Anti-ly6k antibodies and methods of use

Abstract

The present disclosure provides antibodies and polypeptides that specifically bind to lymphocyte antigen 6 family, member K (LY6K). Also provided are compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of using these antibodies to treat subjects.

Description

Cross-references to related applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application Serial No. 63 / 605,977, filed December 4, 2023; U.S. Provisional Patent Application Serial No. 63 / 662,231, filed June 20, 2024; and U.S. Provisional Patent Application Serial No. 63 / 686,563, filed August 23, 2024, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference sequence list This application includes a sequence list, which is submitted electronically in ST.26 format and incorporated herein by reference. The ST.26 copy was created on December 3, 2024, and is named “214967_seqlist.xml”, with a size of 223,249 bytes. Technical Field

[0003] This disclosure relates to antibodies specific to human lymphocyte antigen 6 family member K (LY6K) and methods of using them. Background Technology

[0004] The lymphocyte antigen 6 (LY6) family is a group of cysteine-rich proteins that share a common structure, are expressed in various tissues, and exhibit a variety of cellular functions. LY6 proteins are anchored to or secreted onto the cell membrane via glycosylphosphatidylinositol (GPI), and they are involved in cell proliferation, cell migration, cell-cell interactions, immune cell maturation, macrophage activation, and cytokine production. Dysregulation of LY6 family members is associated with tumorigenesis and autoimmune diseases, and many LY6 proteins are used as tumor markers and potential therapeutic targets. Lymphocyte antigen 6 family member K (LY6K) is a cell surface protein upregulated in several types of cancer, including esophageal squamous cell carcinoma, bladder cancer, and breast cancer, and contributes to cell growth, migration, invasion, and immune evasion.

[0005] Therefore, therapies targeting LY6K are needed. Summary of the Invention

[0006] This disclosure provides antibodies and peptides that specifically bind to LY6K (e.g., human LY6K). Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for preparing these antibodies, and methods for treating subjects using these antibodies are also provided. The antibodies provided herein are particularly advantageous because they specifically bind to cancer cells expressing LY6K, allowing for targeted delivery of therapeutic and diagnostic agents, and thus are useful in the treatment and diagnosis of cancer (e.g., cancers expressing LY6K).

[0007] In one aspect, this article provides an antibody that specifically binds to human LY6K, comprising: VH containing the VH amino acid sequence of CDRH1, CDRH2, and CDRH3 as shown in any one of SEQ ID NO: 1-34; and VL containing the VL amino acid sequence of CDRL1, CDRL2, and CDRL3 as shown in any one of SEQ ID NO: 35-68.

[0008] In some embodiments, the VH amino acid sequences and VL amino acid sequences are as follows: SEQ ID NO: 1 and 35, 2 and 36, 3 and 37, 4 and 38, 5 and 39, 6 and 40, 7 and 41, 8 and 42, 9 and 43, 10 and 44, 11 and 45, 12 and 46, 13 and 47, 14 and 48, 15 and 49, 16 and 50, 17 and 51, 18 and 52, 19 and 53, 20 and 54, 21 and 55, 22 and 56, 23 and 57, 24 and 58, 25 and 59, 26 and 60, 27 and 61, 28 and 62, 29 and 63, 30 and 64, 31 and 65, 32 and 66, 33 and 67, or 34 and 68.

[0009] In some embodiments, the antibody comprises the amino acid sequences of CDRH1, CDRH2, and CDRH3, respectively, shown in the following: SEQ ID NO: 69, 90, and 124; 70, 91, and 125; 71, 92, and 126; 72, 93, and 127; 69, 94, and 124; 73, 95, and 128; 74, 96, and 129; 75, 97, and 130; 74, 98, and 131; 76, 99, and 132; 77, 100, and 133; 78, 101, and 134; 79, 102, and 135; 74, 103, and 136; 80, 104, and 137; 74, 105, and 138; 74, 106, and 139; 81, 107, and 140. ;81, 108 and 141; 82, 109 and 142; 83, 110 and 143; 84, 99 and 144; 85, 111 and 145; 81, 112 and 146; 86, 113 and 147; 87, 114 and 148; 74, 115 and 149; 74, 116 and 150; 74, 117 and 151; 88, 118 and 152; 89, 119 and 153; 81, 120 and 154; 74, 121 and 155; 85, 122 and 156; or 86, 123 and 157.

[0010] In some embodiments, the antibody comprises the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in the following: SEQ ID NO: 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 159, WAS, and SEQ ID NO: 199; SEQ ID NO: 160, STT, and SEQ ID NO: 200; SEQ ID NO: 161, WAS, and SEQ ID NO: 201; SEQ ID NO: 162, AAS, and SEQ ID NO: 202; SEQ ID NO: 163, 185, and 203; SEQ ID NO: 164, 185, and 204; SEQ ID NO: 165, 185, and 205; SEQ ID NO: 166, 186, and 206; SEQ ID NO: 167, 187, and 207; SEQ ID NO: 168, 188, and 208; SEQ ID NO: SEQ ID NO: 169, 186, and 209; SEQ ID NO: 170, 185, and 198; SEQ ID NO: 169, 189, and 210; SEQ ID NO: 170, 185, and 211; SEQ ID NO: 171, 187, and 212; SEQ ID NO: 172, 187, and 213; SEQ ID NO: 173, 190, and 214; SEQ ID NO: 174, 191, and 215; SEQ ID NO: 175, 186, and 216; SEQ ID NO: 176, 186, and 217; SEQ ID NO: 170, 192, and 198; SEQ ID NO: 171, 193, and 218; SEQ ID NO: 177, 187, and 219; SEQ ID NO: 178, 189, and 220; SEQ ID NO: SEQ ID NO: 179, 187, and 221; SEQ ID NO: 165, 185, and 222; SEQ ID NO: 180, 187, and 223; SEQ ID NO: 181, 194, and 224; SEQ ID NO: 182, 195, and 199; SEQ ID NO: 172, 187, and 225; SEQ ID NO: 183, 196, and 226; or SEQ ID NO: 184, 197, and 227.

[0011] In some embodiments, the antibody comprises the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 shown in the following: SEQ ID NO: 69, 90, 124, 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 70, 91, 125, 159, WAS, and SEQ ID NO: 199; SEQ ID NO: 71, 92, 126, 160, STT, and SEQ ID NO: 200; SEQ ID NO: 72, 93, 127, 161, WAS, and SEQ ID NO: 201; SEQ ID NO: 69, 94, 124, 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 73, 95, 128, 162, AAS, and SEQ ID NO: 202; SEQ ID NO: SEQ ID NO: 74, 96, 129, 163, 185, and 203; SEQ ID NO: 75, 97, 130, 164, 185, and 204; SEQ ID NO: 74, 98, 131, 165, 185, and 205; SEQ ID NO: 76, 99, 132, 166, 186, and 206; SEQ ID NO: 77, 100, 133, 167, 187, and 207; SEQ ID NO: 78, 101, 134, 168, 188, and 208; SEQ ID NO: 79, 102, 135, 169, 186, and 209; SEQ ID NO: 74, 103, 136, 170, 185, and 198; SEQ ID NO: 80, 104, 137, 169, 189, and 210; SEQ ID SEQ ID NO: 74, 105, 138, 170, 185, and 211; SEQ ID NO: 74, 106, 139, 171, 187, and 212; SEQ ID NO: 81, 107, 140, 172, 187, and 213; SEQ ID NO: 81, 108, 141, 173, 190, and 214; SEQ ID NO: 82, 109, 142, 174, 191, and 215; SEQ ID NO: 83, 110, 143, 175, 186, and 216; SEQ ID NO: 84, 99, 144, 176, 186, and 217; SEQ ID NO: 85, 111, 145, 170, 192, and 198; SEQ ID NO: 81, 112, 146, 171, 193 and 218; SEQ ID NO: 86, 113, 147, 177, 187 and 219; SEQ ID NO: 87, 114, 148, 178, 189 and 220;SEQ ID NO: 74, 115, 149, 179, 187 and 221; SEQ ID NO: 74, 116, 150, 165, 185 and 222; SEQ ID NO: 74, 117, 151, 180, 187 and 223; SEQ ID NO: 88, 118, 152, 181, 194 and 224; SEQ ID NO: 89, 119, 153, 182, 195 and 199; SEQ ID NO: 81, 120, 154, 172, 187 and 225; SEQ ID NO: 74, 121, 155, 165, 185 and 222; SEQ ID NO: 85, 122, 156, 183, 196 and 226; or SEQ ID NO: 86, 123, 157, 184, 197, and 227.

[0012] In some embodiments, the antibody comprises the VH amino acid sequence shown in any one of SEQ ID NO: 1-34. In some embodiments, the antibody comprises a heavy chain constant region, optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some embodiments, the antibody comprises a heavy chain constant region as a variant of the wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to FcγR with a lower affinity than the wild-type heavy chain constant region. In some embodiments, the antibody comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 228 or 229.

[0013] In some embodiments, the antibody comprises the VL amino acid sequence shown in any one of SEQ ID NO: 35-68. In some embodiments, the antibody comprises a light chain constant region containing the amino acid sequence of SEQ ID NO: 230 or 231.

[0014] In some embodiments, VH and VL comprise amino acid sequences shown in the following: SEQ ID NO: 1 and 35, 2 and 36, 3 and 37, 4 and 38, 5 and 39, 6 and 40, 7 and 41, 8 and 42, 9 and 43, 10 and 44, 11 and 45, 12 and 46, 13 and 47, 14 and 48, 15 and 49, 16 and 50, 17 and 51, 18 and 52, 19 and 53, 20 and 54, 21 and 55, 22 and 56, 23 and 57, 24 and 58, 25 and 59, 26 and 60, 27 and 61, 28 and 62, 29 and 63, 30 and 64, 31 and 65, 32 and 66, 33 and 67, or 34 and 68.

[0015] In another aspect, this document provides an antibody that specifically binds to an amino acid sequence selected from the group consisting of SEQ ID NO: 233-238. In another aspect, this document provides an antibody that specifically binds to an amino acid sequence consisting of SEQ ID NO: 233 and 239. In another aspect, this document provides an antibody that specifically binds to an amino acid sequence consisting of SEQ ID NO: 234 and 240. In another aspect, this document provides an antibody that specifically binds to an amino acid sequence consisting of SEQ ID NO: 237 and 247.

[0016] In some embodiments, the antibody provided herein is a multispecific antibody containing a CD3-binding region, optionally wherein the CD3-binding region contains a single-chain variable region (scFv) that specifically binds to CD3, optionally human CD3.

[0017] On the other hand, this document provides a polypeptide comprising a VH containing the CDRH1, CDRH2, and CDRH3 amino acid sequences of any one of the VH amino acid sequences shown in SEQ ID NO: 1-34. In some embodiments, the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in the following: SEQ ID NO: 69, 90 and 124; 70, 91 and 125; 71, 92 and 126; 72, 93 and 127; 69, 94 and 124; 73, 95 and 128; 74, 96 and 129; 75, 97 and 130; 74, 98 and 131; 76, 99 and 132; 77, 100 and 133; 78, 101 and 134; 79, 102 and 135; 74, 103 and 136; 80, 104 and 137; 74, 105 and 138; 74, 106 and 139; 81, 107 and 140 ;81, 108 and 141; 82, 109 and 142; 83, 110 and 143; 84, 99 and 144; 85, 111 and 145; 81, 112 and 146; 86, 113 and 147; 87, 114 and 148; 74, 115 and 149; 74, 116 and 150; 74, 117 and 151; 88, 118 and 152; 89, 119 and 153; 81, 120 and 154; 74, 121 and 155; 85, 122 and 156; or 86, 123 and 157.

[0018] This article also provides a polypeptide comprising VL containing the VL amino acid sequences of CDRL1, CDRL2, and CDRL3, which are the VL amino acid sequences shown in any one of SEQ ID NO: 35-68. In some embodiments, VL comprises the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in the following items, respectively: SEQ ID NO: 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 159, WAS, and SEQ ID NO: 199; SEQ ID NO: 160, STT, and SEQ ID NO: 200; SEQ ID NO: 161, WAS, and SEQ ID NO: 201; SEQ ID NO: 162, AAS, and SEQ ID NO: 202; SEQ ID NO: 163, 185, and 203; SEQ ID NO: 164, 185, and 204; SEQ ID NO: 165, 185, and 205; SEQ ID NO: 166, 186, and 206; SEQ ID NO: 167, 187, and 207; SEQ ID NO: 168, 188, and 208; SEQ ID NO: SEQ ID NO: 169, 186, and 209; SEQ ID NO: 170, 185, and 198; SEQ ID NO: 169, 189, and 210; SEQ ID NO: 170, 185, and 211; SEQ ID NO: 171, 187, and 212; SEQ ID NO: 172, 187, and 213; SEQ ID NO: 173, 190, and 214; SEQ ID NO: 174, 191, and 215; SEQ ID NO: 175, 186, and 216; SEQ ID NO: 176, 186, and 217; SEQ ID NO: 170, 192, and 198; SEQ ID NO: 171, 193, and 218; SEQ ID NO: 177, 187, and 219; SEQ ID NO: 178, 189, and 220; SEQ ID NO: SEQ ID NO: 179, 187, and 221; SEQ ID NO: 165, 185, and 222; SEQ ID NO: 180, 187, and 223; SEQ ID NO: 181, 194, and 224; SEQ ID NO: 182, 195, and 199; SEQ ID NO: 172, 187, and 225; SEQ ID NO: 183, 196, and 226; or SEQ ID NO: 184, 197, and 227.

[0019] This article also provides a polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-68.

[0020] In some embodiments, the antibodies or peptides provided herein are conjugated with cytotoxic agents, cell growth inhibitors, toxins, radionuclides, or detectable markers.

[0021] On the other hand, this document provides a polynucleotide encoding the VH, VL, heavy chain, and / or light chain of an antibody or polypeptide described herein. A vector comprising the polynucleotide described herein is also provided. A recombinant host cell is also provided, comprising: the polynucleotide described herein; the vector described herein; a polynucleotide encoding the heavy chain variable region or heavy chain of an antibody described herein and a second polynucleotide encoding the light chain variable region or light chain of an antibody described herein; and / or a first vector comprising the first polynucleotide encoding the heavy chain variable region or heavy chain of an antibody described herein and a second vector comprising the second polynucleotide encoding the light chain variable region or light chain of an antibody described herein.

[0022] On the other hand, this article provides a composition comprising the antibodies, peptides, polynucleotides, vectors and / or host cells described herein, as well as pharmaceutically acceptable loaders or excipients.

[0023] On the other hand, this article provides a method for generating antibodies, which includes culturing the host cells described herein under suitable conditions to express polynucleotides and generate antibodies.

[0024] This article also provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of the following: an antibody as described herein; a peptide as described herein; a polynucleotide as described herein; a carrier as described herein; a host cell as described herein; or a composition as described herein. Attached Figure Description

[0025] Figure 1 The exemplary anti-LY6K antibody provided herein selectively binds to LY6K according to various aspects of this disclosure. Plate readings of an ELISA assay measuring the binding of the indicated anti-LY6K antibody to LY6K, LY6E, LY6D, LY6H, and BSA (negative control) are depicted.

[0026] Figure 2 The illustration shows the detection of LY6K using exemplary anti-LY6K antibodies provided herein according to various aspects of this disclosure. The left figure depicts the results of a capture ELISA measuring the sensitivity of commercially available anti-LY6K antibodies used to detect LY6K, along with positive controls from R&D Systems and Abcam. The table in the right figure indicates the sensitivity and half-maximum effective concentration (EC50) of each indicated antibody.

[0027] Figure 3 The in vivo in vivo in cells is illustrated according to various aspects of this disclosure of the anti-LY6K antibody. A representative fluorescence image of anti-LY6K antibody 3082 labeled with a pH-sensitive dye is depicted. Upon in vivo in OVCAR-8 cells, the antibody is endocytosed and releases a fluorescent signal. Results for the negative control huIgG1 and the positive control trastuzumab (a known commercially available in vivo in vivo agent) are also depicted.

[0028] Figure 4 The detection of LY6K with anti-LY6K antibodies provided herein according to various aspects of this disclosure is illustrated. Representative IHC images are depicted, showing the detection of LY6K in OVCAR-8 cells, 293 LY6K cells, and 293 cells (negative control) using anti-LY6K antibody 2660, a commercially available positive control anti-LY6K antibody (Abcam), and an IgG negative control.

[0029] Figure 5 The killing of tumor cells by anti-LY6K bispecific T-cell adaptors provided herein according to various aspects of this disclosure is illustrated. A graph depicting the percentage of OVCAR-8 tumor cell killings by adding increased concentrations of L2K-formatted anti-LY6K bispecific T-cell adaptors (3785, 3520, 2660, 3741) based on indicated anti-LY6K antibodies and, as a comparison, a 4D5-based 1+1 T-cell adaptor targeting Her2. Detailed Implementation

[0030] This disclosure provides anti-LY6K antibodies and peptides. Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for preparing these antibodies, and methods for treating subjects using these antibodies are also provided. The antibodies disclosed herein are particularly useful for diagnosing and / or treating cancer in subjects.

[0031] definition As used herein, the term "LY6K" refers to Lymphocyte Antigen 6 family member K, a membrane-anchoring protein highly expressed in cancer cells and tissues. As used herein, the term "human LY6K" refers to the protein encoded by the wild-type human LY6K gene (e.g., the gene shown in NM_017527.4). An exemplary human LY6K protein (as shown in RefSeq NP_059997.3) has the following amino acid sequence: MALLALLLVVALPRVWTDANLTARQRDPEDSQRTDEGDNRVWCHVCERENTFECQNPRRCKWTEPYCVIAAVKIFPRFFMVAKQCSAGCAAMERPKPEEKRFLLEEPMPFFYLKCCKIRYCNLEGPPINSSVFKEYAGSMGSCGGLWLAILLLLASIAAGLSLS (SEQ ID NO: 232).

[0032] As used herein, the term "antibody" includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDR, VH region, and / or VL region. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramer antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, heteroconjugated antibodies, antibody-drug conjugates, bispecific T-cell adaptors (BiTE), chimeric antigen receptors, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camel-derived antibodies, affinity molecules, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In some embodiments, the antibody described herein refers to a population of polyclonal antibodies. An antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of an immunoglobulin molecule. In some embodiments, the antibody described herein is an IgG antibody or a class (e.g., human IgG1 or IgG4) or subclass thereof. In one specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

[0033] A "multispecific antibody" is an antibody that specifically binds to two or more different antigens or two or more different regions of the same antigen (e.g., a bispecific antibody). Multispecific antibodies include bispecific antibodies containing two different antigen-binding sites (excluding the Fc region). Multispecific antibodies can include, for example, recombinant antibodies, human antibodies, humanized antibodies, surface-reconstructed antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramer antibodies containing two heavy chains and two light chains, antibody light chain monomers, heteroconjugated antibodies, linked single-chain antibodies or linked single-chain Fv (scFv), camelized antibodies, affinity molecules, linked Fab fragments, F(ab')2 fragments, chemically linked Fvs, and disulfide-linked Fvs (sdFvs). Multispecific antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecules. In some embodiments, the multispecific antibody described herein is an IgG antibody or its class (e.g., human IgG1, IgG2, or IgG4) or subclass.

[0034] As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site found within the variable region of heavy and light chain polypeptides. These specific regions have been described, for example, by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), by Chothia et al., J. Mol. Biol. 196: 901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), all of which are incorporated herein by reference in their entirety, where definitions include overlap or subsets of amino acid residues when compared to one another. In some implementations, the term "CDR" is used as described by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," Antibody EngineeringIn some embodiments, the term "CDR" is defined as in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In some embodiments, different conventions are used to define the heavy chain CDR and light chain CDR of the antibody. In some embodiments, the heavy chain CDR and / or light chain CDR are defined by performing structural analysis of the antibody and identifying residues in the variable region that are predicted to contact the epitope region of the target molecule (e.g., human LY6K). CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0035] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, usually a portion of the light or heavy chain, typically about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain, which are widely sequence-dependent between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while more highly conserved regions within the variable region are called frame regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises a rodent or mouse CDR and a human frame region (FR). In some embodiments, the variable region is a primate (e.g., a non-human primate) variable region. In some implementations, the variable region includes the rodent or mouse CDR and the primate (e.g., non-human primate) frame region (FR).

[0036] As used herein, the terms “VH” and “VL” refer to the variable regions of the antibody heavy and light chains, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety.

[0037] As used herein, the term "constant region" is common in the art. A constant region is an antibody moiety, such as the carboxyl-terminal portion of the light chain and / or heavy chain, which does not directly participate in the binding of the antibody to the antigen but can exhibit various effector functions, such as interaction with Fc receptors (e.g., Fcγ receptors).

[0038] As used herein, the term “heavy chain” when referring to antibody use can refer to any different type based on the amino acid sequence of the constant region, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (µ), which produce IgA, IgD, IgE, IgG, and IgM antibodies, including subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4.

[0039] As used herein, the term "light chain," when referring to antibody use, can refer to any different type based on the amino acid sequence of the constant region, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0040] As used herein, the term "specific binding" refers to the specificity of a binding molecule (e.g., an antibody) for an antigen, as understood by those skilled in the art. Binding molecules that specifically bind to an antigen typically have a binding density of less than 1 × 10⁻⁶. -6 The equilibrium dissociation constant (KD) of M is bound to the antigen, as measured by, for example, an ELISA assay, surface plasmon resonance, or other suitable assays known in the art. Those skilled in the art will understand that in some embodiments, the binding molecule can specifically bind to different antigens, such as different antigens sharing a common epitope recognized by the binding molecule.

[0041] As used herein, the term "affinity" refers to the strength of the sum of 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 members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

[0042] As used herein, the term “EU numbering system” refers to the EU numbering convention for antibody constant regions, as described in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0043] As used herein, the term “treatment” refers to the therapeutic or preventative measures described herein. A “treatment” method involves administering antibodies to a subject who has a disease or condition or is susceptible to such a disease or condition, in order to prevent, cure, delay, reduce the severity of one or more symptoms of the disease or condition or a recurrent disease or condition, or to improve one or more symptoms of such a disease or condition, or to prolong the subject’s survival beyond what would be expected in the absence of such treatment.

[0044] As used herein, the term "pharmaceutically active substance" refers to a molecule or portion intended to achieve beneficial outcomes in subjects. Beneficial outcomes include, but are not limited to, diagnosis, prognosis, treatment, cure, and prevention (prevention) of diseases and / or symptoms and / or health problems.

[0045] As used herein, in the context of administering therapy to a subject, the term "effective amount" refers to the amount of therapy that achieves the desired preventive or therapeutic effect.

[0046] As used herein, the term "subject" includes any human or non-human animal. In some embodiments, the subject is a human or a non-human mammal. In some embodiments, the subject is a human.

[0047] As used herein with respect to antibodies, peptides, or polynucleotides, the term "isolated" means an antibody, peptide, or polynucleotide isolated from one or more contaminants (e.g., peptides, polynucleotides, lipids, or carbohydrates) present in the natural source (e.g., in mice or humans). Furthermore, "isolated" antibodies, peptides, or polynucleotides may be substantially free of other cell material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the language "substantially free" includes preparations of antibodies, peptides, or polynucleotides having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (especially less than about 10%) of other materials (e.g., cell material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals). All examples of "isolated antibodies" described herein are also contemplated as may, but not necessarily, isolated antibodies. All examples of "isolated peptides" described herein are also contemplated as may, but not necessarily, isolated peptides. All examples of "isolated polynucleotides" described herein are also contemplated as may, but not necessarily, isolated polynucleotides. All instances of “antibody” as described herein are also contemplated as may, but not necessarily, isolated antibodies. All instances of “peptide” as described herein are also contemplated as may, but not necessarily, isolated polypeptides. All instances of “polynucleotide” as described herein are also contemplated as may, but not necessarily, isolated polynucleotides.

[0048] The determination of the "percentage identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. A specific, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is incorporated herein by reference in its entirety. This algorithm is also incorporated into the NBLAST and XBLAST procedures of Altschul SF et al., (1990) J Mol Biol 215: 403, which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set, e.g., score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST procedure parameter set, for example, a score of 50 and a word length of 3, to obtain amino acid sequences homologous to the protein molecules described herein. For vacancy alignments to be obtained for comparative purposes, vacancy BLAST can be used as described in Altschul SF et al., (1997) NucAcids Res 25: 3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used for iterative searches that detect distant relationships between molecules (ibid.). When using BLAST, vacancy BLAST, and PSI BLAST procedures, the default parameters of the respective procedures (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4: 11-17, which is incorporated herein by reference in its entirety. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4 can be used.

[0049] The percentage of identity between two sequences can be determined using techniques similar to those described above, with or without gaps allowed. When calculating percentage identity, only exact matches are typically counted.

[0050] Anti-LY6K antibody In one aspect, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K). The VH, VL, CDRH, and CDRL amino acid sequences of exemplary antibodies are shown in Tables 1 through 4, respectively.

[0051] Table 1. Exemplary VH amino acid sequences of anti-LY6K antibodies .

[0052]

[0053]

[0054]

[0055] Table 2. Example VL amino acid sequences of anti-LY6K antibodies .

[0056]

[0057]

[0058]

[0059] Table 3. Exemplary CDRH amino acid sequences of anti-LY6K antibodies .

[0060]

[0061] Table 4. Exemplary CDRL amino acid sequences of anti-LY6K antibodies .

[0062]

[0063] The individual CDRs of the antibodies disclosed herein can be determined based on any CDR numbering scheme known in the art.

[0064] In some implementations, one or more of the CDRs of the antibodies disclosed herein may be determined based on Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immune interest (1991), each of which is incorporated herein by reference in its entirety.

[0065] In some embodiments, the antibodies provided herein comprise CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence as shown in any one of SEQ ID NO: 1-34 as determined by the Kabat numbering scheme. In some embodiments, the antibodies provided herein comprise CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence as shown in any one of SEQ ID NO: 35-68 as determined by the Kabat numbering scheme.

[0066] In some embodiments, one or more of the CDRs of the antibodies disclosed herein may be determined according to the Chothia numbering scheme, which relates to the location of the immunoglobulin structural loop (see, for example, Chothia C and Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226, all of which are incorporated herein by reference in their entirety).

[0067] In some embodiments, the antibodies provided herein comprise CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence as shown in any one of SEQ ID NO: 1-34 as determined by the Chothia numbering system. In some embodiments, the antibodies provided herein comprise CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence as shown in any one of SEQ ID NO: 35-68 as determined by the Chothia numbering system.

[0068] In some implementations, one or more of the CDRs of the antibodies disclosed herein may be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745, which is incorporated herein by reference in its entirety. See also, for example, Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” Antibody Engineering Kontermann and Dübel (eds.), Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety.

[0069] In some embodiments, the antibodies provided herein comprise CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence as shown in any one of SEQ ID NOs: 1-34 as determined by the MacCallum numbering system. In some embodiments, the antibodies provided herein comprise CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence as shown in any one of SEQ ID NOs: 35-68 as determined by the MacCallum numbering system.

[0070] In some implementations, the CDR of the antibody disclosed herein may be determined according to the IMGT numbering system as described in the following references: Lefranc MP, (1999) The Immunologist 7: 132-136; Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212, each of which is incorporated herein by reference in its entirety; and Lefranc MP et al., (2009) Nucleic Acids Res 37: D1006-D1012.

[0071] In some embodiments, the antibodies provided herein comprise CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence as shown in any one of SEQ ID NO: 1-34 as determined by the IMGT numbering system. In some embodiments, the antibodies provided herein comprise CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence as shown in any one of SEQ ID NO: 35-68 as determined by the IMGT numbering system.

[0072] In some implementations, the CDR of the antibody disclosed herein can be determined according to the AbM numbering scheme, which involves the AbM hypervariable region, represents a compromise between the Kabat CDR and the Chothia structural loop, and is used by the AbM antibody modeling software of Oxford Molecular (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety.

[0073] In some embodiments, the antibodies provided herein comprise CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence as shown in any of SEQ ID NO: 1-34 as determined by the AbM numbering scheme. In some embodiments, the antibodies provided herein comprise CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence as shown in any of SEQ ID NO: 35-68 as determined by the AbM numbering scheme.

[0074] In some implementations, the CDR of the antibodies disclosed herein can be determined according to the AHo numbering system, as described in Honegger and Plückthun, A., J. Mol. Biol. 309:657-670 (2001), which is incorporated herein by reference in its entirety.

[0075] In some embodiments, the antibodies provided herein comprise CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence as shown in any one of SEQ ID NO: 1-34 as determined by the AHo numbering system. In some embodiments, the antibodies provided herein comprise CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence as shown in any one of SEQ ID NO: 35-68 as determined by the AHo numbering system.

[0076] In some embodiments, the individual CDRs of the antibodies disclosed herein are determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in the variable region predicted to contact the epitope region of LY6K.

[0077] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), the antibody comprising VH containing the VH amino acid sequence of CDRH1, CDRH2, and CDRH3 as shown in any one of SEQ ID NO: 1-34 and VL containing the VL amino acid sequence of CDRL1, CDRL2, and CDRL3 as shown in any one of SEQ ID NO: 35-68, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in a variable region predicted to contact an epitope region of LY6K (e.g., human LY6K).

[0078] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), the antibody comprising VH containing the VH amino acid sequence of CDRH1, CDRH2, and CDRH3 as shown in any one of SEQ ID NO: 1-34 and VL containing the VL amino acid sequence of CDRL1, CDRL2, and CDRL3 as shown in any one of SEQ ID NO: 35-68, wherein the VH and VL amino acid sequences are derived from the same antibody (i.e., as shown in Tables 1 and 2), and wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in the variable region predicted to contact the epitope region of LY6K (e.g., human LY6K).

[0079] In some embodiments, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K), wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in the following: SEQ ID NO: 1 and 35; SEQ ID NO: 2 and 36; SEQ ID NO: 3 and 37; SEQ ID NO: 4 and 38; SEQ ID NO: 5 and 39; SEQ ID NO: 6 and 40; SEQ ID NO: 7 and 41; SEQ ID NO: 8 and 42; SEQ ID NO: 9 and 43; SEQ ID NO: 10 and 44; SEQ ID NO: 11 and 45; SEQ ID NO: 12 and 46; SEQ ID NO: 13 and 47; SEQ ID NO: 14 and 48; SEQ ID NO: 15 and 49; SEQ ID NO: 16 and 50; SEQ ID NO: 17 and 51; SEQ ID SEQ ID NO: 18 and 52; SEQ ID NO: 19 and 53; SEQ ID NO: 20 and 54; SEQ ID NO: 21 and 55; SEQ ID NO: 22 and 56; SEQ ID NO: 23 and 57; SEQ ID NO: 24 and 58; SEQ ID NO: 25 and 59; SEQ ID NO: 26 and 60; SEQ ID NO: 27 and 61; SEQ ID NO: 28 and 62; SEQ ID NO: 29 and 63; SEQ ID NO: 30 and 64; SEQ ID NO: 31 and 65; SEQ ID NO: 32 and 66; SEQ ID NO: 33 and 67; or SEQ ID NO: 34 and 68.

[0080] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), wherein the antibody comprises a VH containing the CDRH1, CDRH2, and / or CDRH3 amino acids shown in Table 3. In some embodiments, the antibody comprises a VH containing the CDRH1 amino acid sequence shown in any one of SEQ ID NO: 69-89. In some embodiments, the antibody comprises a VH containing the CDRH2 amino acid sequence shown in any one of SEQ ID NO: 90-123. In some embodiments, the antibody comprises a VH containing the CDRH3 amino acid sequence shown in any one of SEQ ID NO: 124-157.

[0081] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), wherein the antibody comprises a VH containing the amino acid sequences of CDRH1, CDRH2, and CDRH3 of any of the antibodies in Table 3. In some embodiments, the antibody comprises a VH containing the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in the following: SEQ ID NO: 69, 90, and 124; SEQ ID NO: 70, 91, and 125; SEQ ID NO: 71, 92, and 126; SEQ ID NO: 72, 93, and 127; SEQ ID NO: 69, 94, and 124; SEQ ID NO: 73, 95, and 128; SEQ ID NO: 74, 96, and 129; SEQ ID NO: 75, 97, and 130; SEQ ID NO: 74, 98, and 131; SEQ ID NO: 76, 99, and 132; SEQ ID NO: 77, 100, and 133; SEQ ID NO: 78, 101, and 134; SEQ ID NO: 79, 102, and 135; SEQ ID NO: 74, 103, and 136; SEQ SEQ ID NO: 80, 104, and 137; SEQ ID NO: 74, 105, and 138; SEQ ID NO: 74, 106, and 139; SEQ ID NO: 81, 107, and 140; SEQ ID NO: 81, 108, and 141; SEQ ID NO: 82, 109, and 142; SEQ ID NO: 83, 110, and 143; SEQ ID NO: 84, 99, and 144; SEQ ID NO: 85, 111, and 145; SEQ ID NO: 81, 112, and 146; SEQ ID NO: 86, 113, and 147; SEQ ID NO: 87, 114, and 148; SEQ ID NO: 74, 115, and 149; SEQ ID NO: 74, 116, and 150; SEQ ID NO: 74, 117, and 151; SEQ ID NO: 88, 118 and 152; SEQ ID NO: 89, 119 and 153; SEQ ID NO: 81, 120 and 154; SEQ ID NO: 74, 121 and 155; SEQ ID NO: 85, 122 and 156; or SEQ ID NO: 86, 123 and 157.

[0082] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), wherein the antibody comprises a VL containing the CDRL1, CDRL2, and / or CDRL3 amino acids shown in Table 4. In some embodiments, the antibody comprises a VL containing the CDRL1 amino acid sequence shown in any one of SEQ ID NO: 158-184. In some embodiments, the antibody comprises a VL containing the CDRL2 amino acid sequence shown in any one of KIS, WAS, STT, AAS, and SEQ ID NO: 185-197. In some embodiments, the antibody comprises a VL containing the CDRL3 amino acid sequence shown in any one of SEQ ID NO: 198-227.

[0083] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), wherein the antibody comprises a VL containing the amino acid sequences of CDRL1, CDRL2, and CDRL3 of any of the antibodies in Table 4. In some embodiments, the antibody comprises a VL containing the amino acid sequences of CDRL1, CDRL2, and CDRL3 shown in the following: SEQ ID NO: 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 159, WAS, and SEQ ID NO: 199; SEQ ID NO: 160, STT, and SEQ ID NO: 200; SEQ ID NO: 161, WAS, and SEQ ID NO: 201; SEQ ID NO: 162, AAS, and SEQ ID NO: 202; SEQ ID NO: 163, 185, and 203; SEQ ID NO: 164, 185, and 204; SEQ ID NO: 165, 185, and 205; SEQ ID NO: 166, 186, and 206; SEQ ID NO: 167, 187, and 207; SEQ ID NO: 168, 188, and 208; SEQ ID NO: SEQ ID NO: 169, 186, and 209; SEQ ID NO: 170, 185, and 198; SEQ ID NO: 169, 189, and 210; SEQ ID NO: 170, 185, and 211; SEQ ID NO: 171, 187, and 212; SEQ ID NO: 172, 187, and 213; SEQ ID NO: 173, 190, and 214; SEQ ID NO: 174, 191, and 215; SEQ ID NO: 175, 186, and 216; SEQ ID NO: 176, 186, and 217; SEQ ID NO: 170, 192, and 198; SEQ ID NO: 171, 193, and 218; SEQ ID NO: 177, 187, and 219; SEQ ID NO: 178, 189, and 220; SEQ ID NO: SEQ ID NO: 179, 187, and 221; SEQ ID NO: 165, 185, and 222; SEQ ID NO: 180, 187, and 223; SEQ ID NO: 181, 194, and 224; SEQ ID NO: 182, 195, and 199; SEQ ID NO: 172, 187, and 225; SEQ ID NO: 183, 196, and 226; or SEQ ID NO: 184, 197, and 227.

[0084] In some embodiments, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K), wherein the antibody comprises a VH containing the amino acid sequences of CDRH1, CDRH2 and / or CDRH3 as shown in Table 3 and a VL containing the amino acid sequences of CDRL1, CDRL2 and / or CDRL3 as shown in Table 4, wherein 2, 3, 4, 5 or 6 amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 are derived from the same antibody (i.e., as shown in Tables 3 and 4). In some embodiments, the antibody comprises a VH containing CDRH1, CDRH2, and CDRH3 regions and a VL containing CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise amino acid sequences shown in the following: SEQ ID NO: 69, 90, 124, 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 70, 91, 125, 159, WAS, and SEQ ID NO: 199; SEQ ID NO: 71, 92, 126, 160, STT, and SEQ ID NO: 200; SEQ ID NO: 72, 93, 127, 161, WAS, and SEQ ID NO: 201; SEQ ID NO: 69, 94, 124, 158, KIS, and SEQ ID NO: 198; SEQ ID NO: SEQ ID NO: 73, 95, 128, 162, AAS and SEQ ID NO: 202; SEQ ID NO: 74, 96, 129, 163, 185 and 203; SEQ ID NO: 75, 97, 130, 164, 185 and 204; SEQ ID NO: 74, 98, 131, 165, 185 and 205; SEQ ID NO: 76, 99, 132, 166, 186 and 206; SEQ ID NO: 77, 100, 133, 167, 187 and 207; SEQ ID NO: 78, 101, 134, 168, 188 and 208; SEQ ID NO: 79, 102, 135, 169, 186 and 209; SEQ ID NO: SEQ ID NOs: 74, 103, 136, 170, 185, and 198; SEQ ID NOs: 80, 104, 137, 169, 189, and 210; SEQ ID NOs: 74, 105, 138, 170, 185, and 211; SEQ ID NOs: 74, 106, 139, 171, 187, and 212; SEQ ID NOs: 81, 107, 140, 172, 187, and 213;SEQ ID NO: 81, 108, 141, 173, 190 and 214; SEQ ID NO: 82, 109, 142, 174, 191 and 215; SEQ ID NO: 83, 110, 143, 175, 186 and 216; SEQ ID NO: 84, 99, 144, 176, 186 and 217; SEQ ID NO: 85, 111, 145, 170, 192 and 198; SEQ ID NO: 81, 112, 146, 171, 193 and 218; SEQ ID NO: 86, 113, 147, 177, 187 and 219; SEQ ID NO: 87, 114, 148, 178, 189 and 220; SEQ ID NO: 74, 115, 149, 179, 187, and 221; SEQ ID NO: 74, 116, 150, 165, 185, and 222; SEQ ID NO: 74, 117, 151, 180, 187, and 223; SEQ ID NO: 88, 118, 152, 181, 194, and 224; SEQ ID NO: 89, 119, 153, 182, 195, and 199; SEQ ID NO: 81, 120, 154, 172, 187, and 225; SEQ ID NO: 74, 121, 155, 165, 185, and 222; SEQ ID NO: 85, 122, 156, 183, 196, and 226; or SEQ ID NO: 86, 123, 157, 184, 197, and 227.

[0085] In some embodiments, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K), the antibodies comprising a VH containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in any one of SEQ ID NO: 1-34. In some embodiments, the amino acid sequence of the VH consists of the amino acid sequence shown in any one of SEQ ID NO: 1-34.

[0086] In some embodiments, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K), the antibodies comprising a VL containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in any one of SEQ ID NO: 35-68. In some embodiments, the amino acid sequence of the VL consists of the amino acid sequence shown in any one of SEQ ID NO: 35-68.

[0087] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), the antibody comprising a VH containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in any one of SEQ ID NO: 1-34, and a VL containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in any one of SEQ ID NO: 35-68. In some embodiments, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K), the antibodies comprising a VH containing the amino acid sequence of any one of SEQ ID NO: 1-34 and a VL containing the amino acid sequence of any one of SEQ ID NO: 35-68. In some embodiments, the amino acid sequence of VH consists of the amino acid sequence shown in any one of SEQ ID NO: 1-34, and the amino acid sequence of VL consists of the amino acid sequence shown in any one of SEQ ID NO: 35-68.

[0088] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), the antibody comprising a VH containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in any one of SEQ ID NO: 1-34, and a VL containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in any one of SEQ ID NO: 35-68, wherein the amino acid sequence shown in any one of SEQ ID NO: 1-34 and SEQ ID NO: The amino acid sequences shown in any one of SEQ ID NO: 1-34 are derived from the same antibody (i.e., as shown in Tables 1 and 2). In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), comprising a VH containing the amino acid sequence of any one of SEQ ID NO: 1-34 and a VL containing the amino acid sequence of any one of SEQ ID NO: 35-68, wherein the amino acid sequences shown in any one of SEQ ID NO: 1-34 and any one of SEQ ID NO: 35-68 are derived from the same antibody (i.e., as shown in Tables 1 and 2). In some embodiments, the amino acid sequence of VH consists of the amino acid sequence shown in any one of SEQ ID NO: 1-34, and the amino acid sequence of VL consists of the amino acid sequence shown in any one of SEQ ID NO: 35-68, wherein the amino acid sequences shown in any one of SEQ ID NO: 1-34 and any one of SEQ ID NO: 35-68 are derived from the same antibody (i.e., as shown in Tables 1 and 2).

[0089] In some embodiments, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K), wherein the antibody comprises VH and VL containing the amino acid sequences shown in the following: SEQ ID NO: 1 and 35; SEQ ID NO: 2 and 36; SEQ ID NO: 3 and 37; SEQ ID NO: 4 and 38; SEQ ID NO: 5 and 39; SEQ ID NO: 6 and 40; SEQ ID NO: 7 and 41; SEQ ID NO: 8 and 42; SEQ ID NO: 9 and 43; SEQ ID NO: 10 and 44; SEQ ID NO: 11 and 45; SEQ ID NO: 12 and 46; SEQ ID NO: 13 and 47; SEQ ID NO: 14 and 48; SEQ ID NO: 15 and 49; SEQ ID NO: 16 and 50; SEQ ID NO: 17 and 51; SEQ ID NO: 18 and 52; SEQ ID NO: 19 and 53; SEQ ID SEQ ID NO: 20 and 54; SEQ ID NO: 21 and 55; SEQ ID NO: 22 and 56; SEQ ID NO: 23 and 57; SEQ ID NO: 24 and 58; SEQ ID NO: 25 and 59; SEQ ID NO: 26 and 60; SEQ ID NO: 27 and 61; SEQ ID NO: 28 and 62; SEQ ID NO: 29 and 63; SEQ ID NO: 30 and 64; SEQ ID NO: 31 and 65; SEQ ID NO: 32 and 66; SEQ ID NO: 33 and 67; or SEQ ID NO: 34 and 68.

[0090] In some embodiments, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K), wherein the antibody comprises VH and VL consisting of amino acid sequences shown in the following: SEQ ID NO: 1 and 35; SEQ ID NO: 2 and 36; SEQ ID NO: 3 and 37; SEQ ID NO: 4 and 38; SEQ ID NO: 5 and 39; SEQ ID NO: 6 and 40; SEQ ID NO: 7 and 41; SEQ ID NO: 8 and 42; SEQ ID NO: 9 and 43; SEQ ID NO: 10 and 44; SEQ ID NO: 11 and 45; SEQ ID NO: 12 and 46; SEQ ID NO: 13 and 47; SEQ ID NO: 14 and 48; SEQ ID NO: 15 and 49; SEQ ID NO: 16 and 50; SEQ ID NO: 17 and 51; SEQ ID NO: 18 and 52; SEQ ID NO: 19 and 53; SEQ ID SEQ ID NO: 20 and 54; SEQ ID NO: 21 and 55; SEQ ID NO: 22 and 56; SEQ ID NO: 23 and 57; SEQ ID NO: 24 and 58; SEQ ID NO: 25 and 59; SEQ ID NO: 26 and 60; SEQ ID NO: 27 and 61; SEQ ID NO: 28 and 62; SEQ ID NO: 29 and 63; SEQ ID NO: 30 and 64; SEQ ID NO: 31 and 65; SEQ ID NO: 32 and 66; SEQ ID NO: 33 and 67; or SEQ ID NO: 34 and 68.

[0091] In some embodiments, this disclosure provides antibodies that cross-competitively bind to LY6K (e.g., human LY6K) with any of the antibodies described above. In some embodiments, this disclosure provides antibodies that bind to the same or overlapping epitopes of LY6K (e.g., epitopes of human LY6K) as the antibodies described above.

[0092] In some implementations, the epitopes of the antibody can be determined by, for example, NMR spectroscopy, surface plasmon resonance (BIAcore) methods. ®The crystals can be determined by X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange combined mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any method known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are incorporated herein by reference in their entirety). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and their structures can be refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; U.S. Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323, all of which are incorporated herein by reference in their entirety). Mutagenic mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenic techniques, including alanine scanning mutagenesis, see, for example, Champe M et al., (1995), ibid. and Cunningham BC & Wells JA (1989), ibid. In one specific embodiment, alanine scanning mutagenesis is used to determine the epitope of an antibody. Alternatively, conventional techniques such as immunoassays, for example by demonstrating the ability of one antibody to block the binding of another antibody to the target antigen—i.e., competitive binding assays—can be used to identify antibodies that recognize and bind to the same or overlapping epitopes of LY6K (e.g., human LY6K). Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity to the epitope.Competitive binding can be determined in an assay in which the immunoglobulin to be tested inhibits the reference antibody and specifically binds to a common antigen such as LY6K (e.g., human LY6K). Several types of competitive binding assays are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137: 3614-9); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow E&Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 labeling (see Morel GA et al., (1988) Mol Immunol 25(1): 7-15); solid-phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176: 546-52); and directly labeled RIAs (see Moldenhauer G et al., (1990) Scand J Immunol 32: 77-82), all of which are incorporated herein by reference in their entirety. Typically, this assay involves the use of a purified antigen (e.g., LY6K, such as human LY6K) that binds to a solid surface or cell carrying either the unlabeled test immunoglobulin or a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label that binds to the solid surface or cell in the presence of the test immunoglobulin. Typically, an excess of the test immunoglobulin is present. Typically, when an excess of the competitive antibody is present, it will inhibit the specific binding of the reference or antibody to the common antigen by at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, or more. Competitive binding assays can be configured using labeled antigens or labeled antibodies in a wide variety of different forms. In a common form of this assay, the antigen is immobilized on a 96-well plate. Then, radioactive labeling or enzyme labeling is used to measure the ability of unlabeled antibodies to block the binding of labeled antibodies to antigens.For further details, see, for example, Wagener C et al., (1983) J Immunol 130: 2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407; and Antibodies: A Laboratory Manual, edited by Harlow E & Lane D, ibid., pp. 386-389, all of which are incorporated herein by reference in their entirety.

[0093] The anti-LY6K antigen-binding molecule disclosed herein can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof can be functionally linked to one or more other molecular entities (such as another antibody or antibody fragment) (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to produce a bispecific or multispecific antibody (e.g., a bispecific T-cell adaptor [BiTE] or a biaffinity retargeting antibody [DART]) having a second or additional binding specificity. In some embodiments, the bispecific or multispecific antibody has binding specificity to molecules on effector cells (e.g., CD3, CD16, CD137). In some embodiments, the antibody provided herein is a multispecific antibody. In some embodiments, the multispecific antibody provided herein comprises a CD3-binding region. In some embodiments, the CD3-binding region comprises a sequence from an antibody known to bind CD3 (e.g., OKT3, UTCH1, L2K, TR66, etc.). In some embodiments, the CD3-binding region comprises a sequence from an L2K anti-CD3 antibody. In some embodiments, the CD3-binding region comprises a sequence from a deimmunized L2K anti-CD3 antibody. In some embodiments, the CD3-binding region comprises a variable single-chain fragment (scFv) that specifically binds to CD3. In some embodiments, the multispecific antibody provided herein specifically binds to human CD3.

[0094] In some embodiments, the antibodies disclosed herein are conjugated to a pharmaceutically active substance. Pharmaceutically active substances include, but are not limited to, cytotoxic agents, cell growth inhibitors, toxins, radionuclides (e.g., radioisotopes), peptides, polynucleotides, detectable markers, and combinations thereof. In some embodiments, the pharmaceutically active substance is a cytotoxic agent. In some embodiments, the cytotoxic agent is capable of inducing cell death or destruction in contact with it. In some embodiments, the pharmaceutically active substance is a cell growth inhibitor. In some embodiments, the cell growth inhibitor is capable of preventing or substantially reducing the proliferation of cells in contact with it and / or inhibiting their activity or function. In some embodiments, the cytotoxic agent or cell growth inhibitor is a chemotherapeutic agent.

[0095] In some embodiments, the pharmaceutically active substance is a radionuclide. Suitable radionuclides include, but are not limited to, beta emitters, Auger emitters, converted electron emitters, alpha emitters, and low-photon-energy emitters. In some embodiments, the radionuclide is selected from... 3 H, 14 C 32 P, 35 S, 36 Cl、 45 Ca, 51 Cr 57 Co、 58 Co、 59 Fe、 67 Cu、 67 Ga、 76 As、 77 As、 89 Sr、 90 Y、 99 Tc, 99m Tc, 105 Rh、 111 In、 114m In、 117 Lu、 121 I, 123 I, 124 I, 125 I, 131 I, 149 Tb, 153 Sm、 161 Tb, 166 Ho、 177 Lu、 198 Au、 201 Tl、 211 At、 212 Pb, 213 Bi、 225 Ac、 186 Re、 188 Re、 212Bi、 213 Bi、 221 At、 223 Ac、 223 Ra、 225 Ac、 255 Fm and their combinations.

[0096] In some embodiments, the pharmaceutically active substance is a detectable marker. In some embodiments, the detectable marker includes a fluorescent portion, a click chemical handle, or a combination thereof.

[0097] In some embodiments, the pharmaceutically active substance is a drug. Suitable drugs include, but are not limited to, anticancer agents, anti-inflammatory agents, and anti-infective agents (e.g., antifungal agents, antibacterial agents, antiparasitic agents, and antiviral agents). Suitable anticancer agents include, but are not limited to, alkylating agents, antimetabolites, spindle toxin plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, kinase inhibitors, antihormonal agents, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides (e.g., those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation), ribozymes (e.g., VEGF expression inhibitors and HER2 expression inhibitors), vaccines (e.g., gene therapy vaccines), topoisomerase 1 inhibitors, antiangiogenic agents, pharmaceutically acceptable salts, acids, solvates, and derivatives of any of the above, and any combination thereof.

[0098] In some embodiments, the pharmaceutically active substance is a toxin. Suitable toxins include, but are not limited to, protein toxins (e.g., bacterial-derived and plant-derived toxins), toxins targeting tubulin filaments, DNA-targeting toxins, and RNA-targeting toxins. Examples of protein toxins include saponins, caryophyllin, ricin, modeccin, abrin, volkensin, viscumin, shiga toxin, shiga-like toxin, pseudomonad exotoxin (PE, also known as exotoxin A), diphtheria toxin (DT), and cholera toxin. Examples of toxins targeting tubulin filaments include maytansine alkaloids (e.g., DM1 and DM4), oliquistatins (e.g., monomethyloliquistatin E (MMAE) and monomethyloliquistatin F (MMAF)), toxoids, tubulolysins, cryptophycins, and rhizobium toxins. Examples of DNA-targeting toxins include chachiin: N-acetyl-γ-chachiin, CC-1065 analogs, buprofen, doxorubicin, methotrexate, benzodiazepines, camptothecin analogs, and anthracyclines. Examples of RNA-targeting toxins are muscarinic acid, spliceostatins, and thailanstatins.

[0099] In some embodiments, the pharmaceutically active substance is a polypeptide. Suitable polypeptides include, but are not limited to, Cas9; toxins (e.g., saponins, caryophyllin, white tree toxin, (de)bouganin, agrostin, ricin (toxin A chain); pokeweed antiviral protein, apoptin, diphtheria toxin, Pseudomonas exotoxin); metabolic enzymes (e.g., argininosuccinate lyase, argininosuccinate synthase); coagulation cascade enzymes; repair enzymes; enzymes for cell signaling; cell cycle regulators; gene regulators (e.g., transcription factors such as NF-κB or gene repressors such as methionine repressors).

[0100] In some embodiments, the pharmaceutically active substance is a polynucleotide. In some embodiments, the polynucleotide contains coding information. In some embodiments, the polynucleotide is a gene or open reading frame encoding a protein. In some embodiments, the polynucleotide contains regulatory information. In some embodiments, the polynucleotide is a promoter, a regulatory element binding region, or a sequence encoding microRNA. Suitable polynucleotides include natural and artificial nucleic acids. Artificial nucleic acids include, but are not limited to, peptide nucleic acids (PNAs), morpholino and locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threonine nucleic acids (TNAs). Each of these is distinguished from naturally occurring DNA or RNA by changes in its molecular backbone. Suitable polynucleotides include, but are not limited to, vectors; genes (e.g., transgenes that induce cell suicide); single-stranded DNA; linear double-stranded DNA; circular double-stranded DNA (e.g., plasmids); small circular DNA; DNA aptamers; single-stranded RNA; linear double-stranded RNA; mRNA; tRNA; rRNA; short interfering RNA (siRNA); microRNA (miRNA); antisense RNA; antisense oligonucleotides; peptide nucleic acids (PNA); aminophosphomorpholino oligomers (PMO); locked nucleic acids (LNA); bridged nucleic acids (BNA); 2'-deoxy-2'-fluoroarabinose nucleic acid (FANA); 2'-O-methoxyethyl-RNA (MOE); 2'-O,4'-aminoethylene-bridged nucleic acids; 3'-fluorohexetol nucleotides (FHNA); RNA aptamers; and combinations thereof.

[0101] Any immunoglobulin (Ig) constant region may be used in the antibodies disclosed herein. In some embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b).

[0102] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), the antibody comprising a heavy chain constant region optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

[0103] In some embodiments, this disclosure provides an antibody that specifically binds to LY6K (e.g., human LY6K), the antibody comprising a heavy chain constant region as a variant of the wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to FcγR with a lower affinity than the wild-type heavy chain constant region binds to FcγR.

[0104] In some embodiments, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K), the antibodies comprising a heavy chain constant region containing the amino acid sequence shown in Table 5. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 228 or 229. In some embodiments, the heavy chain constant region consists of the amino acid sequence of SEQ ID NO: 228 or 229.

[0105] Table 5. Exemplary amino acid sequences of the heavy chain constant region of anti-LY6K antibodies .

[0106]

[0107] In some embodiments, this disclosure provides antibodies that specifically bind to LY6K (e.g., human LY6K), the antibodies comprising a light chain constant region containing the amino acid sequence shown in Table 6. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 230 or 231. In some embodiments, the heavy chain constant region consists of the amino acid sequence of SEQ ID NO: 230 or 231.

[0108] Table 6. Amino acid sequences of the light chain constant region of exemplary anti-LY6K antibodies .

[0109]

[0110] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1, according to the EU numbering system) and / or the hinge region (residues 216-230, according to the EU numbering system) of the antibody to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cytotoxicity.

[0111] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the antibody described herein, such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Patent No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region can be altered to, for example, facilitate the assembly of light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody.

[0112] In one specific embodiment, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge-Fc fragment) to alter (e.g., reduce or increase) the in vivo half-life of the antibody. Examples of mutations that alter (e.g., reduce or increase) the in vivo half-life of the antibody can be found, for example, International Publications WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631; and U.S. Patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated herein by reference in their entirety. In some embodiments, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge-Fc fragment) to reduce the in vivo half-life of the antibody. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the constant region of IgG or its FcRn binding fragment (preferably an Fc or hinge Fc fragment) to increase the in vivo half-life of the antibody. In one specific embodiment, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1) according to the EU numbering system. In one specific embodiment, the constant region of IgG1 of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256 according to the EU numbering system. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety. This type of mutant IgG, termed the “YTE mutant,” has been shown to exhibit a fourfold increase in half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In some embodiments, the antibody comprises an IgG constant region containing one, two, three, or more amino acid residues substituted at positions 251-257, 285-290, 308-314, 385-389, and 428-436 according to the EU numbering system.

[0113] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1, according to the EU numbering system) and / or the hinge region (residues 216-230, according to the EU numbering system) of the antibody described herein to increase or decrease the antibody's affinity for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of antibodies that decrease or increase the antibody's affinity for Fc receptors and techniques for introducing such mutations into the Fc receptor or fragments thereof are known to those skilled in the art. Examples of mutations that can alter the affinity of an antibody for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Patent No. 6,737,056, and International Publications Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, all of which are incorporated herein by reference in their entirety.

[0114] In some embodiments, the antibody comprises a heavy chain constant region as a variant of the wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to FcγRIIB with a higher affinity than the wild-type heavy chain constant region. In some embodiments, the variant heavy chain constant region is a variant human heavy chain constant region, such as the variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In some embodiments, the variant human IgG heavy chain constant region comprises one or more amino acid mutations from the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In some embodiments, the constant region of the variant human IgG heavy chain contains a set of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F, according to the EU numbering system. In some embodiments, FcγRIIB is expressed on cells selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0115] In another embodiment, one, two, or more amino acid substitutions are introduced into the Fc region of the IgG constant region to alter the effector function of the antibody. For example, one or more amino acid residues selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered according to the EU numbering system, can be replaced with different amino acid residues, such that the antibody has altered affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or a C1 component of complement. This method is further described in detail in U.S. Patents 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. For descriptions of mutations that result in the deletion or inactivation of the constant region and thereby increase tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276: 6591-604, which is incorporated herein by reference in its entirety). In various implementations, one or more of the following mutations, numbered according to the EU numbering system, may be generated in the constant region of the antibody described herein: N297A substitution; N297Q substitution; L234A substitution; L234F substitution; L235A substitution; L235F substitution; L235V substitution; L237A substitution; S239D substitution; E233P substitution; L234V substitution; C236 deletion; P238A substitution; F243L substitution; D265A substitution; S267E substitution; L328F substitution; R292P substitution; Y300L substitution; A327Q substitution; P329A substitution; A330L substitution; I332E substitution; or P396L substitution.

[0116] In some embodiments, mutations selected from the group consisting of D265A, P329A, and combinations thereof according to the EU numbering system can be generated in the constant region of the antibody described herein. In some embodiments, mutations selected from the group consisting of L235A, L237A, and combinations thereof according to the EU numbering system can be generated in the constant region of the antibody described herein. In some embodiments, mutations selected from the group consisting of S267E, L328F, and combinations thereof according to the EU numbering system can be generated in the constant region of the antibody described herein. In some embodiments, mutations selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof according to the EU numbering system can be generated in the constant region of the antibody described herein. In some embodiments, mutations selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof according to the EU numbering system can be generated in the constant region of the antibody described herein. In some implementations, mutations selected from groups consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, can be generated in the constant region of the antibody described herein.

[0117] In one specific embodiment, the antibody described herein comprises a constant region of IgG1 having amino acid substitutions of N297Q or N297A according to the EU numbering system. In some embodiments, the antibody described herein comprises a constant region of IgG1 having mutations selected from the group consisting of D265A, P329A, and combinations thereof according to the EU numbering system. In another embodiment, the antibody described herein comprises a constant region of IgG1 having mutations selected from the group consisting of L234A, L235A, and combinations thereof according to the EU numbering system. In yet another embodiment, the antibody described herein comprises a constant region of IgG1 having mutations selected from the group consisting of L234F, L235F, N297A, and combinations thereof according to the EU numbering system. In some embodiments, the amino acid residues (according to the EU numbering system) at positions L234, L235, and D265 in the constant region of the antibody described herein are not L, L, and D, respectively. This method is described in detail in International Publication No. WO 14 / 108483, which is incorporated herein by reference in its entirety. In some embodiments, the amino acids corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively numbered according to the EU numbering system.

[0118] In some embodiments, one or more amino acid residues 329, 331, and 322 (according to EU numbering system) in the constant region of the antibody described herein may be replaced with different amino acid residues, such that the antibody exhibits altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is further described in detail in U.S. Patent No. 6,194,551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In some embodiments, one or more amino acid residues at positions 231 to 238 in the N-terminal region of the CH2 domain of the antibody described herein are altered, thereby altering the antibody's ability to fix complement, according to EU numbering system. This method is further described in International Publication No. WO 94 / 29351, which is incorporated herein by reference in its entirety. In some embodiments, the Fc region of the antibody described herein is modified by mutating one or more amino acids (e.g., introducing amino acid substitutions) at the following positions according to the EU numbering system to increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) and / or increase the antibody's affinity for the Fcγ receptor: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 28 6, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This method is further described in International Publication No. WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0119] In some embodiments, the antibody described herein comprises a modified constant region of IgG1, wherein the modification increases the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC). In some embodiments, 0.1, 1, or 10 µg / ml of antibody is capable of inducing cell death in at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of LY6K-expressing cells within 1, 2, or 3 hours, as evaluated by methods described herein and / or known to those skilled in the art. In some embodiments, the modified constant region of IgG1 comprises S239D and I332E substitutions according to the EU numbering system. In some embodiments, the modified constant region of IgG1 comprises S239D, A330L, and I332E substitutions according to the EU numbering system. In some embodiments, the modified constant region of IgG1 comprises L235V, F243L, R292P, Y300L, and P396L substitutions according to the EU numbering system. In some embodiments, the antibody is capable of inducing cell death in effector T cells and Tregs, wherein the percentage of Tregs undergoing cell death is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times the percentage of effector T cells undergoing cell death.

[0120] In some embodiments, the antibody described herein comprises a constant region of an IgG4 antibody, and the serine residue at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is replaced by a proline residue.

[0121] In some implementations, any constant region mutation or modification described herein may be introduced into one or both heavy chain constant regions of the antibody described herein, which has two heavy chain constant regions.

[0122] polypeptide On the other hand, this document provides polypeptides comprising one or more sequences shown in Tables 1 to 4 above. In some embodiments, the polypeptide comprises CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence shown in any of SEQ ID NO: 1-34 as determined by any of the methods discussed above. In some embodiments, the polypeptide comprises VH containing the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in any of SEQ ID NO: 1-34. In some embodiments, the polypeptide comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in the following items, respectively: SEQ ID NO: 1-34. NO: 69, 90 and 124; 70, 91 and 125; 71, 92 and 126; 72, 93 and 127; 69, 94 and 124; 73, 95 and 128; 74, 96 and 129; 75, 97 and 130; 74, 98 and 131; 76, 99 and 132; 77, 100 and 133; 78, 101 and 134; 79, 102 and 135; 74, 103 and 136; 80, 104 and 137; 74, 105 and 138; 74, 106 and 139; 81, 107 and 14 0; 81, 108 and 141; 82, 109 and 142; 83, 110 and 143; 84, 99 and 144; 85, 111 and 145; 81, 112 and 146; 86, 113 and 147; 87, 114 and 148; 74, 11 or 86, 123 and 157.

[0123] In some embodiments, the polypeptide comprises CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence shown in any of SEQ ID NO: 35-68 as determined by any of the methods discussed above. In some embodiments, the polypeptide comprises VL containing the CDRL1, CDRL2, and CDRL3 amino acid sequences shown in any of SEQ ID NO: 35-68.In some embodiments, the polypeptide comprises the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in the following items, respectively: SEQ ID NO: 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 159, WAS, and SEQ ID NO: 199; SEQ ID NO: 160, STT, and SEQ ID NO: 200; SEQ ID NO: 161, WAS, and SEQ ID NO: 201; SEQ ID NO: 162, AAS, and SEQ ID NO: 202; SEQ ID NO: 163, 185, and 203; SEQ ID NO: 164, 185, and 204; SEQ ID NO: 165, 185, and 205; SEQ ID NO: 166, 186, and 206; SEQ ID NO: 167, 187, and 207; SEQ ID NO: 168, 188, and 208; SEQ ID NO: SEQ ID NO: 169, 186, and 209; SEQ ID NO: 170, 185, and 198; SEQ ID NO: 169, 189, and 210; SEQ ID NO: 170, 185, and 211; SEQ ID NO: 171, 187, and 212; SEQ ID NO: 172, 187, and 213; SEQ ID NO: 173, 190, and 214; SEQ ID NO: 174, 191, and 215; SEQ ID NO: 175, 186, and 216; SEQ ID NO: 176, 186, and 217; SEQ ID NO: 170, 192, and 198; SEQ ID NO: 171, 193, and 218; SEQ ID NO: 177, 187, and 219; SEQ ID NO: 178, 189, and 220; SEQ ID NO: SEQ ID NO: 179, 187, and 221; SEQ ID NO: 165, 185, and 222; SEQ ID NO: 180, 187, and 223; SEQ ID NO: 181, 194, and 224; SEQ ID NO: 182, 195, and 199; SEQ ID NO: 172, 187, and 225; SEQ ID NO: 183, 196, and 226; or SEQ ID NO: 184, 197, and 227.

[0124] In some embodiments, the polypeptide comprises: VH containing the VH amino acid sequence of CDRH1, CDRH2, and CDRH3 as shown in any one of SEQ ID NO: 1-34; and VL containing the VL amino acid sequence of CDRL1, CDRL2, and CDRL3 as shown in any one of SEQ ID NO: 35-68. In some embodiments, the polypeptide comprises the VH and VL amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 as shown in the following: SEQ ID NO: 1 and 35, 2 and 36, 3 and 37, 4 and 38, 5 and 39, 6 and 40, 7 and 41, 8 and 42, 9 and 43, 10 and 44, 11 and 45, 12 and 46, 13 and 47, 14 and 48, 15 and 49, 16 and 50, 17 and 51, 18 and 52 , 19 and 53, 20 and 54, 21 and 55, 22 and 56, 23 and 57, 24 and 58, 25 and 59, 26 and 60, 27 and 61, 28 and 62, 29 and 63, 30 and 64, 31 and 65, 32 and 66, 33 and 67, or 34 and 68.

[0125] In some embodiments, the polypeptides provided herein comprise the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively, shown in the following items: SEQ ID NO: 69, 90, 124, 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 70, 91, 125, 159, WAS, and SEQ ID NO: 199; SEQ ID NO: 71, 92, 126, 160, STT, and SEQ ID NO: 200; SEQ ID NO: 72, 93, 127, 161, WAS, and SEQ ID NO: 201; SEQ ID NO: 69, 94, 124, 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 73, 95, 128, 162, AAS, and SEQ ID NO: 202; SEQ ID NO: SEQ ID NO: 74, 96, 129, 163, 185, and 203; SEQ ID NO: 75, 97, 130, 164, 185, and 204; SEQ ID NO: 74, 98, 131, 165, 185, and 205; SEQ ID NO: 76, 99, 132, 166, 186, and 206; SEQ ID NO: 77, 100, 133, 167, 187, and 207; SEQ ID NO: 78, 101, 134, 168, 188, and 208; SEQ ID NO: 79, 102, 135, 169, 186, and 209; SEQ ID NO: 74, 103, 136, 170, 185, and 198; SEQ ID NO: 80, 104, 137, 169, 189, and 210; SEQ ID SEQ ID NO: 74, 105, 138, 170, 185, and 211; SEQ ID NO: 74, 106, 139, 171, 187, and 212; SEQ ID NO: 81, 107, 140, 172, 187, and 213; SEQ ID NO: 81, 108, 141, 173, 190, and 214; SEQ ID NO: 82, 109, 142, 174, 191, and 215; SEQ ID NO: 83, 110, 143, 175, 186, and 216; SEQ ID NO: 84, 99, 144, 176, 186, and 217; SEQ ID NO: 85, 111, 145, 170, 192, and 198; SEQ ID NO: 81, 112, 146, 171, 193 and 218; SEQ ID NO: 86, 113, 147, 177, 187 and 219;SEQ ID NO: 87, 114, 148, 178, 189, and 220; SEQ ID NO: 74, 115, 149, 179, 187, and 221; SEQ ID NO: 74, 116, 150, 165, 185, and 222; SEQ ID NO: 74, 117, 151, 180, 187, and 223; SEQ ID NO: 88, 118, 152, 181, 194, and 224; SEQ ID NO: 89, 119, 153, 182, 195, and 199; SEQ ID NO: 81, 120, 154, 172, 187, and 225; SEQ ID NO: 74, 121, 155, 165, 185, and 222; SEQ ID NO: 85, 122, 156, 183, 196, and 226; or SEQ ID NO: 86, 123, 157, 184, 197, and 227.

[0126] Pharmaceutical Composition This document provides compositions comprising the disclosed anti-LY6K antibody of desired purity in a physiologically acceptable carrier, excipient, or stabilizer (see, for example, Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dose and concentration employed and includes buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10). (10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. ™ PLURONICS ™ Or polyethylene glycol (PEG).

[0127] In one specific embodiment, the pharmaceutical composition comprises the disclosed anti-LY6K antibody in a pharmaceutically acceptable carrier and optionally one or more other prophylactic or therapeutic agents. In some embodiments, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein can be used to reduce or block LY6K (e.g., human LY6K) activity and to treat conditions such as cancer. In some embodiments, this disclosure relates to a pharmaceutical composition of this disclosure comprising the disclosed anti-LY6K antibody, used as a medicine. In another embodiment, this disclosure relates to a pharmaceutical composition of this disclosure used in a method of treating cancer.

[0128] Pharmaceutically acceptable carriers for parenteral preparations include aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, multivalent chelating agents or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous media include sodium chloride injection, Ringer's solution injection, isotonic glucose injection, sterile water injection, and glucose and lactated Ringer's solution injection. Non-aqueous parenteral media include fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at antibacterial or antifungal concentrations may be added to parenteral preparations packaged in multi-dose containers, including phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride. Isotonic agents include chlorides and glucose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspensions and dispersants include sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (TWEEN). ® 80). Multivalent chelating agents or chelating agents for metal ions include EDTA. Drug carriers also include ethanol, polyethylene glycol, and propylene glycol for water-miscible media; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0129] Pharmaceutical compositions can be formulated for administration to a subject via any route. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral administration. Parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection is also contemplated herein. Injectables can be prepared in conventional forms, as liquid solutions or suspensions, as solid forms suitable for dissolving or suspending in a liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextran, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, and other such agents, such as, for example, sodium acetate, sorbitol monolaurate, triethanolamine oleate, and cyclodextrin.

[0130] Preparations for parenteral administration of antibodies include sterile solutions for injection, sterile dried soluble products such as lyophilized powders (including subcutaneous tablets) prepared for immediate use in combination with a solvent, sterile suspensions for injection, sterile dried insoluble products prepared for immediate use in combination with a mediator, and sterile emulsions. Solutions may be aqueous or non-aqueous.

[0131] If administered intravenously, suitable carriers include physiological saline or phosphate-buffered saline (PBS), as well as solutions containing thickeners and solubilizers, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0132] Prepare the antibody-containing topical mixture as described for local and systemic application. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated into creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, rinses, sprays, suppositories, bandages, skin patches, or any other formulation suitable for topical application.

[0133] The anti-LY6K antibodies disclosed herein can be formulated as aerosols for topical application, such as by inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids that can be used to treat inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). These formulations for respiratory application can be in the form of aerosols or solutions for nebulizers, or as fine powders for inhalation, alone or in combination with an inert carrier such as lactose. In this context, in some embodiments, the particles of the formulation will have a diameter of less than 50 micrometers, and in some embodiments, less than 10 micrometers.

[0134] The anti-LY6K antibody disclosed herein can be formulated into gels, creams, and lotions for topical or external application, such as for application to the skin and mucous membranes, such as the eyes, and for application to the eyes or for intracranial or intraspinal applications. Topical application is envisioned for transdermal delivery, as well as for application to the eyes or mucous membranes, or for inhalation therapy. Nasal solutions of the antibody, alone or in combination with other pharmaceutically acceptable excipients, can also be administered.

[0135] Transdermal patches, including iontophoresis and electrophoresis devices, are well known to those skilled in the art and can be used to administer antibodies. Such patches are disclosed, for example, in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entirety.

[0136] In some embodiments, the pharmaceutical composition comprising the antibody described herein is a lyophilized powder that can be reconstituted for administration as a solution, emulsion, and other mixture. It can also be reconstituted and formulated into a solid or gel. The lyophilized powder is prepared by dissolving the antibody described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacological components. Excipients that may be used include, but are not limited to, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. In some embodiments, the solvent may also contain a buffer at approximately a neutral pH, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art. The solution is then sterilely filtered and subsequently lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In some embodiments, the resulting solution is dispensed into vials for lyophilization. Each vial will contain a single or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as at approximately 4°C to room temperature. Reconstitute the lyophilized powder with water for injection to provide a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or another suitable carrier. The precise amount depends on the compound selected. This amount can be determined empirically.

[0137] The anti-LY6K antibodies disclosed herein and other compositions provided herein can also be formulated to target specific tissues, receptors, or other regions of the body of a subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions of the invention. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entirety. In one specific embodiment, the antibody described herein targets a tumor.

[0138] Compositions intended for in vivo administration can be sterile. This can be easily achieved through filtration, for example, using a sterile filter membrane.

[0139] Usage and Purpose In another aspect, this disclosure provides a method of treating a subject using the anti-LY6K antibody disclosed herein. The anti-LY6K antibody disclosed herein can be used to treat any disease or condition in a subject characterized by overexpression of LY6K (e.g., human LY6K). In some embodiments, the disease or condition is resistant to a checkpoint target agent (e.g., an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, or an antagonist anti-PD-1 antibody). In some embodiments, the disease or condition relapses after treatment with a checkpoint target agent (e.g., an antagonist anti-CTLA-4 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, or an antagonist anti-PD-1 antibody).

[0140] In some embodiments, this disclosure provides a method of treating a subject for cancer, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as disclosed herein.

[0141] Cancers that can be treated with the anti-LY6K antibodies or pharmaceutical compositions disclosed herein include, but are not limited to, solid tumors, hematologic malignancies (e.g., leukemia, lymphoma, myeloma, such as multiple myeloma), and metastatic lesions. In some embodiments, the cancer is a solid tumor. Examples of solid tumors include malignancies such as sarcomas and carcinomas, such as adenocarcinomas of various organ systems, such as those involving the lungs, breast, ovaries, lymph nodes, gastrointestinal tract (e.g., colon), anus, genital and genitourinary tracts (e.g., kidneys, urothelial cells, bladder cells, prostate), pharynx, CNS (e.g., brain, nerves, or glial cells), head and neck, skin (e.g., melanoma), and pancreas, as well as adenocarcinomas including malignancies such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), small bowel cancer, and esophageal cancer. The cancer may be in an early, intermediate, late, or metastatic stage. In some embodiments, the cancer is resistant to checkpoint-targeting agents (e.g., antagonist anti-CTLA-4 antibody, antagonist anti-PD-L1 antibody, antagonist anti-PD-L2 antibody, or antagonist anti-PD-1 antibody). In some embodiments, the cancer recurs after treatment with checkpoint-targeting agents (e.g., antagonist anti-CTLA-4 antibody, antagonist anti-PD-L1 antibody, antagonist anti-PD-L2 antibody, or antagonist anti-PD-1 antibody).

[0142] In some implementations, the cancer is selected from lung cancer (e.g., lung adenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC or NSCLC adenocarcinoma with squamous and / or non-squamous histology)), melanoma (e.g., advanced melanoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), myeloid carcinoma (e.g., multiple myeloid carcinoma), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of estrogen receptor, progesterone receptor, or Her2 / neu, e.g., triple-negative breast cancer), ovarian cancer, colorectal cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer (e.g., esophageal squamous cell carcinoma), mesothelioma, nasopharyngeal carcinoma, thyroid cancer, cervical cancer, epithelial cancer, peritoneal cancer, or lymphoproliferative disorders (e.g., post-transplant lymphoproliferative disorders).

[0143] In some implementations, the cancer is a hematologic cancer, such as leukemia, lymphoma, or myeloma. In other implementations, the cancer is a type of leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In some implementations, the cancer is a lymphoma, such as B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) diffuse large B-cell lymphoma, germinal center B-cell (GCB) diffuse large B-cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, relapsed follicular non-Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In some implementations, the cancer is a myeloma, such as multiple myeloma.

[0144] In another implementation, the cancer is selected from carcinoma (e.g., advanced or metastatic cancer), melanoma, or lung cancer, such as non-small cell lung cancer.

[0145] In some implementations, the cancer is lung cancer, such as lung adenocarcinoma, non-small cell lung cancer, or small cell lung cancer.

[0146] In some embodiments, the cancer is melanoma, such as advanced melanoma. In some embodiments, the cancer is advanced or unresectable melanoma that is unresponsive to other therapies. In other embodiments, the cancer is melanoma with a BRAF mutation (e.g., BRAF V600 mutation). In other embodiments, the anti-LY6K antibody or pharmaceutical composition disclosed herein is administered after treatment with an anti-CTLA-4 antibody (e.g., ipilimumab) in combination with or without a BRAF inhibitor (e.g., vemurafenib or dabrafenib).

[0147] In another implementation, the cancer is liver cancer, such as advanced liver cancer, with or without viral infection, such as chronic viral hepatitis.

[0148] In another implementation, the cancer is prostate cancer, such as advanced prostate cancer.

[0149] In yet another implementation, the cancer is myeloma, such as multiple myeloma.

[0150] In yet another implementation, the cancer is kidney cancer, such as renal cell carcinoma (RCC) (e.g., metastatic RCC, clear cell renal cell carcinoma (CCRCC), or papillary renal cell carcinoma).

[0151] In yet another implementation scheme, the cancer is selected from lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, leukemia, or metastatic lesions of cancer.

[0152] In some embodiments, these methods further include administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapy agent, or a checkpoint target. In some embodiments, the chemotherapeutic agent is a hypomethylating agent (e.g., azacitidine). In some embodiments, the chemotherapeutic agent is a DNA damage inducer (e.g., gemcitabine). In some embodiments, the checkpoint target agent is selected from the group consisting of: antagonistic anti-CTLA-4 antibody, antagonistic anti-PD-L1 antibody, antagonistic anti-PD-L2 antibody, antagonistic anti-PD-1 antibody, antagonistic anti-TIM-3 antibody, antagonistic anti-LAG-3 antibody, antagonistic anti-VISTA antibody, antagonistic anti-CD96 antibody, antagonistic anti-CEACAM1 antibody, agonistic anti-CD137 antibody, agonistic anti-GITR antibody, and agonistic anti-OX40 antibody. In some embodiments, the checkpoint target is selected from the group consisting of: antagonistic anti-CTLA-4 antibodies, antagonistic anti-PD-L1 antibodies, antagonistic anti-PD-L2 antibodies, and antagonistic anti-PD-1 antibodies, wherein the LY6K (e.g., human LY6K) antibody or pharmaceutical composition disclosed herein synergizes with the checkpoint target.

[0153] In some embodiments, this disclosure relates to the antibody and / or pharmaceutical composition of this disclosure used in the methods of this disclosure, wherein the method further includes administering an additional therapeutic agent to a subject. In some embodiments, this disclosure relates to (a) the antibody and / or pharmaceutical composition of this disclosure and (b) an additional therapeutic agent used as a medicine. In some embodiments, this disclosure relates to (a) the antibody and / or pharmaceutical composition of this disclosure and (b) an additional therapeutic agent used in a method of treating cancer. In another embodiment, this disclosure relates to a pharmaceutical composition, kit, or combination kit comprising (a) the antibody and / or pharmaceutical composition of this disclosure and (b) an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapy agent, or a checkpoint targeting agent.

[0154] In some embodiments, the anti-PD-1 antibody is used in the methods disclosed herein. In some embodiments, the anti-PD-1 antibody is nivolumab, developed by Bristol-Myers Squibb, also known as BMS-936558 or MDX1106. In some embodiments, the anti-PD-1 antibody is pembrolizumab, developed by Merck & Co, also known as lambolizumab or MK-3475. In some embodiments, the anti-PD-1 antibody is pidilizumab, developed by CureTech, also known as CT-011. In some embodiments, the anti-PD-1 antibody is MEDI0680, developed by MedImmune, also known as AMP-514. In some embodiments, the anti-PD-1 antibody is PDR001, developed by Novartis Pharmaceuticals. In some embodiments, the anti-PD-1 antibody is REGN2810, developed by Regeneron Pharmaceuticals. In some embodiments, the anti-PD-1 antibody is PF-06801591 developed by Pfizer. In some embodiments, the anti-PD-1 antibody is BGB-A317 developed by BeiGene. In some embodiments, the anti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. In some embodiments, the anti-PD-1 antibody is SHR-1210 developed by Hengrui.

[0155] Other non-limiting examples of anti-PD-1 antibodies that can be used in the treatment methods disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety for all purposes: U.S. Patent No. 6,808,710; U.S. Patent No. 7,332,582; U.S. Patent No. 7,488,802; U.S. Patent No. 8,008,449; U.S. Patent No. 8,114,845; U.S. Patent No. 8,168,757; U.S. Patent No. 8,354,509; U.S. Patent No. 8,686,119; U.S. Patent No. 8,735,553; U.S. Patent No. 8,747,847; U.S. Patent No. 8,779,105; U.S. Patent No. 8,927,697; U.S. Patent No. 8,993,731; U.S. Patent No. 9,102,727; U.S. Patent No. 9,205,148; U.S. Publication No. US US Patent Application No. 2013 / 0202623 A1; US ​​Publication No. 2013 / 0291136 A1; US ​​Publication No. 2014 / 0044738 A1; US ​​Publication No. 2014 / 0356363 A1; US ​​Publication No. 2016 / 0075783 A1; PCT Publication No. 2013 / 033091 A1; PCT Publication No. 2015 / 036394 A1; PCT Publication No. 2014 / 179664 A2; PCT Publication No. 2014 / 209804 A1; PCT Publication No. 2014 / 206107 A1; PCT Publication No. 2015 / 058573 A1; PCT Publication No. 2015 / 085847 A1; PCT Publication No. WO 2015 / 200119 A1; PCT Publication No. WO 2016 / 015685 A1; and PCT Publication No. WO2016 / 020856 A1.

[0156] In some embodiments, the anti-PD-L1 antibody is used in the methods disclosed herein. In some embodiments, the anti-PD-L1 antibody is atezolizumab, developed by Genentech. In some embodiments, the anti-PD-L1 antibody is durvalumab, developed by AstraZeneca, Celgene, and MedImmune. In some embodiments, the anti-PD-L1 antibody is avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In some embodiments, the anti-PD-L1 antibody is MDX-1105, developed by Bristol-Myers Squibb. In some embodiments, the anti-PD-L1 antibody is AMP-224, developed by Amplimmune and GSK.

[0157] Non-limiting examples of anti-PD-L1 antibodies that can be used in the treatment methods disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety for all purposes: U.S. Patent No. 7,943,743; U.S. Patent No. 8,168,179; U.S. Patent No. 8,217,149; U.S. Patent No. 8,552,154; U.S. Patent No. 8,779,108; U.S. Patent No. 8,981,063; U.S. Patent No. 9,175,082; U.S. Publication No. US 2010 / 0203056 A1; U.S. Publication No. US 2003 / 0232323 A1; U.S. Publication No. US 2013 / 0323249A1; U.S. Publication No. US 2014 / 0341917 A1; U.S. Publication No. US 2014 / 0044738 ... US Publication No. 2015 / 0203580 A1; US ​​Publication No. 2015 / 0225483 A1; US ​​Publication No. 2015 / 0346208A1; US ​​Publication No. 2015 / 0355184 A1; PCT Publication No. 2014 / 100079 A1; PCT Publication No. 2014 / 022758 A1; PCT Publication No. 2014 / 055897 A2; PCT Publication No. 2015 / 061668 A1; PCT Publication No. 2015 / 109124 A1; PCT Publication No. 2015 / 195163 A1; PCT Publication No. 2016 / 000619 A1; and PCT Publication No. 2016 / 030350 A1.

[0158] In some embodiments, an anti-CTLA-4 antibody is used in the methods disclosed herein. In some embodiments, the anti-CTLA-4 antibody is ipilimumab developed by Bristol-Myers Squibb.

[0159] In some embodiments, the anti-LY6K antibody disclosed herein is administered to the subject in combination with a compound that targets immunomodulatory enzymes such as IDO (indoleamine-(2,3)-dioxygenase) and / or TDO (tryptophan 2,3-dioxygenase). Therefore, in some embodiments, an additional therapeutic agent is a compound that targets an immunomodulatory enzyme, such as an inhibitor of indoleamine-(2,3)-dioxygenase (IDO). In some embodiments, such compounds are selected from epacadostat (IncyteCorp; see, for example, WO 2010 / 005958, which is incorporated herein by reference in its entirety), F001287 (FlexusBiosciences / Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In some embodiments, the compound is epacadostat. In another embodiment, the compound is F001287. In yet another embodiment, the compound is indoximod. In another embodiment, the compound is NLG919. In one specific embodiment, the anti-LY6K antibody disclosed herein is administered in combination with an IDO inhibitor to a subject for the treatment of cancer. The IDO inhibitors for the treatment of cancer as described herein are available in solid dosage forms of pharmaceutical compositions, such as tablets, pills, or capsules, wherein the pharmaceutical composition comprises an IDO inhibitor and a pharmaceutically acceptable excipient. Therefore, the antibody as described herein and the IDO inhibitor as described herein can be administered alone, sequentially, or simultaneously as separate dosage forms. In some embodiments, the antibody is administered parenterally, and the IDO inhibitor is administered orally. In some embodiments, the inhibitor is selected from the group consisting of epaccaldo (Incyte Corporation), F001287 (Flexus Biosciences / Bristol-Myers Squibb), indomod (NewLink Genetics), and NLG919 (NewLink Genetics). Epicadol has been described in PCT Publication WO 2010 / 005958, which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the inhibitor is epicadol. In another embodiment, the inhibitor is F001287. In another embodiment, the inhibitor is indomod. In another embodiment, the inhibitor is NLG919.

[0160] In some implementations, the anti-LY6K antibody disclosed herein is administered in combination with a vaccine to a subject. The vaccine may be, for example, a peptide vaccine, a DNA vaccine, or an RNA vaccine.

[0161] In some implementations, the anti-LY6K antibody disclosed herein is administered to a subject in combination with an adjuvant. Various adjuvants may be used depending on the treatment context. Non-limiting examples of suitable adjuvants include, but are not limited to, Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), montanide ISA (Sebik incomplete adjuvant), Ribi adjuvant system (RAS), TiterMax, muramyl peptide, Syntex adjuvant formulation (SAF), alum (aluminum hydroxide and / or aluminum phosphate), aluminum salt adjuvants, Gerbu... ® Adjuvants, nitrocellulose-adsorbed antigens, encapsulated or entrapped antigens, 3-de-O-acylated monophosphoryllipid A (3D-MPL), immunostimulatory oligonucleotides, Toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, and immunostimulatory complexes such as saponins, Quil A, QS-21, QS-7, ISCOMATRIX, etc. Other adjuvants include CpG oligonucleotides and double-stranded RNA molecules such as poly(A) and poly(U). Combinations of the above adjuvants may also be used. See, for example, U.S. Patent Nos. 6,645,495, 7,029,678, and 7,858,589, all of which are incorporated herein by reference in their entirety. In some embodiments, the adjuvant used herein is QS-21 STIMULON.

[0162] In some embodiments, the anti-LY6K antibody disclosed herein is administered to a subject in combination with an additional therapeutic agent comprising a TCR. In some embodiments, the additional therapeutic agent is a soluble TCR. In some embodiments, the additional therapeutic agent is cells expressing a TCR. Therefore, in some embodiments, this disclosure relates to the antibody and / or pharmaceutical compositions of this disclosure in combination with an additional therapeutic agent comprising a TCR, used as a medicament and / or in methods of treating cancer.

[0163] In some embodiments, the anti-LY6K antibody disclosed herein is administered to a subject in combination with cells expressing a chimeric antigen receptor (CAR). In some embodiments, the cells are T cells.

[0164] In some embodiments, the anti-LY6K antibody disclosed herein is administered in combination with a TCR mimic antibody to a subject. In some embodiments, the TCR mimic antibody is an antibody that specifically binds to a peptide-MHC complex. For non-limiting examples of TCR mimic antibodies, see, for example, U.S. Patent No. 9,074,000 and U.S. Publications Nos. US 2009 / 0304679 A1 and US 2014 / 0134191 A1, all of which are incorporated herein by reference in their entirety.

[0165] In some embodiments, the anti-LY6K antibody disclosed herein is administered to a subject in combination with a bispecific T-cell adaptor (BiTE) (e.g., as described in WO2005061547A2, which is incorporated herein by reference in its entirety) and / or a biaffinity retargeting antibody (DART) (e.g., as described in WO2012162067A2, which is incorporated herein by reference in its entirety). In some embodiments, the BiTE and / or DART specifically bind to tumor-associated antigens (e.g., peptides overexpressed in tumors, peptides derived from tumor viruses, peptides containing tumor-specific post-translational modifications, peptides specifically mutated in tumors) and molecules on effector cells (e.g., CD3 or CD16). In some embodiments, the tumor-associated antigen is EGFR (e.g., human EGFR), optionally wherein the BiTE and / or DART comprises the VH and VL sequences of cetuximab. In some embodiments, the tumor-associated antigen is Her2 (e.g., human Her2), optionally wherein BiTE and / or DART comprise the VH and VL sequences of trastuzumab. In some embodiments, the tumor-associated antigen is CD20 (e.g., human CD20).

[0166] Anti-LY6K antibodies and other therapeutic agents (e.g., chemotherapeutic agents, radiotherapy agents, checkpoint targets, IDO inhibitors, vaccines, adjuvants, soluble TCRs, TCR-expressing cells, chimeric antigen receptor-expressing cells, and / or TCR mimic antibodies) can be administered alone, sequentially, or simultaneously as separate dosage forms. In some embodiments, the anti-LY6K antibody is administered parenterally, and the IDO inhibitor is administered orally.

[0167] The antibody or pharmaceutical compositions described herein can be delivered to subjects via a variety of routes. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intraarterial, and subcutaneous routes. Lung administration is also possible, for example, through the use of inhalers or nebulizers, and formulations containing aerosolizers used as nebulizers. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered subcutaneously or intravenously. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered intraarterially. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered intratumorally. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered to tumor-draining lymph nodes.

[0168] The amount of antibody or composition that will effectively treat and / or prevent the symptoms will depend on the nature of the disease and can be determined using standard clinical techniques.

[0169] The precise dosage used in the composition will also depend on the route of administration and the severity of the infection or disease it causes, and should be determined based on the physician's judgment and the individual subject's circumstances. For example, the effective dosage can also vary depending on the route of administration, target site, patient's physiological state (including age, weight, and health status), whether the patient is human or animal, and whether other drugs or treatments administered are prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including genetically modified mammals, can also be treated. The therapeutic dosage is optimally titrated to optimize safety and efficacy.

[0170] The anti-LY6K described herein can also be used to determine the level of LY6K (e.g., human LY6K) protein in biological samples using classical immunohistochemical methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay markers are known in the art and include enzyme markers such as glucose oxidase; radioactive isotopes such as iodine (… 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In) and technetium ( 99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, as well as biotin. Such labels can be used to label the antibodies described herein. Alternatively, a second antibody recognizing the anti-LY6K antibody described herein can be labeled and used in combination with the anti-LY6K antibody to detect LY6K (e.g., human LY6K) protein levels. Thus, in some embodiments, this disclosure relates to the use of the anti-LY6K antibody of this disclosure for in vitro detection of LY6K (e.g., human LY6K) protein in biological samples. In another embodiment, this disclosure relates to the use of the anti-LY6K antibody of this disclosure for in vitro determination and / or detection of LY6K (e.g., human LY6K) protein levels in biological samples, optionally wherein the anti-LY6K antibody is conjugated with a radionuclide or a detectable label, and / or carries a label described herein, and / or is used using immunohistochemical methods.

[0171] Determining the expression level of LY6K (e.g., human LY6K) protein aims to include qualitatively or quantitatively measuring or estimating the level of LY6K (e.g., human LY6K) protein in a first biological sample, either directly (e.g., by determining or estimating an absolute protein level) or relatively (e.g., by comparing it with disease-related protein levels in a second biological sample). The expression level of LY6K (e.g., human LY6K) peptide in the first biological sample can be measured or estimated and compared with a standard LY6K (e.g., human LY6K) protein level, which is taken from, for example, a second biological sample obtained from a disease-free individual, or determined by averaging levels over a population of disease-free individuals. As understood in the art, once the “standard” LY6K (e.g., human LY6K) peptide level is known, it can be repeatedly used as a standard for comparison. This disclosure relates to an in vitro method for determining and / or detecting the level of LY6K protein (e.g., human LY6K protein) in a biological sample, the method comprising qualitatively or quantitatively measuring or estimating the level of LY6K protein (e.g., human LY6K protein) in the biological sample by immunohistochemical methods.

[0172] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells that potentially express LY6K (e.g., human LY6K). Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Biological samples include peripheral blood mononuclear cells (PBMCs).

[0173] The anti-LY6K antibody described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications well-known and standard to those skilled in the art and based on this specification. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro assessment and evaluation of immune system status, immune response, and / or cancer (e.g., tumorigenesis) can be used to predict, diagnose, and monitor patient samples, including those known to have or suspected of having immune system dysfunction or cancer, or regarding anticipated or expected immune system responses, antigen responses, or vaccine responses. Assessment and evaluation of immune system status, immune response, and / or cancer can also be used to determine a patient's suitability for clinical trials of drugs or for a specific chemotherapeutic agent, radiotherapy agent, or antibody (including combinations thereof) relative to the administration of different agents or antibodies. This type of prognostic and diagnostic monitoring and assessment has already been utilized in practice using antibodies against the HER2 protein in breast cancer (HercepTest). ™ The assay was performed in Dako, where it was also used to evaluate the use of Herceptin. ® This disclosure relates to antibody therapy for patients. In vivo application includes targeted cell therapy and immunomodulation, as well as radiographic imaging of immune responses. Therefore, in some embodiments, this disclosure relates to the anti-LY6K antibody and / or pharmaceutical composition of this disclosure used as a diagnostic agent. In some embodiments, this disclosure relates to the anti-LY6K antibody and / or pharmaceutical composition of this disclosure used in methods for predicting, diagnosing, and / or monitoring subjects with or suspected of having immune system dysfunction, cancer, and / or regarding anticipated or desired immune system responses, antigen responses, or vaccine responses. In another embodiment, this disclosure relates to the use of the anti-LY6K antibody of this disclosure for predicting, diagnosing, and / or monitoring subjects and / or regarding anticipated or desired immune system responses, antigen responses, or vaccine responses by in vitro determination and / or detection of human LY6K protein levels in biological samples of subjects with or suspected of having immune system dysfunction, cancer.

[0174] In some embodiments, anti-LY6K antibodies can be used for immunohistochemistry of biopsy samples. In some embodiments, this method is an in vitro method. In another embodiment, anti-LY6K antibodies can be used to detect levels of LY6K (e.g., human LY6K), or the levels of cells containing LY6K (e.g., human LY6K) on their membrane surface, the levels of which can then be correlated with certain disease symptoms. The anti-LY6K antibodies described herein may carry a detectable or functional label and / or may be conjugated to a radionuclide or a detectable label. When fluorescent labeling is used, the specific binding member can be identified and quantified using currently available microscopy and fluorescence activated cell sorter analysis (FACS) or a combination of two methodological procedures known in the art. The anti-LY6K antibodies described herein may carry a fluorescent label or may be conjugated to a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein and Texas Red, Alexa Fluor dye, Cy dye, and DyLight dye. Anti-LY6K antibodies may carry a radiolabel or a radionuclide or may be conjugated to a radiolabel or radionuclide, such as an isotope. 3 H, 14 C 32 P, 35 S, 36 Cl、 51 Cr 57 Co、 58 Co、 59 Fe、 67 Cu、 90 Y、 99 Tc, 111 In、 117 Lu、 121 I, 124 I, 125 I, 131 I, 198 Au、 211 At、 213 Bi、 225 Ac and 186Re. When using radiolabeling, currently available counting procedures known in the art can be used to identify and quantify the specific binding of anti-LY6K antibodies to LY6K (e.g., human LY6K). In the case of an enzyme label, detection can be performed by any currently used colorimetric, spectrophotometric, fluorescence spectrophotometric, galvanic, or gas analysis technique known in the art. This can be achieved by contacting a sample or control sample with the anti-LY6K antibody under conditions that allow for the formation of a complex between the anti-LY6K antibody and LY6K (e.g., human LY6K). Any complexes formed between the anti-LY6K antibody and LY6K (e.g., human LY6K) are detected and compared in the sample and control. Given the specific binding of the anti-LY6K antibody described herein to LY6K (e.g., human LY6K), the anti-LY6K antibody can be used for the specific detection of LY6K (e.g., human LY6K). The anti-LY6K antibody described herein can also be used to purify LY6K (e.g., human LY6K) via immunoaffinity purification. This article also includes assay systems, which can be prepared in the form of test kits, reagent kits, or combination kits, for the quantitative analysis of the presence of, for example, LY6K (e.g., human LY6K) / LY6K (e.g., human LY6K) ligand complexes. The systems, test kits, reagent kits, or combination kits may contain labeled components, such as labeled antibodies, and one or more additional immunochemical reagents.

[0175] Polynucleotides, vectors, and methods for antibody production On the other hand, this document provides: a polynucleotide comprising a nucleotide sequence encoding an antibody described herein, or a portion thereof or a fragment thereof (e.g., VL and / or VH; and light and / or heavy chains) specifically binding to the LY6K (e.g., human LY6K) antigen; and a vector, such as a vector comprising such a polynucleotide, for recombinant expression in host cells (e.g., *E. coli* and mammalian cells). This document provides polynucleotides comprising nucleotide sequences encoding the heavy and / or light chains of any antibody provided herein, and vectors comprising such polynucleotide sequences, such as expression vectors for their efficient expression in host cells (e.g., mammalian cells). In some embodiments, nucleic acid molecules encoding antibodies described herein are isolated or purified.

[0176] In certain respects, this document provides an antibody that encodes a polypeptide that specifically binds to LY6K (e.g., human LY6K) and contains an amino acid sequence as described herein, as well as (e.g., in a dose-dependent manner) a polynucleotide sequence that competes with such an antibody for binding to the LY6K polypeptide (e.g., human LY6K) or an antibody that binds to the same epitope as such an antibody.

[0177] In some aspects, this document provides polynucleotides comprising nucleotide sequences encoding a light chain or heavy chain of an antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain containing a VL FR and CDR of an antibody described herein (see, for example, Tables 2 and 4) or a nucleotide sequence encoding a heavy chain containing a VH FR and CDR of an antibody described herein (see, for example, Tables 1 and 3). In some embodiments, the polynucleotide encodes the VH, VL, heavy chain, and / or light chain of the antibody described herein. In another embodiment, the polynucleotide encodes a first VH and a first VL of the antibody described herein. In another embodiment, the polynucleotide encodes a second VH and a second VL of the antibody described herein. In another embodiment, the polynucleotide encodes a first heavy chain and a first light chain of the antibody described herein. In another embodiment, the polynucleotide encodes a second heavy chain and a second light chain of the antibody described herein. In yet another embodiment, the polynucleotide encodes the VH and / or VL, or the heavy chain and / or light chain of the antibody described herein.

[0178] This document also provides polynucleotides encoding anti-LY6K antibodies, which are optimized, for example, through codon / RNA optimization, replacement with heterologous signaling sequences, and elimination of unstable elements in the mRNA. Methods for generating optimized nucleic acids encoding anti-LY6K antibodies or fragments thereof (e.g., light chains, heavy chains, VH domains, or VL domains) for recombinant expression by introducing codon changes and / or eliminating repressive regions in the mRNA can be performed by correspondingly modifying the optimization methods described in, for example, U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splicing sites and unstable elements within the RNA (e.g., A / T- or A / U-rich elements) can be mutated without altering the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These alterations exploit the degeneracy of the genetic code, such as using alternative codons for the same amino acids. In some implementations, it may be necessary to change one or more codons to encode conserved mutations, such as similar amino acids having similar chemical structure and properties and / or functions to the original amino acid. Such methods can increase the expression of anti-LY6K antibodies or fragments thereof by at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times or more relative to the expression of anti-LY6K antibodies encoded by unoptimized polynucleotides.

[0179] In some embodiments, an optimized polynucleotide sequence encoding an anti-LY6K antibody or a fragment thereof (e.g., a VL domain and / or a VH domain) described herein may hybridize with an antisense (e.g., complementary) polynucleotide sequence encoding an unoptimized polynucleotide sequence encoding an anti-LY6K antibody or a fragment thereof (e.g., a VL domain and / or a VH domain). In a specific embodiment, an optimized nucleotide sequence encoding an anti-LY6K antibody or a fragment thereof described herein hybridizes with an antisense polynucleotide sequence encoding an unoptimized polynucleotide sequence encoding an anti-LY6K antibody or a fragment thereof described herein under high-strict hybridization conditions. In one specific embodiment, an optimized nucleotide sequence encoding an anti-LY6K antibody or a fragment thereof described herein hybridizes with an antisense polynucleotide sequence encoding an unoptimized polynucleotide sequence encoding an anti-LY6K antibody or a fragment thereof described herein under high-strict, medium-strict, or low-strict hybridization conditions. Information regarding hybridization conditions has been described, see, for example, U.S. Patent Application Publication No. 2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference in its entirety.

[0180] Polynucleotides can be obtained and their nucleotide sequences determined by any method known in the art. The nucleotide sequences encoding antibodies described herein (e.g., antibodies described in Tables 1 through 4 and modified forms of these antibodies) can be determined using methods known in the art, i.e., assembling in a manner known to encode specific amino acids in order to generate nucleic acids encoding antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6, which is incorporated herein by reference in its entirety, and which simply involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding an antibody, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR).

[0181] Alternatively, the polynucleotide encoding the antigen-binding region of the antibody described herein can be generated from nucleic acids from a suitable source (e.g., hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells that produce the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the light chain and / or heavy chain of the antibody. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding variable light chain regions and / or variable heavy chain regions of the antibody. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning.

[0182] If a clone of a nucleic acid containing a specific antigen-binding region or antibody is unavailable, but the sequence of the antigen-binding region or antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from any tissue or cell expressing an antibody, or nucleic acid isolated from any tissue or cell expressing an antibody, preferably polyA+ RNA, such as hybridoma cells selected to express the antibodies described herein) using any method well known in the art. The amplified nucleic acid generated by PCR can then be cloned into a reproducible cloning vector using any method known in the art.

[0183] DNA encoding the anti-LY6K (e.g., human LY6K) antibody described herein can be readily isolated and sequenced using conventional methods, such as by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the anti-LY6K (e.g., human LY6K) antibody. Hybridoma cells can serve as a source of this DNA. After isolation, the DNA can be placed in an expression vector and then transfected into host cells that do not otherwise produce immunoglobulins, such as *E. coli* cells, simian COS cells, and Chinese hamster ovary (CHO) cells (e.g., from the CHO GS System). ™ (Lonza) CHO cells) or myeloma cells, to obtain the synthesis of anti-LY6K antibodies in recombinant host cells.

[0184] To generate complete antibodies or antigen-binding regions, PCR primers comprising the VH or VL nucleotide sequence, restriction sites, and flanking sequences protecting the restriction sites can be used to amplify the VH or VL sequence in the scFv clone. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region (e.g., human γ1 or human γ4 constant region), and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region (e.g., human κ or λ constant region). In some embodiments, the vector for expressing the VH or VL domain includes an EF-1α promoter, a secretion signal, a cloning site for the variable region, a constant region, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a vector expressing the essential constant region. The heavy chain transformation vector and the light chain transformation vector are then co-transfected into cell lines using techniques known to those skilled in the art to generate stable or transient cell lines expressing full-length antibodies (e.g., IgG).

[0185] DNA can also be modified, for example, by replacing mouse sequences with coding sequences of human heavy and light chain constant regions, or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence.

[0186] Also provided are polynucleotides that hybridize with polynucleotides encoding the antibodies described herein under high-strict, moderate-strict, or low-strict hybridization conditions. In specific embodiments, the polynucleotides described herein hybridize with polynucleotides encoding the VH domain and / or VL domain provided herein under high-strict, moderate-strict, or low-strict hybridization conditions.

[0187] Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve hybridization of DNA bound to a filter in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC / 0.1% SDS at about 50–65°C; hybridization under highly stringent conditions may involve hybridization of nucleic acids bound to a filter in 6x SSC at about 45°C, followed by one or more washes in 0.1x SSC / 0.2% SDS at about 68°C. Hybridization under other stringent conditions is known to those skilled in the art and has been described, see, for example, Ausubel FM et al. (eds.), (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pp. 6.3.1–6.3.6 and 2.10.3, which is incorporated herein by reference in its entirety.

[0188] In some aspects, this document provides (e.g., recombinantly) cells (e.g., host cells) that express antibodies described herein that specifically bind to LY6K (e.g., human LY6K), along with associated polynucleotides and expression vectors. This document provides vectors (e.g., expression vectors) for recombinant expression in host cells, preferably mammalian cells (e.g., CHO cells), which contain polynucleotides encoding a nucleotide sequence encoding an anti-LY6K antibody or fragment. This document also provides host cells containing such vectors for recombinant expression of the anti-LY6K antibody (e.g., human or humanized antibody) described herein. In one particular aspect, this document provides a method for generating the antibodies described herein, the method comprising expressing the antibody from a host cell.

[0189] Recombinant expression of antibodies described herein that specifically bind to LY6K (e.g., human LY6K), including full-length antigen-binding regions or the heavy and / or light chains of the antibody described herein, typically involves the construction of expression vectors containing polynucleotides encoding antibodies. Once polynucleotides encoding antibody molecules described herein, the heavy and / or light chains of antibodies, or fragments thereof (e.g., variable regions of the heavy and / or light chains) are obtained, vectors for generating antibody molecules can be produced using recombinant DNA techniques well known in the art. Therefore, methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies or antibody fragments (e.g., light or heavy chains) are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding antibodies or antibody fragments (e.g., light or heavy chains) and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Replicable vectors are also provided containing nucleotide sequences operatively linked to a promoter encoding antibody molecules described herein, the heavy or light chains of antibodies, variable regions of the heavy or light chains of antibodies, or fragments thereof, or CDRs of the heavy or light chains. Such a vector may, for example, include a nucleotide sequence encoding a constant region of an antibody molecule (see, for example, International Publications WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464, which are incorporated herein by reference in their entirety), and the variable region of the antibody may be cloned into such a vector to express the entire heavy chain, the entire light chain, or both the entire heavy chain and the light chain.

[0190] In some embodiments, the vector comprises polynucleotides encoding the VH, VL, heavy chain, and / or light chain of the antibody described herein. In another embodiment, the vector comprises polynucleotides encoding the VH and VL of the antibody described herein. In yet another embodiment, the vector comprises polynucleotides encoding the heavy chain and light chain of the antibody described herein.

[0191] Expression vectors can be transferred to cells (e.g., host cells) using conventional techniques, and the resulting cells can then be cultured using conventional techniques to produce cells containing the antibodies or fragments thereof described herein. Therefore, this document provides host cells containing a polynucleotide encoding an antibody or fragment thereof described herein, or a heavy or light chain thereof, or a single-chain antibody described herein, operatively linked to a promoter for expressing such a sequence in the host cell.

[0192] In some embodiments, the host cell contains polynucleotides encoding VH and VL of the antibody described herein. In another embodiment, the host cell contains a vector containing polynucleotides encoding VH and VL of the antibody described herein. In yet another embodiment, the host cell contains a first polynucleotide encoding VH of the antibody described herein and a second polynucleotide encoding VL of the antibody described herein. In yet another embodiment, the host cell contains a first vector and a second vector, the first vector containing a first polynucleotide encoding VH of the antibody described herein and the second vector containing a second polynucleotide encoding VL of the antibody described herein.

[0193] In specific embodiments, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form the anti-LY6K (e.g., human LY6K) antibody described herein. In some embodiments, a host cell population comprising such a first host cell and such a second host cell is provided herein.

[0194] In some embodiments, this document provides a vector family comprising a first vector and a second vector, the first vector comprising a polynucleotide encoding a light chain / light chain variable region of an anti-LY6K (e.g., human LY6K) antibody described herein, and the second vector comprising a heavy chain / heavy chain variable region of a polynucleotide encoding a heavy chain / heavy chain variable region of an anti-LY6K (e.g., human LY6K) antibody described herein.

[0195] Various host expression vector systems can be used to express the antibody molecules described herein (see, for example, U.S. Patent No. 5,807,715, which is incorporated herein by reference in its entirety). Such host expression systems represent vectors by which the coding sequence of interest can be generated and subsequently purified, but also represent cells that can express the antibody molecules described herein in situ when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with, for example, recombinant phage DNA, plasmid DNA, or coliform DNA expression vectors containing antibody coding sequences (e.g., *Escherichia coli* and *Bacillus subtilis*). B. subtilis Yeast transformed with, for example, a recombinant yeast expression vector containing an antibody coding sequence (e.g., *Saccharomyces* genus). Saccharomyces ) and Pichia pastoris ( PichiaInsect cell systems infected with, for example, recombinant viral expression vectors containing antibody-encoding sequences (e.g., baculoviruses); plant cell systems infected with, for example, recombinant viral expression vectors containing antibody-encoding sequences (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with, for example, recombinant plasmid expression vectors containing antibody-encoding sequences (e.g., Ti plasmids) (e.g., green algae, such as Chlamydomonas reinhardtii); or mammalian cell systems having, for example, recombinant expression constructs (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH). The recombinant expression construct, comprising 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells, contains a promoter derived from a mammalian cell genome (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In one specific embodiment, the cells used to express the antibody described herein are Chinese hamster ovary (CHO) cells, such as those from the CHO GS System. ™ (Lonza) CHO cells. In some embodiments, the heavy and / or light chains of the antibody produced by CHO cells may have an N-terminal glutamine or glutamate residue replaced by pyroglutamic acid. In some embodiments, the cells used to express the antibody described herein are human cells, such as human cell lines. In one specific embodiment, the mammalian expression vector is pOptiVEC. ™ Or pcDNA3.3. In some embodiments, bacterial cells such as *Escherichia coli* or eukaryotic cells (e.g., mammalian cells), particularly those intended for expressing complete recombinant antibody molecules, are used to express the recombinant antibody molecules. For example, mammalian cells such as CHO cells, bound to a major intermediate early gene promoter element from a vector such as human cytomegalovirus, are efficient antibody expression systems (Foecking MK & Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8(7): 662-7, each of which is incorporated herein by reference in its entirety). In some embodiments, the antibodies described herein are produced by CHO cells or NSO cells. In one specific embodiment, the expression of the nucleotide sequence encoding the antibody described herein that specifically binds to LY6K (e.g., human LY6K) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0196] In bacterial systems, a number of expression vectors can be advantageously selected based on the intended use of the expressed antibody molecule. For example, when producing large quantities of such antibodies, a vector that directs the expression of easily purified, high-level fusion protein products may be desirable in order to generate a pharmaceutical composition of the antibody molecule. Such vectors include, but are not limited to: the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the coding sequence can be individually ligated into the vector along with the lac Z coding region in a frame-compliant manner to produce the fusion protein; the pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509); and so on, all of which are incorporated herein by reference in their entirety. For example, the pGEX vector can also be used to express exogenous peptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a matrix of glutathione agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites, allowing the cloned target gene product to be released from the GST moiety.

[0197] In insect systems, for example, the alfalfa silver-striped noctuid moth ( Autographa californica Nucleopolyhedrovirus (AcNPV) can be used as a vector to express exogenous genes. The virus is found in the fall armyworm (AcNPV). Spodoptera frugiperda It grows in cells. The coding sequence can be cloned separately into non-essential regions of the virus (such as the polyhedrin gene) and placed under the control of AcNPV promoters (such as the polyhedrin promoter).

[0198] In mammalian host cells, numerous virus-based expression systems can be utilized. When adenovirus is used as the expression vector, the coding sequence of interest can be linked to the adenoviral transcription / translation control complex, such as the late promoter and triple leader sequence. This chimeric gene can then be inserted into the adenoviral genome via in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) will produce a viable recombinant virus capable of expressing the molecule in an infected host (see, for example, Logan J & Shenk T (1984) PNAS 81(12):3655-9, which is incorporated herein by reference in its entirety). Effective translation of the inserted coding sequence may also require specific start signals. These signals include the ATG start codon and adjacent sequences. Furthermore, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by including appropriate transcriptional enhancer elements, transcription terminators, etc. (see, for example, Bitter G et al., (1987) Methods Enzymol. 153: 516-544, which is incorporated herein by reference in its entirety).

[0199] Furthermore, host cell lines can be selected that regulate the expression of inserted sequences or modify and process gene products in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for protein function. Different host cells possess characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the proper modification and processing of expressed exogenous proteins. For this purpose, eukaryotic host cells with cellular mechanisms for the proper processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In some embodiments, the anti-LY6K (e.g., human LY6K) antibody described herein is produced in mammalian cells such as CHO cells.

[0200] In one specific embodiment, the antibody described herein has a reduced fucose content or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibody can be expressed in cells with defective or absent fucosylation capacity. In one specific example, a cell line with two knockout alleles of α1,6-fucosyltransferase can be used to produce an antibody with a reduced fucose content. ® The Lonza system is an example of such a system that can be used to generate antibodies with reduced fucose content.

[0201] To achieve long-term, high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines that stably express the anti-LY6K (e.g., human LY6K) antibody described herein can be engineered. In specific embodiments, the cells provided herein stably express light chain / light chain variable regions and heavy chain / heavy chain variable regions, which associate to form the antigen-binding region or antibody described herein.

[0202] In some respects, instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA and selectivity markers controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). Following the introduction of exogenous DNA / polynucleotides, engineered cells can be allowed to grow in enrichment media for 1–2 days before being transferred to selectivity media. The selectivity markers in the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into their chromosome and grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing anti-LY6K (e.g., human LY6K) or fragments thereof as described herein. Such engineered cell lines are particularly useful in screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0203] Many selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-32), hypoxanthine-guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034) and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23) genes in tk-, hgprt-, or aprt- cells, all of which are incorporated herein by reference in their entirety. Additionally, antimetabolite resistance can be used as a basis for selecting the following genes: dhfrThis confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS78: 1527-31). gpt , which conferred resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo, which conferred resistance to aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro This confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-56), all of which are incorporated herein by reference in their entirety. Methods commonly known in the field of recombinant DNA technology can be routinely applied to select desired recombinant clones, and such methods are described, for example, in the following literature: Ausubel FM et al., (ed.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al., (ed.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) JMol Biol 150: 1-14, all of which are incorporated herein by reference in their entirety.

[0204] The expression level of antibody molecules can be increased by vector amplification (see Bebbington CR & Henschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). When the biomarker in the vector system is amplifiable, an increase in the level of inhibitors present in the host cell culture will increase the copy number of the biomarker gene. Protein production will also increase because the amplified region is associated with the gene of interest (Crouse GF et al., (1983) Mol CellBiol 3: 257-66, which is incorporated herein by reference in its entirety).

[0205] Host cells can be co-transfected with two or more expression vectors described herein, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. Both vectors may contain the same selectivity markers, enabling equal expression of heavy and light chain polypeptides. Host cells can be co-transfected with varying amounts of the two or more expression vectors. For example, host cells can be transfected with any of the following ratios of first and second expression vectors: approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0206] Alternatively, a single vector encoding and capable of expressing both the heavy and light chain polypeptides can be used. In this case, the light chain should be placed before the heavy chain to avoid excessive toxicity of the free heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and Köhler G (1980) PNAS 77: 2197-2199, each of which is incorporated herein by reference in its entirety). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA. The expression vector may be monocistronic or polycistronic. Polycistronic nucleic acid constructs may encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more gene / nucleotide sequences, or in the range of 2 to 5, 5 to 10, or 10 to 20 gene / nucleotide sequences. For example, bicistronic nucleic acid constructs may contain a promoter, a first gene (e.g., the heavy chain of the antibody described herein), and a second gene (e.g., the light chain of the antibody described herein) in the following order. In this expression vector, transcription of both genes can be driven by promoters, while translation of mRNA from the first gene can be carried out via a cap-dependent scanning mechanism, and translation of mRNA from the second gene can be carried out via a cap-independent mechanism (e.g., via IRES).

[0207] Once the antibody molecules described herein are generated through recombinant expression, they can be purified using any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after protein A, and size column chromatography), centrifugation, differential dissolution, or any other standard technique for protein purification. Furthermore, the antibodies described herein can be fused with heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0208] In specific embodiments, the antibodies described herein are isolated or purified. In some embodiments, the isolated antibody is an antibody substantially free of other antibodies having an antigen specificity different from that of the isolated antibody. For example, in some embodiments, the antibody preparations described herein are substantially free of cellular material and / or chemical precursors. The term “substantially free of cellular material” includes antibody preparations in which the antibody is isolated from the cellular components of the cells from which the antibody was isolated or recombined to produce the antibody. Thus, antibodies substantially free of cellular material include antibody preparations having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as “contaminating proteins”) and / or antibody variants (e.g., different post-translational modifications of the antibody or other different forms of the antibody, such as antibody fragments). When antibodies are recombined to produce the antibody, they are generally also substantially free of culture medium, i.e., the culture medium constitutes less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When antibodies are produced by chemical synthesis, they are generally substantially free of chemical precursors or other chemicals, i.e., they are isolated from chemical precursors or other chemicals involved in protein synthesis. Therefore, preparations of such antibodies contain less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In one specific embodiment, the antibody described herein is isolated or purified.

[0209] Anti-LY6K (e.g., human LY6K) antibodies or fragments thereof can be produced by any method known in the art for synthesizing proteins or antibodies, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise stated, the methods described herein employ conventional techniques from molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art. These techniques are described, for example, in the references cited herein and are fully explained therein. See, for example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual update); Current Protocols in Immunology, John Wiley & Sons (1987 and annual update); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRLPress; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (ed.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of these references are incorporated into this article in their entirety by way of citation.

[0210] In one specific embodiment, the antibody described herein is prepared, expressed, generated, or isolated by any means involving, for example, the synthesis or genetic engineering of a DNA sequence. In some embodiments, such an antibody comprises a sequence (e.g., a DNA sequence or amino acid sequence) that is not naturally present in an antibody germline library of animals or mammals (e.g., humans).

[0211] In one aspect, this document provides a method for preparing an anti-LY6K (e.g., human LY6K) antibody, the method comprising culturing the cells or host cells described herein. In some embodiments, the method is performed in vitro. In some aspects, this document provides a method for preparing an anti-LY6K (e.g., human LY6K) antibody, the method comprising expressing (e.g., recombinantly expressing) the antibody using the cells or host cells described herein (e.g., cells or host cells containing a polynucleotide encoding an antibody described herein). In some embodiments, the cells are isolated cells. In some embodiments, exogenous polynucleotides have been introduced into the cells. In some embodiments, the method further includes the step of purifying the antibody obtained from the cells or host cells.

[0212] In some embodiments, antibodies are generated by expressing polynucleotides encoding VH and VL of the antibody described herein in cells under suitable conditions, such that the polynucleotides are expressed and antibodies are generated. In another embodiment, antibodies are generated by expressing polynucleotides encoding the heavy and light chains of the antibody described herein in cells under suitable conditions, such that the polynucleotides are expressed and antibodies are generated. In some embodiments, antibodies are generated by expressing a first polynucleotide encoding VH of the antibody described herein and a second polynucleotide encoding VL of the antibody described herein in cells under suitable conditions, such that the polynucleotides are expressed and antibodies are generated. In some embodiments, antibodies are generated by expressing a first polynucleotide encoding the heavy chain of the antibody described herein and a second polynucleotide encoding the light chain of the antibody described herein in cells under suitable conditions, such that the polynucleotides are expressed and antibodies are generated.

[0213] The methods used to generate polyclonal antibodies are known in the art (see, for example, Chapter 11, in: Short Protocols in Molecular Biology, (2002) 5th Edition, edited by Ausubel FM et al., John Wiley and Sons, New York, which is incorporated herein by reference in its entirety).

[0214] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, hybridoma techniques can be used to generate monoclonal antibodies, including those known in the art and taught, for example, in Harlow E & Lane D, *Antibodies: A Laboratory Manual*, (Cold SpringHarbor Laboratory Press, 2nd edition, 1988); and Hammerling GJ et al., *Monoclonal Antibodies and T-Cell Hybridomas 563 681* (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma techniques. For example, monoclonal antibodies can be recombinantly generated from host cells expressing exogenous antibodies or fragments thereof (e.g., light and / or heavy chains of such antibodies).

[0215] In specific embodiments, as used herein, a “monoclonal antibody” is an antibody produced by a single cell (e.g., a hybridoma or host cell that produces a recombinant antibody) that specifically binds to LY6K (e.g., human LY6K), as determined, for example, by an ELISA or other antigen-binding or competitive binding assay known in the art or provided herein. In some embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In some embodiments, the monoclonal antibody is a monovalent or multivalent (e.g., bivalent) antibody. In some embodiments, the monoclonal antibody is a monospecific or multispecific antibody (e.g., a bispecific antibody). The monoclonal antibodies described herein may be prepared, for example, by a hybridoma method as described in Kohler G & Milstein C (1975) Nature 256: 495, which is incorporated herein by reference in its entirety, or may be isolated from a phage library, for example, using techniques as described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed therefrom are well known in the art (see, for example, Chapter 11, see: Short Protocols in Molecular Biology, (2002) 5th edition, Ausubel FM et al., ibid.).

[0216] As used herein, an antibody is multivalent (e.g., bivalent) to an antigen when it contains at least two (e.g., two or more) monovalent binding regions, each capable of binding to an epitope on the antigen. Each monovalent binding region may bind to the same or different epitopes on the antigen.

[0217] Methods for generating and screening specific antibodies using hybridoma technology are routine and well-known in the art. For example, in hybridoma methods, mice or other suitable host animals such as sheep, goats, rabbits, rats, hamsters, or macaques are immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to proteins used for immunization (e.g., LY6K (e.g., human LY6K)). Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, pp. 59–103 (Academic Press, 1986), which is incorporated herein by reference in its entirety). Additionally, RIMMS (Repeated Immunization Multiple Sites) technology can be used to immunize animals (Kilpatrick KE et al., (1997) Hybridoma 16:381–9, which is incorporated herein by reference in its entirety).

[0218] In some implementations, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) may be immunized with an antigen (e.g., LY6K, e.g., human LY6K), and once an immune response is detected, such as the detection of antibodies specific to the antigen in mouse serum, the mouse spleen is harvested and spleen cells are isolated. The spleen cells are then fused with any suitable myeloma cells using well-known techniques, such as those from the American Type Culture Collection (ATCC). ® Cells from the SP20 cell line (Manassas, VA) were used to form hybridomas. Hybridomas were selected and cloned through limiting dilution. In some embodiments, lymph nodes from immunized mice were harvested and fused with NSO myeloma cells.

[0219] The hybridoma cells thus prepared are seeded in a suitable culture medium and allowed to grow, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium used for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (“HAT medium”), which prevent the growth of HGPRT-deficient cells.

[0220] The specific implementation scheme employs myeloma cells that are efficiently fused, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. These myeloma cell lines include mouse myeloma cell lines such as the NS0 cell line, or cell lines derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells, available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human heterologous myeloma cell lines for producing human monoclonal antibodies are also described (Kozbor D (1984) J Immunol 133:3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), each of which is incorporated herein by reference in its entirety).

[0221] The production of monoclonal antibodies against LY6K (e.g., human LY6K) in the culture medium in which hybridoma cells are grown is measured. The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, such as immunoprecipitation, or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0222] Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity have been identified, clonal subclones can be grown using a limiting dilution procedure and standard methods (Goding JW (edited), Monoclonal Antibodies: Principles and Practice, ibid.). Suitable media for this purpose include, for example, D-MEM or RPMI 1640. Furthermore, hybridoma cells can be grown in vivo as ascites tumors in animals.

[0223] Monoclonal antibodies secreted by subclones can be appropriately separated from culture medium, ascites fluid, or serum using conventional immunoglobulin purification procedures such as protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0224] The antibodies described herein include, for example, antibody fragments that recognize LY6K (e.g., human LY6K) and can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be generated by proteolytic cleavage of an immunoglobulin molecule using an enzyme such as papain (to produce the Fab fragment) or pepsin (to produce the F(ab')2 fragment). The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of the antibody molecule linked by disulfide bonds in the hinge region.

[0225] Furthermore, the antibodies described herein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle carrying a polynucleotide sequence encoding them. Specifically, DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of the affected tissue). The DNA encoding the VH and VL domains is recombined with an scFv adapter by PCR and cloned into a phage particle vector. The vector is electroporated into *E. coli*, and *E. coli* is infected with a helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are typically recombined and fused to phage gene III or gene VIII. Phages expressing antigen-binding regions that bind to specific antigens can be selected or identified using antigens, such as labeled antigens or antigens that bind to or capture on solid surfaces or beads. Examples of phage display methods that can be used to prepare the antibodies described herein include those disclosed in the following literature: Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT application number PCT / GB91 / 001134; International Publications WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO93 / 1 WO 95 / 15982, WO 95 / 20401 and WO 97 / 13844; and U.S. Patents 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108, all of which are incorporated herein by reference in their entirety.

[0226] As described in the references above, after phage selection, antibody-coding regions from phages can be isolated and used to generate complete antibodies, including human antibodies or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described below. Methods known in the art, such as those disclosed in PCT Publication WO 92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; and Better M et al., (1988) Science 240: 1041-1043, can also be used to generate antibody fragments such as Fab, Fab', and F(ab')2 fragments through recombinant synthesis, all of which are incorporated herein by reference in their entirety.

[0227] In some embodiments, to generate complete antibodies, the VH or VL sequence can be amplified from a template (e.g., scFv clone) using PCR primers comprising the VH or VL nucleotide sequence, a restriction site, and flanking sequences protecting the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing the VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing the VL constant region (e.g., the human κ or λ constant region). Alternatively, both the VH and VL domains can be cloned into a single vector expressing the essential constant region. The heavy chain transformation vector and the light chain transformation vector are then co-transfected into cell lines using techniques known to those skilled in the art to generate stable or transient cell lines expressing full-length antibodies (e.g., IgG).

[0228] Chimeric antibodies are molecules in which different portions of an antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain a variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody. Methods for generating chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, all of which are incorporated herein by reference in their entirety.

[0229] Humanized antibodies are capable of binding to predetermined antigens and comprise a framework region having essentially the amino acid sequence of human immunoglobulins and a core region (CDR) having essentially the amino acid sequence of non-human immunoglobulins (e.g., mouse immunoglobulins). In some embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins. The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotypes, including IgG1, IgG2, IgG3, and IgG4.Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to CDR transplantation (European Patent EP 239400; International Publication WO 91 / 09967; and US Patents 5,225,539, 5,530,101 and 5,585,089), faceting or surface repair (European Patents EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5): 489-498; Studnicka GM et al., (1994) Prot Engineering 7(6):805-814; and Roguska MA et al., (1994) PNAS 91: 969-973), chain truncation (US Patent No. 5,565,332), and techniques disclosed in, for example, the following documents: US Patent No. 6,407,213, US Patent No. 5,766,886; International Publication No. WO 93 / 17105; Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10): 895 904; Couto JR et al., (1995) Cancer Res. 55 (23 Supplement): 5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10; and Pedersen JT et al., (1994) J Mol Biol 235(3): 959-73, all of which are incorporated herein by reference in their entirety. See also U.S. Application Publication No. US 2005 / 0042664 A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0230] Methods for preparing multispecific antibodies (e.g., bispecific antibodies) have been described, see, for example, U.S. Patent Nos. 7,951,917, 7,183,076, 8,227,577, 5,837,242, 5,989,830, 5,869,620, 6,132,992 and 8,586,713, all of which are incorporated herein by reference in their entirety.

[0231] Bispecific bivalent antibodies and methods for their preparation are described, for example, in U.S. Patent Nos. 5,731,168, 5,807,706, and 5,821,333 and U.S. Application Publications Nos. 2003 / 020734 and 2002 / 0155537, each of which is incorporated herein by reference in its entirety. Bispecific tetravalent antibodies and methods for their preparation are described, for example, in International Application Publications Nos. WO02 / 096948 and WO 00 / 44788, the disclosures of which are incorporated herein by reference in their entirety. See also International Application Publications Nos. 93 / 17715, 92 / 08802, 91 / 00360 and 92 / 05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Patents Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920 and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992); each of these documents is incorporated herein by reference in its entirety.

[0232] The bispecific antibodies described herein can be generated using the DuoBody technology platform (Genmab A / S) as described, for example, in International Publications Nos. WO 2011 / 131746, WO2011 / 147986, WO 2008 / 119353 and WO 2013 / 060867, and in Labrijn AF et al., (2013) PNAS 110(13): 5145-5150. The DuoBody technology can be used to combine half of a first monospecific antibody or a first antigen-binding region containing two heavy chains and two light chains with half of a second monospecific antibody or a second antigen-binding region containing two heavy chains and two light chains. The resulting heterodimer contains one heavy chain and one light chain from the first antibody or the first antigen-binding region, which are paired with one heavy chain and one light chain from the second antibody or the second antigen-binding region. The resulting heterodimer is a bispecific antibody when the two monospecific antibodies or antigen-binding regions recognize different epitopes on different antigens.

[0233] DuoBody technology requires that each monospecific antibody or antigen-binding region include a heavy chain constant region with a single-point mutation in the CH3 domain. The point mutation allows for stronger interactions between the CH3 domains in the resulting bispecific antibody than between the CH3 domains in the monospecific antibody or antigen-binding region. The single-point mutation in each monospecific antibody or antigen-binding region is located at residues 366, 368, 370, 399, 405, 407, or 409 in the CH3 domain of the heavy chain constant region, according to the EU numbering system, as described, for example, in International Publication No. WO 2011 / 131746. Furthermore, the single-point mutation is located at a different residue in a monospecific antibody or antigen-binding region compared to another monospecific antibody or antigen-binding region. For example, one monospecific antibody or antigen-binding region may contain the mutant F405L (i.e., a mutation from phenylalanine to leucine at residue 405), while another monospecific antibody or antigen-binding region may contain the mutant K409R (i.e., a mutation from lysine to arginine at residue 409), numbered according to the EU numbering system. The heavy chain constant region or antigen-binding region of a monospecific antibody may be an IgG1, IgG2, IgG3, or IgG4 isotype (e.g., human IgG1 isotype), and bispecific antibodies produced via DuoBody technology can retain Fc-mediated effector function.

[0234] Another method for generating bispecific antibodies is known as the "mortar and pestle" strategy (see, for example, International Publication WO2006 / 028936). In this technique, mismatches in the Ig heavy chains are reduced by mutating selected amino acids at the interface that forms the CH3 domain in IgG. At the site where the two heavy chains interact directly within the CH3 domain, an amino acid with a small side chain (mortar) is introduced into the sequence of one heavy chain, and an amino acid with a large side chain (mortar) is introduced into the corresponding interacting residue site on the other heavy chain. In some embodiments, the compositions of this disclosure have immunoglobulin chains in which the CH3 domain has been modified by mutating selected amino acids that interact at the interface between the two polypeptides to preferentially form bispecific antibodies. The bispecific antibodies may consist of immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or different subclasses (e.g., IgG1 and IgG3, or IgG3 and IgG4).

[0235] In some cases, bispecific antibodies may contain heterodimers of IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG3, or IgG1 and IgG3 chains. Such heterodimer heavy chain antibodies can routinely be engineered, for example, by modifying selected amino acids at the interface that forms the CH3 domain in human IgG4 and IgG1 or IgG3 to facilitate the formation of heterodimer heavy chains.

[0236] In some embodiments, the antibodies described herein that bind to the same epitope of LY6K (e.g., human LY6K) as the anti-LY6K (e.g., human LY6K) antibodies described herein are human antibodies. In some embodiments, the antibodies described herein that competitively block (e.g., in a dose-dependent manner) the binding of any of the antibodies described herein to LY6K (e.g., human LY6K) are human antibodies. Human antibodies can be produced using any method known in the art. For example, transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. By homologous recombination, mouse heavy chain and light chain immunoglobulin genes can become nonfunctional separately from or simultaneously with the introduction of human immunoglobulin gene loci. In particular, J H The homozygous absence of the region prevents the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous offspring expressing human antibodies. The transgenic mice are immunized in a normal manner with a selected antigen, such as all or part of the antigen (e.g., LY6K (e.g., human LY6K)). Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes present in the transgenic mice undergo rearrangement during B cell differentiation, followed by class switching and somatic mutations. Therefore, using this technique, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technique for generating human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of this technique and the protocols used to generate human antibodies and human monoclonal antibodies, see, for example, International Publications WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are incorporated herein by reference in their entirety. Examples of mice capable of generating human antibodies include XenoMouse. ™ (Abgenix, Inc.; U.S. Patents Nos. 6,075,181 and 6,150,184), Huab-Mouse ™(Medarex, Inc. / Gen Pharm; U.S. Patents Nos. 5,545,806 and 5,569,825), TransChromo mice ™ (Kirin) and KM mice ™ (Medarex / Kirin), all of these references are incorporated into this paper in their entirety by way of citation.

[0237] Human antibodies that specifically bind to LY6K (e.g., human LY6K) can be prepared by a variety of methods known in the art, including the phage display method described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patents 4,444,887, 4,716,111, and 5,885,793; and International Publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, all of which are incorporated herein by reference in their entirety.

[0238] In some embodiments, human antibodies can be generated using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to generate mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to identify hybridomas that secrete human monoclonal antibodies that specifically bind to the target antigen (e.g., LY6K (e.g., human LY6K)). Such methods are known and described in the art, see, for example, Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31, each of which is incorporated herein by reference in its entirety.

[0239] Reagent test kit Kits containing one or more antibodies or pharmaceutical compositions or conjugates described herein are also provided. In one specific embodiment, this document provides pharmaceutical packages or kits comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, such as one or more antibodies provided herein. In some embodiments, the kit contains the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein. In some embodiments, the kit may contain T-cell mitogens, such as phytohemagglutinin (PHA) and / or myristoyl phorbol ethyl ester (PMA), or TCR complex stimulating antibodies, such as anti-CD3 antibodies and anti-CD28 antibodies. Optionally, the association with such containers may be a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, reflecting that agency's approval for the manufacture, use, or sale for human administration.

[0240] Kits for use with the methods described above are also provided. In some embodiments, the kit contains the antibody described herein, preferably purified antibody, in one or more containers. In one specific embodiment, the kit described herein contains substantially isolated LY6K (e.g., human LY6K) antigen as a control. In another specific embodiment, the kit described herein also contains a control antibody that does not react with the LY6K (e.g., human LY6K) antigen. In yet another specific embodiment, the kit described herein contains one or more elements for detecting the binding of the antibody to the LY6K (e.g., human LY6K) antigen (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, enzyme substrate, radioactive compound, or luminescent compound, or a second antibody recognizing the first antibody may be conjugated to a detectable substrate). In specific embodiments, the kits provided herein may include recombinantly generated or chemically synthesized LY6K (e.g., human LY6K) antigen. The LY6K (e.g., human LY6K) antigen provided in the kit may also be attached to a solid support. In a more specific embodiment, the detection tool of the above kit includes a solid support to which the LY6K (e.g., human LY6K) antigen is attached. This kit may also include an unattached reporter-labeled anti-human antibody or an anti-mouse / rat antibody. In this embodiment, the binding of the antibody to the LY6K (e.g., human LY6K) antigen can be detected by the binding of the reporter-labeled antibody. In some embodiments, this disclosure relates to the use of the kit of this disclosure for the in vitro determination and / or detection of the LY6K (e.g., human LY6K) antigen in biological samples.

[0241] Example The following examples are provided in an illustrative rather than limiting manner.

[0242] Example 1: Generation of anti-LY6K antibody Human LY6K was used as an immunogen, and anti-LY6K antibodies were generated via mouse immunization at 10 μg / site using a standard RIMMS protocol. Bone marrow and spleen were harvested when a test hemorrhage titer greater than 5000 on LY6K was achieved by ELISA. Plasma B cells and antigen-reactive B cells were sorted into single wells by FACS staining with dye-labeled LY6K and standard B cells, and plasma B markers and VH and VL sequences were amplified by PCR. Then, transient transfection with CHO and ELISA with unique VH and VL sequences were used to select mAbs that reacted with LY6K by ELISA but not with unrelated Her2-His-labeled proteins.

[0243] Example 2: Selectivity of Exemplary Anti-LY6K Antibody for Human LY6K The selectivity of anti-LY6K antibodies for human LY6K was measured using ELISA. Human LY6K (“hLY6K”), cynomolgus monkey LY6K (“cyLY6K”), rat LY6K (“rLY6K”), and human LY6E (“hLY6E”) derived from R&D Systems were coated onto highly binding ELISA plates in PBS and incubated overnight. The plates were washed and blocked in PBS 1% BSA, and probed with anti-LY6K antibodies formatted as human IgG1 monoclonal antibodies at 10 μg / ml in PBS 1% BSA. After 1 hour, the plates were washed and probed for 1 hour with either anti-human F(ab)2HRPO conjugate (for hLY6K, cyLY6K, and rLY6K) or anti-human Ig FcγHRPO conjugate (for hLY6E), followed by washing, addition of TMB substrate, and plate reading. The combined data for hLY6K, cyLY6K, rLY6K, and hLY6E are shown in Table 7 (expressed as a percentage of the maximum value of commercial anti-LY6K mAb against human LY6K).

[0244] Table 7. Binding of anti-LY6K antibody to indicated proteins .

[0245]

[0246] ND: Undetermined The selectivity of a subset of anti-LY6K antibodies for LY6K relative to various LY6 family members was measured using an ELISA. LY6K, LY6E, LY6D, and LY6H, derived from R&D Systems, were coated overnight on a high-binding ELISA plate in PBS with BSA as a negative control. The plate was washed and blocked in PBS 1% BSA, and probed with a subset of anti-LY6K antibodies formatted as human IgG1 monoclonal antibodies at 10 μg / ml in PBS 1% BSA. After 1 hour, the plate was washed and probed with an anti-human F(ab)2HRPO conjugate for 1 hour, followed by washing, addition of TMB substrate, and plate reading. Figure 1 As shown, each anti-LY6K antibody selectively binds to LY6K.

[0247] Example 3: Affinity of an exemplary anti-LY6K antibody to human LY6K The apparent affinity of subsets of anti-LY6K antibodies for cell surface-anchored LY6K was measured using flow cytometry. Primary anti-LY6K mAb was incubated in 96-well U-plates with 10(5) target cells at 4°C for 30 min, followed by two washes at 1500 RPM and the addition of a stage 2 goat F(ab)2 anti-human Fcγ dye-labeled polyclonal antibody (Southern goat 1012-09) at a 1:200 dilution for 30 min. Cells were then washed and mean fluorescence was analyzed on FACSCAN, and these values ​​were used in Prism to calculate the apparent affinity of each subset of anti-LY6K antibodies for cell surface-anchored LY6K, expressed as half-maximum effective concentration (EC50), as shown in Table 8. The apparent affinity of a commercially available anti-LY6K antibody (#MAB6648, referred to as “R&D” in Table 8) was also determined for comparison.

[0248] Table 8. Epigenetic affinity (EC50) of exemplary anti-LY6K antibodies for LY6K anchored on the cell surface. .

[0249]

[0250] The apparent affinity of subsets of anti-LY6K antibodies for LY6K was also measured using an ELISA assay, where individual antibodies were HRP-labeled using an Abcam kit (ab102890). High-binding ELISA plates were coated overnight with 2 μg / mL recombinant human LY6K (Wuxi), washed, and blocked in PBS with 1% BSA. HRPO-linked anti-LY6K mAb was then added as a titrant for 1 hour, followed by washing the plates, adding TMB, and reading at 450 nM on a GloMax plate reader. The apparent affinity of each subset of anti-LY6K antibodies generated using GraphpadPrism and expressed as half-maximum effective concentration (EC50) for LY6K is shown in Table 9. The apparent affinity of commercially available anti-LY6K antibodies from R&D Systems (“R&D”) was also determined for comparison.

[0251] Table 9. Exemplary anti-LY6K antibody apparent affinity for LY6K (EC50) .

[0252]

[0253] The affinity of each subset of anti-LY6K antibodies for LY6K (“hLY6K”), cynomolgus monkey LY6K (“cyLY6K”), and rat LY6K (“rLY6K”) was measured using surface plasmon resonance. For measuring binding kinetics, capture and binding assays were performed on a Carterra LSA system. Briefly, amine-conjugated anti-human IgG mcocytes were generated using an HC30M chip with an anti-human IgG-Fc specific antibody (Jackson Immuno-Research). All test antibodies were expressed in CHO cells at a 1 mL scale for 6 days, and cells were removed from the supernatant by centrifugation. The CHO supernatant was diluted to approximately 1 μg / mL of antibody and injected onto the capture chip to generate a captured antibody array. Non-regenerative kinetics were measured by injecting hLY6K-his, hLY6K-mFc, cyLY6K-mFc, or rLY6K-mFc, starting at a concentration of 1.37E-9M and increasing by 3-fold up to 1.00E-6M. Each analyte was injected through the array, followed by a 5-minute association phase and a 10-minute buffer dissociation phase. The relative response units (RUs) of each antibody bound to the surface were measured and collected. KD values ​​were determined using Carterra software. Results are shown in Table 10.

[0254] Table 10. Affinities of exemplary anti-LY6K antibodies to hLY6K, cyLY6K, and rLY6K .

[0255]

[0256] N / A: Low / no binding, KD unmeasurable. Example 4: Sensitivity of an exemplary anti-LY6K antibody for detecting LY6K Capture ELISA was used to determine the subset of anti-LY6K antibodies for LY6K detection sensitivity. Two commercially available anti-LY6K antibodies were used as references (R&D Systems MAB6648 and Abcam ab246486). The capture antibodies were diluted to 2 μg / mL and added to a plate (MSD L55XB-3). Human LY6K (2 μg / mL Wuxi LY^KGLY138(1-38TR)_8His) was used. A commercial polyclonal anti-hLY6K antibody (R&D Systems, AF6648) was sulfonated (R31AA-1) according to the manufacturer's protocol and used for detection at 200 ng / mL. Results are shown in... Figure 2 The EC50 of LY6K was determined using nonlinear regression with Prism software. Sensitivity refers to the limit of detection, which was determined using MSD Discover benchtop software and calculated as the concentration above the calibration minimum (without antigen) by 2.5 standard deviations.

[0257] Example 5. Internalization of anti-LY6K antibody .

[0258] Temperature-sensitive internalization of the exemplary LY6K antibody was tested in OVCAR-8 cells. 2,000,000 OVCAR-8 cells were stained with 1 μg / mL anti-LY6K antibody at 4°C or 37°C for 6 hours. IgG isotypes were used as negative controls (Jackson 015-000-003, 009-000-003), and trastuzumab (absolute antibody T1722A01) was used as a positive control. Cells were washed twice and incubated with the fluorescent secondary antibody on ice for 40 minutes (Jackson 109-116-190 1:200 dilution, Southern Biotech 1012-09 1:100 dilution). Cells were washed twice and fixed with fixation buffer (BD554655). Cells were washed again and fluorescence was recorded using Attune Nxt (Invitrogen A24858). The percentage of internalization was calculated based on the geometric mean fluorescence fold increase for each antibody, with 4 degrees Celsius set as the maximum fluorescence.

[0259] pH-sensitive internalization of the exemplary LY6K antibody was tested in cells expressing OVCAR-8 and HEKLY6K. Cells were seeded at 30,000 cells / well (50 μL) into tissue culture plates and incubated overnight in phenol red-free medium supplemented with FBS (Gibco 11835-030). Fabfluor-pH-sensitive antibody (Satorius 4722) was incubated with either anti-LY6K antibody alone or control antibody trastuzumab (absolute antibody T1722A01) and huIgG1 (Wuxi, at a 1:3 molar ratio and incubated for 20 min). The Fabfluor antibody mixture (50 μL) was added to the cell plate at a final concentration of 2 μg / mL (13.33 nM) and incubated for 18 h on OVCAR-8 cells or 2 h on HEKLY6K cells. After incubation, cells were washed twice with PBS in the plate. Cells were removed from the plate using a versene (ThermoFischer Scientific 15040066) and washed twice. Fluorescence was recorded on an Attune Nxt (Invitrogen A24858). The percentage of internalization on OVCAR-8 cells was calculated based on the geometric mean fluorescence for each antibody, with trastuzumab set as the maximum fluorescence. The percentage of internalization on HEKLY6K cells was calculated based on the geometric mean fluorescence for each antibody, with the highest mean fluorescence intensity (MFI) of 3035 for the group set as the maximum fluorescence (100% internalization).

[0260] Exemplary temperature-sensitive and pH-sensitive internalization results of anti-LY6K antibodies are shown in Table 11, and a representative fluorescence diagram of antibody 3082 labeled with a pH-sensitive dye is shown in Table 11. Figure 3 The figure shows that the anti-LY6K antibody 3082 (solid clear line) undergoes internalization and endocytosis, releasing a fluorescent signal exceeding that of the negative control huIgG1 (shaded black line), due to the increased MFI caused by the decrease in pH in OVCAR-8 cells. Trastuzumab, a known commercially available internalizer (dashed line), is used as a positive control.

[0261] Table 11. Exemplary internalization percentage of anti-LY6K antibodies .

[0262]

[0263] Example 6. Exemplary epitope binning of anti-LY6K antibody Epitope binning of a subset of anti-LY6K antibodies was performed using a competitive immunoassay. Exemplary anti-LY6K antibodies and commercial anti-LY6K antibodies (R&D MAB6648; “R&D” in Table 12) were HRP-conjugated using the Lightning Chain HRP Conjugation Kit (Abcam ab10289). HRP-conjugated antibodies were titrated and EC80 values ​​were determined. Each individually purified anti-LY6K antibody had a corresponding HRP-conjugated antibody used to calculate the percentage of competition against itself. If the antigen of a non-HRP-capture mAb prevented 60% of the competitive HRP-conjugated antibody from binding to human LY6K (2 μg / mL Wuxi LY^KGLY138(1-38TR)_8His), these mAbs were considered to bind similar or overlapping epitopes. Conversely, if the binding of unconjugated antibodies to LY6K did not interfere with the binding of other HRP-conjugated antibodies, they were considered to bind different, non-overlapping epitopes. Binding results are shown in Table 12.

[0264] Table 12. Epitope binning of exemplary anti-LY6K antibodies .

[0265]

[0266] Example 7. Detection of LY6K via immunohistochemistry using an exemplary anti-LY6K antibody. Immunohistochemistry (IHC) was used to detect LY6K overexpression in 293 cells, OVCAR-8 cells, and 293 cells (negative control) using an exemplary anti-LY6K antibody. Briefly, sections were treated by deparaffining and rehydration, incubated at 4°C for 20 minutes, washed, placed in Bloxall for 10 minutes, then in normal serum for 30 minutes, and stained overnight with anti-LY6K antibody formatted with human IgG1 monoclonal antibody or Abcam (ab246486) anti-LY6K peptide (positive control) at 5 μg / ml in 5% mouse serum in PBS. Afterwards, staining was performed with biotinylated anti-rabbit or anti-human IgG for 30 minutes, followed by AB staining for 30 minutes, and then hematoxylin staining. An exemplary IHC image of antibody 2660 is shown in [image description missing]. Figure 4 The images include those of the positive control (Abcam anti-LY6K) and the negative control (IgG). The IHC results for the remaining anti-LY6K antibodies are summarized in Table 13.

[0267] Table 13. IHC results of exemplary anti-LY6K antibodies .

[0268]

[0269] (++) Best; (+) Positive; (+ / -) Marginal or diffuse staining; (-) No staining or nonspecific staining; (NA) If the 293 LY6K result is negative, do not stain OVCAR-8; (ND) Undetermined. Example 8. Killing tumor cells using a T-cell adaptor based on anti-LY6K antibody. An exemplary anti-LY6K antibody was used to prepare an L2K-formatted bispecific T-cell adaptor targeting CD3. Th1 lymphocytes were generated by activation with Miltenyi Transact beads in medium (RPMI 10% FBS+NEAA and P / S) for 4 days, followed by culturing in medium + 5 ng / ml IL2 for 9 days. These were then placed at 10(6) cells / ml in RPMI 5% FBS+NEAA+P / S and at 10(5) cells / ml in an OVCAR-8 GFP luciferase system. Anti-LY6K T-cell adaptors were added at various concentrations as a comparison using a 4D5-based 1+1 T-cell adaptor targeting Her2. After 18 hours, luciferin was added to 200 μg / ml, and luminescence readings were taken. Results are shown in... Figure 5 middle.

[0270] Example 9. Exemplary epitope mapping of anti-LY6K antibody Epitopes of five exemplary anti-LY6K antibodies (2660, 3481, 3501, 3520, and 3741) were mapped using linear and conformational epitope mapping.

[0271] Peptide synthesis: To reconstruct the LY6K epitope, a library of linear peptide-based mimics was synthesized using Fmoc-based solid-phase peptide synthesis. Grafting with a proprietary hydrophilic polymer formulation followed by reaction with dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt) with tert-butyloxycarbonyl-hexamethylenediamine (BocHMDA), and subsequent cleavage of the Boc- group with trifluoroacetic acid (TFA), yielded an amino-functionalized polypropylene support. Peptides were synthesized on the amino-functionalized solid support using standard Fmoc-peptide synthesis via a custom-modified JANUS liquid processing station (Perkin Elmer). Three groups of linear peptides were synthesized. The first group comprised a 7-residue peptide derived from a human LY6K sequence (SEQ ID NO:232) with a one-residue offset. The second group comprised a 10-residue peptide derived from a human LY6K sequence (SEQ ID NO:232) with a one-residue offset. The third group includes a peptide of length 15, which is derived from a human LY6K sequence (SEQ ID NO:232) with a residue offset.

[0272] Using scaffold chemically linked peptides (CLIPS) ™ Biosynth technology is used to synthesize conformational epitope structure mimics. See, for example, Timmerman et al., 2007. J. Mol.Recognit.20:283-299 and Langedijk et al., 2011, Analytical Biochemistry 417:149-155. CLIPS technology allows peptides to adopt thermodynamically favorable conformations, including monocyclic, bicyclic, tricyclic, sheet-like, helical, and combinations thereof. CLIPS scaffolds are coupled with cysteine ​​residues. Multiple cysteine ​​side chains in the peptide are coupled to CLIPS scaffolds having two or three reactive groups. For example, a 0.5 mM solution of P2 CLIPS (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8) / acetonitrile (1:3 (v / v)), and this solution is added to a peptide array, wherein the CLIPS template is bound to the side chains of two cysteine ​​residues in the immobilized peptide introduced into the peptide array. The peptide array is gently shaken in the solution for 30 to 60 minutes while being completely covered by the solution. Finally, the peptide array was thoroughly washed with excess H2O and sonicated at 70°C in a disruption buffer (1% SDS / 0.1% PBS solution of 2,2'-(ethylenedioxy)diethylthiol (pH 7.2)) for 30 min, followed by sonication in H2O for another 45 min. T3 CLIPS carrying the peptides were prepared in a similar manner using three cysteine ​​residues. Three sets of conformational structure mimic peptides were synthesized. The first set consisted of a constrained monocyclic peptide of length 15, where positions 2-14 were 13-mer peptides derived from the human LY6K sequence (SEQ ID NO: 232) with a one-residue offset. Cys residues were inserted at positions 1 and 15 and linked via mP2 CLIPS to generate the ring mimic. Native Cys residues were replaced with Cys-acm. The second group comprises a β-turn peptide mimic of length 22, wherein positions 2-21 are 20-mer peptides derived from the human LY6K sequence (SEQ ID NO: 232) with a one-residue offset. Residues at positions 11 and 12 are replaced with a “PG” motif to induce β-turn formation. Cys residues are inserted at positions 1 and 22 and linked via mP2 CLIPS to stabilize the mimic. Native Cys residues are replaced with Cys-acm. The third group comprises an α-helical peptide mimic of length 19, derived from the human LY6K sequence (SEQ ID NO: 232) with a one-residue offset. Cys residues are inserted at positions 1 and 5 and linked via mP2 CLIPS to nucleate the α-helical structure. Native Cys residues are replaced with Cys-acm (represented as “2”).

[0273] To verify the quality of the synthesized peptides, a separate set of positive and negative control peptides were synthesized in parallel. These peptides were screened using commercial antibodies 3C9 and 57.9 (see Posthumus et al., 1990). J. Virol. 64:3304–3309).

[0274] ELISA screening The antibody binding to each synthetic peptide was detected by ELISA. The peptide array was incubated overnight at 4°C with the primary antibody solution. After washing, the peptide array was incubated for one hour at 25°C with a 1 / 1000 diluted goat anti-human HRP conjugated antibody (Southern Biotech, catalog number 2010-05). After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzothiazoline sulfonate (ABTS) and 20 µl / ml of 3% H2O2 were added. After one hour, the color development was measured. The color development was quantified using a charge-coupled device (CCD) camera and image processing system. The values ​​obtained from the CCD camera ranged from 0 to 3000 mAU, similar to a standard 96-well plate ELISA reader. Occasionally, air bubbles were present in the wells, resulting in false positive values. These false positives were scored as 0 by manual inspection.

[0275] Optimize sample sieving for each sample concentration and blocking conditions until a clear signal exceeding the background value is observed. Typically, a peak is defined as at least twice the background value (defined as the value screened for isotype controls for each peptide of a specific peptide mimic). In the absence of an isotype control, a single antibody sample is used as an internal control, and a distinctly different binding region is observed. Binding events are only noted when multiple overlapping peaks are present within this binding region. The putative core epitope is identified from adjacent peptides, with an intensity similar to or up to 20% lower than the highest peak within the binding region. Furthermore, if the peak profile is distinct and contains multiple overlapping peptides, lower-intensity peaks are also included. To comprehensively evaluate the putative epitope, information is obtained from multiple mimic types where possible.

[0276] All five antibodies exhibited distinct binding peaks in different regions of the classical human LY6K sequence. The putative core epitopes are listed in Table 14. The residue number indicates the location of the putative core epitope sequence within the classical human LY6K sequence shown in SEQ ID NO: 232. Underlined sequences represent the most probable core epitope sequences.

[0277] Table 14. Proposed core epitopes of exemplary anti-LY6K antibodies .

[0278]

[0279] As demonstrated in Examples 2 and 3, some exemplary anti-LY6K antibodies (including 2660, 3520, and 3741) are cross-reactive and bind to both human LY6K and cynomolgus monkey LY6K (“cyLY6K”). Furthermore, based on the putative epitopes shown in Table 14 and the binning data described in Example 6, antibodies 2660, 3520, and 3741 appear to bind to different epitopes within LY6K. The amino acid sequence of cyLY6K was compared with the sequence of human LY6K (i.e., via BLAST alignment) to evaluate whether the exemplary cross-reactive anti-LY6K antibodies could bind to similar epitopes. As shown in Table 15, the cyLY6K amino acid sequences corresponding to the putative epitope regions in Table 14 are highly similar to the human LY6K epitope sequences of the cross-reactive anti-LY6K antibodies. As in Table 14, the underlined sequences in Table 15 represent the most likely core epitope sequences. Regions corresponding to the putative epitopes of the 2660 anti-LY6K antibody from rat LY6K (“rLY6K”) and mouse LY6K (“mLY6K”) are also shown in Table 15. As demonstrated in Examples 2 and 3, the 2660 antibody does not react with rLY6K.

[0280] Table 15. Proposed cross-reactive epitopes of exemplary anti-LY6K antibodies .

[0281]

[0282] The antibody showed cross-reactivity with cyLY6K (see Examples 2 and 3).

[0283] The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention, in addition to those described, will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

[0284] All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein in their entirety and for all purposes, to the extent that each individual reference (e.g., publications, patents, or patent applications) is specifically and individually indicated to be incorporated herein in its entirety for all purposes.

[0285] Other embodiments are defined in the following claims.

Claims

1. An antibody that specifically binds to human LY6K, said antibody comprising: VH containing the VH amino acid sequence of CDRH1, CDRH2 and CDRH3 as shown in any one of SEQ ID NO:1-34; and VL containing the VL amino acid sequence of CDRL1, CDRL2 and CDRL3 as shown in any one of SEQ ID NO:35-68.

2. The antibody according to claim 1, wherein the VH amino acid sequence and the VL amino acid sequence are respectively shown in the following: SEQ ID NO: 1 and 35, 2 and 36, 3 and 37, 4 and 38, 5 and 39, 6 and 40, 7 and 41, 8 and 42, 9 and 43, 10 and 44, 11 and 45, 12 and 46, 13 and 47, 14 and 48, 15 and 49, 16 and 50, 17 and 51, 18 and 52, 19 and 53, 20 and 54, 21 and 55, 22 and 56, 23 and 57, 24 and 58, 25 and 59, 26 and 60, 27 and 61, 28 and 62, 29 and 63, 30 and 64, 31 and 65, 32 and 66, 33 and 67, or 34 and 68.

3. The antibody according to any one of the preceding claims, wherein the antibody comprises the amino acid sequences of CDRH1, CDRH2 and CDRH3 respectively shown in the following: SEQ ID NO: 69, 90 and 124; 70, 91 and 125; 71, 92 and 126; 72, 93 and 127; 69, 94 and 124; 73, 95 and 128; 74, 96 and 129; 75, 97 and 130; 74, 98 and 131; 76, 99 and 132; 77, 100 and 133; 78, 101 and 134; 79, 102 and 135; 74, 103 and 136; 80, 104 and 137; 74, 105 and 138; 74, 106 and 139; 81, 107 and 14 0; 81, 108 and 141; 82, 109 and 142; 83, 110 and 143; 84, 99 and 144; 85, 111 and 145; 81, 112 and 146; 86, 113 and 147; 87, 114 and 148; 74, 11 or 86, 123 and 157.

4. The antibody according to any one of the preceding claims, wherein the antibody comprises the amino acid sequences of CDRL1, CDRL2 and CDRL3 respectively shown in the following: SEQ ID NO: 158, KIS and SEQ ID NO: 198; SEQ ID NO: 159, WAS and SEQ ID NO: 199; SEQ ID NO: 160, STT and SEQ ID NO: 200; SEQ ID NO: 161, WAS and SEQ ID NO: 201; SEQ ID NO: 162, AAS and SEQ ID NO: 202; SEQ ID NO: 163, 185 and 203; SEQ ID NO: 164, 185 and 204; SEQ ID NO: 165, 185 and 205; SEQ ID NO: 166, 186 and 206; SEQ ID NO: 167, 187 and 207; SEQ ID NO: 168, 188 and 208; SEQ ID NO: SEQ ID NO: 169, 186, and 209; SEQ ID NO: 170, 185, and 198; SEQ ID NO: 169, 189, and 210; SEQ ID NO: 170, 185, and 211; SEQ ID NO: 171, 187, and 212; SEQ ID NO: 172, 187, and 213; SEQ ID NO: 173, 190, and 214; SEQ ID NO: 174, 191, and 215; SEQ ID NO: 175, 186, and 216; SEQ ID NO: 176, 186, and 217; SEQ ID NO: 170, 192, and 198; SEQ ID NO: 171, 193, and 218; SEQ ID NO: 177, 187, and 219; SEQ ID NO: 178, 189, and 220; SEQ ID NO: SEQ ID NO: 179, 187, and 221; SEQ ID NO: 165, 185, and 222; SEQ ID NO: 180, 187, and 223; SEQ ID NO: 181, 194, and 224; SEQ ID NO: 182, 195, and 199; SEQ ID NO: 172, 187, and 225; SEQ ID NO: 183, 196, and 226; or SEQ ID NO: 184, 197, and 227.

5. The antibody according to any one of the preceding claims, wherein the antibody comprises the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 respectively shown in the following: SEQ ID NO: 69, 90, 124, 158, KIS and SEQ ID NO: 198; SEQ ID NO: 70, 91, 125, 159, WAS and SEQ ID NO: 199; SEQ ID NO: 71, 92, 126, 160, STT and SEQ ID NO: 200; SEQ ID NO: 72, 93, 127, 161, WAS and SEQ ID NO: 201; SEQ ID NO: 69, 94, 124, 158, KIS and SEQ ID NO: 198; SEQ ID NO: 73, 95, 128, 162, AAS and SEQ ID NO: 202; SEQ ID NO: SEQ ID NO: 74, 96, 129, 163, 185, and 203; SEQ ID NO: 75, 97, 130, 164, 185, and 204; SEQ ID NO: 74, 98, 131, 165, 185, and 205; SEQ ID NO: 76, 99, 132, 166, 186, and 206; SEQ ID NO: 77, 100, 133, 167, 187, and 207; SEQ ID NO: 78, 101, 134, 168, 188, and 208; SEQ ID NO: 79, 102, 135, 169, 186, and 209; SEQ ID NO: 74, 103, 136, 170, 185, and 198; SEQ ID NO: 80, 104, 137, 169, 189, and 210; SEQ ID SEQ ID NO: 74, 105, 138, 170, 185, and 211; SEQ ID NO: 74, 106, 139, 171, 187, and 212; SEQ ID NO: 81, 107, 140, 172, 187, and 213; SEQ ID NO: 81, 108, 141, 173, 190, and 214; SEQ ID NO: 82, 109, 142, 174, 191, and 215; SEQ ID NO: 83, 110, 143, 175, 186, and 216; SEQ ID NO: 84, 99, 144, 176, 186, and 217; SEQ ID NO: 85, 111, 145, 170, 192, and 198; SEQ ID NO: 81, 112, 146, 171, 193 and 218; SEQ ID NO: 86, 113, 147, 177, 187 and 219;SEQ ID NO: 87, 114, 148, 178, 189, and 220; SEQ ID NO: 74, 115, 149, 179, 187, and 221; SEQ ID NO: 74, 116, 150, 165, 185, and 222; SEQ ID NO: 74, 117, 151, 180, 187, and 223; SEQ ID NO: 88, 118, 152, 181, 194, and 224; SEQ ID NO: 89, 119, 153, 182, 195, and 199; SEQ ID NO: 81, 120, 154, 172, 187, and 225; SEQ ID NO: 74, 121, 155, 165, 185, and 222; SEQ ID NO: 85, 122, 156, 183, 196, and 226; or SEQ ID NO: 86, 123, 157, 184, 197, and 227.

6. The antibody according to any one of the preceding claims, wherein the antibody comprises the VH amino acid sequence shown in any one of SEQ ID NO: 1-34.

7. The antibody according to any one of the preceding claims, wherein the antibody comprises a heavy chain constant region, the heavy chain constant region optionally being selected from the group consisting of: human IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

8. The antibody according to any one of the preceding claims, wherein the antibody comprises a heavy chain constant region as a variant of the wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with a lower affinity than the wild-type heavy chain constant region.

9. The antibody according to any one of the preceding claims, wherein the antibody comprises a heavy chain constant region containing an amino acid sequence of SEQ ID NO: 228 or 229.

10. The antibody according to any one of the preceding claims, wherein the antibody comprises the VL amino acid sequence shown in any one of SEQ ID NO: 35-68.

11. The antibody according to any one of the preceding claims, wherein the antibody comprises a light chain constant region containing the amino acid sequence of SEQ ID NO: 230 or 231.

12. The antibody according to any one of the preceding claims, wherein the VH and VL each comprise the amino acid sequences shown in the following: SEQ ID NO: 1 and 35, 2 and 36, 3 and 37, 4 and 38, 5 and 39, 6 and 40, 7 and 41, 8 and 42, 9 and 43, 10 and 44, 11 and 45, 12 and 46, 13 and 47, 14 and 48, 15 and 49, 16 and 50, 17 and 51, 18 and 52, 19 and 53, 20 and 54, 21 and 55, 22 and 56, 23 and 57, 24 and 58, 25 and 59, 26 and 60, 27 and 61, 28 and 62, 29 and 63, 30 and 64, 31 and 65, 32 and 66, 33 and 67, or 34 and 68.

13. An antibody that specifically binds to an amino acid sequence selected from the group consisting of SEQ ID NO: 233-238.

14. An antibody that specifically binds to the amino acid sequence of SEQ ID NO: 233 and 239.

15. An antibody that specifically binds to the amino acid sequence of SEQ ID NO: 234 and 240.

16. An antibody that specifically binds to the amino acid sequence of SEQ ID NO: 237 and 247.

17. The antibody according to any one of the preceding claims, wherein the antibody is a multispecific antibody comprising a CD3-binding region, optionally wherein the CD3-binding region comprises a single-stranded variable region (scFv) that specifically binds to CD3, optionally human CD3.

18. A polypeptide comprising VH containing the VH amino acid sequences of CDRH1, CDRH2 and CDRH3 as shown in any one of SEQ ID NO: 1-34.

19. The polypeptide of claim 18, wherein the VH comprises the amino acid sequences of CDRH1, CDRH2 and CDRH3 respectively shown in the following: SEQ ID NO: 69, 90 and 124; 70, 91 and 125; 71, 92 and 126; 72, 93 and 127; 69, 94 and 124; 73, 95 and 128; 74, 96 and 129; 75, 97 and 130; 74, 98 and 131; 76, 99 and 132; 77, 100 and 133; 78, 101 and 134; 79, 102 and 135; 74, 103 and 136; 80, 104 and 137; 74, 105 and 138; 74, 106 and 139; 81, 107 and 140 ;81, 108 and 141; 82, 109 and 142; 83, 110 and 143; 84, 99 and 144; 85, 111 and 145; 81, 112 and 146; 86, 113 and 147; 87, 114 and 148; 74, 115 and 149; 74, 116 and 150; 74, 117 and 151; 88, 118 and 152; 89, 119 and 153; 81, 120 and 154; 74, 121 and 155; 85, 122 and 156; or 86, 123 and 157.

20. A polypeptide comprising a VL containing the VL amino acid sequences of CDRL1, CDRL2, and CDRL3 as shown in any one of SEQ ID NO: 35-68.

21. The polypeptide of claim 20, wherein the VL comprises the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in the following: SEQ ID NO: 158, KIS, and SEQ ID NO: 198; SEQ ID NO: 159, WAS, and SEQ ID NO: 199; SEQ ID NO: 160, STT, and SEQ ID NO: 200; SEQ ID NO: 161, WAS, and SEQ ID NO: 201; SEQ ID NO: 162, AAS, and SEQ ID NO: 202; SEQ ID NO: 163, 185, and 203; SEQ ID NO: 164, 185, and 204; SEQ ID NO: 165, 185, and 205; SEQ ID NO: 166, 186, and 206; SEQ ID NO: 167, 187, and 207; SEQ ID NO: 168, 188, and 208; SEQ ID NO: SEQ ID NO: 169, 186, and 209; SEQ ID NO: 170, 185, and 198; SEQ ID NO: 169, 189, and 210; SEQ ID NO: 170, 185, and 211; SEQ ID NO: 171, 187, and 212; SEQ ID NO: 172, 187, and 213; SEQ ID NO: 173, 190, and 214; SEQ ID NO: 174, 191, and 215; SEQ ID NO: 175, 186, and 216; SEQ ID NO: 176, 186, and 217; SEQ ID NO: 170, 192, and 198; SEQ ID NO: 171, 193, and 218; SEQ ID NO: 177, 187, and 219; SEQ ID NO: 178, 189, and 220; SEQ ID SEQ ID NO: 179, 187 and 221; SEQ ID NO: 165, 185 and 222; SEQ ID NO: 180, 187 and 223; SEQ ID NO: 181, 194 and 224; SEQ ID NO: 182, 195 and 199; SEQ ID NO: 172, 187 and 225; SEQ ID NO: 183, 196 and 226; or SEQ ID NO: 184, 197 and 227.

22. A polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-68.

23. The antibody or polypeptide according to any one of the preceding claims, wherein the antibody or polypeptide is conjugated with a cytotoxic agent, a cell growth inhibitor, a toxin, a radionuclide, or a detectable label.

24. A polynucleotide encoding: the VH, VL, heavy chain and / or light chain of an antibody according to any one of claims 1 to 17; or a polypeptide according to any one of claims 18 to 22.

25. A vector comprising the polynucleotide according to claim 24.

26. A recombinant host cell, said recombinant host cell comprising: (a) The polynucleotide according to claim 24; (b) The carrier according to claim 25; (c) Encoding the heavy chain variable region or a first polynucleotide of the heavy chain of the antibody according to any one of claims 1 to 17 and a second polynucleotide encoding the light chain variable region or a light chain of the antibody according to any one of claims 1 to 17; and / or (d) A first vector comprising a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody according to any one of claims 1 to 17 and a second vector comprising a second polynucleotide encoding a light chain variable region or a light chain of an antibody according to any one of claims 1 to 17.

27. A composition comprising an antibody according to any one of claims 1 to 17 or 23, a polypeptide according to any one of claims 18 to 23, a polynucleotide according to claim 24, a vector according to claim 25 or a host cell according to claim 26, and a pharmaceutically acceptable carrier or excipient.

28. A method for producing an antibody, the method comprising culturing a host cell according to claim 26 under suitable conditions to express the polynucleotide and produce the antibody.

29. A method of treating a subject's cancer, the method comprising administering to the subject an effective amount of the following: The antibody according to any one of claims 1 to 17 or 23; the polypeptide according to any one of claims 18 to 23; The polynucleotide according to claim 24; the vector according to claim 25; The host cell according to claim 26; or the composition according to claim 27.

30. The method of claim 29, further comprising administering an additional therapeutic agent to the subject.

31. The method of claim 30, wherein the additional therapeutic agent is a chemotherapeutic agent, a radiotherapy agent, or a checkpoint targeting agent.

32. The method of claim 31, wherein the checkpoint target is selected from the group consisting of: antagonistic anti-PD-1 antibody, antagonistic anti-PD-L1 antibody, antagonistic anti-PD-L2 antibody, antagonistic anti-CTLA-4 antibody, antagonistic anti-TIM-3 antibody, antagonistic anti-LAG-3 antibody, antagonistic anti-VISTA antibody, antagonistic anti-TIGIT antibody, antagonistic anti-CD96 antibody, antagonistic anti-CEACAM1 antibody, agonistic anti-CD137 antibody, agonistic anti-GITR antibody, and agonistic anti-OX40 antibody.

33. Use of the antibody according to any one of claims 1 to 17 or 23, the polypeptide according to any one of claims 18 to 23, the polynucleotide according to claim 24, the carrier according to claim 25, the host cell according to claim 26, or the composition according to claim 27, for the manufacture of a medicament for treating cancer.

34. The antibody according to any one of claims 1 to 17 or 23; the polypeptide according to any one of claims 18 to 23; The polynucleotide according to claim 24; the vector according to claim 25; The host cell according to claim 26; or the composition according to claim 27, used in a pharmaceutical.

35. The antibody according to any one of claims 1 to 17 or 23; the polypeptide according to any one of claims 18 to 23; The polynucleotide according to claim 24; the vector according to claim 25; The host cell according to claim 26; or the composition according to claim 27, for treating cancer in a subject.

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