CDH6‑targeting binding molecule and use thereof

Abstract

Provided are an antibody or an antigen-binding fragment thereof that binds to CDH6 and has internalization activity, a derivative comprising the antibody or the antigen-binding fragment thereof, and an antibody-drug conjugate. Also disclosed is a related use of the antibody or the antigen-binding fragment thereof in the treatment, detection and diagnosis of CDH6-related diseases.

Description

Binding molecules targeting CDH6 and their applications

[0001] This application claims priority to Chinese patent application 2024118856689, filed on December 19, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of immunology, and more specifically, relates to CDH6-binding molecules and their applications, including antibody molecules that bind to CDH6 and have internalization activity, antigen-binding fragments thereof, derivatives comprising said antibody molecules or their antigen-binding fragments, pharmaceutical compositions, and their related applications in the treatment of CDH6-related diseases. Background Technology

[0003] Cadherins are glycoproteins on the cell membrane surface that function as cell-cell adhesion molecules or as signaling molecules responsible for cell-cell interactions through calcium-dependent binding of their N-terminal extracellular domain. Classical cadherins are single-transmembrane proteins belonging to the cadherin family, composed of three distinct domains: a five-cadherin extracellular domain, a transmembrane domain, and an intracellular domain. Classical cadherins are further classified into type I and type II families based on their amino acid homology.

[0004] cadherin 6 (CDH6) is a type II cadherin composed of 790 amino acids. It shares 75% homology with its family members CDH9 and CDH10. Literature reports that CDH6 plays a role in intercellular adhesion and β-catenin interactions. CDH6 contains an RGD motif that can interact with integrins, promoting cancer cell proliferation, invasion, and metastasis through mechanisms such as promoting EMT and inhibiting autophagy. CDH6 is specifically expressed during developmental stages; its expression is limited in normal adult tissues, but it is highly expressed in renal cell carcinoma (RCC) and ovarian cancer (OVC). It is associated with poor prognosis in renal cell carcinoma. Furthermore, literature and databases show that CDH6 is expressed to varying degrees in thyroid cancer, gastric cancer, and cholangiocarcinoma.

[0005] The etiology of renal cell carcinoma (RCC) remains unclear, but its development is associated with factors such as obesity, genetics, and smoking. Based on clinical staging and patient tolerance, current treatment methods mainly include surgery, chemotherapy, radiotherapy, and drug therapy. Current clinical drug therapy guidelines primarily include immune checkpoint inhibitors, targeted therapy, and cytokine therapy. Although first-line drug therapy has shown good efficacy, the median progression-free survival and disease control rate for RCC patients who have failed first-line therapy remain limited, indicating unmet clinical needs.

[0006] Due to the current lack of effective screening and early diagnosis measures for ovarian cancer, the vast majority of patients already have local or distant metastasis at the time of diagnosis, with a 5-year survival rate of approximately 46%. Based on its histopathological characteristics, ovarian cancer is mainly divided into three categories: epithelial ovarian cancer, germ cell tumors, and sex cord-stromal tumors. Ovarian cancer has a high recurrence rate; approximately 70%-80% of patients with advanced ovarian cancer experience disease recurrence after receiving standard platinum-based chemotherapy, and drug resistance is easily developed after recurrence. For recurrent epithelial ovarian cancer, treatment options for platinum-resistant ovarian cancer are currently limited, mainly including single-agent chemotherapy, but the activity of single-agent chemotherapy is limited (ORR, 4%–13%), and these patients do not benefit from existing treatment regimens. 65%-85% of ovarian cancer patients express CDH6, which is associated with poor prognosis. Currently, no CDH6-targeted cancer therapies are approved for the treatment of any cancer. Therefore, developing a drug targeting CDH6 holds promise as a new and effective treatment option.

[0007] Invention Overview

[0008] This disclosure provides an antibody or antigen-binding fragment thereof that binds to CDH6, wherein the antibody or antigen-binding fragment thereof specifically binds to CDH6 and has internalization activity.

[0009] In some embodiments, CDH6 is a polypeptide having the sequence shown in SEQ ID NO:33 (Uniprot ID: P55285).

[0010] In one embodiment, this disclosure provides an antibody against CDH6 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment being capable of specifically binding to CDH6. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), said heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, said light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as follows:

[0011] (1) SEQ ID NO: 5, 6, 7, 8, 9 and 10; (2) SEQ ID NO: 11, 12, 13, 14, 15 and 16; or (3) SEQ ID NO: 11, 37, 13, 14, 15 and 16;

[0012] According to one aspect of this disclosure, the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the following groups of heavy chain variable regions and light chain variable regions:

[0013] (1) SEQ ID NO:1 and 2; (2) SEQ ID NO:17 and 18; (3) SEQ ID NO:19 and 20; (4) SEQ ID NO:21 and 22; (5) SEQ ID NO:23 and 24; (6) SEQ ID NO:25 and 26; (7) SEQ ID NO:3 and 4; (8) SEQ ID NO:27 and 28; (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32.

[0014] According to one aspect of this disclosure, the anti-CDH6 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region and the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are selected from the following sequences: HCDR1, HCDR2, and HCDR3 in the heavy chain variable region and LCDR1, LCDR2, and LCDR3 in the light chain variable region:

[0015] (1) SEQ ID NO:1 and 2; (2) SEQ ID NO:17 and 18; (3) SEQ ID NO:19 and 20; (4) SEQ ID NO:21 and 22; (5) SEQ ID NO:23 and 24; (6) SEQ ID NO:25 and 26; (7) SEQ ID NO:3 and 4; (8) SEQ ID NO:27 and 28; (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32.

[0016] In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region includes a natural Fc or a modified variant Fc. More preferably, the Fc is derived from a mouse or a human.

[0017] In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0018] In a preferred embodiment, the heavy chain constant region comprises natural Fc or modified variant Fc.

[0019] In a preferred embodiment, Fc is derived from mice or humans.

[0020] In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment described in this disclosure is a murine antibody, a chimeric antibody, a fully human antibody, or a humanized antibody.

[0021] In a preferred embodiment, the anti-CDH6 antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, or a multispecific antibody or its antigen-binding fragment.

[0022] In some preferred embodiments, the anti-CDH6 antibody is a full-length antibody.

[0023] In some preferred embodiments, the anti-CDH6 antibody or its antigen-binding fragment described in this disclosure is in the form of IgG1, IgG2, IgG3 or IgG4.

[0024] In some preferred embodiments, the antigen-binding fragments against CDH6 described in this disclosure are Fab, Fv, scFv, F(ab')2, linear antibodies, and single-domain antibodies.

[0025] In some embodiments, this disclosure provides a conjugate formed by coupling the aforementioned anti-CDH6 antibody or its antigen-binding fragment with a capture marker or detection marker. The detection marker includes, but is not limited to, radionuclides, luminescent substances (e.g., fluorescein), colored substances, or enzymes.

[0026] In some embodiments, this disclosure provides an antibody-drug conjugate (ADC) formed by conjugating the aforementioned anti-CDH6 antibody or its antigen-binding fragment to other bioactive molecules. Preferably, the anti-CDH6 antibody or its antigen-binding fragment is conjugated to the other bioactive molecules via a linker. Preferably, the other bioactive molecules are small molecule drugs, such as antitumor drugs; more preferably, the antitumor drug is an antitumor compound.

[0027] In some embodiments, this disclosure provides a fusion protein comprising an anti-CDH6 antibody of this disclosure or an antigen-binding fragment thereof.

[0028] In some embodiments, this disclosure provides a bispecific antibody or a multispecific antibody, or an antigen-binding fragment thereof, wherein one antigen-binding domain of the bispecific antibody or multispecific antibody, or an antigen-binding fragment thereof, comprises the anti-CDH6 antibody of this disclosure or an antigen-binding fragment thereof.

[0029] In some embodiments, this disclosure provides a bispecific antibody or a multispecific antibody, or an antigen-binding fragment thereof, wherein the bispecific antibody or multispecific antibody comprises at least two antigen-binding domains; one of the antigen-binding domains comprises the anti-CDH6 antibody of this disclosure or an antigen-binding fragment thereof.

[0030] In some embodiments, this disclosure provides nucleic acids encoding any of the aforementioned anti-CDH6 antibodies or antigen-binding fragments thereof. In one embodiment, this disclosure also provides a recombinant vector comprising said nucleic acid.

[0031] In some embodiments, this disclosure provides a host cell comprising the recombinant vector described herein or a nucleic acid containing an antibody encoding the anti-CDH6 antibody or an antigen-binding fragment thereof. In some preferred embodiments, the host cell may be a prokaryotic cell, such as *Escherichia coli*; or a eukaryotic cell, such as yeast; or a mammalian cell, such as CHO cells or HEK293 cells.

[0032] In some embodiments, this disclosure provides a method for preparing the anti-CDH6 antibody or an antigen-binding fragment thereof, comprising: culturing the host cells of this disclosure under suitable conditions and purifying the expression product from the cells.

[0033] In some embodiments, this disclosure provides the use of the antibody against CDH6 or an antigen-binding fragment thereof for the detection, diagnosis or treatment of diseases associated with CDH6 expression.

[0034] In some embodiments, this disclosure provides a method for detecting CDH6 in a sample, comprising: contacting the sample with the aforementioned anti-CDH6 antibody or its antigen-binding fragment; detecting the formation of a complex of the anti-CDH6 antibody or its antigen-binding fragment with CDH6; optionally, the anti-CDH6 antibody or its antigen-binding fragment is detectably labeled.

[0035] In some embodiments, this disclosure provides a pharmaceutical composition comprising an effective amount of the disclosed anti-CDH6 antibody or its antigen-binding fragment, or an effective amount of nucleic acid encoding the anti-CDH6 antibody or its antigen-binding fragment, or an effective amount of a recombinant vector containing encoding nucleic acid, or an effective amount of host cell containing encoding nucleic acid, or an effective amount of the disclosed antibody-drug conjugate, or an effective amount of the disclosed fusion protein, or an effective amount of the disclosed bispecific or multispecific antibody. In some more preferred embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0036] In some embodiments, this disclosure provides a pharmaceutical composition comprising an antibody against CDH6 of this disclosure or an antigen-binding fragment thereof, or comprising a nucleic acid encoding the antibody against CDH6 or an antigen-binding fragment thereof, or comprising a recombinant vector encoding a nucleic acid, or comprising a host cell encoding a nucleic acid, or comprising an antibody-drug conjugate of this disclosure, or comprising a fusion protein of this disclosure, or comprising a bispecific antibody or a multispecific antibody of this disclosure. In some more preferred embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0037] In some specific embodiments, the pharmaceutical composition further comprises one or more other therapeutic agents. The additional therapeutic agents are pharmaceutical formulations that can be administered to a subject in combination with the pharmaceutical compositions of this disclosure.

[0038] In some embodiments, this disclosure provides a method for treating a disease in a subject associated with abnormal CDH6 expression, the method comprising administering an effective amount of the pharmaceutical composition of this disclosure to a subject in need. In some preferred embodiments, the disease is a tumor, preferably, the tumor is associated with CDH6 expression. In other preferred embodiments, the disease is renal cell carcinoma and ovarian cancer. In some more preferred embodiments, the method further includes administering additional therapeutic agents to the subject.

[0039] In some embodiments, this disclosure provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating a disease. In some preferred embodiments, the disease is a disease associated with abnormal CDH6 expression. In other preferred embodiments, the disease associated with abnormal CDH6 expression is a tumor. In some more preferred embodiments, the disease is renal cell carcinoma or ovarian cancer.

[0040] In some embodiments, this disclosure provides the above-described pharmaceutical composition for treating a disease. In some preferred embodiments, the disease is associated with abnormal CDH6 expression. In other preferred embodiments, the disease associated with abnormal CDH6 expression is a tumor. In some more preferred embodiments, the disease is renal cell carcinoma or ovarian cancer. Attached Figure Description

[0041] The accompanying drawings further illustrate the novel features disclosed herein. A better understanding of the features and advantages disclosed herein will be achieved by referring to these drawings; however, it should be understood that these drawings are for illustrating specific embodiments of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0042] Figure 1A shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to CDH6-overexpressing cells (OVCAR3).

[0043] Figure 1B shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to cells with low CDH6 expression (PA1).

[0044] Figure 1C shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to cyno CDH6-expressing cells (CHOK1-cyno CDH6).

[0045] Figure 2A shows the internalization of the mouse-derived anti-CDH6 chimeric antibody of this disclosure in CDH6-overexpressing cells (OVCAR3).

[0046] Figure 2B shows the internalization of the mouse-derived anti-CDH6 chimeric antibody of this disclosure in a CDH6-low-expressing tumor cell line (PA-1).

[0047] Figure 3A shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to the family-related protein CDH9.

[0048] Figure 3B shows the binding of the mouse-derived anti-CDH6 chimeric antibody of this disclosure to the family-related protein CDH10.

[0049] Figures 4A-4B show the binding of the mouse-derived anti-CDH6 humanized antibody of this disclosure to CDH6-overexpressing cells (OVCAR3).

[0050] Figures 4C-4D show the binding of the mouse-derived anti-CDH6 humanized antibody of this disclosure to cells with low CDH6 expression (PA1).

[0051] Figures 4E-4F show the binding of the mouse-derived anti-CDH6 humanized antibody of this disclosure to cyno CDH6-expressing cells (CHOK1-cyno CDH6).

[0052] Figures 5A-5B show the internalization of the mouse-derived anti-CDH6 humanized antibody of this disclosure in CDH6-overexpressing cells (OVCAR3).

[0053] Figures 5C-5D show the internalization of the mouse-derived anti-CDH6 humanized antibody of this disclosure in low-expressing CDH6 cells (PA-1).

[0054] Figure 6A shows the binding level of the mouse-derived anti-CDH6 humanized antibody of this disclosure to the mouseCDH6 protein.

[0055] Figure 6B shows the binding level of the mouse-derived anti-CDH6 humanized antibody of this disclosure to the RatCDH6 protein.

[0056] Figure 7A shows the binding level of the mouse-derived anti-CDH6 humanized antibody of this disclosure to the CDH9 protein, a member of the same family.

[0057] Figure 7B shows the binding level of the mouse-derived anti-CDH6 humanized antibody of this disclosure to the CDH10 protein, a member of the same family. Detailed Implementation

[0058] the term

[0059] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated that each individual publication, patent or patent application is incorporated by reference.

[0060] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0061] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for the purpose of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. For example, a description of a range from 1 to 6 should be considered as specifically disclosing subranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6, and specific numerical points within those ranges, such as 1, 2, 3, 4, 5, and 6. The above principles apply equally regardless of the breadth of the numerical values. When a range description is used, the range includes the endpoints of the range.

[0062] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0063] The three-letter and single-letter codes for amino acids used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0064] The term "antibody" in this article may include complete antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragment (i.e., antigen-binding part) or its single chain, and may also include products with antigen-specific binding ability formed by modifying complete antibodies or their antigen-binding fragments or their single chains (e.g., linking other peptides, rearranging functional units, etc.).

[0065] As used herein, the term "antibody" typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies possess this structure. Each light chain contains a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH), or heavy chain constant region (CH). Five main classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM, with corresponding heavy chain constant domains referred to as α, δ, ε, γ, and μ, respectively. IgG and IgA can be further subdivided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be identified as one of two distinct types based on the amino acid sequence of their constant domains, termed κ and λ.

[0066] In the case of IgG, IgA, and IgD antibodies, this heavy chain constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length segment rich in proline and cysteine. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0067] As used herein, the terms "anti-CDH6 antibody" or "CDH6-binding antibody" refer to antibodies that are able to bind to CDH6 with sufficient affinity.

[0068] The term "variable region" or "variable domain" indicates a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light / heavy chain pair forms the antigen-binding site, giving the complete IgG antibody two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVR), or more commonly, complementarity-determining regions (CDR). Each VH and VL has four backbone regions (FR), denoted as FR1, FR2, FR3, and FR4, respectively. Therefore, the CDR and FR sequences typically appear in the following sequence of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0069] The term "Fc" is used herein to define the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0070] As used herein, the broad category of "antibody" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including recombinant human antibodies), recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-individual genotype antibodies, synthetic antibodies (including mutant proteins and their variants), etc.

[0071] The terms “full-length antibody,” “complete antibody,” and “intact antibody” may be used interchangeably in this document to refer to antibodies whose structure is substantially similar to that of natural antibodies or that contain an Fc region.

[0072] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced from a single cell clone that targets only a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above.

[0073] The term "chimeric antibody" is a construct in which a portion of the heavy and / or light chains is identical or homologous to a corresponding sequence in an antibody from a specific species or belonging to a specific antibody class or subclass, while the remainder of this or these chains is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, or in fragments of such antibodies. In a narrower sense, a chimeric antibody comprises all or most of selected murine heavy and light chain variable regions operatively linked to human light and heavy chain constant regions. Constant region sequences, or variants or derivatives thereof, can be operatively associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide a full-length anti-CDH6 antibody that can be used on its own or incorporated into this disclosure.

[0074] The term "humanized antibody" refers to a hybrid immunoglobulin, immunoglobulin chain, or fragment thereof containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) where residues of the receptor's core sequence (CDR) are replaced by residues of a CDR from a non-human species (donor antibody) possessing the desired specificity, affinity, and performance, such as mice, rats, rabbits, or primates. In some cases, framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In certain circumstances, "reversion mutations" can be introduced into humanized antibodies where residues in one or more frame regions (FRs) of the variable region of the recipient human antibody are replaced by corresponding residues from a non-human species donor antibody. Such reversion mutations can help maintain the appropriate three-dimensional conformation of one or more grafted CDRs and thus improve affinity and antibody stability. Antibodies from a variety of donor species can be used, including but not limited to mice, rats, rabbits, or non-human primates. Additionally, humanized antibodies may contain novel residues not found in the recipient antibody or the donor antibody to further improve antibody performance.

[0075] It should be noted that the division of the CDR and FR in the variable region of the monoclonal antibody disclosed herein is determined according to the Kabat definition. Other nomenclature and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any nomenclature system based on the monoclonal antibody sequence of this disclosure are explicitly kept within the scope of this disclosure.

[0076] The terms "sequence identity," "sequence similarity," or "sequence homology" refer to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after aligning the sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0077] The term "antibody fragment" includes at least a portion of a complete antibody. As used herein, a "fraction" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its epitope, or a fusion protein product further derived from such fragment, or a product conjugated with other compounds, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-domain antibodies (VHH), linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.

[0078] The term "Fab fragment" or "Fab" includes the variable regions of the heavy chain and the light chain, and also includes the constant region of the light chain and the first constant region CH1 of the heavy chain, which is a monovalent antibody fragment. The term "F(ab')2 fragment" contains two Fab fragments and a hinge region, which is a bivalent antibody fragment.

[0079] The term "Fd fragment" generally includes the heavy chain variable region and the constant region CH1; the term "Fv fragment" contains the antibody heavy chain variable region and the light chain variable region, but no constant region, and is the smallest antibody fragment with all antigen binding sites.

[0080] The term "scFv" refers to a fusion protein comprising at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it originates. Unless otherwise specified, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0081] The term "fusion protein" refers to a larger molecule formed by linking different polypeptides / proteins together through genetic recombination or chemical methods. Linkers can be used for this linking, or not.

[0082] The term "multispecific antibody" refers to a novel antibody construct formed by functionally linking an antibody to one or more other binding molecules (e.g., chemical conjugation, gene fusion, non-covalent binding, or other methods) to bind to two or more different sites and / or targets. Among these, "bispecific antibody" is more commonly used, specifically referring to an antibody construct that is specific to two different antigens. Typically, bispecific or multispecific antibodies include at least two antigen-binding domains.

[0083] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or its antigen-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including single epitopes, multiple epitopes, single domains, multiple domains, or intact extracellular domains (ECDs) or proteins. Peptides, proteins, glycoproteins, polysaccharides, and lipids, as well as portions thereof, can constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen, or cells or preparations containing that antigen, can be used to generate antibodies specific to the antigenic determinant. An antigen can be an isolated full-length protein, a cell surface protein (e.g., used for immunization with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., used for immunization with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the foregoing antigens can be used alone or in combination with one or more immunogenic adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform cells expressing the antigen, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0084] The term "epitope," also known as an "antigenic determinant," refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed through ternary folding are typically lost after treatment with denaturing solvents. Epitopes typically consist of 3–15 amino acid residues. Methods for determining the epitope bound to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0085] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear, cyclic, or branched, may contain modified amino acids, particularly conserved modified amino acids, and may be interrupted by non-amino acid components. The term also includes amino acid polymers that have been modified, for example, by glycosylation, esterification, acetylation, phosphorylation, methylation, or any other manipulation such as conjugation with a labeled component. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. “Derived from” a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0086] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. "Amino acid substitution" or "replacement" means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid. For example, substitution of S32A means that the serine at position 32 is replaced by alanine.

[0087] The sequence identity or homology between the variable region of the humanized antibody and the variable region of the human receptor can be determined as discussed herein, and when such a determination is made, preferably at least 60% or 65% sequence identity will be shared, more preferably at least 70%, 75%, 80%, 85%, or 90% sequence identity, and even more preferably at least 93%, 95%, 98%, or 99% sequence identity. Preferably, the different residue positions are due to conserved amino acid substitutions. A “conserved substitution” is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of the protein. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the CDR region or frame region of the disclosed antibody can be replaced with amino acid residues of other similar side chains. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the sequence identity percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution.

[0088] During monoclonal antibody production, various physicochemical factors can easily generate post-translational modification (PTM) variants, such as glycosylation, oxidation, glycation, deamidation, isomerization, and terminal cyclization. These PTMs can cause changes in the physicochemical properties of antibodies, alter their interaction with the antibody Fc receptor, and affect their binding activity to the target antigen. Some PTMs can even reduce antibody stability and induce immunogenicity (JARASCH et al., JOURNAL OF PHARMACEUTICAL SCIENCES, 2015). The negative effects of PTMs can be eliminated by modifying the amino acid sites, such as through conserved substitutions. Amino acid substitutions of antibody CDRs for the purpose of modifying PTMs are also explicitly kept within the scope of this disclosure.

[0089] The antibodies disclosed herein may also include substitutions or modifications to constant regions (e.g., Fc), including but not limited to amino acid residue substitutions, mutations, and / or modifications, which produce compounds having preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased yield, altered binding to Fc receptors (FcRs), enhanced or weakened ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity.

[0090] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a specific antibody-antigen interaction. Binding affinity can be determined using a variety of techniques known in the art, such as surface plasmon resonance, biolayer interferometry, bipolar interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0091] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, wherein the active ingredients contained herein are present in a biologically effective form and do not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation. When a "pharmaceutical composition" exists as a combination of individual formulations containing two or more different active ingredients, it can be administered simultaneously, sequentially, separately, or at intervals, with the aim of exerting the biological activity of multiple active ingredients together for the treatment of a disease.

[0092] The term "antibody-drug conjugate" (ADC) refers to an antibody covalently conjugated to a bioactive molecule, such as a therapeutic active substance or active pharmaceutical ingredient (API), so that the therapeutic active substance or active pharmaceutical ingredient (API) can target the antibody's binding target to exhibit its pharmacological function. The therapeutic active substance or active pharmaceutical ingredient can be a cytotoxic agent capable of killing cells targeted by the ADC, preferably malignant or cancerous cells. The covalent conjugation of the therapeutic active substance, active pharmaceutical ingredient, or cytotoxic agent can be performed in a non-site-specific manner using standard chemical linkers that conjugate the payload to lysine or cysteine ​​residues, or preferably, the conjugation is performed in a site-specific manner, which allows complete control over the conjugation site and the drug-to-antibody ratio of the resulting ADC. The ADCs described herein can be used to deliver cytotoxic agents or other payloads to target sites (e.g., tumorigenic cells and / or cells expressing CDH6). As used herein, the terms "drug" or "warhead" are used interchangeably and refer to a biologically active or detectable molecule or compound, including anticancer agents. A "payload" can comprise a drug or warhead in combination with an optional linker compound. The warhead can contain peptides, polypeptides, proteins, precursor drugs that are metabolized into active agents in the body, polymers, nucleic acid molecules, small molecules, binders, mimics, synthetic drugs, inorganic molecules, organic molecules, and radioactive isotopes.

[0093] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0094] The term “pharmaceutical carrier” or “pharmaceuticalally acceptable carrier” refers to a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), excipient, or medium that is administered with a therapeutic agent.

[0095] The term "effective dose" refers to a dosage of a pharmaceutical formulation of the antibody or its antigen-binding fragment disclosed herein, which, when administered to a patient in a single or multiple doses, produces the intended effect in the treated patient. The effective dose can be readily determined by an attending physician skilled in the art by considering a variety of factors, such as: racial differences; weight, age, and health status; the specific disease involved; the severity of the disease; the individual patient's response; the specific antibody administered; the administration modality; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0096] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in terms of nucleic acid content and may contain mutations. This document includes mutant progeny that have the same function or biological activity as those screened or selected in the initially transformed cells.

[0097] As used in this article, the term "transfection" refers to the introduction of exogenous nucleic acids into eukaryotic cells. Transfection can be achieved through a variety of techniques known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0098] The term "stable transfection" or "stable transformation" refers to the introduction and integration of exogenous nucleic acids, DNA, or RNA into the genome of transfected cells. The term "stable transfectant" refers to a cell in which foreign DNA is stably integrated into the genomic DNA.

[0099] The terms "isolated polynucleotide" or "isolated nucleic acid" refer to nucleic acid molecules, DNA, or RNA that have been removed from their natural environment. For example, for the purposes of this disclosure, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or (partially or substantially) purified polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain such polynucleotide molecules, but which are present outside the chromosome or at a chromosomal location other than their natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, as well as positive-stranded, negative-stranded, and double-stranded forms.

[0100] The terms "nucleic acid molecule encoding," "encoding DNA sequence," and "encoding DNA" refer to the sequence of deoxyribonucleotides along a deoxyribonucleic acid (DNA) chain. This sequence of deoxyribonucleotides determines the sequence of amino acids along a polypeptide (protein) chain. Therefore, a nucleic acid sequence encodes an amino acid sequence.

[0101] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and available in the prior art, such as in Cold Spring Harbor's *Antibody Laboratory Techniques Guide*, Chapters 5-8 and 15. The antibodies or antigen-binding fragments described in this invention are genetically engineered to add one or more human FR regions to a non-human CDR region. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from the journal *Immunoglobulins*, (2001) ISBN: 012441351.

[0102] The engineered antibodies or antigen-binding fragments thereof disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly 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 scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange.

[0103] As used herein, the terms “individual” or “subject” refer to any animal, such as a mammal or marsupial. Individuals disclosed herein include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0104] As used herein, the terms “disease,” “symptom,” or “disorder,” etc., refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. For example, “disease” includes, but is not limited to: tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or deficiencies in immune tolerance (such as transplant rejection).

[0105] As used in this article, the term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion into other tissues or organs. Tumor growth is typically uncontrolled and progressive, neither inducing nor inhibiting the proliferation of normal cells.

[0106] The term "inflammation" as used in this article refers to the basic pathological process that occurs when the body is stimulated, primarily as a defensive response.

[0107] As used in this article, the term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in a clinicopathological process. Treatment effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0108] As used herein, the term "combination" refers to a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect. These therapies can be physical, such as radiation therapy, or chemical, such as administering a drug to the subject, including combination drugs. "Combination drugs" refers to a combination of two or more pharmaceutical preparations, each containing an active ingredient, that are administered to a subject in combination. The active ingredients may be mixed together to form a single dosing unit or may be administered separately as independent dosing units; during administration, the different pharmaceutical preparations may be administered substantially synchronously, simultaneously, or sequentially.

[0109] The negative control antibody in the examples is an irrelevant antibody and is used only as an IgG control.

[0110] Example

[0111] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions (such as those described in "J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002") or as recommended by the manufacturer.

[0112] Example 1. Human CDH6 and cynomolgus monkey CDH6 antigen information

[0113] The full-length amino acid sequence of human CDH6 used in the examples (SEQ ID NO:33) (Uniprot ID: P55285) is shown below and was purchased from Acro Bio (catalog number CA6-H5229).

[0114] Note: Double underlined parts are signal peptides (1-18); underlined parts are the extracellular region of CDH6 (19-615); dotted underlined parts are transmembrane regions (616-636); italicized parts are intracellular regions (637-790).

[0115] The full-length amino acid sequence (SEQ ID NO:34) (Uniprot ID: A0A2K5TW62_MACFA) of the cynomolgus monkey CDH6 (cyno-CDH6) used in the examples is shown below. It was purchased from Acro Bio (catalog number CA6-C52H3).

[0116] Note: Double underlined parts are signal peptides (1-21); underlined parts are the extracellular region of CDH6 (22-614); dotted underlined parts are transmembrane regions (615-636); italicized parts are intracellular regions (637-790).

[0117] Example 2. Antigen information of family members CDH9 and CDH10

[0118] The full-length amino acid sequence of human CDH9 used in the examples (SEQ ID NO:35) (Uniprot ID: Q9ULB4) is shown below, which was purchased from Acro Bio (catalog number CA9-H52H6).

[0119] Note: Double underlined parts are signal peptides (1-21); underlined parts are the extracellular region of CDH9 (22-615); dotted underlined parts are transmembrane regions (616-636); italicized parts are intracellular regions (636-789).

[0120] The full-length amino acid sequence of human CDH10 used in the examples (SEQ ID NO:36) (Uniprot ID: Q9Y6N8) is shown below, and it was purchased from Acro Bio (catalog number CA0-H52H5).

[0121] Note: Double underlined parts are signal peptides (1-22); underlined parts are CDH10 extracellular regions (23-613); dotted underlined parts are transmembrane regions (614-634); italicized parts are intracellular regions (634-788).

[0122] Example 3. Obtaining anti-CDH6 antibodies derived from mouse hybridomas

[0123] Anti-CDH6 monoclonal antibodies were produced by immunizing mice. Female SJL mice (Shanghai Lingchang Biotechnology Co., Ltd.) were used in the experiment. The mice were housed in SPF-grade environments. After purchase, the mice were housed in a laboratory environment for one week with a 12 / 12-hour light / dark cycle, temperature 20-25℃, and humidity 40-60%. Immunization was performed using both gene and cell methods, with vaccinations on days 0, 14, 28, 42, 56, and 70, and a booster immunization 3 days before spleen cell fusion. During this period, mouse serum antibody titers were measured using ELISA and FACS methods. After the sixth immunization, mice with high and plateauing antibody titers in their serum were selected for spleen cell fusion. An optimized electrofusion procedure was used to fuse spleen lymphocytes with myeloma Sp2 / 0 cells (…). CRL-8287 TM Hybridoma cells are obtained by fusing them together.

[0124] After culturing hybridoma cells for 7-14 days, the culture supernatant was collected. ELISA was performed using CDH6 recombinant protein to screen for antibodies in the hybridoma supernatant. Positive antibody strains were further screened using HEK-293 cells stably expressing CDH6, compared with blank HEK293 cells to exclude non-specific antibody-binding hybridoma strains. Flow cytometry was used to select hybridomas that bound both the recombinant protein and the cell-expressed antigen. Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript). TM Reverse Transcriptase (Takara#2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced to obtain the amino acid sequences of the variable regions of the two monoclonal antibodies disclosed in this invention, as shown in Table 1A.

[0125] Table 1A shows the amino acid sequences of the variable regions of two monoclonal antibodies against CDH6 derived from mouse hybridomas.

[0126] Based on the above amino acid sequence, the CDR and FR of the antibody variable region were divided using the Kabat / IMGT numbering rule. The composition of the 6 CDR sequences of each antibody is shown in Table 1B below.

[0127] Table 1B CDR sequences of two monoclonal antibodies against CDH6 derived from mouse hybridoma.

[0128] Example 4. Construction of mouse hybridoma-derived anti-CDH6 chimeric antibody and its transient transfection expression in eukaryotic cells.

[0129] The target gene fragment generated by splicing the coding nucleic acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody of this disclosure, which have been sequenced, with the coding nucleic acid sequences of the IgG1 heavy chain constant region and κ light chain constant region, was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0130] Expi293F cultured in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C, 8% CO2, and 125 rpm. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. Feeding was performed on days 1 and 3 post-transfection. After 6 days of cell culture, the expression supernatant was collected, cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was washed with PBS until the A column was reached. 280 The reading dropped to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5), and neutralized with 1 M Tris-HCl (pH 8.0-9.0). After appropriate concentration of the eluted sample, the buffer was changed to PBS, filtered for sterilization, and dispensed. The final purified chimeric antibody was analyzed for purity by SDS-PAGE and HPLC. 280 Concentration determination.

[0131] Example 5. In vitro cell binding verification of mouse hybridoma-derived anti-CDH6 chimeric antibody

[0132] OVCAR3, PA-1, and CHOK1-cyno CDH6 cells were cultured. OVCAR3 cells were cultured in RPMI 1640 + 10% FBS + Insulin (0.01 mg / ml). PA-1 cells were cultured in MEM + 10% FBS + 1% NEAA + 1% NAP. CHOK1-cyno CDH6 cells were cultured in F12K + 10% FBS + 200 μg / ml Hygromycin. All cells were cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. Before use, cells were washed with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0133] Centrifuge the digested cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and rehydrate with 100 μL of 5% FBS (in DPBS with Ca). 2+Resuspend cells. Count cells and adjust the cell density to 1E6 / mL. Spread cells into 96-well round-bottom plates (corning 3799), centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant, and store at 4°C for later use. Incubate with 5% FBS (in DPBS with Ca). 2+ Dilute the antibody sample to be tested, starting at a concentration of 100 nM, and then dilute 4-fold down to 8 concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. 160 μL of 5% FBS (in DPBS with Ca) 2+ Resuspend and wash, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Use 5% FBS (in DPBS with Ca) 2+ Dilute the secondary antibody (goat anti-human IgG Fc APC) 1:500 according to the instructions, resuspend the cells in the diluted secondary antibody at 100 μL / well, and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Add 160 μL of 5% FBS (in DPBS with Ca) to each well. 2+ Resuspend and wash, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Add 100 μL of 5% FBS (in DPBS with Ca) to the solution. 2+ Cells were resuspended, filtered through 300-mesh gauze, and the average fluorescence intensity of the APC channel was detected by flow cytometry.

[0134] Export the FCS file from the flow cytometer, analyze the mean fluorescence intensity (MFI) of the APC channel for each sample using software, and import the obtained mean fluorescence intensity into Graphpad to analyze the half-maximal binding concentration (EC) of the antibody to cells. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 2 and Figures 1A-1C.

[0135] Table 2. Binding of mouse hybridoma-derived anti-CDH6 chimeric antibodies to CDH6-expressing cells.

[0136] Example 6. Internalization of mouse hybridoma-derived anti-CDH6 chimeric antibody

[0137] PA-1 and OVCAR3 cells in good growth condition were collected by centrifugation and seeded into 96-well white transparent plates at a certain cell density for overnight culture. The candidate antibody and positive control H01L02 were mixed with DT3C at a certain ratio and incubated at 37°C for 30 minutes to form mAb-DT3C conjugates. The mAb-DT3C conjugates were serially diluted. The diluted mAb-DT3C conjugates were added to 96-well white transparent plates and incubated at 37°C with 5% CO2 for 3 days. After incubation, the 96-well plates were removed and equilibrated at room temperature for 30 minutes. A certain amount of CellTiter-GloReagent was added to each well, and the plates were incubated with shaking at room temperature for 10 minutes. The luminescence signal (RLU) was detected using a microplate reader. The internalization ability of each candidate antibody was calculated using the following formula. The results are shown in Table 3 and Figures 2A and 2B. Internalization rate (%) = (1 - RLU) 实验组 / RLU 对照组 )×100%

[0138] Table 3. Internalization of anti-CDH6 chimeric antibodies derived from mouse hybridomas in CDH6-expressing cells.

[0139] The heavy chain amino acid sequence of the positive control H01L02 is as follows:

[0140] The light chain amino acid sequence is as follows:

[0141] Example 7. Binding experiment of mouse hybridoma-derived anti-CDH6 chimeric antibody to family proteins CDH9 and CDH10.

[0142] Dissolve the antigen protein human CDH9 (ACRO, catalog number CA0-H52H6) or human CDH10 (ACRO, catalog number CA0-H52H5) in 1X DPBS (with Ca2+). 2+ The antigen was added to a high-affinity ELISA plate (Biolegend, catalog number 423501) at a concentration of 1 μg / mL. The plate was then incubated overnight at 4°C. The antigen was washed three times with 300 μL of DPBS (infused with 0.05% Tween). The plate was then blocked with 200 μL of 2% BSA (in DPBS) and incubated at 37°C for 1.5 hours.

[0143] Dilute the antibody to be tested with 2% BSA (in DPBS) to an initial concentration of 100 nM, and then dilute 4-fold down to 8 concentrations. Wash the plate with DPBS (with 0.05% Tween added), 300 μL / well, three times. Add the diluted antibody to the ELISA plate, 100 μL / well, and incubate at room temperature for 2 hours. Wash the antibody with DPBS (with 0.05% Tween added), 300 μL / well, three times. Dilute the secondary antibody (Goat Anti-Human IgG Fc(HRP) for CDH9) 1:20000 with 2% BSA (in DPBS). Add 100 μL of the diluted secondary antibody to each ELISA plate and incubate at room temperature for 1 hour. Wash the plate 6 times with 300 μL of DPBS (with 0.05% Tween added). Prepare the chromogenic solution (TMA and TMB mixed 1:1) and add 100 μL of the solution to each well, incubating in the dark for 5 minutes. Add 50 μL of ELISA stop solution to the plate and mix well.

[0144] Read OD on envision 450 , using OD 450 Plot the results on GraphPad. The results are shown in Figures 3A and 3B, indicating that the tested antibody did not bind nonspecifically to either CDH9 or CDH10, which are proteins of the same family.

[0145] Example 8. Humanization of anti-CDH6 antibody derived from mouse hybridoma

[0146] After expression purification tests, cell-level binding and internalization tests, the two chimeric antibodies were designed to be humanized.

[0147] Humanization of murine anti-human CDH6 monoclonal antibodies was performed using methods published in numerous publications in the field. In short, a human constant domain was used to replace the parental (murine antibody) constant domain, and the human antibody sequence was selected based on the homology between the murine and human antibodies. Based on the obtained typical VH / VL CDR structure of the murine antibody, the heavy and light chain variable region sequences were compared with a human antibody germline database to obtain a highly homologous human germline template.

[0148] The CDR region of the murine antibody was transplanted onto a selected humanized template, replacing the humanized variable region, and then recombined with the IgG constant region (preferably IgG1 for the heavy chain and κ for the light chain). Based on the three-dimensional structure of the murine antibody, reverse mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH. Antibodies composed of the following humanized light and heavy chain variable region sequences were designed: 5 humanized antibodies were designed using 32G-12A10 (H1L1 / H2L1 / H3L1 / H4L1 / H5L1), and 3 humanized antibodies were designed using 26B-5F7 (H6L2 / H7L3 / H8L4). The heavy and light chain variable regions are shown in Table 4.

[0149] Table 4. Amino acid sequences of the variable regions of eight humanized antibodies derived from mouse hybridomas.

[0150] The amino acid sequence of the heavy chain variable region HCDR2 of H6L2, H7L3 and H8L4 is QIYPGDGDTNYQGKFKG (SEQ ID NO:37).

[0151] Example 9. Preparation of humanized anti-CDH6 antibodies derived from hybridoma

[0152] Referring to Example 4, the target gene fragment generated by splicing the variable region of the humanized antibody with the constant region of human IgG1 was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0153] Expi293F cultured in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C with 8% CO2. Cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flasks. After 6 days of cell culture, the expression supernatant was collected, centrifuged at high speed to remove cell debris, and then purified using a Protein A column for affinity purification. The column was washed with PBS until A... 280 The reading dropped to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). After appropriate concentration, the eluted sample was transferred to PBS for aliquoting. The final purified humanized antibody was analyzed for purity by SDS-PAGE and HPLC. 280 Concentration determination.

[0154] Example 10. In vitro cell binding verification of mouse hybridoma-derived anti-CDH6 humanized antibody

[0155] OVCAR3, PA-1, and CHOK1-cyno CDH6 cells were cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. Before use, cells were washed with sterile DPBS, digested with 0.25% trypsin and EDTA for approximately 5 minutes, and then the culture was stopped with complete culture medium.

[0156] Centrifuge the digested cells at 1000 rpm at room temperature for 5 minutes, discard the supernatant, and rehydrate with 100 μL of 5% FBS (in DPBS with Ca). 2+ Resuspend cells. Count cells and adjust the cell density to 1E6 / mL. Plate cells into 96-well round-bottom plates (corning 3799), centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant, and store at 4°C for later use. Dilute the antibody sample to be tested with 5% FBS (in DPBS with Ca2+), starting at 50 nM, and then dilute 4-fold down to 7 concentrations. Resuspend cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant. 160 μL of 5% FBS (in DPBS with Ca2+) is added to each well. 2+ Resuspend and wash, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Use 5% FBS (in DPBS with Ca) 2+ Dilute the secondary antibody (goat anti-human IgG Fc APC) 1:500 according to the instructions, resuspend the cells in the diluted secondary antibody at 100 μL / well, and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Add 160 μL of 5% FBS (in DPBS with Ca) to each well. 2+ Resuspend and wash, centrifuge at 1500 rpm for 5 minutes at 4°C, and discard the supernatant. Add 30 μL of 5% FBS (in DPBS with Ca) 2+ Resuspend cells, flow cytometry 3. Detect the average fluorescence intensity of the APC channel.

[0157] The average fluorescence intensity obtained from the analysis was imported into Graphpad to analyze the half-maximal binding concentration (EC50) of the antibody to cells. 50 The results of the highest average fluorescence intensity (Top MFI) are shown in Table 5 and Figures 4A-4F.

[0158] Table 5. Binding of mouse hybridoma-derived anti-CDH6 humanized antibodies to CDH6-expressing cells.

[0159] Example 11. Verification of the internalization of anti-CDH6 humanized antibody derived from mouse hybridoma.

[0160] PA-1 and OVCAR3 cells in good growth condition were collected by centrifugation and seeded into 96-well white transparent plates at a certain cell density for overnight culture. The candidate antibody and positive control disclosed herein were mixed with DT3C at a certain ratio and incubated at 37°C for 30 minutes to form mAb-DT3C conjugates. The mAb-DT3C conjugates were serially diluted. The diluted mAb-DT3C conjugates were added to 96-well white transparent plates and incubated at 37°C with 5% CO2 for 3 days. After incubation, the 96-well plates were removed and equilibrated at room temperature for 30 minutes. A certain amount of CellTiter-GloReagent was added to each well, and the plates were incubated with shaking at room temperature for 10 minutes. The luminescence signal (RLU) was detected using a microplate reader. The internalization ability of each candidate antibody was calculated. The results are shown in Table 6 and Figures 5A-5D. Internalization rate (%) = (1-RLU) 实验组 / RLU 对照组 )×100%

[0161] Table 6. Internalization of anti-CDH6 humanized antibodies derived from mouse hybridomas in CDH6-expressing cells.

[0162] Example 12. Biacore affinity assay for mouse hybridoma-derived anti-CDH6 humanized antibody

[0163] The affinity and kinetic properties of the anti-CDH6 humanized antibody with human CDH6 were analyzed using a Biacore 8K instrument. The CM5 chip was first activated with EDC and NHS, then immobilized with a mouse monoclonal antibody against human Fc, and finally blocked with ethanolamine.

[0164] To determine the affinity and kinetic properties with human CDH6, the humanized CDH6 antibody was diluted to 10 μg / mL with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM CaCl2) buffer and captured at a flow rate of 10 μL / min for 60 s. Human CDH6 was serially diluted two-fold to a series of concentrations (400 nM - 1.56 nM), bound at a flow rate of 30 μL / min for 120 s, and dissociated for 600 s.

[0165] After each round of experiments, the chip was washed with 3M MgCl2 solution at a flow rate of 30 μL / min for 30 s to remove the captured antibodies along with the antigens, thus completing the chip regeneration. The raw data were analyzed and fitted using software, and the results are shown in Tables 7A and 7B.

[0166] Table 7A Affinity assay results of mouse hybridoma-derived anti-CDH6 humanized antibody and human CDH6 antigen protein

[0167] Table 7B shows the affinity assay results between the mouse hybridoma-derived anti-CDH6 humanized antibody and the CynoCDH6 antigen protein.

[0168] Example 13. Binding assay of mouse hybridoma-derived anti-CDH6 humanized antibody with Mouse and RatCDH6.

[0169] Dissolve the antigen protein Mouse CDH6 (ACRO, catalog number CA6-M52H8) or Rat CDH6 (Sino, catalog number 80277-R08H) in 1X DPBS (with Ca2+). 2+ The antigen was added to a high-affinity ELISA plate (Biolegend, catalog number 423501) at a concentration of 1 μg / mL. The plate was then incubated overnight at 4°C. The antigen was washed three times with 300 μL of DPBS (infused with 0.05% Tween). The plate was then blocked with 200 μL of 2% BSA (in DPBS) and incubated at 37°C for 1.5 hours.

[0170] Dilute the antibody to be tested with 2% BSA (in DPBS) to an initial concentration of 200 nM, and then dilute 4-fold down to 10 concentrations. Wash the plate with DPBS (with 0.05% Tween added), 300 μL / well, three times. Add the diluted antibody to the ELISA plate, 100 μL / well, and incubate at room temperature for 2 hours. Wash the antibody with DPBS (with 0.05% Tween added), 300 μL / well, three times. Dilute the secondary antibody (Goat Anti-Human IgG Fc(HRP) for CDH9) 1:20000 with 2% BSA (in DPBS). Add 100 μL of the diluted secondary antibody to each ELISA plate and incubate at room temperature for 1 hour. Wash the plate 6 times with 300 μL of DPBS (with 0.05% Tween added). Prepare the chromogenic solution (TMA and TMB mixed 1:1) and add 100 μL of the solution to each well, incubating in the dark for 5 minutes. Add 50 μL of ELISA stop solution to the plate and mix well.

[0171] Read OD on envision 450 , using OD 450 Plotting the results on GraphPad. The results are shown in Figures 6A and 6B, demonstrating that the tested antibodies specifically bind to both MouseCDH6 and RatCDH6.

[0172] Example 14. Binding experiment of mouse hybridoma-derived anti-CDH6 humanized antibody with its family proteins CDH9 and CDH10.

[0173] Dissolve the antigen protein human CDH9 (ACRO, catalog number CA0-H52H6) or human CDH10 (ACRO, catalog number CA0-H52H5) in 1X DPBS (with Ca2+). 2+ The antigen was added to a high-affinity ELISA plate (Biolegend, catalog number 423501) at a concentration of 1 μg / mL. The plate was then incubated overnight at 4°C. The antigen was washed three times with 300 μL of DPBS (infused with 0.05% Tween). The plate was then blocked with 200 μL of 2% BSA (in DPBS) at 37°C for 1.5 hours. Subsequent experimental procedures were performed as described in Example 13. The results were plotted on a GraphPad. Figures 7A and 7B show that the antibody did not specifically bind to either CDH9 or CDH10, both members of the same family.

[0174] Example 15. Study on drug metabolism in rats

[0175] The in vivo metabolism of the chimeric antibody 32G-12A10 derived from mouse hybridomas and the humanized antibodies H2L1, H3L1, and H4L1 was determined. SD rats with an average weight of approximately 150g were selected as experimental animals, and four rats (half male and half female) were administered each antibody. The administration was a single, single dose of 5 mg / kg, administered in a volume of 5 mL / kg. Serum samples were collected and separated at different time points after administration (0, 5 min, 1 h, 2 h, 4 h, 6 h, 24 h, 72 h, 120 h, 144 h, 240 h, 336 h, 504 h, and 672 h). The data are shown in Table 8.

[0176] Table 8. Data from PK studies in rats.

[0177] The embodiments described above are merely exemplary, and any person skilled in the art will recognize or be able to identify numerous equivalents of specific compounds, materials, and operations without the need for extraordinary experimentation. All such equivalents are within the scope of this disclosure and are encompassed by the claims.

Claims

1. An antibody against CDH6 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to CDH6, comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, said light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the amino acid sequences of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively selected from the group consisting of: (1) SEQ ID NO: 5, 6, 7, 8, 9 and 10; (2) SEQ ID NO: 11, 12, 13, 14, 15 and 16; or (3) SEQ ID NO: 11, 37, 13, 14, 15 and 16; Alternatively, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are respectively selected from the heavy chain variable region and light chain variable region HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 sequences of the group consisting of: (1) SEQ ID NO:1 and 2; (2) SEQ ID NO:17 and 18; (3) SEQ ID NO:19 and 20; (4) SEQ ID NO:21 and 22; (5) SEQ ID NO:23 and 24; (6) SEQ ID NO:25 and 26; (7) SEQ ID NO:3 and 4; (8) SEQ ID NO:27 and 28; (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32.

2. The antibody or its antigen-binding fragment as described in claim 1, wherein, The heavy chain variable region and the light chain variable region each comprise an amino acid sequence having 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 with any of the following groups of heavy chain variable regions and light chain variable regions: (1) SEQ ID NO:1 and 2; (2) SEQ ID NO:17 and 18; (3) SEQ ID NO:19 and 20; (4) SEQ ID NO:21 and 22; (5) SEQ ID NO:23 and 24; (6) SEQ ID NO:25 and 26; (7) SEQ ID NO:3 and 4; (8) SEQ ID NO:27 and 28; (9) SEQ ID NO:29 and 30; or (10) SEQ ID NO:31 and 32.

3. The anti-CDH6 antibody or its antigen-binding fragment as described in claim 1 or 2, further comprising one or more of the following characteristics: (1) The anti-CDH6 antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region contains an Fc; and / or, the Fc is derived from a mouse or a human; and / or, the sequence of the Fc is a natural or modified variant. (2) The antibody against CDH6 or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody; (3) The antibody against CDH6 or its antigen-binding fragment is a monoclonal antibody; or a full-length antibody, or its antigen-binding fragment is Fab, Fv, scFv, F(ab')2, a linear antibody, or a single-domain antibody; (4) The antibody against CDH6 or its antigen-binding fragment is in the form of IgG1, IgG2, IgG3 or IgG4.

4. An antibody-drug conjugate formed by conjugating an anti-CDH6 antibody or its antigen-binding fragment as described in any one of the preceding claims with other bioactive molecules, preferably, the other bioactive molecules being small molecule drugs, such as antitumor drugs, preferably antitumor compounds, and preferably, the antibody and other bioactive molecules being linked by a linker.

5. A fusion protein, wherein, The fusion protein comprises an anti-CDH6 antibody or its antigen-binding fragment as described in any one of claims 1-3.

6. A bispecific antibody or a multispecific antibody, wherein, The bispecific or multispecific antibody comprises at least two antigen-binding domains; one of the antigen-binding domains comprises an anti-CDH6 antibody or an antigen-binding fragment thereof as described in any one of claims 1-3.

7. A nucleic acid encoding an antibody against CDH6 as described in any one of claims 1-3 or an antigen-binding fragment thereof, a fusion protein as described in claim 5, or a bispecific or multispecific antibody as described in claim 6.

8. A recombinant vector comprising the nucleic acid as described in claim 7.

9. A host cell comprising the recombinant vector as claimed in claim 8 or the nucleic acid as claimed in claim 7.

10. The host cell of claim 9, wherein it is a prokaryotic cell, such as Escherichia coli; or a eukaryotic cell, such as yeast or a mammalian cell, such as a CHO cell or a HEK293 cell.

11. A method for preparing an anti-CDH6 antibody or its antigen-binding fragment as described in any one of claims 1-3, a fusion protein as described in claim 5, or a bispecific antibody or multispecific antibody as described in claim 6, comprising: The host cells as described in claim 9 or 10 are cultured under suitable conditions, and the expression product is purified from said cells.

12. A method for detecting CDH6 in a sample, comprising: (1) Contact the sample with the anti-CDH6 antibody or its antigen-binding fragment as described in any one of claims 1-3, the fusion protein as described in claim 5, or the bispecific antibody or multispecific antibody as described in claim 6; (2) Detect the formation of a complex of an anti-CDH6 antibody or its antigen-binding fragment, fusion protein, bispecific antibody or multispecific antibody with CDH6; optionally, the anti-CDH6 antibody or its antigen-binding fragment, fusion protein, bispecific antibody or multispecific antibody is detectably labeled.

13. A pharmaceutical composition comprising an anti-CDH6 antibody or an antigen-binding fragment thereof as described in any one of claims 1-3, or an antibody-drug conjugate as described in claim 4, or a fusion protein as described in claim 5, or a bispecific or multispecific antibody as described in claim 6, or a nucleic acid as described in claim 7, or a recombinant vector as described in claim 8, or a host cell as described in claim 9 or 10.

14. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 13 or 14, further comprising one or more other therapeutic agents.

16. A method for treating a disease associated with abnormal CDH6 expression in a subject, the method comprising administering to a subject in need an effective amount of the pharmaceutical composition as described in any one of claims 13-15.

17. The method of claim 16, wherein the disease is a tumor, preferably kidney cancer or ovarian cancer.

18. The method of claim 16 or 17, further comprising administering other therapeutic agents to the subject.

19. Use of the pharmaceutical composition according to any one of claims 13-15 in the preparation of a medicament for treating a disease, preferably, the disease being a disease associated with abnormal CDH6 expression, more preferably, the disease associated with abnormal CDH6 expression being a tumor, preferably renal cancer or ovarian cancer.

20. A pharmaceutical composition according to any one of claims 13-15 for treating a disease, preferably, the disease being a tumor associated with abnormal CDH6 expression, more preferably, the disease associated with abnormal CDH6 expression is a tumor, preferably a solid tumor, preferably renal cancer or ovarian cancer.

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