Anti-CDH6 antibodies and uses thereof

Abstract

The invention provides an antibody specifically combined with CDH6 (cadherin 6) and application thereof, and particularly discloses mouse and humanized antibodies combined with CDH6 as well as a preparation method and application thereof, and the mouse and humanized antibodies have better affinity with CDH6 protein and better endocytosis activity, so that the mouse and humanized antibodies can be applied to preparation of medicines for treating tumors and the like.

Description

Anti-CDH6 antibodies and uses thereof

[0001] This disclosure claims priority to a Chinese patent application filed with the Patent Office of China on February 10, 2023, with application number 202310109927.8 and invention name “Anti-CDH6 Antibodies and Uses Thereof,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the fields of bioengineering and biomedicine, and primarily relates to an anti-CDH6 antibody or an antigen-binding fragment thereof, its encoding nucleic acid, expression vector and expression cell, preparation method, pharmaceutical composition, and their use in treating diseases, such as treating tumors. Background Art

[0003] Cadherins play an important role in tissue homeostasis and are primarily responsible for cell-cell adhesion during embryogenesis, tissue morphogenesis, differentiation, and tumorigenesis. Any dysfunction or instability of the cadherin-catenin complex may lead to tumor progression.

[0004] CDH6 is a type II classical cadherin, also known as K-cadherin. CDH6 is a single transmembrane protein consisting of 790 amino acids, with an extracellular domain divided into five regions. Studies have found that CDH6 is highly expressed in tumor tissues such as renal cancer, ovarian cancer, and thyroid cancer, while its expression in normal tissues is very low (Cancer Discov; 2017, 7(9): 1030-45; the contents of which are incorporated herein by reference). Like other members of the cadherin superfamily, CDH6 protein is localized to the basolateral membrane of epithelial cells and mediates calcium-dependent cell-cell adhesion, with the characteristic of rapid internalization. Therefore, CDH6, as a highly recognizable tumor-specific marker, has become an extremely attractive target in tumor treatment and can be developed for the treatment of cancers such as ovarian cancer and renal cancer.

[0005] Despite recent progress in the treatment of ovarian and renal cancer, significant unmet clinical needs remain. Currently, few drugs targeting CDH6 have been developed, and none have been approved for marketing. Therefore, developing drugs targeting this target holds broad clinical and therapeutic potential.

[0006] Summary of the Invention

[0007] The present invention provides antibodies or antigen-binding fragments that specifically bind to CDH6, nucleic acids encoding these antibodies and antigen-binding fragments thereof, pharmaceutical compositions and kits comprising the antibodies and antigen-binding fragments thereof, and the preparation of drugs that can be used to treat tumors.

[0008] In the first aspect, the present invention provides an antibody or antigen-binding fragment that specifically binds to CDH6, wherein the antibody or antigen-binding fragment specifically binds to one or more amino acid sequence fragments of the extracellular region EC1, EC1-EC2, EC2, EC2-EC3, EC3, EC4 or EC5 of the CDH6 protein; preferably, the antibody or antigen-binding fragment specifically binds to one or more amino acid sequence fragments shown in any one of SEQ ID NOs: 168-172; preferably, the antibody or antigen-binding fragment has endocytosis activity.

[0009] In a second aspect, the present invention provides an antibody or antigen-binding fragment that specifically binds to CDH6, wherein the antibody or antigen-binding fragment comprises:

[0010] (a) the HCDR1, HCDR2, and HCDR3 of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 136, 137, 138, 139, 150, 151, 152, 160, 161, 162, 163, 164, or 165; and / or, (b) the LCDR1, LCDR2, and LCDR3 of any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 133, 134, 135, 145, 146, 147, 148, 149, 157, 158, or 159;

[0011] Preferably, the HCDR1-3 and / or the LCDR1-3 are encoded according to the popular analysis method of KABAT, IMGT or Chothia.

[0012] In a specific embodiment, the HCDR1, HCDR2 and HCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination:

[0013] and,

[0014] The LCDR1, LCDR2 and LCDR3 have a sequence combination selected from any of the following sequence combinations or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination:

[0015] Preferably, the substitution is a conservative amino acid substitution.

[0016] In another specific embodiment, the antibody or antigen-binding fragment comprises a heavy chain CDRs and light chain CDRs combination selected from the group consisting of VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5, VH6+VL6, VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, or VH14+VL14, as well as CDRs combinations having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence of the heavy and light chain CDRs combination;

[0017] Preferably, the substitution is a conservative amino acid substitution.

[0018] In another specific embodiment, the framework regions of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment are derived from a human germline heavy chain template and a human germline light chain template, wherein:

[0019] (1) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-69*02 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-69*02 as shown in SEQ ID NO: 143 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 144;

[0020] (2) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-46*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-46*01 as shown in SEQ ID NO: 156 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 144;

[0021] (3) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-3*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-3*01 as shown in SEQ ID NO: 166 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 144;

[0022] (4) The framework region sequence is derived from the combined sequence of human germline light chains IGKV4-1*01 and IGKJ4*01; it comprises the FR1, FR2, and FR3 regions of IGKV4-1*01 as shown in SEQ ID NO: 140 and the FR4 region of IGKJ4*01 as shown in SEQ ID NO: 142;

[0023] (5) The framework region sequence is derived from the combined sequence of human germline light chains IGKV1-33*01 and IGKJ4*01; it comprises the FR1, FR2, and FR3 regions of IGKV1-33*01 as shown in SEQ ID NO: 141 and the FR4 region of IGKJ4*01 as shown in SEQ ID NO: 142;

[0024] (6) The framework sequence is derived from the combined sequence of human germline light chains IGKV1-NL1*01 and IGKJ2*01; it comprises the FR1, FR2, and FR3 regions of IGKV1-NL1*01 as shown in SEQ ID NO: 153 and the FR4 region of IGKJ2*01 as shown in SEQ ID NO: 155; or

[0025] (7) The framework region sequence is derived from the combined sequence of human germline light chains IGKV2-28*01 and IGKJ2*01; it includes the FR1, FR2, and FR3 regions of IGKV2-28*01 shown in SEQ ID NO: 154 and the FR4 region of IGKJ2*01 shown in SEQ ID NO: 155.

[0026] In a specific embodiment, the framework region of the antibody or antigen-binding fragment further comprises one or more mutations selected from the following group according to the Kabat numbering system, wherein:

[0027] (1) The framework region of the heavy chain variable region includes: G27Y, S30T, A40R, I70L or A72V; preferably includes G27Y and A72V; or preferably includes G27Y, S30T and A72V; or preferably includes G27Y, S30T, I70L and A72V; or preferably includes G27Y, S30T, A40R and A72V;

[0028] (2) the framework region of the heavy chain variable region comprises: G42R, M70L, R72V or T74K; preferably comprises R72V and T74K; or preferably comprises M70L, R72V and T74K; or preferably comprises G42R, R72V and T74K;

[0029] (3) the framework regions of the heavy chain variable region include: V2I, V5Q, R44G, I70L, R72V, Y95L or R98S; preferably include R72V and R98S; or preferably include V2I, R72V and R98S; or preferably include V2I, I70L, R72V and R98S; or preferably include R44G, R72V and R98S; or preferably include V5Q, R72V and R98S; or preferably include R72V, Y95L and R98S;

[0030] (4) the framework region of the light chain variable region includes: P44S, A47P or G72E; preferably includes G72E; or preferably includes P44S and G72E; or preferably includes A47P and G72E;

[0031] (5) The framework region of the light chain variable region includes: Q42K, A43S, K45Q, L48V, I48V or T85R; preferably includes A43S; or preferably includes A43S and L48V; or preferably includes A43S, K45Q and L48V; or preferably includes A43S, L48V and T85R; or preferably includes Q42K and I48V; or,

[0032] (6) The framework region of the light chain variable region includes: K42G, P43T, P44F or Y49S; preferably includes P44F and Y49S; or preferably includes K42G and Y49S; or preferably includes P43T and Y49S.

[0033] In a specific embodiment, the antibody or antigen-binding fragment comprises:

[0034] (1) a heavy chain variable region having a sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 136, 137, 138, 139, 150, 151, 152, 160, 161, 162, 163, 164, or 165;

[0035] (2) the light chain variable region has the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 133, 134, 135, 145, 146, 147, 148, 149, 157, 158 or 159;

[0036] (3) An amino acid sequence that is at least 90% identical to the sequence of any one of (1) to (2) above, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

[0037] In another specific embodiment, the heavy chain variable region and light chain variable region are selected from the following groups:

[0038] (1) having VH shown in SEQ ID NO. 2 and VL shown in SEQ ID NO. 3;

[0039] (2) having VH shown in SEQ ID NO. 4 and VL shown in SEQ ID NO. 5;

[0040] (3) having VH shown in SEQ ID NO. 6 and VL shown in SEQ ID NO. 7;

[0041] (4) having VH shown in SEQ ID NO. 8 and VL shown in SEQ ID NO. 9;

[0042] (5) having the VH shown in SEQ ID NO. 10 and the VL shown in SEQ ID NO. 11;

[0043] (6) having the VH shown in SEQ ID NO. 12 and the VL shown in SEQ ID NO. 13;

[0044] (7) having VH shown in SEQ ID NO. 14 and VL shown in SEQ ID NO. 15;

[0045] (8) having VH shown in SEQ ID NO. 16 and VL shown in SEQ ID NO. 17;

[0046] (9) having VH shown in SEQ ID NO. 18 and VL shown in SEQ ID NO. 19;

[0047] (10) having VH shown in SEQ ID NO. 20 and VL shown in SEQ ID NO. 21;

[0048] (11) having VH shown in SEQ ID NO. 22 and VL shown in SEQ ID NO. 23;

[0049] (12) having VH shown in SEQ ID NO. 24 and VL shown in SEQ ID NO. 25;

[0050] (13) having VH shown in SEQ ID NO. 26 and VL shown in SEQ ID NO. 27;

[0051] (14) having VH shown in SEQ ID NO. 28 and VL shown in SEQ ID NO. 29;

[0052] (15) having VH shown in SEQ ID NO. 136 and VL shown in SEQ ID NO. 133;

[0053] (16) having VH shown in SEQ ID NO. 137 and VL shown in SEQ ID NO. 133;

[0054] (17) having VH shown in SEQ ID NO. 138 and VL shown in SEQ ID NO. 133;

[0055] (18) having VH shown in SEQ ID NO. 139 and VL shown in SEQ ID NO. 133;

[0056] (19) having VH shown in SEQ ID NO. 136 and VL shown in SEQ ID NO. 134;

[0057] (20) having VH shown in SEQ ID NO. 137 and VL shown in SEQ ID NO. 134;

[0058] (21) having VH shown in SEQ ID NO. 138 and VL shown in SEQ ID NO. 134;

[0059] (22) having VH shown in SEQ ID NO. 139 and VL shown in SEQ ID NO. 134;

[0060] (23) having VH shown in SEQ ID NO. 136 and VL shown in SEQ ID NO. 135;

[0061] (24) having VH shown in SEQ ID NO. 137 and VL shown in SEQ ID NO. 135;

[0062] (25) having VH shown in SEQ ID NO. 138 and VL shown in SEQ ID NO. 135;

[0063] (26) having VH shown in SEQ ID NO. 139 and VL shown in SEQ ID NO. 135;

[0064] (27) having VH shown in SEQ ID NO. 150 and VL shown in SEQ ID NO. 145;

[0065] (28) having VH shown in SEQ ID NO. 150 and VL shown in SEQ ID NO. 146;

[0066] (29) having VH shown in SEQ ID NO. 150 and VL shown in SEQ ID NO. 147;

[0067] (30) having VH shown in SEQ ID NO. 150 and VL shown in SEQ ID NO. 148;

[0068] (31) having VH shown in SEQ ID NO. 150 and VL shown in SEQ ID NO. 149;

[0069] (32) having VH shown in SEQ ID NO. 151 and VL shown in SEQ ID NO. 145;

[0070] (33) having VH shown in SEQ ID NO. 151 and VL shown in SEQ ID NO. 146;

[0071] (34) having VH shown in SEQ ID NO. 151 and VL shown in SEQ ID NO. 147;

[0072] (35) having VH shown in SEQ ID NO. 151 and VL shown in SEQ ID NO. 148;

[0073] (36) having VH shown in SEQ ID NO. 151 and VL shown in SEQ ID NO. 149;

[0074] (37) having VH shown in SEQ ID NO. 152 and VL shown in SEQ ID NO. 145;

[0075] (38) having VH shown in SEQ ID NO. 152 and VL shown in SEQ ID NO. 146;

[0076] (39) having VH shown in SEQ ID NO. 152 and VL shown in SEQ ID NO. 147;

[0077] (40) having VH shown in SEQ ID NO. 152 and VL shown in SEQ ID NO. 148;

[0078] (41) having VH shown in SEQ ID NO. 152 and VL shown in SEQ ID NO. 149;

[0079] (42) having VH shown in SEQ ID NO. 160 and VL shown in SEQ ID NO. 157;

[0080] (43) having VH shown in SEQ ID NO. 160 and VL shown in SEQ ID NO. 158;

[0081] (44) having VH shown in SEQ ID NO. 160 and VL shown in SEQ ID NO. 159;

[0082] (45) having VH shown in SEQ ID NO. 161 and VL shown in SEQ ID NO. 157;

[0083] (46) having VH shown in SEQ ID NO. 161 and VL shown in SEQ ID NO. 158;

[0084] (47) having VH shown in SEQ ID NO. 161 and VL shown in SEQ ID NO. 159;

[0085] (48) having VH shown in SEQ ID NO. 162 and VL shown in SEQ ID NO. 157;

[0086] (49) having VH shown in SEQ ID NO. 162 and VL shown in SEQ ID NO. 158;

[0087] (50) having VH shown in SEQ ID NO. 162 and VL shown in SEQ ID NO. 159;

[0088] (51) having VH shown in SEQ ID NO. 163 and VL shown in SEQ ID NO. 157;

[0089] (52) having VH shown in SEQ ID NO. 163 and VL shown in SEQ ID NO. 158;

[0090] (53) having VH shown in SEQ ID NO. 163 and VL shown in SEQ ID NO. 159;

[0091] (54) having VH shown in SEQ ID NO. 164 and VL shown in SEQ ID NO. 157;

[0092] (55) having VH shown in SEQ ID NO. 164 and VL shown in SEQ ID NO. 158;

[0093] (56) having VH shown in SEQ ID NO. 164 and VL shown in SEQ ID NO. 159;

[0094] (57) having VH shown in SEQ ID NO. 165 and VL shown in SEQ ID NO. 157;

[0095] (58) having VH shown in SEQ ID NO. 165 and VL shown in SEQ ID NO. 158;

[0096] (59) having VH shown in SEQ ID NO.165 and VL shown in SEQ ID NO.159; or

[0097] (60) A VH and VL combination having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to any of the sequence combinations of (1) to (59) above.

[0098] In a specific embodiment, the dissociation constant (KD) of the antibody or antigen-binding fragment binding to human CDH6 protein is no greater than 2×10 -7 M.

[0099] In another specific embodiment, the antibody or antigen-binding fragment is:

[0100] (1) Chimeric antibodies or fragments thereof;

[0101] (2) humanized antibodies or fragments thereof;

[0102] (3) fully human antibodies or fragments thereof;

[0103] Preferably, the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

[0104] In another specific embodiment, the antibody comprises the sequence of the constant region of any one of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it comprises the sequence of the constant region of human or murine antibodies IgG1, IgG2, IgG3 or IgG4.

[0105] In another specific embodiment, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies and antibody minimal recognition units.

[0106] In a third aspect, the antibody or antigen-binding fragment of the present invention is further coupled to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug or an immunomodulatory agent, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent or a photosensitizer.

[0107] In a fourth aspect, the present invention further provides a multispecific antigen-binding molecule, comprising a first antigen-binding moiety and a second antigen-binding moiety, wherein the first antigen-binding moiety is the antibody or antigen-binding fragment according to any one of the first or second aspects above, and the second antigen-binding moiety specifically binds to an antigen other than CDH6 or binds to a CDH6 antigen epitope different from that of the first antigen-binding moiety;

[0108] Preferably, the other antigens are selected from CD3, CD7, CD16, CD16A, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD66(ad), CD74, CD80, CD126, CD138, BCMA, HLA-DR, HER2, VEGF, P1GF, HER3 / ERBB3, HER4 / ERBB4, IL-2, IL-6, PD-1, PD-L1, TRAIL-R1 or TRAIL-R2;

[0109] Preferably, the multispecific antigen-binding molecule is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

[0110] In the fifth aspect, the present invention also provides a chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises the CDH6 antibody or antigen binding fragment described in the first or second aspect above.

[0111] In a sixth aspect, the present invention further provides an immune effector cell, comprising the chimeric antigen receptor described in the fifth aspect or a nucleic acid fragment encoding the chimeric antigen receptor described in the fifth aspect;

[0112] Preferably, the immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells; the T cells can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Treg) or helper T cells;

[0113] Preferably, the immune effector cells are allogeneic immune effector cells or autologous immune cells.

[0114] In the seventh aspect, the present invention also provides an isolated nucleic acid molecule, which encodes the antibody, antigen-binding fragment, or any combination described in the first and second aspects above, the antibody or antigen-binding fragment conjugated with a tracer described in the third aspect, the multispecific antigen-binding molecule of the fourth aspect, or the chimeric antigen receptor of the fifth aspect.

[0115] In an eighth aspect, the present invention further provides an expression vector comprising the isolated nucleic acid molecule according to the seventh aspect.

[0116] In the ninth aspect, the present invention provides an isolated host cell of the isolated nucleic acid molecule described in the seventh aspect, or the expression vector described in the eighth aspect; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

[0117] In the tenth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled to a tracer described in the third aspect, or the multispecific antigen-binding molecule described in the fourth aspect, by incubating the host cell described in the ninth aspect under appropriate conditions, and isolating the antibody or antigen-binding fragment or multispecific antigen-binding molecule.

[0118] In the eleventh aspect, the present invention provides a method for preparing the immune effector cells described in the sixth aspect above, the method comprising introducing a nucleic acid fragment encoding the CAR described in the fifth aspect into the immune effector cells, and optionally, the method further comprises initiating the immune effector cells to express the CAR described in the fifth aspect.

[0119] In the twelfth aspect, the present invention provides a pharmaceutical composition, comprising the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth or eleventh aspect; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0120] In the thirteenth aspect, the present invention provides the use of the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth or eleventh aspect, or the pharmaceutical composition described in the twelfth aspect in the preparation of a medicament for preventing and / or treating a tumor disease; preferably, the tumor disease is selected from a solid tumor, preferably, the solid tumor is a solid tumor expressing CDH6 protein, more preferably, the solid tumor is selected from renal cancer, ovarian cancer, thyroid cancer, bile duct cancer, lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, bile duct cancer, Wilms' tumor or neuroblastoma.

[0121] In the fourteenth aspect, the present invention provides a method for preventing and / or treating a tumor disease, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth or eleventh aspect, or the pharmaceutical composition described in the twelfth aspect; preferably, the tumor disease is selected from solid tumors, preferably, the solid tumor is a solid tumor expressing CDH6 protein, more preferably, the solid tumor is selected from renal cancer, ovarian cancer, thyroid cancer, bile duct cancer, lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, bile duct cancer, Wilms' tumor or neuroblastoma.

[0122] In the fifteenth aspect, the present invention provides the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth or eleventh aspect, or the pharmaceutical composition described in the twelfth aspect for use in and / or treatment of tumor diseases; preferably, the tumor disease is selected from solid tumors, preferably, the solid tumor is a solid tumor expressing CDH6 protein, more preferably, the solid tumor is selected from renal cancer, ovarian cancer, thyroid cancer, bile duct cancer, lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, bile duct cancer, Wilms' tumor or neuroblastoma.

[0123] In the sixteenth aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment described in the first or second aspect above, the antibody or antigen-binding fragment conjugated to a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth or eleventh aspect, or the pharmaceutical composition described in the twelfth aspect, and instructions for use.

[0124] Beneficial effects: The present invention aims to develop monoclonal antibodies that specifically bind to CDH6, which have good binding activity and internalization ability, and can be used for the subsequent development of drugs such as CDH6-ADC, and for the treatment of diseases such as ovarian cancer and renal cancer.

[0125] Definitions and Explanations of Terms

[0126] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including, but not limited to, chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies, diabodies, triabodies, and tetrabodies, antibody conjugates), and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments). In addition, unless otherwise indicated, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules that are capable of specifically binding to a target protein, as well as incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack the Fc fragment of an intact antibody (which is cleared more rapidly from the animal circulation) and therefore lack Fc-mediated effector functions (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein by reference).

[0127] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0128] The term "monospecific" as used herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

[0129] The term "multispecific" herein refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0130] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0131] As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. An antibody fragment can be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb comprising both VH and VL domains; (vi) a dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546, 1989; the contents of which are incorporated herein by reference); (vii) a dAb consisting of either a VH or VL domain; (viii) isolated complementarity determining regions (CDRs); and (ix) combinations of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, the two domains can be joined using recombinant methods via a linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; the contents of which are incorporated herein by reference). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.

[0132] As used herein, the term "CDH6", also known as "cadherin 6", refers to a cell adhesion molecule, which is a member of the cadherin family of cell-cell adhesion molecules. CDH6 is a single transmembrane protein consisting of 790 amino acids, which is classified as a type II cadherin family, and the protein has an N-terminal extracellular and C-terminal intracellular domain. The CDH6 protein used in the present invention can be directly purified from cells expressing CDH6 of humans or non-human mammals (such as rats, mice or monkeys), and can be used subsequently, or the cell membrane fraction of the above-mentioned cells can be prepared and can be used as CDH6 protein. Alternatively, CDH6 can also be obtained by allowing host cells to produce CDH6 by in vitro synthesis or by genetic manipulation. According to this genetic manipulation, CDH6 protein can be obtained, specifically, CDH6 cDNA is incorporated into a vector capable of expressing CDH6 cDNA, and then CDH6 is synthesized in a solution containing enzymes, substrates and energy materials required for transcription and translation, or by transforming other prokaryotes or eukaryotic host cells, thereby allowing them to express CDH6. CDH6-expressing cells or CDH6-expressing cell lines based on the above genetic manipulations can also be used to present CDH6 protein. Alternatively, an expression vector incorporating CDH6 cDNA can be directly administered to an animal to be immunized, and CDH6 can be expressed in the animal thus immunized.

[0133] As used herein, the term "bispecific antibody" refers to an antibody, typically a human or humanized antibody, that has monoclonal binding specificities for at least two different antigens. In the present invention, one of the binding specificities can be detected for an antigenic epitope of CDH6, and the other can be detected for another antigenic epitope of CDH6 or any other antigen other than CDH6, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a viral protein, a virally encoded envelope protein, a bacterial protein, or a bacterial surface protein.

[0134] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences of an immunoglobulin from one source organism (e.g., rat or mouse) and constant regions of an immunoglobulin from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art.

[0135] As used herein, the term "complementarity determining region" (CDR) refers to the hypervariable region found in both the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). As understood in the art, the amino acid position representing the hypervariable region of an antibody can vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered as hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of standards (such as IMGT or KABAT), while being considered to be outside the hypervariable region under different sets of standards (such as KABAT or IMGT). One or more of these positions can also be found in the hypervariable region of extension. The present invention includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a lamellae configuration, connected by three CDRs (CDR1, CDR2, and CDR3), which form a loop connecting the lamellae structure and, in some cases, form a part of the lamellae structure. The CDRs in each chain are closely held together by the FR region in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen binding site of the antibody with the CDRs from other antibody chains (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference). For example, in this article, CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, second CDR and third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or its variable region); CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and third CDR of the light chain variable region (VL), respectively, and these three CDRs constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).

[0136] As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation to the method by which the antibody is produced.

[0137] As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain (including the heavy chain of Fv, scFv or Fab) of an antibody. The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv or Fab).

[0138] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least one of the following: a CHI domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter only comprises "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CHI domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CHI domain. For example, a typical "heavy chain constant region fragment" can be selected from the CHI, Fc, or CH3 domains.

[0139] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0140] The term "Fc" herein refers to the antibody carboxyl terminal portion of a complete antibody that is hydrolyzed by papain, and typically, it comprises the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at Cys226 position or from Pro230 to its carboxyl terminal. The C-terminal lysine (according to the residue 447 of the EU numbering system) in the Fc region can, for example, be present in the production or purification process of an antibody, or removed by recombinant engineering of a nucleic acid encoding an antibody heavy chain. Therefore, the Fc region may include or may not include Lys447.

[0141] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0142] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein are not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been transplanted to human framework sequences.

[0143] The term "naked antibody" herein refers to an antibody that is not connected, fused or conjugated to another agent or molecule (e.g., a label or drug), peptide or polypeptide. In specific embodiments, naked antibodies expressed by mammalian host cells can be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell. In certain embodiments, naked antibodies are not glycosylated when expressed by host cells that do not have their own glycosylation machinery (e.g., glycosylase). In certain embodiments, naked antibodies are intact antibodies, while in other embodiments, naked antibodies are antigen-binding fragments of intact antibodies, such as Fab antibodies.

[0144] The term "conjugated antibody" herein refers to an antibody that can be associated with a pharmaceutically acceptable carrier or diluent, which can be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody.

[0145] The term "diabody" herein refers to a bivalent, bispecific antibody that can bind to different epitopes on the same or different antigens.

[0146] The term "minimum antibody recognition unit" herein refers to the smallest unit of an antigen that can be recognized by an antibody in an antigen-antibody binding reaction.

[0147] The term "nanoantibody" in this article refers to the natural heavy chain antibody lacking light chain in camels. Cloning its variable region can produce a single domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0148] As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced into one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored). For the purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires maximum alignment over the full length of the compared sequences. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0149] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0150] (1) Acidic amino acids: Asp (D) and Glu (E);

[0151] (2) Basic amino acids: Lys (K), Arg (R), and His (H);

[0152] (3) hydrophilic uncharged amino acids: Ser (S), Thr (T), Asn (N), and Gln (Q);

[0153] (4) Aliphatic uncharged amino acids: Gly (G), Ala (A), Val (V), Leu (L) and Ile (I);

[0154] (5) Non-polar uncharged amino acids: Cys (C), Met (M), and Pro (P);

[0155] (6) Aromatic amino acids: Phe (F), Tyr (Y) and Trp (W).

[0156] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; which is incorporated herein by reference).

[0157] As used herein, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by an antibody or its antigen-binding fragment. An antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or its antigen-binding fragment that does not exhibit specific binding to a specific antigen or epitope thereof will exhibit a KD of greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that specific antigen or epitope. Various immunoassays can be used to select antibodies that specifically immunoreact with a specific protein or carbohydrate. For example, solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe immunoassay formats and conditions that can be used to determine specific immunoreactivity (which are incorporated herein by reference).

[0158] As used herein, the term "antibody conjugate" refers to a coupling / conjugate formed by chemically bonding an antibody molecule directly or through a linker to another molecule, such as an antibody-drug conjugate (ADC), wherein the drug molecule is the other molecule.

[0159] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for directly expressing the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP2 101 823B1; the contents of which are incorporated herein by reference).

[0160] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors of the present invention contain polynucleotide sequences and, for example, additional sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct the efficient transcription of genes carried on the expression vector. The expression vectors of the present invention may also contain the following polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0161] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0162] The term "pharmaceutical composition" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0163] As used herein, the terms "subject," "object," and "patient" refer to an organism that is being treated for a particular disease or condition, such as cancer or an infectious disease, as described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovidae family (such as cattle, bison, buffalo, elk, and yaks, etc.), sheep, and horses, etc., that are being treated for a disease or condition, such as a cell proliferative disorder, such as cancer or an infectious disease.

[0164] As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as the progression of a cell proliferative disorder (such as cancer or an infectious disease). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a disease or disease, as well as subjects susceptible to a disease or disease, or subjects intending to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0165] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0166] The term "appropriate conditions" herein refers to conditions suitable for culturing various host cells, wherein the host cells include eukaryotic cells and prokaryotic cells.

[0167] The term "tumor" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0168] The term "anti-tumor agent" herein refers to anti-tumor drugs, which are a class of drugs for treating tumor diseases, such as chemotherapy drugs, biological agents, etc.

[0169] The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0170] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0171] Figures 1 and 2 show the binding activity of hCDH6-1 humanized antibody to human CDH6 protein.

[0172] Figures 3 and 4 show the binding activity of hCDH6-1 humanized antibody to OVCAR3 cells.

[0173] Figures 5 and 6 show the binding activity of hCDH6-6 humanized antibody to human CDH6 protein.

[0174] Figures 7 and 8 show the binding activity of hCDH6-6 humanized antibody to OVCAR3 cells.

[0175] Figures 9-11 show the binding activity of hCDH6-14 humanized antibody to human CDH6 protein.

[0176] Figures 12 and 13 show the binding activity of hCDH6-14 humanized antibody to PA-1 cells. DETAILED DESCRIPTION

[0177] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0178] The embodiments of the present invention are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0179] Example 1 Preparation of anti-human CDH6 mouse monoclonal antibody

[0180] 1.1 Animal immunization

[0181] 1.1.1 Protein Immunization

[0182] Five 6- to 8-week-old SJL mice (mouse numbers 2401-2405) or five MRL / lpr mice (mouse numbers 2136-2140) (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were immunized. Mice were housed under SPF conditions. For the primary immunization, an Fc-tagged human CDH6 extracellular domain protein (hCDH6 ECD-hFc, conventionally synthesized; the hCDH6 ECD sequence is derived from Uniprot #P55285 (19-615), as shown in SEQ ID NO: 1, conventionally synthesized) was mixed with Titermax (purchased from Sigma, catalog number T2684) and oligonucleotide CPG (ODN 1826, synthesized from Shanghai Sangon Biotechnology) and emulsified, then injected into the footpad and back. hCDH6 ECD-hFc protein was mixed with Imject Alum (purchased from Thermo Fisher, catalog number 77161) and CPG and injected intraperitoneally. 50 μg of antigen was injected per mouse. For the first booster immunization, hCDH6 ECD-hFc protein, Imject Alum, and CPG were mixed and injected into the footpad and back. For the second booster immunization, hCDH6 ECD-hFc protein, Titer Max, and CPG were emulsified and injected into the back. Subsequent booster immunizations alternated with the first and second booster immunizations, with 25 μg of antigen injected per mouse each time, and 7 days between immunizations. Blood was collected from mice five days after the second and fourth booster immunizations, and serum was isolated for determination of specific antibody titers using enzyme-linked immunosorbent assay (ELISA).

[0183] hCDH6 ECD, SEQ ID NO: 1 (Uniprot#P55285(19-615))

[0184] The ELISA assay was performed as follows: His-tagged human CDH6 extracellular domain protein (hCDH6 ECD-his, conventionally synthesized; the hCDH6 ECD sequence is derived from Uniprot #P55285 (19-615), as shown in SEQ ID NO: 1) was diluted with PBS to a final concentration of 1 μg / mL. 100 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the plate was washed three times with plate wash buffer (PBS + 0.01% (v / v) Tween 20) and blocked with blocking buffer (PBS + 0.01% (v / v) Tween 20 + 2% (w / w) BSA) at room temperature for 2 hours. The blocking buffer was discarded and the plate was washed three times with plate wash buffer. Mouse serum was diluted with 1% BSA + PBS, and 50 μl was added to each well of a 96-well ELISA plate. After incubation at 37°C for 2 hours, the plate was washed three times with plate wash buffer. Add 50 μL of a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody (Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L), purchased from Jackson Immuno, Cat. No. 115-035-003) to each well. Incubate at 37°C for 1 hour, then wash the plate three times with plate washer buffer. Add 50 μL of TMB substrate to each well, incubate at room temperature for 10 minutes, and then add 50 μL of stop solution (1.0 N HCl) to each well. OD 450 nm values ​​were read using a microplate reader (Biotek).

[0185] The ELISA results are shown in Table 1. After five immunizations, the serum from the immunized mice showed high titers of binding to the human CDH6 protein, demonstrating an antigen-antibody reaction. TB2 refers to the serum from mice on day 5 after five immunizations (primary immunization + four booster immunizations). The data in the table are OD450nm values.

[0186] Table 1 ELISA detection of serum titers of mice immunized with hCDH6 ECD-hFc protein

[0187] 1.1.2 Cell and protein immunization

[0188] Three 6-8 week old SJL mice (mouse numbers #2453, 2454, 2456) or five MRL / lpr mice (mouse numbers #2448-2452) (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were used for immunization. The mice were housed under SPF conditions. For the first immunization, HEK293T cells overexpressing human CDH6 protein (HEK293T-hCDH6, conventional construction, HEK293T cells purchased from the Chinese Academy of Sciences Cell Bank, catalog number SCSP-502) were mixed with Titer max (purchased from Sigma, catalog number T2684) and injected intraperitoneally. Each mouse was injected with 5×10 6For the first booster immunization, HEK293T-hCDH6 cells were mixed with CPG and injected intraperitoneally; for the second booster immunization, HEK293T-hCDH6 cells were mixed with Titer max and injected intraperitoneally. Subsequent booster immunizations were performed alternately according to the first and second booster immunizations, with each mouse injected with 5×10 6 Each immunization was performed with a 7-day interval. Blood was collected from mice on day 5 after the second and fourth booster immunizations, and serum was isolated. Serum-specific antibody titers were determined using ELISA. Based on the titers, mice were immunized with hCDH6 ECD-hFc protein (protein source as in 1.1.1) after five rounds of cell-based immunization (primary immunization + four booster immunizations).

[0189] Enzyme-linked immunosorbent assay (ELISA) was used to determine specific antibody titers in serum. The ELISA results are shown in Table 2. After five rounds of cell-mediated immunization and three rounds of protein immunization, serum from immunized mice showed high titers of binding to human CDH6 protein, demonstrating an antigen-antibody reaction. TB4 refers to serum from mice on day 5 after eight rounds of immunization. Data in the table represent OD450nm values.

[0190] Table 2 ELISA detection of serum titers in mice immunized with HEK293T-hCDH6 cells and hCDH6 ECD-hFc protein

[0191] 1.2 Hybridoma Preparation

[0192] Mice with high titers were selected and injected with 50 μg of hCDH6 ECD-hFc protein (protein source as described in 1.1.1) intraperitoneally, in the plantar aspect, and in the dorsal aspect. Three days later, the mice were sacrificed, and splenocytes and lymphocytes were harvested. After centrifugation at 1500 rpm / min, the supernatant was discarded, and ACK lysis buffer (Gibco, Catalog No. A1049201) was added to the cells to lyse any erythrocytes contaminating the cells, yielding a cell suspension. The cells were washed three times with DMEM basal medium (Gibco, Catalog No. 10569044) at 1500 rpm / min, then mixed with mouse myeloma SP2 / 0 cells (ATCC, Catalog No. CRL-1581) at a ratio of 2:1 viable cell count. Cell fusion was performed using electrofusion (BTX 2001+ instrument). The fused cells were diluted into DMEM medium containing 20% ​​(w / w) fetal bovine serum (purchased from ExCell Bio, product number FND500), 1×HAT (purchased from Sigma, product number H0262-10VL), bovine insulin (purchased from Yeason, product number 40107ES25), and NEAA (purchased from Gibco, product number 11140050), and then cultured at 5×104 200 μL of each cell culture medium was added to each well of a 96-well cell culture plate and cultured in a 5% (v / v) CO2, 37°C incubator.

[0193] After 7 days, the supernatants from the fusion plates were screened by ELISA to determine binding activity to human CDH6 protein. Supernatants from positive clones were also assayed for binding activity to monkey CDH6 protein, mouse CDH6 protein, and cells overexpressing human CDH6 by ELISA. Based on the screening results, eligible positive clones were selected and subcloned using semi-solid culture medium (purchased from Stemcell, Catalog No. 03810). Seven days later, the clones were individually transferred to 96-well culture plates and expanded in DMEM medium supplemented with 10% (w / w) fetal bovine serum and 1×HT (purchased from Sigma, Catalog No. H0137-10VL). One day later, preliminary screening was performed by ELISA, and single clones with human CDH6 binding activity were selected and expanded to 24-well plates for further culture. Three days later, the culture supernatants were further assayed to evaluate their binding activity to monkey CDH6 protein, mouse CDH6 protein, and their endocytosis activity in cells overexpressing human CDH6. Based on the results of the 24-well plate sample test, the optimal clone was selected and expanded in DMEM medium containing 10% (w / w) FBS at 37°C and 5% (v / v) CO2. The optimal hybridoma cells were frozen in liquid nitrogen and used for subsequent antibody production and purification.

[0194] Example 2 Preparation and identification of chimeric antibodies

[0195] 2.1 Preparation of chimeric antibodies

[0196] The hybridoma clones with better activity in Example 1 were selected for sequencing. The sequences of the positive control anti-human CDH6 antibodies DS-H01L02 and NOV0712 were derived from patent WO2018212136A1. The corresponding sequence information is shown in Tables 3 and 4 below, where Table 3 shows the VH and VL sequences of the mouse monoclonal antibody, and Table 4 shows the CDRs of the mouse monoclonal antibody molecule (KABAT analysis).

[0197] Table 3 VH and VL sequences of murine monoclonal antibodies and positive control antibodies and constant region sequences of chimeric antibodies

[0198] Table 4 CDRs of mouse monoclonal antibody molecules and positive control antibodies (KABAT analysis)

[0199] Based on the above sequences, heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding the antibody VH and VL were recombined into an expression vector containing a signal peptide (SEQ ID NO: 132, MGWSWILLFLLSVTAGVHS) and heavy chain constant region / light chain constant region sequences to obtain recombinant plasmids expressing VH-CH1-Fc / VL-CL). The corresponding heavy chain and light chain plasmids of the antibody and the transfection reagent PEI (purchased from Polysciences, Catalog No.: 24765-1) were added to OPTI-ME M (purchased from Gibco, Catalog No.: 11058021), mixed, and allowed to stand for 15 minutes. The cells were then added to Expi293 cells (purchased from Thermofisher, Catalog No.: A14527) and cultured in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (purchased from Shanghai Aopuma, Catalog No.: F081918-001) and glucose (purchased from Sigma, Catalog No.: G7528) were added. On the sixth day of transfection, the cell supernatant was collected and purified with Protein A (purchased from GE, Catalog No.: 28985254). The eluted sample was dialyzed into PBS buffer (pH 7.4) to obtain the chimeric antibody and positive control antibody.

[0200] 2.2 Enzyme-linked immunosorbent assay (ELISA) detection of the binding of chimeric antibodies to human CDH6, monkey CDH6, and mouse CDH6 proteins

[0201] Human CDH6 protein (hCDH6 ECD-his, conventionally synthesized), monkey CDH6 protein (cynoCDH6 ECD-his, conventionally synthesized; the cynoCDH6 ECD sequence is derived from Uniprot #A0A2K5TW62 (19-615)), and mouse CDH6 protein (mCDH6 ECD-his, conventionally synthesized; the mCDH6 ECD sequence is derived from Uniprot #P97326 (19-615)) were diluted with PBS to a final concentration of 1 μg / mL and 100 μl was added to each well of a 96-well ELISA plate. The plates were incubated overnight at 4°C. The next day, the plates were washed three times with plate wash buffer (PBS + 0.01% (v / v) Tween 20) and blocked with blocking buffer (PBS + 0.01% (v / v) Tween 20 + 2% (w / w) BSA) at room temperature for 2 hours. The blocking buffer was discarded and the plates were washed three times. Chimeric antibodies were diluted in 1% BSA + PBS (Cytiva, Catalog No. SH30264.01), and 50 μl was added to each well of a 96-well plate. After incubation at 37°C for 2 hours, the plates were washed three times with plate washer buffer. A horseradish peroxidase-conjugated secondary antibody, Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson Immuno, Catalog No. 109-035-098), was diluted 1:5000 in 1% BSA and 50 μl was added to each well of the 96-well plate. After incubation at 37°C for 1 hour, the plates were washed five times with plate washer buffer. TMB substrate (Seracare, Catalog No. 5120-0077) (50 μl) was added to each well, and after incubation at room temperature for 10 minutes, 50 μl of stop solution (1.0 N HCl) was added to each well. OD 450 nm was read using a microplate reader (Biotek). GraphPad Prism software was used for data analysis and EC50 calculation. The ELISA results are shown in Table 5. The chimeric antibodies tested strongly bound to human CDH6 protein, monkey CDH6 protein, and mouse CDH6 protein.

[0202] Table 5 Binding activity of chimeric antibodies to human CDH6, monkey CDH6 and mouse CDH6 proteins Note: “ / ” indicates no binding or weak binding, and EC50 value cannot be fitted or EC50>10nM.

[0203] 2.3 Flow cytometry (FACS) assay to detect the binding of chimeric antibodies to CDH6-expressing tumor cells

[0204] The binding activity of the antibody to the cells was detected using OVCAR3 cells with high CDH6 expression and PA-1 cells with moderate CDH6 expression. OVCAR3 cells (purchased from ATCC, #HTB-161) or PA-1 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., #CBP60800) were cultured to 90% confluence, the culture medium was aspirated, and the cells were washed once with PBS. Then, the cells were treated and collected with enzyme-free cell dissociation solution (Versene, purchased from Gibco, product number 15040-066). The cells were washed once with PBS, counted, and resuspended in FACS buffer (PBS + 2% FBS) to 2×10 6 Cells / mL, 50 μl per well was added to a 96-well plate. Chimeric antibody was diluted with FACS buffer (PBS + 2% FBS), 50 μl per well was added to a 96-well plate, and incubated at 4°C for 1 hour. Washed 3 times with PBS, and secondary antibody Alexa Fluor was diluted 1:800 with FACS buffer. 647AffiniPure Goat Anti-Human IgG, Fcγfragment specific (purchased from Jackson Immuno, Cat. No. 109-605-098), 50 μl per well, was added to a 96-well plate and incubated at 4°C for 1 hour. Cells were washed twice with PBS by centrifugation and resuspended in FACS buffer. Mean fluorescence intensity (MFI) was measured using a FACS Canton II (BD Biosciences). GraphPad Prism software was used for analysis, data fitting, and EC50 values ​​were calculated. The FACS results are shown in Table 6. The chimeric antibody exhibited strong binding to both OVCAR3 and PA-1 cells.

[0205] Table 6 Binding activity of chimeric antibodies to OVCAR3 and PA-1 cells

[0206] 2.4 Endocytic activity detection of chimeric antibodies

[0207] OVCAR3 cells were cultured to 90% confluence, the medium was aspirated, and the cells were washed once with PBS. Then, the cells were treated and collected with enzyme-free cell dissociation solution (Versene, purchased from Gibco, product number 15040-066). The cells were washed once with PBS, counted, and resuspended in FACS buffer (PBS + 2% FBS) to 2 × 10 6Cells / mL, 50 μl per well was added to two 96-well plates. Chimeric antibodies were diluted to 10 μg / ml with FACS buffer (PBS + 2% FBS), and 50 μl per well was added to two 96-well plates, and incubated at 4°C for 1 hour. After washing the cells 3 times with PBS, FACS buffer was added to resuspend the cells. One 96-well plate was incubated at 4°C for 1 hour; the other 96-well plate was incubated at 37°C for 1 hour. After washing the cells 3 times with PBS, secondary antibody Alexa 647AffiniPure Goat Anti-Human IgG, Fcγfragment specific (purchased from Jackson Immuno, Cat. No. 109-605-098) was incubated at 4°C for 1 hour. Cells were washed twice with PBS by centrifugation and resuspended in FACS buffer. Mean fluorescence intensity (MFI) was measured using a FACS Canton II (BD Biosciences). The results are shown in Table 7. All tested chimeric antibodies exhibited excellent endocytic activity.

[0208] Table 7 Endocytic activity of chimeric antibodies

[0209] 2.5 Biacore detection of affinity between chimeric antibody and human CDH6 protein

[0210] The affinity of antibodies and antigens was determined by a multi-cycle kinetic method using a BIAcore 8K instrument. In each cycle, the antibody to be tested was first captured using a Protein A chip (purchased from GE, catalog number: 29-1275-56). Then the antigen human CDH6 protein (hCDH6-his, conventional synthesis) was injected, and the binding and dissociation processes of the antibody and antigen protein were recorded. Finally, the chip was regenerated with Glycine pH 1.5. The mobile phase was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 1 mM Ca), the flow rate was 30 μL / min, the association time was 240 s, the dissociation time was 300 s, the regeneration time was 30 s, the detection temperature was 25°C, and the antigen concentration ranged from 31.25 to 1000 nM. The data were analyzed using Biacore 8K analysis software (version 2.0) based on a 1:1 binding model, and the antibody-antigen binding kinetic parameters, including the association rate constant ka, the dissociation rate constant kd, and the equilibrium dissociation constant KD, were fitted. The results are shown in Table 8. The affinity value of the control antibody DS-H01L02 was undetectable. Antibodies not shown in the table have undetectable affinity values.

[0211] Table 8 Affinity of chimeric antibodies to human CDH6 protein Note: “ / ” indicates that the value cannot be detected.

[0212] Example 3 Antibody Humanization

[0213] By comparing the IMGT (http: / / imgt.cines.fr) database of human antibody heavy and light chain variable region germline genes, we selected heavy and light chain variable region germline genes with high homology to mouse antibodies as templates. The CDRs of the mouse antibodies, as delineated according to Kabat, were transplanted into the corresponding human templates, resulting in a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the antibody's three-dimensional structure, we backmutated buried residues, residues that directly interact with the CDR regions, and residues in the framework regions that significantly influence VL and VH conformations, resulting in humanized monoclonal antibodies.

[0214] 3.1 Humanization of CDH6-1

[0215] The humanized light chain templates for the murine antibody CDH6-1 are IGKV4-1*01 / IGKV1-33*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01. The CDRs of the murine antibody CDH6-1 were transplanted into their humanized templates, leaving the CDR sequences unchanged, to create the corresponding humanized antibody hCDH6-1. As needed, key amino acids in the FR region sequence of the humanized hCDH6-1 antibody were backmutated to their murine counterparts to maintain the original affinity. Detailed backmutation designs are shown in Table 9.

[0216] Table 9 Back mutation design of humanized antibody of hCDH6-1

[0217] Note: Graft represents the grafting of mouse antibody CDRs into the human germline template FR sequence; G72E indicates the mutation of G at position 72 of Graft to E, and so on. The backmutated amino acids are numbered in natural order.

[0218] The specific sequence of the hCDH6-1 humanized antibody variable region is as follows (mutations are shown in bold):

[0219] The amino acid sequence of hCDH6-1.VL1 is shown in SEQ ID NO: 133:

[0220] The amino acid sequence of hCDH6-1.VL2 is shown in SEQ ID NO: 134:

[0221] The amino acid sequence of hCDH6-1.VL3 is shown in SEQ ID NO: 135:

[0222] The amino acid sequence of hCDH6-1.VH1 is shown in SEQ ID NO: 136:

[0223] The amino acid sequence of hCDH6-1.VH2 is shown in SEQ ID NO: 137:

[0224] The amino acid sequence of hCDH6-13.VH3 is shown in SEQ ID NO: 138:

[0225] The amino acid sequence of hCDH6-1.VH4 is shown in SEQ ID NO: 139:

[0226] The amino acid sequence of the human germline light chain template IGKV4-1*01 is shown in SEQ ID NO: 140:

[0227] The amino acid sequence of the human germline light chain template IGKV1-33*01 is shown in SEQ ID NO: 141:

[0228] The amino acid sequence of the human germline light chain template IGKJ4*01 is shown in SEQ ID NO: 142:

[0229] The amino acid sequence of the human germline heavy chain template IGHV1-69*02 is shown in SEQ ID NO: 143:

[0230] The amino acid sequence of the human germline heavy chain template IGHJ6*01 is shown in SEQ ID NO: 144:

[0231] From the above-mentioned backmutation designs of the light and heavy chain variable regions of the hCDH6-1 humanized antibody, different light and heavy chain sequences were selected for cross-combination to ultimately obtain multiple hCDH6-1 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0232] Table 10 Amino acid sequences corresponding to the variable regions of hCDH6-1 antibody

[0233] Based on the above sequences, heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding the antibody VH and VL were recombined into an expression vector carrying a signal peptide (SEQ ID NO: 132, MGWSWILLFLLSVTAGVHS) and heavy chain constant region / light chain constant region sequences to obtain a recombinant plasmid expressing VH-CH1-Fc / VL-CL). Following the expression and purification method described in Example 2, the hCDH6-1 humanized antibody was obtained, and the binding activity of the humanized antibody was assayed by ELISA and FACS (see 2.2 and 2.3 for methods). The results are shown in Table 11 and Figures 1-4 . The binding activity of the hCDH6-1 humanized antibody to human CDH6 protein and OVCAR3 cells was comparable to that of the chimeric antibody.

[0234] Table 11 Binding of hCDH6-1 humanized antibody to human CDH6 protein and OVCAR3 cells

[0235] Four hCDH6-1 humanized antibodies with fewer back mutations were selected, and their affinity to human CDH6 protein was further confirmed using Biacore. The results are shown in Table 12. The selected hCDH6-1 humanized antibodies had an affinity comparable to that of the chimeric antibody.

[0236] Table 12 Affinity of hCDH6-1 humanized antibody to human CDH6 protein

[0237] 3.2 Humanization of CDH6-6

[0238] The humanized light chain templates for the murine antibody CDH6-6 are IGKV1-NL1*01 / IGKV2-28*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-46*01 and IGHJ6*01. The CDRs of the murine antibody CDH6-6 were transplanted into their humanized templates, leaving the CDR sequences unchanged, to produce the corresponding humanized version, hCDH6-6. As needed, key amino acids in the FR region of the humanized hCDH6-6 antibody were backmutated to their murine counterparts to maintain the original affinity. Detailed backmutation designs are shown in Table 13.

[0239] Table 13 Design of humanized antibody back mutation of hCDH6-6 Note: Graft represents the grafting of mouse antibody CDRs into the human germline template FR sequence; A43S indicates the mutation of A to S at position 43 of Graft, and so on. The backmutated amino acids are numbered in natural order.

[0240] The specific sequence of the hCDH6-6 humanized antibody variable region is as follows (mutations are shown in bold):

[0241] The amino acid sequence of hCDH6-6.VL1 is shown in SEQ ID NO: 145:

[0242] The amino acid sequence of hCDH6-6.VL2 is shown in SEQ ID NO: 146:

[0243] The amino acid sequence of hCDH6-6.VL3 is shown in SEQ ID NO: 147:

[0244] The amino acid sequence of hCDH6-6.VL4 is shown in SEQ ID NO: 148:

[0245] The amino acid sequence of hCDH6-6.VL5 is shown in SEQ ID NO: 149:

[0246] The amino acid sequence of hCDH6-6.VH1 is shown in SEQ ID NO: 150:

[0247] The amino acid sequence of hCDH6-6.VH2 is shown in SEQ ID NO: 151:

[0248] The amino acid sequence of hCDH6-6.VH3 is shown in SEQ ID NO: 152:

[0249] The amino acid sequence of the human germline light chain template IGKV1-NL1*01 is shown in SEQ ID NO: 153:

[0250] The amino acid sequence of the human germline light chain template IGKV2-28*01 is shown in SEQ ID NO: 154:

[0251] The amino acid sequence of the human germline light chain template IGKJ2*01 is shown in SEQ ID NO: 155:

[0252] The amino acid sequence of the human germline heavy chain template IGHV1-46*01 is shown in SEQ ID NO: 156:

[0253] The amino acid sequence of the human germline heavy chain template IGHJ6*01 is shown in SEQ ID NO: 144:

[0254] From the above-mentioned backmutation designs of the light chain and heavy chain variable regions of the hCDH6-6 humanized antibody, different light chain and heavy chain sequences were selected for cross-combination, ultimately obtaining a variety of hCDH6-6 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0255] Table 14 Amino acid sequences corresponding to the variable regions of hCDH6-6 antibody

[0256] Based on the above sequences, heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding the antibody VH and VL were recombined into an expression vector carrying a signal peptide (SEQ ID NO: 132, MGWSWILLFLLSVTAGVHS) and heavy chain constant region / light chain constant region sequences to obtain a recombinant plasmid expressing VH-CH1-Fc / VL-CL). Following the expression and purification method described in Example 2, the hCDH6-6 humanized antibody was obtained, and the binding activity of the humanized antibody was assayed by ELISA and FACS (see 2.2 and 2.3 for methods). The results are shown in Table 15 and Figures 5-8 . The binding activity of the hCDH6-6 humanized antibody to human CDH6 protein and OVCAR3 cells was comparable to that of the chimeric antibody.

[0257] Table 15 Binding of hCDH6-6 humanized antibody to human CDH6 protein and OVCAR3 cells

[0258] Four hCDH6-6 humanized antibodies with fewer back mutations were selected, and their affinity to human CDH6 protein was further confirmed using Biacore. The results are shown in Table 16. The affinity of the four selected hCDH6-6 humanized antibodies was comparable to that of the chimeric antibody.

[0259] Table 16 Affinity of hCDH6-6 humanized antibody to human CDH6 protein

[0260] 3.3 Humanization of CDH6-14

[0261] The humanized light chain templates for the murine antibody CDH6-14 are IGKV1-33*01 / IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDRs of the murine antibody CDH6-14 were transplanted into their humanized templates, leaving the CDR sequences unchanged, to create the corresponding humanized version, hCDH6-14. As needed, key amino acids in the FR region sequence of the humanized hCDH6-14 antibody were backmutated to their murine counterparts to maintain the original affinity. Detailed backmutation designs are shown in Table 17.

[0262] Table 17 Humanized antibody backmutation design of hCDH6-14 Note: Graft represents the grafting of murine antibody CDRs into the human germline template FR sequence; P44F indicates the mutation of P at position 44 of Graft to F, and so on. The backmutated amino acids are numbered in natural order.

[0263] The specific sequence of the hCDH6-14 humanized antibody variable region is as follows (mutations are shown in bold):

[0264] The amino acid sequence of hCDH6-14.VL1 is shown in SEQ ID NO: 157:

[0265] The amino acid sequence of hCDH6-14.VL2 is shown in SEQ ID NO: 158:

[0266] The amino acid sequence of hCDH6-14.VL3 is shown in SEQ ID NO: 159:

[0267] The amino acid sequence of hCDH6-14.VH1 is shown in SEQ ID NO: 160:

[0268] The amino acid sequence of hCDH6-14.VH2 is shown in SEQ ID NO: 161:

[0269] The amino acid sequence of hCDH6-14.VH3 is shown in SEQ ID NO: 162:

[0270] The amino acid sequence of hCDH6-14.VH4 is shown in SEQ ID NO: 163:

[0271] The amino acid sequence of hCDH6-14.VH5 is shown in SEQ ID NO: 164:

[0272] The amino acid sequence of hCDH6-14.VH6 is shown in SEQ ID NO: 165:

[0273] The amino acid sequence of the human germline light chain template IGKV1-33*01 is shown in SEQ ID NO: 141:

[0274] The amino acid sequence of the human germline light chain template IGKV4-1*01 is shown in SEQ ID NO: 140:

[0275] The amino acid sequence of the human germline light chain template IGKJ4*01 is shown in SEQ ID NO: 142:

[0276] The amino acid sequence of the human germline heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 166:

[0277] The amino acid sequence of the human germline heavy chain template IGHJ6*01 is shown in SEQ ID NO: 144:

[0278] From the backmutation designs of the light and heavy chain variable regions of the hCDH6-14 humanized antibody, different light and heavy chain sequences were selected for cross-combination, ultimately obtaining multiple hCDH6-14 humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0279] Table 18 Amino acid sequences corresponding to the variable regions of hCDH6-14 antibody

[0280] Based on the above sequences, heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding the antibody VH and VL were recombined into an expression vector carrying a signal peptide (SEQ ID NO: 132, MGWSWILLFLLSVTAGVHS) and heavy chain constant region / light chain constant region sequences to obtain a recombinant plasmid expressing VH-CH1-Fc / VL-CL). Following the expression and purification method described in Example 2, the humanized hCDH6-14 antibody was obtained, and the binding activity of the humanized antibody was assayed by ELISA and FACS (see 2.2 and 2.3 for methods). The results are shown in Table 19 and Figures 9-13 . The binding activity of the hCDH6-14 humanized antibody to human CDH6 protein and PA-1 cells was comparable to that of the chimeric antibody.

[0281] Table 19 Binding of hCDH6-14 humanized antibody to human CDH6 protein and PA-1 cells

[0282] 3.4 Endocytosis activity detection of humanized antibodies

[0283] Some antibodies were selected for endocytosis activity testing. The endocytosis activity testing method of humanized antibodies in OVCAR3 cells was the same as 2.4. The results are shown in Table 20. All humanized antibodies tested had good endocytosis activity.

[0284] Table 20 Endocytic activity of humanized antibodies

[0285] Example 4 Epitope Identification of CDH6 Antibody

[0286] The full-length human CDH6 protein consists of 790 amino acids (SEQ ID NO: 167), of which the extracellular region includes EC1 (SEQ ID NO: 168), EC2 (SEQ ID NO: 169), EC3 (SEQ ID NO: 170), EC4 (SEQ ID NO: 171), and EC5 (SEQ ID NO: 172). To confirm the binding epitope of the CDH6 antibody, the sequences after removing these five regions were constructed into the pcDNA3.1 vector (purchased from Youbao Bio, product number VT1001) and transfected into HEK293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences, product number SCSP-502), obtaining five CDH6 truncation cell lines: HEK293T-hCDH6-EC1deletion, HEK293T-hCDH6-EC2deletion, HEK293T-hCDH6-EC3deletion, HEK293T-hCDH6-EC4deletion and HEK293T-hCDH6-EC5deletion.

[0287] Human CDH6 protein full-length sequence (Uniprot#P55285) SEQ ID NO: 167

[0288] EC1 SEQ ID NO:168

[0289] EC2 SEQ ID NO:169

[0290] EC3 SEQ ID NO:170

[0291] EC4 SEQ ID NO:171

[0292] EC5 SEQ ID NO:172

[0293] The binding activity of CDH6 chimeric antibodies to these five cell lines was detected by FACS. The results are shown in Table 21. Based on the test results, the binding epitopes of the antibodies can be inferred: CHI-CDH6-1 and CHI-CDH6-7 do not bind to EC1-deletion cells, indicating that their binding epitope is EC1; CHI-CDH6-5 does not bind to EC1-deletion cells and binds very weakly to EC2-deletion cells, indicating that its binding epitope may be between EC1 and EC2; CHI-CDH6-4 does not bind to EC2-deletion cells, indicating that its binding epitope is EC2; CHI-CDH6-14 binds very weakly to EC2-deletion and EC3-deletion cells, indicating that its binding epitope may be between EC2 and EC3; CHI-CDH6-3 binds very weakly to EC3-deletion cells, indicating that its binding epitope is EC3; CHI-CDH6-12 does not bind to EC3-deletion cells, indicating that its binding epitope is EC3.

[0294] Table 21 Binding epitopes of CDH6 chimeric antibodies to human CDH6 Note: MFI higher than 1.5 times the MFI of negative control hIgG1 is considered to be binding.

Claims

1. An antibody or antigen-binding fragment that specifically binds to CDH6, wherein the antibody or antigen-binding fragment specifically binds to one or more amino acid sequence fragments in the EC1 region, EC1-EC2 region, EC2 region, EC2-EC3 region, EC3 region, EC4 region or EC5 region of the extracellular region of the CDH6 protein; preferably, the antibody or antigen-binding fragment specifically binds to one or more amino acid sequence fragments shown in any one of SEQ ID NOs: 168-172; preferably, the antibody or antigen-binding fragment has endocytosis activity.

2. An antibody or antigen-binding fragment that specifically binds to CDH6, It is characterized in that The antibody or antigen-binding fragment comprises: (a) HCDR1, HCDR2 and HCDR3 of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 136, 137, 138, 139, 150, 151, 152, 160, 161, 162, 163, 164 or 165; and / or, (b) LCDR1, LCDR2 and LCDR3 of any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 133, 134, 135, 145, 146, 147, 148, 149, 157, 158 or 159; Preferably, the HCDR1-3 and / or the LCDR1-3 are encoded according to the popular analysis method of KABAT, IMGT or Chothia.

3. The antibody or antigen-binding fragment according to claim 2, It is characterized in that The HCDR1, HCDR2 and HCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination: and, The LCDR1, LCDR2 and LCDR3 have a sequence combination selected from any of the following sequence combinations or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination: Preferably, the substitution is a conservative amino acid substitution.

4. The antibody or antigen-binding fragment according to claim 3, It is characterized in that It comprises a heavy chain CDRs and light chain CDRs combination selected from the following: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5, VH6+VL6, VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, or VH14+VL14, and a CDRs combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence of the heavy chain CDRs and light chain CDRs combination; Preferably, the substitution is a conservative amino acid substitution.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, It is characterized in that The framework regions of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment are derived from a human germline heavy chain template and a human germline light chain template, wherein: (1) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-69*02 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-69*02 as shown in SEQ ID NO: 143 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 144; (2) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-46*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-46*01 as shown in SEQ ID NO: 156 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 144; (3) The framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-3*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-3*01 as shown in SEQ ID NO: 166 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 144; (4) The framework region sequence is derived from the combined sequence of human germline light chain IGKV4-1*01 and IGKJ4*01; it comprises the FR1, FR2, and FR3 regions of IGKV4-1*01 as shown in SEQ ID NO: 140 and the FR4 region of IGKJ4*01 as shown in SEQ ID NO: 142; (5) The framework region sequence is derived from the combined sequence of human germline light chain IGKV1-33*01 and IGKJ4*01; it comprises the FR1, FR2, and FR3 regions of IGKV1-33*01 as shown in SEQ ID NO: 141 and the FR4 region of IGKJ4*01 as shown in SEQ ID NO: 142; (6) The framework sequence is derived from the combined sequence of human germline light chain IGKV1-NL1*01 and IGKJ2*01; it comprises the FR1, FR2, and FR3 regions of IGKV1-NL1*01 as shown in SEQ ID NO: 153 and the FR4 region of IGKJ2*01 as shown in SEQ ID NO: 155; or, (7) The framework region sequence is derived from the combined sequence of human germline light chains IGKV2-28*01 and IGKJ2*01; it includes the FR1, FR2, and FR3 regions of IGKV2-28*01 shown in SEQ ID NO: 154 and the FR4 region of IGKJ2*01 shown in SEQ ID NO:

155.

6. The antibody or antigen-binding fragment according to claim 5, It is characterized in that According to the Kabat numbering system, the framework regions of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment further include one or more mutations selected from the following group, wherein: (1) the framework region of the heavy chain variable region comprises: G27Y, S30T, A40R, I70L or A72V; preferably comprises G27Y and A72V; or preferably comprises G27Y, S30T and A72V; or preferably comprises G27Y, S30T, I70L and A72V; or preferably comprises G27Y, S30T, A40R and A72V; (2) The framework region of the heavy chain variable region includes: G42R, M70L, R72V or T74K; preferably includes R72V and T74K; or preferably including M70L, R72V and T74K; or preferably including G42R, R72V and T74K; (3) the framework region of the heavy chain variable region includes: V2I, V5Q, R44G, I70L, R72V, Y95L or R98S; preferably includes R72V and R98S; or preferably includes V2I, R72V and R98S; or preferably includes V2I, I70L, R72V and R98S; or preferably includes R44G, R72V and R98S; or preferably includes V5Q, R72V and R98S; or preferably includes R72V, Y95L and R98S; (4) the framework region of the light chain variable region includes: P44S, A47P or G72E; preferably includes G72E; or preferably includes P44S and G72E; or preferably includes A47P and G72E; (5) the framework region of the light chain variable region includes: Q42K, A43S, K45Q, L48V, I48V or T85R; preferably includes A43S; or preferably includes A43S and L48V; or preferably includes A43S, K45Q and L48V; or preferably includes A43S, L48V and T85R; or preferably includes Q42K and I48V; or, (6) The framework region of the light chain variable region includes: K42G, P43T, P44F or Y49S; preferably includes P44F and Y49S; or preferably includes K42G and Y49S; or preferably includes P43T and Y49S.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, It is characterized in that The antibody or antigen-binding fragment comprises: (1) the heavy chain variable region has the sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 136, 137, 138, 139, 150, 151, 152, 160, 161, 162, 163, 164 or 165; (2) the light chain variable region has the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 133, 134, 135, 145, 146, 147, 148, 149, 157, 158 or 159; (3) An amino acid sequence that is at least 90% identical to the sequence of any one of (1) to (2) above, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

8. The antibody or antigen-binding fragment according to claim 7, It is characterized in that The heavy chain variable region and the light chain variable region are selected from the following groups: (1) having VH shown in SEQ ID NO.2 and VL shown in SEQ ID NO.3; (2) having VH shown in SEQ ID NO.4 and VL shown in SEQ ID NO.5; (3) having VH shown in SEQ ID NO.6 and VL shown in SEQ ID NO.7; (4) having VH shown in SEQ ID NO.8 and VL shown in SEQ ID NO.9; (5) having VH shown in SEQ ID NO.10 and VL shown in SEQ ID NO.11; (6) having VH shown in SEQ ID NO.12 and VL shown in SEQ ID NO.13; (7) having VH shown in SEQ ID NO.14 and VL shown in SEQ ID NO.15; (8) having VH shown in SEQ ID NO.16 and VL shown in SEQ ID NO.17; (9) having VH shown in SEQ ID NO.18 and VL shown in SEQ ID NO.19; (10) having VH shown in SEQ ID NO.20 and VL shown in SEQ ID NO.21; (11) having VH shown in SEQ ID NO.22 and VL shown in SEQ ID NO.23; (12) having VH shown in SEQ ID NO.24 and VL shown in SEQ ID NO.25; (13) having VH shown in SEQ ID NO.26 and VL shown in SEQ ID NO.27; (14) having VH shown in SEQ ID NO.28 and VL shown in SEQ ID NO.29; (15) having VH shown in SEQ ID NO.136 and VL shown in SEQ ID NO.133; (16) having VH shown in SEQ ID NO.137 and VL shown in SEQ ID NO.133; (17) having VH shown in SEQ ID NO.138 and VL shown in SEQ ID NO.133; (18) having VH shown in SEQ ID NO.139 and VL shown in SEQ ID NO.133; (19) having VH shown in SEQ ID NO.136 and VL shown in SEQ ID NO.134; (20) having VH shown in SEQ ID NO.137 and VL shown in SEQ ID NO.134; (21) having VH shown in SEQ ID NO.138 and VL shown in SEQ ID NO.134; (22) having VH shown in SEQ ID NO.139 and VL shown in SEQ ID NO.134; (23) having VH shown in SEQ ID NO.136 and VL shown in SEQ ID NO.135; (24) having VH shown in SEQ ID NO.137 and VL shown in SEQ ID NO.135; (25) having VH shown in SEQ ID NO.138 and VL shown in SEQ ID NO.135; (26) having VH shown in SEQ ID NO.139 and VL shown in SEQ ID NO.135; (27) having VH shown in SEQ ID NO.150 and VL shown in SEQ ID NO.145; (28) having VH shown in SEQ ID NO.150 and VL shown in SEQ ID NO.146; (29) having VH shown in SEQ ID NO.150 and VL shown in SEQ ID NO.147; (30) having VH shown in SEQ ID NO.150 and VL shown in SEQ ID NO.148; (31) having VH shown in SEQ ID NO.150 and VL shown in SEQ ID NO.149; (32) having VH shown in SEQ ID NO.151 and VL shown in SEQ ID NO.145; (33) having VH shown in SEQ ID NO.151 and VL shown in SEQ ID NO.146; (34) having VH shown in SEQ ID NO.151 and VL shown in SEQ ID NO.147; (35) having VH shown in SEQ ID NO.151 and VL shown in SEQ ID NO.148; (36) having VH shown in SEQ ID NO.151 and VL shown in SEQ ID NO.149; (37) having VH shown in SEQ ID NO.152 and VL shown in SEQ ID NO.145; (38) having VH shown in SEQ ID NO.152 and VL shown in SEQ ID NO.146; (39) having VH shown in SEQ ID NO.152 and VL shown in SEQ ID NO.147; (40) having VH shown in SEQ ID NO.152 and VL shown in SEQ ID NO.148; (41) having VH shown in SEQ ID NO.152 and VL shown in SEQ ID NO.149; (42) having VH shown in SEQ ID NO.160 and VL shown in SEQ ID NO.157; (43) having VH shown in SEQ ID NO.160 and VL shown in SEQ ID NO.158; (44) having VH shown in SEQ ID NO.160 and VL shown in SEQ ID NO.159; (45) having VH shown in SEQ ID NO.161 and VL shown in SEQ ID NO.157; (46) having VH shown in SEQ ID NO.161 and VL shown in SEQ ID NO.158; (47) having VH shown in SEQ ID NO.161 and VL shown in SEQ ID NO.159; (48) having VH shown in SEQ ID NO.162 and VL shown in SEQ ID NO.157; (49) having VH shown in SEQ ID NO.162 and VL shown in SEQ ID NO.158; (50) having VH shown in SEQ ID NO.162 and VL shown in SEQ ID NO.159; (51) having VH shown in SEQ ID NO.163 and VL shown in SEQ ID NO.157; (52) having VH shown in SEQ ID NO.163 and VL shown in SEQ ID NO.158; (53) having VH shown in SEQ ID NO.163 and VL shown in SEQ ID NO.159; (54) having VH shown in SEQ ID NO.164 and VL shown in SEQ ID NO.157; (55) having VH shown in SEQ ID NO.164 and VL shown in SEQ ID NO.158; (56) having VH shown in SEQ ID NO.164 and VL shown in SEQ ID NO.159; (57) having VH shown in SEQ ID NO.165 and VL shown in SEQ ID NO.157; (58) having VH shown in SEQ ID NO.165 and VL shown in SEQ ID NO.158; (59) having VH shown in SEQ ID NO.165 and VL shown in SEQ ID NO.159; or, (60) A VH and VL combination having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to any of the sequence combinations of (1) to (59) above.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, It is characterized in that The dissociation constant (KD) of its binding to human CDH6 protein is no more than 2×10 -7 M.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, It is characterized in that The antibody or antigen-binding fragment is: (1) Chimeric antibodies or fragments thereof; (2) humanized antibodies or fragments thereof; (3) fully human antibodies or fragments thereof; Preferably, the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

11. The antibody or antigen-binding fragment according to any one of claims 1 to 10, It is characterized in that The antibody comprises the sequence of any one of the constant regions of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably comprises the sequence of the constant region of human or mouse antibody IgG1, IgG2, IgG3 or IgG4.

12. The antibody or antigen-binding fragment according to any one of claims 1 to 11, It is characterized in that The antigen binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimum recognition unit.

13. The antibody or antigen-binding fragment according to any one of claims 1 to 12, It is characterized in that The antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioactive isotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent or a photosensitizer.

14. A multispecific antigen-binding molecule, It is characterized in that The multispecific antigen-binding molecule comprises a first antigen-binding moiety and a second antigen-binding moiety, wherein the first antigen-binding moiety comprises the antibody or antigen-binding fragment according to any one of claims 1 to 13, and the second antigen-binding moiety specifically binds to an antigen other than CDH6 or binds to a CDH6 antigen epitope different from that of the first antigen-binding moiety; Preferably, the other antigens are selected from CD3, CD7, CD16, CD16A, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD66 (ad), CD74, CD80, CD126, CD138, BCMA, HLA-DR, HER2, VEGF, P1GF, HER3 / ERBB3, HER4 / ERBB4, IL-2, IL-6, PD-1, PD-L1, TRAIL-R1 or TRAIL-R2; Preferably, the multispecific antigen-binding molecule is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

15. A chimeric antigen receptor (CAR), It is characterized in that The chimeric antigen receptor comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises the CDH6 antibody or antigen-binding fragment according to any one of claims 1-13.

16. An immune effector cell, It is characterized in that The immune effector cell comprises the chimeric antigen receptor according to claim 15 or comprises a nucleic acid fragment encoding the chimeric antigen receptor according to claim 15; Preferably, the immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells; the T cells can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Treg) or helper T cells; Preferably, the immune effector cells are allogeneic immune effector cells or autologous immune cells.

17. An isolated nucleic acid molecule, It is characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment according to any one of claims 1 to 13, or any combination thereof, the multispecific antigen-binding molecule according to claim 14, or the chimeric antigen receptor according to claim 15.

18. An expression vector comprising the isolated nucleic acid molecule according to claim 17.

19. An isolated host cell comprising the isolated nucleic acid molecule according to claim 17 or the expression vector according to claim 18; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

20. A method for preparing the antibody or antigen-binding fragment according to any one of claims 1 to 13 or the multispecific antigen-binding molecule according to claim 14, It is characterized in that The host cell of claim 19 is cultured under appropriate conditions, and the antibody or antigen-binding fragment or multispecific antigen-binding molecule is isolated.

21. A method for preparing the immune effector cell according to claim 16, It is characterized in that The method comprises introducing a nucleic acid fragment encoding the chimeric antigen receptor according to claim 15 into an immune effector cell. Optionally, the method further comprises activating the immune effector cell to express the chimeric antigen receptor according to claim 15.

22. A pharmaceutical composition, It is characterized in that The composition comprises the antibody or antigen-binding fragment according to any one of claims 1-13, the multispecific antigen-binding molecule according to claim 14, the chimeric antigen receptor according to claim 15, the immune effector cell according to claim 16, the isolated nucleic acid molecule according to claim 17, the expression vector according to claim 18, the cell according to claim 19, or the product prepared by the method according to claim 20 or 21; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.

23. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 13, the multispecific antigen-binding molecule according to claim 14, the chimeric antigen receptor according to claim 15, the immune effector cell according to claim 16, the isolated nucleic acid molecule according to claim 17, the expression vector according to claim 18, the cell according to claim 19, the product prepared by the method according to claim 20 or 21, or the pharmaceutical composition according to claim 22 in the preparation of a medicament for preventing and / or treating a tumor disease; Preferably, the tumor disease is selected from solid tumors, preferably, the solid tumor is a solid tumor expressing CDH6 protein, more preferably, the solid tumor is selected from renal cancer, ovarian cancer, thyroid cancer, bile duct cancer, lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, bile duct cancer, Wilms tumor or neuroblastoma.

24. A method for preventing and / or treating a tumor disease, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment according to any one of claims 1 to 13, the multispecific antigen-binding molecule according to claim 14, the chimeric antigen receptor according to claim 15, the immune effector cell according to claim 16, the isolated nucleic acid molecule according to claim 17, the expression vector according to claim 18, the cell according to claim 19, the product prepared by the method according to claim 20 or 21, or the pharmaceutical composition according to claim 22; Preferably, the tumor disease is selected from solid tumors. Preferably, the solid tumor is a solid tumor expressing CDH6 protein. More preferably, the solid tumor is selected from renal cancer, ovarian cancer, thyroid cancer, bile duct cancer, lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, bile duct cancer, Wilms' tumor or neuroblastoma.

25. The antibody or antigen-binding fragment of any one of claims 1-13, the multispecific antigen-binding molecule of claim 14, the chimeric antigen receptor of claim 15, the immune effector cell of claim 16, the isolated nucleic acid molecule of claim 17, the expression vector of claim 18, the cell of claim 19, the product prepared by the method of claim 20 or 21, or the pharmaceutical composition of claim 22, It is characterized in that For use and / or treatment of neoplastic diseases; Preferably, the tumor disease is selected from solid tumors, preferably, the solid tumor is a solid tumor expressing CDH6 protein, more preferably, the solid tumor is selected from renal cancer, ovarian cancer, thyroid cancer, bile duct cancer, lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, bile duct cancer, Wilms tumor or neuroblastoma.

26. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, a multispecific antigen-binding molecule according to claim 14, a chimeric antigen receptor according to claim 15, an immune effector cell according to claim 16, an isolated nucleic acid molecule according to claim 17, an expression vector according to claim 18, a cell according to claim 19, a product prepared by the method according to claim 20 or 21, or a pharmaceutical composition according to claim 22; optionally, further comprising instructions for use.

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