Antigen-binding molecule, drug conjugate thereof and medical use thereof

Abstract

Provided are an anti-Cadherin6 antibody, a drug conjugate thereof, and a preparation method therefor. The anti-Cadherin6 antibody and the antibody-drug conjugate thereof can be used for treating diseases or disorders, such as cancers.

Description

Antigen binding molecules, drug conjugates thereof and medical uses thereof Technical Field

[0001] The present disclosure relates to anti-CDH6 antibodies, drug conjugates thereof, and preparation methods thereof. The anti-CDH6 antibodies and antibody-drug conjugates thereof can be used to prepare drugs for treating diseases or conditions. Background Art

[0002] Cadherins are glycoproteins on the cell membrane surface that play a role in cell-cell adhesion through calcium-dependent binding of the N-terminal extracellular domain. Classical cadherins are divided into type I cadherins and type II cadherins, represented by E-cadherin and N-cadherin, based on amino acid sequence homology. Human CDH6 (also known as Cadherin6, K cadherin) is a member of the type II cadherin family. It is a single transmembrane protein containing 790 amino acids, including five N-terminal extracellular domains from EC1 to EC5, a transmembrane domain, and a C-terminal intracellular domain. CDH6 is highly expressed in renal and ovarian cancers, and its expression in normal tissues is relatively limited, mainly in the kidneys, and at a lower level in the bile duct, with a small amount of expression in the brain and respiratory system. CDH6 has been reported to have a certain degree of heterogeneity in the expression of PDX models of renal and ovarian cancer (Cancer Discov. 2017; 7(9): 1030-1045.). CDH6 is a tumor-associated antigen (TAA) target, and its expression is negatively correlated with tumor prognosis (Cancer Cell Int. 2021; 21(1): 493).

[0003] The Cadherin family is involved in maintaining intercellular spaces and polarity, and plays a regulatory role in tissue development and EMT. The RGD motif in EC1 of CDH6 is crucial for interaction with α2β1 integrin and activation of the posterior integrin pathway in cancer metastatic cells, while the HAV motif in EC5 is associated with stability. Cadherin has a certain degree of endocytic activity, and P120 can regulate its endocytic activity. There are a few reports on the role of CDH6 in tumor development. It can interact with downstream signaling proteins to promote tumor EMT and mediate tumor proliferation (Oncogene. 2017; 36(5): 667-677.).

[0004] There are currently no anti-CDH6 antibody-drug conjugates on the market. The present disclosure provides anti-human CDH6 antibodies with high affinity activity and high endocytosis activity, and in particular provides antibodies targeting the human CDH6 EC1 or EC3 domains. Antibodies targeting EC1 are not yet available in the prior art. The anti-CDH6 antibodies disclosed herein can be coupled with toxins to form ADCs, such as inhibitors of topoisomerase I, for the treatment of various tumors with high CDH6 expression. The anti-CDH6 antibodies and anti-CDH6 antibody-drug conjugates disclosed herein have the characteristics of high endocytosis activity, high tumor inhibitory activity, good stability, and good drugability, and have high potential for clinical treatment of tumors. Summary of the Invention

[0005] The present disclosure provides CDH6-binding molecules, antibody-drug conjugates, and medical uses and preparation methods, as well as pharmaceutical compositions comprising the CDH6-binding molecules and antibody-drug conjugates, and methods for treating and preventing diseases.

[0006] CDH6 binding molecules

[0007] I:

[0008] In some embodiments, the present disclosure provides CDH6 binding molecules that specifically bind to the EC1 domain of CDH6. The amino acid sequence of the EC1 is, for example, as shown in SEQ ID NO: 71. In some embodiments, the binding molecule has internalization ability that allows cellular uptake. In some embodiments, the binding molecule that specifically binds to the EC1 domain of CDH6 has a stronger cellular internalization ability than the binding molecule that specifically binds to the EC1 domain of CDH6.

[0009] In some embodiments, the present disclosure provides CDH6-binding molecules comprising a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and / or HCDR3 of the amino acid sequence shown in SEQ ID NO: 1, 17, 62, or 63, and the VL comprises LCDR1, LCDR2, and / or LCDR3 of the amino acid sequence shown in SEQ ID NO: 2, 18, 64, or 65, and the CDRs are defined according to Kabat, IMGT, Chothia, AbM, or Contact numbering conventions. In some specific embodiments, the CDRs are defined according to the Kabat numbering convention.

[0010] In some embodiments, a CDH6 binding molecule is provided, comprising a VH and / or a VL, wherein:

[0011] 1-1) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 2;

[0012] 1-2) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18;

[0013] 1-3) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 62, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 64 or 65; or,

[0014] 1-4) The VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 64 or 65.

[0015] In some embodiments, a CDH6 binding molecule is provided, which comprises VH and / or VL, wherein the VH comprises HCDR1, HCDR2 and / or HCDR3, wherein HCDR1, HCDR2, HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 5, 6, and 7, respectively; and the VL comprises LCDR1, LCDR2 and / or LCDR3, wherein LCDR1, LCDR2, LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 24, 25, and 10, respectively.

[0016] In some embodiments, a CDH6 binding molecule is provided, which comprises VH and / or VL, wherein the VH comprises HCDR1, HCDR2 and / or HCDR3, wherein HCDR1, HCDR2, HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 5, 6, and 7, respectively; and the VL comprises LCDR1, LCDR2 and / or LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 8 or 21, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9 or 22, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 10.

[0017] In some embodiments, a CDH6-binding molecule is provided, comprising a VH and / or a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein:

[0018] 1-1) LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively;

[0019] 1-2) LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 21, 22, and 10, respectively.

[0020] In some embodiments, the CDRs in the present disclosure may also be CDR sequences defined by other numbering systems. The following examples include HCDR1, HCDR2, and HCDR3 in the VH shown in SEQ ID NO: 17, and LCDR1, LCDR2, and LCDR3 in the VL shown in SEQ ID NO: 18, as defined by the IMGT, Chothia, AbM, or Contact numbering systems.

[0021] Exemplarily, the CDR sequences defined by the IMGT numbering system are as follows:

[0022] HCDR1:GYTFTNYW(SEQ ID NO:31)

[0023] HCDR2:IAPNSGGT(SEQ ID NO:32)

[0024] HCDR3:TSYQYVSSQYYFDY(SEQ ID NO:33)

[0025] LCDR1: STVGSSY (SEQ ID NO: 34)

[0026] LCDR2: ST (SEQ ID NO: 35)

[0027] LCDR3:HQWNTYPFT(SEQ ID NO:10)

[0028] Exemplarily, the CDR sequences defined by the Chothia numbering system are as follows:

[0029] HCDR1: GYTFTNY (SEQ ID NO: 36)

[0030] HCDR2: APNSGG (SEQ ID NO: 37)

[0031] HCDR3:YQYVSSQYYFDY(SEQ ID NO:7)

[0032] LCDR1:TASSTVGSSYLY(SEQ ID NO:21)

[0033] LCDR2: STSNLAT (SEQ ID NO: 22)

[0034] LCDR3:HQWNTYPFT(SEQ ID NO:10)

[0035] Illustratively, the CDR sequences defined by the AbM numbering system are as follows:

[0036] HCDR1:GYTFTNYWMH(SEQ ID NO:38)

[0037] HCDR2:RIAPNSGGTK(SEQ ID NO:39)

[0038] HCDR3:YQYVSSQYYFDY(SEQ ID NO:7)

[0039] LCDR1:TASSTVGSSYLY(SEQ ID NO:21)

[0040] LCDR2: STSNLAT (SEQ ID NO: 22)

[0041] LCDR3:HQWNTYPFT(SEQ ID NO:10)

[0042] For example, the CDR sequence defined by the Contact numbering system is as follows:

[0043] HCDR1: TNYWMH (SEQ ID NO: 40)

[0044] HCDR2:WIGRIAPNSGGTK(SEQ ID NO:41)

[0045] HCDR3:TSYQYVSSQYYFD(SEQ ID NO:42)

[0046] LCDR1:GSSYLYWF(SEQ ID NO:43)

[0047] LCDR2:LWIYSTSNLA(SEQ ID NO:44)

[0048] LCDR3:HQWNTYPF(SEQ ID NO:45)

[0049] In some embodiments, the VH and VL in the aforementioned CDH6 binding molecules are humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced, and / or antibody isomerization reduced.

[0050] In some embodiments, the heavy chain framework region of the human germline template used in the humanization process for the VH in the aforementioned CDH6 binding molecules is derived from IGHV7-4, IGHV4-4, IGHV1-69, or IGHJ4*01, and the light chain framework region of the human germline template used in the humanization process for the VL is derived from IGKV6-21, IGKV4-1, IGKV6-21, or IGKJ2*01. In some specific embodiments, in the heavy chain framework region of the human germline template used in the humanization process for the VH in the CDH6 binding molecule, FR1, FR2, and FR3 are respectively derived from IGHV7-4, IGHV4-4, and IGHV1-69, and FR4 is derived from IGHJ4*01; in the light chain framework region of the human germline template used in the humanization process for the VL, FR1, FR2, and FR3 are respectively derived from IGKV6-21, IGKV4-1, and IGKV6-21, and FR4 is derived from IGKJ2*01.

[0051] In some embodiments, a CDH6 binding molecule is provided, which comprises a VH and / or a VL, wherein the VH comprises an amino acid sequence as shown in SEQ ID NO: 1, 17, 62 or 63, or at least 80%, at least 90% identical thereto, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 2, 18, 64 or 65, or at least 80%, at least 90% identical thereto.

[0052] In some embodiments, the CDH6 binding molecule comprises the following VH and VL:

[0053] 1-1) VH comprises an amino acid sequence as set forth in SEQ ID NO: 1, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO: 2, or at least 80% or at least 90% identical thereto;

[0054] 1-2) VH comprises an amino acid sequence as set forth in SEQ ID NO: 17, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO: 18, or at least 80% or at least 90% identical thereto;

[0055] 1-3) VH comprises an amino acid sequence as set forth in SEQ ID NO: 62, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO: 64 or 65, or at least 80% or at least 90% identical thereto;

[0056] 1-4) VH comprises an amino acid sequence as shown in SEQ ID NO: 63, or at least 80%, at least 90% identical thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO: 64 or 65, or at least 80%, at least 90% identical thereto.

[0057] In the present disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity; "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0058] In some specific embodiments, the CDH6 binding molecule comprises a VH and a VL, wherein:

[0059] The VH is shown in SEQ ID NO: 1, and the VL is shown in SEQ ID NO: 2;

[0060] The VH is shown in SEQ ID NO: 17, and the VL is shown in SEQ ID NO: 18;

[0061] The VH is shown in SEQ ID NO: 62, and the VL is shown in SEQ ID NO: 64 or 65;

[0062] The VH is shown in SEQ ID NO: 63, and the VL is shown in SEQ ID NO: 64 or 65.

[0063] II:

[0064] In some embodiments, the present disclosure provides CDH6 binding molecules that specifically bind to the EC3 domain of CDH6. The amino acid sequence of the EC3 domain is, for example, as shown in SEQ ID NO: 73. In some embodiments, the binding molecules have internalization capability that allows cellular uptake.

[0065] In some embodiments, the present disclosure provides CDH6-binding molecules comprising a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and / or HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, 19, 66, or 67, and the VL comprises LCDR1, LCDR2, and / or LCDR3 of the amino acid sequence shown in SEQ ID NO: 4, 20, 68, or 69, and the CDRs are defined according to Kabat, IMGT, Chothia, AbM, or Contact numbering conventions. In some specific embodiments, the CDRs are defined according to the Kabat numbering convention.

[0066] In some embodiments, a CDH6 binding molecule is provided, comprising a VH and / or a VL, wherein:

[0067] 2-1) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 3, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 4;

[0068] 2-2) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 19, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 20;

[0069] 2-3) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 66, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 68 or 69; or,

[0070] 2-4) The VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 67, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 68 or 69.

[0071] In some embodiments, a CDH6 binding molecule is provided, which comprises VH and / or VL, wherein the VH comprises HCDR1, HCDR2 and / or HCDR3, wherein HCDR1, HCDR2, HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 11, 12, and 13, respectively; and the VL comprises LCDR1, LCDR2 and / or LCDR3, wherein LCDR1, LCDR2, LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 26, 15, and 16, respectively.

[0072] In some embodiments, a CDH6 binding molecule is provided, which comprises VH and / or VL, wherein the VH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 11, 12, and 13, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NOs: 14, 23, or 70, and LCDR2 and LCDR3 comprise the amino acid sequences shown in SEQ ID NOs: 15 and 16, respectively.

[0073] The following examples illustrate HCDR1, HCDR2, and HCDR3 in the VH of SEQ ID NO: 19, and LCDR1, LCDR2, and LCDR3 in the VL of SEQ ID NO: 20, as defined by the IMGT, Chothia, AbM, or Contact numbering systems.

[0074] Exemplarily, the CDR sequences defined by the IMGT numbering system are as follows:

[0075] HCDR1:GFSLSSYG(SEQ ID NO:46)

[0076] HCDR2: IWSGGST (SEQ ID NO: 47)

[0077] HCDR3:ARYSNSYAMDY(SEQ ID NO:48)

[0078] LCDR1:QSIVHSTGNTY(SEQ ID NO:49)

[0079] LCDR2:KV (SEQ ID NO:50)

[0080] LCDR3:FQTSHVPYT(SEQ ID NO:16)

[0081] Exemplarily, the CDR sequences defined by the Chothia numbering system are as follows:

[0082] HCDR1: GFSLSSY (SEQ ID NO: 51)

[0083] HCDR2: WSGGS (SEQ ID NO: 52)

[0084] HCDR3:YSNSYAMDY(SEQ ID NO:13)

[0085] LCDR1:RSSQSIVHSTGNTYLE(SEQ ID NO:23)

[0086] LCDR2: KVSKRFS (SEQ ID NO: 15)

[0087] LCDR3:FQTSHVPYT(SEQ ID NO:16)

[0088] Illustratively, the CDR sequences defined by the AbM numbering system are as follows:

[0089] HCDR1:GFSLSSYGVH(SEQ ID NO:53)

[0090] HCDR2:MIWSGGSTD(SEQ ID NO:54)

[0091] HCDR3:YSNSYAMDY(SEQ ID NO:13)

[0092] LCDR1:RSSQSIVHSTGNTYLE(SEQ ID NO:23)

[0093] LCDR2: KVSKRFS (SEQ ID NO: 15)

[0094] LCDR3:FQTSHVPYT(SEQ ID NO:16)

[0095] For example, the CDR sequence defined by the Contact numbering system is as follows:

[0096] HCDR1: SSYGVH (SEQ ID NO: 55)

[0097] HCDR2: WIGMIWSGGSTD(SEQ ID NO:56)

[0098] HCDR3:ARYSNSYAMD(SEQ ID NO:57)

[0099] LCDR1:VHSTGNTYLEWY(SEQ ID NO:58)

[0100] LCDR2:LLIYKVSKRF(SEQ ID NO:59)

[0101] LCDR3:FQTSHVPY(SEQ ID NO:60)

[0102] In some embodiments, the VH and VL in the aforementioned CDH6 binding molecules are humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced, and / or antibody isomerization reduced.

[0103] In some embodiments, the heavy chain framework region of the VH in the aforementioned CDH6-binding molecules is derived from IGHV4-30 and IGHJ4*01 in the human germline template used in the humanization process, and the light chain framework region of the VL in the human germline template used in the humanization process is derived from IGKV2-28 and IGKJ2*01. In some specific embodiments, in the heavy chain framework region of the VH in the CDH6-binding molecules, FR1, FR2, and FR3 are derived from IGHV4-30, and FR4 is derived from IGHJ4*01 in the human germline template used in the humanization process; and in the light chain framework region of the VL in the human germline template used in the humanization process, FR1, FR2, and FR3 are derived from IGKV2-28, and FR4 is derived from IGKJ2*01.

[0104] In some embodiments, a CDH6 binding molecule is provided, which comprises a VH and / or a VL, wherein the VH comprises an amino acid sequence as shown in SEQ ID NO: 3, 19, 66 or 67, or at least 80%, at least 90% identical thereto, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 4, 20, 68 or 69, or at least 80%, at least 90% identical thereto.

[0105] In some embodiments, the CDH6 binding molecule comprises the following VH and VL:

[0106] 2-1) VH comprises an amino acid sequence as set forth in SEQ ID NO: 3, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO: 4, or at least 80% or at least 90% identical thereto;

[0107] 2-2) VH comprises an amino acid sequence as set forth in SEQ ID NO: 19, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO: 20, or at least 80% or at least 90% identical thereto;

[0108] 2-3) VH comprises an amino acid sequence as set forth in SEQ ID NO: 66, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO: 68 or 69, or at least 80% or at least 90% identical thereto;

[0109] 2-4) VH comprises an amino acid sequence as shown in SEQ ID NO: 67, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO: 68 or 69, or at least 80% or at least 90% identical thereto.

[0110] In some specific embodiments, the CDH6 binding molecule comprises a VH and a VL, wherein:

[0111] The VH is shown in SEQ ID NO: 3, and the VL is shown in SEQ ID NO: 4;

[0112] The VH is shown in SEQ ID NO: 19, and the VL is shown in SEQ ID NO: 20;

[0113] The VH is shown in SEQ ID NO: 66, and the VL is shown in SEQ ID NO: 68 or 69;

[0114] The VH is shown in SEQ ID NO: 67, and the VL is shown in SEQ ID NO: 68 or 69.

[0115] In the above I and II:

[0116] In some embodiments, the CDH6 binding molecule comprises a VH and a VL, wherein:

[0117] The VH shown has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the VH shown in any of the preceding items; and / or, the VL shown has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to the VL shown in any of the preceding items.

[0118] In some specific embodiments, the amino acid mutations in VH or VL are conservative substitutions.

[0119] In some embodiments, the CDH6 binding molecule is an anti-CDH6 antibody or an antigen-binding fragment thereof.

[0120] In some embodiments, the antibody is selected from a murine antibody, a chimeric antibody, a human antibody, a humanized antibody, or a fragment thereof. In some specific embodiments, the antibody is a humanized antibody, a human antibody, or a fragment thereof.

[0121] In some embodiments, the humanized antibody or fragment thereof further comprises a back mutation to VH or VL. In some embodiments, the back mutation is located in the framework region (FR) of VH and / or VL. In some embodiments, the back mutation is located in the CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) of VH and / or VL.

[0122] In some embodiments, the CDH6 binding molecule is an antigen-binding fragment of an anti-CDH6 antibody, including but not limited to any of the following: Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody and multispecific antibody (bispecific antibody, double-chain antibody (diabody), three-chain antibody (triabody) and four-chain antibody (tetrabody), tandem two-scFv, tandem three-scFv), for example, specifically scFv, Fv, Fab or Fab' fragment.

[0123] In some embodiments, the CDH6 binding molecule further comprises a constant region. For example, the heavy chain constant region is derived from IgG1, IgG2, IgG3, IgG4, or variants thereof, and the light chain constant region is derived from κ, λ, or variants thereof.

[0124] In some specific embodiments, the Fc region of the constant region of the CDH6 binding molecule is derived from human IgG1, IgG2, IgG3 or IgG4. For example, the Fc region of IgG1 comprises the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence having at least 80% sequence identity thereto.

[0125] In some embodiments, a CDH6 binding molecule is provided, comprising a heavy chain and a light chain, wherein:

[0126] The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 27, or a sequence identity thereof of at least 80%, at least 90%, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 28, or a sequence identity thereof of at least 80%, at least 90%; and / or the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 29, or a sequence identity thereof of at least 80%, at least 90%, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 30, or a sequence identity thereof of at least 80%, at least 90%.

[0127] In some embodiments, a CDH6 binding molecule is provided, comprising a heavy chain and a light chain, wherein:

[0128] The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 28; or, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 29, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 30.

[0129] In some embodiments, the CDH6 binding molecule has cell internalization activity. In some specific embodiments, the CDH6 binding molecule specifically binds to human CDH6 and has tumor cell internalization activity. In some specific embodiments, the CDH6 binding molecule specifically binds to the extracellular domain of human CDH6 and has tumor cell internalization activity.

[0130] In some embodiments, any of the aforementioned CDH6-binding molecules has at least one of the following characteristics:

[0131] (1) Possessing internalization capability that allows cellular uptake;

[0132] (2) having different or partially overlapping epitopes with the Daiichi07 antibody;

[0133] (3) With a K less than or equal to 100 nM D Values ​​bind to human CDH6;

[0134] (4) Does not bind to human CDH9 and CDH10.

[0135] In some embodiments, the CDH6 binding molecule binds to CDH6 on the cell surface and is internalized (or endocytosed) into the cell. In some specific embodiments, the CDH6 binding molecule binds to the extracellular domain of CDH6 and is internalized into the cell. In some specific embodiments, compared to the Nov0712 antibody (sequence see patent number: US2016 / 0046711A1, which is incorporated herein by reference in its entirety) and the Daiichi07 antibody (sequence see patent number: US2020 / 0390900A1, which is incorporated herein by reference in its entirety), the CDH6 binding molecule in the present disclosure has an enhanced internalization ability that allows cellular uptake.

[0136] In some embodiments, the CDH6 binding molecule has a K of less than or equal to 100 nM. D The value of binding to human CDH6 is, for example, less than or equal to about 75 nM, about 50 nM, about 25 nM, about 50 nM, about 10 nM, about 5 nM, etc.

[0137] In some embodiments, the CDH6 binding molecules of the present disclosure specifically bind to the EC1 domain of human CDH6, for example, the CDH6 binding molecules described in Section 1 of the present disclosure. In some embodiments, the binding molecules have internalization ability that allows cellular uptake. In some embodiments, the binding molecules that specifically bind to the EC1 domain of CDH6 have a stronger internalization ability that allows cellular uptake than the binding molecules that specifically bind to the EC1 domain of CDH6. In some embodiments, the CDH6 binding molecules described in Section 1 of the present disclosure have enhanced internalization ability that allows cellular uptake compared to the Nov0712 antibody and the Daiichi07 antibody.

[0138] In other embodiments, the CDH6-binding molecules of the present disclosure specifically bind to the EC3 domain of human CDH6, for example, the CDH6-binding molecules described in Section II of the present disclosure. In some embodiments, the binding molecules have internalization ability that allows cellular uptake. In some embodiments, the CDH6-binding molecules described in Section II of the present disclosure have enhanced internalization ability that allows cellular uptake compared to the Nov0712 antibody and the Daiichi07 antibody.

[0139] In some embodiments, the present disclosure provides a CDH6-binding molecule that binds to the same epitope as the aforementioned CDH6-binding molecule.

[0140] In other embodiments, CDH6-binding molecules are provided that cross-block the binding of the aforementioned CDH6-binding molecules to human CDH6.

[0141] In other embodiments, a CDH6-binding molecule is provided, wherein the binding of the molecule to human CDH6 is cross-blocked by a CDH6-binding molecule described above.

[0142] Antibody Drug Conjugates

[0143] In some embodiments, the present disclosure provides an antibody drug conjugate comprising the heavy chain variable region (VH) and the light chain variable region (VL) of any of the aforementioned CDH6-binding molecules.

[0144] In some embodiments, the antibody drug conjugate comprises a CDH6 binding molecule and an effector molecule. In some embodiments, the effector molecule is coupled to the CDH6 binding molecule. In some embodiments, the CDH6 binding molecule is any one of the CDH6 binding molecules provided herein.

[0145] In some embodiments, effector molecules include, but are not limited to, cytotoxic drugs, immunomodulators, and cytostatics. For example, compounds, polypeptides, proteins, nucleic acids, and the like that have cell growth inhibition, cytotoxicity, and / or immunomodulatory functions. Exemplary examples include toxins (such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin), radioactive isotopes, chemotherapeutic drugs, antibiotics, and nucleolytic enzymes.

[0146] The antibody-drug conjugate disclosed herein can bind to the extracellular domain of CDH6 and then undergo endocytosis, with the effector molecules being released within the cell, which can not only effectively exert cell killing and inhibitory effects, but also have a good bystander effect.

[0147] In some embodiments, the effector molecule is exitecan or a derivative thereof.

[0148] In some embodiments, the antibody drug conjugate is an antibody or fragment thereof-exitecan or a derivative thereof conjugate, which is represented by the general formula (Pc-LYD) of any one of Formulas (I) to (V):

[0149] in:

[0150] Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CR a R b ) m -CR 1 R 2 -C(O)-、-S-(CR a R b ) m -CR 1 R 2 -C(O)- and is absent; optionally, when of formula (V), Y is absent;

[0151] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group; or, R a and Rb Together with the carbon atom to which it is attached, they form a cycloalkyl group and a heterocyclyl group;

[0152] R 1 R is selected from hydrogen, halogen, haloalkyl, alkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl groups; 2 is selected from hydrogen, halogen, haloalkyl, alkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0153] Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0154] m is an integer from 0 to 4;

[0155] n is 1 to 10, n is a decimal or an integer, preferably, n is 2 to 8 or 5 to 9;

[0156] L is the joint unit;

[0157] Pc is any one of the CDH6-binding molecules disclosed herein.

[0158] In some embodiments, the antibody drug conjugate is an antibody or fragment thereof-exitecan or a derivative thereof conjugate, which is represented by the general formula (Pc-LYD) of formula (I):

[0159] in:

[0160] Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2-C(O)-;

[0161] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group; or, R a and R b Together with the carbon atom to which it is attached, they form a cycloalkyl group and a heterocyclyl group;

[0162] R 1 R is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl groups; 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0163] Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0164] m is an integer from 0 to 4;

[0165] n is 1 to 10, n is a decimal or an integer, preferably, n is 2 to 8 or 5 to 9;

[0166] L is the joint unit;

[0167] Pc is any one of the CDH6-binding molecules disclosed herein.

[0168] In some embodiments, the antibody or fragment thereof-exitecan or derivative conjugate of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, wherein -Y- is -O-(CR a R b )m-CR 1 R 2 -C(O)-;

[0169] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;

[0170] R 1 C 1-3 Alkyl, C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl group, hydrogen atom;

[0171] R 2Selected from hydrogen atom, halogenated alkyl, C 1-3 Alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0172] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0173] m is 0 or 1.

[0174] In some embodiments, the antibody or fragment thereof-exitecan or derivative conjugate of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, wherein -Y- is -O-(CR a R b )m-CR 1 R 2 -C(O)-;

[0175] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;

[0176] R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl group, hydrogen atom;

[0177] R 2 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0178] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0179] m is 0 or 1.

[0180] In some embodiments, the conjugate of the antibody or fragment thereof-exitecan or its derivative disclosed herein, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0181] R 1 C 1-3 Alkyl, C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl group, hydrogen atom;

[0182] R 2 Selected from hydrogen atom, halogenated alkyl, C 1-3 Alkyl or C 3-6 Cycloalkyl;

[0183] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0184] m is 0 or 1.

[0185] In some embodiments, the conjugate of the antibody or fragment thereof-exitecan or its derivative disclosed herein, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0186] R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl group, hydrogen atom;

[0187] R 2 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl;

[0188] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0189] m is 0 or 1.

[0190] In some embodiments, the conjugate of the antibody or fragment thereof-exitecan or its derivative disclosed herein, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0191] R 1 C 1-3 Alkyl, C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl group, hydrogen atom;

[0192] R 2 is a hydrogen atom;

[0193] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0194] m is 0 or 1.

[0195] In some embodiments, the conjugate of the antibody or fragment thereof-exitecan or its derivative disclosed herein, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0196] R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl group, hydrogen atom;

[0197] R 2 is a hydrogen atom;

[0198] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0199] m is 0 or 1.

[0200] In some embodiments, the conjugate of the antibody or fragment thereof-exitecan or its derivative disclosed herein, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0201] R 1 C 1-3 Alkyl, C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl group, hydrogen atom;

[0202] R 2 is a hydrogen atom;

[0203] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0204] m is 0.

[0205] In some embodiments, the conjugate of the antibody or fragment thereof-exitecan or its derivative disclosed herein, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0206] R 1 C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl group, hydrogen atom;

[0207] R 2 is a hydrogen atom;

[0208] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0209] m is 0.

[0210] In some embodiments, the conjugate of the antibody or fragment thereof-exitecan or its derivative disclosed herein, wherein the structural unit -Y- is -O-(CH2)m-CR 1 R 2 -C(O)-;

[0211] R 1 is a hydrogen atom;

[0212] R 2 is a hydrogen atom;

[0213] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0214] m is 0.

[0215] In some embodiments, in the conjugate of the antibody or fragment thereof-exitecan or its derivatives disclosed herein, Y is selected from:

[0216] The O end of Y is connected to the linker unit L.

[0217] In other embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, which is represented by the general formula (Pc-L-D1) of Formula (VI):

[0218] in:

[0219] R 1 For hydrogen atoms, C 1-3 Alkyl, C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0220] R 2 Selected from hydrogen atom, halogenated alkyl, C 1-3 Alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0221] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0222] m is 0 or 1;

[0223] n is 1 to 10, and can be an integer or a decimal. Preferably, n is a decimal or integer from 1 to 8 or from 1 to 6. More preferably, n is a decimal or integer from 1 to 5 or from 2 to 4.

[0224] In some embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, which is represented by the general formula (Pc-L-D1) of Formula (VI):

[0225] in:

[0226] R 1 For hydrogen atoms, C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0227] R 2 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0228] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0229] m is 0 or 1;

[0230] n is 1 to 10, and can be an integer or a decimal. Preferably, n is a decimal or integer from 1 to 8 or from 1 to 6. More preferably, n is a decimal or integer from 1 to 5 or from 2 to 4.

[0231] In some specific embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-isotecan or a derivative thereof, wherein n is 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), which can be an integer or a decimal; preferably 1 to 6, which can be an integer or a decimal.

[0232] In some embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0233] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0234] L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20;

[0235] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

[0236] L 4 Selected from-NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6;

[0237] R 3 、R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0238] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0239] In some embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)-, -(succinimide-3-yl-N)-CH2-cyclohexyl-C(O)-, -(succinimide-3-yl-N)-(CH2CH2O)s 2-CH2CH2-C(O)-, -CH2-C(O)-NR 3 -(CH2)s 3 -C(O)- or -C(O)-(CH2)s 4 C(O)-, where s 1 is an integer from 2 to 8, s 2 is an integer from 1 to 3, s 3 is an integer from 1 to 8, s 4 is an integer from 1 to 8; s 1 Preferably 5.

[0240] In some specific embodiments, the present disclosure provides a conjugate of an antibody or a fragment thereof-exitecan or a derivative thereof, wherein the linker unit L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 An integer from 6 to 12.

[0241] In some specific embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein L 4 Selected from-NR 5 (CR 6 R 7 )t-,R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, t is 1 or 2, preferably 2; L 4 Preferably, it is -NR 5 CR 6 R 7 -;L 4 More preferred is -NHCH2-.

[0242] In some specific embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0243] L 1 for s 1 is an integer from 2 to 8;

[0244] L 2 is a chemical bond;

[0245] L 3 is a tetrapeptide residue;

[0246] L4 -NR 5 (CR 6 R 7 )t-,R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.

[0247] In some specific embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0248] L 1 is -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0249] L 2 -NR 4 (CH2CH2O)9CH2C(O)-;

[0250] L 3 is a tetrapeptide residue;

[0251] L 4 -NR 5 (CR 6 R 7 )t-,R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.

[0252] In some specific embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the L 3 The peptide residue is an amino acid residue formed by one, two or more amino acids selected from phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), and aspartic acid (N); preferably, it is an amino acid residue formed by one, two or more amino acids selected from phenylalanine and glycine; more preferably, it is a tetrapeptide residue; and most preferably, it is a tetrapeptide residue of GGFG (glycine-glycine-phenylalanine-glycine).

[0253] In some embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit -L-, wherein L 1 The end is connected to the antibody, L 4 Connect the Y end.

[0254] In some embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the -LY- is:

[0255] L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0256] L 2 -NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 is an integer from 6 to 12;

[0257] L 3 is the tetrapeptide residue of GGFG;

[0258] R 1 For hydrogen atoms, C 1-3 Alkyl, cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0259] R 2 Selected from hydrogen atom, halogenated alkyl, C 1-3 Alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0260] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0261] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0262] s 1 is an integer from 2 to 8; preferably 5;

[0263] m is an integer from 0 to 4.

[0264] In some embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the -LY- is:

[0265] L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1-C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0266] L 2 -NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 is an integer from 6 to 12;

[0267] L 3 is the tetrapeptide residue of GGFG;

[0268] R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0269] R 2 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0270] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0271] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0272] s 1 is an integer from 2 to 8; preferably 5;

[0273] m is an integer from 0 to 4.

[0274] In some embodiments, the conjugate of antibody or fragment thereof-exitecan or its derivatives,

[0275] Wherein -LY- is:

[0276] Preferably:

[0277] L 2 -NR 4 (CH2CH2O)9CH2C(O)-;

[0278] L 3 is the tetrapeptide residue of GGFG;

[0279] R 1 For hydrogen atoms, C 1-3 Alkyl, cycloalkylalkyl or cycloalkyl; preferably C3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0280] R 2 Selected from hydrogen atom, halogenated alkyl, C 1-3 Alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0281] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0282] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0283] m is an integer from 0 to 4.

[0284] In some embodiments, the conjugate of antibody or fragment thereof-exitecan or its derivatives,

[0285] Wherein -LY- is:

[0286] Preferably:

[0287] L 2 -NR 4 (CH2CH2O)9CH2C(O)-;

[0288] L 3 is the tetrapeptide residue of GGFG;

[0289] R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0290] R 2 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0291] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0292] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0293] m is an integer from 0 to 4.

[0294] In some embodiments, the antibody or fragment thereof-exitecan or a derivative thereof conjugate, wherein the antibody or fragment thereof-exitecan or a derivative thereof conjugate comprises a structure represented by formula (VII):

[0295] in:

[0296] L 2 is a chemical bond;

[0297] L 3 is the tetrapeptide residue of GGFG;

[0298] R 1 For hydrogen atoms, C 1-3 Alkyl, C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0299] R 2 Selected from hydrogen atom, halogenated alkyl, C 1-3 Alkyl or C 3-6 Cycloalkyl;

[0300] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0301] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0302] s 1 is an integer from 2 to 8;

[0303] m is an integer from 0 to 4.

[0304] In some embodiments, the antibody or fragment thereof-exitecan or a derivative thereof conjugate, wherein the antibody or fragment thereof-exitecan or a derivative thereof conjugate comprises a structure represented by formula (VII):

[0305] in:

[0306] L 2 is a chemical bond;

[0307] L 3 is the tetrapeptide residue of GGFG;

[0308] R 1 For hydrogen atoms, C 3-6Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0309] R 2 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl;

[0310] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0311] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0312] s 1 is an integer from 2 to 8;

[0313] m is an integer from 0 to 4.

[0314] In some embodiments, the antibody or fragment thereof-exitecan or a derivative thereof conjugate, wherein the antibody or fragment thereof-exitecan or a derivative thereof conjugate comprises a structure represented by the formula (-LY-):

[0315] It can be used to obtain antibody-drug conjugates by connecting effector molecules to antibodies via linker fragments;

[0316] in:

[0317] L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0318] L 2 -NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 is an integer from 1 to 20;

[0319] L 3 is the tetrapeptide residue of GGFG;

[0320] R 1 For hydrogen atoms, C 1-3 Alkyl, cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0321] R 2Selected from hydrogen atom, halogenated alkyl, C 1-3 Alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0322] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0323] R 5 、R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0324] s 1 is an integer from 2 to 8;

[0325] m is an integer from 0 to 4.

[0326] In some embodiments, the antibody or fragment thereof-exitecan or a derivative thereof conjugate, wherein the antibody or fragment thereof-exitecan or a derivative thereof conjugate comprises a structure represented by the formula (-LY-):

[0327] It can be used to obtain antibody-drug conjugates by connecting effector molecules to antibodies via linker fragments;

[0328] in:

[0329] L 1 Selected from -(succinimidyl-3-yl-N)-(CH2)s 1 -C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

[0330] L 2 -NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, p 1 is an integer from 1 to 20;

[0331] L 3 is the tetrapeptide residue of GGFG;

[0332] R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0333] R 2 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0334] Or, R 1and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0335] R 5 、R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0336] s 1 is an integer from 2 to 8;

[0337] m is an integer from 0 to 4.

[0338] In some embodiments, the antibody or fragment thereof-exitecan or a derivative thereof conjugate, wherein the antibody or fragment thereof-exitecan or a derivative thereof conjugate comprises a structure represented by the formula (-LY-):

[0339] in:

[0340] L 2 is a chemical bond;

[0341] L 3 is the tetrapeptide residue of GGFG;

[0342] R 1 For hydrogen atoms, C 1-3 Alkyl, cycloalkylalkyl or cycloalkyl; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0343] R 2 Selected from hydrogen atom, halogenated alkyl, C 1-3 Alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0344] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0345] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0346] s 1 is an integer from 2 to 8;

[0347] m is an integer from 0 to 4.

[0348] In some embodiments, the antibody or fragment thereof-exitecan or a derivative thereof conjugate, wherein the antibody or fragment thereof-exitecan or a derivative thereof conjugate comprises a structure represented by the formula (-LY-):

[0349] in:

[0350] L 2 is a chemical bond;

[0351] L 3 is the tetrapeptide residue of GGFG;

[0352] R 1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably C 3-6 Cycloalkylalkyl or C 3-6 Cycloalkyl;

[0353] R 2 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl; preferably a hydrogen atom;

[0354] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0355] R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and are each independently a hydrogen atom or an alkyl group;

[0356] s 1 is an integer from 2 to 8;

[0357] m is an integer from 0 to 4.

[0358] In some embodiments, the antibody or fragment thereof-exitecan or derivative thereof conjugate is of the general formula (Pc-L) of formula (VIII) a -Y-Dr) shown in the antibody or fragment thereof - ixitecan or its derivative conjugate:

[0359] in:

[0360] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0361] L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20;

[0362] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0363] R 1 is selected from a hydrogen atom, a halogen, an alkyl group, a cycloalkylalkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group;

[0364] R 2 is selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0365] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0366] R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0367] R 6 and R 7 are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

[0368] m is an integer from 0 to 4;

[0369] n is 1 to 10 and can be an integer or a decimal;

[0370] Pc is a CDH6 binding molecule provided by the present disclosure.

[0371] In some embodiments, the antibody or fragment thereof-exitecan or derivative thereof conjugate is of the general formula (Pc-L) of formula (VIII) a -Y-Dr) shown in the antibody or fragment thereof - ixitecan or its derivative conjugate:

[0372] in:

[0373] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0374] L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20;

[0375] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0376] R 1 is selected from hydrogen, halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0377] R 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0378] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0379] R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0380] R 6 and R 7 are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

[0381] m is an integer from 0 to 4;

[0382] n is 1 to 10 and can be an integer or a decimal;

[0383] Pc is a CDH6 binding molecule provided by the present disclosure.

[0384] In some embodiments, the antibody or fragment thereof-exitecan or derivative thereof conjugate is of the general formula (Pc-L) of formula (IX) b -Y-Dr) shown in the antibody or fragment thereof - ixitecan or its derivative conjugate:

[0385] in:

[0386] s 1 is an integer from 2 to 8; preferably 5;

[0387] Pc, R 1 、R 2 、R 5 ~R 7 , m and n are as defined in formula (VIII).

[0388] In some embodiments, the linker unit -LY- of the antibody or fragment thereof-exitecan or derivative thereof conjugate includes, but is not limited to:

[0389] wherein x and y are independently selected from integers of 2-8.

[0390] In some embodiments, the present disclosure provides antibody or fragment thereof-exitecan or derivative conjugates including, but not limited to:

[0391] in:

[0392] n is 1 to 10, and can be an integer or a decimal, such as 2, 3, 4, 5, 6, 7, 8, 9, preferably 7-8;

[0393] Pc is any of the aforementioned CDH6-binding molecules of the present disclosure.

[0394] In some embodiments, n is 4±0.4, 4±0.3, 4±0.2, 4±0.1.

[0395] In some embodiments, n is 6±0.4, 6±0.3, 6±0.2, 6±0.1.

[0396] In some embodiments, n is 7±0.4, 7±0.3, 7±0.2, or 7±0.1.

[0397] In some embodiments, n is 8±0.4, 8±0.3, 8±0.2, 8±0.1.

[0398] In some embodiments, n is a measured value calculated by RP-HPLC, and the RP-HPLC method is conventional in the art.

[0399] Exemplarily, in some embodiments, Pc in the antibody drug conjugate comprises VH and VL, wherein:

[0400] 1-1) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 2;

[0401] 1-2) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 17, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 18;

[0402] 1-3) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 62, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 64 or 65; or,

[0403] 1-4) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence of SEQ ID NO: 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence of SEQ ID NO: 64 or 65;

[0404] 2-1) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 3, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 4;

[0405] 2-2) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 19, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 20;

[0406] 2-3) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 66, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 68 or 69; or,

[0407] 2-4) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence of SEQ ID NO: 67, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence of SEQ ID NO: 68 or 69;

[0408] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions. In some specific embodiments, the CDRs are defined according to the Kabat numbering conventions.

[0409] In some embodiments, the Pc in the antibody drug conjugate comprises VH and VL, wherein:

[0410] 1-1) the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 5, 6, and 7, respectively; the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 8, 9, and 10, respectively;

[0411] 1-2) the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 5, 6, and 7, respectively; the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 21, 22, and 10, respectively;

[0412] 2-1) The VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 11, 12, and 13, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 14, 15, and 16, respectively;

[0413] 2-2) the VH comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 11, 12, and 13, respectively; the VL comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 23, 15, and 16, respectively;

[0414] 2-3) The VH comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 11, 12, and 13, respectively; the VL comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 70, 15, and 16, respectively.

[0415] In some embodiments, Pc in the antibody drug conjugate comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 28; or, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 29, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 30.

[0416] Polynucleotides and vectors

[0417] In some embodiments, the present disclosure provides polynucleotides encoding any of the CDH6-binding molecules (eg, antibodies or antigen-binding fragments thereof) of the present disclosure, or encoding any of the antibodies or antigen-binding fragments thereof in an anti-CDH6 antibody-drug conjugate.

[0418] In some embodiments, the polynucleotides of the present disclosure may be RNA, DNA, or cDNA. In some embodiments, the polynucleotides may be isolated polynucleotides.

[0419] In some embodiments, the aforementioned polynucleotides may also be in the form of a vector, may be present in a vector and / or may be part of a vector. In some embodiments, the vector comprising the polynucleotide may be a eukaryotic vector, a prokaryotic vector, a viral vector, such as a plasmid, a cosmid, a phage, and the like. The vector may be, in particular, an expression vector, i.e., a vector that provides for expression of the CDH6 binding molecule in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector typically comprises at least one nucleic acid of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a particular host.

[0420] The polynucleotides of the present disclosure can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the antibodies or fragments thereof of the present disclosure, and / or can be isolated from suitable natural sources.

[0421] In some embodiments, the polynucleotides and vectors disclosed herein can be used to prepare CDH6-binding molecules. In some embodiments, the polynucleotides and vectors disclosed herein are used to express CDH6-binding molecules in vitro or in vivo for various purposes, such as detection, diagnosis, treatment, and regulation.

[0422] host cells

[0423] In some embodiments, the present disclosure provides a host cell comprising any of the aforementioned vectors; or expressing any of the aforementioned CDH6-binding molecules (e.g., antibodies or antigen-binding fragments thereof), or antibodies or antigen-binding fragments thereof in anti-CDH6 antibody-drug conjugates. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0424] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0425] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0426] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0427] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0428] Composition

[0429] The present disclosure provides compositions comprising the aforementioned CDH6-binding molecules (e.g., antibodies or antigen-binding fragments thereof) or anti-CDH6 antibody-drug conjugates of the present disclosure. For example, pharmaceutical compositions are provided, comprising an effective amount of the aforementioned CDH6-binding molecules or anti-CDH6 antibody-drug conjugates for treating, alleviating, or preventing a disease, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0430] In some embodiments, the unit dosage of the pharmaceutical composition may contain 0.01 to 99 weight % of a CDH6 binding molecule (e.g., an antibody or an antigen-binding fragment thereof) or an anti-CDH6 antibody-drug conjugate, or the amount of the CDH6 binding molecule or anti-CDH6 antibody-drug conjugate in a unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some embodiments, 1-1000 mg.

[0431] In some embodiments, a product or article is provided comprising the aforementioned CDH6 binding molecules (e.g., antibodies or antigen-binding fragments thereof) or anti-CDH6 antibody-drug conjugates. Optionally, the article comprises a container and a label. Containers such as bottles, syringes, and test tubes contain a composition effective for treating a condition. A label on or associated with the container indicates that the composition is used to treat the selected condition.

[0432] In some embodiments, the aforementioned disease is a cell proliferative disease or cancer.

[0433] Preparation method

[0434] In some embodiments, the present disclosure provides a method for preparing a CDH6-binding molecule (eg, an antibody or an antigen-binding fragment thereof), comprising: expressing the antibody or antigen-binding fragment thereof in a host cell as described above, and isolating the antibody or antigen-binding fragment thereof from the host cell.

[0435] In some embodiments, the preparation method may further include a purification step, for example, using an A or G Sepharose FF column containing an adjusted buffer to wash away nonspecifically bound components, then eluting the bound antibody using a pH gradient, detecting it using SDS-PAGE, and collecting it. Alternatively, filtration and concentration may be performed using conventional methods. Soluble mixtures and polymers may also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0436] Methods for producing and purifying antibodies or antigen-binding fragments are well known in the art and can be found in, for example, the Cold Spring Harbor Laboratory Manual (Chapters 5-8 and 15). For example, mice can be immunized with human FcRn or fragments thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods.

[0437] As an example of the preparation method, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves, ion exchange, etc.

[0438] In some embodiments, the present disclosure provides a method for preparing an anti-CDH6 antibody-drug conjugate, comprising: performing a conjugation reaction between any of the aforementioned CDH6 binding molecules (e.g., an antibody or antigen-binding fragment thereof) and an effector molecule to obtain the antibody-drug conjugate. Furthermore, the method further comprises: purifying the antibody-drug conjugate.

[0439] In some embodiments, the method for preparing a conjugate of a ligand-exitecan or a derivative thereof represented by the general formula (Pc-La-YD) comprises the following steps:

[0440] After Pc is reduced, it undergoes a coupling reaction with the general formula (La-YD) to obtain a compound represented by the general formula (Pc-La-YD);

[0441] wherein Pc is a CDH6-binding molecule of the present disclosure (eg, an antibody or an antigen-binding fragment thereof); W, L2, L3, R1, R2, R5-R7, m, and n are as defined in formula (IV).

[0442] The compounds and preparation methods thereof in WO2020063673 and WO2022022508 are incorporated herein in their entirety.

[0443] Medical uses and treatments

[0444] In some embodiments, the present disclosure provides at least one of the following uses of a CDH6-binding molecule (eg, an antibody or antigen-binding fragment thereof), an anti-CDH6 antibody-drug conjugate, a pharmaceutical composition, a polynucleotide, or a vector:

[0445] (1) for preventing or treating diseases or conditions associated with CDH6;

[0446] (2) preparing a medicament for preventing or treating a disease or condition associated with CDH6;

[0447] (3) For the treatment of cancer or tumors;

[0448] (4) Preparation of drugs for treating cancer or tumors;

[0449] (5) For the prevention of cancer or tumors;

[0450] (6) Preparation of drugs for preventing cancer or tumors;

[0451] (7) Used to detect CDH6 protein;

[0452] (8) preparing reagents for detecting CDH6 protein;

[0453] (9) Used for diagnosis of cancer or tumor;

[0454] (10) Preparation of reagents for diagnosing cancer or tumors.

[0455] In the present disclosure, the diseases associated with CDH6 include but are not limited to cancer or tumor.

[0456] In some embodiments, the aforementioned disease is a disease or disorder caused by overexpression of CDH6.

[0457] In some embodiments, the aforementioned disease is a cell proliferative disease or cancer; in some specific embodiments, it is a CDH6-positive cancer.

[0458] In some embodiments, the aforementioned disease is renal cancer, ovarian cancer, renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian serous adenocarcinoma, thyroid cancer, bile duct cancer, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma; in some specific embodiments, it is CDH6-positive renal cancer, ovarian cancer, renal cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, ovarian serous adenocarcinoma, thyroid cancer, bile duct cancer, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma. The anti-CDH6 antibody drug conjugates disclosed herein have tumor targeting, tumor cell toxicity, and bystander killing, and can be used to inhibit the activity of tumor cells in vitro or in vivo in a subject, kill tumor cells, and treat diseases associated with CDH6.

[0459] In some embodiments, the present disclosure provides methods of treating a subject having cancer or a tumor, or a subject at risk of developing cancer, comprising administering to the subject a therapeutically effective amount of a CDH6 binding molecule, an anti-CDH6 antibody drug conjugate, a pharmaceutical composition, a polynucleotide, or a vector.

[0460] In some specific embodiments, the present disclosure provides a method for treating a subject having cancer or a tumor, or a subject at risk of developing cancer, comprising: administering to the subject a therapeutically effective amount of an anti-CDH6 antibody drug conjugate or a pharmaceutical composition thereof.

[0461] In some embodiments, the present disclosure provides a method for killing or inhibiting cells expressing CDH6 in vitro or in a subject, comprising:

[0462] administering an effective amount of a CDH6-binding molecule, anti-CDH6 antibody-drug conjugate, pharmaceutical composition, polynucleotide, or vector of the present disclosure in vitro; or,

[0463] An effective amount of a CDH6-binding molecule, anti-CDH6 antibody drug conjugate, pharmaceutical composition, polynucleotide, or vector of the present disclosure is administered to a subject.

[0464] Detection purpose

[0465] In some embodiments, the present disclosure provides a method for detecting CDH6 in vivo or in vitro, comprising: contacting a sample with a CDH6-binding molecule, polynucleotide, or vector of the present disclosure. The method can be used to detect the presence or amount of CDH6 in a sample.

[0466] In some embodiments, the CDH6 binding molecules of the present disclosure are further detectably labeled. In some specific embodiments, the method further comprises detecting the CDH6 binding molecules and anti-CDH6 antibody-drug conjugates of the present disclosure using a detectably labeled reagent. The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample rather than a sample from a patient).

[0467] In some specific embodiments, the present disclosure provides a method for detecting CDH6 in vivo or in vitro, comprising:

[0468] (1) contacting a CDH6-binding molecule, polynucleotide, or vector disclosed herein with a sample to be tested;

[0469] (2) Detecting the formation of a complex between the CDH6 binding molecule and the sample to be tested.

[0470] In some specific embodiments, the method further comprises:

[0471] (3) contacting a CDH6-binding molecule, polynucleotide, or vector of the present disclosure with a reference sample (e.g., a control sample);

[0472] (4) Determine whether the test sample contains CDH6 by comparing it with a reference sample; or, determine the content of CDH6 in the test sample by comparing it with a reference sample.

[0473] In the context of the present disclosure, a "test sample" refers to any type of sample in which it is desired to determine whether CDH6 is present or the amount of CDH6 is desired to be determined. Exemplarily, the test sample includes cells, cell lysates, blood smears, cell centrifugation preparations, cytological smears, body fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage fluid, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and / or tissue sections (e.g., cryostat tissue sections and / or paraffin-embedded tissue sections).

[0474] The CDH6-binding molecules disclosed herein are capable of specifically binding to CDH6, and thus can be used to detect the presence or level of CDH6 in a sample, and to diagnose whether a subject has a disease associated with CDH6. For example, because CDH6 is expressed at a higher level on tumor cells (or cancer cells) than on healthy cells, the CDH6-binding molecules can be used for the diagnosis, prognosis, and therapeutic efficacy monitoring of cancer or tumors by detecting the expression level of CDH6 protein in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0475] Figure 1 shows the binding activity of the produced recombinant human CDH6 protein (CDH6-his tag) and the anti-CDH6 antibody Nov0712, wherein the control is an irrelevant protein, CDH6-Acro is a control commercial protein (purchased from Acro Biosystems), and CDH6-Sino is a control commercial protein (purchased from Sino Biologics).

[0476] FIG2 shows the binding activity of the constructed cell line overexpressing human CDH6 protein and the cell line overexpressing monkey CDH6 protein to the anti-CDH6 antibody Nov0712, respectively.

[0477] FIG3A shows the binding results of 2061-ChPR0003 and the humanized antibody to 786-O cells, and FIG3B shows the binding results of 2061-ChPR0022 and the humanized antibody to 786-O cells.

[0478] FIG4A shows the results of DT3C-dependent internalization and immunotoxin activity evaluation of 2061-ChPR0003 and the humanized antibody, and FIG4B shows the results of DT3C-dependent internalization and immunotoxin activity evaluation of 2061-ChPR0022 and the humanized antibody.

[0479] Figures 5A-5C show the binding activity of anti-CDH6 antibodies to CDH6 antigen proteins of different species, wherein Figure 5A shows the binding to human CDH6 protein antigen, Figure 5B shows the binding to monkey CDH6 protein antigen, and Figure 5C shows the binding to mouse CDH6 protein antigen.

[0480] 6A-6B show the protein antigen binding activity of anti-CDH6 antibodies to human CDH9 and CDH10, wherein FIG6A shows the binding activity to human CDH9, and FIG6B shows the binding activity to human CDH10.

[0481] Figures 7A-7E show the binding activity of anti-CDH6 antibodies to tumor cell lines with different CDH6 expression levels, wherein Figure 7A shows the binding to OVCAR3 tumor cells, Figure 7B shows the binding to SKOV3 tumor cells, Figure 7C shows the binding to 786-O tumor cells, Figure 7D shows the binding to PA-1 tumor cells, and Figure 7E shows the binding to NCI-N87 tumor cells.

[0482] Figures 8A-8B show the endocytic activity test of anti-CDH6 antibodies in the high-expressing cell line OVCAR3, wherein Figure 8A shows the endocytic result based on the Phrodol method, and Figure 8B shows the endocytic result mediated by the DT3C toxin secondary antibody.

[0483] Figures 9A-9D show the results of DT3C toxin secondary antibody-mediated killing activity of anti-CDH6 antibodies on tumor cell lines with different CDH6 expression levels, among which Figure 9A is a killing activity graph on the high-expressing cell line OVCAR3, Figure 9B is a killing activity graph on the medium- and low-expressing cell line 786-O, Figure 9C is a killing activity graph on the low-expressing cell line PA-1, and Figure 9D is a killing activity graph on the low-expressing cell line NCI-N87.

[0484] Figures 10A-10B are epitope analyses of anti-CDH6 antibodies, wherein Figure 10A is a cell binding diagram of overexpressed CDH6 proteins with different domain deletions, and Figure 10B is an epitope map of the antibody.

[0485] Figures 11A-11B are cell binding activity graphs of anti-CDH6 antibody drug conjugates, wherein Figure 11A is a graph showing the cell binding activity of positive controls ADC-3 and ADC-4, and Figure 11B is a graph showing the cell binding activity of ADC-1 and ADC-2 provided in the present disclosure.

[0486] Figures 12A-12C are graphs showing the cytotoxic activity of anti-CDH6 antibody-drug conjugates against tumor cell lines expressing different amounts of CDH6, wherein Figure 12A shows the cytotoxic activity of ADC-1 (DAR8), Figure 12B shows the cytotoxic activity of ADC-2 (DAR8), and Figure 12C shows the cytotoxic activity of hIgG1 (DAR4).

[0487] Figures 13A-13C show the anti-tumor activity results of the anti-CDH6 antibody-drug conjugate in the OVCAR3 cell xenograft model, wherein Figure 13A shows the comparison of anti-tumor activity at the same dose with the same DAR value, Figure 13B shows the comparison of anti-tumor activity at different DAR values ​​of the same toxin, and Figure 13C shows the weight change of the anti-CDH6 antibody-drug conjugate in the OVCAR3 mouse model.

[0488] Figures 14A-14C show the anti-tumor activity results of the anti-CDH6 antibody drug conjugate in a PA-1 cell xenograft model, wherein Figure 14A compares the anti-tumor activity of low doses with the same DAR value, Figure 14B compares the anti-tumor activity of multiple doses with different DAR values, and Figure 14C shows the body weight changes of the CDH6 ADC in the PA-1 mouse model.

[0489] FIG15 shows the anti-tumor activity of the anti-CDH6 antibody drug conjugate in a 786-O cell xenograft model. DETAILED DESCRIPTION

[0490] definition

[0491] In order to make the present disclosure more easily understood, certain technologies and sciences are specifically defined below. Unless otherwise explicitly defined in the present disclosure, all other technologies and sciences used in the present disclosure have the meanings commonly understood by those skilled in the art in the art to which the present disclosure belongs.

[0492] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0493] "CDH6" (also known as Cadherin6, K cadherin) is a member of the type II cadherin family. It is a single transmembrane protein composed of 790 amino acids, including 5 N-terminal extracellular domains from EC1 to EC5, a transmembrane domain and a C-terminal intracellular domain. The human CDH6 gene can be referenced under accession number P55285 (UniProt), the cynomolgus monkey gene can be referenced under accession number G7MUQ7-1 (UniProt), and the mouse gene can be referenced under accession number P97326 (UniProt). Within the scope of the present disclosure, "CDH6" covers the natural form of CDH6 in nature, naturally occurring variants, artificially expressed forms, and one or more amino acid substitutions, deletions and / or additions of the amino acid sequence of CDH6, and has biological activity equivalent to that of the CDH6 protein. When the context is not specifically stated, in the case of antigen-antibody interaction, CDH6 covers the scope of the complete protein, the extracellular domain and its epitope.

[0494] "CDH6 binding molecule" encompasses any protein, polypeptide, or any molecule comprising the protein or polypeptide that can specifically bind to an antigen (eg, human CDH6), including but not limited to antibodies or antigenic fragments thereof as defined in the present disclosure.

[0495] "Antibodies" encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or so-called immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds, such as IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of antibody heavy and light chains vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0496] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly suitable for use in a number of embodiments. In general, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" refers generally to products such as cells or nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0497] For the determination or definition of CDRs, the deterministic depiction of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions. (See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).In addition, other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or residue group does not significantly affect antigen binding. As used in this disclosure, CDRs can refer to CDRs defined by any method known in the art (including combinations of methods). The correspondence between the various numbering systems is well known to those skilled in the art.

[0498] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0499] For purposes of this disclosure, a "murine antibody" is a monoclonal antibody against human CDH6 or an epitope thereof, prepared according to the knowledge and skill in the art. This antibody is prepared by injecting a test subject with a CDH6 antigen, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional properties. In one embodiment of this disclosure, the murine CDH6 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0500] The term "human antibody" includes antibodies with variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include "humanized antibodies."

[0501] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into the human variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large number of non-human protein components. To avoid a simultaneous decrease in immunogenicity and activity, minimal reverse mutations can be performed on the fully human variable region to maintain activity. Examples of "humanization" include "humanizing" a Camelidae-derived VHH domain by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a VH domain of a conventional human tetrapeptide antibody (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removal of potential post-translational modification sites). A humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain human framework region sequences from IGHV3. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), that is, CDR "grafting" on other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and technologies suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies after affinity maturation of CDRs by phage display. In addition, in order to avoid a decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0502] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for the antigen, compared to a parent antibody that does not possess such alterations. For example, an "affinity matured" TRGV9 binding protein or anti-TRGV9 antibody has one or more changes in one or more CDRs that result in increased affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0503] A "chimeric antibody" is an antibody formed by fusing the variable region of an antibody from a first species with the constant region of an antibody from a second species, which can reduce the immune response induced by the antibody from the first species. As an example, to establish a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies is selected, and then the variable region genes are cloned from the mouse hybridoma cells. Then, the constant region genes of human antibodies are cloned as needed, and the mouse variable region genes and human constant region genes are connected to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, such as a heavy chain constant region comprising human IgG2 or IgG4, or an IgG1 that is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

[0504] "Antigen-binding fragments" include single-chain antibodies (i.e., heavy or light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabodies, tribodies, tetrabodies, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies can comprise multiple specificities, such as bispecifics or monospecifics (see, for example, WO92 / 22583 and WO05 / 113605).

[0505] Typically, the CDH6 binding molecules of the present disclosure will be expressed as preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 - 9 to 10 -10 or lower dissociation constant (K D ), and / or with at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen or target protein (ie, human CDH6). Any -4 M's K DValues ​​are generally considered to indicate nonspecific binding.Specific binding of a binding molecule (e.g., an antibody) to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding as described herein.

[0506] "Epitope" refers to the site on the antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids (non-adjacent amino acids are brought into close proximity in space by the tertiary folding of the protein). Epitopes formed by adjacent amino acids are typically retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are typically lost after treatment with a denaturing solvent. Epitopes typically exist in a unique spatial conformation that includes at least 3-15 amino acids. Methods for determining epitopes are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0507] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the KD The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​were compared with those for non-target interactions (e.g., control antibodies known not to bind IGF-1R or TRGV9). D The value is evaluated.

[0508] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0509] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0510] "Cross-reactivity" refers to the ability of an antibody of the present disclosure to bind to CDH6 from a different species. For example, a CDH6 binding molecule (e.g., an antibody) of the present disclosure may also bind to CDH6 from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA), or binding or functional interaction with cells expressing CDH6. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.

[0511] "Internalization" refers to the transport of a portion from the outside of a cell to the inside. The internalized portion can be located in an intracellular compartment. An "internalized" or "internalized" antigen or antibody refers to an antigen or antibody that is able to be transported from the outside of a target cell to the inside. It is generally understood by those skilled in the art that the process of cellular internalization generally refers to the movement of a cell surface molecule across the plasma membrane from the cell surface to the inside of the cell. After internalization, the endosome can be transported to the lysosome for degradation or recycled to the cell surface. The cellular internalization rate of a given cell surface molecule provides a measure of the kinetics of the movement of the molecule from the cell surface across the plasma membrane to the inside of the cell. Internalization activity or internalization rate of antigens and antibodies can be monitored and / or measured by various techniques known in the art, including acid dissociation (Li N. et al., Methods Mol. Biol., 457:305–17, 2008) and toxin killing assays (Pahara J. et al. Exp Cell Res., 316:2237–50, 2010; and Mazor et al., J. Immunol. Methods, 321:41–59, 2007). Many antibody labeling techniques, dyes, and kits for antibody labeling that can be used to quantify and monitor internalization are commercially available (e.g., pHrodo iFL antibody labeling methods, reagents, and kits sold by Thermo Fisher Scientific). For example, antibody-drug conjugates (ADCs) bind to tumor cell surface antigens through the antibody in the ADC, which is then internalized into endosomes and converted from endosomes to lysosomes. Then, under the action of hydrolases in the lysosomes, the bioactive molecules (e.g., toxins or payloads) are dissociated from the ADC. The dissociated bioactive molecules enter the cytoplasm from the lysosomes and kill tumor cells. The bioactive molecules that escape from the killed tumor cells can further kill surrounding tumor cells that do not express or express low levels of the antigen (the so-called bystander effect).

[0512] "Linker" refers to a fragment or bond that is connected to a ligand at one end and to a drug at the other end, and can also be connected to other linkers before being connected to a ligand or drug. The linker can include one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), and those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"). The linker may include a stretching unit, a spacer unit, an amino acid unit, and an extension unit, and may be synthesized by methods known in the art, such as those described in US 2005-0238649A1. The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, an acid-labile linker (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker may be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0513] "Amino acid unit" means, if there is a stretching unit, the following structure Y R The carbonyl group in is connected to the extension unit. If there is no extension unit, Y R The amino acid directly linked to the cytotoxic drug, in the embodiment of the present disclosure, the amino acid unit is represented by -K k -:

[0514] -K k - is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide, and the -K- units each independently have the following structural formula K a or K b , k is an integer between 0 and 10:

[0515] in:

[0516] R in the above amino acid unit 23 is -H or methyl;

[0517] R 24is H, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

[0518] R 25 It is -aryl-, -alkyl-aryl-, -cycloalkyl-, -alkyl-cycloalkyl-, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-alkyl-, -heterocyclyl-, -alkyl-heterocyclyl-, -heterocyclyl-alkyl-, -alkyl-heterocyclyl-alkyl-, -aryl-, -alkyl-aryl-, -aryl-alkyl-, -alkyl-aryl-alkyl-, -heteroaryl-, -alkyl-heteroaryl-, -heteroaryl-alkyl-, -alkyl-heteroaryl-alkyl-.

[0519] In one embodiment, -K k - is a dipeptide, preferably -valine-citrulline-, -phenylalanine-lysine- or -N-methylvaline-citrulline-, more preferably -valine-citrulline-.

[0520] "Stretcher" refers to a chemical structure segment that is covalently linked to a ligand via a carbon atom at one end and to a cytotoxic drug via a sulfur atom at the other end.

[0521] The "spacer unit" is a bifunctional compound structural fragment that can be used to couple the linker unit and the cytotoxic drug to ultimately form a ligand-cytotoxic drug conjugate. This coupling method can selectively connect the cytotoxic drug to the linker unit.

[0522] "Stretcher unit" refers to a chemical structure that can couple the amino acid unit to the cytotoxic drug when the amino acid unit is present, or can couple the cytotoxic drug to the cytotoxic drug through the carbonyl group on YR when the amino acid unit is absent. q -, q is selected from 0, 1, 2.

[0523] The extension unit in the present disclosure is PAB, which has a structure such as a 4-iminobenzylcarbamoyl fragment, and is connected to D as shown in the following formula:

[0524] abbreviation

[0525] Connector components include but are not limited to:

[0526] MC=6-maleimidocaproyl, the structure is as follows:

[0527] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker)

[0528] Citrulline = 2-amino-5-ureidopentanic acid

[0529] PAB = p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker component)

[0530] Me-Val-Cit = N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to protect it from cleavage by cathepsin B)

[0531] MC(PEG)6-OH = Maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines)

[0532] SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate

[0533] SPDP = N-succinimidyl 3-(2-pyridyldithio) propionate

[0534] SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

[0535] IT = Iminothiolane

[0536] PBS = Phosphate buffered saline

[0537] "Immunoconjugate," "antibody drug conjugate (ADC)," and "ligand drug conjugate" are used interchangeably in this disclosure to refer to an antibody or fragment thereof linked to a drug via a linker (or linking unit). In some embodiments, an "immunoconjugate" in this disclosure refers to an anti-CDH6 antibody or antigen-binding fragment thereof linked to a toxic drug via a linker.

[0538] "Drug loading" refers to the average number of cytotoxic drugs loaded on each ligand in the ADC, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be 1-20, preferably 1-10 cytotoxic drugs (D) connected to each antibody (Pc). In embodiments of the present disclosure, drug loading is expressed as n or k, and exemplary is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two numerical values. Preferably 1-10, more preferably 1-9, or 1-8, or 1-7, or 2-8, or 2-7, or 2-6, or 2-5, or 2-3, or 1-2, or 2-4, or 1-4, or 1-5, or 1-6, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5 mean. Conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, CE-SDS, and HPLC characterization can be used to identify the average amount of drug substance per ADC molecule after the conjugation reaction. The disclosed CE-SDS method can quantitatively determine the purity of a recombinant monoclonal antibody product by capillary electrophoresis (CE-SDS) with ultraviolet detection using sodium dodecyl sulfate (UVD) under reducing and non-reducing conditions, based on molecular weight, according to capillary electrophoresis (Chinese Pharmacopoeia 2015, 0542).

[0539] In one embodiment of the present disclosure, the cytotoxic drug is coupled to the N-terminal amino group and / or the ε-amino group of the lysine residue of the ligand via a linker. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.

[0540] The drug loading of antibody drug conjugates can be controlled by the following non-limiting methods, including:

[0541] (1) Control the molar ratio of the linker and the monoclonal antibody,

[0542] (2) Control reaction time and temperature,

[0543] (3) Select different reaction reagents.

[0544] Although for a particular conjugate molecule, the drug to antibody ratio has an exact value (e.g., n in formula (I)), it will be understood that when used to describe a sample containing many molecules, the value will often be an average value, due to a certain degree of non-uniformity typically associated with the conjugation step. The average loading of an antibody drug conjugate sample is referred to herein as the drug to antibody ratio or "DAR." In some embodiments, the DAR is between about 1 and about 6, and is typically about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0. In some embodiments, at least 50% of the sample by weight is a compound having an average DAR plus or minus 2, and preferably at least 50% of the sample is a conjugate containing an average DAR plus or minus 1. Embodiments include wherein the DAR is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,. , 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 10.1, 10.2, 11.3, 12.4, 13.5, 14.6, 15.7, 16.8, 17.9, 18.0, 19.1, 20.2, 21.3, 22.4, 23.5, 24.6, 25.8, 26.9, 27.0, 28.1, 29.3, 30.5, 31.7, 32.5, 33.6, 34.

[0545] The detection method of DAR, for example, is to extrapolate the DAR value from the LC-MS data of reduced and deglycosylated samples. LC / MS allows the quantification of the average number of payload (drug moiety) molecules connected to the antibody in the ADC. HPLC separates the antibody into light and heavy chains, and also separates the heavy chain (HC) and light chain (LC) according to the number of linker-payload groups of each chain. Mass spectrometry data can identify the types of components in the mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc. Based on the average loading amount of the LC and HC chains, the average DAR of the ADC can be calculated. The DAR of a given immunoconjugate sample represents the average number of drug (payload) molecules connected to a tetrameric antibody containing two light chains and two heavy chains. For example, the DAR detection method in WO2018142322.

[0546] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0547] "Heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

[0548] "Alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same or two different carbon atoms of an alkane group by removing two hydrogen atoms, and is a straight or branched group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). The alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0549] "Alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl are as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0550] "Cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0551] "Heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, the ring contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spirocyclic, fused, and bridged heterocyclyls.

[0552] "Spiro heterocyclyl" refers to a polycyclic heterocyclic group with 5 to 20 members in which the monocyclic rings share one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. For example, it is 6 to 14 members, and for example, it is 7 to 10 members. According to the number of shared spiro atoms between the rings, the spiro heterocyclic group is divided into a monospiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a monospiro heterocyclic group and a dispiro heterocyclic group. For example, it is 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan monospiro heterocyclic group. Non-limiting examples of spiro heterocyclic groups include:

[0553] "Fused heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 20 members, wherein each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, and one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatom, and the remaining ring atoms are carbon. For example, it is 6 to 14 members, and another example is 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, for example, it is bicyclic or tricyclic, and another example is a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

[0554] "Bridged heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 14 members, wherein any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system, and wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. For example, it is 6 to 14 members, and another example is 7 to 10 members. Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic group, for example, a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged heterocyclic groups include:

[0555] The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:

[0556] wait.

[0557] The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0558] "Aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, for example, 6- to 10-membered, such as phenyl and naphthyl, with phenyl being a specific example. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0559] The aryl group may be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0560] "Heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5-membered or 6-membered, for example, furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0561] The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0562] An "amino-protecting group" is a readily removable group used to protect an amino group, thereby preserving the amino group during reactions elsewhere in the molecule. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl. These groups may optionally be substituted with one to three substituents selected from halogen, alkoxy, or nitro. The amino-protecting group is preferably 9-fluorenylmethyloxycarbonyl.

[0563] "Aminoheterocyclyl" refers to a heterocyclyl group substituted by one or more amino groups, preferably by one amino group, wherein heterocyclyl is as defined above, and wherein "amino" refers to -NH2. Representative embodiments of the present disclosure are as follows:

[0564] "Heterocyclylamino" refers to an amino group substituted by one or more heterocyclyl groups, preferably substituted by one heterocyclyl group, wherein amino is as defined above and heterocyclyl is as defined above. Representative embodiments of the present disclosure are as follows:

[0565] "Cycloalkylamino" refers to an amino group substituted by one or more cycloalkyl groups, preferably by one cycloalkyl group, wherein amino is as defined above, and wherein cycloalkyl is as defined above. Representative embodiments of the present disclosure are as follows:

[0566] "Cycloalkylalkyl" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably with one cycloalkyl group, wherein alkyl is as defined above, and wherein cycloalkyl is as defined above.

[0567] "Haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0568] "Deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0569] "Hydroxy" refers to an -OH group.

[0570] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0571] "Amino" refers to -NH2.

[0572] "Nitro" refers to -NO2.

[0573] The abbreviation "Me" in the chemical formula is methyl.

[0574] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms, in a group are replaced independently of one another by substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g., olefinic) bonds.

[0575] "Nucleic acid" or "polynucleotide" are used interchangeably in this disclosure to refer to any DNA or RNA molecule, whether single-stranded or double-stranded, and, in the case of single-stranded, its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0576] "Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporating polynucleotide inserts. Host cells include the progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0577] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0578] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0579] "Treatment" means administering an internal or external therapeutic agent, such as a binding protein or a pharmaceutical composition thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0580] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0581] Excipients are substances added to pharmaceutical preparations in addition to the main drug, also known as excipients. For example, preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations can all be called excipients.

[0582] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" used in phrases such as "A and / or B" in the present disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to." In the context of mutations contained in the Fc region in the present disclosure, " / " means "and," for example, "354C / 366W" means "354C and 366W," that is, the Fc contains 354C and 366W mutations; the amino acid positions of the mutations in the Fc region of the present disclosure are all numbered according to the EU numbering system.

[0583] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans.

[0584] Example

[0585] The following examples further illustrate the present disclosure, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0586] Example 1. Preparation of recombinant CDH6 extracellular domain protein

[0587] The amino acid sequence of the extracellular region (Thr19-Ala615) of the human CDH6 protein (UniProt, accession number: P55285) was selected, and a polyhistidine (His) tag was added to the C-terminus. The antigen gene plasmid was synthesized according to methods well known in the art, and an expression vector containing the antigen gene was obtained by enzyme digestion, ligation and transformation. Clones with the correct insertion sequence were screened by DNA sequencing for large-scale extraction of the plasmid. The extracted expression vectors were transiently expressed in Expi293 cells for 7 days. The expression supernatant was purified and prepared after ELISA detection. The protein was tested for activity using the anti-CDH6 antibody Nov0712 (the sequence of Nov0712 is shown in SEQ ID NOs 234 and 235 of US2016 / 0046711A1). The test results are shown in Figure 1, which shows that the produced human CDH6-His protein has good binding activity with the anti-CDH6 antibody Nov0712.

[0588] The recombinant monkey CDH6-His protein was obtained by fusion of a His tag at the C-terminus of the amino acids in the extracellular region (Thr22-Gly614) of the monkey CDH6 protein (UniProt, accession number: G7MUQ7-1) and purchased from Acro Biosystems.

[0589] Example 2. Construction of a stable cell line expressing recombinant CDH6 protein

[0590] After transfecting CHO-K1 cells with a plasmid encoding recombinant human CDH6 protein (UniProt, accession number: P55285) or a plasmid encoding cynomolgus monkey CDH6 protein (UniProt, accession number: G7MUQ7-1), a cell line stably expressing recombinant human or cynomolgus monkey CDH6 protein was obtained by screening. In short, the nucleotide sequence encoding human or cynomolgus monkey CDH6 protein was cloned and connected to a lentiviral expression vector. The CHO-K1 cell line was transfected using a lentiviral transfection method well known in the industry, and selectively cultured for two weeks using F-12 medium containing Puromycin (purchased from Gibco). Subcloning was performed in a 96-well culture plate using the limiting dilution method, and the cells were cultured in a cell culture incubator. After two weeks, the selected monoclones were detected and screened by flow cytometry. The results in Figure 2 show that the selected cell line hCDH6-CHOK1 can stably express the full-length human CDH6 protein, and CynoCDH6-CHOK1 can stably express the full-length monkey CDH6 protein.

[0591] Example 3. Screening of mouse anti-CDH6 monoclonal antibodies

[0592] The recombinant human CDH6-His described in Example 1 was used as an immunogen to immunize 6-8 week old SJL mice. The primary immunization dose was 50 μg per mouse. Two weeks after the primary immunization, a booster immunization was performed at a dose of 25 μg per mouse. Each subsequent booster immunization was performed two weeks apart. Serum samples were collected one week after each booster immunization, and the antibody activity in the mouse serum was detected by protein ELISA and FACS. The specific detection process of protein ELISA was as follows: the plate was coated with 1 μg / mL recombinant human CDH6-His, incubated overnight at 4°C, blocked with PBST buffer containing 1% BSA for 1 hour, and washed three times. The mouse serum was diluted 10-fold in the blocking buffer starting at 1:100, incubated at 37°C for 1 hour, washed three times, and incubated with a secondary antibody against mouse IgG-Fc-HRP for 1 hour. The wells were washed three times with PBST, 100 μL of TMB substrate was added to each well, and the reaction was terminated with 2M HCl after 15 minutes. The absorbance at 450 nm was read using a microplate reader. The FACS assay procedure is as follows: 1E5 cells / 100 μL per well were plated, centrifuged, and the supernatant discarded. A serial dilution of mouse serum was added. Incubated at 4°C for 1 hour. The cells were washed twice with 1% BSA / PBS buffer, the supernatant discarded, and Alexa Flour 647 goat anti-mouse secondary antibody (purchased from Jackson Immuno Research) was added. The cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with 1% BSA / PBS buffer, resuspended in 200 μL of buffer, and analyzed by FACS. Sera from mice immunized with the recombinant human CDH6-his immunogen showed varying degrees of binding to the immunogen, with the highest serum dilution of 1:100K still showing an antigen-antibody reaction.

[0593] The last immunization was intraperitoneal injection of 25 μg recombinant human CDH6-His. Four days later, the mice were killed and the spleens were removed. Splenocytes were collected by grinding. NH4OH at a final concentration of 1% (w / w) was added to lyse the red blood cells mixed in the spleen cells to obtain a spleen cell suspension. The cells were centrifuged and washed three times at 1000 rpm. Mouse spleen cells were mixed with mouse myeloma cells SP2 / 0 at a ratio of 5:1 in terms of the number of viable cells, and cell fusion was performed using a high-efficiency electrofusion method. The fused cells were diluted with DMEM medium containing 20% ​​fetal bovine serum and 1×HAT (w / w) to a 96-well cell culture plate, with 200 μL per well for a total of 1× 10 5Cells were plated and placed in a 37°C incubator with 5% (v / v) CO2. After 14 days, the fused cells were screened using ELISA or Acumen. Positive clones with an OD450nm > 0.1 were expanded to 24-well cell plates and further cultured at 37°C with 5% (v / v) CO2 in DMEM medium supplemented with 10% (w / w) HT fetal bovine serum. After 3 days, the culture fluid from the 24-well cell plates was centrifuged and the supernatant was collected. Binding activity against recombinant human CDH6-positive cells, recombinant monkey CDH6-positive cells, and related tumor cells was determined by FACS. After initial subclone screening and retesting, excellent positive monoclonal strains were identified using the aforementioned ELISA and FACS assays. The sequenced amino acid sequence of the antibody is shown below. The underlined portion represents the complementary determining region (CDR) sequence (using the Kabat numbering system).

[0594] Table 1. CDR sequences of murine anti-CDH6 antibodies (Kabat numbering system)

[0595] Example 4. Preparation of anti-CDH6 chimeric and humanized antibodies

[0596] To reduce potential immunogenicity, murine antibodies are humanized and the constant regions of the murine monoclonal antibody are replaced to create recombinant chimeric antibodies. The nucleotide sequence encoding the variable region of the murine monoclonal antibody is cloned into a vector containing the human heavy and light chain constant region (human IgG1, kappa) protein sequences and transfected into CHO cells to create a chimeric antibody whose heavy and light chain variable regions are identical to those of the murine antibody, respectively.

[0597] For humanized antibodies, after predicting the structure of the mouse monoclonal antibody through homology modeling, the mouse CDRs were chimerized into a suitable human GermLine framework (Bioinformation. 2014; 10(4): 180–186; Methods Mol Biol. 2019; 1904: 213-230). The variable region framework selection is shown in Table 2. Subsequently, back mutations were introduced at sites that may affect antibody-antigen binding. Finally, the nucleotide sequence encoding the variable region of the humanized monoclonal antibody was cloned into a vector containing the human heavy and light chain constant region (Human IgG1, kappa) protein sequence and transfected into CHO cells to produce the antibody. The VH and VL sequences obtained after humanization are shown below, with the CDR regions of the sequences underlined. The humanized antibodies obtained by combining different VH and VL sequences are shown in Table 3.

[0598] Table 2. Selection of humanized anti-CDH6 antibody variable region frameworks

[0599] Table 3. Corresponding VH and VL of humanized antibodies

[0600] Example 5. Binding activity of anti-CDH6 antibodies to CDH6-expressing tumor cells and endocytosis activity detection

[0601] 1. FACS experiments were used to detect the binding characteristics of anti-CDH6 antibodies to CDH6-expressing tumor cell lines.

[0602] 786-O cells express CDH6 and are cultured in 1640 growth medium supplemented with 10% fetal bovine serum (Gibco, cat#10099141C). The assay is as follows: Step 1: Plate 786-O cells (2E5 cells / well). Step 2: Add the test antibody at 100 μL / well, using a top concentration of 100 nM, followed by 3-fold dilutions for a total of 8 concentrations, and incubate at 4°C for 1 hour. Step 3: Add the secondary antibody, anti-hIgG Alexa Fluor-647 (Jackson, cat#209-605-088), at a dilution ratio of 1:800 and incubate at 4°C for 30 minutes. Step 4: Perform MFI analysis using flow cytometry. Isotype hIgG1 was used as a negative control.

[0603] The binding results of 2061-ChPR0003 and the humanized antibody to 786-O cells are shown in Figure 3A , and the binding results of 2061-ChPR0022 and the humanized antibody to 786-O cells are shown in Figure 3B . The results show that the humanized antibodies still maintain good binding activity.

[0604] 2. DT3C Antibody Internalization and Cytotoxicity Evaluation System

[0605] DT3C is a recombinantly expressed fusion protein with a molecular weight of 70 kDa. It is composed of the fusion of Fragment A of diphtheria toxin (toxin only) and the 3C fragment of group G Streptococcus (IgG binding portion). This protein has a high affinity for the IgG portion of the antibody and enters the cell together with the antibody during endocytosis. Under the action of intracellular furin, DT is released, which is toxic. DT can inhibit the activity of EF2-ADP ribosylation, block protein translation, and ultimately lead to cell death. By using this system, it is possible to simultaneously observe the internalization of the antibody and the cell-killing effect caused by the immunotoxin (Biochem Biophys Res Commun. 2014 Nov 28; 454(4): 600-3.).

[0606] Evaluation of DT3C-dependent internalization and immunotoxin activity: Sterile-filtered DT3C and the chimeric antibody to be tested (DT3C at a molar concentration twice that of the antibody) were mixed at a 1:1 ratio, incubated at 37°C for 30 minutes, and then serially diluted with complete medium. The mixture was added to 786-O cells plated one day earlier (2000 cells / well) and incubated in a 5% CO2 incubator at 37°C for 6 days. CellTiter-Glo was added, incubated at room temperature in the dark for 10 minutes, and chemiluminescence was measured on a PerkinElmer ELISA. As shown in Figures 4A and 4B, both humanized antibodies 2061-ChPR0003 and 2061-ChPR0022 maintained strong anti-cancer activity.

[0607] Example 6. Further modification of humanized antibodies

[0608] After evaluating humanized antibodies, 2061-rH3rL1B and 2061-H2Lptm3 were selected for TCE and PTM risk removal. These antibodies, 2061-1 and 2061-3, were generated, respectively. The heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and light chain CDRs (LCDR1, LCDR2, and LCDR3) are shown in Table 4. CDR regions are underlined.

[0609] Table 4. Variable region sequences of humanized anti-CDH6 antibodies (Kabat numbering system)

[0610] The obtained humanized sequences are as follows:

[0611] In conjunction with Examples 3 to 6, the antibody derived from 2061-ChPR0003 disclosed herein has the following CDRs:

[0612] HCDR1, HCDR2, and HCDR1 are shown in SEQ ID NOs: 5, 6, and 7, respectively.

[0613] LCDR1 is: X1ASSTVGSSYLY (SEQ ID NO: 24), wherein X1 is N or T,

[0614] LCDR2 is: STSNLAX2 (SEQ ID NO: 25), wherein X2 is S or T,

[0615] LCDR3 is shown in SEQ ID NO: 10;

[0616] In some embodiments, LCDR1 is selected from NASSTVGSSYLY (SEQ ID NO: 8) or

[0617] TASSTVGSSYLY (SEQ ID NO: 21), LCDR2 is selected from STSNLAS (SEQ ID NO: 9) or

[0618] STSNLAT (SEQ ID NO: 22).

[0619] The antibody derived from 2061-ChPR0022 of the present disclosure has the following CDRs:

[0620] HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 11, 12, and 13, respectively.

[0621] LCDR1 is: RSSQSIVHSX3X4NTYLE (SEQ ID NO: 26), wherein X3 is N or T, and X4 is G or A;

[0622] LCDR2 and LCDR3 are shown in SEQ ID NOs: 15 and 16, respectively;

[0623] In some embodiments, LCDR1 is selected from RSSQSIVHSNGNTYLE (SEQ ID NO: 14) or RSSQSIVHSTGNTYLE (SEQ ID NO: 23) or RSSQSIVHSNANTYLE (SEQ ID NO: 70; derived from SEQ ID NO: 69).

[0624] An exemplary full-length sequence is as follows, wherein the constant region is in italics:

[0625] >2061-1 heavy chain

[0626] >2061-1 light chain

[0627] >2061-3 heavy chain

[0628] >2061-3 light chain

[0629] >IgG1 Fc region

[0630] Example 7. Binding activity of anti-CDH6 antibodies to CDH6 protein antigens from different species

[0631] ELISA assays were used to test the binding properties of anti-CDH6 antibodies from different species. CDH6 recombinant protein with a His tag was directly coated. After the antibody was added, the antibody-antigen binding activity was detected by adding a secondary antibody (HRP-conjugated anti-primary antibody Fc) and the HRP substrate TMB.

[0632] Coat a 96-well microtiter plate with CDH6-His proteins from different species (Acro, cat#CA6-H5229 for human Cadherin-6; Acro, cat#CA6-C52H3 for Cynomolgus / Rhesus macaque Cadherin-6; Acro, cat#CA6-M52H8 for mouse Cadherin-6). Add 100 μL per well at a concentration of 1 μg / mL and incubate overnight at 4°C. Wash three times with 250 μL per well of Wash Buffer, shaking for 10 seconds between washes to ensure thorough washing. Add 200 μL / well of Blocking Buffer (3% BSA) and incubate at room temperature for 2 hours. Wash three times with 250 μL per well of Wash Buffer, shaking for 10 seconds between washes to ensure thorough washing. Add 100 μL of the anti-CDH6 test antibody diluted in Diluent to each well. Incubate at room temperature for 1 hour, wash three times with the wash buffer, and add 100 μL of HRP-conjugated goat anti-human IgG secondary antibody (BETHYL, Cat#A80-319P-29) diluted 1:20,000 in diluent to each well (250 μL per well). Incubate at room temperature for 1 hour, wash three times with the wash buffer, and add 100 μL of TMB mixture (Biopanda, cat#TMB-S-003, Lot#2022122901 for TMB colorimetric solution A, cat#TMB-S-003, Lot#2022122902 for TMB colorimetric solution B) to each well. Protect the well from light and observe the color reaction. After approximately 5 minutes, add 50 μL of stop solution (Solarbio, cat#C1058) to each well. Read the OD at 450 nm using an Envision microplate reader. Nov0712 antibody was used as a positive control and isotype IgG1 as a negative control.

[0633] The results showed that both 2061-1 and 2061-3 could bind to human and monkey CDH6 antigens well, as shown in Figures 5A, 5B, and 5C. 2061-1 could bind to mouse CDH6 well. Calculation of the binding EC of anti-CDH6 antibodies 50 The results are shown in Table 5.

[0634] Table 5. Affinity EC of each antibody binding to human, monkey, and mouse CDH6 antigens 50 Value and Emax value

[0635] Example 8. Antigen Binding Activity of Anti-CDH6 Antibodies to Human CDH9 and CDH10 Proteins of the Same Family

[0636] The CDH protein family also includes CDH9 and CDH10, which share the highest homology with CDH6. The sequence homology of human CDH9 and CDH10 with each ECD of human CDH6 is shown in Table 6. CDH10 is expressed in the brain, and nonspecific binding to CDH9 or CDH10 may pose unknown toxicity risks. Therefore, binding assays were performed in this example.

[0637] Human CDH9 protein (Acro, cat#CA9-H52H6) and human CDH10 protein (Acro, cat#CA0-H52H5) were coated onto a 96-well microtiter plate at a concentration of 5 μg / mL. 100 μL was added to each well and incubated overnight at 4°C. The plates were washed three times with 250 μL per well of the wash buffer, with vortexing for 10 seconds between washes to ensure thorough washing. 200 μL / well of blocking buffer (3% BSA) was added and incubated at room temperature for 2 hours. The plates were washed three times with 250 μL per well of the wash buffer, with vortexing for 10 seconds between washes to ensure thorough washing. 100 μL of anti-CDH6 antibody to be tested, diluted in diluent, was added to each well. The plates were incubated at room temperature for 1 hour. The plates were then washed three times with 250 μL per well of the wash buffer, and 100 μL of HRP-conjugated goat anti-human IgG secondary antibody (BETHYL, Cat#A80-319P-29) diluted 1:20,000 in diluent was added to each well. Incubate at room temperature for 1 hour, then wash three times with 250 μL per well. Add 100 μL of TMB mixture (Biopanda, cat#TMB-S-003, Lot#2022122901 for TMB colorimetric solution A, cat#TMB-S-003, Lot#2022122902 for TMB colorimetric solution B) to each well. Protect from light and observe the color reaction. After approximately 5 minutes, add 50 μL of stop solution (Solarbio, cat#C1058) per well. Read the OD value at 450 nm using an Envision microplate reader.

[0638] The results showed that, as shown in Figures 6A and 6B, 2061-1, 2061-3, and the control had no antigen binding activity for human CDH9, but the control molecule had binding ability for human CDH10, while 2061-1 and 2061-3 had no binding ability for human CDH10. Calculation of the binding EC of anti-CDH6 antibodies 50 For details, see Table 7.

[0639] Table 6. Sequence homology of human CDH9 and CDH10 with each ECD of human CDH6

[0640] Table 7. EC affinity of each antibody binding to human CDH9 and CDH10 antigens 50 Value and Emax value

[0641] Example 9. Detection of affinity between anti-CDH6 antibodies and CDH6 on the cell membrane surface of tumor cell lines with different CDH6 expression levels

[0642] The binding properties of anti-CDH6 antibodies were tested by FACS against tumor cell lines expressing different levels of CDH6. OVCAR3 cells were cultured in 1640 growth medium supplemented with 0.01 mg / mL bovine insulin (Solarbio, cat#I8040) and 20% fetal bovine serum (Gibco, cat#10099141C). 786-O cells were cultured in 1640 growth medium supplemented with 10% fetal bovine serum (Gibco, cat#10099141C). PA-1 and NCI-N87 cells were cultured in MEM (with ENEAA) growth medium supplemented with 0% fetal bovine serum (Gibco, cat#10099141C). SKOV3 cells were cultured in McCoy's 5A growth medium supplemented with 10% fetal bovine serum (Gibco, cat#10099141C). Among them, OVCAR3 and SKOV3 cells are CDH6-high-expressing cells, and 786-O, PA-1, and NCI-N87 cells are CDH6-low-expressing cells. Nov0712 antibody and Daiichi07 antibody (Daiichi07 sequence is shown in US2020 / 0390900A1, heavy chain variable region is SEQ ID NO 25, light chain variable region is SEQ ID NO 20, isotype full-length antibody is constructed) were used as positive controls, and isotype hIgG1 was used as a negative control.

[0643] The experimental method is the same as the FACS in Example 5. The binding results of anti-CDH6 antibodies to tumor cell lines with different CDH6 expression levels are shown in Figures 7A, 7B, 7C, 7D, and 7E. The anti-CDH6 antibodies disclosed herein have good binding activity against tumor cell lines with high or low CDH6 expression. EC 50 For details of the values ​​and Max MFI values, see Table 8.

[0644] Table 8. EC affinity of each antibody to tumor cell lines with different CDH6 expression levels 50 Value and Emax value

[0645] Example 10. Detection of endocytic activity of anti-CDH6 antibodies

[0646] 1. pHrodo Indicator Reagent to Assess Internalization of Anti-CDH6 Antibodies

[0647] Zenon TM pHrodo TMiFL IgG Indicator Reagent (Invitrogen, Cat. No. Z25611) provides a rapid, real-time, and reliable method for assessing antibody internalization. The pHrodo iFL Green-labeled Fab fragment binds to the Fc portion of intact IgG antibodies, forming a labeled complex within 5 minutes. Once the complex enters the cell via endocytosis, fluorescence increases dramatically as the acidity of the surrounding environment increases. The fluorescence intensity can be recorded in real time using the Incucyte instrument, thereby determining the extent of antibody internalization. Specifically, cells are plated with 2500 OVCAR3 cells per well in 50 μL of the labeled complex. After the cells adhere, 50 μL of the labeled complex is added to each well. In a single centrifuge tube, the labeled complex is prepared: 20 nM antibody and 60 nM pHrodo Green, mixed, and incubated at room temperature for 15 minutes. The plate is placed in the Incucyte instrument (Incucyte S3) and the plate reading conditions are set according to the instrument's operating procedures. After the plate reading is completed, the data is analyzed using the instrument's built-in analysis software. The results showed that, as shown in FIG8A , 2061-1 and 2061-3 had significantly better endocytic activity than the positive controls Daiichi07 and Nov0712, among which 2061-1 had stronger endocytic activity.

[0648] 2. DT3C Antibody Internalization and Cytotoxicity Evaluation System

[0649] The experimental method was as described in Example 5. The results are shown in FIG8B . 2061-1 had stronger internalization and immunotoxin killing activity on high-expressing OVCAR3 cells, and the internalization and immunotoxin killing activity of 2061-3 molecule was comparable to that of the positive control.

[0650] Example 11. Detection of DT3C Killing Activity of Anti-CDH6 Antibodies against Tumor Cell Lines with Different CDH6 Expression Levels

[0651] In order to evaluate the difference in DT3C killing activity of the antibodies disclosed herein on tumor cells with different CDH6 expression levels, tumor cells with different CDH6 expression levels were selected for evaluation of DT3C killing activity, and the method was as described in Example 5. The selected tumor cell lines were OVCAR3, 786-O, PA-1, and NCI-N87. The cell density per well was 2000 (OVCAR3) or 1000 (786-O) or 3000 (PA-1, NCI-N87). The results are shown in Figures 9A, 9B, 9C, and 9D. The antibodies disclosed herein have antigen-dependent cell killing activity, and the killing activity of 2061-1 is better than that of the positive control antibodies Daiichi07 and Nov0712, and the killing activity of 2061-3 is comparable to that of the positive control antibody Daiichi07. Calculate EC 50 See Table 9 for values.

[0652] Table 9. EC of each antibody against DT3C killing of tumor cell lines with different CDH6 expression levels 50 Value and Top viability% value

[0653] Example 12. Epitope differentiation between anti-CDH6 antibodies

[0654] Plasmids with different CDH6 domain deletions were synthesized. The sequences of different EC regions are shown in Table 10. The sequences of EC1 to 5 domains refer to Uniprot's annotations on human CDH6 protein and SEQ1 to 5 sequences in patent US2020 / 0390900A1. Plasmids with different domain deletions were highly expressed in HEK293 cells. After digestion, the HEK293 cells were centrifuged and the supernatant was discarded. The cells were resuspended in 3E 6 / mL, add 50μL / well to 96-well plate. Add 50μL of the test antibody (final concentration 50nM) and incubate at 4℃ for 1 hour. After washing twice, add Alexa Fluor 500 at a dilution ratio of 1:800. 647AffiniPure Mouse Anti-Human IgG (Fcγfragment specific) secondary antibody for flow cytometry (Jackson, 209-605-098) was added and incubated at 4°C for 30 min. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. The cells were washed twice with PBST and resuspended in 150 μL of supernatant per well. Alexa Fluor 500 was detected by flow cytometry. The fluorescence intensity was 647. The results showed that the binding of each antibody was detected by FACS as shown in Figure 10A, and the analysis of each epitope was shown in Figure 10B. 2061-1 bound to the EC1 domain, and 2061-3 bound to the EC3 domain.

[0655] Table 10. Sequences of different domains in the extracellular region of human CDH6 protein

[0656] Example 13. Preparation of Antibody Drug Conjugate (ADC)

[0657] 1. Preparation of Compound 9-A

[0658] Compound 9-A (i.e., compound 9-A of Example 9 of WO2020063676A1) is N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide. WO2020063676 is incorporated into the present disclosure by reference in its entirety.

[0659] 2. Design and Preparation of ADC

[0660] The present disclosure prepares ADCs with the following structures, wherein, when Ab is 2061-1, it is ADC-1; when Ab is 2061-3, it is ADC-2; when Ab is Daiichi07, it is ADC-3; and when Ab is Nov0712, it is ADC-4.

[0661] Preparation of ADC:

[0662] At 37°C, a solution of anti-CDH6 antibody in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 5.0 mL, 342.5 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (10 mM, 75.4 μL, 753.5 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0663] Compound 9-A (3.7 mg, 3424.6 nmol) was dissolved in 250 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted using HiPrep26 / 10 (buffer: PBS pH 7.2-7.4, flow rate 10 mL / min) and concentrated using an ultrafiltration tube to obtain PBS buffer (2.0 mg / mL) of each anti-CDH6 antibody coupled to DAR8 and DAR6, which was refrigerated and stored at 4°C.

[0664] Reference substance ADC-5: Compound ADC-5 was prepared according to the preparation method of the antibody drug conjugate H01L02-Dxd described in Example 1 of US2020 / 0390900A1, with a theoretical DAR value of 8. US2020 / 0390900A1 is incorporated herein by reference in its entirety.

[0665] Table 11. ADC Information

[0666] Example 14. Detection of cell binding activity of ADC

[0667] The cell binding activity of the antibody before and after conjugation with toxin was evaluated in OVCAR3 tumor cells that highly express CDH6. First, OVCAR3 cells were digested and centrifuged, and the supernatant was discarded. The cells were resuspended in 3E 6 / mL, add 50μL / well to 96-well plate. Add 50μL of the test antibody (final concentration up to 100nM, 3-fold dilution) and incubate at 4℃ for 1 hour. After washing twice, add Alexa Fluor 500 at a dilution ratio of 1:800. 647AffiniPure Mouse Anti-Human IgG (Fcγfragment specific) secondary antibody for flow cytometry (Jackson, 209-605-098) was added and incubated at 4°C for 30 min. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. The cells were washed twice with PBST and resuspended in 150 μL of supernatant per well. Alexa Fluor 500 was detected by flow cytometry. The fluorescence intensity was 647. The results showed that, as shown in Figures 11A and 11B, the binding activity of the antibody to CDH6 did not change significantly before and after conjugation.

[0668] Example 15. Cytotoxicity of anti-CDH6 ADC against tumor cell lines with different CDH6 expression levels

[0669] OVCAR3 cells were cultured in 1640 growth medium supplemented with 0.01 mg / mL bovine insulin (Solarbio, cat#I8040) and 20% fetal bovine serum (Gibco, cat#10099141C). 786-O cells were cultured in 1640 growth medium supplemented with 10% fetal bovine serum (Gibco, cat#10099141C). PA-1 and NCI-N87 cells were cultured in MEM (with ENEAA) growth medium supplemented with 10% fetal bovine serum (Gibco, cat#10099141C). Cells were grown overnight in 96-well plates at a density of 2000 (OVCAR3), 1000 (786-O), or 3000 (PA-1 and NCI-N87) cells per well. Equal volumes of serially diluted ADCs were added the following day. After 4 days, the killing of 786-O and PA-1 cells was assessed using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega). After 6 days, the killing of OVCAR3 and NCI-N87 cells was assessed using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega). Cell viability was determined according to the kit instructions. Cell viability was assessed as a percentage of untreated control cells.

[0670] The results show that, as shown in Figures 12A, 12B, and 12C, the ADC disclosed herein exhibits antigen-dependent tumor cell killing activity compared to hIgG1. 50 The values ​​are shown in Table 12.

[0671] Table 12. Cytotoxicity of ADCs in cell lines with different expression levels Note: NA means the value cannot be calculated.

[0672] Example 16. Anti-tumor activity of anti-CDH6 ADC in OVCAR3 cell xenograft model

[0673] In order to evaluate the anti-tumor activity of the ADC disclosed herein in mice, this example evaluated the OVCAR3 ovarian cancer xenograft tumor model that highly expresses CDH6.

[0674] NCG mice were obtained from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. and inoculated with 5×10 OVCAR3 cells. 6 / 0.15mL / mouse (1:1 with Matrigel) was injected subcutaneously into the mouse. When the average tumor volume grew to 80-120mm 3Mice with appropriate tumor volumes were randomly assigned to the main experimental group, with six mice per group. Dosing was intraperitoneal. Dosing was once weekly for a total of three doses. The day of grouping was defined as D0, and the dosing period lasted from D0 to D14. Pharmacodynamic and safety evaluations were conducted using clinical observations such as tumor inhibition rate and body weight.

[0675] The calculation formula is as follows:

[0676] Tumor volume V = 1 / 2 × a × b2, where a and b represent length and width, respectively.

[0677] Relative tumor growth rate (T / C%) = (T - T0) / (C - C0) * 100, where T and C are the tumor volumes of the treatment and control groups at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. Tumor inhibition rate (TGI%) = 1 - T / C%.

[0678] A PBS control group served as a negative control. TGI represents the maximum tumor growth inhibition achieved during the experiment. Data are presented as mean ± standard error (SEM) and were plotted and analyzed using GraphPad Prism 9 software. Significant differences in tumor volume were compared using a one-way analysis of variance, with multiple comparisons performed if the F value was significant. Comparison of tumor volume on day 28 revealed highly significant target-dependent antitumor activity for each ADC tested compared to the vehicle group (P values ​​< 0.0001, respectively).

[0679] The results showed that the disclosed ADC had good tumor inhibition activity in the OVCAR3 cell xenograft model with high CDH6 expression, with no significant differentiation between different molecules. While showing good anti-tumor activity, the disclosed ADC did not cause significant changes in mouse body weight.

[0680] The changes in tumor volume of different ADC molecules in the OVCAR3 xenograft tumor model are shown in Table 13. The anti-tumor activity of different ADC molecules in the OVCAR3 xenograft tumor model is shown in Table 14. Figure 13A shows the comparison of the anti-tumor activity of low doses with the same DAR value, Figure 13B shows the comparison of the anti-tumor activity of the same toxin with different DAR values, and Figure 13C shows the body weight changes of CDH6 ADC in the OVCAR3 mouse model.

[0681] Table 13. Changes in tumor volume of ADC in OVCAR3 xenograft tumor model

[0682] Table 14. Antitumor activity of ADCs in the OVCAR3 xenograft tumor model

[0683] Example 17. Anti-tumor activity of anti-CDH6 ADC in PA-1 cell xenograft model

[0684] Next, the anti-tumor activity of the ADC disclosed herein in mice was further evaluated using a PA-1 xenograft tumor model with low CDH6 expression.

[0685] Human ovarian cancer PA-1 cells (ATCC-CRL-1572) were cultured as monolayers in vitro in a 5% CO2 incubator at 37°C. Twice a week, trypsin was used for routine digestion and passage. When the cell saturation reached 80%-90% and the number reached the required level, the cells were harvested, counted, and inoculated. 0.2 mL (10×10 6 PA-1 cells (1:1 with Matrigel) were subcutaneously inoculated on the right back of each mouse. For the pharmacodynamics experiment, the average tumor volume reached 118 mm 3 The administration frequency was once a week and stopped after 3 doses.

[0686] Female BALB / c nude mice were used. The development of this experimental protocol and any modifications were evaluated and approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai WuXi AppTec Pharmaceuticals Co., Ltd. The use and welfare of experimental animals were carried out in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals were monitored daily. Routine examinations included observations of tumor growth and the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual observation only), and twice-weekly weight changes. Physical signs or other abnormalities were also observed.

[0687] The experimental indicator is to determine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice weekly using a vernier caliper. The formula for calculating tumor volume is shown in Example 16.

[0688] Statistical analysis included the mean and standard error (SEM) of tumor volume at each time point for each group. Statistical analysis was performed to assess intergroup differences based on data from day 21 after group dosing. Comparisons between three or more groups were analyzed using one-way ANOVA, with Dunnett's t (2-sided) method used for multiple comparisons. All data were analyzed using Graphpad Prism 9. P < 0.05 was considered significant. (P values ​​were < 0.05* and < 0.01**, respectively). Relative body weight change was calculated based on the animal's weight at the start of dosing. Data points represent the mean percentage change in body weight within the group, and error bars represent the standard error (SEM).

[0689] A comparison of the anti-tumor activity of each ADC against a female BALB / c nude mouse model bearing subcutaneous human ovarian cancer PA-1 cell xenografts at low doses at the same DAR value is shown in Figure 14A. A comparison of the anti-tumor activity of each ADC against a female BALB / c nude mouse model bearing subcutaneous PA-1 cell xenografts at multiple doses at different DAR values ​​is shown in Figure 14B. The relative body weight changes of tumor-bearing mice after administration of the test drugs are shown in Figure 14C.

[0690] Results showed that at equivalent doses of DAR6, the disclosed ADC-1 and ADC-2 exhibited superior antitumor activity compared to the positive control. At a low dose of 2 mpk (2 mpk), ADC-1 (DAR6) achieved a TGI of 61.57%. Furthermore, the disclosed ADCs exhibited dose-dependent antitumor activity, with high doses completely inhibiting tumor growth. DAR8 and DAR6 showed no significant difference in activity when administered with equivalent toxins. Despite demonstrating excellent antitumor activity, the disclosed ADCs did not cause significant changes in mouse body weight.

[0691] The changes in tumor volume of different ADC molecules in the PA-1 xenograft tumor model are shown in Table 15. The anti-tumor activities of different ADC molecules in the PA-1 xenograft tumor model are shown in Tables 16 and 17.

[0692] Table 15. Changes in tumor volume of ADC in PA-1 xenograft tumor model

[0693] Table 16. Comparison of antitumor activity of ADCs with the same DAR value (low dose)

[0694] Table 17. Comparison of antitumor activity of ADCs with different DAR values ​​at multiple doses

[0695] Example 18. Anti-tumor activity of anti-CDH6 ADC in 786-O cell xenograft model

[0696] This example evaluates the renal cancer cell 786-O xenograft tumor model with low CDH6 expression.

[0697] NOD SCID mice were obtained from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. 786-O cells were cultured in vitro, and tumor cells in the logarithmic growth phase were collected and resuspended in 1:1 PBS and Matrigel to prepare a single-cell suspension according to the desired concentration. 0.2 mL of cell suspension was inoculated subcutaneously in the right upper limb of each NOD SCID mouse (1×10 7 14 days after cell inoculation, 24 mice were selected with an average tumor volume of approximately 149.5 mm 3The mice were randomly divided into 4 groups of 6 mice each. The day of grouping was recorded as "PG-D0" and the drugs were administered according to the schedule in Table 18, with a frequency of once a week and discontinuation after 3 doses.

[0698] Table 18. Dosage and administration schedule Note: ip: intraperitoneal injection; QW: once a week; “ / ” indicates not applicable.

[0699] After the experiment, the tumor inhibition rate (TGI) and body weight change rate (BWC) of each group were analyzed.

[0700] The calculation formula for tumor volume (TV) is: V = L × W 2 / 2. Where L and W represent the measured length and width of the tumor, respectively.

[0701] TGI (%) was calculated as follows: [1-(average tumor volume of a treatment group at the end of administration-average tumor volume of the treatment group at the time of grouping) / (average tumor volume of the solvent control group at the end of treatment-average tumor volume of the solvent control group at the time of grouping)]×100%.

[0702] The calculation formula of the body weight change rate (BWC%) of mice is: (BW c -BW i ) / BW i ×100%,BW c Indicates the animal weight on day c of the experiment, BW i Indicates the weight of animals when grouped.

[0703] Tumor growth curves were plotted with time as the X-axis and tumor volume as the Y-axis. Weight gain curves were plotted with time as the X-axis and animal weight change rate as the Y-axis. Data were analyzed based on raw data, and the results are presented as mean ± SEM. Tumor volume was analyzed using Graphpad Prism software using the T-test method. P < 0.05 was considered significant.

[0704] At the endpoint (PG-D21), the anti-tumor activity of each ADC against the subcutaneous xenograft tumor model of human renal cancer cell 786-O is compared in Figure 15. The average tumor volume of ADC-1 at the doses of 1.5 mg / kg and 5 mg / kg was 322.26 mm, respectively. 3 and 57.77mm 3 , compared with the Vehicle group (528.12mm 3) were statistically significantly different (P<0.001 and P<0.001), with tumor inhibition rates (TGI) of 54% and 124%, respectively. All tumor-bearing mice (6 / 6) in the ADC-1_5mg / kg group experienced tumor regression, demonstrating a good dose-response relationship. The average tumor volume of the control ADC-5 at a dose of 1.5mg / kg was 427.92mm 3 The TGI was 26%, a statistically significant difference compared to the vehicle group (P<0.05). At the same dose (1.5 mg / kg), the mean tumor volume at endpoint in the ADC-1 group was significantly lower than that in the control group (ADC-5) (P<0.05). During the experiment, the mean body weight of mice in each treatment group did not decrease significantly, indicating good tolerance to the test drug.

[0705] In summary, ADC-1 inhibited the growth of 786-O xenograft tumors in an adjuvant-dependent manner. At the same dose (1.5 mg / kg), the tumor inhibition effect of ADC-1 was better than that of ADC-5. The specific results are shown in Table 19 and Figure 15.

[0706] Table 19. Evaluation of the anti-tumor efficacy of ADC on 786-O xenograft tumors Note: a. T-test method was used to compare with the Vehicle group; b. T-test method was used to compare with the ADC-5_1.5mg / kg group.

[0707] Although specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

1. A CDH6 binding molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: 1) the VH comprises HCDR1, HCDR2, and / or HCDR3 of the amino acid sequences shown in SEQ ID NO: 17, 1, 62, or 63, and the VL comprises LCDR1, LCDR2, and / or LCDR3 of the amino acid sequences shown in SEQ ID NO: 18, 2, 64, or 65; or, 2) the VH comprises HCDR1, HCDR2, and / or HCDR3 of the amino acid sequences shown in SEQ ID NO: 3, 19, 66, or 67, and the VL comprises LCDR1, LCDR2, and / or LCDR3 of the amino acid sequences shown in SEQ ID NO: 4, 20, 68, or 69; Preferably, 1-1) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 17, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 18; 1-2) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 2; 1-3) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NO: 62 or 63, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NO: 64 or 65; 2-1) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 4; 2-2) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 19, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 20; or, 2-3) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NO: 66 or 67, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NO: 68 or 69.

2. A CDH6 binding molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: 1) the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NO: 5, 6, and 7 respectively; the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NO: 24, 25, and 10 respectively; or 2) The VH contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 12, and 13 respectively; the VL contains LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 26, 15, and 16 respectively; Preferably, 1-1) The VH contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 5, 6, and 7 respectively; the VL contains LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 21, 22, and 10 respectively; 1-2) The VH contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 5, 6, and 7 respectively; the VL contains LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 8, 9, and 10 respectively; 2-1) The VH contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 12, and 13 respectively; the VL contains LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 14, 15, and 16 respectively; 2-2) The VH contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 12, and 13 respectively; the VL contains LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 23, 15, and 16 respectively; 2-3) The VH contains HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 12, and 13 respectively; the VL contains LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 70, 15, and 16 respectively.

3. The CDH6 binding molecule according to claim 1 or 2, which is an anti-CDH6 antibody or an antigen-binding fragment thereof; preferably, the antibody is selected from murine antibodies, chimeric antibodies, or humanized antibodies; preferably, the antigen-binding fragment is scFv, Fv, Fab, Fab’, (Fab’)2 fragment, linear antibody, single-chain antibody, sdAb, sdFv, nanobody, peptibody, domain antibody, diabody, triabody, tetrabody, tandem di-scFv, or tandem tri-scFv.

4. The CDH6 binding molecule according to any one of claims 1 to 3, which is modified by affinity maturation, removal / reduction of T cell epitopes, reduction of antibody deamidation, and / or reduction of antibody isomerization.

5. The CDH6 binding molecule according to any one of claims 1 to 4, wherein: 1-1) VH comprises an amino acid sequence as shown in SEQ ID NO:17 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:18 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; 1-2) VH comprises an amino acid sequence as shown in SEQ ID NO:1 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto; 1-3) VH comprises an amino acid sequence as shown in SEQ ID NO:62 or 63 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:64 or 65 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; 2-1) VH comprises an amino acid sequence as shown in SEQ ID NO:3 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:4 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; 2-2) VH comprises an amino acid sequence as shown in SEQ ID NO:19 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:20 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; 2-3) VH comprises an amino acid sequence as shown in SEQ ID NO:66 or 67 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:68 or 69 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

6. The CDH6 binding molecule according to any one of claims 1 to 5, which comprises an immunoglobulin constant region; wherein the heavy chain constant region is derived from human IgG1, IgG2, IgG3, IgG4 or variants thereof; and / or the light chain constant region is derived from human antibody kappa, lambda chains or variants thereof; Preferably, the amino acid sequence of the heavy chain constant region is derived from human IgG1 or variants thereof; and / or the light chain constant region is derived from human antibody kappa chain or variants thereof; More preferably, the heavy chain constant region comprises an Fc region having an amino acid sequence as shown in SEQ ID NO: 61 or an amino acid sequence having at least 80% sequence identity thereto.

7. The CDH6 binding molecule according to any one of claims 1 to 6, comprising a heavy chain and a light chain, wherein: 1) the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; 2) the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 30 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

8. The CDH6 binding molecule according to any one of claims 1 to 7, wherein, The CDH6 binding molecule has at least the following properties: (1) having cell internalization activity; preferably, having tumor cell internalization activity; (2) binds to human CDH6 with a K D value less than or equal to 100 nM; (3) not binding to human CDH9 and CDH10.

9. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, which comprises the CDH6 binding molecule according to any one of claims 1 to 8 and an effector molecule, and the effector molecule is conjugated to the CDH6 binding molecule; preferably, the effector molecule is a cytotoxic drug, an immunomodulator or a cell inhibitor..

10. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 9, wherein the effector molecule is selected from camptothecin, auristatin, maytansine alkaloids, vinca alkaloids, pyrrolobenzodiazepines (PBD), calicheamicin, duocarmycin, daunorubicin, doxorubicin, calicheamycin, anthramycin, neomycin, amanitin toxin, halichondrin, eribulin, Tubulysin, and analogs or derivatives thereof; preferably, the effector molecule is selected from Exatecan, MMAE, MMAF, MMAD, SN-38, DM1, DM4, pyrrolobenzodiazepine (PBD) dimer, eribulin, and analogs or derivatives thereof.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 9 or 10, wherein the antibody-drug conjugate is represented by the general formula (Pc-L-Y-D) as shown in any one of formulas (I)-(V): Wherein: Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)-, -S-(CR a R b ) m -CR 1 R 2 -C(O)- and is absent; optionally, when of formula (V), Y is absent; R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group; or, R a and R b together with the carbon atom to which it is attached form a cycloalkyl group and a heterocyclic group; R 1 is selected from a hydrogen atom, a halogen, a haloalkyl, an alkyl, a deuterated alkyl, a cycloalkyl, a cycloalkylalkyl, an alkoxyalkyl, a heterocyclic group, an aryl, and a heteroaryl; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl, an alkyl, a deuterated alkyl, a cycloalkyl, a cycloalkylalkyl, an alkoxyalkyl, a heterocyclic group, an aryl, and a heteroaryl; or, R 1 and R 2 together with the carbon atom to which it is attached form a cycloalkyl or a heterocyclic group; Alternatively, R a and R 2 together with the carbon atom to which it is attached form a cycloalkyl or heterocyclic group; m is an integer from 0 to 4; n is from 1 to 10, n is a decimal or an integer, preferably, n is from 2 to 8 or from 5 to 9, more preferably, n is from 7 to 8, n is a decimal or an integer; L is a linker unit; Pc is a CDH6 binding molecule according to any one of claims 1 to 8; Preferably, The antibody-drug conjugate described above is represented by the general formula (Pc-L-Y-D) of formula (I): wherein: Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group; or, R a and R b together with the carbon atom to which it is attached form a cycloalkyl group and a heterocyclic group; R 1 is selected from a hydrogen atom, a halogen, a haloalkyl, a deuterated alkyl, a cycloalkyl, a cycloalkylalkyl, an alkoxyalkyl, a heterocyclic group, an aryl, and a heteroaryl; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl, a deuterated alkyl, a cycloalkyl, a cycloalkylalkyl, an alkoxyalkyl, a heterocyclic group, an aryl, and a heteroaryl; or, R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl or a heterocyclic group; Alternatively, R a and R 2 together with the carbon atom to which it is attached form a cycloalkyl or heterocyclic group; m is an integer from 0 to 4; n is from 1 to 10, n is a decimal or an integer, preferably, n is from 2 to 8 or from 5 to 9 or from 4 to 8, more preferably, n is from 7 to 8, n is a decimal or an integer; L is a linker unit; Pc is a CDH6 binding molecule according to any one of claims 1 to 8.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 9-11, wherein, Y is -O-(CR a R b )m-CR 1 R 2 -C(O)-; R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group; R 1 is C 1-3 alkyl, C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl, a hydrogen atom; R 2 selected from a hydrogen atom, a haloalkyl group, a C 1-3 alkyl group or a C 3-6 cycloalkyl group; preferably a hydrogen atom; Alternatively, R 1 and R 2 together with the carbon atom to which it is attached form a C 3-6 cycloalkyl group; m is 0 or 1; Preferably, Y is -O-(CR a R b )m-CR 1 R 2 -C(O)-; R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group; R 1 is C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl, a hydrogen atom; R 2 selected from a hydrogen atom, a haloalkyl group, or a C 3-6 cycloalkyl group; preferably a hydrogen atom; Alternatively, R 1 and R 2 together with the carbon atom to which it is attached form a C 3-6 cycloalkyl group; m is 0 or 1.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 9-12, wherein, Y is selected from: wherein the O-terminus of Y is connected to the linker unit L.

14. The antibody-drug conjugate according to any one of claims 11-13, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 For s 1 is an integer from 2 to 8, s 2 is selected from integers from 0 to 8; preferably s 2 is 2; L 2 is a chemical bond; L 3 is a tetrapeptide residue; L 4 is -NR 5 (CR 6 R 7 )t-, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 11-13, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 -(succinimido-3-yl-N)-CH2-cyclohexyl-C(O)-; L 2 is -NR 4 (CH2CH2O)9CH2C(O)-; L 3 is a tetrapeptide residue; L 4 is -NR 5 (CR 6 R 7 )t-, R 5 is selected from a hydrogen atom or an alkyl group, R 6 and R 7 are the same or different and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2.

16. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 14 or 15, wherein the L 3 peptide residue is an amino acid residue formed by one, two or more amino acids selected from phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), aspartic acid (N); preferably an amino acid residue formed by one, two or more amino acids selected from phenylalanine and glycine; more preferably a tetrapeptide residue; most preferably a tetrapeptide residue of GGFG (glycine-glycine-phenylalanine-glycine).

17. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 11-16, wherein the linking unit -L-Y- is selected from: wherein x and y are independently selected from integers from 2 to 8.

18. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 9-17, wherein the antibody-drug conjugate is selected from: wherein: n is from 1 to 10, which can be an integer or a decimal, preferably 2, 3, 4, 5, 6, 7, 8 or 9, more preferably 7 - 8; Pc is a CDH6 binding molecule as described in any one of claims 1 - 8.

19. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 18, wherein the antibody-drug conjugate is selected from: wherein: n is as defined in claim 18; the Pc comprises VH and VL, 1) the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:17, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:18; or 2) the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:19, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:20; Preferably, 1) the VH comprises HCDR1, HCDR2, HCDR3 in the amino acid sequences shown in SEQ ID NO:5, 6, 7 respectively; the VL comprises LCDR1, LCDR2, LCDR3 in the amino acid sequences shown in SEQ ID NO:21, 22, 10 respectively; 2) the VH comprises HCDR1, HCDR2, HCDR3 in the amino acid sequences shown in SEQ ID NO:11, 12, 13 respectively; the VL comprises LCDR1, LCDR2, LCDR3 in the amino acid sequences shown in SEQ ID NO:23, 15, 16 respectively.

20. The antibody - drug conjugate according to claim 19, wherein: 1) the VH comprises an amino acid sequence shown in SEQ ID NO:17 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises an amino acid sequence shown in SEQ ID NO:18 or having at least 90% identity thereto; or 2) the VH comprises an amino acid sequence shown in SEQ ID NO:19 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises an amino acid sequence shown in SEQ ID NO:20 or having at least 90% identity thereto.

21. The antibody - drug conjugate according to claim 20, wherein: the Pc comprises a heavy chain and a light chain, 1) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 27 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or 2) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 29 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 30 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

22. A CDH6 binding molecule that specifically binds to the EC1 domain of the extracellular region of human CDH6; Preferably, the binding molecule has an internalization ability that allows cell uptake; Preferably, the EC1 domain is the amino acid sequence shown in SEQ ID NO: 71; Preferably, the binding molecule is an anti-CDH6 binding molecule or an antigen-binding fragment thereof.

23. A polynucleotide encoding the CDH6 binding molecule according to any one of claims 1 to 8, 22 or the Pc in claims 9 to 21; Preferably, the polynucleotide is DNA or RNA.

24. A vector containing the polynucleotide according to claim 23.

25. A host cell that contains the vector according to claim 24; or expresses the CDH6 binding molecule shown in any one of claims 1 to 8, 22; Preferably, the host cell is a bacterium, yeast or mammalian cell; More preferably, the host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary cells or human embryonic kidney 293 cells.

26. A pharmaceutical composition containing: The CDH6 binding molecule according to any one of claims 1 to 8, 22, the antibody-drug conjugate according to any one of claims 9 to 21, the polynucleotide according to claim 23, or the vector according to claim 24; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents or adjuvants.

27. A method for preparing a CDH6 binding molecule, comprising: Expressing the CDH6 binding molecule according to any one of claims 1 to 8, 22 in a host cell, and Isolating the CDH6 binding molecule from the host cell; Optionally, comprising a purification step for the CDH6 binding molecule.

28. A method for preparing an antibody-drug conjugate, comprising: Coupling the CDH6 binding molecule according to any one of claims 1 to 8, 22 with an effector molecule to obtain the antibody-drug conjugate; Optionally, the method further comprises: purifying the antibody-drug conjugate. Use of the CDH6 binding molecule according to any one of claims 1 to 8 and 22, the antibody conjugate according to any one of claims 9 to 19, the polynucleotide according to claim 23, the vector according to claim 24, or the pharmaceutical composition according to claim 26 in the preparation of a medicament for treating cancer or a tumor; Preferably, the cancer or tumor is CDH6-related or CDH6-positive; Preferably, the cancer or tumor is renal cancer, ovarian cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma, more preferably CDH6-related or CDH6-positive renal cancer, ovarian cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma.

30. A method for treating cancer or a tumor in a subject in need thereof, the method comprising: the CDH6 binding molecule according to any one of claims 1 to 8 and 20, the antibody conjugate according to any one of claims 9 to 19, the polynucleotide according to claim 21, the vector according to claim 22, or the pharmaceutical composition according to claim 24; Preferably, the cancer or tumor is CDH6-related or CDH6-positive; Preferably, the cancer or tumor is renal cancer, ovarian cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma, more preferably CDH6-related or CDH6-positive renal cancer, ovarian cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma.

31. Use of at least one of the CDH6 binding molecule according to any one of claims 1 to 8 and 22, the antibody conjugate according to any one of claims 9 to 21, the polynucleotide according to claim 23, and the vector according to claim 24 as follows: (1) for detecting CDH6 protein; (2) for preparing a reagent for detecting CDH6 protein; (3) for the diagnosis, prognosis, and efficacy monitoring of cancer or a tumor; (4) for preparing a reagent for the diagnosis, prognosis, and efficacy monitoring of cancer or a tumor; Preferably, the cancer or tumor is CDH6-related or CDH6-positive; Preferably, the cancer or tumor is renal cancer, ovarian cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma, more preferably CDH6-related or CDH6-positive renal cancer, ovarian cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, ovarian serous adenocarcinoma, thyroid cancer, cholangiocarcinoma, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma; Optionally, the CDH6 binding molecule also carries a detectable label.

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