Antibodies binding to LY6G6D

Abstract

Disclosed are anti-LY6G6D antibodies, as well as methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use for the treatment of disorders characterized by the expression of LY6G6D.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 496,928, filed April 18, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to antibodies that bind to LY6G6D. The invention further relates to methods for preparing such antibodies, compositions comprising such antibodies (including pharmaceutical compositions), and their use in treating conditions characterized by the expression of LY6G6D. Background Technology

[0004] LY6G6D

[0005] LY6G6D belongs to the leukocyte antigen-6 (LY6) gene cluster located in the major histocompatibility complex (MHC) class III region on chromosome 6. Members of the LY6 superfamily typically contain 70 to 80 amino acids, including 8 to 10 cysteine ​​residues. Most LY6 proteins attach to the cell surface via a glycosylphosphatidylinositol (GPI) anchor, which is directly involved in signal transduction.

[0006] RNA expression analysis from the TCGA database showed that LY6G6D is highly expressed and specific for colorectal cancer. Its expression in other tumor types and normal cells is negligible, making it a highly promising tumor target. LY6G6D expression is associated with cancers that do not respond well to immune checkpoint inhibitors. Therefore, antibody therapy development targeting this target could be effective in treating certain groups of colorectal cancer patients refractory to chemotherapy or immune checkpoint therapy. Summary of the Invention

[0007] This invention relates to LY6G6D binding antibodies. Other aspects of this invention relate to methods for preparing such antibodies, compositions comprising such antibodies, and their use in treating conditions characterized by the expression of LY6G6D.

[0008] In some embodiments, the antibody that binds to LY6G6D comprises a first binding unit comprising: a heavy chain variable region comprising: (a) a CDRH1 sequence of any one of SEQ ID NOs: 1-6; and / or (b) a CDRH2 sequence of any one of SEQ ID NOs: 7-11; and / or (c) a CDRH3 sequence of any one of SEQ ID NOs: 12-15; and a light chain variable region comprising: (d) a CDRL1 sequence of any one of SEQ ID NOs: 16-19; and / or (e) a CDRL2 sequence of any one of SEQ ID NOs: 20-23; and / or (f) a CDRL3 sequence of any one of SEQ ID NOs: 24-26. In some embodiments, the CDRH1, CDRH2, and CDRH3 sequences of the first binding unit are in a human VH framework. In some embodiments, the CDRL1, CDRL2, and CDRL3 sequences of the first binding unit are in a human VL framework.

[0009] In some embodiments, the first binding unit comprises: a heavy chain variable region comprising: (a) a CDRH1 sequence of any one of SEQ ID NOs: 1-6; and (b) a CDRH2 sequence of any one of SEQ ID NOs: 7-11; and (c) a CDRH3 sequence of any one of SEQ ID NOs: 12-15.

[0010] In some embodiments, the first binding unit comprises: a heavy chain variable region comprising: (a) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 7, and a CDRH3 sequence of SEQ ID NO: 12; or (b) a CDRH1 sequence of SEQ ID NO: 2, a CDRH2 sequence of SEQ ID NO: 8, and a CDRH3 sequence of SEQ ID NO: 13; or (c) a CDRH1 sequence of SEQ ID NO: 3, a CDRH2 sequence of SEQ ID NO: 9, and a CDRH3 sequence of SEQ ID NO: 13; or (d) a CDRH1 sequence of SEQ ID NO: 4, a CDRH2 sequence of SEQ ID NO: 10, and a CDRH3 sequence of SEQ ID NO: 14; or (e) a CDRH1 sequence of SEQ ID NO: 5, a CDRH2 sequence of SEQ ID NO: 11, and a CDRH3 sequence of SEQ ID NO: 15; or (f) a CDRH1 sequence of SEQ ID NO: 6, a CDRH2 sequence of SEQ ID NO: 11, and a CDRH3 sequence of SEQ ID NO: 15.

[0011] In some embodiments, the first binding unit comprises: a heavy chain variable region comprising: (a) a CDRH1 sequence of any one of SEQ ID NOs: 1-4; and (b) a CDRH2 sequence of any one of SEQ ID NOs: 5-8; and (c) a CDRH3 sequence of any one of SEQ ID NOs: 9-11.

[0012] In some embodiments, the first binding unit comprises: a heavy chain variable region comprising: (a) a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 5, and a CDRH3 sequence of SEQ ID NO: 9; or (b) a CDRH1 sequence of SEQ ID NO: 2, a CDRH2 sequence of SEQ ID NO: 6, and a CDRH3 sequence of SEQ ID NO: 10; or (c) a CDRH1 sequence of SEQ ID NO: 3, a CDRH2 sequence of SEQ ID NO: 7, and a CDRH3 sequence of SEQ ID NO: 9; or (d) a CDRH1 sequence of SEQ ID NO: 4, a CDRH2 sequence of SEQ ID NO: 8, and a CDRH3 sequence of SEQ ID NO: 11.

[0013] In some embodiments, the first binding unit comprises: (a) a heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 1, a CDRH2 sequence of SEQ ID NO: 7, and a CDRH3 sequence of SEQ ID NO: 12; and a light chain variable region comprising a CDRL1 sequence of SEQ ID NO: 16, a CDRL2 sequence of SEQ ID NO: 20, and a CDRL3 sequence of SEQ ID NO: 24; or (b) a heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 2, a CDRH2 sequence of SEQ ID NO: 8, and a CDRH3 sequence of SEQ ID NO: 13; and a light chain variable region comprising a CDRL1 sequence of SEQ ID NO: 17, a CDRL2 sequence of SEQ ID NO: 21, and a CDRL3 sequence of SEQ ID NO: 25; or (c) a heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 3, a CDRH2 sequence of SEQ ID NO: 9, and a CDRH3 sequence of SEQ ID NO: 13; and a light chain variable region comprising a CDRL1 sequence of SEQ ID NO: 17, a CDRL2 sequence of SEQ ID NO: 21, and a CDRL3 sequence of SEQ ID NO: 25; or (d) a heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 4, a CDRH2 sequence of SEQ ID NO: 10, and a CDRH3 sequence of SEQ ID NO: 14; and a light chain variable region comprising a CDRL1 sequence of SEQ ID NO: 18, a CDRL2 sequence of SEQ ID NO: 22, and a CDRL3 sequence of SEQ ID NO: 24; or (e) a heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 5, a CDRH2 sequence of SEQ ID NO: 11, and a CDRH3 sequence of SEQ ID NO: 15; and a light chain variable region comprising a CDRL1 sequence of SEQ ID NO: 19, a CDRL2 sequence of SEQ ID NO: 23, and a CDRL3 sequence of SEQ ID NO: 26; or (f) a heavy chain variable region comprising a CDRH1 sequence of SEQ ID NO: 6, a CDRH2 sequence of SEQ ID NO: 11, and a CDRH3 sequence of SEQ ID NO: 15; and a light chain variable region comprising a CDRL1 sequence of SEQ ID NO: 19, a CDRL2 sequence of SEQ ID NO: 23, and a CDRL3 sequence of SEQ ID NO: 26.

[0014] In some embodiments, the first binding unit comprises a heavy chain variable region sequence having at least 95% identity with any of SEQ ID NO:27-34.

[0015] In some embodiments, the first binding unit comprises a heavy chain variable region sequence, which includes any one of SEQ ID NO:27-34.

[0016] In some embodiments, the first binding unit comprises a light chain variable region sequence having at least 95% identity with any one of SEQ ID NO:35-42.

[0017] In some embodiments, the first binding unit comprises a light chain variable region sequence, which includes any one of SEQ ID NO:35-42.

[0018] In some embodiments, the first binding unit comprises: (a) the heavy chain variable region sequence of SEQ ID NO:27 and the light chain variable region sequence of SEQ ID NO:35; or (b) the heavy chain variable region sequence of SEQ ID NO:28 and the light chain variable region sequence of SEQ ID NO:36; or (c) the heavy chain variable region sequence of SEQ ID NO:29 and the light chain variable region sequence of SEQ ID NO:37; or (d) the heavy chain variable region sequence of SEQ ID NO:30 and the light chain variable region sequence of SEQ ID NO:38; or (e) the heavy chain variable region sequence of SEQ ID NO:31 and the light chain variable region sequence of SEQ ID NO:39; or (f) the heavy chain variable region sequence of SEQ ID NO:32 and the light chain variable region sequence of SEQ ID NO:40; or (g) the heavy chain variable region sequence of SEQ ID NO:33 and the light chain variable region sequence of SEQ ID NO:41; or (h) the heavy chain variable region sequence of SEQ ID NO:34 and the light chain variable region sequence of SEQ ID NO:35. The light chain variable region sequence of NO:42.

[0019] In some embodiments, the antibody further comprises a heavy chain constant region. In some embodiments, the heavy chain constant region comprises a hinge region, a CH1 region, a CH2 region, and / or a CH3 region. In some embodiments, the heavy chain constant region comprises one or more knock-in-hole (KiH) mutations. In some embodiments, the heavy chain constant region comprises one or more silent mutations. In some embodiments, the heavy chain constant region comprises one or more FcRn binding mutations. In some embodiments, the one or more FcRn binding mutations include a T250Q mutation, an M428L mutation, or both T250Q and M428L mutations. In some embodiments, the heavy chain constant region comprises one or more protein A binding mutations. In some embodiments, the one or more protein A binding mutations include an H435R mutation, a Y436F mutation, or both H435R and Y436F mutations.

[0020] In some embodiments, the antibody further comprises a light chain constant region. In some embodiments, the light chain constant region comprises a CL region. In some embodiments, the antibody is single-specific. In some embodiments, the antibody is multi-specific. In some embodiments, the antibody is bispecific.

[0021] In some embodiments, the antibody further comprises a second binding unit that binds to CD3ε. In some embodiments, the second binding unit comprises: a heavy chain variable region comprising: (a) the CDRH1 sequence of SEQ ID NO:43; and / or (b) the CDRH2 sequence of any one of SEQ ID NO:44-45; and / or (c) the CDRH3 sequence of SEQ ID NO:46; and a light chain variable region comprising: (d) the CDRL1 sequence of SEQ ID NO:47; and / or (e) the CDRL2 sequence of SEQ ID NO:48; and / or (f) the CDRL3 sequence of SEQ ID NO:49. In some embodiments, the CDRH1, CDRH2, and CDRH3 sequences of the second binding unit are present in the human VH frame. In some embodiments, the CDRL1, CDRL2, and CDRL3 sequences of the second binding unit are present in the human VL frame. In some embodiments, the second binding unit comprises: a heavy chain variable region comprising: (a) the CDRH1 sequence of SEQ ID NO:43; and (b) the CDRH2 sequence of any one of SEQ ID NO:44-45; and (c) the CDRH3 sequence of SEQ ID NO:46. In some embodiments, the second binding unit comprises: a heavy chain variable region comprising: (a) the CDRH1 sequence of SEQ ID NO:43, the heavy chain CDRH2 sequence of SEQ ID NO:44, and the heavy chain CDRH3 sequence of SEQ ID NO:46; or (b) the CDRH1 sequence of SEQ ID NO:43, the CDRH2 sequence of SEQ ID NO:45, and the CDRH3 sequence of SEQ ID NO:46.

[0022] In some embodiments, the second binding unit comprises a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:47; the CDRL2 sequence of SEQ ID NO:48; and the CDRL3 sequence of SEQ ID NO:49. In some embodiments, the second binding unit comprises: (a) a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:43, the CDRH2 sequence of SEQ ID NO:44, and the CDRH3 sequence of SEQ ID NO:46; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:47, the CDRL2 sequence of SEQ ID NO:48, and the CDRL3 sequence of SEQ ID NO:49; or (b) a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:43, the CDRH2 sequence of SEQ ID NO:45, and the CDRH3 sequence of SEQ ID NO:46; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:47, the CDRL2 sequence of SEQ ID NO:48, and the CDRL3 sequence of SEQ ID NO:49.

[0023] In some embodiments, the second binding unit comprises a heavy chain variable region sequence having at least 95% identity with any one of SEQ ID NO:50-51. In some embodiments, the second binding unit comprises a heavy chain variable region sequence comprising any one of SEQ ID NO:50-51. In some embodiments, the second binding unit comprises a light chain variable region sequence having at least 95% identity with any one of SEQ ID NO:52-53. In some embodiments, the second binding unit comprises a light chain variable region sequence comprising any one of SEQ ID NO:52-53.

[0024] In some embodiments, the second binding unit comprises: (a) the heavy chain variable region sequence of SEQ ID NO:50 and the light chain variable region sequence of SEQ ID NO:52; or (b) the heavy chain variable region sequence of SEQ ID NO:51 and the light chain variable region sequence of SEQ ID NO:53.

[0025] Aspects of the present invention include antibodies that bind to LY6G6D and CD3ε, said antibodies comprising: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:54; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:56.

[0026] Aspects of the present invention include antibodies that bind to LY6G6D and CD3ε, said antibodies comprising: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:55; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:56.

[0027] Aspects of the present invention include antibodies that bind to LY6G6D and CD3ε, said antibodies comprising: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:58; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:60.

[0028] Aspects of the present invention include antibodies that bind to LY6G6D and CD3ε, said antibodies comprising: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:59; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:60.

[0029] Aspects of the present invention include antibodies that bind to LY6G6D and CD3ε, said antibodies comprising: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:61; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:62.

[0030] Aspects of the present invention include antibodies that bind to LY6G6D and CD3ε, said antibodies comprising: a first light chain subunit comprising SEQ ID NO:69; a first heavy chain subunit comprising SEQ ID NO:64; a second light chain subunit comprising SEQ ID NO:69; and a second heavy chain subunit comprising SEQ ID NO:66.

[0031] Aspects of the present invention include antibodies that bind to LY6G6D and CD3ε, said antibody comprising: a first light chain subunit comprising SEQ ID NO:69; a first heavy chain subunit comprising SEQ ID NO:65; a second light chain subunit comprising SEQ ID NO:69; and a second heavy chain subunit comprising SEQ ID NO:66.

[0032] Aspects of the present invention include antibodies that bind to LY6G6D and CD3ε, said antibodies comprising: a first light chain subunit comprising SEQ ID NO:69; a first heavy chain subunit comprising SEQ ID NO:67; a second light chain subunit comprising SEQ ID NO:69; and a second heavy chain subunit comprising SEQ ID NO:68.

[0033] Aspects of the present invention include pharmaceutical compositions comprising antibodies as described herein.

[0034] Aspects of the present invention include treatment methods comprising administering an effective dose of an antibody or pharmaceutical composition as described herein to an individual in need.

[0035] Aspects of the present invention include methods for treating a condition characterized by the expression of LY6G6D, the methods comprising administering an antibody or pharmaceutical composition as described herein to a subject suffering from the condition.

[0036] Aspects of the invention include the use of antibodies as described herein in the preparation of medicaments for treating conditions characterized by the expression of LY6G6D.

[0037] Aspects of the present invention include antibodies as described herein for treating conditions characterized by the expression of LY6G6D.

[0038] In some implementations, the condition is cancer. In some implementations, the cancer is a solid tumor. In some implementations, the solid tumor is colorectal cancer. In some implementations, the colorectal cancer is refractory to chemotherapy and / or immune checkpoint inhibitor therapy.

[0039] Aspects of the invention include polynucleotides encoding antibodies as described herein, vectors containing such polynucleotides, and cells containing such vectors.

[0040] Aspects of the present invention include a method for producing antibodies as described herein, the method comprising growing cells as described herein under conditions that allow expression of the antibodies, and isolating the antibodies.

[0041] Aspects of the invention include a kit comprising an antibody or pharmaceutical composition as described herein and instructions for use. In some embodiments, the kit further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic drug.

[0042] The present invention includes a diagnostic method for determining whether a subject has a condition characterized by the expression of LY6G6D or is at risk of developing said condition, the method comprising: contacting a biological test sample from the subject with an antibody bound to LY6G6D to generate an LY6G6D-antibody complex, wherein said antibody comprises: (a) a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:1, the CDRH2 sequence of SEQ ID NO:7, and the CDRH3 sequence of SEQ ID NO:12; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:16, the CDRL2 sequence of SEQ ID NO:20, and the CDRL3 sequence of SEQ ID NO:24; or (b) a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:2, the CDRH2 sequence of SEQ ID NO:8, and the CDRH3 sequence of SEQ ID NO:13; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:17, the CDRH2 sequence of SEQ ID NO:20, and the CDRH3 sequence of SEQ ID NO:24; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:17, the CDRH2 sequence of SEQ ID NO:20, and the CDRH3 sequence of SEQ ID NO:24; (c) The heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:21, the CDRH2 sequence of SEQ ID NO:9, and the CDRH3 sequence of SEQ ID NO:13; and the light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:17, the CDRL2 sequence of SEQ ID NO:21, and the CDRL3 sequence of SEQ ID NO:25; or (d) The heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:4, the CDRH2 sequence of SEQ ID NO:10, and the CDRH3 sequence of SEQ ID NO:14; and the light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:18, the CDRL2 sequence of SEQ ID NO:22, and the CDRL3 sequence of SEQ ID NO:24; or (e) The heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:21, the CDRH2 sequence of SEQ ID NO:22, and the CDRL3 sequence of SEQ ID NO:24; or (e) The heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:21, the CDRH2 sequence of SEQ ID NO:22, and the CDRL3 sequence of SEQ ID NO:25; The CDRH1 sequence of SEQ ID NO:5, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15; and the light chain variable region, which includes: the CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:26;Or (f) a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:6, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:26; detecting the concentration of the LY6G6D-antibody complex in the bioassay sample; and comparing the concentration of the LY6G6D-antibody complex with a reference value to determine whether the subject has the condition or is at risk of developing the condition.

[0043] In some implementations, the condition is cancer. In some implementations, the cancer is a solid tumor. In some implementations, the solid tumor is colorectal cancer.

[0044] These and other aspects will be further explained in the remainder of this disclosure, including the embodiments. Attached Figure Description

[0045] Figure 1 A-1D is a series of graphs showing binding data from humanized anti-LY6G6D antibodies to colon cancer cells expressing different levels of LY6G6D.

[0046] Figure 2 A-2B is a series of graphs showing binding data from humanized anti-LY6G6D antibodies to CHO-K1 cells expressing human and rhesus monkey LY6G6D.

[0047] Figure 3 This is a summary from... Figure 1 and Figure 2 EC cell binding data 50 A table of values.

[0048] Figure 4 This is a schematic diagram of the LY6G6D x CD3 multispecific antibody according to an embodiment of the present invention.

[0049] Figure 5 A-5D is a series of graphs showing binding data from the LY6G6DxCD3 antibody to colon cancer cells expressing LY6G6D and T cells expressing CD3.

[0050] Figure 6 This is a summary from... Figure 5 EC cell binding data 50 A table of values.

[0051] Figure 7A-7H is a series of graphs showing cytokine release data from the binding of the LY6G6DxCD3 antibody to colon cancer cells expressing LY6G6D and T cells expressing CD3.

[0052] Figure 8 A-8D is a series of graphs showing T cell activation data from the binding of the LY6G6DxCD3 antibody to colon cancer cells expressing LY6G6D and T cells expressing CD3.

[0053] Figure 9 A-9D is a series of graphs showing cytotoxicity data from the binding of the LY6G6DxCD3 antibody to colon cancer cells expressing LY6G6D and T cells expressing CD3.

[0054] Figure 10 This is a graph showing the antitumor efficacy of the LY6G6Dx CD3 bispecific antibody according to an embodiment of the present invention in an HT55 colon cancer cell model in NSG mice when administered at 0.3 to 1 mg / kg every two weeks.

[0055] Figure 11 This is a graph showing the antitumor efficacy of the LY6G6D xCD3 bispecific antibody according to an embodiment of the present invention in an HT55 colon cancer cell model in NSG mice when administered at 0.3 mg / kg every two weeks.

[0056] Figure 12 This is a graph showing the antitumor efficacy of the LY6G6D x CD3 bispecific antibody according to an embodiment of the present invention in an HT55 colon cancer cell model in NSG mice when administered at 0.1 mg / kg once weekly.

[0057] Figure 13 This is a graph showing the antitumor efficacy of the LY6G6D x CD3 bispecific antibody according to an embodiment of the present invention in a COLO32DM colon cancer cell model in NSG mice when administered intravenously once weekly at a dose of 0.1 to 1 mg / kg.

[0058] Figure 14 This is a graph showing the PK data of LY6G6D x CD3 bispecific antibodies with or without (WT) FcRn binding mutations according to embodiments of the present invention. Detailed Implementation

[0059] Unless otherwise indicated, the practice of this invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. These techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by MJ Gait, 1984); "Animal Cell Culture" (edited by R.R. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (edited by F.M. Usubel et al., 1987, and updated regularly); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001); Harlow, Lane, and Harlow, "Using Antibodies: A Laboratory Manual: Portable Protocol No. I," Cold Spring Harbor. Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0060] When providing a range of values, it should be understood that, unless the context explicitly indicates otherwise, every midpoint between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other specified value or midpoint within the specified range, is covered within this invention. The upper and lower limits of these smaller ranges may be independently included within that smaller range and are also included in this invention, subject to any specifically excluded limits within the stated range. When a stated range includes one or both of the included limits, the range excluding one or both of those included limits is also included in this invention.

[0061] Unless otherwise indicated, antibody residues in this article are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0062] In the following description, numerous specific details are set forth to provide a more complete understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without one or more of these specific details. In other instances, well-known features and processes familiar to those skilled in the art have not been described to avoid obscuring the invention.

[0063] All references cited in this disclosure (including patent applications and publications) are incorporated herein by reference in their entirety.

[0064] I. Definitions

[0065] "Comprising" means that the listed elements are required in the composition / method / kit, but may include other elements to form a composition / method / kit, etc., within the scope of the claims.

[0066] "consisting essentially of..." means to limit the scope of the described composition or method to specified materials or steps that do not substantially affect the essential and novel features of the invention.

[0067] "Composed of" means excluding any element, step or component not specified in the claims from the composition, method or kit.

[0068] The antibody residues in this article are numbered according to the Kabat numbering system and the EU numbering system. When referring to residues in the variable domain (approximately residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is generally used (e.g., Kabat et al., EU index as reported above). “EU index as in Kabat” refers to the residue number of the human IgG1 EU antibody. Unless otherwise specified herein, references to the numbering of residues in the variable domain of an antibody mean residue numbering according to the Kabat numbering system. Unless otherwise stated herein, references to the numbering of residues in the constant domain of an antibody mean residue numbering according to the EU numbering system.

[0069] Antibodies, also known as immunoglobulins, conventionally comprise at least one heavy chain and one light chain, wherein the amino-terminal domains of the heavy and light chains are sequence-variable and are therefore commonly referred to as variable regions or variable heavy (VH) or variable light (VL) domains. The two domains conventionally associate to form a specific binding region; however, as will be discussed herein, specific binding can also be achieved with only the variable heavy chain sequence, and various non-natural antibody conformations are known and used in the art.

[0070] "Functional" or "bioactive" antibodies or antigen-binding molecules (including multispecific (e.g., bispecific) antibodies) are antibodies or antigen-binding molecules capable of exerting one or more of their native activities in structural, regulatory, biochemical, or biophysical events. For example, a functional antibody or other binding molecule may have the ability to specifically bind to an antigen, and said binding may subsequently induce or alter cellular or molecular events, such as signal transduction or enzymatic activity. Functional antibodies or other binding molecules may also block ligand activation of a receptor or act as agonists or antagonists. The ability of an antibody or other binding molecule to exert one or more of its native activities depends on several factors, including the proper folding and assembly of the polypeptide chain.

[0071] In this article, the term "antibody" is used in its broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, monomers, dimers, polymers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, TCAs, single-chain Fvs (scFvs), nanobodies, and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species.

[0072] The term antibody may refer to the full-length heavy chain, full-length light chain, or complete immunoglobulin molecule; or the immunoactive portion of any of these polypeptides, i.e., a polypeptide or portion thereof containing an antigen-binding site that specifically binds to an antigen of a target, including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules, including engineered subclasses with altered Fc moieties that provide reduced or enhanced effector cell activity. The light chain of the subject antibody may be a κ light chain (Vκ) or a λ light chain (Vλ). Immunoglobulins may be derived from any species. On the one hand, immunoglobulins are primarily of human origin.

[0073] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. For example, monoclonal antibodies according to the invention can be prepared by a hybridoma method first described by Kohler et al. (1975) Nature 256:495, or they can also be prepared via a recombinant protein production method (see, for example, U.S. Patent No. 4,816,567).

[0074] The term "variable" used in conjunction with antibodies refers to the fact that certain portions of the antibody's variable domain are significantly different in sequence between antibodies and are used for the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the variable domain of an antibody. In both the light chain and heavy chain variable domains, the variability is concentrated in three segments called hypervariable regions. The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, which mostly adopt a β-sheet configuration linked by the three hypervariable regions, forming loop connections and, in some cases, part of a β-sheet structure. The hypervariable regions in each chain are held together very closely by the FRs and, together with hypervariable regions from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as antibody involvement in antibody-dependent cytotoxicity (ADCC).

[0075] The term "hypervariant region" as used herein refers to the amino acid residues in an antibody responsible for antigen binding. Hypervariant regions generally contain amino acid residues from the complementarity-determining region (CDR) or CDR (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from the hypervariant loop residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some implementations, "CDR" refers to the complementarity-determining region of the antibody, as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). "Frame region" or "FR" residues are those variable domain residues other than the hypervariable region / CDR residues as defined herein.

[0076] Exemplary CDR names are shown in this document; however, those skilled in the art will understand that various CDR definitions are commonly used, including the Kabat definition (see “Zhao et al., A germline knowledge-based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010; 47:694–700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions (Chothia et al., “Conformations of immunoglobulin hypervariable regions.” Nature. 1989; 342:877–883).Alternative definitions of target CDRs include, but are not limited to, those published by: Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001; 309:657–670; Ofran et al., “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes.” J Immunol. 2008; 181:6230–6235; Almagro, “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004; 17:132–143; and Padlan et al., “Identification of specificity-determining residues in antibodies.” Faseb J. 1995; 9: 133–139., each of these references is specifically incorporated into this paper by reference.

[0077] As used herein, an “intact antibody chain” is an antibody chain containing a full-length variable region and a full-length constant region (Fc). For secreted IgG, an intact “standard” antibody contains an intact light chain and a intact heavy chain, as well as a light chain constant domain (CL) and a heavy chain constant domain, CH1, a hinge, CH2, and CH3. Other isotypes (such as IgM or IgA) may have different CH domains. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or a variant of its amino acid sequence. An intact antibody can have one or more “effector functions,” which refer to those biological activities attributable to the antibody’s Fc constant region (native sequence Fc region or amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors. Constant region variants include those that alter the effector profile, binding to Fc receptors, etc.

[0078] Antibodies and various antigen-binding proteins can be provided as different classes depending on the amino acid sequence of their heavy chain Fc (constant domain). There are five main classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant domain of the Fc corresponding to different antibody classes can be referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known. Ig forms include hinged or hingeless forms (Roux et al. (1998) J. Immunol. 161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US2005 / 0048572; US2004 / 0229310). The light chain of an antibody from any vertebrate species can be assigned to one of two types (referred to as κ (kappa) and λ (lambda)) based on the amino acid sequence of its constant structural domain. An antibody according to an embodiment of the invention may comprise either a κ light chain sequence or a λ light chain sequence.

[0079] A “functional Fc region” possesses the “effective function” of the native Fc region. Non-limiting examples of effector functions include C1q binding; CDC; Fc receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions typically require the Fc region to interact with receptors (e.g., FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors, and low-affinity FcRn receptors); and can be assessed using various assays known in the art. A “dead” or “silent” Fc is an Fc that has been mutated to retain activity associated with, for example, prolonged serum half-life, but does not activate high-affinity Fc receptors, or has a reduced affinity for Fc receptors.

[0080] A “natural sequence Fc region” contains an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Natural sequence human Fc regions include, for example, the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as their naturally occurring variants.

[0081] The “variant Fc region” comprises an amino acid sequence that distinguishes it from the native Fc region due to at least one amino acid modification (preferably one or more amino acid substitutions). Preferably, the variant Fc region has at least one amino acid substitution compared to the native Fc region or the Fc region of the parent polypeptide, for example, about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native Fc region or the Fc region of the parent polypeptide. The variant Fc region as used herein will preferably have at least about 80% homology with the native Fc region and / or with the Fc region of the parent polypeptide, and most preferably at least about 90% homology, more preferably at least about 95% homology.

[0082] The variant Fc sequence may include three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563; Hezareh et al., (2001) J. Virology 75:12161; US ​​Patent No. 5,624,821, the disclosures of which are incorporated herein by reference in their entirety). In some embodiments, the variant Fc sequence may include the following amino acid substitutions: L234A; L235A; and G237A. When these three amino acid substitutions are present in the IgG1 Fc sequence, they may be referred to as G1AAA.

[0083] Two amino acid substitutions at complement C1q binding sites at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J.Exp.Med. 178:661 (1993) and Canfield and Morrison, J.Exp.Med. 173:1483 (1991)). Substitutions at human IgG1 or IgG2 residues at positions 233–236 and at IgG4 residues at positions 327, 330, and 331 significantly reduce ADCC and CDC (see, for example, Armour KL et al., 1999 Eur J Immunol. 29(8):2613–24; and Shields RL et al., 2001 J Biol Chem. 276(9):6591–604). The human IgG4 Fc amino acid sequence (UniProtKB number P01861) is provided herein as SEQ ID NO:76. Silent IgG1 is described, for example, in Boesch, AW et al., “Highly parallel characterization of IgG Fc binding interactions.” MAbs, 2014, 6(4): 915-27, the contents of which are incorporated herein by reference in their entirety.

[0084] Other Fc variants are also possible, including, but not limited to, variants that lack a region capable of forming disulfide bonds, or variants that eliminate certain amino acid residues at the N-terminus of the native Fc, or variants that add methionine residues thereto. Thus, in some embodiments, one or more Fc portions of the antibody may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, the antibody may contain an Fc variant.

[0085] Furthermore, Fc variants can be constructed to remove or substantially reduce effector function by substituting (mutating), deleting, or adding amino acid residues to achieve complement binding or Fc receptor binding. For example, but not limited to, deletions can occur at complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publications WO 97 / 34631 and WO 96 / 32478. Additionally, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0086] Furthermore, Fc variants can be constructed to promote heterodimerization of desired heavy chain polypeptide subunits, for example, using a mortar and pestle structure or a KiH mutation. In some embodiments, the heavy chain polypeptide may contain one or more mortar and pestle mutations, such as Y349C, T366S, L368A, and / or Y407V, or any combination thereof. In some embodiments, the heavy chain polypeptide may contain one or more mortar and pestle mutations, such as S354C and / or T366W, or any combination thereof. Antibodies according to embodiments of the present invention can utilize any suitable combination of mortar and pestle residues to promote the formation of desired heterodimers.

[0087] Fc variants can also be constructed to promote half-life extension, for example, through enhanced FcRn binding. In some embodiments, the heavy chain polypeptide may include a T250Q mutation for this purpose. In some embodiments, the heavy chain polypeptide may include an M428L mutation for this purpose. In some embodiments, the heavy chain polypeptide may include both a T250Q mutation and an M428L mutation for this purpose.

[0088] Fc variants can also be constructed to facilitate improved purification procedures, such as through enhanced protein A binding. In some embodiments, the heavy chain polypeptide may include an H435R mutation for this purpose. In some embodiments, the heavy chain polypeptide may include a Y436F mutation for this purpose. In some embodiments, the heavy chain polypeptide may include both H435R and Y436F mutations for this purpose.

[0089] The term "Fc-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Therefore, antibodies containing an Fc region according to the present invention may include antibodies with or without K447.

[0090] The present invention includes antibodies having multispecific conformations, including but not limited to bispecific and trispecific conformations. Various methods and protein conformations are known and used for bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc.

[0091] Various methods for generating multivalent artificial antibodies have been developed by recombinantly fusing variable domains of two or more antibodies. In some embodiments, first and second antigen-binding domains on the peptide are linked by a peptide linker. A non-limiting example of such a peptide linker is the GS linker, which has an amino acid sequence of four glycine residues followed by one serine residue, wherein said sequence is repeated n times, where n is an integer in the range of 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO:84) (n=1) and GGGGSGGGGS (SEQ ID NO:85) (n=2). Further non-limiting examples of linkers include EPKSCDKTHT (SEQ ID NO:86) and EPKSSDKTHT (SEQ ID NO:87), which are derived from the natural hinge region of human IgG1. Other suitable adapters may also be used and are described, for example, in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10):1357-69, the contents of which are incorporated herein by reference in their entirety. Additional adapter sequences may be described elsewhere in this paper and may be incorporated into the subject antibody in any suitable configuration.

[0092] Antibodies (e.g., multispecific antibodies) as described herein may be in dimer form in which two heavy chains are bonded by disulfide bonds or otherwise covalently or non-covalently attached to each other, and may optionally include an asymmetric interface between two or more CH domains in the CH domain to facilitate proper pairing between polypeptide chains (commonly referred to as a "knobs-into-holes" interface). Techniques for engineering knobs-into-holes antibodies for heavy chain heterodimerization are discussed, for example, in Ridgway et al., Protein Eng. 1996 Jul; 9(7):17-21 and U.S. Patent No. 8,216,805, the disclosure of which is incorporated herein by reference in its entirety. Fc regions containing asymmetric interfaces may be referred to herein by the abbreviation "KiH," meaning knobs-into-holes. For example, aspects of the invention include variant Fc region sequences, such as the G1AAA sequence, which contain an asymmetric interface and are referred to herein as "G1AAA KiH."

[0093] As used herein, the term "LY6G6D" refers to G6D, a member of the lymphocyte antigen 6 family, which belongs to the leukocyte antigen-6 (LY6) gene cluster located in the major histocompatibility complex (MHC) class III region on chromosome 6. The term "LY6G6D" includes the LY6G6D protein in all human and non-human animal species, and specifically includes human LY6G6D and LY6G6D in non-human mammals.

[0094] As used herein, the term "human LY6G6D" includes any variant, isotype, and species homolog of human LY6G6D (UniProt A0A1U9X7Z8), regardless of its origin or preparation method. Therefore, "human LY6G6D" includes both naturally expressed human LY6G6D in cells and LY6G6D expressed on cells transfected with the human LY6G6D gene.

[0095] The terms “anti-LY6G6D antibody,” “LY6G6D antibody,” and “LY6G6D binding antibody” are used interchangeably in this document and refer to antibodies that specifically bind to LY6G6D, including human LY6G6D, as defined above.

[0096] The "percentage of amino acid sequence identity (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after sequence alignment and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. To determine the percentage of amino acid sequence identity, alignment can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this document, the sequence comparison computer program ALIGN-2 is used to generate the % amino acid sequence identity value.

[0097] "Isolated" antibodies are antibodies that have been identified, isolated, and / or recovered from components of their native environment. Contaminating components of their native environment are materials that will interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteins or non-protein solutes. In a preferred embodiment, the antibody is purified (1) to a weight greater than 95% by antibody weight, as determined by the Lowry method, and most preferably greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a turn-cup sequencer; or (3) to achieve homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibodies include in situ antibodies from recombinant cells, since at least one component of the antibody's native environment will be absent. However, typically, isolated antibodies are prepared by at least one purification step.

[0098] The antibodies of the present invention include multispecific antibodies. Multispecific antibodies have more than one binding specificity. The term "multispecific" specifically includes "bispecific" and "trispecific," as well as higher-order independent specific binding affinity, such as higher-order multi-epitope specificity, and tetravalent antibodies and antibody fragments. The term "multispecific antibody" is used herein in the broadest sense and covers all antibodies having more than one binding specificity. The multispecific anti-LY6G6D antibody of the present invention specifically includes antibodies (i.e., bivalent and bicomplementary sites) that are immune-specifically bound to two or more non-overlapping epitopes on the LY6G6D protein (such as human LY6G6D). The multispecific anti-LY6G6D antibody of the present invention also specifically includes antibodies (i.e., bivalent and bicomplementary sites) that are immune-specifically bound to epitopes on the LY6G6D protein (such as human LY6G6D) and epitopes on different proteins (such as, for example, CD3 proteins, such as human CD3). The multispecific anti-LY6G6D antibody of the present invention further specifically includes antibodies (i.e., trivalent and bicomplementary sites) that specifically bind to two or more non-overlapping or partially overlapping epitopes on the LY6G6D protein (such as human LY6G6D protein) and epitopes on different proteins (such as, for example, CD3 protein, such as human CD3 protein).

[0099] The antibodies of the present invention include monospecific antibodies having one binding specificity. Monospecific antibodies specifically include antibodies comprising a single binding specificity, as well as antibodies comprising more than one binding unit having the same binding specificity. The term "monospecific antibody" is used herein in the broadest sense and covers all antibodies having one binding specificity. The monospecific anti-LY6G6D antibody of the present invention specifically includes antibodies (monovalent and monospecific) that bind immune-specifically to one epitope on the LY6G6D protein (such as human LY6G6D). The monospecific anti-LY6G6D antibody of the present invention also specifically includes antibodies (e.g., multivalent antibodies) having more than one binding unit that binds immune-specifically to an epitope on the LY6G6D protein (such as human LY6G6D). For example, a monospecific antibody according to an embodiment of the present invention may comprise two variable regions each comprising an antigen-binding domain, wherein each antigen-binding domain binds to the same epitope on the LY6G6D protein (i.e., bivalent and monospecific).

[0100] An epitope is a site on the surface of an antigen molecule that binds to a single antibody molecule. Typically, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.

[0101] Epitope localization is the process of identifying the binding site or epitope of an antibody on its target antigen. Antibody epitopes can be linear epitopes or conformational epitopes. Linear epitopes are formed from a continuous amino acid sequence in a protein. Conformational epitopes are formed from discontinuous amino acids in a protein sequence, but aggregated together when the protein folds into its three-dimensional structure.

[0102] "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. As described above, the present invention specifically includes anti-LY6G6D antibodies with multi-epitope specificity, namely, anti-LY6G6D antibodies that bind to one or more non-overlapping epitopes on the LY6G6D protein (such as human LY6G6D); and anti-LY6G6D antibodies that bind to one or more epitopes on the LY6G6D protein and epitopes on different proteins (such as, for example, CD3 protein). The terms "non-overlapping epitope" or "non-competitive epitope" of an antigen are defined herein as meaning an epitope recognized by one member of a pair of antigen-specific antibodies rather than the other. The antigen-binding regions on paired antibodies or multi-specific antibodies that recognize non-overlapping epitopes and target the same antigen do not compete for binding to that antigen and are capable of binding to the antigen simultaneously.

[0103] When two antibodies recognize the same or spatially overlapping epitopes, the antibody binds to "substantially the same epitope" as the reference antibody. The most widely used and rapid method for determining whether two epitopes bind to the same or spatially overlapping epitopes is a competitive assay, which can be configured using labeled antigens or labeled antibodies in all number of different forms. Typically, the antigen is immobilized on a 96-well plate, and radioactive or enzyme labeling is used to measure the ability of the unlabeled antibody to block the binding of the labeled antibody.

[0104] As used in this article, the term "valence" refers to a specified number of binding sites in an antibody molecule.

[0105] A monovalent antibody has only one binding site. Therefore, a monovalent antibody is also monospecific.

[0106] "Multivalent" antibodies have two or more binding sites. Therefore, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two, three, and four binding sites, respectively. Thus, the bispecific antibody according to the invention is at least bivalent and can be trivalent, tetravalent, or otherwise multivalent. A bivalent antibody according to an embodiment of the invention can have two binding sites targeting the same epitope (i.e., bivalent, monocomplementary) or two different epitopes (i.e., bivalent, bicomplementary).

[0107] Various methods and protein conformations are known and used to prepare bispecific monoclonal antibodies (BsMAB), trispecific antibodies, etc.

[0108] The term “chimeric antigen receptor” or “CAR” is used in its broadest sense herein to refer to an engineered receptor that has the desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) transplanted into transmembrane and intracellular signaling domains. Typically, the specificity of a monoclonal antibody is transplanted onto T cells using said receptor to create a chimeric antigen receptor (CAR). (J Natl Cancer Inst, 2015; 108(7):dvj439; and Jackson et al., Nature Reviews Clinical Oncology, 2016; 13:370–383). CAR-T cells are T cells that have been genetically engineered to produce artificial T cell receptors for immunotherapy. In one embodiment, “CAR-T cell” means a therapeutic T cell expressing a transgene that encodes one or more chimeric antigen receptors, which at least include an extracellular domain, a transmembrane domain, and at least one cytoplasmic domain.

[0109] The term "human antibody" is used herein to refer to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies as used herein may contain amino acid residues not encoded by human germline immunoglobulin sequences, for example, mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo.

[0110] "Chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule containing amino acid sequences from at least two different Ig loci, such as a transgenic antibody containing a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies having a non-human or artificial Fc-region and a human idiotype. Such immunoglobulins can be isolated from the animals of this invention, which have been engineered to produce such chimeric antibodies.

[0111] As used herein, the term "effective cell" refers to immune cells that participate in the effector phase of an immune response (as opposed to the cognitive and activation phases of the immune response). Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells (such as natural killer cells) are capable of inducing antibody-dependent cytotoxicity (ADCC). For example, monocytes and macrophages expressing Fc receptors participate in the specific killing of target cells and the presentation of antigens to other components of the immune system, or bind to antigen-presenting cells. In some embodiments, effector daughter cells may phagocytose target antigens or target cells.

[0112] "Human effector cells" are leukocytes that express receptors (such as T-cell receptors or FcRs) and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; among which NK cells are preferred. Effector cells can be isolated from their natural sources, such as blood or PBMCs as described herein.

[0113] The term “immune cells” is used in the broadest sense herein, including but not limited to cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, and polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0114] Antibody "effector functions" refer to those biological activities attributable to the Fc region of the antibody (either the native Fc region or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR).

[0115] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells and subsequently cause the lysis of the target cells. The primary cells used to mediate ADCC (NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of molecules of interest, in vitro ADCC assays, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337, can be performed. Effector cells useful for such assays include peripheral blood monocytes (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in animal models (such as the animal models disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998)).

[0116] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to cleave its target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (C1q) of the complement system to a molecule (e.g., an antibody) that has a complex with a homologous antigen. To assess complement activation, a CDC assay can be performed, for example, as described by Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0117] "Binding affinity" refers to the strength of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigens slowly and tend to dissociate rapidly, while high-affinity antibodies generally bind antigens more quickly and tend to maintain the binding.

[0118] As used herein, “Kd” or “Kd value” refers to the dissociation constant determined by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetic mode. For example, an anti-mouse Fc sensor was loaded with mouse-Fc fusion antigen and then immersed in wells containing antibody to measure the concentration-dependent association rate (k-association). In the final step, the antibody dissociation rate (k-dissociation) was measured, in which the sensor was immersed in wells containing only buffer. Kd is the ratio of k-dissociation to k-association. (For more details, see Concepcion, J et al., Comb Chem HighThroughput Screen, 12(8), 791-800, 2009).

[0119] The terms “treatment”, “treating,” etc., are generally used herein to mean achieving the desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of the disease or its symptoms and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects attributable to the disease. As used herein, “treatment” encompasses any treatment of mammalian diseases and includes: (a) preventing the disease from occurring in subjects who are susceptible to disease but have not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development; or (c) alleviating the disease, i.e., causing disease remission. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Of particular interest is the treatment of an ongoing disease in which the treatment stabilizes or reduces unwanted clinical symptoms in the patient. It is desirable to perform such treatment before complete loss of function of the affected tissue. Subjective therapies may be administered during the symptomatic phase of the disease and, in some cases, after the symptomatic phase of the disease.

[0120] "Therapeutic effective amount" refers to the amount of active agent necessary for a subject to impart a therapeutic benefit. For example, "therapeutic effective amount" is the amount that induces, improves, or otherwise causes improvement in pathological symptoms, disease progression, or physiological condition associated with a disease, or increases resistance to the condition.

[0121] As used interchangeably in this document, the terms “cancer,” “tumor,” “cancerous,” and “malignant” refer to or describe a physiological disorder in mammals characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinomas, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia.

[0122] The terms “blood cancer,” “blood malignancy,” and “blood cancer” are used interchangeably herein to refer to cancers that originate in blood-forming tissues (including, but not limited to, bone marrow and / or immune system cells). Non-limiting examples of blood cancers include leukemia, lymphoma, myeloma, or myelodysplastic syndromes. In some embodiments, leukemia is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL). In some embodiments, lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma.

[0123] The term "characterized by LY6G6D expression" refers to any disease or condition in which LY6G6D expression is associated with or involved in one or more pathological processes characteristic of the disease or condition. Such conditions include, but are not limited to, colorectal cancer.

[0124] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to a mammal that is being evaluated for and / or undergoing treatment. In the implementation plan, the mammal is a human. The terms “subject,” “individual,” and “patient” cover, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly those that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0125] The term "pharmaceutical formulation" refers to a preparation in a form that allows the bioactivity of the active ingredient to be effective and that does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (carriers, additives) are those that can be reasonably administered to a test mammal to provide an effective dose of the active ingredient used.

[0126] "Sterile" preparations are sterile or contain no or substantially no live microorganisms and their spores. "Frozen" preparations are preparations kept at temperatures below 0°C.

[0127] A “stable” formulation is one in which the protein substantially retains its physical and / or chemical stability and / or biological activity during storage. Preferably, the formulation substantially retains its physical and chemical stability, as well as its biological activity, during storage. The storage period is typically selected based on the expected shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in *Peptide and Protein Drug Delivery*, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and *Jones. A. Adv. Drug Delivery Rev. 10:29-90* (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be assessed qualitatively and / or quantitatively in a variety of ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity and / or by visual inspection); assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary band electrophoresis; N-terminal or C-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessing antibody bioactivity or antigen-binding function; etc. Instability may involve one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), cleavage / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine ​​residues, N-terminal extension, C-terminal processing, differential glycosylation, etc.

[0128] II. Detailed description

[0129] Anti-LY6G6D antibodies

[0130] This invention provides a family of closely related antibodies that bind to human LY6G6D. The antibodies in this family comprise a set of CDRH sequences as defined herein and shown in Table 1, and a set of CDRL sequences as defined herein and shown in Table 2.

[0131] Anti-LY6G6D antibodies according to embodiments of the present invention are exemplified by the provided heavy chain variable region (VH) sequences listed in Table 3 and the provided light chain variable region (VL) sequences listed in Table 4. This antibody family provides numerous benefits contributing to its utility as one or more clinical therapeutic agents. The antibodies include members having a range of binding affinities, thereby allowing selection of specific sequences having the desired binding affinity.

[0132] Table 1: Amino acid sequence of anti-LY6G6D antibody CDRH.

[0133]

[0134] Table 2: Amino acid sequence of anti-LY6G6D antibody CDRL.

[0135]

[0136]

[0137] Table 3. Amino acid sequences of the variable domain of the anti-LY6G6D heavy chain.

[0138]

[0139] Table 4. Amino acid sequences of the variable domain of the anti-LY6G6D light chain.

[0140]

[0141]

[0142] Suitable antibodies may be selected from those provided herein for development and therapeutic or other uses, including but not limited to those used as bispecific antibodies, such as... Figure 4 As shown; or part of a CAR-T structure. Figure 4 This is a schematic diagram of an anti-LY6G6D x anti-CD3ε multispecific antibody, wherein the anti-LY6G6D binding region is located at the N-terminus of the polypeptide subunit, and the CD3ε binding unit is in the form of scFv and is located on one of the heavy chain polypeptide subunits between the variable region and the hinge region. In some embodiments, the two heavy chain polypeptide subunits of the heterodimeric multispecific antibody are paired using, for example, a mortar and pestle (KiH) technique.

[0143] Affinity determination for candidate proteins can be performed using methods known in the art, such as the Biacore measurement. Members of the antibody family exhibit affinity for LY6G6D with a Kd of approximately 10. -6 Up to approximately 10 -11 Including but not limited to: approximately 10 -6 Up to approximately 10 -10 Approximately 10 -6 Up to approximately 10 -9 Approximately 10 -6 Up to approximately 10 -8 Approximately 10 -8 Up to approximately 10 -11 Approximately 10 -8 Up to approximately 10 -10 Approximately 10 -8 Up to approximately 10 -9Approximately 10 -9 Up to approximately 10 -11 Approximately 10 -9 Up to approximately 10 -10 Or any value within these ranges. Affinity selection can be confirmed through biological assessments used to modulate (e.g., block) the biological activity of LY6G6D, including in vitro assays, preclinical models and clinical trials, and assessments of potential toxicity.

[0144] Members of the antibody family described in this article cross-react with the LY6G6D protein of cynomolgus monkeys and can be engineered, if necessary, to provide cross-reactivity with the LY6G6D protein of any other animal species.

[0145] The anti-LY6G6D antibody family described herein comprises one or more binding units containing a VH domain, including CDRH1, CDRH2, and CDRH3 sequences in the human VH framework, and CDRL1, CDRL2, and CDRL3 sequences in the human Vκ or Vλ framework. As an example, for CDR1, CDR2, and CDR3, the CDR sequences may be located in the regions of approximately amino acid residues 26-33; 51-58; and 97-116 of the exemplary variable region sequences provided in Tables 3 and 4, respectively. Those skilled in the art will understand that the CDR sequences may be located in different positions if different framework sequences are chosen, although the sequence order will generally remain unchanged.

[0146] In some embodiments, the anti-LY6G6D antibody comprises the CDRH1 sequence of any one of SEQ ID NO:1-6. In some embodiments, the anti-LY6G6D antibody comprises the CDRH2 sequence of any one of SEQ ID NO:7-11. In some embodiments, the anti-LY6G6D antibody comprises the CDRH3 sequence of any one of SEQ ID NO:12-15.

[0147] In one preferred embodiment, the anti-LY6G6D antibody comprises the CDRH1 sequence of SEQ ID NO:1, the CDRH2 sequence of SEQ ID NO:7, and the CDRH3 sequence of SEQ ID NO:12. In another preferred embodiment, the anti-LY6G6D antibody comprises the CDRH1 sequence of SEQ ID NO:2, the CDRH2 sequence of SEQ ID NO:8, and the CDRH3 sequence of SEQ ID NO:13. In yet another preferred embodiment, the anti-LY6G6D antibody comprises the CDRH1 sequence of SEQ ID NO:2, the CDRH2 sequence of SEQ ID NO:8, and the CDRH3 sequence of SEQ ID NO:13. In yet another preferred embodiment, the anti-LY6G6D antibody comprises the CDRH1 sequence of SEQ ID NO:3, the CDRH2 sequence of SEQ ID NO:9, and the CDRH3 sequence of SEQ ID NO:13. In yet another preferred embodiment, the anti-LY6G6D antibody comprises the CDRH1 sequence of SEQ ID NO:4, the CDRH2 sequence of SEQ ID NO:10, and the CDRH3 sequence of SEQ ID NO:14. In one preferred embodiment, the anti-LY6G6D antibody comprises the CDRH1 sequence of SEQ ID NO:5, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15. In another preferred embodiment, the anti-LY6G6D antibody comprises the CDRH1 sequence of SEQ ID NO:6, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15.

[0148] In some embodiments, the anti-LY6G6D antibody comprises the CDRL1 sequence of any one of SEQ ID NO:16-19. In some embodiments, the anti-LY6G6D antibody comprises the CDRL2 sequence of any one of SEQ ID NO:20-23. In some embodiments, the anti-LY6G6D antibody comprises the CDRL3 sequence of any one of SEQ ID NO:24-26.

[0149] In one preferred embodiment, the anti-LY6G6D antibody comprises the CDRL1 sequence of SEQ ID NO:16, the CDRL2 sequence of SEQ ID NO:20, and the CDRL3 sequence of SEQ ID NO:24. In one preferred embodiment, the anti-LY6G6D antibody comprises the CDRL1 sequence of SEQ ID NO:17, the CDRL2 sequence of SEQ ID NO:21, and the CDRL3 sequence of SEQ ID NO:25. In one preferred embodiment, the anti-LY6G6D antibody comprises the CDRL1 sequence of SEQ ID NO:18, the CDRL2 sequence of SEQ ID NO:22, and the CDRL3 sequence of SEQ ID NO:24. In one preferred embodiment, the anti-LY6G6D antibody comprises the CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:26.

[0150] In a preferred embodiment, the anti-LY6G6D antibody comprises a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:1, the CDRH2 sequence of SEQ ID NO:7, and the CDRH3 sequence of SEQ ID NO:12; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:16, the CDRL2 sequence of SEQ ID NO:20, and the CDRL3 sequence of SEQ ID NO:24.

[0151] In a preferred embodiment, the anti-LY6G6D antibody comprises a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:2, the CDRH2 sequence of SEQ ID NO:8, and the CDRH3 sequence of SEQ ID NO:13; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:17, the CDRL2 sequence of SEQ ID NO:21, and the CDRL3 sequence of SEQ ID NO:25.

[0152] In a preferred embodiment, the anti-LY6G6D antibody comprises a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:3, the CDRH2 sequence of SEQ ID NO:9, and the CDRH3 sequence of SEQ ID NO:13; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:17, the CDRL2 sequence of SEQ ID NO:21, and the CDRL3 sequence of SEQ ID NO:25.

[0153] In a preferred embodiment, the anti-LY6G6D antibody comprises a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:4, the CDRH2 sequence of SEQ ID NO:10, and the CDRH3 sequence of SEQ ID NO:14; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:18, the CDRL2 sequence of SEQ ID NO:22, and the CDRL3 sequence of SEQ ID NO:24.

[0154] In a preferred embodiment, the anti-LY6G6D antibody comprises a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:5, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:26.

[0155] In a preferred embodiment, the anti-LY6G6D antibody comprises a heavy chain variable region comprising: the CDRH1 sequence of SEQ ID NO:6, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15; and a light chain variable region comprising: the CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:26.

[0156] In some embodiments, the anti-LY6G6D antibody comprises any one of the heavy chain variable region amino acid sequences of SEQ ID NO:27-34 (Table 3). In some embodiments, the anti-LY6G6D antibody comprises a heavy chain variable region amino acid sequence having at least about 80% identity with the sequence of any one of SEQ ID NO:27-34 (Table 3), such as about 85%, 90%, 95%, 99%, or 100% identity.

[0157] In some embodiments, the anti-LY6G6D antibody comprises any one of the light chain variable region amino acid sequences of SEQ ID NO:35-42 (Table 4). In some embodiments, the anti-LY6G6D antibody comprises a light chain variable region amino acid sequence having at least about 80% identity with the sequence of any one of SEQ ID NO:35-42 (Table 4), such as about 85%, 90%, 95%, 99%, or 100% identity.

[0158] In some embodiments, the anti-LY6G6D antibody comprises the heavy chain variable region sequence of SEQ ID NO:27 and the light chain variable region sequence of SEQ ID NO:35. In some embodiments, the anti-LY6G6D antibody comprises the heavy chain variable region sequence of SEQ ID NO:28 and the light chain variable region sequence of SEQ ID NO:36. In some embodiments, the anti-LY6G6D antibody comprises the heavy chain variable region sequence of SEQ ID NO:29 and the light chain variable region sequence of SEQ ID NO:37. In some embodiments, the anti-LY6G6D antibody comprises the heavy chain variable region sequence of SEQ ID NO:30 and the light chain variable region sequence of SEQ ID NO:38. In some embodiments, the anti-LY6G6D antibody comprises the heavy chain variable region sequence of SEQ ID NO:31 and the light chain variable region sequence of SEQ ID NO:39. In some embodiments, the anti-LY6G6D antibody comprises the heavy chain variable region sequence of SEQ ID NO:32 and the light chain variable region sequence of SEQ ID NO:40. In some embodiments, the anti-LY6G6D antibody comprises the heavy chain variable region sequence of SEQ ID NO:33 and the light chain variable region sequence of SEQ ID NO:41. In some embodiments, the anti-LY6G6D antibody comprises the heavy chain variable region sequence of SEQ ID NO:34 and the light chain variable region sequence of SEQ ID NO:42.

[0159] In some embodiments, the CDR sequence in the anti-LY6G6D antibody of the present invention contains one or two amino acid substitutions relative to the CDR1, CDR2 and / or CDR3 sequences or the set of CDR1, CDR2 and CDR3 sequences in Table 1 or Table 2.

[0160] In some embodiments, the anti-LY6G6D antibody preferably comprises a heavy chain variable domain (VH) wherein the CDRH3 sequence has greater than or equal to 80%, such as at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity at the amino acid level with the CDRH3 sequence of any antibody provided in Table 1, and binds to LY6G6D.

[0161] In some embodiments, the anti-LY6G6D antibody preferably comprises a heavy chain variable domain (VH) wherein the complete set of CDRH 1, 2 and 3 (combination) has greater than or equal to 85% sequence identity at the amino acid level with the CDRH sequences of the antibody provided in Table 1, and binds to LY6G6D.

[0162] In some embodiments, bispecific or multispecific antibodies are provided, which may have any conformation discussed herein, including but not limited to multispecific antibodies comprising one or more binding units located on a heavy chain polypeptide subunit and positioned between an N-terminal binding unit and a hinge region, such as, for example Figure 4 As shown in the diagram. In some embodiments, this binding unit may comprise an scFv conformation, wherein the VH and VL regions are linked by a linker sequence and together form a binding unit that binds to a target protein (e.g., CD3).

[0163] In some embodiments, a multispecific antibody may comprise a first binding unit that binds to LY6G6D, the first binding unit comprising a heavy chain variable region paired with a light chain variable region; and a second binding unit that binds to a protein other than LY6G6D, the second binding unit comprising a heavy chain variable region paired with a light chain variable region. In some embodiments, a multispecific antibody may comprise first and second binding units, each comprising a heavy chain variable region paired with a light chain variable region, and both binding units binding to LY6G6D; and at least a third binding unit that binds to a protein other than LY6G6D (e.g., CD3). In some embodiments, the third binding unit is located at the C-terminus of one of the light chain polypeptide subunits constituting the antibody. In some embodiments, the third binding unit is located within one of the heavy chain polypeptide subunits, for example, between the variable region and the hinge region of the polypeptide subunit, such as... Figure 4 As depicted in the diagram. In some embodiments, the third binding unit includes an scFv, wherein the heavy chain variable region is connected to the light chain variable region via a connector sequence.

[0164] In some embodiments, the anti-LY6G6D antibody described herein includes a heavy chain constant region sequence, such as CH1, hinge, CH2, CH3, and / or CH4 domains. In some embodiments, the anti-LY6G6D antibody described herein includes a light chain constant region sequence, such as a CL domain. In some embodiments, the anti-LY6G6D antibody described herein includes a heavy chain constant region sequence forming an Fc region.

[0165] As described above, in some embodiments, the multispecific anti-LY6G6D antibody comprises a binding unit that binds to proteins other than LY6G6D, such as, for example, CD3 (e.g., CD3ε). In some embodiments, the CD3 binding unit comprises a set of CDRH sequences as defined herein and shown in Table 5 and a set of CDRL sequences as defined herein and shown in Table 6.

[0166] Table 5: CD3 heavy chain CDR sequences:

[0167]

[0168] Table 6: CD3 light chain CDR sequences:

[0169]

[0170] In some implementations, the CD3 binding unit comprises the heavy chain variable region (VH) sequence listed in Table 7 and the light chain variable region (VL) sequence listed in Table 8. These CD3 binding units provide numerous benefits that contribute to their utility as one or more clinical therapeutic agents.

[0171] Table 7: CD3 Heavy Chain Variable Region Sequence

[0172]

[0173] Table 8: CD3 light chain variable region sequence

[0174]

[0175]

[0176] The multispecific anti-LY6G6D antibody according to an embodiment of the present invention may comprise one or more CD3 binding units, as described herein. Any binding domain of the anti-LY6G6D antibody described herein may be combined with the CD3 binding domain described herein to produce a multispecific antibody that binds to LY6G6D and CD3. Full-length sequences that can be used to constitute anti-LY6G6DxCD3 antibodies are provided in Table 9.

[0177] Table 9: Full-length polypeptide subunit sequence of LY6G6D x CD3 antibody

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] In a preferred embodiment, the multispecific antibody binds to LY6G6D and CD3ε and comprises: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:54; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:56.

[0185] In a preferred embodiment, the multispecific antibody binds to LY6G6D and CD3ε and comprises: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:55; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:56.

[0186] In a preferred embodiment, the multispecific antibody binds to LY6G6D and CD3ε and comprises: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:58; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:60.

[0187] In a preferred embodiment, the multispecific antibody binds to LY6G6D and CD3ε and comprises: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:59; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:60.

[0188] In a preferred embodiment, the multispecific antibody binds to LY6G6D and CD3ε and comprises: a first light chain subunit comprising SEQ ID NO:57; a first heavy chain subunit comprising SEQ ID NO:61; a second light chain subunit comprising SEQ ID NO:57; and a second heavy chain subunit comprising SEQ ID NO:62.

[0189] In a preferred embodiment, the multispecific antibody binds to LY6G6D and CD3ε and comprises: a first light chain subunit comprising SEQ ID NO:69; a first heavy chain subunit comprising SEQ ID NO:64; a second light chain subunit comprising SEQ ID NO:69; and a second heavy chain subunit comprising SEQ ID NO:66.

[0190] In a preferred embodiment, the multispecific antibody binds to LY6G6D and CD3ε and comprises: a first light chain subunit comprising SEQ ID NO:69; a first heavy chain subunit comprising SEQ ID NO:65; a second light chain subunit comprising SEQ ID NO:69; and a second heavy chain subunit comprising SEQ ID NO:66.

[0191] In a preferred embodiment, the multispecific antibody binds to LY6G6D and CD3ε and comprises: a first light chain subunit comprising SEQ ID NO:69; a first heavy chain subunit comprising SEQ ID NO:67; a second light chain subunit comprising SEQ ID NO:69; and a second heavy chain subunit comprising SEQ ID NO:68.

[0192] Various forms of multispecific antibodies are within the scope of this invention, including but not limited to single-chain polypeptides, double-chain polypeptides, triple-chain polypeptides, quadruple-chain polypeptides, and their polyploids. Specifically, multispecific antibodies as used herein include T cell multispecific (e.g., bispecific) antibodies (anti-LY6G6D x anti-CD3ε antibodies) that bind to LY6G6D and CD3ε. Such antibodies induce effective T cell-mediated killing of LY6G6D-expressing cells.

[0193] Preparation of anti-LY6G6D antibodies

[0194] The antibodies of the present invention can be prepared by methods known in the art, such as recombinant DNA technology, by expressing the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (e.g., CHO cells), Escherichia coli, or yeast.

[0195] Antibodies binding to non-overlapping epitopes on the LY6G6D protein can be identified using competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA) or flow cytometry competitive binding assays. For example, competition between a known antibody binding to a target antigen and a target antibody can be used. By using this method, the set of antibodies can be divided into those that compete with a reference antibody and those that do not. Non-competitive antibodies are identified as binding to unique epitopes that do not overlap with the epitopes bound by the reference antibody. Typically, an antibody is immobilized, an antigen is bound, and the ability of a second labeled (e.g., biotinylated) antibody to bind the captured antigen is tested in an ELISA assay. This can also be performed using surface plasmon resonance (SPR) platforms, including the ProteOn XPR36 (BioRad, Inc.), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and the MX96 SPR imager (Ibis technologies BV), as well as biolayer interferometry platforms such as the Octet Red384 and Octet HTX (ForteBio, Pall Inc.).

[0196] Typically, if, as measured by standard techniques such as the competitive binding assay described above, the antibody causes a reduction of approximately 15%–100% in the binding of the reference antibody to the target antigen, then it is considered to be “competing” with the reference antibody. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or higher.

[0197] Pharmaceutical compositions, uses, and methods of treatment

[0198] Another aspect of the invention provides pharmaceutical compositions comprising a mixture of one or more antibodies of the invention with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers as used herein are, for example, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers in the art for holding therapeutic components, or combinations thereof.

[0199] In one embodiment, the pharmaceutical composition comprises an antibody that binds to LY6G6D. In another embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) antibody that binds to two or more non-overlapping epitopes on the LY6G6D protein. In a preferred embodiment, the pharmaceutical composition comprises a multispecific (including bispecific) antibody that binds to LY6G6D and effector cells (e.g., binding targets on T cells, such as, for example, the CD3ε protein on T cells).

[0200] The pharmaceutical composition of the antibody used according to the invention is prepared for storage, such as in the form of a lyophilized formulation or an aqueous solution, by mixing a protein of desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. editor (1980)).

[0201] Pharmaceutical compositions intended for parenteral administration are preferably sterile and substantially isotonic, and manufactured under Good Manufacturing Practices (GMP) conditions. The pharmaceutical compositions may be provided in unit dosage forms (i.e., doses for a single administration). The formulation depends on the chosen route of administration. The antibodies described herein may be administered intravenously, by infusion, or subcutaneously. For injection administration, the antibodies described herein may be formulated in an aqueous solution, preferably in a physiologically compatible buffer solution.

[0202] For example, antibody formulations are disclosed in U.S. Patent No. 9,034,324. Similar formulations can be used for the heavy chain antibodies of this invention, including UniAbs.TM Subcutaneous antibody preparations are described, for example, in US20160355591 and US20160166689.

[0203] Methods of use

[0204] The anti-LY6G6D antibody and pharmaceutical composition described herein may be used to treat diseases and disorders characterized by the expression of LY6G6D, including but not limited to those further described herein.

[0205] LY6G6D belongs to the leukocyte antigen-6 (LY6) gene cluster located in the major histocompatibility complex (MHC) class III region on chromosome 6. Members of the LY6 superfamily typically contain 70 to 80 amino acids, including 8 to 10 cysteine ​​residues. Most LY6 proteins attach to the cell surface via a glycosylphosphatidylinositol (GPI) anchor, which is directly involved in signal transduction.

[0206] RNA expression analysis from the TCGA database showed that LY6G6D is highly expressed and specific for colorectal cancer. Its expression in other tumor types and normal cells is negligible, making it a highly promising tumor target. Therefore, antibody therapy development targeting this target could be effective in treating certain groups of colorectal cancer patients refractory to chemotherapy or immune checkpoint therapy.

[0207] In one aspect, the anti-LY6G6D antibody and pharmaceutical composition described herein can be used to treat conditions characterized by LY6G6D expression, including but not limited to colorectal cancer. In a preferred embodiment, the colorectal cancer is refractory colorectal cancer, wherein the patient receiving treatment has previously received another therapy (e.g., a chemotherapy regimen) and has not achieved remission of the colorectal cancer. In a preferred embodiment, the colorectal cancer is refractory colorectal cancer, wherein the patient receiving treatment has previously received another therapy (e.g., checkpoint inhibitor therapy) and has not achieved remission of the colorectal cancer.

[0208] The effective dosage of the compositions of the present invention for treating diseases varies depending on a number of different factors, including the means of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is preventative or therapeutic. Typically, the patient is human, but non-human mammals such as companion animals (e.g., dogs, cats, horses), laboratory mammals (e.g., rabbits, mice, rats), etc., can also be treated. The therapeutic dosage can be titrated to optimize safety and efficacy.

[0209] Typically, compositions are prepared as injectable liquid solutions or suspensions; they may also be prepared as solid forms suitable for dissolution or suspension in a liquid medium prior to injection. The pharmaceutical compositions described herein may be administered directly or after reconstitution of a solid (e.g., lyophilized) composition for intravenous or subcutaneous administration. The preparations may also be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolic acid, or copolymers, to achieve an enhanced adjuvant effect, as discussed above. (Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997). The pharmaceutical preparations of the present invention may be administered as reservoir injections or implant preparations, which may be formulated to allow for sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practices (GMP) regulations of the U.S. Food and Drug Administration.

[0210] The toxicity of the antibodies and antibody structures described herein can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals, for example, by determining the LD50 (the dose that is lethal to 50% of the population) or LD100 (the dose that is lethal to 100% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate dose ranges that are non-toxic for use in humans. The dosage of the antibodies described herein is preferably within a range of circulating concentrations, which includes effective doses with minimal or no toxicity. The dosage may vary within this range depending on the dosage form and route of administration used. The exact formulation, route of administration, and dosage can be selected by an individual physician based on the patient's condition.

[0211] Compositions intended for administration typically contain antibodies or other ablative agents dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as buffered physiological saline. These solutions are sterile and generally free of unwanted substances. These compositions can be sterilized using conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable adjuvants close to physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and will be selected based primarily on fluid volume, viscosity, body weight, etc., depending on the specific administration modality chosen and the patient's needs (e.g., Remington's Pharmaceutical Science (15th edition, 1980) and Goodman and Gillman, The Pharmacological Basis of Therapeutics (edited by Hardman et al., 1996)).

[0212] Kits containing the active agents and formulations of the present invention, along with instructions for use, are also within the scope of this invention. The kits may also contain at least one additional agent, such as a chemotherapeutic agent, a checkpoint inhibitor, etc. Kits typically include a label indicating the intended use of the kit contents. As used herein, the term "label" includes any written or recorded material provided on or with the kit.

[0213] The present invention includes diagnostic assays, such as diagnostic immunoassays, which can be used to detect the presence of LY6G6D in a test sample. Immunoassays for detecting LY6G6D can be configured in various ways. Immunoassays may include homogeneous and heterogeneous assays, competitive and non-competitive assays, direct and indirect assays, and "sandwich" assays. Useful assays include, but are not limited to, enzyme immunoassays, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), electrochemiluminescence assay, radioimmunoassay, immunofluorescence assay, fluorescence anisotropy assay, immunoprecipitation assay, equilibrium dialysis assay, immunodiffusion assay, immunoblotting assay, agglutination assay, luminescent proximity assay, and turbidimetric assay.

[0214] Regardless of the assay method used, the biological sample is brought into contact with the anti-LY6G6D antibody of the present invention. In some embodiments, the biological sample may be immobilized on a solid support (e.g., a histological slide). In some embodiments, the biological sample is brought into contact with the anti-LY6G6D antibody of the present invention immobilized on a solid support. The solid support may be, for example, a plastic surface, a glass surface, a paper or fiber surface, or a particulate surface. More specifically, the support may include microplates, beads, polyvinylidene fluoride (PVDF) membranes, nitrocellulose membranes, nylon membranes, porous membranes, non-porous membranes, or any equivalent thereof. The composition of the substrate may vary. For example, the substrate or support may include glass, cellulose-based materials, thermoplastic polymers (such as polyethylene, polypropylene, or polyester), sintered structures composed of particulate materials (e.g., glass or various thermoplastic polymers), or cast films composed of nitrocellulose, nylon, or polysulfone. In general embodiments, the substrate can be any surface or support on which the antibody or peptide can be immobilized, including one or more of solid supports (e.g., glass, such as a slide or coated plate, silica, plastic or derivatized plastic, paramagnetic or nonmagnetic metals), semi-solid supports (e.g., polymeric materials, gels, agarose, or other matrices), and / or porous supports (e.g., filters, nylon or nitrocellulose membranes, or other membranes). In some embodiments, synthetic polymers may be used as the substrate, including, for example, polystyrene, polypropylene, polyglycidyl methacrylate, aminated or carboxylated polystyrene, polyacrylamide, polyamide, and polyvinyl chloride.

[0215] In some embodiments, the immunoassay may be a two-antibody "sandwich" assay. A biological sample is contacted with the anti-LY6G6D antibody of the present invention immobilized on a solid support, such as a microtiter plate. The sample and the first antibody are incubated under conditions favorable to specific binding and the formation of the LY6G6D-antibody complex. After the contact step, unbound components of the biological sample are removed. The complex is then contacted with a second antibody. In some embodiments, the second antibody is an anti-LY6G6D antibody. The second antibody may bind to an LY6G6D epitope different from the epitope bound by the first antibody. Therefore, the first and second antibodies do not competitively inhibit each other's binding to LY6G6D. In some embodiments, the second antibody may bind to a portion of the first antibody to facilitate detection.

[0216] Antibody binding can be measured in a variety of ways. For example, the signal generated by the detectable label can be analyzed and quantified (if applicable) using an optical scanner or other image acquisition device and software that allows the measurement of the signal, such as a fluorescence, luminescence, phosphorescence, or radioactivity signal associated with complex formation. Exemplary instruments for measuring the detectable signal may include, but are not limited to, microplate readers, fluorometers, spectrophotometers, and gamma counters.

[0217] The level of LY6G6D in biological samples can be compared with the level in a reference sample. A standard reference level typically represents the mean LY6G6D level derived from an individual population. A reference population may include individuals of similar age, body size, ethnic background, or general health status to the individual in question. Therefore, the LY6G6D level in a patient sample can be compared with values ​​derived from: 1) individuals known to have colorectal cancer who express LY6G6D and whose body fluids contain LY6G6D; and 2) individuals who do not have colorectal cancer and whose body fluids contain low levels of LY6G6D.

[0218] In some implementations, a reference chart can be used to determine whether a specific level of LY6G6D in a sample is elevated relative to a control sample or a larger population. For example, the reference chart may contain the normal range of LY6G6D found in healthy individuals with the same age, ethnic background, or general health status as the individual in question. Using this reference chart, any LY6G6D level measured in the sample can be classified as low, normal, or elevated relative to a control sample or relative to the mean from a larger population. The term "elevated level" is defined as a level that is at least 2% higher, preferably at least 5% higher than a reference level.

[0219] Alternatively or additionally, the level of LY6G6D in a biological sample can be "normalized" against the levels of one or more other biomarkers (e.g., another biomarker whose expression is independent of LY6G6D expression). That is, the levels of the other biomarker can be assessed in parallel with the level of LY6G6D, simultaneously or at separate times. The other biomarker can serve as an internal control for sample preparation, processing, and storage, as well as for routine determination of variability. The values ​​of LY6G6D levels and the other biomarker can be expressed as a ratio, and said ratio can be compared with similar ratios obtained against a reference sample or population. A useful second biomarker could be alpha-fetoprotein (AFP).

[0220] The diagnostic methods disclosed herein can be used to detect colorectal cancer in patients suspected of having colorectal cancer or at risk of developing colorectal cancer. These methods can also be used to analyze samples from patients who have received treatment for colorectal cancer to determine if the patient is at risk of experiencing recurrent or refractory colorectal cancer. The methods can also be used to monitor treatment processes, such as treatment with therapeutic agents like chemotherapy, checkpoint inhibitor therapy, radiation therapy, or surgery, to determine the efficacy of treatment and allow managing clinicians to modify treatment as needed. These methods can also be used to detect, monitor, or analyze patients with any condition associated with regulation (e.g., increased) of LY6G6D levels in biological samples (e.g., blood or serum samples obtained from the patient) or at risk of developing said condition.

[0221] The compositions described herein can be packaged in suitable containers labeled, for example, for the detection, identification, and quantification of LY6G6D in biological samples. The product (also referred to as a “kit” or “assay kit”) may contain the antibodies of the present invention, antigen-binding fragments of the antibodies, or any combination thereof, as well as culture media, purified antigen samples used as positive controls, or any combination thereof. Containers included in the kit may include compositions containing antibodies of the present invention, which specifically or preferentially bind to LY6G6D. Suitable buffers for diluting or reconstituted test samples and antibodies may also be provided. Some components may be provided in dry form and may require reconstituted. Anti-LY6G6D antibodies may be pre-bound to an assay device, such as a microplate. Thus, in one embodiment, a kit for the detection, identification, and quantification of LY6G6D includes anti-LY6G6D antibodies pre-bound to an assay device. The kit may optionally further include a detectable label.

[0222] Therefore, packaged products (e.g., sterile containers containing one or more compositions described herein and packaged at concentrated or ready-to-use concentrations for storage, transport, or sale) and kits (including at least one composition of the present invention (e.g., anti-LY6G6D antibody)) are also within the scope of this invention. Products may include containers (e.g., vials, wide-mouth bottles, flasks, bags, etc.) containing one or more compositions of the present invention. Furthermore, articles may also include, for example, packaging materials, instructions for use, syringes, delivery devices, buffers, or other control reagents for treating or monitoring diseases requiring diagnosis or treatment.

[0223] Reagents for specific types of assays may also be provided in the kits of the present invention. Thus, the kits may include bead populations (e.g., suitable for agglutination assays or lateral flow assays) or plates (e.g., plates suitable for ELISA assays). In other embodiments, the kits include devices such as lateral flow immunoassay devices, analytical rotors, or electrochemical, optical, or photoelectric sensors. Bead populations, plates, and devices can be used to perform immunoassays. For example, they can be used to detect the formation of a first reagent-analyte-second reagent complex.

[0224] In addition, the kit may include various diluents and buffers, labeled conjugates or other reagents for detecting specifically bound antigens or antibodies, and other signal-generating reagents such as enzyme substrates, cofactors, and chromogens. The kit may include one or more reference samples at different concentrations, such as purified recombinant LY6G6D. The kit may also include a positive control, such as cell supernatant from a cell line overexpressing LY6G6D. Other components of the kit may include coating reagents, polyclonal or monoclonal capture antibodies specific to the antigen or analyte to be tested, or mixtures of two or more antibodies, purified or semi-purified extracts of these antigens as standards, monoclonal antibody detection antibodies, anti-mouse, anti-dog, anti-chicken, or anti-human antibodies with indicator molecules conjugated thereto, indicator charts for colorimetric comparison, disposable gloves, decontamination instructions, swabs or containers, sample preparation cups, etc. In one embodiment, the kit contains buffers or other reagents suitable for constituting a reaction medium that allows the formation of peptide-antibody complexes.

[0225] These kits provide clinicians with a convenient and effective way to determine whether a subject has liver cancer or is at risk of developing colorectal cancer. Therefore, in some embodiments, the kit also includes instructions for use. The product may also include illustrations (e.g., printed labels or inserts or other media describing the product's use (e.g., audio or video tapes)). The illustrations may be associated with the container (e.g., attached to the container) and may describe how the assay should be performed, its indications, and other uses.

[0226] The present invention has now been fully described, and it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit or scope of the invention.

[0227] Example

[0228] Example 1: Binding analysis of humanized anti-LY6G6D antibody against LY6G6D-expressing cell lines and primary cells.

[0229] The binding of the humanized anti-LY6G6D antibody to cells expressing LY6G6D was tested. Four colon cancer cell lines expressing different levels of LY6G6D, as well as CHO-K1 cells expressing human and rhesus monkey LY6G6D, were tested. Cells were washed with FACS buffer and resuspended in FACS buffer. Cells were seeded at 1E5 cells / well in 96-well plates. Cells were stained with titrated anti-LY6G6D antibody on ice for 30 min, followed by washing and secondary staining with 1:500 diluted goat anti-human IgG-AF647. After a final wash and the addition of 7-AAD, cells were analyzed by flow cytometry. The geometric mean of fluorescence intensity was plotted against antibody concentration to generate a dose-response curve. Figure 1The binding curves of colon cancer cell lines are shown. Figure 2 The binding curve of CHO-K1 cells is shown. Figure 3 Summary of EC binding to cells 50 .

[0230] Example 2: Binding of LY6G6DxCD3 bispecific antibody to colon cancer cell lines and T cells

[0231] The binding of the LY6G6DxCD3 antibody to LY6G6D-expressing colon cancer cells and CD3-expressing T cells was tested. Cells were washed with FACS buffer and resuspended in FACS buffer. Cells were seeded at 1E5 cells / well in 96-well plates. Cells were stained with titrated anti-LY6G6D antibody on ice for 30 min, followed by washing and secondary staining with 1:500 diluted goat anti-human IgG-AF647. After a final wash and the addition of 7-AAD, cells were analyzed by flow cytometry. The geometric mean of fluorescence intensity was plotted against antibody concentration to generate a dose-response curve. Figure 5 The bonding curve is shown. Figure 6 Summary of EC binding to cells 50 .

[0232] Example 3: Cytokine release mediated by LY6G6DxCD3 bispecific antibody in the presence of colon cancer cell lines.

[0233] The LY6G6DxCD3 bispecific antibody was used in a cytokine release assay. Pan-T cells were isolated from human PBMCs and incubated with the LY6G6DxCD3 bispecific antibody in the presence of LY6G6D-expressing colon cancer cells. After 48 hours, the supernatant was collected for IL-2 and IFNγ release assays using an ELISA kit from R&D Systems. Data are presented in... Figure 7 As shown in the image.

[0234] Example 4: Activation of CD8+ T cells mediated by LY6G6DxCD3 bispecific antibody

[0235] The ability of the LY6G6DxCD3 bispecific antibody to activate T cells in colon cancer cells was tested. Pan-T cells were isolated from PBMCs and co-cultured with colon cancer cell lines and the LY6G6DxCD3 bispecific antibody for 48 hours. Cells were harvested and stained for CD4, CD8, CD25, and CD69 using 7-AAD cell viability dye. The percentages of CD4+ and CD8+ T cells expressing CD25 and CD69 were plotted against antibody concentration. Results are presented in... Figure 8 As shown in the image.

[0236] Example 5: Cytotoxicity of colon cancer cells mediated by LY6G6DxCD3 bispecific antibody

[0237] The efficacy of the LY6G6DxCD3 bispecific antibody in mediating the killing of colon cancer cells was tested. Pan-T cells were isolated from PBMCs and co-cultured with colon cancer cell lines and the LY6G6DxCD3 bispecific antibody for 48 hours. Cytotoxicity was determined using the CytotoxGlo kit from Promega. Results were obtained in... Figure 9 As shown in the image.

[0238] Example 6: In vivo tumor growth inhibition of HT55 colon cancer xenograft

[0239] The ability of the LY6G6DxCD3 bispecific antibody to inhibit the growth of HT55 colon cancer cells in vivo was tested. In a human PBMC remodeling model, HT55 cells were subcutaneously implanted into the flank of NSG mice and allowed to grow to approximately 150 mm3, followed by intraperitoneal injection of human PBMCs. 24–72 hours later, the LY6G6DxCD3 bispecific antibody was administered intravenously. Figure 10 The study showed tumor growth inhibition after administration of 0.3-1 mg / kg every two weeks. Figure 11 The study showed tumor growth inhibition after administration of 0.3 mg / kg every two weeks. Figure 12 The study showed tumor growth inhibition after weekly administration of 0.1 mg / kg.

[0240] Example 7: In vivo tumor growth inhibition of COLO320DM colon cancer xenograft

[0241] The ability of the LY6G6DxCD3 bispecific antibody to inhibit the growth of COL032DM colon cancer cells in vivo was tested. In a human PBMC remodeling model, COLO320DM cells were subcutaneously implanted into the flank of NSG mice and allowed to grow to approximately 150 mm³, followed by intraperitoneal injection of human PBMCs. Twenty-four hours later, the LY6G6DxCD3 bispecific antibody was administered intravenously at 0.1–1 mg / kg once weekly. Results were obtained in… Figure 13 As shown in the image.

[0242] Example 8: PK of LY6G6DxCD3 bispecific antibody with wild-type FcRn binding and T250Q M428L mutation in Tg276 hemizygous human FcRn transgenic mice

[0243] The pharmacokinetics of the LY6G6DxCD3 bispecific antibody with or without the (QL) FcRn binding mutation were evaluated in transgenic mice expressing human FcRn. Single doses of the antibody were administered, and serum samples were collected at different time points up to day 21. Serum antibody concentrations at each time point were determined using ELISA. Results were presented in... Figure 14 As shown in the image.

[0244] Although preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Various modifications, variations, and substitutions will now be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. An antibody that binds to LY6G6D, the antibody comprising a first binding unit, the first binding unit comprising: Heavy chain variable region, the heavy chain variable region comprising: (a) The CDRH1 sequence of any one of SEQ ID NO: 1-6; and / or (b) The CDRH2 sequence of any one of SEQ ID NO:7-11; and / or (c) The CDRH3 sequence of any one of SEQ ID NO: 12-15; and Light chain variable region, the light chain variable region comprising: (d) The CDRL1 sequence of any one of SEQ ID NO: 16-19; and / or (e) the CDRL2 sequence of any one of SEQ ID NO:20-23; and / or (f) CDRL3 sequence of any one of SEQ ID NO:24-26.

2. The antibody of claim 1, wherein the CDRH1, CDRH2 and CDRH3 sequences of the first binding unit are present in the human VH framework.

3. The antibody of claim 1 or 2, wherein the CDRL1, CDRL2 and CDRL3 sequences of the first binding unit are present in the human VL frame.

4. The antibody of any one of claims 1-3, wherein the first binding unit comprises: Heavy chain variable region, the heavy chain variable region comprising: (a) The CDRH1 sequence of any one of SEQ ID NO: 1-6; and (b) The CDRH2 sequence of any one of SEQ ID NO:7-11; and (c) CDRH3 sequence of any one of SEQ ID NO:12-15.

5. The antibody of claim 4, wherein the first binding unit comprises: Heavy chain variable region, the heavy chain variable region comprising: (a) The CDRH1 sequence of SEQ ID NO:1, the CDRH2 sequence of SEQ ID NO:7, and the CDRH3 sequence of SEQ ID NO:12; or (b) The CDRH1 sequence of SEQ ID NO:2, the CDRH2 sequence of SEQ ID NO:8, and the CDRH3 sequence of SEQ ID NO:13; or (c) The CDRH1 sequence of SEQ ID NO:3, the CDRH2 sequence of SEQ ID NO:9, and the CDRH3 sequence of SEQ ID NO:13; or (d) The CDRH1 sequence of SEQ ID NO:4, the CDRH2 sequence of SEQ ID NO:10, and the CDRH3 sequence of SEQ ID NO:14; or (e) the CDRH1 sequence of SEQ ID NO:5, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15; or (f) The CDRH1 sequence of SEQ ID NO:6, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:

15.

6. The antibody of any one of claims 1-5, wherein the first binding unit comprises: Light chain variable region, the light chain variable region comprising: (a) The CDRL1 sequence of any one of SEQ ID NO: 16-19; and (b) The CDRL2 sequence of any one of SEQ ID NO: 20-23; and (c) CDRL3 sequence of any one of SEQ ID NO:24-26.

7. The antibody of claim 6, wherein the first binding unit comprises: Light chain variable region, the light chain variable region comprising: (a) The CDRL1 sequence of SEQ ID NO:16, the CDRL2 sequence of SEQ ID NO:20, and the CDRL3 sequence of SEQ ID NO:24; or (b) The CDRL1 sequence of SEQ ID NO:17, the CDRL2 sequence of SEQ ID NO:21, and the CDRL3 sequence of SEQ ID NO:25; or (c) The CDRL1 sequence of SEQ ID NO:18, the CDRL2 sequence of SEQ ID NO:22, and the CDRL3 sequence of SEQ ID NO:24; or (d) The CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:

26.

8. The antibody of any one of claims 1-7, wherein the first binding unit comprises: (a) A heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:1, the CDRH2 sequence of SEQ ID NO:7, and the CDRH3 sequence of SEQ ID NO:12; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:16, the CDRL2 sequence of SEQ ID NO:20, and the CDRL3 sequence of SEQ ID NO:24; or (b) a heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:2, the CDRH2 sequence of SEQ ID NO:8, and the CDRH3 sequence of SEQ ID NO:13; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:17, the CDRL2 sequence of SEQ ID NO:21, and the CDRL3 sequence of SEQ ID NO:25; or (c) Heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:3, the CDRH2 sequence of SEQ ID NO:9, and the CDRH3 sequence of SEQ ID NO:13; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:17, the CDRL2 sequence of SEQ ID NO:21, and the CDRL3 sequence of SEQ ID NO:25; or (d) Heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:4, the CDRH2 sequence of SEQ ID NO:10, and the CDRH3 sequence of SEQ ID NO:14; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:18, the CDRL2 sequence of SEQ ID NO:22, and the CDRL3 sequence of SEQ ID NO:24; or (e) a heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:5, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:26; or (f) Heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:6, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:

26.

9. The antibody of claim 8, wherein the first binding unit comprises a heavy chain variable region sequence having at least 95% identity with any one of SEQ ID NO:27-34.

10. The antibody of claim 8, wherein the first binding unit comprises a heavy chain variable region sequence, the heavy chain variable region sequence comprising any one of SEQ ID NO:27-34.

11. The antibody of any one of claims 8-10, wherein the first binding unit comprises a light chain variable region sequence having at least 95% identity with any one of SEQ ID NO:35-42.

12. The antibody of any one of claims 8-10, wherein the first binding unit comprises a light chain variable region sequence, the light chain variable region sequence comprising any one of SEQ ID NO:35-42.

13. The antibody of any one of claims 8-12, wherein the first binding unit comprises: (a) The heavy chain variable region sequence of SEQ ID NO:27 and the light chain variable region sequence of SEQ ID NO:35; or (b) The heavy chain variable region sequence of SEQ ID NO:28 and the light chain variable region sequence of SEQ ID NO:36; or (c) The heavy chain variable region sequence of SEQ ID NO:29 and the light chain variable region sequence of SEQ ID NO:37; or (d) The heavy chain variable region sequence of SEQ ID NO:30 and the light chain variable region sequence of SEQ ID NO:38; or (e) The heavy chain variable region sequence of SEQ ID NO:31 and the light chain variable region sequence of SEQ ID NO:39; or (f) The heavy chain variable region sequence of SEQ ID NO:32 and the light chain variable region sequence of SEQ ID NO:40; or (g) The heavy chain variable region sequence of SEQ ID NO:33 and the light chain variable region sequence of SEQ ID NO:41; or (h) The heavy chain variable region sequence of SEQ ID NO:34 and the light chain variable region sequence of SEQ ID NO:

42.

14. The antibody according to any one of claims 1-13, wherein the antibody further comprises a heavy chain constant region.

15. The antibody of claim 14, wherein the heavy chain constant region comprises a hinge region, a CH1 region, a CH2 region, and / or a CH3 region.

16. The antibody of any one of claims 14-15, wherein the heavy chain constant region comprises one or more KiH mutations.

17. The antibody of any one of claims 14-16, wherein the heavy chain constant region comprises one or more silencing mutations.

18. The antibody of any one of claims 14-17, wherein the heavy chain constant region comprises one or more FcRn binding mutations.

19. The antibody of claim 18, wherein the one or more FcRn binding mutations include a T250Q mutation, an M428L mutation, or both a T250Q and M428L mutation.

20. The antibody of any one of claims 14-19, wherein the heavy chain constant region comprises one or more protein A binding mutations.

21. The antibody of claim 20, wherein the one or more protein A binding mutations include H435R mutation, Y436F mutation, or both H435R and Y436F mutations.

22. The antibody according to any one of claims 1-21, wherein the antibody further comprises a light chain constant region.

23. The antibody of claim 22, wherein the light chain constant region comprises the CL region.

24. The antibody according to any one of claims 1-23, wherein the antibody is single-specific.

25. The antibody according to any one of claims 1-23, wherein the antibody is multispecific.

26. The antibody of claim 25, wherein the antibody is bispecific.

27. The antibody of claim 25 or 26, wherein the antibody further comprises a second binding unit that binds to CD3ε.

28. The antibody of claim 27, wherein the second binding unit comprises: Heavy chain variable region, the heavy chain variable region comprising: (a) The CDRH1 sequence of SEQ ID NO:43; and / or (b) The CDRH2 sequence of any one of SEQ ID NO:44-45; and / or (c) The CDRH3 sequence of SEQ ID NO:46; and Light chain variable region, the light chain variable region comprising: (d) The CDRL1 sequence of SEQ ID NO:47; and / or (e) the CDRL2 sequence of SEQ ID NO:48; and / or (f) CDRL3 sequence of SEQ ID NO:

49.

29. The antibody of claim 28, wherein the CDRH1, CDRH2, and CDRH3 sequences of the second binding unit are present in the human VH framework.

30. The antibody of claim 28 or 29, wherein the CDRL1, CDRL2, and CDRL3 sequences of the second binding unit are present in a human VL frame.

31. The antibody of any one of claims 28-30, wherein the second binding unit comprises: Heavy chain variable region, the heavy chain variable region comprising: (a) The CDRH1 sequence of SEQ ID NO:43; and (b) The CDRH2 sequence of any one of SEQ ID NO:44-45; and (c) CDRH3 sequence of SEQ ID NO:

46.

32. The antibody of claim 31, wherein the second binding unit comprises: Heavy chain variable region, the heavy chain variable region comprising: (a) The CDRH1 sequence of SEQ ID NO:43, the heavy chain CDRH2 sequence of SEQ ID NO:44, and the heavy chain CDRH3 sequence of SEQ ID NO:46; or (b) The CDRH1 sequence of SEQ ID NO:43, the CDRH2 sequence of SEQ ID NO:45, and the CDRH3 sequence of SEQ ID NO:

46.

33. The antibody of any one of claims 28-32, wherein the second binding unit comprises: The light chain variable region comprises: the CDRL1 sequence of SEQ ID NO:47; the CDRL2 sequence of SEQ ID NO:48; and the CDRL3 sequence of SEQ ID NO:

49.

34. The antibody of any one of claims 28-33, wherein the second binding unit comprises: (a) A heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:43, the CDRH2 sequence of SEQ ID NO:44, and the CDRH3 sequence of SEQ ID NO:46; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:47, the CDRL2 sequence of SEQ ID NO:48, and the CDRL3 sequence of SEQ ID NO:49; or (b) a heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:43, the CDRH2 sequence of SEQ ID NO:45, and the CDRH3 sequence of SEQ ID NO:46; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:47, the CDRL2 sequence of SEQ ID NO:48, and the CDRL3 sequence of SEQ ID NO:

49.

35. The antibody of claim 34, wherein the second binding unit comprises a heavy chain variable region sequence having at least 95% identity with any one of SEQ ID NO:50-51.

36. The antibody of claim 34, wherein the second binding unit comprises a heavy chain variable region sequence, the heavy chain variable region sequence comprising any one of SEQ ID NO:50-51.

37. The antibody of any one of claims 34-35, wherein the second binding unit comprises a light chain variable region sequence having at least 95% identity with any one of SEQ ID NO:52-53.

38. The antibody of any one of claims 34-35, wherein the second binding unit comprises a light chain variable region sequence, the light chain variable region sequence comprising any one of SEQ ID NO:52-53.

39. The antibody of any one of claims 34-35, wherein the second binding unit comprises: (a) The heavy chain variable region sequence of SEQ ID NO:50 and the light chain variable region sequence of SEQ ID NO:52; or (b) The heavy chain variable region sequence of SEQ ID NO:51 and the light chain variable region sequence of SEQ ID NO:

53.

40. An antibody that binds to LY6G6D and CD3ε, said antibody comprising: Contains a first light chain subunit of SEQ ID NO:57; Contains the first heavy chain subunit of SEQ ID NO:54; The second light chain subunit containing SEQ ID NO:57; and It contains the second heavy chain subunit of SEQ ID NO:

56.

41. An antibody that binds to LY6G6D and CD3ε, said antibody comprising: Contains a first light chain subunit of SEQ ID NO:57; Contains the first heavy chain subunit of SEQ ID NO:55; The second light chain subunit containing SEQ ID NO:57; and It contains the second heavy chain subunit of SEQ ID NO:

56.

42. An antibody that binds to LY6G6D and CD3ε, said antibody comprising: Contains a first light chain subunit of SEQ ID NO:57; Contains the first heavy chain subunit of SEQ ID NO:58; The second light chain subunit containing SEQ ID NO:57; and It contains a second heavy chain subunit of SEQ ID NO:

60.

43. An antibody that binds to LY6G6D and CD3ε, said antibody comprising: a first light chain subunit comprising SEQ ID NO:57; Contains the first heavy chain subunit of SEQ ID NO:59; The second light chain subunit containing SEQ ID NO:57; and It contains a second heavy chain subunit of SEQ ID NO:

60.

44. An antibody that binds to LY6G6D and CD3ε, said antibody comprising: a first light chain subunit comprising SEQ ID NO:57; Contains the first heavy chain subunit of SEQ ID NO:61; The second light chain subunit containing SEQ ID NO:57; and It contains a second heavy chain subunit of SEQ ID NO:

62.

45. An antibody that binds to LY6G6D and CD3ε, said antibody comprising: a first light chain subunit comprising SEQ ID NO:69; Contains the first heavy chain subunit of SEQ ID NO:64; Contains a second light chain subunit of SEQ ID NO:69; and It contains a second heavy chain subunit of SEQ ID NO:

66.

46. ​​An antibody that binds to LY6G6D and CD3ε, said antibody comprising: Contains a first light chain subunit of SEQ ID NO:69; Contains the first heavy chain subunit of SEQ ID NO:65; Contains a second light chain subunit of SEQ ID NO:69; and It contains a second heavy chain subunit of SEQ ID NO:

66.

47. An antibody that binds to LY6G6D and CD3ε, said antibody comprising: Contains a first light chain subunit of SEQ ID NO:69; Contains the first heavy chain subunit of SEQ ID NO:67; Contains a second light chain subunit of SEQ ID NO:69; and It contains a second heavy chain subunit of SEQ ID NO:

68.

48. A pharmaceutical composition comprising the antibody according to any one of claims 1-47.

49. A treatment method comprising administering to an individual in need an effective dose of the antibody of any one of claims 1-47 or the pharmaceutical composition of claim 48.

50. A method for treating a condition characterized by the expression of LY6G6D, the method comprising administering to a subject suffering from the condition the antibody of any one of claims 1-47 or the pharmaceutical composition of claim 48.

51. Use of the antibody as described in any one of claims 1-47 in the preparation of a medicament for treating a condition characterized by the expression of LY6G6D.

52. The antibody according to any one of claims 1-47, wherein the antibody is used to treat a condition characterized by the expression of LY6G6D.

53. The method, use, or antibody according to any one of claims 49-52, wherein the condition is cancer.

54. The method, use, or antibody of claim 53, wherein the cancer is a solid tumor cancer.

55. The method, use, or antibody of claim 54, wherein the solid tumor cancer is colorectal cancer.

56. The method, use, or antibody of claim 55, wherein the colorectal cancer is refractory to chemotherapy and / or immune checkpoint inhibitor therapy.

57. A polynucleotide encoding an antibody according to any one of claims 1-47.

58. A vector comprising the polynucleotide of claim 57.

59. A cell comprising the carrier of claim 58.

60. A method for producing an antibody according to any one of claims 1-47, the method comprising growing cells according to claim 59 under conditions that allow expression of the antibody, and isolating the antibody.

61. A medicine box comprising the antibody of any one of claims 1-47 or the pharmaceutical composition of claim 48 and instructions for use.

62. The medicine box of claim 61, wherein the medicine box further comprises an additional therapeutic agent.

63. The medicament box of claim 62, wherein the additional therapeutic agent comprises a chemotherapeutic agent.

64. A diagnostic method for determining whether a subject has a condition characterized by the expression of LY6G6D or is at risk of developing said condition, the method comprising: A biological test sample from the subject is contacted with an antibody bound to LY6G6D to generate an LY6G6D-antibody complex, wherein the antibody comprises: (a) A heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:1, the CDRH2 sequence of SEQ ID NO:7, and the CDRH3 sequence of SEQ ID NO:12; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:16, the CDRL2 sequence of SEQ ID NO:20, and the CDRL3 sequence of SEQ ID NO:24; or (b) a heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:2, the CDRH2 sequence of SEQ ID NO:8, and the CDRH3 sequence of SEQ ID NO:13; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:17, the CDRL2 sequence of SEQ ID NO:21, and the CDRL3 sequence of SEQ ID NO:25; or (c) Heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:3, the CDRH2 sequence of SEQ ID NO:9, and the CDRH3 sequence of SEQ ID NO:13; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:17, the CDRL2 sequence of SEQ ID NO:21, and the CDRL3 sequence of SEQ ID NO:25; or (d) Heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:4, the CDRH2 sequence of SEQ ID NO:10, and the CDRH3 sequence of SEQ ID NO:14; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:18, the CDRL2 sequence of SEQ ID NO:22, and the CDRL3 sequence of SEQ ID NO:24; or (e) a heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:5, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:26; or (f) Heavy chain variable region, the heavy chain variable region comprising: The CDRH1 sequence of SEQ ID NO:6, the CDRH2 sequence of SEQ ID NO:11, and the CDRH3 sequence of SEQ ID NO:15; and Light chain variable region, the light chain variable region comprising: The CDRL1 sequence of SEQ ID NO:19, the CDRL2 sequence of SEQ ID NO:23, and the CDRL3 sequence of SEQ ID NO:26; The concentration of the LY6G6D-antibody complex in the biological test sample was detected; and The concentration of the LY6G6D-antibody complex was compared with a reference value to determine whether the subject had the condition or was at risk of developing the condition.

65. The diagnostic method of claim 64, wherein the condition is cancer.

66. The diagnostic method of claim 65, wherein the cancer is a solid tumor cancer.

67. The diagnostic method of claim 66, wherein the solid tumor cancer is colorectal cancer.

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