Antibody targeting ly6g6d and use thereof

Abstract

Provided are an antibody targeting Ly6G6D and a use thereof. Specifically, provided are an anti-Ly6G6D (lymphocyte antigen 6 complex, locus G61) antibody and an antibody fragment. Further provided is a use of the antibody or antibody fragment in preparation of a drug for treating an Ly6G6D-positive cancer.

Description

An antibody targeting Ly6G6D and its application Technical Field

[0001] The present invention belongs to the field of antibody engineering. Specifically, the present invention relates to an antibody, and in particular to an antibody targeting Ly6G6D and use thereof in preparing a drug. Background Art

[0002] Colorectal cancer is a malignant tumor that originates in the mucosal epithelium and glands of the colon and rectum. Its incidence is second only to gastric cancer and esophageal cancer among digestive tract malignancies. In recent years, due to changes in dietary habits and structure, as well as an aging population, the incidence and mortality of colorectal cancer in my country have both increased.

[0003] Lymphocyte antigen 6 family member G6D (Ly6G6D) is a leukocyte antigen cluster located in the major histocompatibility complex (MHC) class III region of chromosome 6. It encodes a 133-amino acid protein with a molecular weight of approximately 13.7 kDa. Like most family members, Ly6G6D is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. Numerous studies have demonstrated that members of the LAG6 family are crucial for regulating immune, nervous, and complement functions. In cancer, many LAG6 family members play a key role in numerous cancer types, including gastric, cervical, breast, ovarian, lung, and bladder cancers. Therefore, the LAG6 gene family has the potential to play a significant role in clinical practice, not only as a biomarker for disease prognosis but also as a key target for new drug development. In-depth understanding of their biological functions will be crucial for elucidating the functions of the LAG6 family, analyzing protein interactions within their structure, exploring disease pathogenesis, and discovering new therapeutic targets. Ly6G6D is a new member of the lymphocyte antigen 6 gene family. Studies have shown that Ly6G6D plays an important role in the development of diseases such as colorectal cancer. Currently, there is a Ly6G6D-based bispecific antibody for the treatment of colorectal cancer, which indicates that Ly6G6D is expected to become a potential target for antibody-based therapy. Summary of the Invention

[0004] The present invention provides a fully human anti-Ly6G6D antibody, which has a high affinity for human recombinant Ly6G6D protein and has better low immunogenicity and safety than currently commonly used rabbit- or mouse-derived anti-Ly6G6D antibodies, and even has better effects, and has good application prospects.

[0005] Specifically, in one aspect, the present invention provides an anti-Ly6G6D antibody or antigen-binding fragment, which binds to Ly6G6D, and the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region, and the heavy chain variable region comprises complementarity determining regions: H-CDR1, H-CDR2 and H-CDR3; wherein,

[0006] (1) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 61 or SEQ ID NO: 71; and

[0007] (2) H-CDR2 has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 57 or SEQ ID NO: 72; and

[0008] (3) the H-CDR3 has the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, or SEQ ID NO: 73; and

[0009] Wherein, the light chain comprises a light chain variable region, and the light chain variable region comprises complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3; wherein,

[0010] (1) L-CDR1 has the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 68 or SEQ ID NO: 74; and

[0011] (2) L-CDR2 has the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 69 or SEQ ID NO: 75; and

[0012] (3) L-CDR3 has the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 70 or SEQ ID NO: 76.

[0013] In some embodiments, the heavy chain variable region of the present invention comprises complementarity determining regions: H-CDR1, H-CDR2 and H-CDR3; wherein,

[0014] (1) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 1, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 3; or

[0015] (2) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 7, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 8, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 9; or

[0016] (3) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 13, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 14, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 15; or

[0017] (4) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 18, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 19, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 20; or

[0018] (5) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 22, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 23, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 24; or

[0019] (6) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 28, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 30; or

[0020] (7) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 34, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 35, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 36; or

[0021] (8) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 18, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 40; or

[0022] (9) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 42, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 43, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 44; or

[0023] (10) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 46, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 47, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 48; or

[0024] (11) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 52, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 53, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 54; or

[0025] (12) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 56, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 57, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 58; or

[0026] (13) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 61, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 62; or

[0027] (14) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 18, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 66; or

[0028] (15) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 18, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 62; or

[0029] (16) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 71, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 72, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 73.

[0030] In some embodiments, the light chain variable region of the present invention comprises complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3; wherein,

[0031] (1) L-CDR1 has the amino acid sequence shown in SEQ ID NO: 4, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 5, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 6; or

[0032] (2) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 10, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 11, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 12; or

[0033] (3) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 4, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 16, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 17; or

[0034] (4) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 4, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 16, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 21; or

[0035] (5) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 25, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 26, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 27; or

[0036] (6) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 31, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 32, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 33; or

[0037] (7) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 39; or

[0038] (8) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 41; or

[0039] (9) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 45; or

[0040] (10) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 49, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 50, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 51; or

[0041] (11) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 55; or

[0042] (12) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 59, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 60, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 51; or

[0043] (13) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 63, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 64, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 65; or

[0044] (14) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 67, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 41; or

[0045] (15) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 68, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 69, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 70; or

[0046] (16) L-CDR1 has the amino acid sequence shown in SEQ ID NO: 74, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 75, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 76.

[0047] In some embodiments, the heavy chain variable region of the present invention comprises complementarity determining regions: H-CDR1, H-CDR2, and H-CDR3; and the light chain variable region of the present invention comprises complementarity determining regions: L-CDR1, L-CDR2, and L-CDR3; wherein,

[0048] (1) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 1, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 3; and

[0049] L-CDR1 has the amino acid sequence shown in SEQ ID NO: 4, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 5, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 6; or

[0050] (2) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 7, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 8, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 9; and

[0051] L-CDR1 has the amino acid sequence shown in SEQ ID NO: 10, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 11, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 12; or

[0052] (3) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 13, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 14, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 15; and

[0053] L-CDR1 has the amino acid sequence shown in SEQ ID NO: 4, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 16, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 17; or

[0054] (4) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 19, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 20; and

[0055] L-CDR1 has the amino acid sequence shown in SEQ ID NO: 4, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 16, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 21; or

[0056] (5) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 22, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 23, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 24; and

[0057] L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 25, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 26, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 27; or

[0058] (6) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 28, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 30; and

[0059] L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 31, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 32, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 33; or

[0060] (7) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 34, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 35, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 36; and

[0061] L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 39; or

[0062] (8) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 40; and

[0063] L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 41; or

[0064] (9) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 42, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 43, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 44; and

[0065] L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 45; or

[0066] (10) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 46, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 47, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 48; and

[0067] L-CDR1 has the amino acid sequence set forth in SEQ ID NO:49, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:50, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:51; or

[0068] (11) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 52, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 53, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 54; and

[0069] L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 55; or

[0070] (12) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 56, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 57, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 58; and

[0071] L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 59, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 60, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 51; or

[0072] (13) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 61, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 62; and

[0073] L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 63, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 64, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 65; or

[0074] (14) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 66; and

[0075] L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:67, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:41; or

[0076] (15) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 62; and

[0077] L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 68, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 69, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 70; or

[0078] (16) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 71, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 72, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 73; and

[0079] L-CDR1 has the amino acid sequence shown in SEQ ID NO: 74, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 75, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 76.

[0080] In some embodiments, the CDR sequences of the invention are defined with reference to the Kabat nomenclature system.

[0081] In some embodiments, the antibody of the present invention comprises at least one of a heavy chain framework region sequence and a light chain framework region sequence, and at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0082] In some embodiments, the heavy chain variable region of an antibody described herein comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 77-92.

[0083] In some embodiments, the antibody heavy chain variable region comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs:77-92.

[0084] In some embodiments, the light chain variable region of an antibody described herein comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 93-108.

[0085] In some embodiments, the light chain variable region of an antibody described herein comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 93-108.

[0086] In some embodiments, the heavy chain variable region of the antibody of the present invention comprises the amino acid sequence shown in SEQ ID NO:77 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:93.

[0087] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 77 and a light chain variable region represented by SEQ ID NO: 93 was numbered: 2E7.

[0088] In some embodiments, the heavy chain variable region of the antibody of the present invention comprises the amino acid sequence shown in SEQ ID NO:78 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:94.

[0089] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 78 and a light chain variable region represented by SEQ ID NO: 94 was numbered: 2G10.

[0090] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 79, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 95.

[0091] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 79 and a light chain variable region represented by SEQ ID NO: 95 was numbered: 5C12.

[0092] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 80 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 96.

[0093] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 80 and a light chain variable region represented by SEQ ID NO: 96 was numbered: 6C1.

[0094] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 81 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 97.

[0095] In the present invention, an antibody clone comprising the heavy chain variable region shown in SEQ ID NO: 81 and the light chain variable region shown in SEQ ID NO: 97 was numbered: 7B1.

[0096] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 82, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 98.

[0097] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 82 and a light chain variable region represented by SEQ ID NO: 98 was numbered: 12A1.

[0098] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 83 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 99.

[0099] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 83 and a light chain variable region represented by SEQ ID NO: 99 was numbered: 13A10.

[0100] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 84, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 100.

[0101] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 84 and a light chain variable region represented by SEQ ID NO: 100 was numbered: 14A2.

[0102] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 85, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 101.

[0103] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 85 and a light chain variable region represented by SEQ ID NO: 101 was numbered: 14B5.

[0104] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 86, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 102.

[0105] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 86 and a light chain variable region represented by SEQ ID NO: 102 was numbered: 14G2.

[0106] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 87, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 103.

[0107] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 87 and a light chain variable region represented by SEQ ID NO: 103 was numbered: 16H6.

[0108] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 88, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 104.

[0109] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 88 and a light chain variable region represented by SEQ ID NO: 104 was numbered: 17A11.

[0110] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 89 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 105.

[0111] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 89 and a light chain variable region represented by SEQ ID NO: 105 was numbered: 18A11.

[0112] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 90 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 106.

[0113] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 90 and a light chain variable region represented by SEQ ID NO: 106 was numbered: 19A4.

[0114] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 91 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 107.

[0115] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 91 and a light chain variable region represented by SEQ ID NO: 107 was numbered: 20E2.

[0116] The antibody heavy chain variable region of the present invention comprises the amino acid sequence shown in SEQ ID NO: 92, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 108.

[0117] In the present invention, an antibody clone comprising a heavy chain variable region represented by SEQ ID NO: 92 and a light chain variable region represented by SEQ ID NO: 108 was cloned and numbered: 22A6.

[0118] In some embodiments, the antibodies of the present invention comprise at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

[0119] In some embodiments, the heavy chain constant region and light chain constant region of the antibody of the present invention are both derived from human IgG antibodies or mutants thereof.

[0120] In some embodiments, the heavy chain constant region of the antibody of the present invention is derived from a human IgG1 or IgG4 antibody or a mutant thereof.

[0121] In some embodiments, the light chain constant region of the antibody described herein is derived from a human IgGκ or IgGλ constant region.

[0122] In some embodiments, the heavy chain constant region of the antibody of the present invention comprises the full-length sequence of the constant region, and the light chain constant region of the antibody comprises the full-length sequence of the constant region.

[0123] In some embodiments, the heavy chain of the antibody described in the present invention comprises a heavy chain variable region and a constant region; the heavy chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs:77-92.

[0124] In some embodiments, the heavy chain of the antibody described in the present invention comprises a heavy chain variable region and a constant region; the heavy chain variable region comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs:77-92.

[0125] In some embodiments, the antibody light chain of the present invention comprises a light chain variable region and a constant region; the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs: 93-108.

[0126] In some embodiments, the light chain of the antibody described in the present invention comprises a light chain variable region and a constant region; the light chain variable region comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs: 93-108.

[0127] In some embodiments, the antibodies described herein are monoclonal antibodies.

[0128] In some embodiments, the antibodies described herein are monoclonal human antibodies.

[0129] In some embodiments, the antigen-binding fragment of the antibody of the present invention is selected from the group consisting of: Fab, Fab'-SH, Fv, and (Fab')2 fragments.

[0130] In some embodiments, the antibodies of the present invention are natural whole antibodies, single-chain antibodies (scFv), single-chain Fv-Fc antibodies (scFv-Fc), scFv dimers, and dimers of scFv-Fc antibodies, wherein the dimers include homodimers and heterodimers.

[0131] The single-chain antibody (scFv) as described above comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 77-92 and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 93-108, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the light chain variable region via a connecting peptide Linker, or the C-terminus of the variable region is connected to the N-terminus of the heavy chain variable region via a connecting peptide Linker. It should be noted that the "connecting peptide Linker" of the single-chain antibody described in the present invention is used to connect the heavy chain variable region and the light chain variable region of the antibody, which can be a commonly used connecting peptide Linker for preparing single-chain antibodies, or a connecting peptide Linker modified by scientific researchers. An example of the amino acid sequence of the connecting peptide Linker is: (GnS)m, n is a natural number of 3-5, n is preferably 4, m is a natural number of 1-5, and m is preferably 3.

[0132] As a specific example, the single-chain antibody (scFv) of the present invention comprises the amino acid sequence shown in SEQ ID NO:113.

[0133] In the amino acid sequence shown in SEQ ID NO: 113, the bold portion is the connecting peptide Linker.

[0134] The single-chain antibodies of the present invention can be constructed with reference to the specific examples described above.

[0135] The single-chain Fv-Fc antibody (scFv-Fc) described above comprises an scFv and an Fc fragment, wherein the Fc fragment refers to an antibody heavy chain constant region fragment comprising at least a hinge region, a CH2 domain, and a CH3 domain. The scFv is as described above, and the scFv is directly connected to the Fc fragment or connected via a linker peptide (a specific example of a linker peptide is GGGGSGGGGSGGGGS). In some embodiments, the scFv of the present invention is directly connected to the Fc fragment via the hinge region.

[0136] The Fc fragment as described above is derived from, for example, a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; particularly selected from, for example, a heavy chain constant region of human IgG1, IgG2, IgG3 and IgG4, more particularly selected from the heavy chain constant region of human IgG1 or IgG4; and, the Fc fragment has one or more amino acid substitutions, deletions or additions (e.g., at most 20, at most 15, at most 10, at most 5, at most 3 or at most 1 substitution, deletion or addition) compared to the native sequence from which it is derived.

[0137] Specifically, the Fc in the single-chain Fv-Fc antibody (scFv-Fc) of the present invention has the amino acid sequence shown in SEQ ID NO:114.

[0138] In some embodiments, the antibodies of the present invention are monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0139] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment of the present invention.

[0140] In some embodiments, the nucleic acid molecule of the present invention is DNA.

[0141] On the other hand, the present invention provides a nucleic acid construct, which comprises the nucleic acid molecule described in the present invention; further, the nucleic acid construct comprises one or more control sequences operably linked to the nucleic acid molecule, wherein the one or more control sequences direct the production of the antibody or antigen-binding fragment thereof described in the present invention in an appropriate host cell, and the one or more control sequences are selected from: a promoter, an enhancer, a stop signal, a signal peptide, a leader sequence, a transcription terminator and any group thereof.

[0142] In yet another aspect, the present invention provides an expression vector carrying the nucleic acid molecule of the present invention.

[0143] In some embodiments, the expression vector of the present invention is selected from the group consisting of: a plasmid, a cosmid, a virus, a minichromosome, and an artificial chromosome.

[0144] In some embodiments, the expression vector of the present invention is a eukaryotic expression vector.

[0145] In some embodiments, the eukaryotic expression vector of the present invention is selected from the group consisting of: pCRII, pCR3, pcDNA3.4, pBSII, pET15, pGEX, pEGFP-N1, pETL, pDSR-α, and pFastBacDual.

[0146] In yet another aspect, the present invention provides a host cell comprising the nucleic acid molecule of the present invention or the expression vector of the present invention.

[0147] In some embodiments, the host cell of the present invention is Escherichia coli, yeast or a eukaryotic cell.

[0148] In some embodiments, the host cell of the present invention is a eukaryotic host cell.

[0149] In some embodiments, the eukaryotic host cell of the present invention is selected from: Chinese hamster ovary cells CHO, monkey kidney cells COS cells, human embryonic kidney cells HEK-293, human cervical cancer cells HELA, etc.

[0150] In some embodiments, the host cell is a mammalian host cell.

[0151] In yet another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention or the host cell of the present invention and a pharmaceutically acceptable carrier.

[0152] In yet another aspect, the present invention provides use of the antibody or antigen-binding fragment of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a cell proliferative disease or delaying the progression of a cell proliferative disease.

[0153] In yet another aspect, the present invention provides a method for treating a cell proliferative disease or delaying the progression of a cell proliferative disease, the method comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention.

[0154] In yet another aspect, the present invention provides a method for treating cancer, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention.

[0155] In some embodiments, the administration described herein is oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular.

[0156] In some embodiments, the cell proliferative disorder described herein is cancer.

[0157] In some embodiments, the cancer described herein is a Ly6G6D-positive cancer.

[0158] In some embodiments, the Ly6G6D-positive cancer described in the present invention is colorectal cancer, head and neck cancer, ovarian cancer, cervical cancer, melanoma, gastric cancer, esophageal cancer, small intestine cancer, large intestine cancer or adenocarcinoma, etc.

[0159] In some embodiments, the adenocarcinoma described herein is colorectal adenocarcinoma, gastric adenocarcinoma, or pancreatic adenocarcinoma.

[0160] In yet another aspect, the present invention provides an antibody-drug conjugate comprising the antibody or antigen-binding fragment of the present invention.

[0161] In some embodiments, the drug conjugates of the present invention are preferably small molecule drugs, and the antibody in the antibody-drug conjugate targets the small molecule drug to the therapeutic target through specific binding.

[0162] In yet another aspect, the present invention provides a kit for detecting Ly6G6D, wherein the kit comprises the antibody or antigen-binding fragment of the present invention.

[0163] In some embodiments, the kit of the present invention further comprises a container, instructions for use, a buffer, etc. In the kit of the present invention, the antibody of the present invention can be fixed on a detection plate.

[0164] Definition of terms

[0165] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0166] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0167] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0168] The terms "anti-Ly6G6D antibody" and "antibody that binds to Ly6G6D" refer to an antibody that is capable of binding to Ly6G6D with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting Ly6G6D.

[0169] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., bis-Fab), so long as they exhibit the desired antigen-binding activity.

[0170] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In a nanobody, there are three CDRs, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given nanobody, one skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefrance et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0171] The term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above. The FR of a variable domain generally consists of the following four FR domains: FR1, FR2, FR3, and FR4.

[0172] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0173] The term "affinity" refers to the strength of the sum of the 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 indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0174] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure, or has heavy chains that contain an Fc region as defined herein.

[0175] The term "natural antibody" refers to naturally occurring immunoglobulin molecules with different structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded together by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be classified into one of two types based on the amino acid sequence of its constant domain, which are called kappa (κ) and lambda (λ).

[0176] The terms "antibody fragment" and "antigen-binding fragment" are interchangeable and refer to molecules other than intact antibodies that contain a portion of an intact antibody and bind to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to: diaFab; Fv; Fab; Fab, Fab'-SH; F(ab')2; single-chain antibody molecules (e.g., scFv, ScFab); and multispecific antibodies formed from antibody fragments.

[0177] Fab fragments are antigen-binding fragments produced by papain digestion of antibodies and are composed of an intact L chain and the variable region domain (VH) of the H chain and the first constant domain (CH1) of a heavy chain. Papain digestion of antibodies produces two identical Fab fragments. Pepsin treatment of antibodies produces a single large F(ab')2 fragment, which is roughly equivalent to two Fab fragments connected by disulfide bonds with divalent antigen-binding activity and still capable of cross-linking antigens. The difference between Fab' fragments and Fab fragments is that Fab' fragments have some residues added to the carboxyl terminus of the CH1 domain, which contain one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0178] "Fv" consists of a tight, non-covalently associated dimer of a heavy chain variable region domain and a light chain variable region domain. The folding of these two domains produces six hypervariable loops (3 loops each from the H chain and the L chain), which contribute amino acid residues to achieve antigen binding and provide the antibody with antigen-binding specificity. However, even a single variable domain (or half of an Fv, containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is often lower than that of the entire binding site.

[0179] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which includes a native sequence Fc region and a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. The C-terminal lysine in the Fc region (according to the residue 447 of the EU numbering system) can be removed in, for example, the production or purification process of an antibody or by recombinant design of nucleic acids encoding an antibody heavy chain. Therefore, the composition of a complete antibody can include an antibody population removing all Lys447 residues, an antibody population not removing Lys447 residues, and an antibody population with and without an antibody mixture containing Lys447 residues.

[0180] Light chains (LC) from any vertebrate can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes, or isotypes, based on the amino acid sequence of their heavy chain constant domains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, γ, ε, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0181] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0182] The term "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing a repertoire of human antibodies or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0183] The term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVR and amino acid residues from people's FR. In some aspects, a humanized antibody will comprise substantially all at least one, usually two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of people's antibodies. In some aspects where all or substantially all of the FRs of a humanized antibody correspond to the FRs of people's antibodies, any FR of a humanized antibody can contain one or more amino acid residues from non-human FRs (e.g., one or more vernier position residues of FR). A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from people's antibodies. The antibody of a "humanized form," for example, a non-human antibody, refers to a humanized antibody.

[0184] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using the VH or VL domains from antibodies that bind to that antigen, respectively, to screen for libraries of complementary VL or VH domains. See, e.g., Portolano et al. J. Immunol. 150: 880-887, 1993; Clarkson et al. Nature 352: 624-628, 1991.

[0185] The term "hypervariable region" or "HVR" refers to the individual regions of an antibody variable domain that are hypervariable in sequence (complementarity determining regions or CDRs). Typically, an antibody comprises six CDRs; three in the VH (H-CDR1, H-CDR2, H-CDR3) and three in the VL (L-CDR1, L-CDR2, L-CDR3).

[0186] The term "single-chain Fv," also abbreviated as "sFv" or "scFv," refers to an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired antigen-binding structure.

[0187] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (for example, containing naturally occurring mutations or producing in the production process of monoclonal antibody preparations, such variants are usually presented in small amounts), each antibody comprising the population is identical and / or in conjunction with identical epi-positions. Contrary to the polyclonal antibody preparations typically comprising different antibodies for different determinants (epi-positions), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the feature of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as needing to produce an antibody by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by various techniques, including but not limited to hybridoma method, recombinant DNA method, phage display method, and the method utilizing a transgenic animal containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for the preparation of monoclonal antibodies are described herein.

[0188] The term "bispecific" antibody refers to an artificial antibody or antigen-binding fragment that has proteins derived from two different monoclonal antibodies and can bind to two different epitopes. The two epitopes can be present on the same antigen, or they can be present on two different antigens.

[0189] The term "multispecific antibody" is used in the broadest sense and specifically encompasses antibodies with multiple epitope specificities. In one aspect, a multispecific antibody binds to two different targets (e.g., a bispecific antibody). Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH / VL unit has multiple epitope specificities; antibodies having two or more VL and VH domains, wherein each VH / VL unit binds to a different epitope; antibodies having two or more single variable domains, wherein each single variable domain binds to a different epitope; full-length antibodies; antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, and triabodies, covalently or non-covalently linked antibody fragments.

[0190] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "cell proliferative disease," "proliferative disorder," and "tumor" are not mutually exclusive herein.

[0191] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Aspects of cancer include solid tumor cancers and non-solid tumor cancers. Solid cancer tumors include, but are not limited to, colorectal cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bladder cancer, kidney cancer, ovarian cancer, pancreatic cancer, or prostate cancer, or metastatic forms thereof. The cancer may be an LY6G6D-positive cancer.

[0192] In some aspects, the cancer is colorectal cancer.As used herein, the terms "colorectal cancer," "CRC," "colon cancer," or "bowel cancer" refer to cancer that develops from the large intestine, such as the colon or rectum.

[0193] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0194] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (CIq) to antibodies (of the appropriate subclass) bound to its cognate antigen.

[0195] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells with a cytotoxic agent. Antibodies "arm" the cytotoxic cells and are required for such killing. NK cells, the primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII.

[0196] The term "antibody-drug conjugate (ADC)" refers to an antibody conjugated, preferably chemically, to an effector molecule. The effector molecule is preferably a therapeutically active drug, such as one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0197] As used herein, "delaying progression" of a condition or disease means slowing, hindering, slowing, postponing, stabilizing, and / or postponing the development of a disease or condition (e.g., a cell proliferative disorder, e.g., cancer). Such a delay can be of varying lengths of time, depending on the medical history and / or the individual being treated. It will be apparent to one skilled in the art that a substantial or significant delay can actually encompass prevention, as the individual will not develop the disease. For example, the development of advanced cancers, such as metastases, can be delayed.

[0198] The term "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention intended to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some aspects, the antibodies of the invention (e.g., anti-LY6G6D antibodies of the invention) are used to delay the development of the disease or slow the progression of the disease.

[0199] An "effective amount" of a compound (e.g., an anti-LY6G6D antibody of the invention) or a composition thereof (e.g., a pharmaceutical composition) is at least the minimum amount required to achieve the desired therapeutic or preventive result, such as a measurable improvement or prevention of a particular disorder (e.g., a cell proliferative disorder, such as cancer). The effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit the desired response in the individual. An effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the treatment. For prophylactic use, beneficial or expected results include, for example, elimination or reduction of risk, lessening of severity, or delay in onset of the disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that arise during the course of the disease. For therapeutic use, beneficial or expected results include clinical results, such as reduction of one or more symptoms caused by the disease, improvement in the quality of life of the patient, reduction in the dose of other drugs required to treat the disease, and enhancement of the effects of other drugs (such as by targeting, slowing disease progression, and / or prolonging survival). In the case of cancer or a tumor, an effective amount of a drug may reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down or expect to stop to some extent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down and expect to stop to some extent) tumor metastasis; inhibit the growth of the tumor to some extent; and / or alleviate one or more symptoms associated with the condition to some extent. An effective amount can be administered one or more times. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to prevent or treat, directly or indirectly. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and an effective amount of a single agent may be considered if the desired result can be obtained or achieved in combination with one or more other agents.

[0200] An "isolated" protein or peptide is one that has been separated from a component of its natural environment. In some aspects, the protein or peptide is purified to a purity greater than 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that would normally contain the nucleic acid molecule, but that is present in an extrachromosomal or chromosomal location that is different from its natural chromosomal location.

[0201] The term "expression vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. A vector is called an expression vector when it can express the protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell through transformation, transduction, or transfection, so that the genetic material it carries is expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0202] The term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be a cDNA.

[0203] The term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. A host cell can include a single cell or a cell population.

[0204] The terms "identity" or "sequence identity" are used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0205] The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0206] The term "subject" refers to a mammal, such as a primate mammal, such as a human. In some embodiments, the subject (eg, human) suffers from a disease associated with Ly6G6D.

[0207] The term "administering" means a method of administering a dose of a compound (e.g., an anti-LY6G6D antibody of the present invention) to a subject. In some aspects, the composition used in the methods herein is administered intravenously. The composition used in the methods described herein can be, for example, intramuscularly, intravenously, intradermally, transcutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intracapsularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, topically, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion directly into target cells, by catheter, by lavage, in the form of an emulsion or a lipid composition. The method of administration can vary depending on a variety of factors (e.g., the compound or composition to be administered and the severity of the condition, disease or disorder to be treated).

[0208] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only intended to illustrate the present invention and are not intended to limit the scope of the invention. Based on the following detailed description of the drawings and preferred embodiments, the various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] FIG1 is a graph showing the binding results of candidate antibodies to the hLY6G6D antigen confirmed by ELISA;

[0210] FIG2 is a graph showing the binding results of candidate antibodies to the cynoLY6G6D antigen confirmed by ELISA;

[0211] FIG3 is a graph showing the binding results of HEK23T cells highly expressing hLY6G6D and candidate antibodies detected by flow cytometry;

[0212] FIG4 is a graph showing the results of flow cytometry detection of the binding between HEK23T cells that highly express cynoLY6G6D and candidate antibodies;

[0213] FIG5 is a graph showing the ADCC activity evaluation results of candidate molecules using the Jurkat-NFAT-Luc2-CD16 cell model. DETAILED DESCRIPTION

[0214] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0215] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0216] Example 1: Phage display technology and flow cytometry screening of LY6G6D monoclonal antibodies

[0217] Panning with a natural, fully human phage display library combined with ELISA screening can generate monoclonal phage that strongly bind to LY6G6D at the molecular level. Using Uniprot and NCBI databases, genes encoding the extracellular domains of the humanLY6G6D and cynoLY6G6D proteins were designed and constructed into a mammalian eukaryotic expression system. The extracellular domains of the humanLY6G6D and cynoLY6G6D proteins were expressed and used for natural human antibody library screening. The heavy and light chain variable region sequences of the antibody were amplified from human PBMCs, and the light and heavy chain gene fragments were connected by a linker (specifically, the linker has the amino acid sequence shown in SEQ ID NO: 112) to establish a natural human antibody phage library. The library was panned using the HumanLY6G6D protein. After four rounds of panning, the ELSIA method was used to screen monoclonal phage that could bind to HumanLY6G6D. The binding results of the 16 candidate phages to HumanLY6G6D are shown in Table 1 below. As can be seen from the results in Table 1, all 16 candidate phages of the present invention have good binding to HumanLY6G6D.

[0218] Table 1

[0219] Example 2: Expression and purification of antibodies

[0220] The 16 candidate phages obtained in Example 1 were sequenced to obtain the sequences encoding the variable regions of the light and heavy chains of the antibody candidates. The single-chain antibody (VH-Linker1-VL-Linker2-Fc, wherein VH has an amino acid sequence as shown in any one of SEQ ID NOs: 77-92, VL has an amino acid sequence as shown in any one of SEQ ID NOs: 93-108, Linker1 and Linker2 have an amino acid sequence as shown in SEQ ID NO: 112, and Fc has an amino acid sequence as shown in SEQ ID NO: 114) gene coding sequences were synthesized using whole gene synthesis methods. A series of antibody expression vectors were constructed using molecular cloning methods and recombinant expression was performed in the 293F expression system. After the 293F host cells were revived and cultured with Aupromycin medium, the cells were cultured at a cell density of about 3*10 6 When the cell count reached 100 cells / mL, cells were harvested and transfected with PEI reagent. On the second day of culture, feed was added and glucose was added to the culture concentration to 8 g / L. On the sixth day of culture, the cell culture medium was harvested.

[0221] A series of candidate antibodies were tested at the translational level. The collected cell culture fluid was purified using a Protein A chromatography column, and the absorption peaks were collected for mass spectrometry analysis. Mass spectrometry analysis revealed that the molecular weight of the scFv-Fc of a series of candidate antibodies was approximately 100 kDa, consistent with the theoretical molecular weight, indicating that they were in the form of dimers. The collected samples were also analyzed by 10% SDS-PAGE electrophoresis after both reduction and non-reduction. The reduced SDS-PAGE electrophoresis pattern of the candidate antibody scFv-Fc showed a band at approximately 50 kDa, while the non-reduced SDS-PAGE electrophoresis pattern showed a single band at approximately 100 kDa, consistent with the theoretical band size. The purified samples were dialyzed overnight at 4°C using 0.02 M PBS buffer, pH 7.4.

[0222] Example 3: Detection of binding properties between antibodies and antigens

[0223] Binding of the candidate antibodies obtained in Example 2 to the hLY6G6D antigen was confirmed by ELISA. ELISA plates were coated with 100 ng / well of hLY6G6D and placed in a 37°C incubator for 2 hours. Nonspecific binding sites were then blocked with 5% skim milk powder overnight at 4°C. The plates were incubated with various concentrations of mAb for 1 hour, washed three times with 0.1% PBST, and bound mAb was detected using HRP-mouse anti-human IgG1 Fc. The plates were then washed five times with 0.1% PBST, and TMB substrate (Yunqiao Bio) was added. The reaction was terminated after color development and read at 450 nm. The results are shown in Figure 1 and Tables 2-3. Figure 1A corresponds to Table 2, and Figure 1B corresponds to Table 3. The candidate antibodies exhibited comparable affinity to the positive antibody 20A12. Furthermore, 17A11, 19A4, and 20E2 exhibited superior binding activity to the hLY6G6D antigen compared to the positive antibody 20A12. The positive control antibody 20A12 is derived from patent CN202080086360.1. The heavy chain of antibody 20A12 comprises the amino acid sequence shown in SEQ ID NO: 115, and the light chain of antibody 20A12 comprises the amino acid sequence shown in SEQ ID NO: 116.

[0224] Table 2

[0225] Table 3

[0226] Example 4: Detection of binding properties between antibodies and antigens

[0227] Cross-reactivity testing with monkeys: Binding of the expressed candidate antibodies (obtained in Example 2) to the cynoLY6G6D antigen was confirmed by ELISA. Information for the positive control antibody 20A12 is shown in Example 3. ELISA plates were coated with 100 ng / well of cynoLY6G6D and placed in a 37°C incubator for 2 hours. Nonspecific binding sites were then blocked with 5% skim milk powder overnight at 4°C. The plates were incubated with various concentrations of mAb for 1 hour, washed three times with 0.1% PBST, and bound mAb was detected with HRP-mouse anti-human IgG1 Fc. The plates were then washed five times with 0.1% PBST, and TMB substrate (Yunqiao Bio) was added. The reaction was terminated after color development and read at 450 nm. The results are shown in Figure 2 and Tables 4-5. Figure 2A corresponds to Table 4, and Figure 2B corresponds to Table 5. Candidate antibody 14B5 did not bind to cynoLY6G6D, while the remaining candidate antibodies and the positive antibodies all bound to cynoLY6G6D.

[0228] Table 4

[0229] Table 5

[0230] Example 5: Detection of binding properties between antibodies and antigens

[0231] Flow cytometry was used to detect the binding of HEK23T cells that highly expressed hLY6G6D to the candidate antibodies (obtained in Example 2). The information of the positive control antibody 20A12 is shown in Example 3. 5 The cells were resuspended in a 96-well plate and incubated with different concentrations of mAb for 1 hour. The cells were then washed twice in PBS. PE-Goat anti-human IgG Fc (Jackson ImmunoResearch) was diluted 1:500 and incubated at 4°C for 30 minutes to detect the bound candidate antibodies. The cells were then washed twice in PBS and analyzed on FACS (Beckman). The results are shown in Figure 3 and Tables 6-7. Figure 3A corresponds to Table 6, and Figure 3B corresponds to Table 7. The affinity of the candidate antibodies was comparable to that of the positive antibody 20A12, and the binding activity of 19A4, 20E2, and hLY6G6D to positive HEK293T cells was better than that of the positive antibody 20A12.

[0232] Table 6

[0233] Table 7

[0234] Example 6: Detection of binding properties between antibodies and antigens

[0235] Cross-reaction detection with monkeys: Flow cytometry was used to detect the binding of HEK23T cells that highly expressed cynoLY6G6D to the candidate antibodies (obtained in Example 2). The information of the positive control antibody 20A12 is shown in Example 3. 5 Cells were resuspended in a 96-well plate and incubated with various concentrations of mAb for 1 hour. The cells were then washed twice in PBS and bound antibodies were detected by incubation with PE-Goat anti-human IgG Fc (Jackson ImmunoResearch) at a 1:500 dilution at 4°C for 30 minutes. The cells were then washed twice in PBS and analyzed by FACS (Beckman). The results are shown in Figure 4. Candidate antibody 14B5 did not bind to cynoLY6G6D, while the remaining candidate antibodies and the positive antibodies all bound to cynoLY6G6D.

[0236] Example 7: Evaluation of ADCC activity of candidate molecules using the Jurkat-NFAT-Luc2-CD16 cell model

[0237] A Jurkat-NFAT-Luc2-CD16 reporter gene cell model was constructed using Jurkat cells stably transfected with FcγRIIIa receptor and NFAT (nuclear factor of activated T cells) to evaluate the ADCC activity of candidate molecules.

[0238] Constructed hLY6G6D-HEK293T cells were used as target cells, and Jurkat-NFAT-Luc2-CD16 cells were used as effector cells. The effector cells and target cells were plated in a 384-well plate, with 5,000 cells per well. Information about the positive control antibody 20A12 is provided in Example 3. Different concentrations of the antibody (obtained in Example 2) were added to the 384-well plate, and the effector cells and target cells were co-cultured in a 37°C, 5% carbon dioxide incubator for 14 hours. The antibody can specifically bind to hLY6G6D on the surface of the target cells, and the Fc end of the antibody can bind to the FcγRIIIa receptor on the surface of the effector cells, thereby forming a target cell-antibody-effector cell complex, activating the NFAT-Luc signaling pathway in the effector cells, and causing the effector cells to produce luciferase. Luciferase detection substrate was added, reacted for 3 minutes, and the luminescence value of each well was read using a multifunctional microplate reader. The results are shown in FIG5 and Tables 8-9, FIG5A corresponds to Table 8, and FIG5B corresponds to Table 9. The ADCC activities of the candidate antibodies are comparable to those of the positive antibody 20A12, and the ADCC activities of 17A11 and 20E2 are superior to those of the positive antibody.

[0239] Table 8

[0240] Table 9

[0241] In the description of this specification, the reference terms "some embodiments", "one embodiment", "some embodiments", "examples", "specific examples" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0242] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-Ly6G6D antibody or antigen-binding fragment thereof, characterized in that, The antibody or antigen-binding fragment binds to Ly6G6D, and the antibody comprises a heavy chain and a light chain. The heavy chain comprises a heavy chain variable region, and the heavy chain variable region comprises complementarity-determining regions: H-CDR1, H-CDR2, and H-CDR3. Among them, (1) H-CDR1 has an amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:61, or SEQ ID NO:71; and (2) H-CDR2 has an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:57, or SEQ ID NO:72; and (3) H-CDR3 has an amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:66, or SEQ ID NO:73; and Among them, the light chain comprises a light chain variable region, and the light chain variable region comprises complementarity-determining regions: L-CDR1, L-CDR2, and L-CDR3. Among them, (1) L-CDR1 has an amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:68, or SEQ ID NO:74; and (2) The L-CDR2 has an amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69 or SEQ ID NO:75; and (3) The L-CDR3 has an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:65, SEQ ID NO:70 or SEQ ID NO:

76.

2. The antibody or antigen-binding fragment according to claim 1, wherein The heavy chain variable region comprises complementarity-determining regions: H-CDR1, H-CDR2 and H-CDR3; wherein, (1) The H-CDR1 has an amino acid sequence shown in SEQ ID NO:1, the H-CDR2 has an amino acid sequence shown in SEQ ID NO:2, and the H-CDR3 has an amino acid sequence shown in SEQ ID NO:3; or (2) The H-CDR1 has an amino acid sequence shown in SEQ ID NO:7, the H-CDR2 has an amino acid sequence shown in SEQ ID NO:8, and the H-CDR3 has an amino acid sequence shown in SEQ ID NO:9; or (3) The H-CDR1 has an amino acid sequence shown in SEQ ID NO:13, the H-CDR2 has an amino acid sequence shown in SEQ ID NO:14, and the H-CDR3 has an amino acid sequence shown in SEQ ID NO:15; or (4) The H-CDR1 has an amino acid sequence shown in SEQ ID NO:18, the H-CDR2 has an amino acid sequence shown in SEQ ID NO:19, and the H-CDR3 has an amino acid sequence shown in SEQ ID NO:20; or (5) The H-CDR1 has an amino acid sequence shown in SEQ ID NO:22, the H-CDR2 has an amino acid sequence shown in SEQ ID NO:23, and the H-CDR3 has an amino acid sequence shown in SEQ ID NO:24; or (6) The H-CDR1 has an amino acid sequence shown in SEQ ID NO:28, the H-CDR2 has an amino acid sequence shown in SEQ ID NO:29, and the H-CDR3 has an amino acid sequence shown in SEQ ID NO:30; or (7) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 34, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 35, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 36; or (8) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 40; or (9) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 42, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 43, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 44; or (10) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 46, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 47, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 48; or (11) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 52, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 53, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 54; or (12) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 56, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 57, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 58; or (13) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 61, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 62; or (14) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 66; or (15) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 62; or (16) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 71, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 72, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:

73.

3. The antibody or antigen-binding fragment according to claim 1, wherein, The light chain variable region contains complementary determining regions: L-CDR1, L-CDR2, and L-CDR3; wherein, (1) L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, L-CDR2 has the amino acid sequence shown in SEQ ID NO:5, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:6; or (2) L-CDR1 has the amino acid sequence shown in SEQ ID NO:10, L-CDR2 has the amino acid sequence shown in SEQ ID NO:11, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:12; or (3) L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, L-CDR2 has the amino acid sequence shown in SEQ ID NO:16, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:17; or (4) L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, L-CDR2 has the amino acid sequence shown in SEQ ID NO:16, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:21; or (5) L-CDR1 has the amino acid sequence shown in SEQ ID NO:25, L-CDR2 has the amino acid sequence shown in SEQ ID NO:26, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:27; or (6) L-CDR1 has the amino acid sequence shown in SEQ ID NO:31, L-CDR2 has the amino acid sequence shown in SEQ ID NO:32, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:33; or (7) L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, L-CDR2 has the amino acid sequence shown in SEQ ID NO:38, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:39; or (8) L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, L-CDR2 has the amino acid sequence shown in SEQ ID NO:38, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:41; or (9) L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, L-CDR2 has the amino acid sequence shown in SEQ ID NO:38, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:45; or (10) L-CDR1 has the amino acid sequence shown in SEQ ID NO:49, L-CDR2 has the amino acid sequence shown in SEQ ID NO:50, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:51; or (11) L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, L-CDR2 has the amino acid sequence shown in SEQ ID NO:38, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:55; or (12) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:59, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:60, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:51; or (13) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:63, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:64, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:65; or (14) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:67, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:41; or (15) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:68, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:69, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:70; or (16) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:74, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:75, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:

76.

4. The antibody or antigen-binding fragment according to any one of claims 1-3, characterized in that, The antibody comprises at least one of a heavy chain framework region sequence and a light chain framework region sequence, and at least a part of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

5. The antibody or antigen-binding fragment according to any one of claims 1-3, characterized in that, The variable region of the antibody heavy chain comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs:77 - 92.

6. The antibody or antigen-binding fragment according to any one of claims 1-4, characterized in that, The variable region of the antibody light chain comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs:93 - 108.

7. The antibody or antigen-binding fragment according to any one of claims 1-6, wherein The antibody comprises at least one of a heavy chain constant region and a light chain constant region, and at least a part of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof; Preferably, both the heavy chain constant region and the light chain constant region of the antibody are derived from a human IgG antibody or a mutant thereof; More preferably, the heavy chain constant region of the antibody is derived from a human IgG1 or IgG4 antibody or a mutant thereof; More preferably, the light chain constant region of the antibody is derived from a human IgGκ or IgGλ constant region.

8. The antibody or antigen-binding fragment according to any one of claims 1-7, characterized in that, The antibody heavy chain comprises a heavy chain variable region and a constant region; the heavy chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs:77 - 92.

9. The antibody or antigen-binding fragment according to any one of claims 1-8, characterized in that, The antibody light chain comprises a light chain variable region and a constant region; the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any one of SEQ ID NOs: 93 - 108.

10. The antibody or antigen-binding fragment according to any one of claims 1-9, characterized in that, The antibody is a monoclonal antibody.

11. The antibody or antigen-binding fragment according to any one of claims 1-10, characterized in that, The antigen-binding fragment of the antibody is selected from: Fab, Fab'-SH, Fv and (Fab')2 fragments.

12. The antibody or antigen-binding fragment according to any one of claims 1-11, characterized in that, The antibody is a monospecific antibody, bispecific antibody or multispecific antibody.

13. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the antibody or antigen-binding fragment according to any one of claims 1 - 12.

14. An expression vector, characterized in that, The expression vector carries the nucleic acid molecule according to claim 13.

15. A host cell, characterized in that, The host cell comprises the nucleic acid molecule according to claim 13 or the expression vector according to claim 14.

16. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or antigen-binding fragment according to any one of claims 1 - 12, the nucleic acid molecule according to claim 13, the expression vector according to claim 14, or the host cell according to claim 15 and a pharmaceutically acceptable carrier.

17. Use of the antibody or antigen-binding fragment according to any one of claims 1 - 12, the nucleic acid molecule according to claim 13, the expression vector according to claim 14, the host cell according to claim 15 or the pharmaceutical composition according to claim 16 in the preparation of a medicament for treating a cell proliferative disease or delaying the progression of a cell proliferative disease.

18. The use according to claim 17, wherein The cell proliferative disease is cancer.

19. The use according to claim 18, characterized in that, The cancer is Ly6G6D-positive cancer.

20. The use according to claim 19, characterized in that, The Ly6G6D-positive cancer is colorectal cancer, head and neck cancer, ovarian cancer, cervical cancer, melanoma, gastric cancer, esophageal cancer, small intestine cancer, large intestine cancer or adenocarcinoma, etc.

21. An antibody-drug conjugate, characterized in that, The antibody-conjugate comprises the antibody or antigen-binding fragment according to any one of claims 1 - 12.

22. A kit for detecting Ly6G6D, characterized in that, The kit comprises the antibody or antigen-binding fragment according to any one of claims 1 - 12.

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