WO2025140511A1 - Antigen-binding molecule, drug conjugate thereof and medical use thereof - Google Patents

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

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 disorders.

Background Art

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 cancer and ovarian cancer, 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 cancer 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).

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 essential for the interaction with α2β1 integrin and activation of the posterior integrin pathway in cancer metastatic cells, and the HAV motif in EC5 is associated with stability. Cadherin has a certain 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.).

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 endocytic activity, and in particular provides antibodies targeting the EC1 or EC3 domains of human CDH6. Antibodies targeting EC1 are not yet available in the prior art. The anti-CDH6 antibodies disclosed in the present disclosure 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 in the present disclosure have the characteristics of high endocytic activity, high tumor inhibitory activity, good stability, and good drugability, and have high potential for clinical treatment of tumors.

Summary of the invention

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.

CDH6 binding molecules

I:

In some embodiments, the present disclosure provides a CDH6 binding molecule that specifically binds 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 an 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.

In some embodiments, the present disclosure provides a CDH6 binding molecule comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2 and/or HCDR3 in the amino acid sequence shown in SEQ ID NO: 1, 17, 62 or 63, and the VL comprises LCDR1, LCDR2 and/or LCDR3 in 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 rules. In some specific embodiments, the CDRs are defined according to the Kabat numbering rules.

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

1-1) the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:1, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:2;

1-2) 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;

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

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.

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, 7, respectively; and the VL comprises LCDR1, LCDR2 and/or LCDR3, wherein LCDR1, LCDR2, LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 24, 25, 10, respectively.

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, 7, respectively; 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.

In some embodiments, a CDH6 binding molecule is provided, comprising VH and/or VL, wherein the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 5, 6 and 7, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3, wherein:

1-1) LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively;

1-2) LCDR1, LCDR2, and LCDR3 contain the amino acid sequences shown in SEQ ID NO: 21, 22, and 10, respectively.

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

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

HCDR1：GYTFTNYW(SEQ ID NO:31)

HCDR2：IAPNSGGT(SEQ ID NO:32)

HCDR3：TSYQYVSSQYYFDY(SEQ ID NO:33)

LCDR1：STVGSSY(SEQ ID NO:34)

LCDR2: ST (SEQ ID NO: 35)

LCDR3：HQWNTYPFT(SEQ ID NO:10)

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

HCDR1：GYTFTNY(SEQ ID NO:36)

HCDR2：APNSGG(SEQ ID NO:37)

HCDR3：YQYVSSQYYFDY(SEQ ID NO:7)

LCDR1：TASSTVGSSYLY(SEQ ID NO:21)

LCDR2：STSNLAT(SEQ ID NO:22)

LCDR3：HQWNTYPFT(SEQ ID NO:10)

Exemplarily, the CDR sequences defined by the AbM numbering system are as follows:

HCDR1：GYTFTNYWMH(SEQ ID NO:38)

HCDR2：RIAPNSGGTK(SEQ ID NO:39)

HCDR3：YQYVSSQYYFDY(SEQ ID NO:7)

LCDR1：TASSTVGSSYLY(SEQ ID NO:21)

LCDR2：STSNLAT(SEQ ID NO:22)

LCDR3：HQWNTYPFT(SEQ ID NO:10)

Exemplarily, the CDR sequence defined by the Contact numbering system is as follows:

HCDR1：TNYWMH(SEQ ID NO:40)

HCDR2：WIGRIAPNSGGTK(SEQ ID NO:41)

HCDR3：TSYQYVSSQYYFD(SEQ ID NO:42)

LCDR1：GSSYLYWF(SEQ ID NO:43)

LCDR2：LWIYSTSNLA(SEQ ID NO:44)

LCDR3：HQWNTYPF(SEQ ID NO:45)

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.

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

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

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

1-1) VH comprises an amino acid sequence as shown in SEQ ID NO:1, or having at least 80%, at least 90% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:2, or having at least 80%, at least 90% identity thereto;

1-2) VH comprises an amino acid sequence as shown in SEQ ID NO: 17, or having at least 80%, at least 90% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO: 18, or having at least 80%, at least 90% identity thereto;

1-3) VH comprises an amino acid sequence as shown in SEQ ID NO: 62, or having at least 80%, at least 90% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO: 64 or 65, or having at least 80%, at least 90% identity thereto;

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

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.

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

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

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

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

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

II:

In some embodiments, the present disclosure provides a CDH6 binding molecule that specifically binds to the EC3 domain of CDH6. The amino acid sequence of the EC3 is, for example, as shown in SEQ ID NO: 73. In some embodiments, the binding molecule has internalization capability that allows cellular uptake.

In some embodiments, the present disclosure provides a CDH6 binding molecule comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2 and/or HCDR3 in the amino acid sequence shown in SEQ ID NO: 3, 19, 66 or 67, and the VL comprises LCDR1, LCDR2 and/or LCDR3 in 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 conventions.

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

2-1) the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:3, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:4;

2-2) 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;

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

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.

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: 11, 12, 13, respectively; and the VL comprises LCDR1, LCDR2 and/or LCDR3, wherein LCDR1, LCDR2, LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 26, 15, 16, respectively.

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, HCDR3 comprise the amino acid sequences shown in SEQ ID NO: 11, 12, 13, respectively; the VL comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14 or 23 or 70, and LCDR2, LCDR3 comprise the amino acid sequences shown in SEQ ID NO: 15, 16, respectively.

The following exemplifies HCDR1, HCDR2, HCDR3 in VH shown in SEQ ID NO:19, and LCDR1, LCDR2 and LCDR3 in VL shown in SEQ ID NO:20, as defined by the IMGT, Chothia, AbM or Contact numbering systems.

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

HCDR1：GFSLSSYG(SEQ ID NO:46)

HCDR2：IWSGGST(SEQ ID NO:47)

HCDR3：ARYSNSYAMDY(SEQ ID NO:48)

LCDR1：QSIVHSTGNTY(SEQ ID NO:49)

LCDR2:KV (SEQ ID NO:50)

LCDR3：FQTSHVPYT(SEQ ID NO:16)

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

HCDR1：GFSLSSY(SEQ ID NO:51)

HCDR2:WSGGS (SEQ ID NO:52)

HCDR3：YSNSYAMDY(SEQ ID NO:13)

LCDR1：RSSQSIVHSTGNTYLE(SEQ ID NO:23)

LCDR2:KVSKRFS(SEQ ID NO:15)

LCDR3：FQTSHVPYT(SEQ ID NO:16)

Exemplarily, the CDR sequences defined by the AbM numbering system are as follows:

HCDR1：GFSLSSYGVH(SEQ ID NO:53)

HCDR2：MIWSGGSTD(SEQ ID NO:54)

HCDR3：YSNSYAMDY(SEQ ID NO:13)

LCDR1：RSSQSIVHSTGNTYLE(SEQ ID NO:23)

LCDR2:KVSKRFS(SEQ ID NO:15)

LCDR3：FQTSHVPYT(SEQ ID NO:16)

Exemplarily, the CDR sequence defined by the Contact numbering system is as follows:

HCDR1：SSYGVH(SEQ ID NO:55)

HCDR2：WIIGMIWSGGSTD(SEQ ID NO:56)

HCDR3：ARYSNSYAMD(SEQ ID NO:57)

LCDR1：VHSTGNTYLEWY(SEQ ID NO:58)

LCDR2：LLIYKVSKRF(SEQ ID NO:59)

LCDR3：FQTSHVPY(SEQ ID NO:60)

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.

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 molecule is derived from IGHV4-30 and IGHJ4*01, and the light chain framework region of the human germline template used in the humanization process for the VL is derived from IGKV2-28 and 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 derived from IGHV4-30, 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 derived from IGKV2-28, and FR4 is derived from IGKJ2*01.

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

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

2-1) VH comprises an amino acid sequence as shown in SEQ ID NO:3, or having at least 80%, at least 90% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:4, or having at least 80%, at least 90% identity thereto;

2-2) VH comprises an amino acid sequence as shown in SEQ ID NO: 19, or having at least 80%, at least 90% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO: 20, or having at least 80%, at least 90% identity thereto;

2-3) VH comprises an amino acid sequence as shown in SEQ ID NO: 66, or having at least 80%, at least 90% identity thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO: 68 or 69, or having at least 80%, at least 90% identity thereto;

2-4) VH comprises an amino acid sequence as shown in SEQ ID NO:67, or a sequence that is 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 a sequence that is at least 80% or at least 90% identical thereto.

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

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

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

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

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

In the above I and II:

In some embodiments, a CDH6 binding molecule comprises a VH and a VL, wherein:

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.

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

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

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.

In some embodiments, the humanized antibody or fragment thereof further comprises a back mutation to VH and 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.

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.

In some embodiments, the CDH6 binding molecule further comprises a constant region. Exemplarily, the heavy chain constant region is derived from IgG1, IgG2, IgG3, IgG4 or a variant thereof, and the light chain constant region can be derived from κ, λ chain or a variant thereof.

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. Exemplarily, the Fc of IgG1 comprises an amino acid sequence as shown in SEQ ID NO: 61 or an amino acid sequence having at least 80% sequence identity thereto.

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

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

In some embodiments, a CDH6 binding molecule is provided, comprising 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.

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.

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

(1) Possessing internalization capacity that allows cellular uptake;

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

(3) binds to human CDH6 with a _KD value of less than or equal to 100 nM;

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

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 with the Nov0712 antibody (sequence see Publication No.: US2016/0046711A1, which is incorporated herein by reference in its entirety) and the Daiichi07 antibody (sequence see Publication No.: 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.

In some embodiments, the CDH6 binding molecule binds to human CDH6 with a _KD value of less than or equal to 100 nM, such as less than or equal to about 75 nM, about 50 nM, about 25 nM, about 50 nM, about 10 nM, about 5 nM, etc.

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 shown in Section I 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 shown in Section I of the present disclosure have an enhanced internalization ability that allows cellular uptake compared to the Nov0712 antibody and the Daiichi07 antibody.

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 shown 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 shown in Section II of the present disclosure have enhanced internalization ability that allows cellular uptake compared to the Nov0712 antibody and the Daiichi07 antibody.

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

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

In other embodiments, a CDH6 binding molecule is provided, the binding of which to human CDH6 is cross-blocked by the aforementioned CDH6 binding molecule.

Antibody Drug Conjugates

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

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.

In some embodiments, effector molecules include, but are not limited to, cytotoxic drugs, immunomodulators, and cytostatic agents. For example, compounds, polypeptides, proteins, nucleic acids, etc., that have cell growth inhibition, cytotoxicity, and/or immunomodulatory functions. Exemplarily, toxins (such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants, or animals), radioactive isotopes, chemotherapeutic drugs, antibiotics, and nucleolytic enzymes, etc.

The antibody-drug conjugate disclosed in the present invention can bind to the extracellular domain of CDH6 and then undergo endocytosis, and the effector molecules are released inside the cells, which can not only effectively exert cell killing and inhibitory effects, but also have a good bystander effect.

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

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

in:

Y is selected from ^-O- ( ^{CRaRb )} _m- ^{CR1R2 -C} (O)-, ^-O - ^CR1R2- ( ^CRaRb ) _m- , -O- ^CR1R2- , -NH-(CRaRb) ^m _- ^{CR1R2 - C} (O)-, -S-( ^{CRaRb )} _m- ^CR1R2 - ^C (O)- and absent; optionally, when of formula (V), Y is absent ^;

^Ra and ^Rb 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, ^Ra and ^Rb together with the carbon atom to which they are attached form a cycloalkyl group and a heterocyclic group;

^R1 is selected from the group consisting of hydrogen, halogen, haloalkyl, alkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; ^R2 is selected from the group consisting of hydrogen, halogen, haloalkyl, alkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; or, ^R1 and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group;

Alternatively, ^Ra and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

m is an integer from 0 to 4;

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

L is the joint unit;

Pc is any one of the CDH6 binding molecules disclosed herein.

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

in:

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)-;

^Ra and ^Rb 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, ^Ra and ^Rb together with the carbon atom to which they are attached form a cycloalkyl group and a heterocyclic group;

^R1 is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; ^R2 is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; or, ^R1 and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group;

Alternatively, ^Ra and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

m is an integer from 0 to 4;

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

L is the joint unit;

Pc is any one of the CDH6 binding molecules disclosed herein.

In some embodiments, the conjugate of the antibody or fragment thereof-exitecan or its derivative disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, wherein -Y- is -O-(CR ^a R ^b )m-CR ¹ R ² -C(O)-;

^Ra and ^Rb are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;

^R1 is _C1-3 alkyl, _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, or a hydrogen atom;

^R2 is selected from hydrogen atom, haloalkyl, _C1-3 alkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

m is 0 or 1.

In some embodiments, the conjugate of the antibody or fragment thereof-exitecan or its derivative disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, wherein -Y- is -O-(CR ^a R ^b )m-CR ¹ R ² -C(O)-;

^Ra and ^Rb are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;

^R1 is _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, hydrogen atom;

^R2 is selected from hydrogen atom, haloalkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, R ¹ and R ² together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

m is 0 or 1.

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

^R1 is _C1-3 alkyl, _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, or a hydrogen atom;

^R2 is selected from a hydrogen atom, a haloalkyl group, a _C1-3 alkyl group or a _C3-6 cycloalkyl group;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

m is 0 or 1.

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

^R1 is _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, hydrogen atom;

^R2 is selected from a hydrogen atom, a haloalkyl group or a _C3-6 cycloalkyl group;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

m is 0 or 1.

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

^R1 is _C1-3 alkyl, _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, or a hydrogen atom;

^R2 is a hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

m is 0 or 1.

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

^R1 is _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, hydrogen atom;

^R2 is a hydrogen atom;

Alternatively, R ¹ and R ² together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

m is 0 or 1.

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

^R1 is _C1-3 alkyl, _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, or a hydrogen atom;

^R2 is a hydrogen atom;

Alternatively, R ¹ and R ² together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

m is 0.

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

^R1 is _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, hydrogen atom;

^R2 is a hydrogen atom;

Alternatively, R ¹ and R ² together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

m is 0.

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

^R1 is a hydrogen atom;

^R2 is a hydrogen atom;

Alternatively, R ¹ and R ² together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

m is 0.

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

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

In other embodiments, the present disclosure provides a conjugate of an antibody or a fragment thereof-exitecan or a derivative thereof, which is shown in the general formula (Pc-L-D1) of formula (VI):

in:

^R1 is a hydrogen atom, a _C1-3 alkyl group, a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl, _C1-3 alkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

m is 0 or 1;

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

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):

in:

^R1 is a hydrogen atom, a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, R ¹ and R ² together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;

m is 0 or 1;

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

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

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 -,

L ¹ is selected from -(succinimid-3-yl-N)-WC(O)-, -CH ₂ -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, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from N, O or S, wherein the 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;

L ² is selected from -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-, -S(CH ₂ )p ¹ C(O)-, or a chemical bond, wherein p ¹ is an integer from 1 to 20;

^L3 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;

L ⁴ is selected from -NR ⁵ (CR ⁶ R ⁷ ) _t -, -C(O)NR ⁵ , -C(O)NR ⁵ (CH ₂ ) _t -, or a chemical bond, wherein t is an integer from 1 to 6;

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;

^R6 and ^R7 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.

In some embodiments, the present disclosure provides a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof, wherein the linker unit ^L1 is selected from -(succinimidyl-3-yl-N)-( _CH2 ) ^s1 -C(O)-, -(succinimidyl-3-yl-N) _-CH2- cyclohexyl-C(O)-, -( _succinimidyl -3 _- yl-N)-(CH2CH2O) ^s2 - _{CH2CH2 -} C(O)-, _-CH2- C(O) ^-NR3- ( _CH2 ) ^s3 -C(O)-, or -C(O)-( _CH2 ) ^s4C (O)-, wherein ^s1 is an integer from 2 to 8, ^s2 is an integer from 1 to 3, ^s3 is an integer from 1 to 8, and ^s4 is an integer from 1 to 8; ^s1 is preferably 5.

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 ^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2C (O)- or a chemical bond, ^and _p1 is an integer of 6-12.

In some specific embodiments, the present disclosure provides a conjugate of an antibody or a fragment thereof-exitecan or a derivative thereof, wherein L ⁴ is 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 is preferably -NR ⁵ CR ⁶ R ⁷ -; L ⁴ is more preferably -NHCH ₂ -.

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- is -L ¹ -L ² -L ³ -L ⁴ -,

^L1 is ^s1 is an integer from 2 to 8;

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 are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.

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- is -L ¹ -L ² -L ³ -L ⁴ -,

L ¹ is -(succinimidyl-3-yl-N)-CH ₂ -cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(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 are each independently a hydrogen atom or an alkyl group, and t is 1 or 2.

In some specific embodiments, the present disclosure provides a conjugate of an antibody or a fragment thereof-ixitecan or a derivative thereof, wherein the peptide residue of ^L3 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).

In some embodiments, the present disclosure provides a conjugate of an antibody or a fragment thereof-exitecan or a derivative thereof, wherein the linker unit -L- has its ^L1 end connected to the antibody and its ^L4 end connected to Y.

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

L ¹ is selected from -(succinimidyl-3-yl-N)-(CH2)s ¹ -C(O)- or -(succinimidyl-3-yl-N)-CH2-cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)- or a chemical bond, p ¹ is an integer from 6 to 12;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a _C1-3 alkyl group, a cycloalkylalkyl group or a cycloalkyl group; preferably a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl, _C1-3 alkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

^R5 is selected from a hydrogen atom or an alkyl group, ^R6 and ^R7 are the same or different and are each independently a hydrogen atom or an alkyl group;

s ¹ is an integer from 2 to 8, preferably 5;

m is an integer of 0-4.

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

L ¹ is selected from -(succinimid-3-yl-N)-(CH ₂ )s ¹ -C(O)- or -(succinimid-3-yl-N)-CH ₂ -cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)- or a chemical bond, p ¹ is an integer from 6 to 12;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

^R5 is selected from a hydrogen atom or an alkyl group, ^R6 and ^R7 are the same or different and are each independently a hydrogen atom or an alkyl group;

s ¹ is an integer from 2 to 8, preferably 5;

m is an integer of 0-4.

In some embodiments, the conjugate of antibody or fragment thereof-exitecan or its derivative,

Wherein -LY- is:

Preferably:

L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(O)-;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a _C1-3 alkyl group, a cycloalkylalkyl group or a cycloalkyl group; preferably a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl, _C1-3 alkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

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;

m is an integer of 0-4.

In some embodiments, the conjugate of antibody or fragment thereof-exitecan or its derivative,

Wherein -LY- is:

Preferably:

L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(O)-;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

^R5 is selected from a hydrogen atom or an alkyl group, ^R6 and ^R7 are the same or different and are each independently a hydrogen atom or an alkyl group;

m is an integer of 0-4.

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):

in:

L ² is a chemical bond;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a _C1-3 alkyl group, a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from a hydrogen atom, a haloalkyl group, a _C1-3 alkyl group or a _C3-6 cycloalkyl group;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

^R5 is selected from a hydrogen atom or an alkyl group, ^R6 and ^R7 are the same or different and are each independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer of 0-4.

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):

in:

L ² is a chemical bond;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from a hydrogen atom, a haloalkyl group or a _C3-6 cycloalkyl group;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

^R5 is selected from a hydrogen atom or an alkyl group, ^R6 and ^R7 are the same or different and are each independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer of 0-4.

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-):

It can be used to obtain an antibody-drug conjugate by connecting the effector molecule to the antibody via a connecting fragment;

in:

L ¹ is selected from -(succinimid-3-yl-N)-(CH ₂ )s ¹ -C(O)- or -(succinimid-3-yl-N)-CH ₂ -cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)- or a chemical bond, p ¹ is an integer from 1 to 20;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a _C1-3 alkyl group, a cycloalkylalkyl group or a cycloalkyl group; preferably a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl, _C1-3 alkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

R ⁵ , R ⁶ or R ⁷ are the same or different and are each independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer of 0-4.

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-):

It can be used to obtain an antibody-drug conjugate by connecting the effector molecule to the antibody via a connecting fragment;

in:

L ¹ is selected from -(succinimid-3-yl-N)-(CH ₂ )s ¹ -C(O)- or -(succinimid-3-yl-N)-CH ₂ -cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)- or a chemical bond, p ¹ is an integer from 1 to 20;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

R ⁵ , R ⁶ or R ⁷ are the same or different and are each independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer of 0-4.

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-):

in:

L ² is a chemical bond;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a _C1-3 alkyl group, a cycloalkylalkyl group or a cycloalkyl group; preferably a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl, _C1-3 alkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

^R5 is selected from a hydrogen atom or an alkyl group, ^R6 and ^R7 are the same or different and are each independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer of 0-4.

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-):

in:

L ² is a chemical bond;

L ³ is a tetrapeptide residue of GGFG;

^R1 is a hydrogen atom, a cycloalkylalkyl group or a cycloalkyl group; preferably a _C3-6 cycloalkylalkyl group or a _C3-6 cycloalkyl group;

^R2 is selected from hydrogen atom, haloalkyl or _C3-6 cycloalkyl; preferably hydrogen atom;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a _C3-6 cycloalkyl group;

^R5 is selected from a hydrogen atom or an alkyl group, ^R6 and ^R7 are the same or different and are each independently a hydrogen atom or an alkyl group;

^s1 is an integer from 2 to 8;

m is an integer of 0-4.

In some embodiments, the antibody or fragment thereof-exitecan or a derivative thereof conjugate is a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof represented by the general formula (Pc- _La -Y-Dr) of formula (VIII):

in:

W is selected from C _1-8 alkyl, C _1-8 alkyl-cycloalkyl or straight-chain heteroalkyl of 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from N, O or S, wherein the 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;

L ² is selected from -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-, -S(CH ₂ )p ¹ C(O)- or a chemical bond, and p ¹ is an integer from 1 to 20;

^L3 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;

^R1 is selected from hydrogen, halogen, alkyl, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

^R2 is selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group;

^R4 and ^R5 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;

^R6 and ^R7 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;

m is an integer from 0 to 4;

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

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

In some embodiments, the antibody or fragment thereof-exitecan or a derivative thereof conjugate is a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof represented by the general formula (Pc- _La -Y-Dr) of formula (VIII):

in:

W is selected from C _1-8 alkyl, C _1-8 alkyl-cycloalkyl or straight-chain heteroalkyl of 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from N, O or S, wherein the 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;

L ² is selected from -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-, -S(CH ₂ )p ¹ C(O)- or a chemical bond, and p ¹ is an integer from 1 to 20;

^L3 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;

^R1 is selected from hydrogen, halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

^R2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

Alternatively, ^R1 and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group;

^R4 and ^R5 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;

^R6 and ^R7 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;

m is an integer from 0 to 4;

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

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

In some embodiments, the antibody or fragment thereof-exitecan or a derivative thereof conjugate is a conjugate of an antibody or fragment thereof-exitecan or a derivative thereof represented by the general formula (Pc-L _b -Y-Dr) of formula (IX):

in:

s ¹ is an integer from 2 to 8, preferably 5;

Pc, R ¹ , R ² , R ⁵ to R ⁷ , m and n are as defined in formula (VIII).

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

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

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

in:

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

Pc is any of the aforementioned CDH6 binding molecules of the present disclosure.

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

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

In some embodiments, n is 7±0.4, 7±0.3, 7±0.2, 7±0.1.

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

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

Exemplary, in some embodiments, Pc in the antibody drug conjugate comprises VH and VL, wherein:

1-1) the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:1, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:2;

1-2) 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;

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

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;

2-1) the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:3, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:4;

2-2) 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;

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

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;

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.

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

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;

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

2-1) 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: 14, 15, and 16, respectively;

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

2-3) The VH comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 12, and 13, respectively; the VL comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 70, 15, and 16, respectively.

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

Polynucleotides and vectors

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

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

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 generally 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 specific host.

The polynucleotides of the present disclosure can be prepared or obtained by known means (eg, 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.

In some embodiments, the polynucleotides and vectors of the present disclosure can be used to prepare CDH6 binding molecules. In some embodiments, the polynucleotides and vectors of the present disclosure are used to express CDH6 binding molecules in vitro or in vivo for different purposes such as detection, diagnosis, treatment, and regulation.

Host cells

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.

Bacterial cells include, for example, 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).

Fungal cells include, for example, cells of species of Trichoderma, Neurospora and Aspergillus; or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica) and Hansenula.

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

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.

Composition

The present disclosure provides a composition comprising the CDH6 binding molecule (e.g., antibody or antigen-binding fragment thereof) or anti-CDH6 antibody-drug conjugate of the present disclosure. For example, a pharmaceutical composition is provided, which contains an effective amount of the CDH6 binding molecule or anti-CDH6 antibody-drug conjugate for treating, alleviating or preventing a disease, and at least one pharmaceutically acceptable excipient, diluent or carrier.

In some specific embodiments, the unit dosage of the pharmaceutical composition may contain 0.01 to 99 weight % of CDH6 binding molecules (such as antibodies or antigen-binding fragments thereof) or anti-CDH6 antibody-drug conjugates, or the amount of CDH6 binding molecules or anti-CDH6 antibody-drug conjugates contained in a unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

In some embodiments, a product or product is provided, comprising the aforementioned CDH6 binding molecule (e.g., antibody or antigen-binding fragment thereof) or anti-CDH6 antibody drug conjugate. Optionally, the product comprises a container and a label. The container is such as a bottle, a syringe, and a test tube. The container holds a composition effective for treating a disease. The label on or connected to the container indicates that the composition is used to treat a selected disease.

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

Preparation method

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 the antigen-binding fragment thereof in a host cell as described above, and isolating the antibody or the antigen-binding fragment thereof from the host cell.

In some embodiments, the preparation method may further include a purification step, for example, purification using an A or G Sepharose FF column containing an adjusted buffer, washing away nonspecifically bound components, eluting the bound antibodies using a pH gradient method, detecting using SDS-PAGE, and collecting. Optionally, filtration and concentration are 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 must be immediately frozen, such as at -70°C, or freeze-dried.

Methods for producing and purifying antibodies or antigen-binding fragments are well known and can be found in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Techniques (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 amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods.

As an example of the preparation method, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can stably transfect 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 medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified and collected by conventional techniques. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange, etc.

In some embodiments, the present disclosure provides a method for preparing an anti-CDH6 antibody-drug conjugate, comprising: coupling any one of the aforementioned CDH6 binding molecules (e.g., an antibody or an antigen-binding fragment thereof) with an effector molecule to obtain the antibody-drug conjugate. Further, the method further comprises: purifying the antibody-drug conjugate.

In some embodiments, the method for preparing a conjugate of a ligand-exitecan or a derivative thereof as shown in the general formula (Pc-La-YD) comprises the following steps:

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

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

The compounds and preparation methods thereof in WO2020063673 and WO2022022508 are introduced herein in their entirety.

Medical uses and treatments

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

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

(2) preparing a drug for preventing or treating a disease or condition associated with CDH6;

(3) For the treatment of cancer or tumors;

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

(5) For the prevention of cancer or tumors;

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

(7) Used to detect CDH6 protein;

(8) preparing a reagent for detecting CDH6 protein;

(9) Used for diagnosis of cancer or tumor;

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

In the present disclosure, the disease associated with CDH6 includes but is not limited to cancer or tumor.

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

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

In some embodiments, the aforementioned diseases are 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, 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 conjugate disclosed in the present invention has 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.

In some embodiments, the present disclosure provides a method of treating a subject having cancer or a tumor, or a subject at risk of having 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.

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 having cancer, comprising: administering to the subject a therapeutically effective amount of an anti-CDH6 antibody-drug conjugate or a pharmaceutical composition thereof.

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

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

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.

Detection purpose

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

In some embodiments, the CDH6 binding molecules of the present disclosure also carry a detectable label. In some specific embodiments, the method further comprises using a reagent with a detectable label to detect the CDH6 binding molecules and anti-CDH6 antibody drug conjugates of the present disclosure. The method can be used for diagnostic purposes, or non-diagnostic purposes (for example, the sample is a cell sample, not a sample from a patient).

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

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

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

In some specific embodiments, the method further comprises:

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

(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.

In the context of the present disclosure, "test samples" refer to any type of sample in which it is desired to determine whether CDH6 is contained or the amount of CDH6 is desired to be determined. Exemplarily, test samples include 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).

The CDH6 binding molecules disclosed herein can specifically bind to CDH6, and thus can be used to detect the presence or level of CDH6 in a sample, and to diagnose whether a subject suffers from a disease associated with CDH6. Exemplarily, since the expression of CDH6 on tumor cells (or cancer cells) is higher than that on healthy cells, the CDH6 binding molecules can be used for the diagnosis, prognosis, and efficacy monitoring of cancer or tumors by detecting the expression level of CDH6 protein in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

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).

FIG. 2 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.

FIG3A is a graph showing the binding results of 2061-ChPR0003 and humanized antibodies to 786-O cells, and FIG3B is a graph showing the binding results of 2061-ChPR0022 and humanized antibodies to 786-O cells.

FIG. 4A shows the results of the evaluation of the internalization and immunotoxin activity of 2061-ChPR0003 and the humanized antibody depending on DT3C, and FIG. 4B shows the results of the evaluation of the internalization and immunotoxin activity of 2061-ChPR0022 and the humanized antibody depending on DT3C.

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.

6A-6B show the protein antigen binding activity of anti-CDH6 antibodies to human CDH9 and CDH10, wherein FIG. 6A shows the binding to human CDH9, and FIG. 6B shows the binding to human CDH10.

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

FIG8A-FIG8B are tests of the endocytic activity of anti-CDH6 antibodies in the high-expressing cell line OVCAR3, wherein FIG8A is the endocytic result based on the Phrodol method, and FIG8B is the endocytic result mediated by the DT3C toxin secondary antibody.

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, wherein 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.

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

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

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

Figures 13A-13C show the anti-tumor activity results of anti-CDH6 antibody-drug conjugates 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.

Figures 14A-14C show the anti-tumor activity results of anti-CDH6 antibody drug conjugates in the PA-1 cell xenograft model, wherein Figure 14A shows the comparison of anti-tumor activity at low doses with the same DAR value, Figure 14B shows the comparison of anti-tumor activity at multiple doses with different DAR values, and Figure 14C shows the weight change of CDH6 ADC in the PA-1 mouse model.

FIG. 15 shows the anti-tumor activity results of the anti-CDH6 antibody drug conjugate in the 786-O cell xenograft model.

DETAILED DESCRIPTION

definition

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 to which the present disclosure belongs.

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

"CDH6" (also known as Cadherin6, K cadherin) is a member of the type II cadherin family and is a single-pass 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 amino acid sequence compositions of one or more amino acids substituted, deleted and/or added in the amino acid sequence of the above-mentioned 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.

“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.

"Antibodies" cover various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as 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 consisting 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 region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains by different constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain. The sequences of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conservative sequences. The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

The antibodies disclosed herein may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic or modified forms thereof, wherein monoclonal antibodies are particularly suitable for use in a number of embodiments. In general, the antibodies disclosed herein 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 natural 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 natural (non-recombinant) cell form or express natural genes that are abnormally expressed, underexpressed or not expressed at all.

For the determination or definition of CDR, the deterministic depiction of CDR and the identification of residues comprising the binding site of the antibody can be completed by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be achieved 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 definition, and conformational definition. 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 approaches to model the tertiary structure of antibodies from the primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," 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 conformational definitions, the positions of 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). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDR, although they may be shortened or extended based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in the present disclosure, a CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between the various numbering systems is well known to those skilled in the art.

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

"Mouse antibody" in the present disclosure is a monoclonal antibody against human CDH6 or its epitope prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with CDH6 antigen, and then the hybridoma expressing the antibody with the desired sequence or functional properties is isolated. In one embodiment of the present disclosure, the murine CDH6 antibody or its antigen-binding fragment may further comprise a light chain constant region of a murine κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

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 mutations in vivo). However, the term "human antibody" does not include "humanized antibodies".

"Humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting a non-human CDR sequence into a human antibody variable region framework. The strong immune response induced by chimeric antibodies due to carrying a large amount of non-human protein components can be overcome. In order to avoid a decrease in activity while reducing immunogenicity, the fully human antibody variable region can be subjected to minimal reverse mutations to maintain activity. Examples of "humanization" include "humanizing" a VHH domain derived from Camelidae 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 the VH domain of a human conventional tetrapeptide chain structure antibody (also referred to as "sequence optimization" in this disclosure, in addition to humanization, "sequence optimization" may also cover other modifications to the sequence by one or more mutations that provide improved properties of VHH, such as removing potential post-translational modification sites). The humanized VHH domain may contain one or more completely human framework region sequences, and in some specific embodiments, may contain human framework region sequences of 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" to other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques 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, and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed in the present invention also include humanized antibodies after further affinity maturation of CDR by phage display. In addition, in order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

An "affinity matured" antibody is one that has one or more changes in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such changes, such changes resulting in an improvement in the affinity of the antibody for the antigen. 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 an increase in affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared, for example, by methods known in the art 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): 889896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

"Chimeric antibody" is an antibody formed by fusing the variable region of an antibody of a first species with the constant region of an antibody of a second species, which can reduce the immune response induced by the antibody of 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 gene is cloned from the mouse hybridoma cells, and then the constant region gene of the human antibody is cloned as needed, and the mouse variable region gene and the human constant region gene are connected into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a 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 a variant thereof, such as a heavy chain constant region containing human IgG2 or IgG4, or an IgG1 that is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

"Antigen-binding fragment" includes single-chain antibodies (i.e., heavy chain or light chain); 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, double-chain antibodies (diabody), three-chain antibodies (tribody), four-chain antibodies (tetrabody) and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for producing 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 bond-stabilized form Fab-dsFv was first disclosed in WO2010/035012. The antigen-binding fragments disclosed herein also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171. Multivalent antibodies may comprise multispecificity, such as bispecificity or monospecificity (see, e.g., WO92/22583 and WO05/113605).

Typically, the CDH6 binding molecules of the present disclosure will bind to the antigen or target protein (i.e., human CDH6) to be bound with a dissociation constant ( _KD ) of preferably ^10-7 to ^10-10 moles/liter (M), more preferably ^{10-8 to 10-10} moles/liter, even more preferably ^10-9 to ^10-10 or less, as measured in a Biacore or KinExA or Fortibio assay, and/or with an association constant (KA) of at least ^10-7 M, preferably at least ^10-8 M, more preferably at least ^10-9 M, more preferably at least ^10-10 M. Any _KD value greater than ^10-4 M is generally considered to indicate non-specific 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 in the present disclosure.

"Epitope" refers to a site on an 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 tertiary folding of the protein). Epitopes formed by adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. Epitopes usually exist in a unique spatial conformation, which includes at least 3-15 amino acids. Methods for determining epitopes are well known in the art, including immunoblotting and immunoprecipitation detection analysis, etc. 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, etc.

"Binding affinity" or "affinity" is used in the present 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 quantified by determining the dissociation constant ( _KD ). _KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, the surface plasmon resonance (SPR) method (Biacore). 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. _KD is related to ka and kd by the equation _KD = kd/ka. 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, _KD 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). Other standard assays for assessing the binding ability of an antibody against a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometric analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), such as by using a Biacore ^™ system or KinExA. Binding affinities associated with different molecular interactions, for example, comparison of the binding affinities of different antibodies for a given antigen, can be compared by comparing the _KD values of the individual antibody/antigen complexes. Similarly, the specificity of an interaction can be assessed by determining and comparing the _KD value for an interaction of interest (e.g., a specific interaction between an antibody and an antigen) to the _KD value for a non-interest interaction (e.g., a control antibody known not to bind IGF-1R or TRGV9).

"Conservative substitution" refers to substitution with another amino acid residue having a property 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 replacing an amino acid residue in a group showing similar properties as described above, it will not show a specific change in properties.

"Homology", "identity" or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same nucleotide or amino acid monomer, for example, if every position of the two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percentage of 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, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.

"Cross-reactivity" refers to the ability of the disclosed antibodies to bind to CDH6 from different species. For example, the disclosed CDH6 binding molecules (e.g., antibodies) may also bind to CDH6 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens 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.

"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 can 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 cell surface molecules from the cell surface to the inside of the cell across the plasma membrane. 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 measurement of the kinetics of the movement of the molecule from the cell surface through the plasma membrane to the inside of the cell. The internalization activity or internalization rate of antigens and antibodies can be monitored and/or measured by a variety of 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 (ADC) bind to tumor cell surface antigens through the antibody in the ADC, which is then internalized into endosomes and transformed from endosomes to lysosomes. Then, the bioactive molecules (e.g., toxins or payloads) are dissociated from the ADC under the action of hydrolases in the lysosomes. 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 tumor cells that do not express or express low levels of surrounding antigens (the so-called bystander effect).

"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 a 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 US2005-0238649A1. The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., 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).

"Amino acid unit" refers to an amino acid to which the carbonyl group in the following structural formula Y _R can be connected to the stretching unit if there is a stretching unit, or to which Y _R can be directly connected to the cytotoxic drug if there is no stretching unit. In the embodiments of the present disclosure, the amino acid unit is represented by -K _k -:

-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 _Ka or _Kb , and k is an integer between 0 and 10:

in:

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

_R24 is 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 , -(CH ₂ ) ₄ NHCONH ₂ , -CH ₂ CH ₂ CH(OH)CH ₂ NH ₂ , 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

_R25 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-.

In one embodiment, -K _k - is a dipeptide, preferably -valine-citrulline-, -phenylalanine-lysine- or -N-methylvaline-citrulline-, and more preferably -valine-citrulline-.

"Stretcher" refers to a chemical structure fragment 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.

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.

"Stretcher unit" refers to a chemical structure that can couple the amino acid unit to a cytotoxic drug when the amino acid unit is present, or can couple the cytotoxic drug via the carbonyl group on YR when the amino acid unit is absent. In the disclosed embodiments, the stretcher unit is represented by -Q _q -, where q is selected from 0, 1, and 2.

The stretching unit in the present disclosure is PAB, the structure of which is a 4-iminobenzylcarbamoyl fragment, the structure of which is shown in the following formula, connected to D,

abbreviation

Joint components include but are not limited to:

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

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

Citrulline = 2-amino-5-ureidopentanoic acid

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

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

MC(PEG)6-OH = Maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine)

SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate

SPDP = N-succinimidyl 3-(2-pyridyldithio) propionate

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

IT = Iminothiolane

PBS = Phosphate Buffered Saline

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

"Drug loading" refers to the average number of cytotoxic drugs loaded on each ligand in 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 the 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 light spectroscopy, mass spectrometry, ELISA test, monoclonal antibody molecular size variant determination method (CE-SDS) and HPLC characteristics can be used to identify the average amount of drug substance per ADC molecule after the coupling reaction. The monoclonal antibody molecular size variant determination method (CE-SDS) disclosed in the present invention can use sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method, under reducing and non-reducing conditions, according to the molecular weight, according to the capillary electrophoresis method (2015 edition of the "Chinese Pharmacopoeia" 0542), to quantitatively determine the purity of the recombinant monoclonal antibody product.

In one embodiment of the present disclosure, the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the ε-amino group of the lysine residue through 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.

The drug loading of the antibody drug conjugate can be controlled by the following non-limiting methods, including:

(1) Control the molar ratio of the linking reagent and the monoclonal antibody,

(2) Control the reaction time and temperature,

(3) Select different reaction reagents.

Although for a particular conjugate molecule, the drug to antibody ratio has an exact value (e.g., n in formula (I)), it should 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 with 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,. , 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.4, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0. In some embodiments, a DAR of 'about x' means that the measured value of the DAR is within 20% of x.

The detection method of DAR, for example, extrapolates the DAR value from the LC-MS data of reduced and deglycosylated samples. LC/MS allows the average number of effective load (drug moiety) molecules connected to the antibody in quantitative ADC. HPLC separates the antibody into light chain and heavy chain, and also separates the heavy chain (HC) and light chain (LC) according to the number of linker-effective load 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. According to the average load of LC and HC chains, the average DAR of ADC can be calculated. The DAR of a given immunoconjugate sample represents the average number of drug (effective load) molecules connected to a tetrameric antibody containing two light chains and two heavy chains. For example, the DAR detection method in WO2018142322.

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 having 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, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

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

"Alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane radical by removing two hydrogen atoms, and is a straight or branched group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (—CH ₂ —), 1,1-ethylene (—CH(CH ₃ )—), 1,2-ethylene (—CH ₂ CH ₂ )—, 1,1-propylene (—CH(CH ₂ CH ₃ )—), 1,2-propylene (—CH ₂ CH(CH ₃ )—), 1,3-propylene (—CH ₂ CH ₂ CH ₂ —), 1,4-butylene (—CH ₂ CH ₂ CH ₂ CH ₂ —), and 1,5-butylene (—CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ —), and the like. 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, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo.

"Alkoxy" refers to-O-(alkyl) and-O-(unsubstituted cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described 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, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

"Cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, the cycloalkyl ring containing 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, etc.; polycyclic cycloalkyls include spirocyclic, fused and bridged cycloalkyls.

"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 a heteroatom selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. Preferably, it 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, etc. Polycyclic heterocyclyls include spirocyclic, fused ring and bridged heterocyclyls.

"Spiro heterocyclic group" refers to a polycyclic heterocyclic group in which one atom (called spiro atom) is shared between 5 to 20 monocyclic rings, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), 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 rings, spiro heterocyclic groups are divided into single spiro heterocyclic groups, double spiro heterocyclic groups or multi-spiro heterocyclic groups, preferably single spiro heterocyclic groups and double spiro heterocyclic groups. 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 single spiro heterocyclic groups. Non-limiting examples of spiro heterocyclic groups include:

"Fused heterocyclic group" refers to a polycyclic heterocyclic group of 5 to 20 members, 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, 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, 6 to 14 members, and for example, 7 to 10 members. According to the number of constituent rings, it can be divided into a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, for example, a bicyclic or tricyclic, and for example, a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

"Bridged heterocyclic group" refers to a polycyclic heterocyclic group of 5 to 14 members, in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but none of the rings has a completely conjugated π electron system, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) _m (wherein m is an integer from 0 to 2), and the remaining ring atoms are carbon. For example, it is 6 to 14 members, and for example, it is 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, for example, bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

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:

wait.

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, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

"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 with a conjugated π electron system, for example, 6- to 10-membered, such as phenyl and naphthyl, specifically phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples thereof include:

The aryl group may be substituted or unsubstituted. When substituted, 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, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

"Heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 members, more preferably 5 or 6 members, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, 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:

The heteroaryl group may be optionally substituted or unsubstituted, and 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, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

"Amino protecting group" is to protect the amino group with a group that is easy to remove in order to keep the amino group unchanged when other parts of the molecule react. Non-limiting examples include 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, acetyl, benzyl, allyl and p-methoxybenzyl. These groups can be optionally substituted with 1-3 substituents selected from halogen, alkoxy or nitro. The amino protecting group is preferably 9-fluorenylmethoxycarbonyl.

"Aminoheterocyclyl" refers to a heterocyclyl group substituted by one or more amino groups, preferably by one amino group, wherein the heterocyclyl group is as defined above, and wherein "amino" refers to -NH ₂ . Representative embodiments of the present disclosure are as follows:

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

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

"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.

"Haloalkyl" means an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

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

"Hydroxy" refers to an -OH group.

"Halogen" refers to fluorine, chlorine, bromine or iodine.

"Amino" refers to _-NH2 .

"Nitro" refers to _-NO2 .

The abbreviation "Me" in the chemical formula is methyl.

"Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other 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 (such as olefinic) bonds.

"Nucleic acid" or "polynucleotide" are used interchangeably in this disclosure and refer to any DNA molecule or RNA molecule that is single-stranded or double-stranded and, in the case of single strands, 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.

"Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or intentional mutations. 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 names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny having the same function or biological activity as screened in the initially transformed cell are included.

"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.

"Administering," "applying," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, such as for treatment, pharmacokinetic, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "applying," and "treating" also means in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a combination 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.

"Treatment" means administering an internal or external therapeutic agent, such as any binding protein or pharmaceutical composition thereof disclosed herein, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, 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 disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although the embodiments of the present disclosure (e.g., treatment methods or products) may not be effective in alleviating the symptoms of the target disease in a subject, they 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.

An "effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow 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 method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

"Excipients" are additives other than the main drug in pharmaceutical preparations, and can also be called excipients. For example, preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. "And/or" should be regarded as specifically disclosing that each of the two specified features or components has or does not have the other. Therefore, 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.

The “subject” and “patient” of the present disclosure refer to mammals, especially primates, and especially humans.

Example

The following examples are used to further describe the present disclosure, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify specific conditions are usually carried out according to conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or product manufacturer. Reagents that do not specify specific sources are conventional reagents purchased from the market.

Example 1. Preparation of recombinant CDH6 extracellular domain protein

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 the expression vector containing the antigen gene was obtained by enzyme ligation and transformation. The 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 with the anti-CDH6 antibody Nov0712 (the sequence of Nov0712 is shown in SEQ ID NO 234 and 235 of US2016/0046711A1). The test results are shown in Figure 1. The results show that the produced human CDH6-His protein has good binding activity with the anti-CDH6 antibody Nov0712.

The recombinant monkey CDH6-His protein is a His tag fused to the C-terminus of the amino acids in the extracellular region (Thr22-Gly614) of the monkey CDH6 protein (UniProt, accession number: G7MUQ7-1) and was purchased from Acro Biosystems.

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

After transfecting CHO-K1 cells with a plasmid encoding a recombinant human CDH6 protein (UniProt, accession number: P55285) or a plasmid encoding a cynomolgus monkey CDH6 protein (UniProt, accession number: G7MUQ7-1), a cell line that stably expresses the recombinant human or cynomolgus monkey CDH6 protein was obtained by screening. In short, the nucleotide sequence encoding the human or cynomolgus monkey CDH6 protein is cloned and connected to a lentiviral expression vector. The CHO-K1 cell line was transfected with a lentiviral transfection method well known in the industry, selectively cultured for two weeks using F-12 medium containing Puromycin (purchased from Gibco), subcloned in a 96-well culture plate by limiting dilution, and 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.

Example 3. Screening of mouse anti-CDH6 monoclonal antibodies

The recombinant human CDH6-His in Example 1 was used as an immunogen to immunize 6-8 week old SJL mice. The initial immunization dose was 50 μg per mouse. Two weeks after the initial immunization, booster immunization was performed with an immunization dose of 25 μg per mouse. Each subsequent booster immunization was performed 2 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 is as follows: the plate was coated with 1 μg/mL recombinant human CDH6-His, overnight at 4°C, blocked with PBST buffer containing 1% BSA for 1 hour, and washed 3 times. In the blocking buffer, the mouse serum was diluted 10 times in a gradient starting at 1:100, incubated at 37°C for 1 hour, washed 3 times, and incubated with the secondary antibody of anti-mouse IgG-Fc-HRP for 1 hour. Wash 3 times with PBST, add 100 μL TMB substrate to each well, and terminate the reaction with 2M HCl after 15 minutes. The absorbance at 450nm was read using an enzyme reader. The specific FACS detection process is as follows: 1E5/100uL cells were plated per well of the cells, and the supernatant was discarded after centrifugation and added to the gradient diluted mouse serum. Incubate at 4℃ for 1 hour. After washing twice with 1% BSA/PBS buffer, the supernatant was discarded, and Alexa Flour 647 goat anti-mouse secondary antibody (purchased from Jackson immuno research) was added, and incubated at 4℃ in the dark for 30 minutes. Wash twice with 1% BSA/PBS buffer, resuspend in 200uL buffer, and then detect by FACS. The mouse serum immunized with the immunogen recombinant human CDH6-his has different degrees of binding to the immunogen. When the highest dilution of the serum is 1:100K, it can still show antigen-antibody reaction.

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. NH ₄ OH with a final concentration of 1% (w/w) was added to lyse the mixed red blood cells in the spleen cells to obtain a spleen cell suspension, and the cells were washed by centrifugation at 1000 rpm for 3 times. Mouse spleen cells were mixed with mouse myeloma cells SP2/0 at a ratio of 5:1 in the number of live cells, and cell fusion was performed using a high-efficiency electrofusion method. The fused cells were diluted into a 96-well cell culture plate using DMEM medium containing 20% fetal bovine serum and 1×HAT (w/w), with a total of 1x 10 ⁵ cells per well in 200 μL, and placed in a 37°C incubator with 5% (v/v) CO ₂ . After 14 days, the fused cells were screened by ELISA or Acumen, and the positive clones with OD450nm>0.1 were expanded to 24-well cell plates and continued to be cultured at 37°C, 5% (v/v) CO ₂ using DMEM medium containing 10% (w/w) HT fetal bovine serum. After 3 days, the culture fluid of the 24-well cell plate was centrifuged to collect the supernatant, and the binding activity to recombinant human CDH6-positive cells, recombinant monkey CDH6-positive cells and related tumor cells was determined by FACS. After initial screening and retesting of subclones, excellent positive monoclonal strains were screened using the above-mentioned ELISA and FACS detection methods. The sequenced amino acid sequence of the antibody is as follows, and the underlined part is the complementary determining region CDR sequence (using the Kabat numbering system).

Table 1. CDR sequences of mouse anti-CDH6 antibodies (Kabat numbering system)

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

For chimeric antibodies, in order to reduce possible immunogenicity, the mouse antibody is humanized and the constant region of the mouse monoclonal antibody is replaced to obtain a recombinant chimeric antibody. The nucleotide sequence encoding the variable region of the mouse monoclonal antibody is cloned into a vector containing the protein sequence of the human heavy and light chain constant regions (Human IgG1, kappa) and transfected into CHO cells to obtain a chimeric antibody, whose heavy chain and light chain variable regions are the same as those of the mouse antibody, respectively.

For humanized antibodies, after predicting the structure of mouse monoclonal antibodies by homology modeling, the mouse anti-CDR was 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. Then, 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 prepare antibodies. The VH and VL sequences obtained after humanization are as follows, and the CDR region of the sequence is underlined. The humanized antibodies obtained by combining VH and VL with different sequences are shown in Table 3.

Table 2. Selection of variable region frameworks for humanized anti-CDH6 antibodies

Table 3. Corresponding VH and VL of humanized antibodies

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

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

786-O cells express CDH6 and are cultured in 1640 growth medium containing 10% fetal bovine serum (Gibco, cat#10099141C). The detection method is as follows: Step 1: Plate 786-O cells (2E5/well). Step 2: Then add the antibody to be tested, 100μL/well, the highest concentration point: 100nM, 3-fold dilution, a total of 8 concentrations, incubate at 4℃ for 1h. Step 3: Add secondary antibody anti-hIgG Alexa Fluor-647 (Jackson, cat#209-605-088) at a ratio of 1:800 and incubate at 4℃ for 30min. Step 4: Perform MFI detection using flow cytometry. Isotype hIgG1 is used as a negative control.

The binding results of 2061-ChPR0003 and the humanized antibody to 786-O cells are shown in FIG3A , and the binding results of 2061-ChPR0022 and the humanized antibody to 786-O cells are shown in FIG3B . The results show that the humanized antibodies still maintain good binding activity.

2. DT3C antibody internalization and killing activity evaluation system

DT3C is a recombinantly expressed fusion protein with a molecular weight of 70KD. It is formed by the fusion of Fragment A (toxin part only) of diphtheria toxin and 3C fragment (IgG binding part) of group G streptococcus. This protein has a high affinity with the IgG part of the antibody and enters the cell together with the antibody when it is internalized. Under the action of intracellular furin, DT is released with toxicity. DT can inhibit the activity of EF2-ADP ribosylation, block the protein translation process, and eventually lead to cell death. By using this system, the internalization of the antibody and the cell killing effect caused by the immunotoxin can be observed simultaneously (Biochem Biophys Res Commun. 2014 Nov 28; 454 (4): 600-3.).

Evaluation method for internalization and immunotoxin activity dependent on DT3C: Sterile filtered DT3C and the chimeric antibody to be tested (DT3C molar concentration is twice the antibody molar concentration) are mixed in a 1:1 volume ratio, incubated at 37°C for 30 minutes, and then diluted with complete medium, added to 786-O cells plated one day in advance (2000 cells/well), and incubated at 37°C in a 5% _CO2 incubator for 6 days. CellTiter-Glo was added, incubated at room temperature in the dark for 10 minutes, and chemiluminescence was read on PerkinElmer. As shown in Figures 4A and 4B, the humanized antibodies of 2061-ChPR0003 and 2061-ChPR0022 both maintained strong killing activity.

Example 6. Further modification of humanized antibodies

After evaluation of humanized antibodies, 2061-rH3rL1B and 2061-H2Lptm3 antibodies were selected to remove TCE sites and PTM risk sites. Two antibodies, 2061-1 and 2061-3, were obtained respectively. The heavy chain CDR (HCDR1\HCDR2\HCDR3) and light chain CDR (LCDR1\LCDR2\LCDR3) are shown in Table 4, and the CDR region of the underlined sequence is marked.

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

The obtained humanized sequence is as follows:

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

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

LCDR1 is: X ₁ ASSTVGSSYLY (SEQ ID NO: 24), wherein X ₁ is N or T,

LCDR2 is: STSNLAX ₂ (SEQ ID NO: 25), wherein X ₂ is S or T,

LCDR3 is shown in SEQ ID NO: 10;

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

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

STSNLAT (SEQ ID NO:22).

The antibody derived from 2061-ChPR0022 disclosed in the present invention has the following CDRs:

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

LCDR1 is: RSSQSIVHSX ₃ X ₄ NTYLE (SEQ ID NO: 26), wherein X ₃ is N or T, and X ₄ is G or A;

LCDR2 and LCDR3 are shown in SEQ ID NO: 15 and 16 respectively;

In some specific 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).

An exemplary full-length sequence is as follows, wherein the italics are constant regions:

>2061-1 Heavy Chain

>2061-1 Light Chain

>2061-3 heavy chain

>2061-3 light chain

>IgG1Fc region

Example 7. Anti-CDH6 antibody binding activity to CDH6 protein antigens from different species

ELISA experiments were used to detect the binding properties of anti-CDH6 antibodies from different species. CDH6 recombinant protein with a His tag was directly coated, and after the addition of the antibody, the activity of the antibody binding to the antigen was detected by adding a secondary antibody (HRP-conjugated anti-primary antibody Fc antibody) and the HRP substrate TMB.

Different species of CDH6-His proteins (Acro, cat#CA6-H5229for human Cadherin-6；Acro, cat#CA6-C52H3 for Cynomolgus/Rhesus macaque Cadherin-6；Acro, cat#CA6-M52H8for mouse Cadherin-6) were coated on 96-well ELISA plates, 100μL per well at a concentration of 1μg/mL, and incubated overnight at 4℃. Wash three times with washing solution, 250μL per well, and shake for 10 seconds each time to ensure sufficient washing. Add 200μL/well blocking solution (3% BSA) and incubate at room temperature for 2 hours. Wash three times with washing solution, 250μL per well, and shake for 10 seconds each time to ensure sufficient washing. Add 100μL of anti-CDH6 test antibody diluted with diluent to each well. Incubate at room temperature for 1 hour, wash three times with washing solution, 250μL per well, add 100μL of HRP-labeled goat anti-human IgG secondary antibody (BETHYL, Cat#A80-319P-29) diluted 1:20000 with diluent to each well. Incubate at room temperature for 1 hour, wash three times with washing solution, 250μL per well. Add 100μL of TMB mixed solution (Biopanda, cat#TMB-S-003Lot#2022122901 for TMB colorimetric solution A, cat#TMB-S-003Lot#2022122 902 for TMB colorimetric solution B) to each well, protect from light, observe the color reaction, and after about 5 minutes, add 50μL of stop solution (Solarbio, cat#C1058) to each well. Read the OD value at 450nm with Envision microplate reader. Nov0712 antibody was used as a positive control and isotype IgG1 as a negative control.

The results showed that, as shown in Figures 5A, 5B, and 5C, 2061-1 and 2061-3 can bind to human and monkey CDH6 antigens well. 2061-1 can bind to mouse CDH6 well. The binding EC ₅₀ values of anti-CDH6 antibodies were calculated, and the results are shown in Table 5.

Table 5. _EC50 and Emax values of the affinity of each antibody for human, monkey and mouse CDH6 antigen

Example 8. Anti-CDH6 antibody binding activity to human CDH9 and CDH10 proteins of the same family

The CDH protein family also includes CDH9 and CDH10, which have the highest homology with CDH6 protein. 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 non-specific binding to CDH9 or CDH10 may produce unknown toxicity risks. Therefore, this embodiment performs binding detection.

Human CDH9 protein (Acro, cat#CA9-H52H6) and human CDH10 protein (Acro, cat#CA0-H52H5) were coated on 96-well ELISA plates, 100 μL per well at a concentration of 5 μg/mL, and incubated overnight at 4°C. Wash three times with 250 μL per well, and shake for 10 seconds each time to ensure adequate washing. Add 200 μL/well blocking solution (3% BSA) and incubate at room temperature for 2 hours. Wash three times with 250 μL per well, and shake for 10 seconds each time to ensure adequate washing. Add 100 μL of anti-CDH6 antibody diluted with diluent to each well. Incubate at room temperature for 1 hour, wash three times with 250 μL per well, and add 100 μL of HRP-labeled goat anti-human IgG secondary antibody (BETHYL, Cat#A80-319P-29) diluted 1:20000 with diluent to each well. Incubate at room temperature for 1 hour, wash three times with 250μL per well. Add 100μL TMB mixed solution (Biopanda, cat#TMB-S-003Lot#2022122901 for TMB colorimetric solution A, cat#TMB-S-003Lot#2022122 902 for TMB colorimetric solution B) to each well, protect from light, observe the color reaction, and after about 5 minutes, add 50μL stop solution (Solarbio, cat#C1058) to each well. Read the OD value at 450nm using Envision microplate reader.

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, and 2061-1 and 2061-3 had no binding ability for human CDH10. The binding EC ₅₀ values of the anti-CDH6 antibodies were calculated, as shown in Table 7.

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

Table 7. Affinity _EC50 and Emax values of each antibody binding to human CDH9 and CDH10 antigens

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

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

The experimental method is as in FACS of 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 in the present invention have good binding activity to tumor cell lines with high or low CDH6 expression. EC ₅₀ values and Max MFI values were calculated, as shown in Table 8.

Table 8. Affinity _EC50 and Emax values of each antibody for tumor cell lines with different CDH6 expression levels

Example 10. Detection of endocytic activity of anti-CDH6 antibodies

1. pHrodo indicator reagent to assess internalization of anti-CDH6 antibody

Zenon ^TM pHrodo ^TM iFL IgG indicator reagent (Invitrogen, Cat. No. Z25611) provides a fast, real-time and reliable method to evaluate antibody internalization. The pHrodo iFL Green labeled Fab fragment can bind to the Fc part of the intact IgG antibody to form a labeled complex within 5 minutes. When the complex enters the cell through endocytosis, the fluorescence increases sharply as the acidity of the surrounding environment of the complex increases. The fluorescence intensity can be recorded in real time by the Incucyte instrument to determine the degree of antibody internalization. Specifically, the cells are plated, 2500 OVCAR3 cells per well, 50μL per well. After the cells adhere to the wall, 50μL of the labeled complex is added to each well. In the same centrifuge tube, the labeled complex is configured: antibody 20nM, pHrodo 60nM, mixed and incubated at room temperature for 15 minutes. Put it into the Incucyte instrument (IncucyteS3) and set the plate reading conditions according to the instrument operation steps. After the plate reading is completed, analyze the data according to the steps of the instrument's own 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.

2. DT3C antibody internalization and killing activity evaluation system

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 cells OVCAR3, and the internalization and immunotoxin killing activity of 2061-3 molecule was comparable to that of the positive control.

Example 11. Detection of DT3C killing activity of anti-CDH6 antibodies against tumor cell lines with different CDH6 expression levels

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). As 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 equivalent to that of the positive control antibody Daiichi07. The calculated EC ₅₀ values are shown in Table 9.

Table 9. EC ₅₀ values and Top viability % values of each antibody against DT3C of tumor cell lines with different CDH6 expression levels

Example 12. Epitope differentiation between anti-CDH6 antibodies

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 in patent US2020/0390900A1. Plasmids with different domain deletions were highly expressed on HEK293 cells. After digestion, the HEK293 cells were centrifuged and the supernatant was discarded. The cells were resuspended in 3E ⁶ /mL and added to a 96-well plate at 50 μL/well. After adding 50 μL of the antibody to be tested (final concentration 50 nM), incubate at 4°C for 1 hour. After washing twice, Alexa Fluor 500 was added at a dilution ratio of 1:800. 647AffiniPure Mouse Anti-Human IgG (Fcγfragment specific) secondary antibody (Jackson, 209-605-098) was used for flow cytometry. The cells were 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 was detected by flow cytometry. 647 fluorescence intensity. The results showed that the binding of each antibody detected by FACS is shown in Figure 10A, and the analysis of each epitope is shown in Figure 10B. 2061-1 binds to the EC1 domain, and 2061-3 binds to the EC3 domain.

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

Example 13. Preparation of Antibody Drug Conjugate (ADC)

1. Preparation of Compound 9-A

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.

2. Design and preparation of ADC

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.

Preparation of ADC:

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (10 mM, 75.4 μL, 753.5 nmol) was added to a PBS buffer solution of anti-CDH6 antibody (0.05 M PBS buffer solution at pH = 6.5; 10.0 mg/mL, 5.0 mL, 342.5 nmol), 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.

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

Reference substance ADC-5: Compound ADC-5 was prepared by referring to the preparation method of the antibody drug conjugate H01L02-Dxd in the example of US2020/0390900A1, and the theoretical DAR value was 8. US2020/0390900A1 is incorporated into the present disclosure by reference in its entirety.

Table 11. ADC information

Example 14. Cell Binding Activity Detection of ADC

The cell binding activity of antibodies to CDH6 before and after conjugation to toxin was evaluated in OVCAR3 tumor cells that highly express CDH6. First, the OVCAR3 cells were digested and centrifuged to discard the supernatant, resuspended to 3E ⁶ /mL, and added to a 96-well plate at a volume of 50μL/well. After adding 50μL of the test antibody (final concentration up to 100nM, 3-fold dilution), incubate at 4℃ for 1 hour. After washing twice, Alexa Fluor 500 was added at a dilution ratio of 1:800. 647AffiniPure Mouse Anti-Human IgG (Fcγfragment specific) secondary antibody (Jackson, 209-605-098) was used for flow cytometry. The cells were 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 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 coupling.

Example 15. Cytotoxicity of anti-CDH6 ADC against tumor cell lines expressing different CDH6 levels

OVCAR3 cells were cultured in 1640 growth medium containing 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 containing 10% fetal bovine serum (Gibco, cat#10099141C). PA-1 and NCI-N87 cells were cultured in MEM (with ENEAA) growth medium containing 10% fetal bovine serum (Gibco, cat#10099141C). Cells were grown overnight in 96-well culture plates at a density of 2000 (OVCAR3) or 1000 (786-O) or 3000 (PA-1, NCI-N87) per well. An equal volume of serial dilutions of ADC was added the next day. After 4 days, the killing of 786-O and PA-1 cells was detected using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega), and after 6 days, the killing of OVCAR3 and NCI-N87 cells was detected using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega). Cell viability was determined according to the method in the kit instructions. Cell viability was evaluated as a percentage of control untreated cells.

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. The ADC killing EC ₅₀ values are shown in Table 12.

Table 12. Cytotoxicity of ADC in cell lines with different expression levels

Note: NA means the value cannot be calculated.

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

In order to evaluate the anti-tumor activity of the ADC disclosed in the present invention in mice, this example evaluates the ovarian cancer OVCAR3 transplanted tumor model with high CDH6 expression.

NCG mice were obtained from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. OVCAR3 cells 5×10 ⁶ /0.15mL/mouse (1:1 with matrix gel added) were inoculated subcutaneously. When the average tumor volume grew to 80-120mm ³ , mice with appropriate individual tumor volume were selected to enter the main experimental group and randomly divided into groups of 6 mice per group. The administration method was intraperitoneal administration. The frequency of administration was once a week, for a total of 3 times. The day of grouping was defined as D0, and the administration period was from D0 to D14 days. Pharmacodynamics and safety were evaluated by clinical observation indicators such as tumor inhibition rate and body weight.

The calculation formula is as follows:

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

Relative tumor proliferation rate T/C (%) = (T-T0)/(C-C0)*100, where T and C are the tumor volumes of the treatment group and the control group 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 (%).

The PBS control group was used as a negative control. TGI represents the maximum tumor growth inhibition during the experiment. Experimental data are expressed as mean ± standard error (SEM), and GraphPad Prism 9 software was used for plotting and statistical analysis. Comparison of significant differences in tumor volume. One-way ANOVA was used, and multiple comparisons were performed if F was significant. When comparing tumor volumes on day 28, the tested ADCs had highly significant target-dependent antitumor activity compared with the vehicle group (P values were <0.0001****, respectively).

The results showed that the disclosed ADC had good anti-tumor activity in the OVCAR3 cell xenograft model with high CDH6 expression, and there was no obvious distinction between different molecules. The disclosed ADC did not cause significant changes in the body weight of mice while showing good anti-tumor activity.

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 is a comparison of the anti-tumor activity of low doses with the same DAR value, Figure 13B is a comparison of the anti-tumor activity of different DAR values of the same toxin, and Figure 13C is a graph of the weight change of CDH6ADC in the OVCAR3 mouse model.

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

Table 14. Antitumor activity of ADCs in OVCAR3 xenograft tumor model

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

Next, the anti-tumor activity of the ADC disclosed in the present invention in mice was further evaluated, and a PA-1 transplanted tumor model with low CDH6 expression was selected.

Human ovarian cancer PA-1 cells (ATCC-CRL-1572) were cultured in vitro in a monolayer incubator at 37°C and 5% CO _2. The cells were routinely digested and passaged twice a week with trypsin. When the cell saturation was 80%-90% and the number reached the requirement, the cells were collected, counted, and inoculated. 0.2 mL (10×10 ⁶ cells) of PA-1 cells (1:1 with matrix gel) were subcutaneously inoculated into the right back of each mouse. For the pharmacodynamics experiment, group dosing began when the average tumor volume reached 118 mm ^3. The dosing frequency was once a week, and the dosing was stopped after 3 doses.

The animals used were female BALB/c nude mice. 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 International Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health status and mortality of the animals were monitored daily. Routine examinations included observing the effects of tumor growth and drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake (only visual inspection), weight changes measured twice a week, and physical signs or other abnormal conditions.

The experimental index is to examine whether the tumor growth is inhibited, delayed or cured. The tumor diameter is measured with a vernier caliper twice a week. The calculation formula for the tumor volume is shown in Example 16.

Statistical analysis, including the mean and standard error (SEM) of tumor volume at each time point for each group. Statistical analysis was performed to evaluate intergroup differences based on data from day 21 after group administration. Comparisons between three or more groups were analyzed using one-way ANOVA, and multiple comparisons were performed using the Dunnett t (2-sided) method. All data analyses were performed using Graphpad Prism 9. P < 0.05 was considered significant. (P values were < 0.05*, < 0.01**, respectively). Relative body weight changes were calculated based on the weight of the animals at the start of dosing. Data points represent the mean percentage change in body weight within the group, and error bars represent standard errors (SEM).

The comparison of the anti-tumor activity of each ADC on the female BALB/c nude mouse model with subcutaneous xenograft tumors of human ovarian cancer PA-1 cells at low doses with the same DAR value is shown in Figure 14A, and the comparison of the anti-tumor activity of each ADC on the female BALB/c nude mouse model with subcutaneous xenograft tumors of PA-1 cells at multiple doses with different DAR values is shown in Figure 14B. The relative weight changes of tumor-bearing mice after administration of the test drugs are shown in Figure 14C.

The results showed that under the same dose and DAR6 conditions, the ADC-1 and ADC-2 disclosed in the present invention had better anti-tumor activity than the positive control, and the TGI reached 61.57% at a low dose of ADC-1 (DAR6) 2mpk. At the same time, the ADC disclosed in the present invention had a dose-dependent anti-tumor activity, and a high dose completely inhibited tumor growth. There was no difference in activity between DAR8 and DAR6 under the same toxin conditions. The ADC disclosed in the present invention did not cause significant changes in the weight of mice while showing good anti-tumor activity.

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.

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

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

Table 17. Comparison of antitumor activity of multiple doses of ADC with different DAR values

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

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

NOD SCID mice were obtained from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. 786-O cells were cultured in vitro, tumor cells in the logarithmic growth phase were collected, and single cell suspensions were prepared by resuspending in 1:1 PBS and Matrigel according to the required concentration. 0.2 mL of cell suspension was inoculated subcutaneously in the right upper limb of each NOD SCID mouse (1×10 ⁷ cells/mouse, containing 50% Matrigel). 14 days after cell inoculation, 24 mice with an average tumor volume of approximately 149.5 mm ³ were selected, and the animals were divided into 4 groups by random block method, with 6 mice in each group. The day of grouping was recorded as "PG-D0", and the drug was administered according to the schedule in Table 18, with a dosing frequency of once a week, and the drug was stopped after 3 doses.

Table 18. Dosage and dosing schedule

Note: ip: intraperitoneal injection; QW: once a week; “/” indicates not applicable.

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

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

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

The calculation formula of the mouse body weight change rate (Percent of body weight change, BWC%) is: (BW _c -BW _i )/BW _i × 100%, BW _c represents the animal weight on the cth day of the experiment, and BW _i represents the animal weight when grouped.

The tumor growth curve was drawn with the time point as the X-axis and the tumor volume as the Y-axis; the weight growth curve was drawn with the time point as the X-axis and the animal weight change rate as the Y-axis. The analysis was based on the original data, and the results were expressed as mean ± SEM. The tumor volume was analyzed by statistical T-test method using Graphpad Prism software, and P < 0.05 was considered to be significantly different.

At the end point (PG-D21), the anti-tumor activity of each ADC on the subcutaneous xenograft tumor mouse model of human renal cancer cell 786-O cells is shown in Figure 15. The average tumor volume of ADC-1 at a dose of 1.5 mg/kg and 5 mg/kg was 322.26 mm ³ and 57.77 mm ³ , respectively, which was statistically significantly different from the Vehicle group (528.12 mm ³ ) (P<0.001 and P<0.001), and the tumor inhibition rate (TGI) was 54% and 124%, respectively. All tumor-bearing mice (6/6) in the ADC-1_5 mg/kg group showed tumor regression, and the tumor inhibition effect showed a good dose-effect relationship. The average tumor volume of the control substance ADC-5 at a dose of 1.5 mg/kg was 427.92 mm ³ , and the TGI was 26%, which was statistically significantly different from the Vehicle group (P<0.05). At the same dose (1.5 mg/kg), the average tumor volume at the end point of the ADC-1 group was significantly lower than that of the control ADC-5 group (P<0.05). During the experiment, the average body weight of mice in each administration group did not decrease significantly, and the test drug was well tolerated.

In summary, ADC-1 inhibited the growth of 786-O xenograft tumors in a 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.

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.

Although the specific embodiments of the present disclosure are described above, those skilled in the art should understand that these are only examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the protection scope of the present disclosure is limited by the appended claims.

Claims (31)

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 in the amino acid sequence shown in SEQ ID NO: 17, 1, 62 or 63, and the VL comprises LCDR1, LCDR2 and/or LCDR3 in the amino acid sequence shown in SEQ ID NO: 18, 2, 64 or 65; or, 2) the VH comprises HCDR1, HCDR2 and/or HCDR3 in the amino acid sequence shown in SEQ ID NO: 3, 19, 66 or 67, and the VL comprises LCDR1, LCDR2 and/or LCDR3 in the amino acid sequence shown in SEQ ID NO: 4, 20, 68 or 69; Preferably, 1-1) 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; 1-2) the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:1, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:2; 1-3) the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 62 or 63, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 64 or 65; 2-1) the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:3, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:4; 2-2) 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; or, 2-3) The VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:66 or 67, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:68 or 69. 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, 7, respectively; the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO: 24, 25, 10, respectively; or 2) the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 11, 12 and 13, respectively; the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 26, 15 and 16, respectively; Preferably, 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: 21, 22 and 10, respectively; 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: 8, 9 and 10, respectively; 2-1) the VH comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 11, 12 and 13; the VL comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 14, 15 and 16, respectively; 2-2) the VH comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 11, 12 and 13; the VL comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 23, 15 and 16, respectively; 2-3) The VH comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 12 and 13 respectively; the VL comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 70, 15 and 16 respectively. The CDH6 binding molecule according to claim 1 or 2, which is an anti-CDH6 antibody or an antigen-binding fragment thereof; preferably, wherein the antibody is selected from a murine antibody, a chimeric antibody or a humanized antibody; preferably, the antigen-binding fragment is scFv, Fv, Fab, Fab', (Fab') ₂ fragment, linear antibody, single-chain antibody, sdAb, sdFv, nanobody, peptibody, domain antibody, double-chain antibody (diabody), three-chain antibody (triabody), four-chain antibody (tetrabody), tandem di-scFv or tandem tri-scFv. 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. The CDH6 binding molecule according to any one of claims 1 to 4, wherein: 1-1) VH comprises an amino acid sequence as set forth in SEQ ID NO:17, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:18, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; 1-2) VH comprises an amino acid sequence as set forth 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 set forth 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 set forth in SEQ ID NO:62 or 63, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:64 or 65, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; 2-1) VH comprises an amino acid sequence as set forth in SEQ ID NO:3, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:4, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; 2-2) VH comprises an amino acid sequence as set forth in SEQ ID NO:19, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:20, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; 2-3) VH comprises an amino acid sequence as shown in SEQ ID NO:66 or 67, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:68 or 69, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto. The CDH6 binding molecule according to any one of claims 1 to 5, comprising an immunoglobulin constant region; 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 κ, λ chain or variants thereof; Preferably, the amino acid sequence of the heavy chain constant region is derived from human IgG1 or a variant thereof; and/or the light chain constant region is derived from human antibody κ chain or a variant 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. 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 a 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 a 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 a sequence identity thereto of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:30, or a sequence identity thereto of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The CDH6 binding molecule according to any one of claims 1 to 7, wherein the CDH6 binding molecule has at least the following characteristics: (1) having cell internalization activity; preferably, having tumor cell internalization activity; (2) binds to human CDH6 with a _KD value of less than or equal to 100 nM; (3) Does not bind to human CDH9 and CDH10. An antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprising the CDH6 binding molecule according to any one of claims 1 to 8 and an effector molecule, wherein the effector molecule is coupled to the CDH6 binding molecule; preferably, the effector molecule is a cytotoxic drug, an immunomodulator or a cytostatic agent. 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, maytansinoid, vinca alkaloid, pyrrolobenzodiazepine (PBD), calicheamicin, duokamycin, daunorubicin, doxorubicin, calicheamicin, anthramycin, neomycin, amatoxin, hemitoxin, 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. 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-LYD) of any one of formulas (I) to (V):

in: Y is selected from -O-( ^{CRaRb )} _m- ^{CR1R2 -C} (O)-, ^-O - ^CR1R2- ( ^CRaRb ) _m- , -O- ^CR1R2- , -NH-(CRaRb)m- ^{CR1R2 - C} (O)-, -S-( ^{CRaRb )} _{m -} ^CR1R2 - ^C (O)- and is absent; optionally, when it is formula ⁽ V ⁾ , Y is absent ^{; Ra} and ^Rb 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, ^Ra and ^Rb together with the carbon atom to which they are attached form a cycloalkyl group and a heterocyclic group; ^R1 is selected from the group consisting of hydrogen, halogen, haloalkyl, alkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; ^R2 is selected from the group consisting of hydrogen, halogen, haloalkyl, alkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; or, ^R1 and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group; Alternatively, ^Ra and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group; m is an integer from 0 to 4; n is 1 to 10, n is a decimal or an integer, preferably, n is 2 to 8 or 5 to 9, more preferably, n is 7 to 8, n is a decimal or an integer; L is the joint unit; Pc is the CDH6 binding molecule according to any one of claims 1 to 8; Preferably, The antibody-drug conjugate is shown in the general formula (Pc-LYD) of formula (I):
in: 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)-; ^Ra and ^Rb 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, ^Ra and ^Rb together with the carbon atom to which they are attached form a cycloalkyl group and a heterocyclic group; ^R1 is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; ^R2 is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; or, ^R1 and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group; Alternatively, ^Ra and ^R2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group; m is an integer from 0 to 4; n is 1 to 10, n is a decimal or an integer, preferably, n is 2 to 8 or 5 to 9 or 4 to 8, more preferably, n is 7 to 8, n is a decimal or an integer; L is the joint unit; Pc is the CDH6 binding molecule according to any one of claims 1 to 8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 9 to 11, wherein Y is -O-(CR ^a R ^b )m-CR ¹ R ² -C(O)-; ^Ra and ^Rb are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group; ^R1 is _C1-3 alkyl, _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, or a hydrogen atom; ^R2 is selected from hydrogen atom, haloalkyl, _C1-3 alkyl or _C3-6 cycloalkyl; preferably hydrogen atom; Alternatively, R ¹ and R ² together with the carbon atom to which they are 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)-; ^Ra and ^Rb are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group; ^R1 is _C3-6 cycloalkylalkyl or _C3-6 cycloalkyl, hydrogen atom; ^R2 is selected from hydrogen atom, haloalkyl or _C3-6 cycloalkyl; preferably hydrogen atom; Alternatively, R ¹ and R ² together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group; m is 0 or 1. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 9 to 12, wherein Y is selected from:
The O end of Y is connected to the linker unit L. The antibody drug conjugate according to any one of claims 11 to 13, wherein the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, ^L1 is ^s1 is an integer from 2 to 8, ^s2 is selected from an integer from 0 to 8; preferably ^s2 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 are each independently a hydrogen atom or an alkyl group, and t is 1 or 2. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 11 to 13, wherein the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, L ¹ is -(succinimidyl-3-yl-N)-CH ₂ -cyclohexyl-C(O)-; L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(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 are each independently a hydrogen atom or an alkyl group, and t is 1 or 2. The antibody drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 14 or 15, wherein the peptide residue of ^L3 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, 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). The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 11 to 16, wherein the linking unit -LY- is selected from:






wherein x and y are independently selected from integers of 2-8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 9 to 17, wherein the antibody-drug conjugate is selected from:










in: n is 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 the CDH6 binding molecule according to any one of claims 1-8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 18, wherein the antibody-drug conjugate is selected from:
in: n as defined in claim 18; The Pc comprises VH and VL, 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; 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, 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; 2) The VH comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 12, and 13, respectively; the VL comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 23, 15, and 16, respectively. The antibody-drug conjugate of claim 19, wherein: 1) VH comprises an amino acid sequence as set forth in SEQ ID NO:17, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:18, or at least 90% identical thereto; or 2) VH comprises an amino acid sequence as shown in SEQ ID NO:19, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and VL comprises an amino acid sequence as shown in SEQ ID NO:20, or a sequence that is at least 90% identical thereto. The antibody-drug conjugate of 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 a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:28, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; or 2) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:29, or a sequence identity thereto of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and the light chain comprises an amino acid sequence as shown in SEQ ID NO:30, or a sequence identity thereto of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. A CDH6 binding molecule that specifically binds to the EC1 domain of the extracellular region of human CDH6; Preferably, the binding molecule has internalization capability allowing cellular 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. A polynucleotide encoding the CDH6 binding molecule according to any one of claims 1 to 8 and 22 or the Pc in claims 9 to 21; Preferably, the polynucleotide is DNA or RNA. A vector comprising the polynucleotide according to claim 23. A host cell comprising the vector of claim 24; or expressing the CDH6 binding molecule of any one of claims 1 to 8 and 22; Preferably, the host cell is a bacterial, yeast or mammalian cell; More preferably, the host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary cells or human embryonic kidney 293 cells. A pharmaceutical composition comprising: The CDH6 binding molecule of any one of claims 1 to 8 and 22, the antibody drug conjugate of any one of claims 9 to 21, the polynucleotide of claim 23, or the vector of claim 24; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents or adjuvants. A method for preparing a CDH6 binding molecule, comprising: Expressing the CDH6 binding molecule according to any one of claims 1 to 8 and 22 in a host cell, and isolating the CDH6 binding molecule from the host cell; Optionally, a purification step of the CDH6 binding molecule is included. A method for preparing an antibody drug conjugate, comprising: A coupling reaction of the CDH6 binding molecule according to any one of claims 1 to 8 and 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 tumors; Preferably, the cancer or tumor is CDH6-associated or CDH6-positive; Preferably, the cancer or tumor 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, more preferably CDH6-associated or 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. A method of 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-associated or CDH6-positive; Preferably, the cancer or tumor 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, more preferably CDH6-associated or 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. At least one of the following uses 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: (1) Used to detect CDH6 protein; (2) preparing a reagent for detecting CDH6 protein; (3) Used for the diagnosis, prognosis, and efficacy monitoring of cancer or tumors; (4) Preparation of reagents for diagnosis, prognosis, and efficacy monitoring of cancer or tumors; Preferably, the cancer or tumor is CDH6-associated or CDH6-positive; Preferably, the cancer or tumor 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, more preferably CDH6-associated or 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; Optionally, the CDH6 binding molecule is further detectably labeled.