Ly6K protein in a sample are also provided, in some embodiments, comprising contacting the sample with the antibody or fragment thereof, and detecting the binding which indicates expression of Ly6K in the sample.

[0160] In certain embodiments, provided are uses of the antibodies or antigen-binding fragments thereof of the present disclosure in the manufacture of a kit for detecting expression of a human Ly6K protein in a sample.

[0161] Methods of detecting expression of a human Ly6D protein in a sample are also provided, in some embodiments, comprising contacting the sample with the antibody or fragment thereof, and detecting the binding which indicates expression of Ly6D in the sample. [0162] In certain embodiments, provided are uses of the antibodies or antigen-binding fragments thereof of the present disclosure in the manufacture of a kit for detecting expression of a human Ly6D protein in a sample.

[0163] Methods of inhibiting a human Ly6K protein are also provided, in some embodiments, comprising administering any of the anti-Ly6K antibody or fragment thereof described herein. Methods of inhibiting a human Ly6K protein are also provided, in some embodiments, comprising administering any of the modified/engineered immune cells including anti-Ly6K CARs or anti-Ly6D CARs described herein (e.g., CAR T-cells).

Compositions comprising the antibodies

[0164] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody, and an acceptable carrier. In some embodiments, the composition further includes a second anticancer agent e.g., an immune checkpoint inhibitor).

[0165] In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0166] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington’ s Pharmaceutical Sciences by E. W. Martin, incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0167] In an embodiment, the composition comprising the antibodies or the antigen-binding fragment thereof is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0168] The composition comprising the antibodies or the antigen-binding fragment thereof of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

Treatment methods using the CAR immune cells

[0169] In some embodiments, provided are methods for treating a cancer in a patient in need thereof. The method, in one embodiment, entails administering to the patient an effective amount of anti-Ly6 (for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR- expressing T cell of the present disclosure.

[0170] In some embodiments, provided are uses of anti-Ly6 (for example, anti-Ly6K, anti- Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing T cell of the present disclosure in the manufacture of a medicament for treating a cancer in a patient in need thereof.

[0171] In some embodiments, provided are the anti-Ly6 (for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing T cell of the present disclosure for use in the treatment of a cancer in a patient in need thereof.

[0172] In another aspect, the present disclosure relies on methods for treating patients in need thereof, said method comprising at least one of the following steps: (a) providing an engineered immune-cell obtainable by any one of the methods previously described; (b)Administrating said transformed immune cells to said patient.

[0173] In one embodiment, said T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time. Said treatment can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment. By autologous, it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor. By allogeneic is meant that the cells or population of cells used for treating patients are not originating from said patient but from a donor.

[0174] Said treatment can be used to treat patients diagnosed wherein a pre-malignant or malignant cancer condition characterized by anti-Ly6 (for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing cells, especially by an overabundance of anti-Ly6 (for example, anti-Ly6K, anti-Ly6D, anti-Ly6E, or anti-Ly6H) CAR-expressing cells. [0175] The treatment with the engineered immune cells according to the invention may be in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy. Preferably, the treatment with the engineered immune cells according to the invention may be administered in combination (e.g., before, simultaneously or following) with one or more therapies against cancer selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine, chlorambucil, and combination thereof.

[0176] According to a preferred embodiment of the invention, said treatment can be administrated into patients undergoing an immunosuppressive treatment. Indeed, the present disclosure preferably relies on cells or population of cells, which have been made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. In this aspect, the immunosuppressive treatment should help the selection and expansion of the T-cells according to the invention within the patient.

[0177] The administration of the cells or population of cells according to the present disclosure may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermaly, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present disclosure are preferably administered by intravenous injection.

[0178] The administration of the cells or population of cells can consist of the administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight including all integer values of cell numbers within those ranges. The cells or population of cells can be administrated in one or more doses. In another embodiment, said effective amount of cells are administrated as a single dose. In another embodiment, said effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. In another embodiment, said effective amount of cells or composition comprising those cells are administrated parenterally. Said administration can be an intravenous administration. Said administration can be directly done by injection within a tumor.

[0179] The treatment with the engineered immune cells according to the invention may be in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy. Preferably, the treatment with the engineered immune cells according to the invention may be administered in combination (e.g., before, simultaneously or following) with one or more therapies against cancer selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine ,Novantrone, Mitoxantrone, Vepeside, Etoposide (VP 16), arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine, chlorambucil, and combination thereof.

EXAMPLES

Example 1. Ly6K is a valid CAR T-cell therapeutic target in triple negative breast cancer [0180] Ly6K expression was measured in normal breast tissue, breast cancer tissue (FIG. 1A), and TNBC and non-TNBC tissue (FIG. IB). As shown in FIG. 1, Oncomine data analysis showed (A) increased mRNA expression of Ly6K in breast cancer than normal breast tissue using the cancer genome atlas data set, (B) increased mRNA expression in TNBC vs non-TNBC cases using the Curtis dataset. Two sample T-test, p<0.05 considered significant.

[0181] Samples were divided into high and low Ly6K mRNA expression groups by using median gene expression value as bifurcating point. Using survival data and continuous expression variable, survival analysis was done by fitting cox proportional hazards model using function “coxph” of library survival. Hazard ratio (HR) and log rank p value were retrieved from the fitted model. To create prognostic plot, high and low expression categorical variable was used along with survival data. Plots were created using function ’survfit’ of the same R library. p<0.05 considered significant. FIG. 1C showed increased Ly6K expression is significantly associated with poor survival in TNBC (dataset ID: GSE19783) as seen by the prognostic software ProGgene V2.

[0182] Absolute Ly6K mRNA expression were quantified in TNBC subtypes. Relevant GEO datasets (HGU133 Plus 2.0 arrays) of TNBC patients were normalized using Robust multi averaging RMA method and batch corrected using combat. The analysis was done in R, using packages oligo, sva and limma. n=number of tissue samples. The Welch two sample T-test was applied to determine p values, p<0.05 considered significant (FIG. ID).

[0183] The publicly available data at “The human protein atlas” shows quantification of IHC using a NMR validated, affinity purified using the PrEST-antigen as affinity ligand HPA017770 (Sigma) antibody for Ly6K, in a panel of human normal tissues. The intensity of IHC labeling is shown on the Y-axis, X-axis shows name of organs tested. In the inset, IHC images are shown from normal testis and breast using Ly6K antibody (FIG. IE). The results demonstrated that Ly6K was expressed in testis, but not in normal breast cells.

[0184] FIG. IF shows mRNA expression of Ly6D and Ly6E genes in purified immune cells. The absence of Ly6K mRNA expression is indicated by a sign and the presence is indicated by a ‘+’ sign.

[0185] FIG. 1G shows high over-expression of Ly6K in different types of cancer, such as, cervical, breast, head and neck, bladder, esophageal, lung, and colorectal cancer.

Example 2. Ly6K is required for in vivo tumor growth in syngeneic mammary tumor models.

[0186] 4T1 cells in the presence or absence of Ly6K (FIG. 2A-B) were transplanted into lower mammary fat pad of the BALB/c mice by subcutaneous injection (10 mice per group). Tumor measurements were performed using a Vernier caliper. The tumor volumes were calculated using equation ¹/2*length*(width²). ** indicates p<0.001. two tailed student T-test. (FIG. 2C)

Example 3. Generation of Ly6K mouse monoclonal antibodies (mAbs)

[0187] Ly6K mAbs were generated against an extracellular portion of human Ly6K (hLy6K) using the NCI reagent development project. The sequences associated with extracellular form of the protein (shown in FIG. 3A which shows the structure of Ly6K, SEQ ID NOs:86-89 below) were cloned in pET24.1 HIS tagged vectors respectively. Proteins were expressed in E.coli (BL21DE3) cells and purified using a His tag purification (Ni-NTA beads resulting) kit, Lane 10, resulting in a single product of the expected size of 17kDa (FIG. 3B). The protein was confirmed to be Ly6K by M ALDI/TOF analysis. This protein was used for immunization generating several hybridoma cell lines. The supernatants from these clones were screened with ELISA and Western Blottss to identify antibody producing clones. This process yielded three high affinity Ly6K mAbs, as validated by an ELISA assay, as shown in FIG. 3C. For ELISA, microtiter plates were coated with lOOul recombinant Ly6K protein for overnight. Indicated dilutions of the antibodies were added for 30 min. Bound antibodies were detected using TMB substrate.

[0188] hLy6K-MF-C-HIS-noGPI-pcDNA3.1 (Q17RY6) (SEQ ID NO:86)

MDAMKRGLCCVLLLCGAVFVSPDANLTARQRDPEDSQRTDEGDNRVWCHVCEREN TFECQNPRRCKWTEPYCVIAAVKIFPRFFMVAKQCSAGCAAMERPKPEEKRFLLEEP MPFFYLKCCKIRYCNLEGPPINSSVFKEYAHHHHHH

[0189] hLy6E-MF-C-HIS-noGPI-pcDNA3.1 (Q16553) (SEQ ID NO:87)

MDAMKRGLCCVLLLCGAVFVSPLMCFSCLNQKSNLYCLKPTICSDQDNYCVTVSAS AGIGNLVTFGHSLSKTCSPACPIPEGVNVGVASMGISCCQSFLCNFHHHHHH

[0190] hLy6K-MF-C-HIS-noGPI-pcDNA3.1 (Q17RY6) (SEQ ID NO:88)

MDAMKRGLCCVLLLCGAVFVSPDANLTARQRDPEDSQRTDEGDNRVWCHVCEREN TFECQNPRRCKWTEPYCVIAAVKIFPRFFMVAKQCSAGCAAMERPKPEEKRFLLEEP MPFFYLKCCKIRYCNLEGPPINSSVFKEYAHHHHHH

[0191] hLy6H-MF-C-HIS-noGPI-pcDNA3.1 (094772) (SEQ ID NO:89)

MDAMKRGLCCVLLLCGAVFVSPLWCQDCTLTTNSSHCTPKQCQPSDTVCASVRITD PSSSRKDHSVNKMCASSCDFVKRHFFSDYLMGFINSGILKVDVDCCEKDLCNGAAH HHHHH

[0192] 10 Clones of Ly6 mAbs targeting the above Ly6K, Ly6D or Ly6E proteins were obtained as Ly6K-2/l (5G1/2H5), Ly6K-3 (6E1), Ly6K-4 (3A12), Ly6D-l (10E7), Ly6D-2 (11A3), Ly6D-3 (11F4), Ly6D-4 (1E1), Ly6E-l (1C11), Ly6E-2 (6E2), Ly6E-3 (8H8). Among them, Ly6D-2 (11A3) has two different VLs that pair with the same VH, respectively.

Table 3. Variable region sequences of Ly6 mAbs

Example 4. Ly6K mAbs recognize endogenously expressed Ly6K

[0193] To test whether the antibodies generated from the bacterially expressed peptide will recognize endogenously expressed Ly6K protein on the cell surface, anti-human Ly6K antibody 6E1 (Ly6K-3) was used in IHC studies in paraffin fixed section of human testis tissues. Slides were subjected to serial hydration, permeabilization and acidic antigen retrieval methods. Primary mAb (1:100 overnight 4°C). secondary goat anti-mouse Alexa 568 1:500, 1 h RT. The confocal images were acquired on 40X oil objective on Zeiss LSM700 normal human testis (FIG. 4A). As shown in FIG. 4A, the antibody recognized Ly6K on the basal sperm cell in human testis. Axio Scan was used to scan at 40X the TNBC clinical sample slides for Ly6K stain and corresponding H&E stain (FIG. 4B). As shown in FIG. 4B, staining of clinical samples of TNBC (n=3) with CPTC-Ly6K-3 showed membranous/cytoplasmic staining. Similar results were achieved from the two other anti- Ly6K mAbs from above, on testis samples clinical TNBC samples.

[0194] To see if Ly6K mAbs recognize the cell surface Ly6K, unfixed live TNBC cells were stained with mAb Ly6K in flow cytometry analysis. Flow in live unfixed cells was measured to analyze cell surface Ly6K by new mAbs and secondary goat anti mouse Alexa 488 (FIG. 4C). As shown in FIG. 4C, the antibodies recognized cell surface Ly6K protein in the following human and mouse cancer cell lines: TNBC cell lines BT549, MDA-MB-231 and the cervical cancer line Caski, and mouse syngeneic tumor lines 4T1, B16 and E0771. Example 5. Generation ofLy6K mouse chimeric antigen receptors (CARs)

[0195] T cells were enriched from the PBMCs via negative selection and activated using the CTS Dynabeads CD3/CD28. MSGV retrovirus vector was used to clone the scFv of Ly6K mAh sequences of 6E1 (CPCT-Ly6K-3) from polypeptide sequence of SEQ ID NO. 94. pCL- Ampho Retrovirus Packaging Vector from Novus was used to generate retrovirus. The retrovirus was then transduced to the enriched and activated T cells. The Ly6K CAR T cells were then cultured using CTS Optimizer Pro basal+Supplement-i-IL2 +Glutamax+P/S-i-No additional Serum. Transduced CAR T-cells expressed scFV of Ly6K antibody. The CAR expression was detected using protein L recognized by flow cytometry. T-cells were isolated from PBMC and transduced with retrovirus particle CAR construct. The cells were subjected to protein L staining. Protein L can recognize the kappa chain of scFV which is the in the CD19 scFV and LY6K scFV. More than 30 million CAR T-cells was cryo-preserved. Ly6K CART cells have very good transduction comparable to CD 19 CAR T(more than 40% transduction) as shown in FIG. 5.

Example 6. In vitro CTV based cytotoxicity assay

[0196] 5xl0⁴ HeLa tumor cell lines stained with 5uM of Cell trace violet and coculture in 96- well plates with UT (untransduced T cells) or Ly6K CAR T cells in 1:1 and 1:3 ratio (FIG. 6A). Residual viable cells with CTV positive were quantified by Flow cytometry and represented here with Overlay histograms of CTV dilution (FIG. 6B) quantification of Ly6K CAR T cells induced cytotoxicity of HeLa Tumor cells. P-value was determined by Bonferroni test in two-way ANOVA.

[0197] The CAR T-cells were tested for their cytotoxicity against triple negative breast cancer cells (MDA-MB-231). The MDA-MB-231 cells were stained with cell titer blue (CTV) dye and designated with “CTV+”. Ly6K negative pancreatic cancer cells were left unstained and designated with “CTV

These cells were mixed 1 : 1 ratio and both cell populations were detected in flow cytometry histogram as two peaks marked as CTV+ and CTV- (FIG. 7A). When the cells were culture with T-cells not expressing CAR T protein (untransduced T-cells), both cell population could be observed in the histogram as two peaks (FIG. 7B). When the cells were cultured in the presence of anti-LY6K-CAR-T-cells, the LY6K positive cancer cells (CTV+) population was eliminated and only Ly6K negative cancer cell population CTV- cancer were seen (FIG. 7C). The T-cells were tested for multiple cell line and shown are the effectiveness in triple negative breast cancer cells MDA-MB-231 (FIG. 7D) and BT549 (FIG. 7E) comparing in untransduced vs transduced CAR T-cells, in various ratios of effector cell to target cells (E:T).

Example 7. In vivo antitumor activity of Ly6K CAR T-cells against HS378t Ti ’BC cell line xenograft model

[0198] In vivo anti-tumor activity of Ly6K CAR T-cells against HS378t triple negative breast cancer cells (TNBCs) tumor xenograft model was tested. 0.5X10⁶ HS378t tumor cells were subcutaneously (SC) injected near memory fat pad and measured the tumor volume using Vernier calipers. Tumor volume till day 42 and 48 was measured respectively (FIG. 8A-8B). Arrow mark denoted the CAR T or UT (1 X10⁶) cell injection(IV). P value was determined by Tukey test in two way ANOVA with repeated measure. FIG. 8C showed the excised tumor image from UT (top) and CAR T group (bottom). FIG. 8D-8E showed excised tumor volume and tumor weight measurement. P- value was determined by Mann Whitney Unpaired student T-test. n=3 or 4.

Example 8. Activity of the CARs

[0199] Different effector cell vs. target cell ratios were tested using different Ly6K CAR T- cells. The cell lysis percentage increased along with the E:T ratio (FIG. 9A-9B).

Example 9. Ly6D-specific Antibodies

[0200] Four monoclonal anti-Ly6D antibodies from Table 1 (Ly6D-l, Ly6D-2, Ly6D-3, Ly6D-4) were prepared. For SPR assay, Ly6D monoclonal antibodies were captured onto a CM5 chip surface with the protein A/G immobilization. Purified human Ly6D protein were tested for binding to human Ly6D monoclonal antibodies in a dose-dependent manner. The SPR showed that all four antibodies recognized recombinant human Ly6D protein with high affinity, as shown in FIGS. 10A-D.

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

[0202] Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was conducted for LY6D-1, LY6D-2, and LY6D-3. The target cancer cells (T) were incubated with effector PBMC cells (E) in various E:T ratio 40:1, 20: 1, 1:1, 0: 1. Co-cultured cells were labelled with IncuCyte® caspase3/7 dye and imaged in IncuCyte® S3 Live-cell analysis system to distinguish of caspase activation in the real time in cancer cells and in PBMC. The caspase activation specific to cancer cells was calculated by automated image analysis program and data showed that anti-LY6D antibodies effectively generated ADCC in the presence of PBMC cells. For the ADCC assay, 0.25 ug/ml antibody was used, and the dose might not be efficient in inducing cell death in cancer cells on its own, but in the presence of PBMCs, effectively eliminated cancer cells, as shown in FIGS. 11D-F, showing ADCC activity of the anti-LY6D antibodies.

* * *

[0203] The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

[0204] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

SEQUENCE LISTING

Claims

CLAIMS What is claimed is:

1. An antibody or antigen-binding fragment thereof having specificity to a cell-surface Lymphocyte Antigen 6 Family Member K protein (Ly6K), wherein the antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprise the sequences of:

(a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1,

HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6;

(b) HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14; or

(c) HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 22.

2. The antibody or antigen-binding fragment thereof of 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 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 thereof of 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16 and 24.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein

(a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8;

(b) the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16; or

(c) the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 24.

5. An antibody or antigen-binding fragment thereof having specificity to a cell-surface Lymphocyte Antigen 6 Family Member D protein (Ly6D), wherein the antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and

LCDR3, respectively, comprise the sequences of:

(a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, HCDR3 comprising the amino acid sequence of SEQ ID NO: 27,

LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 30;

(b) HCDR1 comprising the amino acid sequence of SEQ ID NO: 33, HCDR2 comprising the amino acid sequence of SEQ ID NO:34, HCDR3 comprising the amino acid sequence of SEQ ID NO: 35,

LCDR1 comprising the amino acid sequence of SEQ ID NO: 39 or 48, LCDR2 comprising the amino acid sequence of SEQ ID NO: 40 or 49 and LCDR3 comprising the amino acid sequence of SEQ ID NO: 41 or 50;

(c) HCDR1 comprising the amino acid sequence of SEQ ID NO: 45,

HCDR2 comprising the amino acid sequence of SEQ ID NO:46, HCDR3 comprising the amino acid sequence of SEQ ID NO: 47, LCDR1 comprising the amino acid sequence of SEQ ID NO: 48,

LCDR2 comprising the amino acid sequence of SEQ ID NO: 49, and

LCDR3 comprising the amino acid sequence of SEQ ID NO: 50; or

(d) HCDR1 comprising the amino acid sequence of SEQ ID NO: 53,

HCDR2 comprising the amino acid sequence of SEQ ID NO:54, HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, LCDR1 comprising the amino acid sequence of SEQ ID NO: 56,

LCDR2 comprising the amino acid sequence of SEQ ID NO: 57, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 58.

6. The antibody or antigen-binding fragment thereof of 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 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 thereof of 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 44, 52 and 60.

8. The antibody or antigen-binding fragment thereof of any one of claims 5-7, wherein:

(a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32;

(b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, the first light chain variable region comprises the amino acid sequence of SEQ ID NO: 52, and a second light chain variable region comprising the amino acid sequence of SEQ ID NO: 44;

(c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 51, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 52; or

(d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 60.

9. An antibody or antigen-binding fragment thereof having specificity to a cell-surface Lymphocyte Antigen 6 Family Member E protein (Ly6E), wherein the antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprise the sequences of: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 61, HCDR2 comprising the amino acid sequence of SEQ ID NO: 62, HCDR3 comprising the amino acid sequence of SEQ ID NO: 63, LCDR1 comprising the amino acid sequence of SEQ ID NO: 64, LCDR2 comprising the amino acid sequence of SEQ ID NO: 65, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 66;

(b) HCDR1 comprising the amino acid sequence of SEQ ID NO: 69, HCDR2 comprising the amino acid sequence of SEQ ID NO:70, HCDR3 comprising the amino acid sequence of SEQ ID NO: 71, LCDR1 comprising the amino acid sequence of SEQ ID NO: 72, LCDR2 comprising the amino acid sequence of SEQ ID NO: 73, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 74; or

(c) HCDR1 comprising the amino acid sequence of SEQ ID NO: 77, HCDR2 comprising the amino acid sequence of SEQ ID NO:78, HCDR3 comprising the amino acid sequence of SEQ ID NO: 79, LCDR1 comprising the amino acid sequence of SEQ ID NO: 80, LCDR2 comprising the amino acid sequence of SEQ ID NO: 81, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 82.

10. The antibody or antigen-binding fragment thereof of 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 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 thereof of 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 76 and 84.

12. The antibody or antigen-binding fragment thereof of any one of claims 9-11, wherein

(a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 67, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68;

(b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 75, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 76; or

(c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 83, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 84.

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

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

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

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the isotype is IgGl, IgG2, IgG3 or IgG4.

17. A chimeric antigen receptor, comprising the antibody, or antigen binding fragment thereof of any one of claims 1-12.

18. The chimeric antigen receptor of claim 17, further comprising one or more costimulatory domains, and an activation domain.

19. The chimeric antigen receptor of claim 18, wherein the one or more costimulatory domains comprise one or more costimulatory signaling regions derived from the group consisting of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, 2B4, CD3 gamma, CD3 delta, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD 16, 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 a zeta chain of a T cell receptor.

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

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

22. The chimeric antigen receptor of claim 21, wherein the transmembrane domain comprises a transmembrane domain selected from the group consisting of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, 2B4, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD28T, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, NKG2D, or a zeta chain of a T cell receptor, or any combination thereof.

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

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

25. The chimeric antigen receptor of claim 18, wherein the activation 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 of claim 25, wherein the activation domain is mutated to prevent cell apoptosis.

27. An antibody or antigen-binding fragment thereof competing with the antibody or antigen-binding fragment thereof of any one of claims 1-16, or the chimeric antigen receptor of any one of claims 17-26.

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

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

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

31. An isolated cell comprising one or more polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-16 or the bifunctional molecule of claim 28.

32. An engineered immune cell expressing at the cell surface membrane a chimeric antigen receptor according to any one of claims 17-26.

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

34. A method of engineering an immune cell comprising:

(a) providing an immune cell, (b) expressing at the surface of said cell at least one chimeric antigen receptor of any one of claims 17-26.

35. A method of engineering an immune cell, comprising:

(a) providing an immune cell,

(b) introducing into said cell at least one polynucleotide encoding said chimeric antigen receptor of any one of claims 17-26,

(c) expressing said polynucleotide into said immune cell.

36. A method of treating a cancer in a patient in need thereof, comprising administering to the patient the antibody or antigen-binding fragment thereof of any one of claims 1-16 or any combination thereof, the bifunctional molecule of claim 28, or the engineered immune cell of claim 32.

37. The method of 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 of claim 37, wherein the breast cancer is triple negative breast cancer.

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

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

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

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

42. A method of treating a breast cancer comprising administering to the patient the antibody or antigen-binding fragment thereof of any one of claims 1-4 or an engineered immune cell expressing at the cell surface membrane a chimeric antigen receptor comprising the antibody, or antigen binding fragment thereof of any one of claims 1-4.

43. The method of claim 42, wherein the breast cancer is triple negative breast cancer.

44. A method of treating a pancreatic cancer comprising administering to the patient the antibody or antigen-binding fragment thereof of any one of claims 5-8 or an engineered immune cell expressing at the cell surface membrane a chimeric antigen receptor comprising the antibody, or antigen binding fragment thereof of any one of claims 5-8.

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