Antibody and drug conjugate thereof, and use thereof

Abstract

The present invention relates to an antigen-binding molecule and an antibody-drug conjugate, and more specifically to an antibody and an antibody-drug conjugate (ADC) targeting CLDN18.2 and / or CDH17, and a composition containing the antibody or ADC and the therapeutic use thereof.

Description

Antibodies and their drug conjugates and their uses

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to PCT International Patent Application No. PCT / CN2024 / 134539, filed on November 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to antibodies and antibody-drug conjugates, and more particularly to antibodies and antibody-drug conjugates (ADCs) targeting CLDN18.2 and / or CDH17, as well as compositions containing said antibodies or ADCs and their therapeutic applications. Background Technology

[0004] Claudin 18.2 (CLDN18.2) belongs to the Claudin family of tight junction proteins. Tight junction proteins are key proteins that maintain tight junctions between cells. Different Claudin isoforms are expressed in different tissues and are associated with various types of cancer. For example, Claudin 1 is highly expressed in colon cancer, while Claudin 7 is closely associated with recurrence of liver cancer. Unlike other family members, CLDN18.2 expression in normal tissues is very limited, mainly confined to tight junctions of gastric mucosal cells, and almost non-existent in other normal tissues. This specific expression pattern makes CLDN18.2 an ideal target for cancer therapy, especially given its high expression rate of up to 70% in primary gastric adenocarcinoma and its metastases, as well as its expression in pancreatic cancer (50%), esophageal cancer (30%), and non-small cell lung cancer (25%), which typically have poor prognosis and high mortality rates, urgently requiring new treatment methods.

[0005] The CLDN18.2 protein comprises four transmembrane regions, two extracellular loops, and one intracellular loop, with its N-terminus and C-terminus located in the cytoplasm. The presence of the extracellular loops makes CLDN18.2 an ideal target for antibody targeting. However, the CLDN18.2 protein sequence is highly conserved across different species, and its high similarity to its homolog, Claudin 18.1, particularly the mere 8-amino acid difference in the extracellular loop D1, presents additional challenges for the development of specific antibody drugs. Claudin 18.1 is primarily expressed in lung tissue and is extremely similar to CLDN18.2 in sequence, requiring highly specific therapeutic strategies targeting CLDN18.2 to avoid potential impacts on non-target tissues.

[0006] Zolbetuximab (IMAB362), developed by Astellas Pharma, is a human-mouse chimeric IgG1 antibody targeting CLDN18.2. It induces tumor cell apoptosis and inhibits tumor cell proliferation by binding to CLDN18.2 on the surface of tumor cells, activating antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Although Zolbetuximab has been approved for marketing, single-target therapeutic strategies may not adequately address the complexity and heterogeneity of tumors, especially when tumor cells express multiple potential targets.

[0007] CDH17, also known as intestinal adhesion molecule 1 (LI-cadherin), is an adhesion protein specifically expressed on intestinal epithelial cells. It is overexpressed in various digestive system tumors, including colorectal cancer, gastric cancer, and pancreatic cancer. CDH17 overexpression is associated with tumor cell adhesion, spread, and metastasis, and is a key factor in tumor progression and deterioration. Therefore, CDH17 is also considered an important target for cancer therapy.

[0008] Nanobodies are small proteins composed of single-chain antibody molecules. They possess high specificity and affinity, and compared to traditional antibodies, they exhibit smaller size, greater stability, and deeper tissue penetration. This makes them highly promising for cancer therapy, enabling precise treatment by recognizing and targeting specific antigens on the surface of tumor cells, and allowing them to penetrate deep into tumor tissues inaccessible to conventional antibodies. Nanobodies can be designed to deliver drugs or radioisotopes to tumor cells to kill them. Furthermore, they can be used in various other therapeutic modalities such as photodynamic therapy and immunoassay. Therefore, the application of nanobodies in cancer therapy is receiving widespread attention and is expected to become one of the important means of future cancer treatment (Bannas, Hambach, and Koch-Nolte 2017).

[0009] While antibody-drug conjugates (ADCs) and other targeted therapies targeting single targets have achieved some clinical success, they face significant challenges in treating tumors expressing CLDN18.2 and CDH17. Intratumoral and intertumoral heterogeneity leads to resistance to these treatments, limiting their efficacy. Furthermore, single-target strategies fail to adequately address the complex biological characteristics and microenvironment of tumors, resulting in poor therapeutic outcomes. Targeting two different biomarkers can effectively attack different tumor subpopulations, reducing or delaying the development of treatment resistance. Therefore, developing a novel bispecific ADC holds promise not only for providing a more effective treatment for specific tumor types but also for bringing new strategies and hope to cancer therapy.

[0010] Invention Overview

[0011] Analysis of immunohistochemical and bioinformatics data from clinicopathological samples (see, for example, DOI:10.18632 / oncotarget.11638; DOI:10.1007 / s00428-019-02739-x; and DOI:10.1016 / j.prp.2024.155175) revealed that CLDN18.2 and CDH17 targets co-expressed in many tumors, especially gastrointestinal tumor tissue samples, while the co-expression rate was low in normal tissues.

[0012] Therefore, this invention first developed a VHH antibody or its heavy chain antibody specifically binding to CLDN18.2, and a VHH antibody or its heavy chain antibody specifically binding to CDH17. Then, based on these VHH antibodies, antibody molecules simultaneously targeting CLDN18.2 and CDH17 were designed and developed to cover tumor cells expressing CLDN18.2 and / or CDH17 targets, overcoming intratumoral heterogeneity and improving therapeutic efficacy. Building on this, the inventors further proposed an innovative antibody modality based on nanobodies, thereby achieving a higher enrichment ratio of antibody molecules in tumor tissue, further improving therapeutic efficacy. Based on these designs and findings, this invention establishes the multispecific antibodies and their drug conjugates, and their uses, particularly in cancer treatment.

[0013] Therefore, in a first aspect, the present invention provides a VHH antibody that specifically binds to CLDN18.2, a heavy chain antibody comprising thereof, and a multispecific antibody comprising thereof, as well as pharmaceutical compositions thereof and uses thereof.

[0014] In a second aspect, the present invention provides a VHH antibody that specifically binds to CDH17, a heavy chain antibody comprising the same, and a multispecific antibody comprising the same, as well as pharmaceutical compositions thereof and uses thereof.

[0015] In a third aspect, the present invention provides a multispecific antibody that binds CLDN18.2 and CDH17, a pharmaceutical composition thereof, and uses thereof, wherein the antibody comprises at least one antigen-binding domain that specifically binds CLDN18.2 and at least one antigen-binding domain that specifically binds CDH17.

[0016] In a fourth aspect, the present invention provides an antibody-drug conjugate (ADC) comprising the antibody of the present invention, a pharmaceutical composition thereof, and uses thereof.

[0017] The antibody-drug conjugate of the present invention has the following advantages:

[0018] (1) It binds to target cells expressing human CDH17 and / or CLDN18.2 with high affinity;

[0019] (2) It has higher endocytic activity;

[0020] (3) It has a significant bystander effect;

[0021] (4) It can solve the problem of tumor heterogeneity, target tumor cells that are CLDN18.2 single positive, CLND18.2 and CDH17 double positive, and CDH17 single positive, and has a better killing effect on CLND18.2 and CDH17 double positive cells than existing drugs. For example, bispecific antibody drug conjugates can have a better killing effect than the sum of monoclonal antibody drug conjugates (synergistic killing effect).

[0022] (5) It has high anti-tumor efficacy, stronger killing effect on tumor cells, and stronger inhibitory effect on tumor growth, especially in tumors with high expression of CDH17 and / or CLDN18.2; it has significantly improved and even unexpected anti-tumor activity.

[0023] (6) It has better product uniformity.

[0024] (7) It has low toxicity;

[0025] (8) It has good drug-like properties.

[0026] The invention is further illustrated in the following figures and specific embodiments. However, these figures and specific embodiments should not be considered as limiting the scope of the invention, and modifications readily apparent to those skilled in the art will be included within the spirit of the invention and the scope of protection of the appended claims.

[0027] Brief description of the attached diagram:

[0028] Figure 1: Binding curves of exemplary antibody CLDN18.2 VHH-Fc FACS against different cells (CHO-K1-CLDN18.2(A), SNU620(B) and HEK293-CLDN18.1(C)).

[0029] Figure 2: FACS binding curves of the exemplary antibody CDH17 VHH-Fc against different cells (SNU5(A) and SNU620(B));

[0030] Figure 3: Detection of endocytosis of exemplary antibody CLDN18.2 VHH-Fc on CHO-K1-CLDN18.2 cells.

[0031] Figure 4: Detection of endocytosis of exemplary antibody CDH17 VHH-Fc on SNU5(A) and SNU620(B) cells.

[0032] Figure 5: Schematic diagram of the structures of bispecific antibodies V-F1, V-F2, V-F4, V-F5, V-F7, V-F8, V-F9, V-F10 and V-F11 (where asterisks represent cysteine ​​mutations in the hinge region).

[0033] Figure 6: Expression of CLDN18.2 and CDH17 in different tumor cell lines.

[0034] Figure 7: Non-specific binding of exemplary antibodies V-F8, V-F9, V-F10 and V-F11, as well as monospecific antibodies znD05.M5-Fc and dznD05.m5m8-Fc and control antibody to the expression paralog CLDN18.1.

[0035] Figure 8: Exemplary antibodies binding to different tumor cells MKN45-CLDN18.2 (A), SNU620 (B), and SNU5 (C);

[0036] Figure 9: Binding of exemplary humanized bispecific antibodies to different tumor cells MKN45-CLDN18.2 (A), SNU620 (B), and SNU5 (C).

[0037] Figure 10: Binding of deimmunogenic exemplary bispecific antibodies to different tumor cells MKN45-CLDN18.2 (A) and AsPC-1-CLDN18.2 (B).

[0038] Figure 11: Binding of exemplary antibodies to stable HEK293-hu / cyno CDH17 cell lines (HEK293-huCDH17 (A, D), HEK293-cynoCDH17 (B, E) and HEK293 (C, F)).

[0039] Figure 12: Endocytosis of incompletely humanized exemplary bispecific anti-CLDN18.2 / CDH17 antibody molecules by MKN45-CLDN18.2 (A), SNU620 (B), and SNU5 (C) distinct CLDN18.2 / CDH17 double-positive tumor cells.

[0040] Figure 13: Endocytosis of V-F8 and V-F9 by CLDN18.2 / CDH17 double-positive tumor cells.

[0041] Figure 14: In vitro cytotoxicity of V-F1-PEG-EVC-MMAE, etc.

[0042] Figure 15: In vitro cytotoxicity of V-F8-VC-MMAE, etc.

[0043] Figure 16: In vitro cytotoxicity of V-F8-Gluc-Exd and V-F9-Gluc-Exd.

[0044] Figure 17: In vitro cytotoxicity of V-F11-Gluc-Exd and V-F11-RDVT-Exd.

[0045] Figure 18: In vitro toxicity of V-F11-Protein G-MMAE, etc.

[0046] Figure 19: Antitumor effects (A) and weight changes (B) of V-F1-PEG-EVC-MMAE et al. in a mouse SNU5 subcutaneous transplantation model.

[0047] Figure 20: Antitumor effects (A) and body weight changes (B) of V-F8-VC-MMAE et al. in a mouse SNU5 subcutaneous transplantation model.

[0048] Figure 21: Antitumor effects (A) and weight changes (B) of V-F1-PEG-EVC-MMAE et al. in a mouse MKN45-CLDN18.2 subcutaneous transplantation model.

[0049] Figure 22: Antitumor effects (A) and weight changes (B) of V-F8-VC-MMAE et al. in a mouse MKN45-CLDN18.2 subcutaneous transplantation model.

[0050] Figure 23: Antitumor effect (A) and weight change (B) of V-F9-Gluc-Exd et al. in mouse SNU5-CLDN18.2 subcutaneous transplantation model.

[0051] Figure 24: Antitumor effects (A) and weight changes (B) of V-F8-VC-MMAE et al. in a mouse SNU620 subcutaneous transplantation model.

[0052] Figure 25: Antitumor effect (A) and weight change (B) of V-F11-Gluc-Exd et al. in mouse SNU620 subcutaneous transplantation model.

[0053] Figure 26: Antitumor effect (A) and weight change (B) of V-F11-Gluc-Exd et al. in mouse Aspc-1 subcutaneous transplantation model.

[0054] Figure 27: Antitumor effect (A) and weight change (B) of V-F11-Gluc-Exd et al. in mouse CRC human tumor xenograft (PDX) model.

[0055] Figure 28: Bystander effect of V-F11-Gluc-Exd, etc.

[0056] Detailed description of the invention:

[0057] Unless otherwise specified, 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 invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0058] For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate.

[0059] definition

[0060] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0061] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0062] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the variable region consists of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region that “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0063] In this document, the term "antigen-binding molecule" refers to a molecule, such as a protein or peptide, or a molecule derived therefrom, that contains an antigen-binding domain or antigen-binding site capable of binding to a target antigen. In this invention, when the target antigen is CLDN18.2 and / or CDH17, antigen-binding molecules binding CLDN18.2 and / or CDH17 are also referred to as CLDN18.2-binding molecules, CDH17-binding molecules, or CLDN18.2 / CDH17-binding molecules. Antigen-binding molecules include, for example, antibodies and their antigen-binding fragments, as well as various fusions constructed based on antibodies or antigen-binding fragments, such as VHH-Fc antibodies, multi / bispecific antibodies, and chimeric antigen receptors (CARs). As will be apparent to those skilled in the art, the antigen-binding site of an antibody typically contains amino acid residues from a "complementarity-determining region" or "CDR".

[0064] In this document, the term "antibody" refers to a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. This term encompasses a wide range of antibody structures, including, but not limited to, monoclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, heavy chain antibodies, chimeric or humanized antibodies, intact antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0065] In this article, "intact antibody" or "full-length antibody" may be used interchangeably, referring to an immunoglobulin molecule containing at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this article) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL in this article) and a light chain constant region.

[0066] In this document, the terms "antibody fragment" and "antigen-binding fragment" are used interchangeably and refer to a molecule distinct from the intact antibody that contains a portion of the intact antibody and is capable of binding the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; camelid antibodies (heavy chain antibodies) or fragments thereof (e.g., VHH); and monospecific, bispecific, or multispecific antibodies formed from antibody fragments. Unless otherwise stated herein or explicitly contradicted by the context, the term "antibody" as used herein is equivalent to "antibody or an antibody fragment thereof." In some embodiments of the invention, the antibody fragment includes cysteine ​​residue portions for forming interchain disulfide bonds between heavy chains, such as cysteine ​​residues in the antibody hinge region, to provide amino acid residue sites usable for thiol coupling chemistry. In other embodiments of the invention, the antibody fragment includes cysteine ​​residues introduced into the Fc region to provide amino acid residue sites usable for thiol coupling chemistry.

[0067] In this document, the terms "antigen binding site" and "antigen binding domain" are used interchangeably to refer to the region in an antibody molecule that actually binds to the antigen. The antigen binding site of the antibody molecule used in this invention is preferably provided by a variable domain (i.e., "VHH") from a heavy chain antibody or an antibody fragment such as Fab.

[0068] In this document, the term "multispecific" refers to an antigen-binding molecule (e.g., an antibody) having at least two antigen-binding sites, each of which binds to a different antigen or a different epitope, for example, to different epitopes on different antigens or different epitopes on the same antigen. Correspondingly, "single-specific" refers to the ability to bind to only one epitope. "Dual-specific" refers to the ability to bind to two different antigens or epitopes.

[0069] In this article, the antibody-related terms "valence" or "valence number" refer to the total number of antigen-binding sites in an antibody molecule, or the number of antigen-binding sites with the same antigen-binding specificity. For example, a quadrivalent antibody means that the antibody molecule contains a total of 4 antigen-binding sites; the antibody molecule can be a "2+2" type bispecific antibody, that is, the antibody has two different antigen-binding specificities, wherein for each antigen-binding specificity, there are 2 identical antigen-binding sites.

[0070] In this document, unless otherwise stated, the term CLDN18.2 includes any variant of human CLDN18.2, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and covers its species homologs. Furthermore, it should be understood that the term covers not only CLDN18.2 expressed naturally or recombinantly in cells or expressed on natural or recombinant cells, but also fusion proteins containing CLDN18.2 or fragments thereof, such as extracellular fragments. An example of CLDN18.2 is the human CLDN18.2 protein containing the amino acid sequence UniProt:P56856-2. Another example of CLDN18.2 is the monkey CLDN18.2 protein containing the amino acid sequence UniProt:A0A2K5VV62. In this document, unless specifically indicated, the term "CLDN18.2" refers to CLDN18.2 derived from humans. In this paper, "antigen binding specificity against CLDN18.2", that is, "antigen binding domain that specifically binds to CLDN18.2", is preferably provided by the VHH domain.

[0071] In this document, the term "CLDN18.2 positive" cell refers to a cell that expresses CLDN18.2 positively, such as cancer cells, modified cancer cells, or modified non-tumor cells. The expression level of CLDN18.2 on the cell surface can be determined by any conventional method known in the art for determining the expression level of cell surface antigens, such as FACS detection methods or immunofluorescence staining methods. CLDN18.2 has significantly higher expression levels on a variety of tumor cells than on normal tissues / cells, for example, SNU620 (gastric cancer cells). Preferably, in this document, CLDN18.2 positive cells are CLDN18.2 positive tumor cells.

[0072] In this document, unless otherwise specified, the term "CDH17" includes any variant of human CDH17, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and covers its species homologs. Furthermore, it should be understood that the term covers not only CDH17 expressed naturally or recombinantly in cells or expressed on natural or recombinant cells, but also recombinantly expressed fusion proteins containing CDH17 or fragments thereof. An example of CDH17 is the human CDH17 protein containing the amino acid sequence under UniProt:Q12864. Another example of CDH17 is the monkey CDH17 protein containing the amino acid sequence under NCBI:XP_005563762.1. In this document, unless specifically indicated, the term "CDH17" refers to CDH17 derived from humans. In the antibodies of this invention, "antigen-binding specificity against CDH17," i.e., "antigen-binding domain that specifically binds to CDH17," is provided by the VHH domain.

[0073] In this document, the term "CDH17 positive" cell refers to a cell that expresses CDH17 positively on its cell surface, such as cancer cells, modified cancer cells, or modified non-tumor cells. The expression level of CDH17 on the cell surface can be determined by any conventional method known in the art for determining the expression level of cell surface antigens, such as FACS detection methods or immunofluorescence staining methods. CDH17 has significantly higher expression levels on a variety of tumor cells than on normal tissues / cells, for example, SNU5 (gastric cancer cells). Preferably, in this document, CDH17 positive cells are CDH17 positive tumor cells.

[0074] In this paper, 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 antigenic epitope). In this context, "binding affinity" reflects the intrinsic binding affinity of a 1:1 interaction between members of a binding pair. Binding affinity is typically expressed as the binding dissociation equilibrium constant (K0). D It can be described by ) and measured by commonly known methods in the art, such as surface plasmon resonance (SPR) techniques.

[0075] In this paper, the term “avidity” or “binding affinity” refers to the combined strength of the interactions between multiple binding sites of a molecule (antibody) and the same target.

[0076] In this paper, the term "immunoglobulin" refers to a protein with a structure that contains naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, from the N-terminus to the C-terminus, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, from the N-terminus to the C-terminus, followed by a light chain constant domain (CL). Immunoglobulin heavy chains can be classified into one of five categories based on the type of their constant domains, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM). Some of these categories can be further subdivided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can also be classified into one of two types based on the amino acid sequence of their constant domains, called κ and λ.

[0077] In this document, the term "isotype" refers to the antibody type determined by the antibody heavy chain constant region. For example, the antibody according to the invention may be an IgA (e.g., IgA1 or IgA2), IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgE, IgM, and IgD antibody, having a heavy chain constant region of the aforementioned immunoglobulin type. Furthermore, the invention contemplates not only antibodies employing native sequence constant regions but also antibodies containing variant sequence constant regions.

[0078] In this document, the term "variable region" or "variable domain" refers to a domain of the antibody's heavy or light chain involved in antibody-antigen binding. In the case of heavy chain antibodies, such as those derived from camelid heavy chains, a single VH domain (also referred to herein as the VHH domain) may be sufficient to impart antigen-binding specificity. Like the variable regions of the heavy and light chains of conventional IgG antibodies, the VHH domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs), arranged in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. According to some aspects of the invention, one or more residues in the variable region of the antibody can be modified, for example, by modifying one or more CDR regions and / or by modifying one or more framework regions, particularly by substituting conserved residues, to obtain antibody variants that still substantially retain at least one biological property (e.g., antigen-binding ability) of the parent antibody. In other aspects, the antibody variable region can be modified by CDR transplantation. Since the CDR sequence is responsible for most antibody-antigen interactions, recombinant antibody variants that mimic the properties of known antibodies can be constructed. In such antibody variants, a CDR sequence from a known antibody is grafted onto a framework region of a different antibody with different properties, and one to several residues can be mutated as needed, such as reverting to a mutation to refine the desired properties of the antibody. The properties of the mutated and / or modified antibody or ADC conjugate containing it can be evaluated in in vitro or in vivo assays, such as target antigen binding properties or other desired functional properties, such as endocytic activity, pharmacokinetics, and in vivo tumor-killing activity. Therefore, variants of any variable regions (e.g., VHH) given herein are also contemplated in this invention.

[0079] In this document, the term "complementarity-determining region" or "CDR region" or "hypervariant region" refers to a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. In the VHH domain of the antibody of this invention, CDRs are sequentially numbered starting from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. A specific CDR sequence within a VHH domain can be determined using methods known in the art, such as the Kabat, AbM, Chothia, Contact, and IMGT schemes, to define the regional extent of the CDR and combinations thereof. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the aforementioned methods and combinations thereof. Furthermore, it is understood in the art that although CDRs differ between antibodies, only a limited number of amino acid positions within a CDR directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, a minimal overlapping region can be determined, thereby providing a “minimum binding unit” for antigen binding. Such a minimum binding unit can be a sub-part of a CDR. The remaining residues of the CDR sequence, as will be apparent to those skilled in the art, can be determined by the antibody’s structure and protein folding. Therefore, the invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues may be substituted.

[0080] Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region and CDR (including heavy chain variable region residues), it means the numbering position according to the Kabat numbering system.

[0081] In this document, the terms "VHH" and "VHH domain" are used interchangeably to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain, sometimes also called a single variable domain fragment (sVD). Therefore, a VHH differs from the conventional VH of a four-chain immunoglobulin in that it does not require pairing with a light chain variable domain to form an antigen-binding site. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids may also produce naturally occurring heavy chain antibodies lacking a light chain, and these VHHs are also within the scope of this invention. In some cases, for the therapeutic application of antibodies or their derivatives, it is desirable to reduce their immunogenicity.

[0082] Heavy chain antibodies (HcAbs) are novel antibody molecules found in camels and sharks. These antibodies are characterized by the natural absence of light chains, consisting only of heavy chains. Despite the lack of light chains, heavy chain antibodies retain the ability to bind antigens. Other species besides camels can also produce naturally occurring heavy chain antibodies lacking light chains. When heavy chain antibodies are mentioned herein, they generally refer to single-chain antibodies containing VHH and a heavy chain constant region or Fc region. The heavy chain antibodies mentioned herein can also dimerize to form dimerized heavy chain antibodies, which are also included within the scope of "heavy chain antibodies" in this invention.

[0083] In this document, the term "immunoglobulin Fc region," used interchangeably with "Fc region" and "Fc domain," defines 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. Fc regions that can be used in the antibodies of this invention include, but are not limited to, Fc regions of IgG1, IgG2, IgG3, or IgG4 having native or variant sequences. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain 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 Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In this document, the term "Fc region" or "Fc domain" excludes the heavy chain variable region (VH) and light chain variable region (VL) of immunoglobulins, as well as the heavy chain constant region (CH1) and light chain constant region (CL); but may include the CH2 and CH3 domains, and may or may not include an immunoglobulin hinge region. For example, in some instances, the Fc region may consist of or be composed of the CH2 and CH3 domains from the N-terminus to the C-terminus. In other instances, the Fc region may consist of or be composed of the immunoglobulin hinge region or a portion thereof from the N-terminus to the C-terminus, the CH2 and CH3 domains, or may be composed of or be composed of the immunoglobulin hinge region or a portion thereof, the CH2 and CH3 domains.

[0084] In this document, the term "natural sequence Fc region" encompasses the Fc region sequences of various naturally occurring immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or Asn231 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In some embodiments, the human IgG heavy chain Fc region carries at the N-terminus a hinge sequence or a partial hinge sequence of a natural immunoglobulin, such as the sequence E216 to P230 or the sequence D221 to P230 according to EU designations.

[0085] In this document, the term "variant sequence Fc region" refers to a polypeptide containing a modified Fc region relative to the native Fc region sequence. The modification can be the addition, deletion, or substitution of amino acid residues. Substitution can include both naturally occurring and non-natural amino acids. The purpose of the modification can be to alter the binding of the Fc region to its receptor and the resulting effector function, or to prevent undesirable heavy chain mismatches, or to site-directedly introduce amino acid modifications that can be used to conjugate other active molecules.

[0086] In this paper, the term "effective function" refers to those biological activities attributable to the Fc region of immunoglobulins that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include Fc receptor binding, C1q binding and complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Depending on the intended use of the antibody molecule, the Fc region of the antibody can be modified to give it altered effector functions relative to antibody molecules with a wild-type Fc region, such as reduced or eliminated Fcγ receptor binding.

[0087] In this document, the terms “flexible linker” or “connector” or “linker peptide” are used interchangeably to refer to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or from the hinge region of an immunoglobulin.

[0088] In this document, the “percentage of identity (%)” for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification, after comparing the candidate sequence with the specific amino acid sequence shown herein and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a considerable degree of identity with respect to the antibody molecules and their sequences specifically disclosed herein, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. These variants may contain conserved modifications.

[0089] For polypeptide sequences, "conservative modification" includes substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of a certain amino acid with a chemically similar amino acid. Tables providing conserved substitutions of functionally similar amino acids are well known in the art. The following eight groups contain amino acids that are conservedly substituted for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used in particular to refer to amino acid modifications that do not significantly affect or alter the desired properties (e.g., binding characteristics and / or internalization characteristics) of the antibody containing the amino acid sequence.

[0090] In this document, the terms "binding" or "specific binding" mean that the binding interaction is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as detecting the binding ability of an antibody to an antigen using the ELISA assay described in the examples, detecting the binding ability of an antibody to cells expressing an antigen using the FACS assay described in the examples, or detecting the affinity constant K using the SPR technique described in the examples. D .

[0091] In this document, the term "epitope" refers to the antigenic moiety that an antibody specifically binds to. An epitope may consist of continuous and / or discontinuous amino acids forming a conformational spatial unit. Different antibodies binding to the same antigen can be grouped by epitope grouping using a competitive binding assay. An antibody and a reference antibody are considered "competitively binding antibodies" when the test antibody blocks the binding of a reference antibody to an antigen (e.g., CLDN18.2 or CDH17) by 50% or more in a competitive binding assay; and conversely, when the reference antibody blocks the binding of the test antibody to the antigen (e.g., CLDN18.2 or CDH17) by 50% or more in a competitive binding assay. Competitively binding antibodies may bind to the same epitope region as the reference antibody, such as identical epitopes, adjacent epitopes, or overlapping epitopes. The competitive binding assay can be performed by methods known in the art, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay, or the methods described in the examples herein.

[0092] In this document, a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from nonhuman CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) in a humanized antibody correspond to those in nonhuman antibodies, and all or substantially all of the FRs correspond to those in human antibodies. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a nonhuman antibody) refers to an antibody that has been humanized. In some embodiments, the humanized antibody of the present invention has a framework region sequence “derived” from a specific human lineage sequence. Here, “derived” means that the amino acid sequence of the antibody framework region has at least 85% or 90% identity with the corresponding framework region amino acid sequence encoded by the human lineage immunoglobulin gene, and that the antibody retains antigen-binding activity.

[0093] In this document, if an amino acid sequence (e.g., VHH) is specific for two different antigens or antigenic determinants (e.g., CLDN18.2 or CDH17 from different mammalian species, such as human CLDN18.2 or human CDH17, or cynomolgus monkey CLDN18.2 or cynomolgus monkey CDH17), then it is said to be "cross-reactive" to these two different antigens or antigenic determinants. Antibodies exhibiting human-monkey species cross-reactivity, particularly having similar human-monkey antigen binding affinity, is advantageous, as this property can facilitate preclinical drug development of antibodies, such as toxicological assays of antibody-derived antibody-drug conjugates (ADCs). In some embodiments, the antibodies of the present invention preferably exhibit human-monkey species cross-reactivity.

[0094] In this document, the terms "endocytosis" and "internalization" are used interchangeably, referring to the process by which a ligand / receptor complex is internalized and delivered into the cytosol or translocated to a suitable intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface. In some embodiments, the antibodies of the present invention induce endocytosis upon binding to CLDN18.2 or CDH17 expressed on the cell surface. In this document, endocytosis and endocytosis rate can be determined, for example, by the methods described in the examples, to characterize the endocytic activity of the antibody. In some embodiments, the antibodies of the present invention having endocytic activity can be used as a tool for delivering antitumor drugs into cancer cells in the ADCs of the present invention.

[0095] In this document, the term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0096] In this document, the term "expression vector" refers to a vector containing a recombinant polynucleotide and an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including clomids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0097] In this document, the terms "immunoconjugate," "immunofusion," or "immunocoupler" are used interchangeably and generally refer to a molecule formed by conjugating or fusing one or more immunoglobulin-associated molecules or fragments thereof (e.g., antibodies or fragments thereof) with one or more other molecules. In some cases, the other molecules may be protein-like molecules, such as peptides, polypeptides, or proteins. In some cases, the other molecules may also be non-protein-like molecules, such as chemical toxins or drugs like small molecule drugs or antitumor compounds. In some cases, the other molecules may be the same as immunoglobulin-associated molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-associated molecules or fragments thereof. The one or more other molecules may be the same or different from each other. For example, the other molecules may be target-binding elements and / or effector elements, such as chemotherapeutic agents, toxins, drugs (e.g., immunotherapeutic agents), radioactive elements, probes, or signaling molecules, etc.

[0098] In this document, the terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual is a human.

[0099] In this article, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic 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 mitigating the disease state, and alleviating or improving prognosis. In cases involving tumor or cancer treatment, "treatment" encompasses antitumor biological effects that can be induced by artificial intervention (e.g., through the administration of drugs), including but not limited to, reductions in tumor volume, number of tumor cells, proliferation, or survival.

[0100] In this document, the terms “cancer” and “tumor” are used interchangeably to refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and non-solid tumors. In some embodiments, cancers suitable for treatment by the antibodies or immunoconjugates or immunofusions of the present invention include CLDN18.2-positive and / or CDH17-positive tumors / cancers, including their metastatic forms. Examples of cancer include the specific cancers mentioned in the embodiments section herein.

[0101] As used in this article, “antibody-drug conjugate (ADC)” refers to a molecule, substance, or compound in which an antigen-binding molecule is linked to a (small molecule) drug (e.g., an antitumor compound) via a linker.

[0102] "Linker-payload" is a term well known to those skilled in the art, referring to a compound formed by linking a linker to a payload (e.g., a drug).

[0103] The term "linker" refers to a structural segment that connects a drug to an antigen-binding molecule. It should be understood that a linker has functional groups that can form bonds with the functional groups of the antigen-binding molecule before linking to it. Non-limiting examples of linkers include those involved in embodiments and examples of the present invention.

[0104] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 1-16 carbon atoms, i.e., C16-C26. 1-16 Alkyl, more preferably C101-12 Alkyl, C 1-10 Alkyl, C 1-8 Alkyl, C 1-6 Alkyl, C 1-4 Alkyl or C 1-2 Alkyl groups. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0105] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing 2-16 carbon atoms and at least one double bond but no triple bonds. Preferably, the alkenyl group contains 2-12 carbon atoms, i.e., C64. 2-12 Alkenyl, more preferably C 2-10 alkenyl, C 2-8 alkenyl, C 2-6 alkenyl or C 2-4 Alkenyl groups. Representative examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl.

[0106] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing 2-16 carbon atoms and at least one triple bond. The alkynyl group preferably contains 2-12 carbon atoms, i.e., C64. 2-12 The alkynyl group, more preferably C, is a alkynyl group. 2-10 alkynyl group, C 2-8 alkynyl group, C 2-6 alkynyl or C 2-4 Alkynyl group. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0107] The term "haloalkyl" refers to an alkyl group as defined above, which is substituted with one or more (e.g., up to 12, such as 1, 2, 3, 4, 5, 6, 7, or 8) halogen groups. Halogenated alkyl groups include perhalogenated alkyl groups. A perhalogenated alkyl group is an alkyl group in which all hydrogen atoms are replaced by halogen atoms. When a haloalkyl group is a polyhalogenated alkyl group, i.e., containing two or more halogens, the halogens may be the same or different. Non-limiting examples of haloalkyl groups include monofluoromethyl, difluoromethyl, trifluoromethyl, monochloromethyl, dichloromethyl, trichloromethyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, trifluoroethyl, dichloroethyl, trichloroethyl, difluoropropyl, perfluoroethyl, and perfluoropropyl.

[0108] The term "haloalkenyl" refers to an alkenyl group as defined above, which is substituted by one or more (e.g., up to 12, such as 1, 2, 3, 4, 5, 6, 7, or 8) halogen groups. When the haloalkenyl is a polyhalogenated alkenyl, i.e., containing more than two halogens, the halogens may be the same or different.

[0109] The term "haloalkynyl" refers to an alkynyl group as defined above, which is substituted by one or more (e.g., up to 12, such as 1, 2, 3, 4, 5, 6, 7, or 8) halogen groups. When the haloalkynyl group is a polyhaloalkynyl group, i.e., containing more than two halogens, the halogens may be the same or different.

[0110] The term “halogen” or “halogenated” refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0111] The term "hexuronic acid" refers to compounds obtained by oxidizing the primary hydroxyl group of a hexose to a carboxyl group. Hexuronic acids include common uronic acids obtained by oxidizing hexoses, including but not limited to glucuronic acid, mannuronic acid, and galacturonic acid.

[0112] The term "amino acid" as used herein has the meaning conventionally understood in the art. Amino acids can be L or D isomers. The notation of amino acids follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. For ease of understanding, the names of some common amino acids and their corresponding abbreviations are listed in the table below:

[0113] The amino acids in this invention also include other amino acids such as citrulline (Cit; C). It should be understood that citrulline (Cit; C) is frequently used in the linker portion of an ADC. Therefore, unless otherwise specified and consistent with the context, the single-letter abbreviation "C" representing an amino acid in a linker or linker fragment involving an ADC represents Cit; and in a linker or linker fragment not involving an ADC, the single-letter abbreviation "C" representing an amino acid represents Cys.

[0114] Unless otherwise specified, the amino acids in this invention refer to L-amino acids.

[0115] The term "optional" or "optionally" means that the event or condition described below either occurs or does not occur, and the description includes instances where the event or condition occurs as well as instances where the event or condition does not occur. For example, when a group or structure is "optionally substituted," the group or structure may or may not be substituted.

[0116] In this article, "pharmaceutically acceptable" means that it can be administered to an individual or subject without producing biologically or otherwise undesirable side effects, such as serious and intolerable side effects.

[0117] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0118] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the molecules of the present invention (e.g., antibody-drug conjugates), and that such salt is not biologically or otherwise undesirable. The ADC conjugates of the present invention can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable, non-toxic acid addition salt refers to a salt formed by the ADC conjugate of the present invention with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N group.

[0119] The term "solvent" refers to an association formed by one or more solvent molecules with the antibody-drug conjugate of this invention. Solvents that form solvates include solvents commonly used in the pharmaceutical industry, such as water, ethanol, diethyl ether, isopropanol, ethyl acetate, and dimethyl sulfoxide.

[0120] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug portion (D) to the antibody portion in the antibody-drug conjugate molecule (e.g., the compound of formula I) described herein. It should be understood that for antibody-drug conjugate molecules, DAR is an integer; for example, the DAR of the antibody-drug conjugate molecule of the present invention can be an integer from 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, or, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The prepared antibody-drug conjugate typically contains one or more antibody-drug conjugate molecules with different DARs, which are usually characterized by the average DAR, i.e., the overall ratio of the drug fraction (D) conjugated to the Ab fraction described herein to the Ab fraction in the product, as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis and / or HPLC). This DAR is referred to as the average DAR in this document and may be a decimal. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 16, for example 2-16, 4-12, 3-5, 5-7, 7-9, 3-8, 2-6, 4-6, for example 3.0-8.0, 3.5-4.5, 5.5-6.5 or 7.5-8.5, for example 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4 6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 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, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, a range with two of these values ​​as endpoints. As stated above, it should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules containing ADC molecules having the same and / or different DAR values.

[0121] The term “therapeutic agent” as used herein encompasses any substance that is effective in preventing or treating diseases such as tumors, such as cancer, including chemotherapeutic agents, cytotoxic agents, immunomodulators (such as immunosuppressants), other antibodies, small molecule drugs, angiogenesis inhibitors, or cytokines.

[0122] "Chemotherapy agents" include chemical compounds that are useful in treating cancer or immune system diseases.

[0123] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0124] As used herein, the term "immunomodulator" refers to a natural or synthetic active agent or drug that inhibits or modulates (e.g., activates) an immune response. An immune response can be humoral or cellular. Immunomodulators include immunosuppressants or immune agonists, such as immune checkpoint inhibitors or immune checkpoint agonists.

[0125] The term "drug" refers to a compound that can regulate biological processes, particularly altering or preventing pathological processes. In this article, "drug" preferably refers to antitumor compounds.

[0126] The term "small molecule drug" refers to a low molecular weight drug that can regulate biological processes, particularly altering or preventing pathological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa, more preferably less than 500 kDa. Small molecule drugs include, but are not limited to, organic molecules having the molecular weights defined above, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0127] "Antitumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773 and US5658920, such as camptothecin compounds eczetidine (a topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative), and auristatin compounds such as monomethyl auristatin E (MMAE) and MMAF.

[0128] The term "effective amount" refers to such an amount or dose of the antigen-binding molecule or ADC molecule or composition or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. Depending on the intended effect, it may include "therapeutic effective amount" and "preventive effective amount".

[0129] "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. Therapeutic effective dose can vary depending on various factors such as disease state, individual age, sex, and weight. Therapeutic effective dose is the amount at which any toxic or harmful effects are less than the beneficial therapeutic effect. Relative to untreated subjects, "therapeutic effective dose" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%. The ability of the antibody of the present invention to inhibit measurable parameters (e.g., tumor volume) can be evaluated in animal model systems that predict efficacy in human tumors.

[0130] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered in subjects before or at an early stage of the disease, the prophylactic effective dose is less than the therapeutic effective dose.

[0131] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0132] The term "pharmaceutical excipient" refers to diluents, adjuvants (such as Freund's adjuvants (complete and incomplete)), carriers, or stabilizers that are applied together with the active substance.

[0133] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0134] The terms “drug combination,” “combination product,” “drug conjugate,” or “combination product” refer to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term “non-fixed combination” means that the active ingredients (e.g., (i) the antigen-binding molecule or ADC molecule of the present invention, including its pharmaceutically acceptable salt, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of two or more active agents. In some embodiments, the antigen-binding molecule or ADC molecule of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when used alone. The term “fixed combination” means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in a separate formulation, and their formulations may be the same or different.

[0135] The terms "combination therapy" or "treatment in combination" refer to the administration of two or more therapeutic agents or modalities of treatment (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0136] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0137] When used herein, the terms “molecule of the invention” or “molecule according to the invention” include antigen-binding molecules (including bispecific antibodies or antigen-binding fragments thereof), immunoconjugates, immunofusions, antibody-drug conjugates, or pharmaceutically acceptable salts or solvates thereof as defined herein (especially in the Embodiments and Examples sections).

[0138] I. Antigen-binding molecules, nucleic acids, and their preparation methods

[0139] In some embodiments, the present invention relates to antigen-binding molecules that specifically bind to CLDN18.2 and / or CDH17, such as VHH antibodies, heavy chain antibodies, full-length antibodies, or multispecific antibodies. The antigen-binding molecules of the present invention also comprise TCR molecules or CAR molecules that specifically bind to CLDN18.2 and / or CDH17.

[0140] I. CLDN18.2 single-domain antibody and its heavy chain antibody

[0141] One aspect of the present invention relates to a CLDN18.2 antigen-binding molecule that specifically binds to CLDN18.2. In some embodiments, the antigen-binding molecule is a single-domain antibody that binds to CLDN18.2, a heavy-chain antibody comprising said single-domain antibody, or a multispecific antibody. In some embodiments, the antigen-binding molecule of the present invention specifically binds to mammalian CLDN18.2, such as human CLDN18.2 or cynomolgus monkey CLDN18.2.

[0142] In some embodiments, the anti-CLDN18.2 single-domain antibody of the present invention is a VHH antibody containing or composed of a heavy chain variable region, wherein the heavy chain variable region typically has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; and VHH CDR1 to VHH CDR3 refer to complementarity-determining regions 1 to 3. The CDR sequence in the VHH variable region can be determined according to any CDR definition scheme, for example, according to AbM, Chothia, Kabat, IMGT, or any combination thereof; more preferably, the CDR is defined according to Kabat or AbM, or a combination thereof; and more preferably, the CDR is defined according to AbM. In this document, the anti-CLDN18.2 single-domain antibody or VHH antibody will also be referred to as VHH. CLDN18.2 .

[0143] In some embodiments, the VHH antibody against CLDN18.2 of the present invention comprises the three complementarity-determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO:1-19; preferably, the CDR sequences are defined according to ABM.

[0144] In some embodiments, the VHH antibody against CLDN18.2 of the present invention comprises or is composed of a heavy chain variable region, said heavy chain variable region comprising the three complementarity-determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO:1-19; preferably, said CDR sequence is defined according to ABM.

[0145] In some embodiments, the anti-CLDN18.2 VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-CLDN18.2 VHH of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0146] The VHH CDR1 comprises, or is composed of, an amino acid sequence selected from SEQ ID NO:20, 21, or 22, or is composed of said amino acid sequence, or the VHH CDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO:20, 21, or 22; or

[0147] The VHH CDR2 comprises, or is composed of, the amino acid sequence of SEQ ID NO:23, or comprises, an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:23; or

[0148] The VHH CDR3 comprises or is composed of an amino acid sequence selected from any one of SEQ ID NO:24-35, or the VHH CDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from any one of SEQ ID NO:24-35.

[0149] In some embodiments, the anti-CLDN18.2 VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-CLDN18.2 VHH of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0150] (i) The VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:20, the VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and the VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:24.

[0151] (ii) The VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:21, the VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:23, and the VHH CDR3 comprises or is composed of the amino acid sequence shown in any one of SEQ ID NO:24-35; or

[0152] (iii) The VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:22, the VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and the VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:24.

[0153] In some embodiments, the VHH antibody against CLDN18.2 of the present invention comprises or is composed of a heavy chain variable region, said heavy chain variable region

[0154] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 1-19; or

[0155] (ii) Contains or consists of an amino acid sequence selected from any one of SEQ ID NO:1-19; or

[0156] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from any of SEQ ID NO:1-19, preferably, the amino acid changes do not occur in the CDR region.

[0157] In some embodiments, the VHH antibody against CLDN18.2 of the present invention comprises or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:1-19.

[0158] In some embodiments, the VHH antibody against CLDN18.2 of the present invention comprises a CDR amino acid sequence and / or a framework (FR) amino acid sequence derived from a camel heavy chain antibody produced by immunizing a camel (e.g., an alpaca). In some embodiments, the VHH monoclonal antibody of the present invention derived from a camel heavy chain antibody can be engineered, for example, to comprise a framework region sequence derived from a human amino acid sequence (i.e., a human antibody) or other non-camel mammal species. Therefore, in one embodiment, the VHH antibody of the present invention is a chimeric antibody.

[0159] In one embodiment, the VHH antibody against CLDN18.2 of the present invention is a humanized antibody. For example, the original VHH sequence is humanized using a "best-matching" method, which includes...

[0160] (i) The amino acid sequence of the VHH framework region was compared and analyzed using the human pedigree V gene database to select the best pedigree sequence;

[0161] (ii) The best-matching human CDR sequence is replaced with a VHH CDR sequence, and optionally multiple residues in the mutated frame region are removed by post-translational modification (PTM) to generate a humanized VHH sequence;

[0162] (iii) Optionally, the immunogenicity of the humanized VHH sequence can be analyzed using software such as WeMol, and mutations that reduce immunogenicity can be introduced at specific residues;

[0163] (iv) Optional sequencing of VHH antibodies.

[0164] Typically, humanization is performed in a manner that preserves the favorable binding properties of single-domain antibodies. Assays for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art, in order to identify and select suitable mutations or combinations of humanized residues.

[0165] In another aspect of the invention, the invention also provides a heavy chain antibody comprising the heavy chain variable region of the anti-CLDN18.2 VHH antibody of the invention.

[0166] In some embodiments, the anti-CLDN18.2 single-domain antibody or VHH (e.g., camel-derived VHH or its humanized form) of the present invention can be linked to a constant region or a portion thereof, such as the Fc region, of a human antibody to generate a heavy chain antibody comprising a VHH-constant region, VHH-CH1-Fc, or VHH-Fc. In one embodiment, the heavy chain antibody comprises the VHH antibody of the present invention and an Fc region located at its C-terminus. In one embodiment, the anti-CLDN18.2 heavy chain antibody comprises an Fc portion derived from a camel (e.g., an alpaca). In one embodiment, the heavy chain antibody is generated and isolated by immunizing the camel, such as an alpaca. Various methods are known in the art for immunizing camel animals and isolating the generated VHH antibody or heavy chain antibody against the target antigen.

[0167] In some embodiments, the anti-CLDN18.2 heavy chain antibody contains a constant region derived from human or non-human primate (e.g., cynomolgus monkey) antibodies, such as a constant region derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0168] In some embodiments, the anti-CLDN18.2 heavy chain antibody comprises an Fc region derived from a human or non-human primate (e.g., a cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, such as a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region. In some embodiments, the Fc region linked to the VHH antibody comprises the entire hinge region, such as the hinge region shown in SEQ ID NO:90. In some embodiments, the Fc region linked to the VHH antibody comprises a partial hinge region, such as the hinge region shown in SEQ ID NO:93. In some embodiments, the Fc region linked to the VHH antibody comprises the entire hinge region containing the C220S mutation, such as the hinge region shown in SEQ ID NO:89.

[0169] In one embodiment, the heavy chain antibody that specifically binds to CLDN18.2 according to the present invention can dimerize with another polypeptide chain (e.g., another heavy chain antibody, the same or different) containing the Fc region via the Fc region. Therefore, in one embodiment, the present invention also provides homologous or heteromeric multimer proteins comprising the heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0170] The Fc region described herein is applicable to the heavy chain antibody of this invention that specifically binds to CLDN18.2.

[0171] In some embodiments, the Fc region is derived from IgG1 and comprises the entire hinge region, containing or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 85 or 86. In some embodiments, the Fc region is derived from IgG1, comprises the entire hinge region, and contains a C220S mutation, containing or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91 or 92.

[0172] The single-domain antibody VHH that specifically binds to CLDN18.2, or a heavy-chain antibody containing it, such as VHH-Fc, may have one or more of the following properties:

[0173] (i) It binds with high affinity to cells expressing CLDN18.2, such as tumor cells, and does not bind nonspecifically to cells expressing CLDN18.1;

[0174] (ii) exhibits species cross-reactivity with cynomolgus monkey CLDN18.2; and / or

[0175] (iii) Has endocytic activity on CLDN18.2 positive cells, for example, higher than antibodies known to be CLDN18.2.

[0176] In some aspects, the single-domain antibody VHH that specifically binds to CLDN18.2, or a heavy-chain antibody containing it such as VHH-Fc, exhibits high binding affinity to tumor cells expressing CLDN18.2. The EC50 value and / or maximum binding amount of the antibody against CLDN18.2-positive tumor cells can be determined by FACS or ELSA assays (e.g., the assays described in the examples) and optionally compared to a reference antibody to reflect the cell-binding affinity of the antibody. Exemplary methods are described in the examples, particularly in the materials and methods section.

[0177] In some embodiments, the multispecific antibody of the present invention exhibits cross-reactivity with human and monkey CLDN18.2.

[0178] In some embodiments, the multispecific antibody of the present invention possesses CLDN18.2-mediated endocytic activity. The endocytic activity of the antibody can be evaluated in cell-based assays, such as those described in the examples. Exemplary endocytosis assays are described in the examples, particularly in the materials and methods described.

[0179] In some embodiments, after one-step purification of the antibody product generated from recombinant mammalian cells using protein A affinity chromatography, the multispecific antibody of the present invention can achieve a purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher, as determined by SEC-HPLC.

[0180] II. CDH17 single-domain antibodies and their heavy chain antibodies

[0181] One aspect of the present invention relates to an antigen-binding molecule that specifically binds to CDH17. In some embodiments, the antigen-binding molecule is a single-domain antibody that binds to CDH17, or a heavy-chain antibody or multispecific antibody comprising said single-domain antibody. In some embodiments, the antigen-binding molecule of the present invention specifically binds to mammalian CDH17, such as human CDH17 or cynomolgus monkey CDH17.

[0182] In some embodiments, the anti-CDH17 single-domain antibody of the present invention is a VHH antibody comprising or composed of a heavy chain variable region, wherein the heavy chain variable region typically has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; and VHH CDR1 to VHH CDR3 refer to complementarity-determining regions 1 to 3. The CDR sequence in the VHH variable region can be determined according to any CDR definition scheme, for example, according to AbM, Chothia, Kabat, IMGT, or any combination thereof; more preferably, the CDR is defined according to Kabat or AbM, or a combination thereof; and more preferably, the CDR is defined according to AbM. In this document, the anti-CDH17 single-domain antibody or VHH antibody is also referred to as VHH. CDH17 .

[0183] In some embodiments, the anti-CDH17 VHH antibody of the present invention comprises the three complementarity-determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO:36-51 or 61-70; preferably, the CDR sequences are defined according to ABM.

[0184] In some embodiments, the anti-CDH17 VHH antibody of the present invention comprises or is composed of a heavy chain variable region, said heavy chain variable region comprising the three complementarity-determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO:36-51 or 61-70; preferably, said CDR sequence is defined according to ABM.

[0185] In some embodiments, the anti-CDH17 VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-CDH17 VHH of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0186] The VHH CDR1 comprises, or is composed of, an amino acid sequence selected from SEQ ID NO:52, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO:52; or

[0187] The VHH CDR2 contains, or is composed of, the amino acid sequence of SEQ ID NO:53, or contains an amino acid sequence that has one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:53; or

[0188] The VHH CDR3 comprises or is composed of an amino acid sequence selected from any one of SEQ ID NO:54-60, or the VHH CDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from any one of SEQ ID NO:54-60.

[0189] In some embodiments, the anti-CDH17 VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-CDH17 VHH of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0190] The VHH CDR1 comprises, or is composed of, an amino acid sequence selected from SEQ ID NO: 71 or 95, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO: 71 or 95; or

[0191] The VHH CDR2 comprises, or is composed of, the amino acid sequence of SEQ ID NO:72, or comprises, an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:72; or

[0192] The VHH CDR3 comprises or is composed of an amino acid sequence selected from any of the amino acid sequences shown in SEQ ID NO:73, or the VHH CDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from any of the amino acid sequences shown in SEQ ID NO:73.

[0193] In some embodiments, the anti-CDH17 VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-CDH17 VHH of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0194] (i) The VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:52, the VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:53, and the VHH CDR3 comprises or is composed of the amino acid sequences shown in SEQ ID NO:54-60; or

[0195] (ii) The VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:71, the VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:72, and the VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:73; or

[0196] (iii) The VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:95, the VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:72, and the VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:73.

[0197] In some embodiments, the anti-CDH17 VHH antibody of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region

[0198] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 36-51 or 61-70; or

[0199] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 36-51 or 61-70; or

[0200] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any one of SEQ ID NO:36-51 or 61-70, preferably, the amino acid changes do not occur in the CDR region.

[0201] In some embodiments, the anti-CDH17 VHH antibody of the present invention comprises or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:36-51 or 61-70.

[0202] In some embodiments, the anti-CDH17 VHH antibody of the present invention comprises a CDR amino acid sequence and / or a framework (FR) amino acid sequence derived from a camel heavy chain antibody produced by immunizing a camel (e.g., an alpaca). In some embodiments, the VHH monoclonal antibody of the present invention derived from a camel heavy chain antibody can be engineered, for example, to comprise a framework region sequence derived from a human amino acid sequence (i.e., a human antibody) or other non-camel mammal species. Therefore, in one embodiment, the VHH antibody of the present invention is a chimeric antibody.

[0203] In one embodiment, the anti-CDH17 VHH antibody of the present invention is a humanized antibody. For example, the original VHH sequence is humanized using a "best-matching" method, which includes...

[0204] (i) The amino acid sequence of the VHH framework region was compared and analyzed using the human pedigree V gene database to select the best pedigree sequence;

[0205] (ii) The best-matching human CDR sequence is replaced with a VHH CDR sequence, and optionally multiple residues in the mutated frame region are removed by post-translational modification (PTM) to generate a humanized VHH sequence;

[0206] (iii) Optionally, the immunogenicity of the humanized VHH sequence can be analyzed using software such as WeMol, and mutations that reduce immunogenicity can be introduced at specific residues;

[0207] (iv) Optional sequencing of VHH antibodies.

[0208] Typically, humanization is performed in a manner that preserves the favorable binding properties of single-domain antibodies. Assays for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art, in order to identify and select suitable mutations or combinations of humanized residues.

[0209] In another aspect of the invention, the invention also provides a heavy chain antibody comprising the heavy chain variable region of the anti-CDH17 VHH antibody of the invention.

[0210] In some embodiments, the anti-CDH17 single-domain antibody or VHH (e.g., camel-derived VHH or its humanized form) of the present invention can be linked to a constant region or a portion thereof, such as the Fc region, of a human antibody to produce a heavy chain antibody comprising a VHH-constant region, VHH-CH1-Fc, or VHH-Fc. In one embodiment, the heavy chain antibody comprises the VHH antibody of the present invention and an Fc region located at its C-terminus.

[0211] In one embodiment, the anti-CDH17 heavy chain antibody comprises the Fc portion derived from a camel (e.g., an alpaca). In one embodiment, the heavy chain antibody is generated and isolated by immunizing the camel, such as an alpaca. Various methods are known in the art for immunizing camel animals and isolating VHH antibodies or heavy chain antibodies against the target antigen.

[0212] In some embodiments, the anti-CDH17 heavy chain antibody contains a constant region derived from human or non-human primate (e.g., cynomolgus monkey) antibodies, such as a constant region derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0213] In some embodiments, the anti-CDH17 heavy chain antibody comprises an Fc region derived from a human or non-human primate (e.g., a cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, such as a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region. In some embodiments, the Fc region linked to the VHH antibody comprises the entire hinge region, such as the hinge region shown in SEQ ID NO:90. In some embodiments, the Fc region linked to the VHH antibody comprises a partial hinge region, such as the hinge region shown in SEQ ID NO:93. In some embodiments, the Fc region linked to the VHH antibody comprises the entire hinge region containing the C220S mutation, such as the hinge region shown in SEQ ID NO:89.

[0214] In one embodiment, the heavy chain antibody that specifically binds to CDH17 according to the invention can dimerize with another polypeptide chain (e.g., another heavy chain antibody, the same or different) containing the Fc region via the Fc region. Therefore, in one embodiment, the invention also provides homologous or heteromeric proteins comprising the heavy chain antibody of the invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0215] The Fc regions described herein are all applicable to the heavy chain antibodies of this invention that specifically bind to CDH17.

[0216] In some embodiments, the Fc region is derived from IgG1 and comprises the entire hinge region, containing or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 85 or 86. In some embodiments, the Fc region is derived from IgG1, comprises the entire hinge region, and contains a C220S mutation, containing or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91 or 92.

[0217] The single-domain antibody VHH that specifically binds to CDH17, or a heavy-chain antibody containing it, such as VHH-Fc, may have one or more of the following properties:

[0218] (i) It binds to CDH17-expressing cells, such as tumor cells, with high affinity;

[0219] (ii) exhibits species cross-reactivity with cynomolgus monkey CDH17; and / or

[0220] (iii) Having endocytic activity on CDH17-positive cells, for example, antibodies with higher levels of known CDH17.

[0221] In some embodiments, the single-domain antibody VHH that specifically binds to CDH17, or a heavy-chain antibody containing it, such as VHH-Fc, specifically binds to human CHD17 or cynomolgus monkey CDH17. In some embodiments, the antibody binds to human CDH17 at a Kc of less than or equal to about 600 nM. D Specifically binds to human CDH17, such as the K D Less than or equal to approximately 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, or 6 nM. In some embodiments, the antibody specifically binds to K+, a component of human CDH17. D Greater than or equal to approximately 0.5 nM or 1 nM. In some embodiments, the antibody specifically binds to K+, a precursor of human CDH17. D Within any of the above values. The binding affinity between the antibody and its antigen can be determined by SPR, for example as described in the examples, particularly the materials and methods.

[0222] In some aspects, the CDH17-specific single-domain antibody VHH of the present invention, or a heavy-chain antibody containing it such as VHH-Fc, exhibits high binding affinity to CDH17-expressing tumor cells. The cell-binding affinity of the antibody to CDH17-positive tumor cells can be reflected by FACS or ELSA assays (e.g., the assays described in the examples).

[0223] In some embodiments, the multispecific antibody of the present invention exhibits cross-reactivity with human and monkey CDH17.

[0224] In some embodiments, the multispecific antibody of the present invention possesses CDH17-mediated endocytic activity. The endocytic activity of the antibody can be evaluated in cell-based assays, such as those described in the examples. Exemplary endocytosis assays are described in the examples, particularly in the materials and methods described.

[0225] In some embodiments, after one-step purification of the antibody product generated from recombinant mammalian cells using protein A affinity chromatography, the multispecific antibody of the present invention can achieve a purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher, as determined by SEC-HPLC.

[0226] III. The multispecific antibody of the present invention

[0227] In another aspect, the antigen-binding molecule of the present invention is a multispecific antibody that specifically binds to CLDN18.2 and CDH17, wherein the antibody comprises at least one antigen-binding domain specifically binding to CLDN18.2 and at least one antigen-binding domain specifically binding to CDH17. In some aspects, to increase the half-life of the antibody of the present invention in animals, the antibody of the present invention also comprises other domains, such as immunoglobulin Fc regions, for antibody dimerization and / or half-life extension. The antibody of the present invention can take any suitable form, such as an IgG-like multispecific antibody comprising two or more polypeptide chains, wherein the domains located on the same chain are linked by a linker or directly as needed.

[0228] In some embodiments, the present invention relates to a multispecific antibody comprising

[0229] (i) Specifically binds to the antigen-binding domain of CLDN18.2; and

[0230] (ii) Specifically binds to the antigen-binding domain of CDH17.

[0231] In some embodiments, the valence (i.e., the total number of antigen-binding domains) of the multispecific antibody according to the invention is quadrivalent.

[0232] In some embodiments, the multispecific antibody comprises two antigen-binding domains, such as VHH, that specifically bind to CLDN18.2 and two antigen-binding domains, such as VHH, that specifically bind to CDH17. Preferably, the two antigen-binding domains that specifically bind to CLDN18.2 are the same, and / or the two antigen-binding domains that specifically bind to CDH17 are the same.

[0233] In some embodiments, the multispecific antibody of the present invention is a bispecific antibody that specifically binds to CLDN18.2 and specifically binds to CDH17.

[0234] The components of the multispecific antibody of the present invention are described in detail below. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of this invention. Furthermore, those skilled in the art will understand that, unless the context clearly indicates otherwise, the antibody of the present invention (including any form of antibody) may contain any such combination of features.

[0235] Specifically binds to the CLDN18.2 antigen-binding domain

[0236] In some embodiments of the multispecific antibody according to the present invention, preferably, the antigen-binding domain that specifically binds to CLDN18.2 comprises or is composed of an anti-CLDN18.2 antibody or its antigen-binding fragment, as long as it can specifically bind to CLDN18.2, including but not limited to, for example, full-length antibodies, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy chain antibodies that specifically bind to CLDN18.2.

[0237] In some embodiments, the antigen-binding domain that specifically binds to CLDN18.2 is the anti-CLDN18.2 VHH (VHH) as defined herein (e.g., Part I, “CLDN18.2 Single-Domain Antibodies and Their Heavy Chain Antibodies”). CLDN18.2 ).

[0238] Specifically binds to the CDH17 antigen-binding domain

[0239] In some embodiments of the multispecific antibody according to the present invention, preferably, the antigen-binding domain that specifically binds to CDH17 comprises or is composed of an anti-CDH17 antibody or its antigen-binding fragment, as long as it can specifically bind to CDH17, including but not limited to, for example, full-length antibodies that specifically bind to CDH17, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy chain antibodies, etc.

[0240] In some embodiments, the antigen-binding domain that specifically binds to CDH17 is the anti-CDH17 VHH (VHH) as defined herein (e.g., Part II, “CDH17 Single-Domain Antibodies and Their Heavy Chain Antibodies”). CDH17 ).

[0241] Immunoglobulin Fc region

[0242] In some embodiments, multispecific antibodies may comprise immunoglobulin Fc regions. The immunoglobulin Fc region can be any immunoglobulin Fc region. The Fc region is the C-terminal constant domain of an immunoglobulin that interacts with cell surface Fc receptors and some proteins of the complement system. Immunoglobulin Fc regions typically contain two or three heavy-chain constant domains (designated CH2, CH3, and CH4) and a hinge region, and are typically present in a dimerized form. The two chains in a dimerized Fc region can be linked by disulfide bonds within the hinge region. In some embodiments, Fc regions from immunoglobulin isotypes IgG1, IgG2, and IgG4 are capable of binding to FcRn receptors and undergoing FcRn-mediated recycling to provide a long circulating half-life. The interaction site between IgG and FcRn has been identified in the Fc region of the covered CH2 and CH3 domains.

[0243] The immunoglobulin Fc region used in the multispecific antibody of the present invention can be derived from any immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region includes at least an immunoglobulin CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin Fc region also includes all or part of a hinge region, for example, a partial hinge region being the amino acid sequence shown in SEQ ID NO:93, or a complete hinge region being the amino acid sequence shown in SEQ ID NO:90. In some embodiments, the immunoglobulin Fc region, from the N-terminus to the C-terminus, includes the complete or partial hinge region of immunoglobulin, the CH2 domain, and the CH3 domain, or is composed of thereof. In some embodiments, the immunoglobulin Fc region, from the N-terminus to the C-terminus, includes the CH2 domain and the CH3 domain, or is composed of thereof. In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, or IgG4, or a subtype thereof. Preferably, the immunoglobulin Fc region includes an Fc region sequence derived from humans.

[0244] The immunoglobulin Fc region can fuse to other domains (i.e., the CLDN18.2 or CDH17 binding domain, such as VHH). CLDN18.2 or VHH CDH17 Immunoglobulin Fc is located at the C or N terminus of the protein. It can fuse to other domains via adaptors or directly to other domains.

[0245] In some cases, immunoglobulin Fc regions containing hinge region sequences are preferred, which can, for example, promote the dimerization of antibody polypeptide chains and / or provide cysteine ​​residues for coupling with other active molecules. Such hinge sequences may substantially or partially correspond to the hinge regions of IgG1, IgG2, IgG3, or IgG4. For example, the hinge region sequence may comprise all or part of a core hinge region and all or part of a lower hinge region. The core hinge region has the amino acid sequence CPPC in IgG1, IgG2, and IgG3, and the CPSC sequence in IgG4. Preferably, the hinge region contains at least one disulfide bond connecting two Fc chains. In some embodiments, the hinge region sequence comprises the hinge region sequence from E216 to P230 of IgG1 (e.g., the amino acid sequence shown in SEQ ID NO:90) or the hinge region sequence from D221 to P230 (e.g., the amino acid sequence shown in SEQ ID NO:93) (according to EU numbers), or a corresponding hinge region sequence from other immunoglobulin isotypes. In this document, when referring to a complete hinge region, it generally refers to the hinge region sequence corresponding to E216 to P230 of IgG1 (according to EU numbering), for example, the complete hinge region of the IgG1 Fc region is the amino acid sequence shown in SEQ ID NO:90. In this document, when referring to a partial hinge region, it generally refers to the hinge region sequence corresponding to D221 to P230 of IgG1 (according to EU numbering), for example, the partial hinge region of the IgG1 Fc region is the amino acid sequence shown in SEQ ID NO:93. In some embodiments, when the Fc region of an immunoglobulin is connected to a VHH domain, such as its C-terminus, via its N-terminus, preferably the Fc region comprises the entire hinge region sequence, for example, comprising the hinge region sequence from E216 to T225 of IgG1 (e.g., the amino acid sequence shown in SEQ ID NO:90).

[0246] In some embodiments, the Fc region is a human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of an amino acid sequence SEQ ID NO:85 or SEQ ID NO:86 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with said amino acid sequence.

[0247] The immunoglobulin Fc region of the multispecific antibody used in this invention can be the natural Fc region sequence. Alternatively, the Fc region can contain mutations relative to the natural Fc sequence. Mutations include substitutions, insertions, and / or deletions. Such mutations can be made for the purpose of introducing desired therapeutic properties.

[0248] In some embodiments, the Fc region may have a cysteine ​​residue mutated at position 220, for example, to serine. In some embodiments, the amino acid sequence of the hinge region containing C220S is as shown in SEQ ID NO:89. In some embodiments, the Fc region contains the hinge region shown in SEQ ID NO:89. In some embodiments, the Fc region containing 220S contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:91 or 92. In some embodiments, the Fc region containing 220S contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:91 or 92 and contains the hinge region shown in SEQ ID NO:89. In some embodiments, the Fc region containing 220S contains or is composed of the amino acid sequence shown in SEQ ID NO:91 or 92.

[0249] For example, the amino acid sequence of the hinge region of the Fc region can retain native cysteine, such as the native cysteine ​​at position 220, for use in constructing disulfide bonds or in adding cysteine ​​coupling sites to improve the DAR of the ADC using the antibody. In some embodiments, the amino acid at position 220 of the hinge region is cysteine.

[0250] In some embodiments, if the multispecific antibody comprises dissimilar chains, such as an asymmetric structure, a Knob-into-Hole (KiH) mutation can be introduced into the CH3 domain to promote heterodimerization. This technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996); and Carter, J Immunol Meth 248,7-15 (2001). In this case, one Fc chain is designed to contain a large protruding residue (i.e., Knob), while the other Fc chain is designed to contain a complementary pocket (i.e., Hole). Suitable locations for the KiH mutation are known in the art.

[0251] Furthermore, depending on the specific application of the antibody or antibody-based molecule, the Fc region may also contain mutations that alter effector function. For example, where effector function is not required, the Fc region may contain mutations that reduce or eliminate effector function. In some cases (e.g., when the antibody of the present invention is used as an ADC carrier), preferably, the Fc region contains mutations that reduce or eliminate the interaction between the Fc region and the Fcγ receptor, such as the LALA mutation (L234A / L235A) where lysine (L) at positions 234 and 235 of the Fc region is replaced with alanine (A), to reduce Fcγ receptor-mediated off-target cytotoxicity. Alternatively or additionally, mutations may be introduced into the Fc region to increase binding to FcRn and / or remove protease sites, and / or introduce amino acid modifications that can be used for conjugation to active molecules. Alternatively or additionally, the Fc region may be mutated for antibody production purposes, for example, by removing or replacing amino acids that may undergo post-translational modifications (e.g., glycosylation), to provide improved drugability and developability of the therapeutic antibody.

[0252] Therefore, in a specific embodiment, the multispecific antibody of the present invention comprises an Fc region containing a mutation that reduces or eliminates the Fc region and the Fcγ receptor, such as the LALA mutation. For example, the Fc region containing the LALA mutation contains an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 87 or 88, such as containing or consisting of the amino acid sequence shown in SEQ ID NO: 87 or 88.

[0253] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises an Fc region containing a mutation that reduces or eliminates the Fc region and the Fcγ receptor, such as the LALA mutation, and retains a cysteine ​​residue at position 220 in the hinge region. For example, the Fc region containing the LALA mutation and the cysteine ​​residue at position 220 contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:88, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:88.

[0254] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises an Fc region containing a mutation that reduces or eliminates the Fc region and the Fcγ receptor, such as the LALA mutation, and a mutation at position 220 of the hinge region to a serine residue. For example, the Fc region containing the LALA mutation and C220S contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:87, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:87.

[0255] In one embodiment, the Fc region of the multispecific antibody suitable for use in this invention comprises or is composed of the amino acid sequence shown in SEQ ID NO:87. In some embodiments, the Fc region of the multispecific antibody suitable for use in this invention comprises or is composed of the amino acid sequence shown in SEQ ID NO:88.

[0256] In one embodiment, the multispecific antibody of the present invention comprises two identical Fc regions, and each Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO:87 or SEQ ID NO:88.

[0257] connector

[0258] In the multispecific antibody according to the invention, antibody components (e.g., antigen-binding domain and Fc region) can be connected by adapters.

[0259] There are no specific limitations on the linkers that can be used in the antibodies of this invention. Linker sequences are generally flexible. They can consist primarily of amino acids with large side chains, such as glycine, alanine, and serine, which do not have the large side chains that might limit flexibility. Alternatively, they can consist of sequences derived from the hinge region of immunoglobulins. Depending on the attachment site and the component to be attached, those skilled in the art can readily determine the available linker sequence or optimal length.

[0260] Suitable linker lengths can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, or longer. In some cases, the linker sequence length can be shorter, for example less than about 20 or 15 amino acids, such as 2–15 amino acids or 5–10 amino acids.

[0261] Suitable connector sequences include, but are not limited to, G4S; (G4S)2; (G4S)3; GGGSG; GGSGG; GSGGG; GSGGGP; GGEPS; GGEGGGP and GGEGGGSEGGGS; and (G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; TS(G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; G(G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; (G4)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; (GRPGS)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5. The linkers that can be used for the antibody molecules of this invention can also be, for example, but not limited to, the following amino acid sequences: (G3S)2, (G4S)2, (G3S)3, (G4S)3, (G3S)4, (G4S)4, (G3S)5, (G4S)5, (G3S)6, (G4S), GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP, and GSTGSGSGKPGSGEGSTKG. Alternatively, suitable flexible linker peptides can be rationally designed by simulating the three-dimensional structure of proteins and peptides using computer programs or by using phage display methods.

[0262] In some embodiments, the linker used in the antibody of the present invention is a flexible linker peptide of 5-50 amino acids, preferably comprising a linker peptide with glycine (G) and / or serine (S) and / or threonine residues (T). In one embodiment, the linker has a length of 5-50 amino acids, for example, 5, 10, 15, 20, 25, or 30 amino acids, or an amino acid length falling between any two integers. In some embodiments, the linker comprises an amino acid sequence (G4S). n , where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7.

[0263] In some preferred embodiments, in the multispecific antibody according to the invention, the linker for connecting the antigen-binding domain comprises the amino acid sequence G4S or (G4S)2 or (G4S)3.

[0264] Structure and examples of multispecific antibodies

[0265] In some implementations, the multispecific antibody is a bispecific antibody with a “2+2” symmetrical structure, such as any of the structures shown in the Format schematic diagram in Figure 5.

[0266] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to CLDN18.2 and CDH17, and comprises or consists of the following polypeptide chains from the N-terminus to the C-terminus:

[0267] VHH A -VHH B -Fc, Equation (X)

[0268] VHH A and VHH B The symbols represent the VHH domains that bind antigens A and B, respectively, wherein A and B are distinct from each other and independently selected from CLDN18.2 and CDH17; wherein the symbol "-" indicates linkage via a linker or direct linkage, preferably representing a linker of 5-15 amino acids in length.

[0269] In some embodiments, the bispecific antibody comprises, or is composed of, two polypeptide chains as shown above, wherein the two polypeptide chains are the same or different.

[0270] In some embodiments, the bispecific antibody comprises, or is composed of, two polypeptide chains of formula (X), wherein the two polypeptide chains are the same or different.

[0271] In some specific embodiments, the bispecific antibody that specifically binds to CLDN18.2 and CDH17 comprises, or is composed of, two polypeptide chains as shown below, wherein the polypeptide chains, from the N-terminus to the C-terminus, respectively comprise or consist of the following: VHH CLDN18.2 -VHH CDH17 -Fc, Equation (X-1)

[0272] VHH CLDN18.2 It specifically binds to the VHH domain of CLDN18.2, as described in sections "I. CLDN18.2 Single-Domain Antibodies and Their Heavy Chain Antibodies" or "III. CLDN18.2 Antigen-Binding Structures Specifically Binding to CLDN18.2"; and VHH CDH17It specifically binds to the VHH domain of CDH17, as described in sections “II. MSLN single-domain antibodies and their heavy chain antibodies” or “III. CDH17 antigen-binding structures specifically binding to CDH17”.

[0273] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker with a length of 5-15 amino acids.

[0274] In some specific embodiments, the bispecific antibody that specifically binds to CLDN18.2 and CDH17 comprises, or is composed of, two polypeptide chains as shown below, wherein the polypeptide chains, from the N-terminus to the C-terminus, respectively comprise or consist of the following: VHH CDH17 -VHH CLDN18.2 -Fc, Equation (X-2)

[0275] The symbol "-" indicates a connection via a connector or a direct connection, preferably a connector with a length of 5-15 amino acids.

[0276] In the above-mentioned multispecific antibodies, preferably, each antigen-binding domain on the polypeptide chain (e.g., VHH) CDH17 Domain and VHH CLDN18.2 The domains are connected by a linker. Preferably, the linker has a length of 5-15 amino acids. More preferably, when VHH CDH17 Domain and VHH CLDN18.2 When connecting structural domains, such as VHH CDH17 The C-terminus of the structural domain and VHH CLDN18.2 When the N-terminus of the structural domain is connected, or VHH CLDN18.2 The C-terminus of the structural domain and VHH CDH17 When the N-terminus of the structural domain is connected, the linker preferably contains a G4S amino acid sequence or a (G4S)3 amino acid sequence. More preferably, when VHH CDH17 Domain or VHH CLDN18.2 When a structural domain is connected to the Fc region, for example, VHH CDH17 Domain or VHH CLDN18.2 When the C-terminus of the structural domain is directly connected to the N-terminus of the Fc region, the Fc region contains a hinge region, such as a complete hinge region (e.g., a hinge region containing the amino acid sequence shown in SEQ ID NO: 89 or 90 or composed thereof) or a partial hinge region (e.g., a hinge region containing the amino acid sequence shown in SEQ ID NO: 93 or composed thereof). More preferably, when VHH CDH17 Domain or VHH CLDN18.2 When the structural domain is connected to the Fc region, VHH CDH17 Domain or VHH CLDN18.2The C-terminus of the structural domain is directly connected to the N-terminus of the Fc region, which contains the complete hinge region.

[0277] Two immunoglobulin Fc regions can associate to form a homodimer through dimerization. In some embodiments, the Fc region contains an amino acid sequence from human IgG1, IgG2, or IgG4. In some embodiments, the Fc region contains a mutation that reduces or eliminates Fcγ receptor binding, such as the LALA mutation. In some embodiments, the Fc region retains a cysteine ​​residue at position 220 of the hinge region. In some embodiments, the Fc region contains an LALA mutation and retains a cysteine ​​residue at position 220 of the hinge region.

[0278] In some embodiments, the bispecific antibody comprises or is composed of two identical polypeptide chains, wherein the polypeptide chains comprise or are composed of the amino acid sequence shown in any one of SEQ ID NO:74-82, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with it.

[0279] Characteristics of the multispecific antibody of this invention

[0280] The multispecific antibodies of this invention may have one or more of the following properties:

[0281] (i) Specific binding to tumor cells expressing CLDN18.2 and / or CDH17 antigens, particularly with high affinity binding to tumor cells co-expressing CLDN18.2 and CDH17 antigens, such as with synergistic binding to tumor cells co-expressing CLDN18.2 and CDH17 antigens;

[0282] (ii) It exhibits species cross-reactivity with cynomolgus monkeys CLDN18.2 and CDH17;

[0283] (iii) Possessing CLDN18.2 and / or CDH17-mediated endocytic activity, particularly specifically binding synergistically to tumor cells that co-express or overexpress CLDN18.2 and CDH17, such as tumor cells that highly express CDH17 and tumor cells that express (e.g., low or high) CLDN18.2; and / or

[0284] (iv) Its molecular weight is only about 70% of that of conventional four-chain antibodies, thereby achieving a higher proportion of antibody molecules enriched in tumor tissue, which further improves the therapeutic effect.

[0285] In some aspects, the multispecific antibodies of the present invention exhibit high binding affinity to cells expressing CLDN18.2 and / or CDH17 antigens, such as tumor cells. The cell-binding affinity of the antibodies to CLDN18.2 and / or CDH17-positive tumor cells can be determined by FACS or ELSA assays (e.g., the assays described in the examples), and optionally compared with a reference antibody. Exemplary endocytosis assays are described in the examples, particularly in the materials and methods described.

[0286] In some embodiments, the multispecific antibody of the present invention is capable of specifically binding to CLDN18.2 and CDH17, such as human or cynomolgus monkey CLDN18.2 and CDH17. The binding affinity of the antibody to its antigen can be detected by SPR assay, for example as described in the examples, particularly the materials and methods.

[0287] In some embodiments, the multispecific antibodies of the present invention exhibit cross-reactivity with human and monkey CLDN18.2 and / or CDH17. In some embodiments, the KD value of the antibody of the present invention binding to human or cynomolgus monkey CDH17 is approximately equivalent to the KD value of the antibody binding to monkey CDH17, for example, the ratio of the two KD values ​​is between 1 and 10, for example between 1 and 5, approximately between 1 and 4, or approximately between 1 and 3.

[0288] In some embodiments, the multispecific antibody of the present invention specifically binds to CLDN18.2 and CDH17 positive tumor cells with high affinity (synergistic effect).

[0289] In some embodiments, the bispecific antibody of the present invention that specifically binds to CLDN18.2 and CDH17 has a synergistic effect with the binding of the CLDN18.2 target and the CDH17 target, i.e., higher binding to CLDN18.2 positive and CDH17 positive tumor cells.

[0290] In some embodiments, the multispecific antibodies of the present invention are capable of being internalized by cells expressing CLDN18.2 and / or CDH17. In particular, the multispecific antibodies of the present invention exhibit greater endocytic activity, such as co-endocytic activity, in CLDN18.2-positive and CDH17-positive cells, such as CLDN18.2-positive and CDH17-positive tumor cells. The endocytic activity of the antibodies can be evaluated in cell-based assays, such as those described in the embodiments. Exemplary methods are described in the embodiments, particularly in the materials and methods.

[0291] IV. Other antigen-binding molecules

[0292] The antigen-binding molecule of the present invention can also be a TCR molecule or a CAR molecule for use in TCR therapy or CAR therapy. Methods for constructing TCR or CAR molecules using antigen-binding domains are known in the art.

[0293] V. Production and purification of the antibodies of this invention

[0294] In another aspect, the present invention provides a method for producing the antigen-binding molecules of the present invention, such as antibodies. To produce the antigen-binding molecules of the present invention, such as antibodies, polypeptide chains of the antigen-binding molecules of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production, and then assembled under suitable conditions.

[0295] For recombinant production, polynucleotides encoding any one or more polypeptide chains of the antigen-binding molecule, such as an antibody, can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. The polynucleotides can be easily isolated and sequenced using conventional methods. In one embodiment, polynucleotides encoding one or more polypeptide chains of the antigen-binding molecule, such as an antibody, of the present invention are provided. In yet another embodiment, the present invention provides vectors comprising one or more polynucleotides of the present invention, preferably expression vectors. Thus, in one embodiment, the present invention provides a method for producing the antigen-binding molecule, such as an antibody, of the present invention, the method comprising: culturing host cells containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the antigen-binding molecule, such as an antibody; and assembling the polypeptide chain to produce the antigen-binding molecule, such as an antibody, under conditions suitable for assembling the polypeptide chain into the antigen-binding molecule, such as an antibody.

[0296] Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YACs). Preferably, the expression vector is pCDNA, such as pCDNA3.4.

[0297] In one embodiment, the present invention also provides a host cell comprising one or more of the polynucleotides of the present invention. In some embodiments, a host cell comprising the expression vector of the present invention is provided. Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63, and Sp2 / O, etc. In a preferred embodiment, the host cell is a CHO or HEK293 cell.

[0298] Antigen-binding molecules, such as antibodies, prepared by the methods described herein can be purified using known techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. After purification, the purity of the antigen-binding molecules, such as antibodies, of the present invention can be determined using any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC. The physical / chemical properties and / or biological activity of the antibodies provided herein can be identified, screened, or characterized using a variety of assays known in the art.

[0299] In a preferred embodiment, the antigen-binding molecules of the present invention, such as antibodies, exhibit good production properties when recombinantly produced in mammalian host cells, such as CHO cells, especially good expression yield and a good byproduct profile.

[0300] II. Immunofusions and Immunoconjugates

[0301] In one aspect, the present invention provides immunofusions or immunoconjugates produced by fusing or conjugating the antigen-binding molecule of the present invention to a heterologous molecule.

[0302] In one embodiment, in the immunofusion, the antigen-binding molecule of the present invention, such as an antibody (or an antigen-binding fragment thereof), is linked to a heterologous peptide or polypeptide molecule directly or via an amino acid linker. Heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that impart another functional activity to the fusion, or tagged peptides that facilitate the purification or detection of the immunofusion.

[0303] In one embodiment, in the immunoconjugate, the antigen-binding molecule of the present invention, such as an antibody (or an antigen-binding fragment thereof), is conjugated to a therapeutic agent, diagnostic agent, or detectable agent. In the conjugate, linkers can be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a "cleavable linker" that facilitates the release of the polypeptide upon delivery to the target site. For example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used.

[0304] In embodiments where a therapeutic agent is conjugated, the therapeutic agents suitable for the conjugation include, but are not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), cell agonists, protein degraders, pharmaceuticals, or radioisotopes.

[0305] In embodiments conjugated with diagnostic or detectable agents, such conjugates can be used as part of clinical testing methods (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnostics and detections can be achieved by conjugating antibodies to detectable agents, including but not limited to a variety of enzymes such as horseradish peroxidase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0306] In some preferred embodiments, the immunoconjugate according to the invention is an antibody-drug conjugate (ADC).

[0307] In some embodiments, the present invention provides an antibody-drug conjugate having formula (I): Ab-(LD) p (I)

[0308] Or its pharmaceutically acceptable salts or solvates,

[0309] in:

[0310] Ab is an antigen-binding molecule as defined above, such as an antibody or fragment thereof (e.g., an antigen-binding fragment) that specifically binds to CLDN18.2 and / or CDH17 (e.g., human CLDN18.2 and / or human CDH17);

[0311] L is the connector;

[0312] D is a drug, such as an anti-tumor compound; and

[0313] p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

[0314] It should be understood that p refers to the number of -LDs linked to Ab in the antibody-drug conjugate molecule of formula (I), also known as DAR. In some embodiments, D in formula (I) of the present invention can be any antitumor compound, as long as it has antitumor effects and has substituents or partial structures that can be linked to the linker structure, preferably small molecule antitumor compounds. In some embodiments, D is a therapeutic agent suitable for the conjugate as described above.

[0315] In some implementations, the antitumor compound may be, for example, a cytotoxic agent, such as a camptothecin compound such as Exatecan, Dxd, or an auristatin compound such as monomethyl auristatin E (MMAE) or MMAF.

[0316] In some implementations, D has the structure shown in formula (D-1a) or formula (D-1b):

[0317] Where R 1a Selected from H and C1-C6 alkyl groups;

[0318] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ;

[0319] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0320] R 4a and R 5a Independently selected from H and C1-C4 alkyl groups;

[0321] Where R 1b R 2b R 3b R 4b R 5b and R 8b Each was independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl;

[0322] R 6b and R7b Each was independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy;

[0323] R 9b Selected from C 1-8 Alkyl groups and COOH; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and

[0324] R 10b Selected from OH and H.

[0325] It should be understood that a key with a wavy line in the structure indicates that the key is connected to another structure or segment.

[0326] In some implementation schemes,

[0327] In some implementation schemes, R 1a For H; R 2a It is a C1-C6 alkyl group; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl.

[0328] In some implementation schemes, R 1b R 4b and R 8b Each was independently selected from C 1-2 Alkyl; preferably methyl;

[0329] R 2b R 3b and R 5b Each was independently selected from C 3-4 alkyl;

[0330] R 6b and R 7b Each was independently selected from C 1-2 alkoxy groups; and

[0331] R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H.

[0332] In some implementations, D has the structure shown in formula (D-2a) or formula (D-2b):

[0333] Where R 1a R 2a R 3a and R 4a As defined above; or

[0334] Where R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined above.

[0335] In some implementations, D has the structure shown in formula (D-3a) or (D-3b):

[0336] In some implementations, the unspecified chiral neutrals are each independently of the R or S configuration.

[0337] In some implementations, D has the structure shown in formula (D-4a) or (D-4b):

[0338] In some implementations, -L- has the following structure: -Z-L1-L2-L3-

[0339] in

[0340] Z is selected from Where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0341] L1 is selected from non-existent, Where n1 and m1 are independently integers selected from 0 to 20, for example, integers selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0342] L2 is a single amino acid or a peptide residue selected from 2-8 amino acids; and

[0343] L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from: C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6.

[0344] It should be understood that in the above -Z-L1-L2-L3-, Z is connected to the S on Ab, and L3 is connected to D. Unless otherwise specified, the divalent groups mentioned herein are connected in the directions shown. That is, the left side of the Z group (maleimide moiety) is connected to Ab, and the right side (e.g., carbonyl group) is connected to L1, and when L1 is absent, it is connected to L2; the left side of L3 (amino group) is connected to L2, and the right side (carbonyl group) is connected to D.

[0345] In some implementation schemes, Z is selected from Where m is 1, 2, 3, 4, 5, 6, 7 or 8.

[0346] In some implementation schemes, Z is selected from

[0347] In some implementations, L1 is selected from non-existent, Where n1 is an integer independently selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0348] In some implementations, L1 is selected from non-existent,

[0349] In some implementations, L2 is a single amino acid or a peptide residue consisting of 2, 3, 4, 5, 6, or 7 amino acids.

[0350] The amino acid in L2 is preferably an L-amino acid. In addition to α-amino acids, it can also be an amino acid with structures such as β-alanine, ε-aminohexanoic acid, and γ-aminobutyric acid. Furthermore, it can also be a non-natural amino acid, such as an N-methylated amino acid. It should be understood that the amino acid in L2 exists as an amino acid residue linked to two end groups.

[0351] The amino acids in L2 can be independently selected from the amino acids defined in the user-defined section, such as phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), glycine (Gly), alanine (Ala), valine (Val), lysine (Lys), citrulline (Cit), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), threonine (Thr), and glutamine (Gln).

[0352] In some implementations, L2 is selected from: -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-, and -Arg-Asp-Val-Thr- (i.e., RDVT).

[0353] In some implementations, L2 is selected from -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, and -Arg-Asp-Val-Thr-;

[0354] It should be understood that L2 is connected to L1 or Z through the amino group of the amino acid on the left, and to L3 through the carbonyl group of the amino acid on the right, which is consistent with the explanation below.

[0355] In some implementations, L3 is Among them, Su and R 1c As defined above ； n2 and n5 are independently 0 or 1; preferably 0.

[0356] In some implementations, L3 is Su is defined as above.

[0357] In some embodiments, Su is selected from xylose, arabinose, xyuronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid, and mannuronic acid.

[0358] In some implementation schemes, Su is selected from

[0359] In some implementation schemes, Su is selected from

[0360] In some implementations, Su is

[0361] In some implementations, Su is

[0362] In some implementations, L3 is selected from:

[0363] It should be understood that L3 is connected to L2 via the amino group on the left and to D via the carbonyl group on the right, which is consistent with the explanation below.

[0364] In some implementations, -Z-L1-L2-L3- is selected from the following structures

[0365] ,

[0366] It should be understood that, unless otherwise specified and without contradiction in the context, for the ADC of the present invention, the left-hand bond of the divalent group shown herein is connected to Ab or a group near the Ab end, and the right-hand bond of the divalent group is connected to D or a group near the D end. For example, when L2 is -Val-Ala-, the amino group on the left side of Val is connected to L1, and the carbonyl group on the right side of Ala is connected to L3;

[0367] In some implementations, Ab is covalently linked to L via a sulfur atom, that is, linked to L via -S-.

[0368] It should be understood that the sulfur (-S-) mentioned above is sulfur derived from Ab.

[0369] In some embodiments, the antibody-drug conjugate is selected from...

[0370] Where Ab is the antigen-binding molecule defined in the text; and p is as defined above.

[0371] In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 3-5, 5-7, 6-9, 6-10, 4-8, 7-9, or 2-4.

[0372] It should be understood that when the maleimide portion of the linker in the ADC is linked to an Ab, it is linked to the Ab through an S atom, which originates from the Ab. The Ab, under the action of a reducing agent such as TCEP, opens disulfide bonds, especially interchain disulfide bonds, to generate a thiol group (-SH), which then links to the terminal functional group of the linker, such as the maleimide portion.

[0373] In some implementations, the S atom attached to Ab comes from the cysteine ​​residue of Ab.

[0374] III. Pharmaceutical Combinations, Drug Conjugates, and Reagent Kits

[0375] In one aspect, the present invention provides compositions, such as pharmaceutical compositions, comprising an antigen-binding molecule, for example, comprising the antigen-binding molecule described herein formulated with a pharmaceutically acceptable excipient. As used herein, "pharmaceutically acceptable excipient" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the molecule of the present invention, such as the antigen-binding molecule of the present invention or the immunoconjugate or immunofusion of the present invention, is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise the molecule of the present invention (encompassing the antigen-binding molecule, immunoconjugate, immunofusion, antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof) with one or more therapeutic agents.

[0376] In another aspect, the present invention also provides a pharmaceutical combination comprising the molecules of the present invention with one or more therapeutic agents.

[0377] The therapeutic agents applicable to the pharmaceutical compositions and combinations thereof of the present invention may be therapeutic agents selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; and (iv) drugs that have antitumor effects.

[0378] The pharmaceutical compositions of the present invention may contain a "therapeutic effective amount" or a "preventive effective amount" of the molecules of the present invention.

[0379] Kits containing the antibodies described herein are also within the scope of this invention. Kits may include one or more other elements, such as: instructions for use; other reagents, such as markers or conjugation agents; pharmaceutically acceptable carriers or excipients; and devices or other materials for administration to a subject.

[0380] IV. Uses and Methods

[0381] Based on the excellent targeting properties of the molecules of the present invention (e.g., the antigen-binding molecules, immunoconjugates, immunofusions, antibody-drug conjugates, or pharmaceutically acceptable salts or solvates thereof) to tumor cells expressing CLDN18.2 and / or CDH17, as well as the other superior properties mentioned above, the present invention also provides the molecules of the present invention and their applications and methods in the treatment and prevention of CLDN18.2 and / or CDH17-related diseases.

[0382] CLDN18.2 and CDH17 are overexpressed in cancer tissues from various sources (e.g., on cell surfaces), and are therefore suitable targets for developing cancer immunotherapies. In one aspect, the present invention provides the use of the molecules of the invention for the prevention and / or treatment of CLDN18.2 and / or CDH17-positive tumors in a subject. In said use, the molecules of the invention may be administered to the subject as the sole active agent or may be administered to the subject in combination with other therapies or therapeutic agents. These other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells to eliminate tumors by binding to and / or blocking these molecules; and drugs that activate the subject's immune system, prompting it to spontaneously eliminate tumors.

[0383] In another aspect, the present invention also provides a method for preventing or treating CLDN18.2 and / or CDH17-related diseases or tumors such as cancer in a subject, comprising administering the molecules of the present invention to a subject in need. In some embodiments, the CLDN18.2 and / or CDH17-related disease is a tumor such as cancer. In some embodiments, the tumor such as cancer is a CLDN18.2 and / or CDH17-positive tumor or cancer, such as a CLDN18.2-positive tumor or cancer, or a CDH17-positive tumor or cancer, or a CLDN18.2-positive and CDH17-positive tumor or cancer.

[0384] The tumors suitable for the methods and applications of this invention can be selected from various solid tumors, such as digestive system cancers like gastric cancer, pancreatic cancer, colorectal cancer, or other tumors like ovarian cancer, head and neck cancer, etc. In some embodiments, the solid tumor is a CLDN18.2 and / or CDH17 positive tumor, particularly a CLDN18.2 and CDH17 positive tumor.

[0385] The CLDN18.2 and / or CDH17 positive tumors suitable for the methods and applications of this invention can be early, intermediate, or late-stage or metastatic cancers. Furthermore, the CLDN18.2 and / or CDH17 positive tumors suitable for the methods and applications of this invention can be tumors that have previously received treatment and have experienced immune escape, drug resistance, or recurrence.

[0386] In some embodiments, CLDN18.2 and / or CDH17 positive tumors suitable for prevention or treatment according to the method of the present invention have overexpression of CLDN18.2 and / or CDH17, for example, overexpression of CLDN18.2 and / or CDH17 in their tumor samples (e.g., tumor cells or tissues), for example, compared to the corresponding tissues or cells of a healthy subject and / or to adjacent healthy tissues or healthy cells of the same subject's tumor tissues or cells.

[0387] In some embodiments, the CLDN18.2 and / or CDH17 positive tumors treated according to the method of the present invention have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% CLDN18.2 and / or CDH17 positive cells. In some embodiments, the CLDN18.2 positive tumors have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% CLDN18.2 positive cells. In some embodiments, CDH17-positive tumors have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% CDH17-positive cells. In some embodiments, CLDN18.2-positive and CDH17-positive tumors have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% CLDN18.2 and CDH17-positive cells. The expression levels of CLDN18.2 and / or CDH17 on tumor biopsies can be assessed by immunohistochemistry. High percentages of CLDN18.2 and / or CDH17-positive cells have been detected in biopsies from various cancers, such as the tumors or cancers described above.

[0388] Preferably, in some embodiments, the method according to the invention is used to treat tumors having a high percentage of CLDN18.2 and / or CDH17 positive cells, for example, tumors having at least 25%, 50%, 75%, or 100% CLDN18.2 and / or CDH17 positive cells. In some embodiments, the tumors having a high percentage of CLDN18.2 positive cells have at least 25%, 50%, 75%, or 100% CLDN18.2 positive cells. In some embodiments, the tumors having a high percentage of CDH17 positive cells have at least 25%, 50%, 75%, or 100% CDH17 positive cells. In some embodiments, the tumors having a high percentage of both CLDN18.2 and CDH17 positive cells have at least 25%, 50%, 75%, or 100% CLDN18.2 and CDH17 double positive cells.

[0389] In some embodiments, the method according to the invention can also be used to treat cancers with a CLDN18.2 and / or CDH17 positive cell percentage of less than 25% or 20%, preferably, the tumor has a CLDN18.2 positive cell percentage of less than 25% or 20% but is a tumor with a high percentage of CDH17 positive cells; or the tumor has a CDH17 positive cell percentage of less than 25% or 20% but is a tumor with a high percentage of CLDN18.2 positive cells. In some embodiments, the tumor has a CLDN18.2 positive cell percentage of less than 25% or 20% and a CDH17 positive cell percentage of less than 25% or 20%.

[0390] In some embodiments, the application of the method of the present invention to said cancer results in tumor growth inhibition. In some embodiments, the application of the method of the present invention to said cancer induces tumor regression.

[0391] In any of the above embodiments of the method of the present invention, the application of the molecules according to the present invention may include 1) a therapeutic measure that cures, slows, alleviates, reduces or stops the progression of a diagnosed pathological condition or disease; or 2) a preventive or preventative measure that prevents and / or slows the development of a pathological condition or disease. Therefore, in the method of the present invention, the subject may be an individual who already suffers from a disease, an individual susceptible to a disease, or an individual wishing to prevent a disease. The individual will benefit from the therapeutic or preventative measures and, compared to an individual who has not received the treatment, exhibit a reduction or improvement in the occurrence, recurrence, or development of the disease, condition, symptom, and / or symptoms. In some embodiments, the present invention relates to the treatment of a disease or condition; in other embodiments, the present invention relates to the prevention of a disease or condition.

[0392] The molecules according to the invention, and optionally other therapeutic agents used in combination therewith, can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Various dosing schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0393] For the prevention or treatment of disease, the appropriate dosage of the molecule according to the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the specific type of drug used, the severity and course of the disease, whether the drug is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0394] On the other hand, the present invention provides molecules of the invention for therapeutic purposes, such as for the prevention and / or prevention of CLDN18.2 and / or CDH17-related diseases as defined herein.

[0395] On the other hand, the present invention provides molecules of the invention that are used as medicines, such as medicines for preventive or therapeutic purposes of the present invention, for example, medicines for treating and / or preventing CLDN18.2 and / or CDH17-related diseases as defined herein.

[0396] On the other hand, the present invention provides the use of the molecules of the present invention for the preparation of medicaments, wherein the medicaments are used for the preventive or therapeutic purposes of the present invention, such as for the treatment and / or prevention of CLDN18.2 and / or CDH17-related diseases as defined herein.

[0397] V. Preparation of the ADC molecule of the present invention

[0398] Another aspect of the present invention provides a method for preparing an ADC of formula (I) of the present invention using the antigen-binding molecule of the present invention. The method includes conjugating the antigen-binding molecule (Ab) of the present invention to a drug (D) via a linker (L).

[0399] In some implementations, the method includes the following steps:

[0400] (a) The antigen-binding molecule (Ab) of the present invention is placed in a buffer solution, a reducing agent and an optional metal salt are added, and the mixture is incubated.

[0401] (b) The linker-loaded component is added to the reaction solution from step (a) for coupling to obtain the crude product; and

[0402] (c) Optionally purify the crude product to obtain the antibody-drug conjugate of the present invention;

[0403] Ab is defined as above.

[0404] In some embodiments, the metal salt is a hydrochloride or sulfate of Zn, Cd, and Hg, preferably ZnCl2.

[0405] In some implementations, the reducing agent in step a) is TCEP.

[0406] In some embodiments, the buffer solution in step a) is a NaOAc-HAc buffer solution, preferably with a pH of 5.0-9.0, for example 5.0-7.0.

[0407] In some implementations, the connector-payload has the following structure: Z'-L1-L2-L3-D, where L1, L2, L3, and D are as defined herein, and Z' is... m is as defined above.

[0408] In some implementations, for the synthesis of Z is The ADC method of formula I also includes an additional hydrolysis step that opens the ring of maleimide.

[0409] In some implementations, the steps are performed under the specific reaction conditions disclosed in the embodiments of the present invention.

[0410] It should be noted that embodiments obtained by varying the range or specific values ​​of the specific reaction conditions disclosed in the embodiments by 100%, 90%, 80%, 70%, 60%, 40%, 20%, or 10% are also considered in this invention. Embodiments obtained by limiting the embodiments described above to the specific conditions and ranges in the embodiments are also included within the scope of this invention.

[0411] It should be understood that, given that the linker-load portion (-LD) in the compound of Formula I is well defined, those skilled in the art can clearly understand the structure of the linker-load used before connection. VI. Specific Implementation Plan

[0412] In some aspects, the present invention relates to the following specific embodiments:

[0413] A1. A VHH antibody that specifically binds to CLDN18.2, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises the three complementarity-determining regions (CDRs) contained in the VH as shown in any one of SEQ ID NO:15, 1-14 and 16-19; preferably, the CDR sequence is defined according to ABM.

[0414] A2. The VHH antibody described in embodiment A1, comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprising or composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0415] (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:21, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:31; or

[0416] (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:20, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:24; or

[0417] (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:21, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of any one of the amino acid sequences shown in SEQ ID NO:24-35; or

[0418] (iv) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:22, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:24.

[0419] A3. The VHH antibody according to embodiment A1 or A2, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 15, 1-14, and 16-19.

[0420] A4. The VHH antibody according to any one of embodiments A1-A3, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from or consisting of any one of SEQ ID NO: 15, 1-14 and 16-19.

[0421] A5. A heavy chain antibody that specifically binds to CLDN18.2, comprising the VHH antibody as described in any one of embodiments A1-A4.

[0422] A6. The heavy chain antibody of embodiment A5, comprising the VHH antibody of any one of embodiments A1-A5 linked to the constant region or Fc region of the antibody.

[0423] A7. The heavy chain antibody of embodiment A6, wherein the constant region or Fc region of the antibody is derived from human IgG1, human IgG2, human IgG3 or human IgG4, preferably from the Fc region of human IgG1 or IgG4.

[0424] A8. A heavy chain antibody according to implementation scheme A6 or A7, wherein the Fc region contains a complete hinge region and is directly linked to the VHH antibody.

[0425] A9. The heavy chain antibody according to any one of embodiments A6-A8, wherein the Fc region is a cysteine ​​residue at position 220 or is mutated to a serine residue at position 220.

[0426] A10, the heavy chain antibody according to any one of embodiments A6-A9, wherein the Fc region comprises the amino acid sequence shown in SEQ ID NO:89 or the amino acid sequence shown in SEQ ID NO:90.

[0427] A11, the heavy chain antibody according to any one of embodiments A6-A10, wherein the Fc region contains an LALA mutation.

[0428] A12, the heavy chain antibody according to any one of embodiments A6-A11, wherein the Fc region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 85 or 86.

[0429] A13, the heavy chain antibody according to any one of embodiments A6-A12, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:85 or 86.

[0430] A14, the heavy chain antibody according to any one of embodiments A6-A13, wherein the Fc region contains a C220S mutation in the hinge region, and the Fc region containing the C220S mutation contains or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91 or 92.

[0431] A15, the heavy chain antibody according to embodiment A14, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 91 or 92.

[0432] A16, the heavy chain antibody according to any one of embodiments A6-A15, wherein the Fc region contains an LALA mutation, and the Fc region containing the LALA mutation contains or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:88.

[0433] A17. The heavy chain antibody according to embodiment A16, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:88.

[0434] A18, the heavy chain antibody according to any one of embodiments A6-A17, wherein the Fc region comprises the LALA mutation and the C220S mutation, and the Fc region comprising the LALA mutation and the C220S mutation comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:87.

[0435] A19. A VHH antibody of any one of embodiments A1-A5, or a heavy chain antibody of any one of embodiments A6-A18, wherein the antibody is a chimeric antibody or a humanized antibody.

[0436] A20. An antigen-binding molecule comprising a VHH antibody of any one of embodiments A1-A5, or a heavy chain antibody of any one of embodiments A6-A18, for example, the antigen-binding molecule being a multispecific antibody such as a bispecific antibody.

[0437] A21. An antigen-binding molecule according to embodiment A20, wherein the antigen-binding molecule specifically binds CLDN18.2 and CDH17, and comprises at least one antigen-binding domain specifically binding CLDN18.2 and at least one antigen-binding domain specifically binding CDH17.

[0438] A22, the antigen-binding molecule of embodiment A21, wherein the antigen-binding domain that specifically binds to CLDN18.2 is a VHH antibody as described in any of embodiments A1-A5.

[0439] A23, the antigen-binding molecule of embodiment A22, wherein the VHH antibody that specifically binds to CLDN18.2 comprises the three complementarity-determining regions (CDRs) contained in the VH shown in SEQ ID NO:15; preferably, the CDR sequence is defined according to ABM.

[0440] A24. An antigen-binding molecule according to embodiment A22 or A23, wherein the VHH antibody that specifically binds to CLDN18.2 comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:21, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:31.

[0441] A25. An antigen-binding molecule of any one of embodiments A22-A24, wherein the VHH antibody that specifically binds to CLDN18.2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15.

[0442] A26. The antigen-binding molecule of embodiment A22, wherein the VHH antibody that specifically binds to CLDN18.2 comprises the three complementarity-determining regions (CDRs) contained in the VH shown in SEQ ID NO:6; preferably, the CDR sequence is defined according to ABM.

[0443] A27. An antigen-binding molecule according to embodiment A22 or A26, wherein the VHH antibody that specifically binds to CLDN18.2 comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:21, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:24.

[0444] A28, an antigen-binding molecule of any one of embodiments A22 and A26-A27, wherein the VHH antibody that specifically binds to CLDN18.2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:6.

[0445] A29. An antigen-binding molecule of any one of embodiments A21-A28, wherein the antigen-binding domain that specifically binds to CDH17 is a VHH antibody as described in any one of embodiments B1-B5.

[0446] A30, the antigen-binding molecule of any one of embodiments A20-A29, wherein the antigen-binding molecule further comprises an Fc region, for example, comprising two identical Fc regions.

[0447] A31. The antigen-binding molecule described in embodiment A30, wherein the Fc region is the Fc region as defined in any one of embodiments A7-A18.

[0448] A32. The antigen-binding molecule according to any one of embodiments A20-A31, wherein the antigen-binding domains are connected to each other or to the Fc region via a connector;

[0449] Preferably, the connector comprises (G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5.

[0450] B1. A VHH antibody that specifically binds to CDH17, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises the three complementarity-determining regions (CDRs) contained in the VH as shown in any one of SEQ ID NO: 50, 36-49, 51 or 61-70; preferably, the CDR sequence is defined according to ABM.

[0451] B2. A VHH antibody that specifically binds to CDH17, comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0452] (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:58; or (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 contains or is composed of any one of the amino acid sequences shown in SEQ ID NO:54-60; or

[0453] (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:71, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:72, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:73; or

[0454] (uv)VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:95, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:72, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:73.

[0455] B3. The VHH antibody according to embodiment B1 or B2, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 50, 36-49, 51, or 61-70.

[0456] B4. The VHH antibody according to any one of embodiments B1-B3, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from or consisting of any one of SEQ ID NO: 50, 36-49, 51 or 61-70.

[0457] B5. A heavy chain antibody that specifically binds to CLDN18.2, comprising the VHH antibody described in any one of embodiments B1-B4.

[0458] B6. The heavy chain antibody of embodiment B5, comprising the VHH antibody of any one of embodiments B1-B5 linked to the constant region or Fc region of the antibody.

[0459] B7. The heavy chain antibody of embodiment B6, wherein the constant region or Fc region of the antibody is derived from human IgG1, human IgG2, human IgG3 or human IgG4, preferably from the Fc region of human IgG1 or IgG4.

[0460] B8. The heavy chain antibody of embodiment B6 or B7, wherein the Fc region contains a complete hinge region and is directly linked to the VHH antibody.

[0461] B9. The heavy chain antibody according to any one of embodiments B6-B8, wherein the Fc region is a cysteine ​​residue at position 220 or is mutated to a serine residue at position 220.

[0462] B10, the heavy chain antibody according to any one of embodiments B6-B9, wherein the Fc region comprises the amino acid sequence shown in SEQ ID NO:89 or the amino acid sequence shown in SEQ ID NO:90.

[0463] B11, the heavy chain antibody according to any one of embodiments B6-B10, wherein the Fc region contains an LALA mutation.

[0464] B12, the heavy chain antibody according to any one of embodiments B6-B11, wherein the Fc region comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 85 or 86.

[0465] B13, the heavy chain antibody according to any one of embodiments B6-B12, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:85 or 86.

[0466] B14. The heavy chain antibody according to any one of embodiments B6-B13, wherein the Fc region contains a C220S mutation in the hinge region, and the Fc region containing the C220S mutation contains or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91 or 92.

[0467] B15. The heavy chain antibody according to embodiment B14, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 91 or 92.

[0468] B16, the heavy chain antibody according to any one of embodiments B6-B15, wherein the Fc region contains an LALA mutation, and the Fc region containing the LALA mutation contains or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:88.

[0469] B17. The heavy chain antibody according to embodiment B16, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:88.

[0470] B18, the heavy chain antibody according to any one of embodiments B6-B17, wherein the Fc region comprises the LALA mutation and the C220S mutation, and the Fc region comprising the LALA mutation and the C220S mutation comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:87.

[0471] B19. A VHH antibody of any one of embodiments B1-B5, or a heavy chain antibody of any one of embodiments B6-B18, wherein the antibody is a chimeric antibody or a humanized antibody.

[0472] B20, an antigen-binding molecule comprising a VHH antibody of any one of embodiments B1-B5, or a heavy chain antibody of any one of embodiments B6-B18, for example, the antigen-binding molecule being a multispecific antibody such as a bispecific antibody.

[0473] B21. The antigen-binding molecule of embodiment B20, wherein the antigen-binding molecule specifically binds CLDN18.2 and CDH17, and comprises at least one antigen-binding domain specifically binding CLDN18.2 and at least one antigen-binding domain specifically binding CDH17.

[0474] B22, the antigen-binding molecule of embodiment B21, wherein the antigen-binding domain that specifically binds to CDH17 is a VHH antibody as described in any of embodiments B1-B5.

[0475] B23, the antigen-binding molecule of embodiment B22, wherein the VHH antibody that specifically binds to CDH17 comprises the three complementarity-determining regions (CDRs) contained in the VH shown in SEQ ID NO:50; preferably, the CDR sequence is defined according to ABM.

[0476] B24. An antigen-binding molecule according to embodiment B22 or B23, wherein the VHH antibody that specifically binds to CLDN18.2 comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:58.

[0477] B25. An antigen-binding molecule of any one of embodiments B22-B24, wherein the VHH antibody that specifically binds to CLDN18.2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:50.

[0478] B26, the antigen-binding molecule of embodiment B22, wherein the VHH antibody that specifically binds to CLDN18.2 comprises the three complementarity-determining regions (CDRs) contained in the VH shown in SEQ ID NO:36; preferably, the CDR sequence is defined according to ABM.

[0479] B27. An antigen-binding molecule according to embodiment B22 or B26, wherein the VHH antibody that specifically binds to CLDN18.2 comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:54.

[0480] B28, an antigen-binding molecule of any one of embodiments B22 and B26-B27, wherein the VHH antibody that specifically binds to CLDN18.2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:36.

[0481] B29, the antigen-binding molecule of embodiment B22, wherein the VHH antibody that specifically binds to CLDN18.2 comprises the three complementarity-determining regions (CDRs) contained in the VH shown in SEQ ID NO:61; preferably, the CDR sequence is defined according to ABM.

[0482] B30, the antigen-binding molecule of embodiment B22 or B29, wherein the VHH antibody that specifically binds to CLDN18.2 comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:71, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:72, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:73.

[0483] B31, an antigen-binding molecule of any one of embodiments B22 and B29-B30, wherein the VHH antibody that specifically binds to CLDN18.2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:61.

[0484] B32. An antigen-binding molecule of any one of embodiments B21-B31, wherein the antigen-binding domain that specifically binds to CLDN18.2 is a VHH antibody as described in any one of embodiments A1-A5.

[0485] B33, the antigen-binding molecule of any one of embodiments B20-B32, wherein the antigen-binding molecule further comprises an Fc region, for example, comprising two identical Fc regions.

[0486] B34. The antigen-binding molecule described in embodiment B33, wherein the Fc region is the Fc region as defined in any one of embodiments B7-B18.

[0487] B35. An antigen-binding molecule according to any one of embodiments B20-B34, wherein the antigen-binding domains are connected to each other or to the Fc region via a connector.

[0488] Preferably, the connector comprises (G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5.

[0489] C1. A multispecific antibody that specifically binds to CLDN18.2 and CDH17, preferably a bispecific antibody, and comprising, or composed of, two polypeptide chains as shown below, wherein the polypeptide chains, from the N-terminus to the C-terminus, respectively comprise or consist of the following: VHH CLDN18.2 -VHH CDH17 -Fc, Equation (X-1)

[0490] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker with a length of 5-15 amino acids.

[0491] C2. A multispecific antibody that specifically binds to CLDN18.2 and CDH17, preferably a bispecific antibody, and comprising, or composed of, two polypeptide chains as shown below, wherein the polypeptide chains, from the N-terminus to the C-terminus, respectively comprise or consist of the following: VHH CDH17 -VHH CLDN18.2 -Fc, Equation (X-2)

[0492] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker with a length of 5-15 amino acids.

[0493] C3. The multispecific antibody described in implementation scheme C1 or C2, wherein VHH CLDN18.2 It is the VHH antibody as described in any one of embodiments A1-A5.

[0494] C4. The multispecific antibody according to any one of implementation schemes C1-C3, wherein VHH CDH17 It is the VHH antibody as described in any one of embodiments B1-B5.

[0495] C5. A multispecific antibody according to any one of embodiments C1-C4, wherein Fc is the Fc region as defined in any one of embodiments A7-A18 or B7-B18.

[0496] C6. A multispecific antibody according to any one of embodiments C1-C5, wherein the Fc region contains a complete hinge region and is a cysteine ​​residue at position 220 or a serine residue mutated at position 220.

[0497] C7. The multispecific antibody according to any one of C1-C6 in the implementation plan, wherein the Fc region contains the LALA mutation.

[0498] C8. A multispecific antibody according to any one of embodiments C1-C7, wherein the Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:87.

[0499] C9. The multispecific antibody according to any one of embodiments C1-C8, wherein the Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:88.

[0500] C10, the multispecific antibody according to any one of embodiments C1-C9, wherein VHH CDH17 Domain and VHH CLDN18.2 The structural domains are connected by joints.

[0501] C11. A multispecific antibody according to any one of embodiments C1-C10, wherein when VHH CDH17 Domain and VHH CLDN18.2 When connecting structural domains, such as VHH CDH17 The C-terminus of the structural domain and VHH CLDN18.2 When the N-terminus of the structural domain is connected, or VHH CLDN18.2 The C-terminus of the structural domain and VHH CDH17 When the N-terminus of the structural domain is connected, the linker preferably contains a G4S amino acid sequence or a (G4S)3 amino acid sequence.

[0502] C12, the multispecific antibody according to any one of embodiments C1-C11, wherein the VHH CDH17Domain or VHH CLDN18.2 The C-terminus of the structural domain is directly connected to the N-terminus of the Fc region.

[0503] C13. The multispecific antibody according to any one of embodiments C1-C12, wherein the VHH CLDN18.2 It includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:15; preferably, the CDR sequence is defined according to ABM.

[0504] C14. The multispecific antibody according to any one of embodiments C1-C13, wherein the VHH CLDN18.2 It contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of heavy chain variable regions, wherein the heavy chain variable regions contain complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:21, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:31.

[0505] C15. The multispecific antibody according to any one of embodiments C1-C14, wherein the VHH CLDN18.2 It contains or consists of the amino acid sequence shown in SEQ ID NO:15.

[0506] C16. The multispecific antibody according to any one of embodiments C1-C12, wherein the VHH CLDN18.2 It includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:6; preferably, the CDR sequence is defined according to ABM.

[0507] C17. A multispecific antibody according to any one of embodiments C1-C12 and C16, wherein the VHH CLDN18.2 It contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of heavy chain variable regions, wherein the heavy chain variable regions contain complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:21, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:24.

[0508] C18, the multispecific antibody according to any one of embodiments C1-C12 and C16 and C17, wherein the VHH CLDN18.2 It contains or consists of the amino acid sequence shown in SEQ ID NO:6.

[0509] C19. The multispecific antibody according to any one of embodiments C1-C18, wherein the VHH CDH17 It includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:50; preferably, the CDR sequence is defined according to ABM.

[0510] C20, the multispecific antibody according to any one of embodiments C1-C19, wherein the VHH CDH17 It contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of heavy chain variable regions, wherein the heavy chain variable regions contain complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:58.

[0511] C21. A multispecific antibody according to any one of embodiments C1-C20, wherein the VHH CDH17 It contains or consists of the amino acid sequence shown in SEQ ID NO:50.

[0512] C22. The multispecific antibody according to any one of embodiments C1-C18, wherein the VHH CDH17 It includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:36; preferably, the CDR sequence is defined according to ABM.

[0513] C23, the multispecific antibody according to any one of embodiments C1-C18 and C22, wherein the VHH CDH17It contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of heavy chain variable regions, wherein the heavy chain variable regions contain complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:54.

[0514] C24. A multispecific antibody according to any one of embodiments C1-C18 and C22-C23, wherein the VHH CDH17 It contains or consists of the amino acid sequence shown in SEQ ID NO:36.

[0515] C25. The multispecific antibody according to any one of embodiments C1-C18, wherein the VHH CDH17 It includes the three complementary determination regions (CDRs) contained in the VH shown in SEQ ID NO:61; preferably, the CDR sequence is defined according to ABM.

[0516] C26. A multispecific antibody according to any one of embodiments C1-C18 and C25, wherein the VHH CDH17 It contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of heavy chain variable regions, wherein the heavy chain variable regions contain complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:71, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:72, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:73.

[0517] C27. A multispecific antibody according to any one of embodiments C1-C18 and C25-C26, wherein the VHH CDH17 It contains or consists of the amino acid sequence shown in SEQ ID NO:61.

[0518] C28. The multispecific antibody according to any one of embodiments C1-C27, comprising or composed of two polypeptide chains, optionally the two polypeptide chains being identical.

[0519] C29. The multispecific antibody according to any one of embodiments C1-C28, wherein the polypeptide chain comprises, or is composed of, an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 82, 74-81.

[0520] C30, the multispecific antibody according to any one of embodiments C1-C29, wherein the polypeptide chain comprises, or is composed of, the amino acid sequence shown in any one of SEQ ID NO:82 and 74-81.

[0521] D1. A nucleic acid molecule encoding an antibody as described in any one of embodiments A1-A19 and B1-B19, or an antigen-binding molecule as described in any one of embodiments A20-A32 and B20-B35, or a multispecific antibody as described in any one of embodiments C1-C30.

[0522] D2. An expression vector comprising the nucleic acid molecule of embodiment D1, preferably pCDNA, such as pCDNA3.4.

[0523] D3. A host cell comprising the nucleic acid molecule described in embodiment D1 or the expression vector described in embodiment D2. Preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells.

[0524] D4. A method for preparing the antibody according to any one of embodiments A1-A19 and B1-B19, or the antigen-binding molecule according to any one of embodiments A20-A32 and B20-B35, or the multispecific antibody according to any one of embodiments C1-C30, the method comprising culturing a host cell containing the nucleic acid molecule according to embodiment D1 or the expression vector according to embodiment D2 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0525] D5. An immunoconjugate or immunofusion compound comprising any of the antibodies described in any of embodiments A1-A19 and B1-B19, or any of the antigen-binding molecules described in any of embodiments A20-A32 and B20-B35, or any of the multispecific antibodies described in any of embodiments C1-C30.

[0526] D6. Antibody-drug conjugate shown in formula (I): Ab-(LD) p (I)

[0527] Or its pharmaceutically acceptable salts or solvates,

[0528] in:

[0529] Ab is the antibody described in any one of embodiments A1-A19 and B1-B19, or the antigen-binding molecule described in any one of embodiments A20-A32 and B20-B35, or the multispecific antibody described in any one of embodiments C1-C30;

[0530] L is the connector;

[0531] D is a drug, such as an anti-tumor compound; and

[0532] p is an integer selected from 1 to 16, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12.

[0533] D7. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment D6, wherein the antitumor compound is a cytotoxic agent, such as camptothecin compounds, auroretamine compounds, such as eczema, Dxd, MMAE or MMAF.

[0534] D8. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment D6, wherein D has the structure shown in formula (D-1a) or formula (D-1b):

[0535] Where R 1a Selected from H and C1-C6 alkyl groups;

[0536] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ;

[0537] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0538] R 4a and R 5a Independently selected from H and C1-C4 alkyl groups;

[0539] or

[0540] Where R 1b R 2b R 3b R 4b R 5b and R 8b Each was independently selected from C 1-8 Alkyl groups; such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl;

[0541] R 6b and R 7b Each was independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy;

[0542] R 9b Selected from C 1-8 Alkyl groups and COOH; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and

[0543] R 10b Selected from OH and H.

[0544] D9. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in implementation scheme D8, wherein...

[0545] R 1a For H; R 2a It is a C1-C6 alkyl group; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl;

[0546] R 1b R 4b and R 8b Each was independently selected from C 1-2 Alkyl; preferably methyl;

[0547] R 2b R 3b and R 5b Each was independently selected from C 3-4 alkyl;

[0548] R 6b and R 7b Each was independently selected from C 1-2 alkoxy groups; and

[0549] R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H.

[0550] D10. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 8, wherein D has the structure shown in formula (D-2a) or formula (D-2b):

[0551] Where R 1a R 2a R 3a and R 4aAs defined in implementation scheme D8 or D9; or

[0552] Where R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined in implementation scheme D8 or D9.

[0553] D11. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment D8, wherein D has the structure shown in formula (D-3a) or (D-3b):

[0554] D12. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment D8, wherein D has the structure shown in formula (D-4a) or (D-4b):

[0555] D13. An antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in any one of embodiments D6-D12, wherein...

[0556] -L- has the following structure: -Z-L1-L2-L3-

[0557] in

[0558] Z is selected from Where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0559] L1 is selected from non-existent, Where n1 and m1 are independently selected from 0 to 20 integers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0560] L2 is a single amino acid or a peptide residue selected from 2-8 amino acids; and

[0561] L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6.

[0562] Preferably, L2 is selected from: -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-, and -Arg-Asp-Val-Thr-.

[0563] D14. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in implementation scheme D12, wherein...

[0564] Z is selected from Where m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0565] L1 is selected from non-existent, Where n1 is an integer independently selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0566] L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6.

[0567] D15. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in implementation scheme D14, wherein...

[0568] Z is selected from

[0569] L1 is selected from non-existent or

[0570] L2 is selected from -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, and -Arg-Asp-Val-Thr-;

[0571] L3 is Among them, Su and R 1c As defined in implementation scheme D14; n2 and n5 are independently 0 or 1.

[0572] D16. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in implementation scheme D15, wherein L3 is... Where Su is as defined in implementation scheme D15,

[0573] Preferably, Su is selected from

[0574] D17. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in implementation scheme D16, wherein...

[0575] Su selected

[0576] D18. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvation as described in implementation scheme D17, wherein...

[0577] Su

[0578] D19. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in implementation scheme D18, wherein...

[0579] Su

[0580] Preferably, L3 is selected from:

[0581] D20. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in implementation scheme D13, wherein...

[0582] -Z-L1-L2-L3- is selected from the following structures

[0583] D21 is the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in embodiment D6, wherein...

[0584] The antibody-drug conjugate is selected from...

[0585] Ab and p are as defined in implementation scheme D6.

[0586] D22. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments D6-D21, wherein the antibody-drug conjugate has an average DAR of 2-10, for example 4-8.

[0587] D23. A pharmaceutical composition comprising an antibody according to any one of embodiments A1-A19 and B1-B19, or an antigen-binding molecule according to any one of embodiments A20-A32 and B20-B35, or a multispecific antibody according to any one of embodiments C1-C30, an immunoconjugate or immunofusion of embodiment D5, an antibody-drug conjugate according to any one of embodiments D6-D22, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.

[0588] D24. Use of an antibody according to any one of embodiments A1-A19 and B1-B19, or an antigen-binding molecule according to any one of embodiments A20-A32 and B20-B35, or a multispecific antibody according to any one of embodiments C1-C30, an immunoconjugate or immunofusion of embodiment D5, an antibody-drug conjugate according to any one of embodiments D6-D22, or a pharmaceutically acceptable salt or solvate thereof, for the preparation of a medicament.

[0589] D25. The use according to embodiment D24, wherein the drug is used to treat and / or prevent cancer in an individual.

[0590] D26 is used according to the implementation plan D25, wherein the cancer is a CLDN18.2 positive cancer.

[0591] D27. The use according to implementation plan D25 or D26, wherein the cancer is a CDH17 positive cancer.

[0592] D28. Use according to any one of embodiments D25-D27, wherein the cancer is CLDN18.2 positive and CDH17 positive cancer.

[0593] D29. Use according to any one of embodiments D25-D28, wherein the cancer is selected from digestive system cancers such as stomach cancer, pancreatic cancer, colorectal cancer or other tumors such as ovarian cancer, head and neck cancer, etc.

[0594] D30, the use of any one of the embodiments D24-D29, wherein the drug is administered in combination with one or more therapeutic agents.

[0595] D31. The use described in Implementation Scheme D30, wherein the therapeutic agent is selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor-suppressing effects.

[0596] D32. An antibody according to any one of embodiments A1-A19 and B1-B19, or an antigen-binding molecule according to any one of embodiments A20-A32 and B20-B35, or a multispecific antibody according to any one of embodiments C1-C30, an immunoconjugate or immunofusion of embodiment D5, an antibody-drug conjugate according to any one of embodiments D6-D22, or a pharmaceutically acceptable salt or solvate thereof, for use in therapy.

[0597] D33. An antibody, antigen-binding molecule, multispecific antibody, immunoconjugate or immunofusion or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof for use according to embodiment D32, wherein the drug is used to treat and / or prevent cancer in an individual.

[0598] D34. An antibody, antigen-binding molecule, multispecific antibody, immunoconjugate or immunofusion, or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof for use according to embodiments D32 or D33, wherein the cancer is a CLDN18.2 positive cancer.

[0599] D35. An antibody, antigen-binding molecule, multispecific antibody, immunoconjugate or immunofusion, or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof for use according to embodiments D33 or D34, wherein the cancer is a CDH17-positive cancer.

[0600] D36. An antibody, antigen-binding molecule, multispecific antibody, immunoconjugate or immunofusion or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof for use according to any one of embodiments D33-D35, wherein the cancer is CLDN18.2 positive and CDH17 positive cancer.

[0601] D37. An antibody, antigen-binding molecule, multispecific antibody, immunoconjugate or immunofusion compound or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof for use according to any one of embodiments D33-D36, wherein the cancer is selected from digestive system cancers such as gastric cancer, pancreatic cancer, colorectal cancer or other tumors such as ovarian cancer, head and neck cancer, etc.

[0602] D38. An antibody, antigen-binding molecule, multispecific antibody, immunoconjugate or immunofusion, or antibody-drug conjugate or its pharmaceutically acceptable salt or solvation for use according to any one of embodiments D33-D37, administered in combination with one or more therapeutic agents.

[0603] D39. An antibody, antigen-binding molecule, multispecific antibody, immunoconjugate or immunofusion, or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof for use according to embodiment D38, wherein the therapeutic agent is selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have antitumor effects.

[0604] D40. Treating and / or preventing cancer in an individual, comprising administering an antibody according to any one of embodiments A1-A19 and B1-B19, or an antigen-binding molecule according to any one of embodiments A20-A32 and B20-B35, or a multispecific antibody according to any one of embodiments C1-C30, an immunoconjugate or immunofusion of embodiment D5, an antibody-drug conjugate according to any one of embodiments D6-D22, or a pharmaceutically acceptable salt or solvation thereof.

[0605] D41 The method according to implementation plan D40, wherein the cancer is a CLDN18.2 positive cancer.

[0606] D42. The method according to implementation scheme D40 or D41, wherein the cancer is a CDH17 positive cancer.

[0607] D43. The method according to any one of embodiments D40-D42, wherein the cancer is CLDN18.2 positive and CDH17 positive cancer.

[0608] D44. The method of any one of embodiments D40-D43, wherein the cancer is selected from digestive system cancers such as stomach cancer, pancreatic cancer, colorectal cancer or other tumors such as ovarian cancer, head and neck cancer, etc.

[0609] D45. The method of any one of embodiments D40-D44, wherein the antibody, antigen-binding molecule, multispecific antibody, immunoconjugate or immunofusion compound or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is administered in combination with one or more therapeutic agents.

[0610] D46. The method described in implementation scheme D45, wherein the therapeutic agent is selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor-suppressive effects.

[0611] Any or all of the features described above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention; however, it should be understood that the examples are for illustrative purposes only and should not be construed as constituting any limitation.

[0612] Example

[0613] Materials and methods

[0614] Reference antibody and its preparation

[0615] In this embodiment, reference antibody 2C6.9-hz21 (also known as V-BMK1), reference antibody PTA001_A4 (also known as V-BMK2) and reference antibody CM311 (also known as V-BMK3) are used.

[0616] "Reference antibody V-BMK1" is an anti-CLDN18.2 antibody constructed based on the amino acid sequences of the heavy and light chain variable regions of the monospecific bivalent antibody 2C6.9-hz21 disclosed in US20230338565A1 (SEQ ID NO 19 and 20). In the context of reference antibody V-BMK1, unless otherwise explicitly stated, the reference antibody will have a Fab antigen-binding domain and a constant region of the human IgG1 heavy chain (SEQ ID NO: 94) and a kappa light chain (SEQ ID NO: 83).

[0617] "Reference antibody V-BMK2" is an anti-CDH17 antibody constructed based on the amino acid sequences of the heavy and light chain variable regions of the monospecific bivalent antibody PTA001_A4 disclosed in US20160039933A1 (SEQ ID NO 38 and 49 in US20160039933A1). In the context of reference antibody V-BMK2, unless otherwise explicitly stated, the reference antibody will have a Fab antigen-binding domain as well as a constant region of the human IgG1 heavy chain (SEQ ID NO: 84) and a kappa light chain (SEQ ID NO: 83).

[0618] "Reference antibody V-BMK3" is an anti-CLDN18.2 antibody constructed based on the amino acid sequences of the heavy and light chain variable regions of the monospecific bivalent antibody CM311 disclosed in US20230381336A1 (SEQ ID NO 65 and 66 in US20230381336A1). In the context of reference antibody V-BMK3, unless otherwise explicitly stated, the reference antibody will have a Fab antigen-binding domain as well as a constant region of the human IgG1 heavy chain (SEQ ID NO: 84) and a kappa light chain (SEQ ID NO: 83).

[0619] The “Isotype control antibody” is constructed based on the sequence of the Anti-Rotavirus single-domain antibody 2B10 published in US20120141503A1 (SEQ ID NO:1 in US20120141503A1). In the context of the reference antibody Isotype, unless otherwise expressly stated, the Isotype antibody will have a VHH antigen-binding domain and human IgG1 Fc (SEQ ID NO:88) to form a bivalent fusion antibody protein.

[0620] The reference antibody and isotype control antibody were prepared as follows: The coding sequence of the reference antibody was synthesized by Genewiz (Shanghai, China), cloned into the pcDNA 3.4 expression vector, and transfected into Expi293F cells. The culture supernatant of the transfected cells was collected, and the antibody was separated and purified by protein A column. The concentration of the purified antibody was measured by Nano Drop; and the protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80℃ for later use.

[0621] Antigens and their preparation

[0622] The extracellular sequence information of human CDH17 (UniProt_Q12864) (Gln23-Met787) and cynomolgus monkey CDH17 (NCBI_XP_005563762.1) (Glu23-Thr784) was retrieved from the database. A His tag was added to the C-terminus of each gene, and after optimization according to human codon preferences, the genes were synthesized and subcloned into the pcDNA3.4 vector. After verification by Sanger sequencing, plasmids were extracted for later use. These constructed eukaryotic expression vectors were transiently transfected into Expi293F cells. The protein expression supernatant was collected from the transfected cell culture, and the target protein was purified using a nickel column. SDS-PAGE was performed to determine protein purity, which was >95%. Using the aforementioned reference anti-CDH17 antibody (V-BMK2), the activities of the prepared human CDH17-His recombinant antigen protein (human-CDH17-his) and cynomolgus monkey CDH17-His recombinant antigen protein (Cyno-CDH17-His) were confirmed by ELISA.

[0623] Recombinant antigen-expressing engineered cell lines and their preparation

[0624] The CHOK1 engineered cell line expressing human CLDN18.2 was prepared as follows.

[0625] The full-length gene of human CLDN18.2 (UniProt: P56856-2) was inserted into the lentiviral expression vector pLVX-puro. Lentiviral virus was packaged in 293T cells and used to infect CHOK1 cells. Cells were cultured in medium containing 8 μg / ml puromycin for selection pressure to obtain the polyclonal cell line CHO-K1-CLDN18.2. FACS analysis using the reference antibody V-BMK1 showed that the antigen expression positivity rate of the stable cell line was >90%.

[0626] Prepare HEK293 engineered cell lines expressing human CLDN18.2 as described below.

[0627] The full-length gene of human CLDN18.2 (UniProt: P56856-2) was inserted into the lentiviral expression vector pLVX-puro. Lentiviral virus was packaged in 293T cells and used to infect HEK293 cells. Cells were cultured in medium containing 1 μg / ml puromycin for selection pressure to obtain polyclonal cell lines. Further limiting dilutions were used to select stable HEK293-human CLDN18.2 cell lines (HEK293-CLDN18.2) that highly expressed the target antigen. FACS analysis using the reference antibody V-BMK1 showed that the antigen expression positivity rate of the stable cell lines was >90%.

[0628] Prepare the HEK293 engineered cell line expressing human CLDN18.1 as described below.

[0629] The full-length gene of human CLDN18.1 (UniProt: P56856-1) was inserted into the lentiviral expression vector pLVX-puro. Lentiviral virus was packaged in 293T cells and used to infect HEK293 cells. Cells were cultured in medium containing 1 μg / ml puromycin for selection pressure to obtain polyclonal cell lines. Further limiting dilutions were used to select stable HEK293-human CLDN18.1 cell lines (HEK293-CLDN18.1) that highly expressed the target antigen. FACS analysis using the positive antibody anti-CLDN18.1 PC (from iCareAb, lot #20230425) showed that the antigen expression positivity rate of the stable cell lines was >90%.

[0630] HEK293 engineered cell lines expressing human and monkey CDH17 were prepared as follows.

[0631] The full-length genes of human CDH17 (UniProt: Q12864) and monkey CDH17 (NCBI: XP_005563762.1) were inserted into the lentiviral expression vector pLVX-puro, respectively. Lentiviral viruses were packaged in 293T cells and used to infect HEK293 cells. Cells were cultured in medium containing 1 μg / ml puromycin for selection to obtain polyclonal cell lines. Through limiting dilution, stable HEK293-human CDH17 cell lines (HEK293-huCDH17) and HEK293-monkey CDH17 cell lines (HEK293-cynoCDH17) expressing high levels of the target antigen were selected. FACS analysis using the reference antibody V-BMK2 showed that the antigen expression positivity rate of the stable cell lines was >90%.

[0632] Prepare MKN45 and SNU5 engineered cell lines expressing human CLDN18.2 as described below.

[0633] The full-length gene of human CLDN18.2 (UniProt: P56856-2) was inserted into the lentiviral expression vector pLVX-puro. Lentiviral virus was packaged in 293T cells and used to infect MKN45 (CBP60488, Nanjing Kebai Biotechnology Co., Ltd.) or SNU5 cells (CBP60505, Nanjing Kebai Biotechnology Co., Ltd.). Cells were cultured in media with a selection pressure of 4 μg / ml puromycin and 2 μg / ml puromycin, respectively, to obtain polyclonal cell lines. Stable transgenic MKN45-human CLDN18.2 cell lines (MKN45-CLDN18.2) and SNU5-human CLDN18.2 cell lines (SNU5-CLDN18.2) expressing the target antigen were selected through limiting dilution. FACS analysis using the reference antibody V-BMK1 showed that the antigen expression positivity rate of the stable transgenic cell lines was >90%.

[0634] Prepare an AsPC-1 engineered cell line expressing human CLDN18.2 as described below.

[0635] The full-length gene of human CLDN18.2 (UniProt: P56856-2) was inserted into the lentiviral expression vector pLVX-puro. Lentiviral virus was packaged in 293T cells and used to infect AsPC-1 cells (CBP60546, Nanjing Kebai Biotechnology Co., Ltd.). Cells were cultured in medium containing 2 μg / ml puromycin for selection pressure to obtain polyclonal cell lines. Stable AsPC-1-human CLDN18.2 cell lines (AsPC-1-CLDN18.2) expressing the target antigen were selected through limiting dilution. FACS analysis using the reference antibody V-BMK1 showed that the antigen expression positivity rate of the stable cell lines was >90%.

[0636] ELISA combination assay

[0637] Dilute the antigen to 0.5-1.0 μg / ml with PBS, apply 100 μl / well to the plate, and incubate overnight at 2-8℃ or for 2 hours at 37℃. Wash the plate 3 times with 0.05% PBST, add 250 μl / well of blocking buffer (1% BSA or 8% skim milk powder in PBS), and incubate at room temperature for at least 1 hour. Dilute the sample to be tested to the appropriate concentration with blocking buffer. Wash the plate 3 times with 0.05% PBST, add 100 μl / well of the diluted sample, and incubate at room temperature for about 1 hour. Wash the plate 3 times with 0.05% PBST, add 100 μl / well of secondary antibody-HRP diluted to the target dilution, and incubate at room temperature for about 0.5 or 1 hour. Wash the plate 3 times with 0.05% PBST, add 100 μl / well of TMB, observe the color change, and immediately add 100 μl / well of stop buffer when the color reaches the appropriate level. Read the OD. 450- OD 650 .

[0638] FACS Combined Test

[0639] Target cells at 1-5 × 10⁶ cells per well 5 Seed cells at a density of 100 μg / well in 96-well plates and centrifuge at 300 g for 5 minutes at 4°C. Add the test sample diluted to the appropriate concentration and incubate at 2-8°C for approximately 1 hour. Centrifuge at 4°C, remove the supernatant, wash twice with 200 μl / well FACS buffer (1% BSA or 2% FBS in PBS), and centrifuge at 4°C. Add 100 μl / well of flow cytometry secondary antibody diluted to the target dilution, resuspend the cells, and incubate at 2-8°C in the dark for approximately 0.5 h or 1 h. Wash twice with 200 μl / well FACS buffer, resuspend the cells in 100 μl / well FACS buffer, and perform flow cytometry analysis. Measure the MFI of the cells using a Beckman Coulter flow cytometer.

[0640] endocytosis test

[0641] Target cells at 1-5 × 10⁶ cells per well 5Plate cells into plates, add the test sample diluted to the appropriate concentration, and incubate at 2-8℃ for approximately 0.5 h to allow the test sample to bind to the cells. Centrifuge at 800xg for 3 min at 4℃, remove the supernatant, and wash the cells 2-3 times with pre-chilled 200 μl / well FACS buffer to remove excess unbound test sample. Resuspend the cells with pre-chilled 100 μl / well FACS buffer. Divide the cells into two groups and incubate at 4℃ and 37℃ for 4 h, respectively. After incubation, immediately add ice-cold FACS buffer to terminate the endocytosis experiment. Centrifuge at 800xg for 3 min at 4℃, and wash the cells 2-3 times with pre-chilled 200 μl / well FACS buffer. Immediately add 100 μl / well of flow cytometry secondary antibody diluted to the target dilution, resuspend the cells, and incubate at 2-8℃ in the dark for approximately 30 min to 1 h. Wash cells 2-3 times with 200 μL / well FACS buffer, then resuspend cells in 100 μL / well FACS buffer for flow cytometry analysis. Measure the MFI of cells using a Beckman Coulter flow cytometer. Calculate the internalization level of antibodies bound to the cell surface using the following formula: Internalization = MFI of sample incubated at 4°C - MFI of sample incubated at 37°C. Internalization rate % = 100% - (MFI of sample incubated at 37°C / MFI of sample incubated at 4°C) × 100%.

[0642] SPR measurement

[0643] The binding affinity between antibodies and antigens is detected using surface plasmon resonance (SPR) technology.

[0644] Antibody immobilization for antigen detection: 10 μg / mL of antibody was immobilized on a Protein A chip. Diluted antigen was injected at a flow rate of 30 μL / min for 120 seconds followed by a dissociation time of 200 seconds. After each dissociation phase, 10 mM glycine (pH 2.0) was used for chip regeneration. Experimental data were analyzed using a 1:1 binding model.

[0645] Antigen immobilization and antibody detection: The antigen concentration was 10 μg / mL, immobilized on a CM5 chip. Diluted antibodies were injected at a flow rate of 30 μL / min, with a binding time of 120 seconds followed by a dissociation time of 200 seconds. After each dissociation, regeneration was performed using 10 mM glycine (pH 2.0). Experimental data were analyzed using a 1:1 binding model.

[0646] SEC-HPLC

[0647] At ambient column temperature, an appropriate amount of protein sample was loaded onto a TSK-gel G3000SWxL column (Tosoh Corporation) or a Zenix-C SEC-300 column (Sepax Technologies). Using an Agilent 1260 HPLC system, the sample was eluted isocratically for 20 minutes at a flow rate of 0.8 mL / min using a mobile phase consisting of 0.05 M sodium phosphate, 0.3 M sodium chloride, and pH 6.8 ± 0.1. The eluted protein was detected using UV absorbance at 280 nm.

[0648] HIC-HPLC

[0649] An appropriate amount of protein sample was loaded onto a MAbPac™ HIC-Butyl column (Thermo SCIENTIFIC) at ambient column temperature. Using an Agilent 1260 HPLC system, the sample was eluted at a gradient flow rate of 0.8 mL / min for 35 min using mobile phases A and B. Mobile phase A consisted of 1.5 M ammonium sulfate, 0.05 M sodium phosphate, and 5% isopropanol, pH 6.0 ± 0.1. Mobile phase B consisted of 0.05 M sodium phosphate and 5% isopropanol, pH 6.0 ± 0.1. The eluted protein was detected using UV absorbance at 280 nm.

[0650] Example 1. Preparation and Validation of VHH Antibody

[0651] Example 1.1 VHH Screening

[0652] Alpaca immunization and magnetic sorting techniques were used to screen candidate VHH sequences binding CLDN18.2 and CDH17 through preliminary characterization. In short, alpacas were immunized using the engineered cell line HEK293-CLDN18.2 prepared above, with an immunization interval of 21 days. Peripheral blood was collected 10 days after the last immunization, and serum was separated for ELISA to detect the immunization effect. Alpaca were then immunized using the human CDH17-His prepared above, with an immunization interval of 14 days. Starting from the second immunization, peripheral blood was collected seven days after each immunization, and the serum titer was monitored using ELISA binding assays.

[0653] After the serum immunogenicity reached the standard for blood collection and bank establishment, peripheral blood was collected from immunized alpacas, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted from PBMCs, and PrimeScript was used as a template to extract RNA. TMII. Reverse transcription was performed using the 1st Strand cDNA Synthesis Kit (Takara) to prepare cDNA. Using the cDNA as a template, a first-round PCR amplification produced nucleic acid fragments of conventional IgG (containing VH) and pure heavy chain IgG lacking the CH1 domain (containing VHH). These two types of nucleic acids were separated on an agarose gel. The VHH-encoding nucleic acid was extracted and purified, followed by a second-round PCR amplification. The VHH fragment was then isolated and purified by gel electrophoresis. The recovered VHH gene fragment was mixed with the linearized yeast display vector pDisaplay and co-transformed into competent yeast cells by electroporation to generate a yeast display library displaying VHH antibodies on the yeast cell surface. Yeast cells bound to the target antigen were enriched from the constructed library by magnetic sorting using streptavidin beads that had been pre-incubated with and thus bound to the target antigen.

[0654] Yeast culture obtained after magnetic bead sorting was plated on SDCAA plates, and single-clonal cells were picked and cultured. After 48 hours of induction, the single-clonal cell cultures were incubated sequentially with biotin-antigen and PE-Streptavidin. After incubation, flow cytometry (FACS) was performed to identify positive single-clonal yeast cells that bound the target antigen. Genomic DNA was extracted from the cultures of the obtained positive yeast cell clones for PCR amplification of antibody sequences and sequencing.

[0655] Based on the sequencing results, candidate VHH sequences with significant sequence differences were selected and ligated into the expression vector pcDNA3.4 in the form of C-terminal fusion with human IgG1Fc sequences. After the vectors were verified by sequencing, they were transiently transfected into HEK-293F cells (hereinafter referred to as "293F cells"). The culture supernatant was used to characterize the binding and endocytic properties of the expressed antibodies, and the anti-CLDN18.2 VHH antibody and anti-CDH17 VHH antibody were finally screened and obtained as shown in Table 1 below:

[0656] Table 1. VHH antibodies and their variable region sequences

[0657] Example 1.2. In vitro characterization of candidate VHH

[0658] Candidate VHH-Fc antibody expression and purification

[0659] The gene encoding the VHH antibody sequence described above was synthesized and inserted into the expression vector pcDNA3.4, fusing the hIgG1 Fc sequence (SEQ ID NO: 91) at its C-terminus. The constructed expression vector was transiently transfected into 293F cells. After culturing transfected cells for 7 days, the culture supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was transferred to sterile centrifuge tubes, and the antibody was purified using a Protein A column. The purity of the antibody product was determined by SEC-HPLC.

[0660] FACS antigen binding property detection

[0661] The binding of the anti-CLDN18.2 VHH-Fc candidate antibody molecule D05 to target cells was detected using a FACS binding assay. The assay was performed under the following conditions: CHO-K1-CLDN18.2 or SNU620 target cells (CBP60508, Nanjing Kebai Biotechnology Co., Ltd.) (1.5 × 10⁻⁶ cells). 5 / well) + VHH-Fc or reference antibody (200nM, 5× dilution, 4℃ 1h) + anti-hu IgG Fc-PE secondary antibody (12-4998-82, Invitrogen; 1:500, 4℃ 0.5h). FACS binding results are shown in Figure 1. The D05 antibody exhibited good target cell binding properties in different target cells, with lower EC5 values ​​than the reference antibody V-BMK1. 50 value.

[0662] Simultaneously, the binding of the CLDN18.2 VHH-Fc candidate antibody molecule D05-Fc to CLDN18.1 cells expressing paraprotein (HEK293-CLDN18.1) was detected using the same implementation conditions. The results are shown in Figure 1. The D05-Fc antibody did not bind nonspecifically to HEK293-CLDN18.1.

[0663] The binding of anti-CDH17 VHH-Fc candidate antibody molecules A004 and C063 to target cells was detected using FACS binding assays. The assays were performed under the following conditions: SNU5 or SNU620 target cells (3 × 10⁶ cells / year). 5 / well) + A004-Fc or C063-Fc antibody or reference antibody (100nM, 4× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (12-4998-82, Invitrogen; 1:500, 4℃ 0.5h). FACS binding results showed (Figure 2) that both candidate antibodies A004-Fc and C063-Fc exhibited good target cell binding properties.

[0664] endocytosis detection

[0665] Based on preliminary experiments using FACS binding, a sample concentration of 20 μg / ml was selected as the concentration used for endocytosis detection. An endocytosis assay was performed on candidate antibody D05 in engineered CHO-K1-CLDN18.2 cells. In this assay, a concentration of 1.5 × 10⁻⁶ μg / ml was used. 5 Add 500 nM, 4x serially diluted VHH-Fc antibody to be tested to the target cells in each well and incubate at 4°C for 1.0 h; then divide the cells into two groups and incubate at 4°C for 1.0 h and 37°C for 4 h, respectively; after incubation, incubate with anti-hIgG-Fc-PE (12-4998-82, Invitrogen; 1:250 dilution) at 4°C in the dark for 30 minutes, fix the cells with 1% paraformaldehyde (PFA), and perform FACS detection.

[0666] As shown in Figure 3, for the candidate anti-CLDN18.2 D05-Fc antibody tested, endocytosis on CHO-K1-CLDN18.2 cells was superior to V-BMK1.

[0667] endocytosis detection

[0668] The candidate anti-CDH17 VHH-Fc antibody was endocytosed on SNU5 and SNU620 tumor cells using FACS binding assays. In this assay, the antibody was tested at 3 × 10⁻⁶ cells. 5 100 nM of serially diluted VHH-Fc antibody was added to the target cells in each well and incubated at 4°C for 1.0 h. The cells were then divided into two groups and incubated at 4°C for 2.5 h and 37°C for 4 h, respectively. After incubation, the cells were incubated at 4°C in the dark for 30 min with anti-hIgG-Fc-PE (1:500 dilution), fixed with 1% paraformaldehyde (PFA), and then subjected to FACS detection.

[0669] As shown in Figure 4, for the candidate anti-CDH17 VHH-Fc antibodies A004-Fc and C063-Fc, regardless of whether they were in SNU5 or SNU620, the endocytosis rate at low concentrations was better than that of V-BMK2, while the endocytosis rate at saturation concentrations was comparable to that of V-BMK2.

[0670] Example 1.3. Sequence optimization and characterization of VHH components

[0671] Sequence optimization of CLDN18.2 VHH-resistant components

[0672] The anti-CLDN18.2 VHH sequence D05 was selected and its sequence optimized. The original VHH sequence was humanized using the "best-matching method". Amino acid sequences of the VHH framework region were compared and analyzed using a human germline V gene database to select the optimal germline sequence. The best-matching human CDR sequence was replaced with the VHH CDR sequence to generate a humanized VHH sequence. The immunogenicity of the humanized VHH sequence was analyzed using WeMol software. Mutant residues were introduced into the humanized sequence D05.m5 to obtain a deimmunogenic sequence, such as D05.m5m8. This sequence was synthesized by Genewiz (Shanghai, China). It was then constructed into the pcDNA 3.4 expression vector, and expressed as human IgG1 Fc sequence (SEQ ID NO: 91) fused to the C-terminus to generate human IgG1, thereby obtaining the VHH antibody protein.

[0673] Table 2 below shows the VHH sequences of the D05 parent and the sequence-optimized antibody.

[0674] Table 2. Humanized sequence of anti-CLDN18.2 VHH D05

[0675] Anti-CLDN18.2 VHH D05.m5 deimmunogenic sequence

[0676] Table 3 below shows the expression and purification results of humanized and deimmunogenic antibodies expressed in the form of VHH-Fc antibody (bivalent).

[0677] Table 3. Results of expression and purification of D05 maternal antibody and humanized antibody

[0678] Results of expression and purification of D05.m5 maternal antibody and deimmunogenic antibody

[0679] The obtained D05 optimized antibody was subjected to FACS detection. The binding of the optimized antibody to target cells CHO-K1-CLDN18.2 and SNU620 was similar to that of the parent antibody (Tables 4 and 5). The FACS binding assay conditions were as follows: target cells (1×10⁻⁶ cells / cells). 5 / well) + sample (200nM, 5X dilution, incubated at 4℃ for 1h) + anti-hu IgG Fc-PE (1:500, incubated at 4℃ for 0.5h).

[0680] Table 4. Binding activity of D05 humanized antibody to target cells

[0681] Table 5. Binding activity of D05.m5 deimmunogenic antibody to target cells.

[0682] Sequence optimization of CDH17 VHH-resistant components

[0683] The anti-CDH17 VHH sequences A004 and C063 (hereinafter referred to as "A004" and "C063") were selected and their sequences were optimized. The original VHH sequences were humanized using the "best-matching method". The amino acid sequences of the VHH framework region were compared and analyzed using a human germline V gene database to select the best germline sequence. The best-matching human CDR sequence was replaced with the VHH CDR sequence to generate the humanized VHH sequence. Several residues in the reversed mutant framework region were removed by post-translational modification (PTM). The humanized sequence was sent to Genewiz (Shanghai, China) for gene synthesis, and the base sequence was optimized. The optimized sequence was synthesized into the pcDNA 3.4 expression vector, and expressed as human VHHs in the form of human IgG1 by C-terminal fusion with the human IgG1 Fc sequence (SEQ ID NO: 91), thereby obtaining the VHH antibody protein.

[0684] Table 6 below shows the humanized and PTM-removed VHH sequences and their corresponding original VHH sequences.

[0685] Table 6. Parent stock and optimized sequence of VHH A004 and C063 VHH

[0686] Table 7 below shows the expression and purification results of A004 and C063 antibodies expressed in VHH-Fc form and after sequence optimization.

[0687] Table 7. Expression and purification results of A004 maternal antibody and sequence-optimized antibody.

[0688] The obtained antibodies were subjected to SPR assays to examine the binding affinity kinetics between the antibodies and the human CDH17 antigen, which showed binding affinity comparable to that of the parent antibody (Tables 8 and 9).

[0689] Table 8. Binding affinity kinetics of antibodies A004 and C063, as well as humanized and PTM-removed antibodies, to human CDH17 antigen.

[0690] Table 9. Binding affinity kinetics of A004 and humanized and PTM-removed antibodies with human CDH17 antigen.

[0691] The sequence-optimized antibodies were subjected to FACS binding assays. When binding to target cells SNU5 or SNU620, both the humanized and PTM-removed VHH-Fc antibodies showed similar or better binding to the parent A004-Fc (Table 10). The FACS assays used the following conditions: SUN5 or SNU620 target cells (2 × 10⁶ cells / year). 5 / well) + antibody sample (200nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (1:500, 4℃ 1h).

[0692] Based on the above data, the humanized sequence and the PTM-removed sequence were combined to obtain further optimized sequences pznA004.m4m11, pznA004.m6m11, and pznA004.m7m11 (Table 6). The expression and purification results of the Fc form bivalent antibody constructed from these further optimized sequences are shown in Table 7, the affinity kinetics are shown in Table 9, and the FACS binding experiment results are shown in Table 11. All of them exhibited similar characteristics to the parent antibody and can be used for the construction of the final antibody.

[0693] The antibody with optimized C063 sequence was subjected to FACS binding assays. Upon binding to target cells SNU5 or SNU620, the humanized sequence showed cell binding similar to that of C063-Fc (Table 10). The FACS assays used the following conditions: SUN5 or SNU620 target cells (2 × 10⁶ cells / year). 5 / well) + antibody sample (200nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (1:500, 4℃ 1h).

[0694] Table 10 Binding activities of humanized and PTM-depleted antibodies A004 and C063 to target cells

[0695] Table 11 Binding activity of A004 humanized and PTM-removed antibodies to target cells.

[0696] Example 1.4 Generation of multispecific anti-CLDN18.2 / CDH17 antibody molecules

[0697] This embodiment describes the structure of an exemplary anti-CLDN18.2 / CDH17 bispecific antibody (BsAb) and the design and construction of its expression vector.

[0698] Multispecific antibody molecule design

[0699] A bispecific antibody molecule construct, as shown in Figure 5, was designed. It is a symmetrical double-stranded structure composed of two identical polypeptide chains. Each chain contains, or is composed of, the following structure from the N-terminus to the C-terminus:

[0700] VHH A -VHH B -Fc, where VHH A and VHH B The symbols represent the VHH domains that bind antigens A and B, respectively, wherein A and B are distinct from each other and independently selected from CLDN18.2 and CDH17; wherein the symbol "-" indicates linkage by a linker or direct linkage, preferably representing a linker with a length of 5-15 amino acids.

[0701] Specifically, VHH A and VHH B The two polypeptide chains are linked by a linker linker (G4S)3, with a direct linker between the VHH domain and the Fc domain. Due to the dimerization of the immunoglobulin Fc region, the two polypeptide chains can associate to form a homodimer, thereby producing a double-chain multispecific binding molecule. The structure of an exemplary bispecific antibody constructed is shown in Figure 5.

[0702] Construction of multispecific antibody molecules

[0703] The exemplary multi-chain bispecific antibodies constructed as shown in Tables 12, 13, and 14 below have their corresponding amino acid sequences provided in the sequence listing.

[0704] In Table 12, humanized and non-humanized VHH sequences are used as building blocks, where VHH... CLDN18.2 The amino acid sequence is derived from D05.m5 (i.e., znD05.m5) (hereinafter referred to as F1), and its amino acid sequence is shown in SEQ ID NO:6; VHH CDH17 The amino acid sequences are from C063 (hereinafter referred to as S1) and A004 (hereinafter referred to as S2), and their amino acid sequences are shown in SEQ ID NO:61 and SEQ ID NO:36. The Fc region of the immunoprotein, which is the half-life extension domain (HLE), is from human IgG1, specifically containing the LALA mutant hIgG1 sequence (Fc region, Fc-LALA-220S, with LALA mutation and S at position 220, see SEQ ID NO:87; Fc' region, Fc-LALA-220C, with LALA mutation and C at position 220, see SEQ ID NO:88). The symbol "-" indicates that the two domains are connected by a linker or directly.

[0705] Table 12. Exemplary Multispecific Antibodies Not Fully Humanized

[0706] Note: The two VHH domains are connected by connector (G4S)3, and the VHH domain and the Fc domain are directly connected.

[0707] In Table 13, humanized VHH sequences and humanized and PTM-removed binding sequences were used as building blocks, where VHH... CLDN18.2 The amino acid sequence is from D05.m5 (hereinafter referred to as F1), and its amino acid sequence is shown in SEQ ID NO:6; VHH CDH17 The amino acid sequence is from A004.m6m11 (i.e., pznA004.m6m11) (hereinafter referred to as S3), and its amino acid sequence is shown in SEQ ID NO:50; the Fc region of the immunoprotein, which is the half-life extension domain (HLE), is from human IgG1, specifically containing the LALA mutant hIgG1 sequence (Fc region, Fc-LALA-220S, with LALA mutation and S at position 220, see SEQ ID NO:87; Fc region', Fc-LALA-220C, with LALA mutation and C at position 220, see SEQ ID NO:88); the symbol "-" indicates that the two domains are connected by a linker or directly.

[0708] Table 13. Multispecific antibodies after sequence humanization and PTM removal

[0709] The two VHH domains are connected via connector (G4S)3, and the VHH domain and the Fc domain are directly connected.

[0710] In Table 14, the final optimized sequence is used as the building blocks, where VHH CLDN18.2 The amino acid sequence is derived from D05.m5m8 (i.e., dznD05.m5m8) (hereinafter referred to as F2), and its amino acid sequence is shown in SEQ ID NO:15; VHH CDH17 The amino acid sequence is from A004.m6m11 (hereinafter referred to as S3), and its amino acid sequence is shown in SEQ ID NO:50; the Fc region of the immunoprotein, which is the half-life extension domain (HLE), is from human IgG1, specifically containing the LALA mutant hIgG1 sequence (Fc region, Fc-LALA-220S, with LALA mutation and S at position 220, see SEQ ID NO:87; Fc' region, Fc-LALA-220C, with LALA mutation and C at position 220, see SEQ ID NO:88); the symbol "-" indicates that the two domains are connected by a linker or directly.

[0711] Table 14 Multispecific antibodies with sequence composition after immunogenicity removal.

[0712] The two VHH domains are connected via connector (G4S)3, and the VHH domain and the Fc domain are directly connected.

[0713] The first / second polypeptide chains of the Fc dimer double-stranded antibodies shown in Tables 12, 13, and 14 were constructed into the pcDNA3.4 expression vector (if the antibody had a symmetrical structure, only one vector was generated), and transfected into HEK293F cells. Cells were cultured for 3 days, and the culture supernatant was collected and loaded into a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody was eluted with acetate-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. Antibody concentration was measured using Nano Drop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and the protein was then stored at -80°C.

[0714] Example 1.5 Characterization of multispecific anti-CLDN18.2 / CDH17 antibody molecules

[0715] Analysis of target antigen expression levels on tumor cells

[0716] Using reference antibodies V-BMK1 and V-BMK2, the expression of CLDN18.2 and CDH17 antigens on various target tumor cells SNU5, SNU620, SNU5-CLDN18.2 and MKN45-CLDN18.2 was detected by FACS binding assay.

[0717] The test results are shown in Figure 6. On the tested tumor cells MKN45-CLDN18.2, the CLDN18.2 antigen showed high expression levels, while CDH17 showed extremely low expression levels, indicating CLDN18.2 single-positive cells or CLDN18.2 positive cells. +++ Cells; on the tested SNU620 tumor cells, CLDN18.2 antigen was positively expressed, and CDH17 showed a high expression level, indicating that these were CLDN18.2 and CDH17 double-positive cells or CLDN18.2 cells. + CDH17 ++ Cells; on the tested SNU5 tumor cells, CLDN18.2 antigen was expressed at extremely low levels, while CDH17 was expressed at high levels, indicating CDH17 monopositive cells or CDH17 positive cells. +++ Cells; on the tested SNU5-CLDN18.2 tumor cells, CLDN18.2 antigen and CDH17 were highly expressed, indicating CLDN18.2 and CDH17 double-positive cells or CLDN18.2 cells.++++ CDH17 +++ cell.

[0718] Nonspecific binding analysis

[0719] The binding of the aforementioned multispecific antibody molecules to the CLDN18.1 antigen homolog of the CLDN18.2 antigen was detected by FACS binding assay (an Isotype control was used as the negative control). The FACS binding assay was performed under the following conditions: target cells HEK293-CLDN18.1 (1×10⁻⁶ cells). 5 ( / well) + sample (100nM, 5× dilution, 4℃ 1h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5h).

[0720] Figure 7 shows that the multispecific antibody molecules did not bind specifically to HEK 293-CLDN18.1 cells.

[0721] Tumor cell binding activity

[0722] Based on the above analysis of antigen expression on tumor cells, cell lines MKN45-CLDN18.2, SNU620, and SNU5 with different target antigen expression levels were selected. The binding ability of the bispecific antibody (hereinafter referred to as "VBsAb") of the present invention on different target cells was tested by FACS binding assay.

[0723] FACS was performed in conjunction with experimental conditions: target cells (3 × 10⁶). 5 / well) + sample (200nM, 5× dilution, 4℃ 1h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5h).

[0724] The detection results of the non-humanized bispecific antibody are shown in Figure 8.

[0725] In the humanized exemplary bispecific antibodies, V-F8 and V-F9 showed comparable binding ability to CLDN18.2 monopositive cells MKN45-CLDN18.2, CLDN18.2 and CDH17 bipositive cells SNU620 and CDH17 monopositive cells SNU5 (Figure 9).

[0726] In the exemplary deimmunogenic bispecific antibodies, V-F10 showed comparable binding ability to V-F8 cells and V-F11 showed comparable binding ability to V-F9 cells on CLDN18.2 monopositive cells MKN45-CLDN18.2, CLDN18.2 and CDH17 bipositive cells AsPC-1-CLDN18.2 (Figure 10).

[0727] FACS examination of immune cross-reactivity

[0728] The immunoreactivity of the humanized bispecific antibodies V-F8 and V-F9 of this invention was detected using an engineered HEK293 cell line stably expressing human / monkey CDH17 via FACS binding assay. The experimental conditions were as follows: target cells (1×10⁻⁶ cells / year)... 5 / well) + sample (100nM, 5× dilution, 4℃ 1h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5h). The tested bispecific antibody showed similar binding activity to human monkey antigen, exhibiting cross-reactivity with monkey antigen, and did not bind nonspecifically to HEK293 cells (Figure 11).

[0729] Internalization assay

[0730] The internalization capacity of the bispecific antibody molecule V-F5 Ab in different target cell lines (MKN45-CLDN18.2, SNU620, and SNU5) was tested using an endocytosis assay (Figure 12). As shown in Figure 12, V-F5 exhibited the best endocytic activity in CLDN18.2 and CDH17 double-positive cells (SNU620) and CDH17 single-positive cells (SNU5). (MFI: 4°C - 37°C)

[0731] As shown in Figure 13, the humanized antibodies V-F8 and V-F9 exhibited comparable endocytic activity in CLDN18.2 / CDH17 double-positive cells, and were superior to the parental control and positive control molecules.

[0732] Combined with kinetic SPR measurement

[0733] The affinity of bispecific antibody molecules V-F8 and V-F9 for human and cynomolgus monkey CDH17 antigens (Sinochem) was determined using the SPR method. 10 μg / mL antibody was captured using a Protein A chip for 15 s, followed by the injection of a 2-fold serially diluted antigen at a flow rate of 30 μL / min. The binding time was 120 s, followed by a dissociation time of 200 s. After each dissociation phase, 10 mM glycine (pH 2.0) was used for chip regeneration. Experimental data were analyzed using a 1:1 binding model.

[0734] The affinity results of candidate molecules V-F8 and V-F9 for human and cynomolgus monkey CDH17 antigens are shown in Table 15. The results indicate that candidate molecules V-F8 and V-F9 cross-react with cynomolgus monkey CDH17 antigens, and exhibit similar binding and dissociation rates with both human and cynomolgus monkey CDH17 antigens.

[0735] Table 15. Affinity of V-F8 and V-F9 to human and cynomolgus monkey CDH17 antigens

[0736] In addition, the affinity of bispecific antibody molecules V-F10 and V-F11 for human and cynomolgus monkey CDH17 antigens was also detected by SPR method, and the results are shown in Table 16 below.

[0737] Table 16. Affinity of V-F10 and V-F11 to human and cynomolgus monkey CDH17 antigens

[0738] Example 2: Linker-payload Synthesis

[0739] Example 2.1 Synthesis of Mc-RDVT-EXD (HM-2100D_11)

[0740] Example 2.1.1 Preparation of HM-2100D_2

[0741] Weigh 10 g (6.67 mmol, ~1.5 mmol / g) of HM-2100C_2 (triphenylchloromethane resin, CAS No.: 42074-68-0) into a 500 mL peptide tube, add 200 mL of DMF solution containing 7.94 g (20 mmol) of HM-2100D_1, and then add DIEA (4.3 g, 33.35 mmol). Shake on a shaker for 16 hours. Add 30 mL of MeOH to the peptide tube, shake for 1 hour, and then dry. Wash the resin sequentially with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100D_2. Take a small sample and treat it with HFIP (hexafluoroisopropanol) / DCM (1 / 4, volume ratio), remove the resin, and send it to LCMS to confirm resin incorporation.

[0742] LC-MS (ESI) m / z: 342.1 [M-56+H] + .

[0743] Example 2.1.2 Preparation of HM-2100D_3

[0744] (1) Add 20% Piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100D_2 (6.67 mmol), shake on a shaker for 30 minutes and then dry. Add another 20% Piperidine / DMF (150 mL), shake on a shaker for 30 minutes and then dry. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence.

[0745] (2) Add a DMF (150 mL) solution of HM-297D_1 (6.78 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100D_3, which is not considered in the yield. Take a small sample and treat it with HFIP / DCM (1 / 4), cut off the resin, and send it to LCMS to confirm the incorporation of HM-297D_1.

[0746] LC-MS (ESI) m / z: 497.3 [M+H] + .

[0747] Example 2.1.3 Preparation of HM-2100D_4

[0748] (1) Add 20% Piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100D_3 (6.67 mmol), shake on a shaker for 30 minutes and then dry. Add another 20% Piperidine / DMF (150 mL), shake on a shaker for 30 minutes and then dry. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence.

[0749] (2) Add a DMF (150 mL) solution of HM-588_3A (8.22 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100D_4, which is not considered in the yield. Take a small sample and treat it with HFIP / DCM (1 / 4), cut off the resin, and send it to LCMS to confirm the attachment of HM-588_3A.

[0750] LC-MS (ESI) m / z: 668.3 [M+H] + .

[0751] Example 2.1.4 Preparation of HM-2100D_5

[0752] (1) Add 20% Piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100D_4 (6.67 mmol), shake on a shaker for 30 minutes and then dry. Add another 20% Piperidine / DMF (150 mL), shake on a shaker for 30 minutes and then dry. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence.

[0753] (2) Add a 150 mL solution of DMF containing HM-2100C_7 (10.52 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100D_5, which is not considered in the yield. Take a small sample and treat it with HFIP / DCM (1 / 4), cut off the resin, and send it to LCMS to confirm the incorporation of HM-2100C_7.

[0754] LC-MS (ESI) m / z: 954.5 [M+H] + .

[0755] Example 2.1.5 Preparation of HM-2100D_6

[0756] HFIP / DCM (1 / 4) (100 mL) was added to a peptide tube containing HM-2100D_5 (6.67 mmol). The mixture was shaken on a shaker for 2 hours, filtered, washed with DCM (200 mL x 4), concentrated, and the residual HFIP was removed with DCM (400 mL x 4) to obtain the target product HM-2100D_6 (15.5 g, yield not considered, light pink solid). LC-MS (ESI) m / z: 954.5 [M+H] + .

[0757] Example 2.1.6 Preparation of HM-2100D_7

[0758] Under ice bath conditions (5°C), COMU (7.41 g, 17.3 mmol) and 2,6-Lutidine (5.05 g, 47.19 mmol) were added to a DMF (150 mL) solution of HM-2100D_6 (15 g, 15.73 mmol) and HM-297D_10 (2.13 g, 17.3 mmol). The mixture was stirred at room temperature (15°C) for 1 hour. The reaction solution was poured into water (1 L), the precipitated solid was filtered, collected, dissolved in ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain the target product HM-2100D_7 (8.8 g, yield: 53%, light white solid). LC-MS (ESI) m / z: 1059.5 [M+H] + .

[0759] Example 2.1.7 Preparation of HM-2100D_8

[0760] Under ice bath conditions (5°C), DIEA (1.6 g, 12.18 mmol) was added to an 86 mL DMF solution of HM-2100D_7 (8.6 g, 8.12 mmol) and NPC (5 g, 16.24 mmol), and the mixture was stirred at room temperature (15°C) for 16 hours. The reaction mixture was poured into water (1 L), extracted with ethyl acetate, concentrated, and the residue was dissolved in a small amount of ethyl acetate and added to MTBE (1 L). The precipitated solid was filtered, collected, and dried to obtain the target product HM-2100D_8 (8.5 g, yield: 85%, light white solid). LC-MS (ESI) m / z: 1224.5 [M+H] + .

[0761] Example 2.1.8 Preparation of HM-2100D_9

[0762] At room temperature (15°C), DIEA (263 mg, 2.034 mmol) was added to a mixture of HM-582_10 (450 mg, 0.8475 mmol), HM-2100D_8 (1.25 g, 1.017 mmol), Py (4.5 mL), and HOAt (115 mg, 0.8475 mmol) in DMF (18 mL). The mixture was stirred at room temperature (15°C) for 16 hours. The reaction solution was poured into water (200 mL), the precipitated solid was filtered, collected, redissolved in DCM, dried, and directly subjected to silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain the target product HM-2100D_9 (1.1 g, yield: 85%, light green solid).

[0763] LC-MS (ESI) m / z: 1520.6 [M+H] + .

[0764] Example 2.1.9 Preparation of HM-2100D_10

[0765] At room temperature (15°C), 10 mL of TFA was added to a mixture of HM-2100D_9 (500 mg, 0.329 mmol) and DCM (10 mL), and the mixture was stirred at room temperature (15°C) for 2 hours. The reaction solution was concentrated, and the residue was purified by reverse-phase column chromatography (ACN / H2O with 0.1% TFA) and lyophilized to obtain the target product HM-2100D_10 (360 mg, yield: 93%, yellow solid, TFA salt). LC-MS (ESI) m / z: 1056.6 [M+H] + .

[0766] Example 2.1.10 Preparation of HM-2100D_11

[0767] At room temperature (15°C), DIEA (50 mg, 0.384 mmol) was added to a 3 mL solution of DMF containing HM-2100D_10 (150 mg, 0.128 mmol) and HM-297Q_6 (60 mg, 0.193 mmol), and the mixture was stirred at room temperature (15°C) for 2 hours. The reaction solution was neutralized with HOAc, purified by reverse-phase reaction (ACN / H2O with 0.1% TFA), and lyophilized to obtain the target product HM-2100D_11 (25 mg, yield: 15.6%, yellow solid). LC-MS (ESI) m / z: 1249.4 [M+H] + .

[0768] HM-2100D_11

[0769] 1H NMR (400MHz, DMSO) δ12.41 (s, 1H), 9.83 (s, 1H), 8.33 (d, J = 7.4Hz, 1H), 8.12-7 .99(m,2H),7.85(d,J=8.0Hz,1H),7.80(d,J=10.9Hz,1H),7.73(d,J=8.6Hz,1H ),7.62(d,J=8.5Hz,2H),7.45(t,J=5.6Hz,1H),7.39(d,J=8.4Hz,2H),7.33(s, 1H),7.26-6.72(m,5H),6.55(s,1H),5.47(s,2H),5.30(d,J=4.0Hz,3H),5.17- 4.91(m,3H),4.66-4.56(m,1H),4.36–4.24(m,3H),4.12–4.04(m,1H),3.33–3. 21(m,2H),3.17–3.05(m,3H),2.79-2.69(m,1H),2.57(d,J=8.0Hz,1H),2.40(s ,3H),2.26–2.10(m,4H),2.09-1.99(m,1H),1.97-1.83(m,2H),1.66(s,1H),1. 54–1.44(m,7H),1.24-1.16(m,2H),1.10(d,J=6.3Hz,3H),0.95–0.78(m,10H).

[0770] MS(ES-API): 1249.4(M+H) +

[0771] Example 2.2 Synthesis of MAL-PEG8-EVC-PAB-MMAE

[0772] Step 1:

[0773] (1) Compounds HM-2039_1 (CAS:13726-84-6) (100mg, 0.33mmol, 1eq) and HN-078A_7 (Catalog:HY-100374, CAS:644981-35-1) (388.86mg, 0.346, 1.05eq) were added to DMF (6mL), along with HATU (188mg) and triethylamine (66.7mg). The mixture was reacted under N2 protection for 4h.

[0774] (2) After the reaction was monitored by LCMS, the reaction solution was dropped into water and extracted twice with EA (30 mL * 2). The organic phases were combined and washed three times with saturated saline (30 mL * 3). After drying with anhydrous sodium sulfate, the solution was concentrated to obtain 410 mg (88.29% yield) of white solid.

[0775] Step Two:

[0776] (1) Compound HM-2039_2 (410 mg, 0.291 mmol, 1 eq) was added to DCM (4 mL), TFA (1 mL) was added, and the mixture was reacted for 1 h under nitrogen protection (0-10 °C).

[0777] (2) The reaction was monitored by LCMS until it ended. The crude product was obtained by low-temperature concentration to remove DCM. The crude product was passed through a reverse phase column to obtain the product: 200 mg, 54.86% yield white solid.

[0778] Step 3:

[0779] (1) Dissolve HM-2039_3 (0.1g, 79.84umol, 1eq) and HM-2039_4 (CAS: 1818294-46-0) (51.86mg, 83.83umol, 1.05eq) in DMF and stir the reaction solution for 2h.

[0780] (2) The reaction was monitored by LCMS until it ended. The reaction solution was directly prepared into an aqueous solution of TFA (ACN) to obtain 30 mg (23.54% yield) of the target product as a white solid with MW 1756.08 and purity 99.18%.

[0781] 1H NMR (400MHz, DMSO) δ10.04(s,1H),8.35-8.25(m,0.28H),8.25-8.15(m,1H),8.13-8.03(m,1.5H),7.94-7.87(m,0.4H),7.76-7.70(m,1H), 7.67-7.63(m,0.4H),7.62-7.57(m,1.7H),7.38-7.25(m,7H),7.22-7 .14(m,1H),7.04(s,2H),5.99(s,1H),5.66–5.20(m,2H),5.15-4.95( s,2H),4.80–4.15(m,8H),4.10-3.90(m,2H),3.63-3.56(m,5H),3.52 -3.48(m,28H),3.28-3.24(m,4H),3.22-3.18(m,5H),3.16-3.10(m,2 H),3.10–2.84(m,9H),2.50-2.40(m,2H),2.30-2.20(m,3H),2.20-1. 69(m,10H),1.67-1.30(m,7H),1.08–0.98(m,7H),0.89–0.76(m,26H).

[0782] Example 3. Preparation and characterization of antibody-drug conjugates (ADCs)

[0783] The connectors-payloads used in some embodiments of the present invention are known in the prior art, commercially available, and / or prepared according to the above embodiments. When the drawn structure is inconsistent with the actual situation, modifications to the structure should be allowed according to the actual situation.

[0784] Materials and methods

[0785] General Synthesis Method A

[0786] Place 2.62 mg / ml of antibody (e.g., the corresponding bispecific antibody prepared according to the antibody preparation example above) in a Biofil tube in 50 mM NaOAc-HAc (pH 5.5). Add 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 5 mM concentration) to the antibody buffer (TCEP:antibody = 6:1). Place the Biofil tube containing the reaction mixture on a shaker (x500 rpm) and react at 37°C for 2 hours. Add another 6 molar equivalents of TCEP (5 mM) to the mixture. Place the reaction mixture on a shaker (500 rpm) and react at 37°C for another 2 hours. Then, ultrafilter (using a MWCO 30 kDa filter membrane) at least 6 times, replacing approximately half the volume each time, and replenishing to the initial volume with 10 mM His-Hac (pH 6.2) buffer to remove excess TCEP. Six molar equivalents of linker-load (MAL-PEG8-EVC-PAB-MMAE) (5 mg / ml, in DMA) were added dropwise to the completely reduced antibody. The reaction was incubated at 25°C on a shaker for 2 hours (linker-load:antibody = 6:1). The conversion rate was assessed using HIC-HLPC, and purification was performed immediately after conversion.

[0787] Ultrafiltration purification method: Transfer the reaction mixture to an ultrafiltration tube (MWCO 30kd), add 20% DMA / 10mM His-HAc (pH 6.2) to the maximum allowable volume of the ultrafiltration tube. Centrifuge the sample at 3000 rpm for 5 minutes to remove half of the solution, and replenish the initial volume with 20% DMA / 10mM His-HAc. Discard the flow-through. Repeat the washing step 10 times. Then, replace the solution with 10mM His-HAc (pH 6.2) 10 times until the organic solvent DMA is completely replaced. Transfer the remaining solution, add an appropriate amount of 10mM His-HAc (pH 6.2), rinse the filter membrane twice, and combine to obtain the final coupling product. Measure the concentration using Nanodrop or BCA methods, and dilute or concentrate the ADC solution to the required concentration as needed.

[0788] Protein A Purification Method: Add an appropriate amount of Protein A (stored in 20% ethanol / pure water, 40 g / L capacity) to a gravity column. Wash Protein A with 10 volumes of PBS, then load the sample to be purified onto a resin-filled gravity column. Wash with at least 10 CV of additional 10 mM His-HAc (pH 6.2) buffer containing 10%–20% DMA. Wash with at least 50 CV of 10 mM His-HAc (pH 6.2) buffer (to completely remove free payload and organic solvent). Elute with acetic acid solution (50 mM) at pH 3 and immediately neutralize to pH 5.5–6.0 with 2 M Tris solution (pH 12.0). Combine the neutralized eluents. Determine the concentration using Nanodrop or BCA methods, and dilute or concentrate the ADC solution to the required concentration as needed. Determine purity using SEC-HPLC. The average DAR value and residual free linker-effective load content were determined by HIC and RP-HPLC methods.

[0789] General Synthesis Method B

[0790] Method 1: For the preparation of heterogeneous DAR4, place 1.42 mg / ml antibody (e.g., the corresponding bispecific antibody prepared according to the antibody preparation example above) in 50 mM NaOAc-HAc (pH 5.5) in a Biofil tube. Add 6 molar equivalents of TCEP (5 mM concentration) and 3 molar equivalents of ZnCl2 (5 mM concentration) antibody buffer (TCEP:antibody:ZnCl2 = 6:1:3). Place the Biofil tube containing the reaction mixture on a shaker (x500 rpm) at 37°C for 2 hours. Ultrafilter (using a MWCO 30kd filter membrane) at least 6 times, replacing approximately half the volume each time, and replenish to the initial volume with 10 mM His-HAc (pH 6.2) buffer to remove excess TCEP. Add 12 molar equivalents of linker-payload (MC-Val-Cit-PAB-MMAE, 5 mg / ml, in DMA) dropwise to the completely reduced antibody. The reaction was incubated at 25°C on a shaker for 2 hours (linker-load:antibody = 12:1). Conversion was assessed using HIC-HLPC, and purification was performed immediately upon completion of conversion. An appropriate amount of Protein A (stored in 20% ethanol / pure water, 40 g / L capacity) was added to a gravity column. Protein A was washed with 10 volumes of PBS, and the sample to be purified was then loaded onto a resin-filled gravity column. Washing was performed with at least 10 CV of additional 10 mM His-HAc (pH 6.2) buffer containing 10%–20% DMA. Washing was then performed with at least 50 CV of 10 mM His-HAc (pH 6.2) buffer (to completely remove free load and organic solvent). The eluent was then eluted with 50 mM acetic acid solution at pH 3 and immediately neutralized to pH 5.5–6.0 with 2 M Tris solution (pH 12.0). The neutralized eluents were combined. Measure the concentration using the Nanodrop or BCA method, and dilute or concentrate the ADC solution to the required concentration as needed.

[0791] Method 2: Place 4.6 mg / ml of antibody (e.g., the corresponding bispecific antibody prepared according to the antibody preparation example above) in a Biofil tube in 50 mM NaOAc-HAc (pH 5.5). Add 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 5 mM concentration) to the antibody buffer (TCEP:antibody = 6:1). Place the Biofil tube containing the reaction mixture on a shaker (x500 rpm) and react at 37°C for 2 hours. Add another 6 molar equivalents of TCEP (5 mM) to the mixture (TCEP:antibody = 6:1). Repeat the above reaction conditions. Ultrafilter (using a MWCO 30 kDa filter membrane) at least 6 times, replacing approximately half the volume of buffer each time, and replenishing to the initial volume with 10 mM His-HAc (pH 6.2) buffer to remove excess TCEP. Add 6 molar equivalents of linker-payload (MC-Val-Cit-PAB-MMAE) (5 mg / ml in DMA) dropwise to the fully reduced antibody, adjusting with buffer as needed to maintain a DMA concentration of at least 20% (v / v). Incubate the reaction at room temperature on a shaker for 2 hours (linker-payload:antibody = 6:1). Purify immediately after transformation. Add an appropriate amount of Protein A (stored in 20% ethanol / pure water, 40 g / L capacity) to a gravity column. Wash Protein A with 10 volumes of PBS, then load the sample to be purified onto a resin-filled gravity column. Wash with at least 10 CV of additional 10 mM His-HAc (pH 6.2) buffer containing 10%–20% DMA. Wash with at least 50 CV of 10 mM His-HAc (pH 6.2) buffer (to completely remove free payload and organic solvent). Then elute with acetic acid solution at pH 3 (50 mM) and immediately neutralize to pH 5.5–6.0 with 2 M Tris solution (pH 12.0). Combine the neutralized eluents and determine the concentration using the Nanodrop or BCA method. Dilute or concentrate the ADC solution as required to adjust it to the desired concentration.

[0792] General Synthesis Method C

[0793] Place 4.99 mg / ml of antibody (e.g., the corresponding bispecific antibody prepared according to the antibody preparation example above) in a Biofil tube in 50 mM NaOAc-HAc (pH 5.5). Add 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 5 mM concentration) to the antibody buffer (TCEP:antibody = 6:1). Place the Biofil tube containing the reaction mixture on a shaker (x500 rpm) and react at 37°C for 2 hours. Add another 6 molar equivalents of TCEP (5 mM) to the mixture. Repeat the above reaction conditions. Ultrafilter (using a MWCO 30 kDa filter membrane) at least 6 times, replacing approximately half the volume of buffer each time, and replenishing to the initial volume with 10 mM His-HAc (pH 6.2) buffer to remove excess TCEP. Add 6 molar equivalents of linker-loador (Mal-Gly-Exatecan-D-glucuronic acid, CAS No.:2763252-25-9) (5 mg / ml, in DMA) dropwise to the fully reduced antibody, adjusting the reaction volume as needed to maintain the DMA concentration below 20% (v / v). Incubate the reaction mixture at room temperature for 2 hours on a shaker (linker-loador:antibody = 6:1). Ultrafiltration purification: Transfer the reaction mixture to an ultrafiltration tube (MWCO 30kd) and add 20% DMA / 10mM His-HAc (pH 6.2) to the maximum volume of the ultrafiltration tube. Centrifuge the sample at 3000 rpm for 5 minutes. When approximately half the maximum volume of the ultrafiltration tube remains, replenish the solution with 20% DMA / 10mM His-HAc to the initial volume. Discard the flow-through. Repeat the washing step 10 times. The solution was then replaced 10 times with 50 mM NaOAc-HAc (pH 5.5) to completely remove the organic solvents DMA and His-HAc. The remaining solution was transferred, and an appropriate amount of 50 mM NaOAc-HAc (pH 5.5) was added. The filter membrane was rinsed twice, and the solutions were combined to obtain the final coupling product. The concentration was determined using Nanodrop or BCA methods. The ADC solution was diluted or concentrated as required to adjust it to the desired concentration. The purity was determined by SEC-HPLC. The average DAR value and the free linker-loaded content were determined by HIC and RP-HPLC methods.

[0794] General Synthesis Method D

[0795] Place 6.46 mg / ml of antibody (e.g., the corresponding bispecific antibody prepared according to the antibody preparation example above) in 50 mM NaOAc-HAc (pH 5.5) into a Biofil tube. Add 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 5 mM concentration) to the antibody buffer (TCEP:antibody = 6:1). Place the Biofil tube containing the reaction mixture on a shaker (x500 rpm) and react at 37°C for 2 hours. Add another 6 molar equivalents of TCEP (5 mM) (TCEP:antibody = 6:1) to the mixture. Repeat the above reaction conditions. Ultrafilter (using a MWCO 30 kDa filter membrane) at least 6 times, replacing approximately half the volume of buffer each time, and replenishing to the initial volume with 10 mM His-HAc (pH 6.2) buffer to remove excess TCEP. Add 8 molar equivalents of linker-load (MC-RDVT-PAB-Exd or Mal-Gly-PAB-Exatecan-D-glucuronic acid) (5 mg / ml in DMA) dropwise to the fully reduced antibody, adjusting the reaction volume as needed to maintain the DMA concentration below 20% (v / v). Incubate the reaction mixture at room temperature on a shaker for 2 hours (linker-load:antibody = 8:1). Purify immediately after transformation. Pro A purification method: Add an appropriate amount of Protein A (stored in 20% ethanol / pure water, 40 g / L capacity) to a gravity column. Wash Protein A with 10 volumes of PBS, then load the sample to be purified onto a resin-filled gravity column. Wash with at least 10 CV of additional 10 mM His-HAc (pH 6.2) buffer containing 10%–20% DMA. Wash with at least 50 CV of 10 mM His-HAc (pH 6.2) buffer (to completely remove free payload and organic solvent). Then elute with 50 mM acetic acid solution at pH 3 and immediately neutralize to pH 5.5–6.0 with 2 M Tris solution (pH 12.0). Combine the neutralized eluates. Determine the concentration using Nanodrop or BCA methods, and dilute or concentrate the ADC solution to the required concentration as needed. Determine purity by SEC-HPLC. Determine the average DAR value and free linker-payload content by HIC and RP-HPLC methods.

[0796] General Synthesis Method E

[0797] Place 7.42 mg / ml of antibody (e.g., the corresponding bispecific antibody prepared according to the antibody preparation example above) in a Biofil tube in 20 mM HIS-HAc buffer (pH 5.5). Add 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 5 mM concentration) to the antibody buffer (TCEP:antibody = 6:1). Place the Biofil tube containing the reaction mixture on a shaker (x500 rpm) and react at 37°C for 2 hours. Add another 6 molar equivalents of TCEP (5 mM) (TCEP:antibody = 6:1) to the mixture. Repeat the above reaction conditions. Ultrafilter (using a MWCO 30 kDa filter membrane) at least 6 times, replacing approximately half the volume of buffer each time, and replenishing to the initial volume with 10 mM His-HAc (pH 6.2) buffer to remove excess TCEP. Add 8 molar equivalents of linker-load (MC-RDVT-PAB-Exd or Mal-Gly-PAB-Exatecan-D-glucuronic acid) (5 mg / ml in DMA) dropwise to the fully reduced antibody, adjusting the reaction volume as needed to maintain the DMA concentration below 20% (v / v). Incubate the reaction mixture at room temperature on a shaker for 2 hours (linker-load:antibody = 8:1). Purify immediately after transformation. Ultrafiltration purification method: Transfer the reaction mixture to an ultrafiltration tube (MWCO 30kd), add 10% DMA / 20mM NaOAcs-HAc, 150mM Arginine, and 8% trehalose (pH 5.5) to the maximum allowable volume of the ultrafiltration tube. Centrifuge the sample at 3000 rpm for 5 minutes to remove approximately half of the solution, and replenish the initial volume with 10% DMA / 20mM NaOAcs-HAc, 150mM Arginine, and 8% trehalose. Discard the flowthrough. Repeat the washing step 10 times. Then, change the solution 10 times with 20 mM NaOAcs-HAc, 150 mM Arginine, and 8% trehalose (pH 5.5) until the organic solvent DMA is completely removed. Transfer the remaining solution and add an appropriate amount of 20 mM NaOAcs-HAc, 150 mM Arginine, and 8% trehalose (pH 5.5) to rinse the filter membrane twice. Combine the solutions to obtain the final coupling product. Measure the concentration using the Nanodrop or BCA method, and dilute or concentrate the ADC solution to the required concentration as needed.

[0798] Purity was determined by SEC-HPLC. The average DAR value and free linker-load content were determined by HIC and RP-HPLC methods.

[0799] General methods and / or parameters for determining or detecting ADCs

[0800] Size exclusion chromatography (SEC-HPLC) method (for total ADC detection).

[0801] SEC-HPLC method parameters

[0802] Reversed-phase HPLC (RP HPLC) method (for the detection of free drugs)

[0803] RP HPLC parameters

[0804] Perform elution according to the table below.

[0805] HIC-HPLC method (for the detection of free antibodies and DAR distribution)

[0806] HIC-HPLC conditions.

[0807] Perform elution according to the table below.

[0808] Example 3.1 Preparation of V-F1-PEG-EVC-MMAE

[0809] Ab is a bispecific antibody V-F1; p is mainly 4, and the obtained V-F1-PEG-EVC-MMAE assay has an average DAR of 3.82.

[0810] V-F1-PEG-EVC-MMAE was prepared using the bispecific antibody V-F1 prepared according to the method described in the above examples, and the linker-loaded vector MAL-PEG8-EVC-PAB-MMAE, in accordance with general synthesis method A.

[0811] Example 3.2 Preparation of V-F5-VC-MMAE

[0812] Ab is the bispecific antibody V-F5; p is mainly 4, and the obtained V-F5-VC-PAB-MMAE assay has an average DAR of 3.96.

[0813] Following the general synthesis method B, Method 1, V-BMK3-VC-MMAE was prepared using the control antibody V-BMK3 prepared according to the method described in the above examples and the linker-loaded MC-Val-Cit-PAB-MMAE (CAS No.: 646502-53-6; MCE, HY-15575). The product purity was 95.3%.

[0814] Following the general synthesis method B, Method 2, V-F5-VC-MMAE was prepared using the bispecific antibody V-F5 prepared according to the method described in the above examples and the linker-loaded MC-Val-Cit-PAB-MMAE (CAS No.: 646502-53-6; MCE, HY-15575). The product purity was 83.8%.

[0815] Example 3.3 Preparation of V-F8-Gly-Exatecan-D-glucuronic acid (also referred to as "V-F8-Gluc-Exd")

[0816] Ab is the bispecific antibody V-F8; p is mainly 4, and the obtained V-F8-Gluc-Exd assay has an average DAR of 4.0.

[0817] Following general synthetic method C, V-F8-Gly-PAB-Exatecan-D-glucuronic acid was prepared using the bispecific antibody V-F8 prepared according to the method described in the above examples and the linker-loaded product Mal-Gly-Exatecan-D-glucuronic acid (CAS No.: 2763252-25-9; MCE, HY-153179). The product had an average DAR value of 4.0 and a SEC-HPLC purity of 93.1%.

[0818] Example 3.4 Preparation of V-F9-Gly-PAB-Exatecan-D-glucuronic acid (also referred to as "V-F9-Gluc-Exd")

[0819] Ab is a bispecific antibody V-F9; p is mainly 6, and the obtained V-F9-Gluc-Exd assay has an average DAR of 6.0.

[0820] Following general synthetic method D, V-F9-Gly-PAB-Exatecan-D-glucuronic acid was prepared using the bispecific antibody V-F9 prepared according to the method described in the above examples and the linker-loaded polymer Mal-Gly-PAB-Exatecan-D-glucuronic acid (CAS No.: 2763252-25-9; MCE, HY-153179). The product had an average DAR value of 6.0 and a SEC-HPLC purity of 98.4%.

[0821] Example 3.5 Preparation of V-F11-RDVT-EXD

[0822] Ab is the bispecific antibody V-F11; p is mainly 6, and the obtained V-F11-RDVT-EXD assay has an average DAR of 6.0.

[0823] Following general synthetic method E, V-F11-RDVT-EXD was prepared using the bispecific antibody V-F11 prepared according to the method described in the above examples and the linker-loaded MC-RDVT-PAB-Exd (i.e., HM-2100D_11). The product had an average DAR value of 6.0 and a SEC-HPLC purity of 95.5%.

[0824] Example 3.6 Preparation of another ADC

[0825] Following the method described in Example 3.1, but using different antibodies (V-F2, V-F3, V-F4, V-F5, or Isotype) instead of V-F1, V-F2-PEG-EVC-MMAE, V-F3-PEG-EVC-MMAE, V-F4-PEG-EVC-MMAE, V-F5-PEG-EVC-MMAE, or Isotype-PEG-EVC-MMAE were prepared respectively.

[0826] The method described in Example 3.2 was followed, but different antibodies (V-F8, V-BMK3, A004-Fc, D05.m5-Fc or Isotype) were used instead of V-F5 to prepare V-F8-VC-MMAE, A004-VC-MMAE, D05.m5-VC-MMAE or Isotype-VC-MMAE.

[0827] Isotype-Gluc-Exd was prepared by following the method described in Example 3.3, but using a different antibody (Isotype-Fc) instead of V-F8.

[0828] Following the method described in Example 3.4, but using different antibodies (V-F11, Isotype-Fc) instead of V-F9, V-F11-Gly-PAB-Exatecan-D-glucuronic acid (V-F11-Gluc-Exd) and Isotype-Gluc-Exd were prepared.

[0829] Following the method described in Example 3.5, but using different antibodies (V-F9, Isotype-Fc) instead of V-F11, V-F9-RDVT-EXD and Isotype-RDVT-Exd were prepared.

[0830] Some relevant information about the ADCs prepared in the embodiments of this application and the control ADC is provided in the table below (Table 17).

[0831] Table 17 Basic Information of ADCs

[0832] Example 4. In vitro killing experiment of ADC

[0833] 1) Target cells SNU-5 or SNU620 or MKN45-CLDN18.2 (prepared as described above) or OCUM-1 (CBP60494, Nanjing Kebai Biotechnology Co., Ltd.) or AsPC-1-CLDN18.2 (prepared as described above) at 1x10 4 Or 3x10 3 1) Spread 100 μL of the sample to a plate in 96 wells and incubate overnight at 37°C with 5% CO2. Dilute the sample to be tested to 200 nM with complete culture medium, perform a 4-fold serial dilution, and add 100 μL / well to each well of the pre-spread 96-well plate. Incubate at 37°C with 5% CO2 for 3-6 days. 2) Add 20 μL / well of CCK-8 (Cell Counting Kit-8) and incubate at 37°C with 5% CO2 for 1-5 hours. 3) Read the OD. 450

[0834] As shown in Figures 14(A)(B)(C), V-F5-VC-MMAE exhibits the strongest killing ability against the three different tumor cell lines (E). max (Maximum), other ADCs have comparable killing power, but all are superior to Isotype control ADC.

[0835] As shown in Figures 15(A)(B)(C), the killing ability of V-F8-VC-MMAE against the CLDN18.2-positive tumor cell line MKN45-CLDN18.2 is comparable to that of the positive control V-BMK3-VC-MMAE. However, it is superior to the positive control V-BMK3-VC-MMAE in the CLDN18.2 and CDH17-positive cell line SNU620 and the CDH17-positive cell line SNU5. Furthermore, as the expression level of CDH17 increases, the expression level of CLDN18.2 decreases, and the killing advantage of V-F8-VC-MMAE becomes more and more obvious. It can also be seen that the killing effect of dual-target ADCs is better than that of their corresponding single-target ADCs.

[0836] As shown in Figures 16(A)(B)(C), V-F9-Gluc-Exd has comparable killing ability against CLDN18.2 monopositive tumor cell line MKN45-CLDN18.2 to V-F8-Gluc-Exd, but is superior to V-F8-Gluc-Exd in CLDN18.2 and CDH17 bipositive cell line SNU620 and CDH17 monopositive cell line SNU5.

[0837] As shown in Figures 17(A)(B)(C), V-F11-Gluc-Exd and V-F11-RDVT-EXD exhibited similar cell killing effects in the CLDN18.2 and CDH17 double-positive cell lines SNU620 and ASPC-1-CLDN18.2, as well as the CLDN18.2 single-positive tumor cell line OCUM-1.

[0838] 2) Target cells MKN45-CLDN18.2 (prepared as described above), SNU620 (CBP60488, Nanjing Kebai Biotechnology Co., Ltd.), or SNU16 (CBP60502, Nanjing Kebai Biotechnology Co., Ltd.) were seeded at 5000 cells / well, 100 μL / well, and incubated overnight at 37°C in a 5% CO2 incubator. rProtein G-MMAE (Levena; LEV-GME-100) was diluted to 200 nM with complete culture medium, and the test sample was also diluted to 200 nM with complete culture medium. The test sample and rProtein G-MMAE were mixed at a volume ratio of 1:1 (molar concentration ratio of 1:1) and incubated at room temperature for about 1 hour. The test sample and rProtein G-MMAE mixture were diluted 1:2.5 with complete culture medium, serially diluted, and 100 μL / well was added to the pre-coated 96-well culture plate and incubated at 37℃ in a 5% CO2 incubator for 3-6 days; 20 μL / well was added to CCK-8 (Cell Counting Kit-8) and incubated at 37℃ in a 5% CO2 incubator for 1-5 h; OD 450 was read.

[0839] As shown in Figures 18(A)(B)(C), the killing ability of V-F11-Protein G-MMAE in the CLDN18.2 single-positive tumor cell line MKN45-CLDN18.2 is comparable to that of the positive control molecule and the CLDN18.2 parental control molecule.

[0840] V-F11-Protein G-MMAE showed superior killing effect in the double-positive cell line SNU620 compared to the positive control molecules V-BMK3-Protein G-MMAE, CLDN18.2 parental dznD05.m5m8-Protein G-MMAE, and CDH17 parental control molecule pznA004-Protein G-MMAE.

[0841] V-F11-Protein G-MMAE was also superior to V-BMK3-Protein G-MMAE, CLDN18.2 parental dznD05.m5m8-Protein G-MMAE control molecule, and CDH17 parental control molecule pznA004-Protein G-MMAE in the CDH17 single-positive cell line SNU16.

[0842] Example 5. In vivo effects experiment of ADC

[0843] Unless otherwise specified and without contradiction with the context, the abbreviations used have the following meanings.

[0844] Example 5.1 Antitumor effect of ADCs in mouse SNU-5 subcutaneous transplantation model

[0845] This experiment was used to evaluate the efficacy of test products (including the ADCs of this invention) in a BALB / c nude mouse model of subcutaneous transplantation of human gastric cancer cells SNU-5.

[0846] Experiment A

[0847] Model building and grouping

[0848] Model construction: 7-8 week old female BALB / c nude mice (Balb / c nude, Shanghai Lingchang Biotechnology Co., Ltd.) were used. After acclimatization for 1 week, the cells were resuspended in SNU-5PBS (5×10⁻⁶) and injected into the right scapula. 6 (cells / animals), until the tumor volume grows to 100-200 mm 3 The tumors were randomly divided into 6 groups (n=4) based on their average tumor volume. The day of grouping was defined as D0, and the test drug was administered intravenously on D0 as a single dose.

[0849] The configuration information of the test sample is shown in the table below:

[0850] Dosage volume: Adjusted according to mouse body weight (dosage volume for mice = 10 μL / g × mouse body weight (g))

[0851] Data collection: After the start of drug administration, the mice were weighed twice a week, the tumor volume was measured twice a week, and the animals were observed twice a day.

[0852] Trial endpoint: based on tumor volume (1500-2000 mmHg) 3 The endpoint is determined by the animal's condition. At the endpoint, all surviving animals are euthanized and tumors are collected. The tumors are photographed, weighed, and then processed for further treatment.

[0853] Endpoint Analysis

[0854] At the end of the experiment, the following indicators were analyzed:

[0855] Tumor volume change (TGI) TV )

[0856] weight change

[0857] TGI TV Calculation formula:

[0858] TGI TV ={1-[(V t -V0) / (C t -C0)]}×100%

[0859] V t : The average tumor volume of mice in the test drug administration group on day t;

[0860] V0: The average tumor volume of mice in the test drug administration group on day 0;

[0861] C t : Average tumor volume of the solvent group mice on day t;

[0862] C0: Average tumor volume of the solvent group mice on day 0.

[0863] When TGI is calculated as described above TV When the value is >100%, the TGI calculation formula is as follows:

[0864] TGI TV =100%-[(V t -V0) / V0)]×100%

[0865] Statistical analysis

[0866] Analysis, processing, and reporting: Quantitative indicators are expressed as mean ± standard error (Mean ± SEM / SD; unit mm). 3 Description. Quantitative indicators were analyzed using one-way ANOVA or two-way ANOVA. For inter-group comparisons, a t-test was used, and p < 0.05 was considered statistically significant. Both statistical and biological significance were considered in the analysis of results.

[0867] The ADC molecules of this invention exhibit good tumor-suppressive effects, while also showing steady weight gain and good safety. Experimental results are detailed in Figures 19(A) and (B) and the table below.

[0868] Experiment B

[0869] Model construction, drug administration, data collection and analysis were the same as in Experiment A, but the model was divided into 9 groups (n=5). The day of grouping was defined as D0. The test drug was administered intravenously on D0 as a single dose.

[0870] Data processing was performed according to Experiment A. The experimental results are shown in Figure 20 (A) and (B) and the table below.

[0871] At the same dose, V-F8-VC-MMAE showed significantly superior efficacy compared to the control drug V-BMK3-VC-MMAE. At the same dose, V-F9-Gluc-Exd was equivalent to V-F8-Gluc-Exd, both achieving strong tumor suppression effects. At 3 mpk and 8 mpk, the TGI in the V-F8-Gluc-Exd treatment group was 154% and 176%, respectively, while the TGI in the V-F9-Gluc-Exd treatment group was 131% and 171%, respectively.

[0872] Example 5.2 Antitumor effect of V-ADCs in MKN45-CLDN18.2 model

[0873] Model construction: 7-8 week old female BALB / c nude mice (Balb / c nude, Shanghai Lingchang Biotechnology Co., Ltd.) were acclimatized for 1 week, and then injected into the right scapula with MKN45-CLDN18.2 (MKN-45, CBP60488, Nanjing Kebai Biomedical Technology Co., Ltd.; MKN-45-CLDN18.2 was prepared as described above) and the cell suspension was resuspended in PBS (5×10⁻⁶). 6 (cells / animals), until the tumor volume grows to 100-200 mm 3 Tumors were grouped according to their average volume.

[0874] The preparation information of the test sample is shown in the table below:

[0875] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)). Data collection: After the start of administration, weigh the mice twice a week, measure the tumor volume twice a week, and observe the animals twice a day.

[0876] Experiment A

[0877] Mice inoculated with MKN45-CLDN18.2 were randomly divided into 6 groups (n=4), with the day of grouping defined as D0. The test drug was administered intravenously on D0 as a single dose. The dosage for each group is shown in Figure 21(A).

[0878] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 21 and the table below. At the same dose, V-F5-VC-MMAE is significantly superior to other ADCs.

[0879] Experiment B

[0880] Mice inoculated with MKN45-CLDN18.2 were randomly divided into 8 groups (n=5), with the day of grouping defined as D0. The test drug was administered intravenously on D0 as a single dose. The dosage for each group is shown in Figure 22(A).

[0881] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 22(A) and the table below. At a dose of 1 mpk, V-F8-VC-MMAE was more effective than V-BMK3-VC-MMAE. In terms of overall efficacy, V-F9-Gluc-Exd was equivalent to or slightly more effective than V-F8-Gluc-Exd or V-F8-VC-MMAE.

[0882] Example 5.3 Antitumor effect of V-ADCs in SNU5-CLDN18.2 model

[0883] Model construction: 7-8 week old female NSG mice (NSG, Shanghai Southern Model Biotechnology Co., Ltd.) were acclimatized for 1 week, and then injected into the right scapula with SNU5-CLDN18.2 (SNU-5, CBP60505, Nanjing Kebai Biomedical Technology Co., Ltd.; SNU5-CLDN18.2 was prepared as described above) and the cells were resuspended in PBS (5×106 cells / animal). The tumor volume was allowed to grow to 100-200 mm. 3 Tumors were grouped according to their average volume.

[0884] The preparation information of the test sample is shown in the table below:

[0885] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)). Data collection: After the start of administration, weigh the mice twice a week, measure the tumor volume twice a week, and observe the animals twice a day.

[0886] Mice inoculated with SNU5-CLDN18.2 were randomly divided into 5 groups (n=5), with the day of grouping defined as D0. The test drug was administered intravenously on D0 as a single dose. The dosage for each group is shown in Figure 23(A).

[0887] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 23 and the table below. It can be seen that both V-F9-Gluc-Exd and V-F9-RDVT-EXD have significant tumor-suppressive effects, which are dose-dependent, and the animals tolerated the drugs well after administration.

[0888] Example 5.4 Antitumor effect of V-ADCs in SNU620 model

[0889] Model construction: 7-8 week old female NSG mice (NSG, from Shanghai Southern Model Biotechnology Co., Ltd.) were acclimatized for 1 week and then injected into the right scapula with SNU620 (CBP60508, Nanjing Kebai Biomedical Technology Co., Ltd.) and PBS mixed with 30% matrix gel to resuspend the cells (1×10⁻⁶). 7 (cells / animals), until the tumor volume grows to 100-200 mm 3 Tumors were grouped according to their average volume.

[0890] The preparation information of the test sample is shown in the table below:

[0891] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)). Data collection: After the start of administration, weigh the mice twice a week, measure the tumor volume twice a week, and observe the animals twice a day.

[0892] Experiment A

[0893] Mice inoculated with SNU620 were randomly divided into 9 groups (n=5), with the day of grouping defined as D0. The test drug was administered intravenously on D0 as a single dose. The dosage for each group is shown in Figure 24(A).

[0894] Data processing was performed according to Example 5.1, and the experimental results are shown in Figures 24(A) & (B) and the table below. It can be seen that at the same dose, V-F9-Gluc-Exd is superior to other ADCs and has good safety (body weight, Figure B).

[0895] Experiment B

[0896] Mice inoculated with SNU620 were randomly divided into 8 groups (n=5), with the day of grouping defined as D0. The test drug was administered intravenously on D0 as a single dose. The dosage for each group is shown in Figure 25(A).

[0897] Data processing was performed according to Example 5.1, and the experimental results are shown in Figures 25(A) & (B) and the table below. It can be seen that at the same dosage, V-F11-Gluc-Exd has similar efficacy to V-F11-RDVT-Exd and good safety.

[0898] Example 5.5 Antitumor effect of V-ADCs in the AsPC-1 model

[0899] Model establishment: 7-8 week old female BALB / c nude (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were injected into the right scapula with AsPC-1 (CBP60546, Nanjing Kebai Biomedical Technology Co., Ltd.) and PBS-resuspended cell solution (5×10⁻⁶). 6 (cells / animals), until the tumor volume grows to 100-200 mm 3 Mice were grouped according to the average tumor volume. Mice inoculated with AsPC-1 were randomly divided into 8 groups (n=5), with the day of grouping defined as D0. The test drug was administered intravenously on D0, with administration occurring every two weeks (QWx2). Dosage for each group is shown in Figure 26(A).

[0900] The preparation information of the test sample is shown in the table below:

[0901] Data processing was performed according to Example 5.1, and the experimental results are shown in Figures 26(A) & (B).

[0902] Example 5.6 Antitumor effects of V-ADCs in a human colorectal cancer (CRC) xenograft model

[0903] Model construction: 7-8 week old female NU / NU mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 1 week. Human tumor tissue was then subcutaneously inoculated into immunodeficient mice. Observation continued until tumor formation and a tumor volume of approximately 500-800 mmHg were achieved. 3 At that time, the tumor tissue was dissected and evenly cut into small tissue pieces of approximately 3mm × 3mm × 3mm. These small tumor tissue pieces were then inoculated subcutaneously into mice, and tumor growth was observed. When the average tumor volume reached approximately 100-200mm... 3 (average value 120±30mm) 3Tumor-bearing mice were randomly divided into 3 groups (n=5), with the day of grouping defined as D0. The test drug was administered intravenously on D0, and the administration was repeated QWx3. The dosage for each group is shown in Figure 27(A).

[0904] The preparation information of the test sample is shown in the table below:

[0905] Data processing was performed according to Example 5.1, and the experimental results are shown in Figures 27(A) & (B). It can be seen that on day 28 after administration, at a dose of 8 mg / kg, V-F11-RDVT-Exd (TGI: 179%) and V-F11-Gluc-Exd (TGI: 176%) had similar efficacy and good safety.

[0906] Example 6: In vitro side-by-side killing experiment of ADC

[0907] 1) Positive cells SNU620 (CBP60508, Nanjing Kebai Biotechnology Co., Ltd.) or SNU5-CLDN18.2 (prepared as described above) and negative cells MKN45 (CBP60488, Nanjing Kebai Biotechnology Co., Ltd.) were stained with fluorescent dyes CFSE (Invitrogen, 65-0850-84) and CellTrace Violet (Thremo, C34557 A), respectively. Positive cells SNU620 or SNU5-CLDN18.2 were seeded at 75,000 cells / 250 μL / well, and negative cells MKN45 were seeded at 25,000 cells / 250 μL / well. The two cell types were cultured together and incubated overnight at 37°C in a 5% CO2 incubator. 1) The test sample was diluted to 4 nM with complete culture medium, serially diluted 2-fold, and four doses were added at 500 μL / well to a pre-coated 24-well culture plate. The plate was incubated at 37°C for 3 days in a 5% CO2 incubator. 2) After 3 days of culture, all cells were collected into deep-well plates, and counting beads (Invitrogen, C36995) were added at 3 μL / well. The plates were centrifuged at 400g for 5 minutes, and then the cells were washed twice with DPBS. The collected cells were stained with Live / Dead dye (Thremo, L34976). 3) After staining, the cells were washed twice with 2% FBS-DPBS buffer, resuspended, and analyzed by flow cytometry (Beckman, CytoFLEX). Fluorescence signals were recorded in the V450 / B525 / R780 channels.

[0908] As shown in Figure 28, after V-F11-Gluc-Exd and V-F11-RDVT-Exd were internalized by different positive cells SNU5-CLDN18.2(A) and SNU620(B), the small molecule toxicity released by enzyme cleavage showed a significant bystander killing effect.

[0909] Sequence List Overview

[0910] 1. The antigen-binding molecule CDR of this invention

[0911] 2. VHH sequence

[0912] 3. Multispecific antibody sequences

[0913] 4. Other sequences

Claims

1. A VHH antibody that specifically binds to CLDN18.2, comprising or consisting of a heavy chain variable region, said heavy chain variable region comprising the three complementarity-determining regions (CDRs) contained in the VH as shown in any one of SEQ ID NO:15, 1-14 and 16-19; preferably, said CDR sequence according to ABM definition.

2. A VHH antibody that specifically binds to CLDN18.2, comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprising or consisting of a heavy chain variable region, wherein... The heavy chain variable region includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; among which (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:21, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:31; or (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:20, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:24; or (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:21, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of any one of the amino acid sequences shown in SEQ ID NO:24-35; or (iv) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:22, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

24.

3. The VHH antibody of claim 1 or 2, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 15, 1-14, and 16-19; or (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:15, 1-14 and 16-19.

4. A heavy chain antibody that specifically binds to CLDN18.2, comprising the VHH antibody according to any one of claims 1-3.

5. The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 1-3 linked to an antibody constant region or Fc region, preferably wherein the antibody constant region or Fc region is derived from human IgG1, human IgG2, human IgG3, or human IgG4; optionally, the Fc region comprises a hinge region or a portion thereof, for example, the Fc region being a cysteine ​​residue at position 220 or mutated to a serine residue at position 220; for example, the Fc region comprising the amino acid sequence shown in SEQ ID NO:89 or SEQ ID NO:90; optionally, the Fc region comprising an LALA mutation.

6. The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 1-3 linked to an antibody Fc region, wherein the Fc region is an Fc region derived from human IgG1 or IgG4, preferably, The Fc region (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 85 or 86; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:85 or 86; Optionally, the Fc region contains a C220S mutation in the hinge region, for example, an Fc region containing a C220S mutation. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91 or 92; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO: 91 or 92; Optionally, the Fc region contains an LALA mutation, for example, an Fc region containing an LALA mutation. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:88; (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:88; or Optionally, the Fc region contains both LALA and C220S mutations, for example, an Fc region containing both LALA and C220S mutations. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:87; (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

87.

7. The VHH antibody of any one of claims 1-3, or the heavy chain antibody of any one of claims 4-6, wherein the antibody is a chimeric antibody or a humanized antibody.

8. An antigen-binding molecule comprising the VHH antibody of any one of claims 1-3 and 7 or the heavy chain antibody of any one of claims 4-7, for example, the antigen-binding molecule being a multispecific antibody such as a bispecific antibody.

9. A multispecific antibody that specifically binds to CDH17 and CLDN18.2, wherein the antibody comprises an antigen-binding domain that specifically binds to CDH17 and an antigen-binding domain that specifically binds to CLDN18.2, wherein... The antigen-binding domain that specifically binds to CLDN18.2 is the VHH domain that specifically binds to CLDN18.

2. CLDN18.2 Preferably, the VHH CLDN18.2 It is a VHH that specifically binds to CLDN18.2 as described in any one of claims 1-3; Preferably, the multispecific antibody is a bispecific antibody.

10. The multispecific antibody of claim 9, wherein the antigen-binding domain that specifically binds to CDH17 is the VHH that specifically binds to CDH17, i.e., VHH CDH17 .

11. The multispecific antibody of claim 10, wherein the VHH that specifically binds to CDH17 comprises or is composed of a heavy chain variable region, the heavy chain variable region comprising the three complementarity-determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 50, 36-49, 51 or 61-70; preferably, the CDR sequence is defined according to ABM.

12. The multispecific antibody of claim 10 or 11, wherein the VHH that specifically binds to CDH17 comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprises or is composed of a heavy chain variable region. The heavy chain variable region includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; among which (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:58; or (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 contains or is composed of any one of the amino acid sequences shown in SEQ ID NO:54-60; or (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:71, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:72, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:73; or (iv) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:95, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:72, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

73.

13. The multispecific antibody of any one of claims 10-12, wherein the VHH that specifically binds to CDH17 comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 50, 36-49, 51, or 61-70; or (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:50, 36-49, 51 or 61-70.

14. The multispecific antibody according to any one of claims 9-13, wherein the multispecific antibody further comprises an Fc region, for example, comprising two identical or different Fc regions.

15. The multispecific antibody of claim 14, wherein the Fc region is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc; optionally, the Fc region comprises a hinge region or a portion thereof, for example, the Fc region is a cysteine ​​residue at position 220 or is mutated to a serine residue at position 220, for example, the Fc region comprises the amino acid sequence shown in SEQ ID NO:89 or the amino acid sequence shown in SEQ ID NO:90, optionally, the Fc region comprises an LALA mutation.

16. The multispecific antibody according to claim 14 or 15, wherein The Fc region (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 85 or 86; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:85 or 86; Optionally, the Fc region contains a C220S mutation in the hinge region, for example, an Fc region containing a C220S mutation. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91 or 92; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO: 91 or 92; Optionally, the Fc region contains an LALA mutation, for example, an Fc region containing an LALA mutation. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:88; (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:88; or Optionally, the Fc region contains both LALA and C220S mutations, for example, an Fc region containing both LALA and C220S mutations. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:87; (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

87.

17. The multispecific antibody according to any one of claims 9-16, which is a bispecific antibody and specifically binds to CLDN18.2 and CDH17, comprising a polypeptide chain having, from the N-terminus to the C-terminus, respectively comprising or consisting of the following: VHH A -VHH B -Fc, formula (X) VHH A and VHH B The symbols represent the VHH domains that bind antigens A and B, respectively, wherein A and B are distinct from each other and independently selected from CLDN18.2 and CDH17; wherein the symbol "-" indicates linkage via a linker or direct linkage, preferably representing a linker of 5-15 amino acids in length.

18. The multispecific antibody of claim 17, wherein antigen A is CLDN18.2 and antigen B is CDH17; or antigen A is CDH17 and antigen B is CLDN18.2, wherein VHH CLDN18.2 It is a VHH antibody that specifically binds to CLDN18.2 as defined in any one of claims 1-3; and / or VHH CDH17 It is a VHH that specifically binds to CDH17 as defined in any one of claims 11-13; Preferably, the VHH CLDN18.2 The compound contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of heavy chain variable regions, wherein the heavy chain variable regions contain complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:21, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and VHH... CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:31; and the VHH CDH17 It contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of heavy chain variable regions, wherein the heavy chain variable regions contain complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:58; More preferably, the VHH CLDN18.2 Contains or consists of the amino acid sequence shown in SEQ ID NO:15, and the VHH CDH17 It contains or consists of the amino acid sequence shown in SEQ ID NO:

50.

19. The multispecific antibody of any one of claims 9-18, wherein the antigen-binding domains are connected to each other or to the Fc region via a linker, for example, the linker comprising (G4S)n, wherein n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5.

20. The multispecific antibody of claim 18 or 19, comprising or consisting of two polypeptide chains, optionally the two polypeptide chains being identical.

21. The multispecific antibody of any one of claims 18-20, wherein the polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO:74-82, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

22. A VHH antibody that specifically binds to CDH17, comprising or consisting of a heavy chain variable region, said heavy chain variable region comprising the three complementarity-determining regions (CDRs) contained in the VH as shown in any one of SEQ ID NO: 50, 36-49, 51 or 61-70; preferably, said CDR sequence according to the definition of ABM.

23. The VHH antibody of claim 22, comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprising or consisting of a heavy chain variable region, wherein... The heavy chain variable region includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; among which (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:58; or (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:52, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and VHH CDR3 contains or is composed of any one of the amino acid sequences shown in SEQ ID NO:54-60; or (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:71, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:72, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:73; or (iv) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:95, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:72, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

73.

24. The VHH antibody of claim 22 or 23, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 50, 36-49, 51, or 61-70; or (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:50, 36-49, 51 or 61-70.

25. A heavy chain antibody that specifically binds to CDH17, comprising the VHH antibody according to any one of claims 22-24.

26. The heavy chain antibody of claim 25, comprising the VHH antibody of any one of claims 22-24 linked to an antibody constant region or Fc region, preferably wherein the antibody constant region or Fc region is derived from human IgG1, human IgG2, human IgG3, or human IgG4, optionally wherein the Fc region comprises a hinge region or a portion thereof, for example, the Fc region being a cysteine ​​residue at position 220 or mutated to a serine residue at position 220, for example, the Fc region comprising the amino acid sequence shown in SEQ ID NO:89 or SEQ ID NO:90, optionally, the Fc region comprising an LALA mutation.

27. The heavy chain antibody of claim 26, comprising the VHH antibody of any one of claims 1-3 linked to an antibody Fc region, wherein the Fc region is an Fc region derived from human IgG1 or IgG4, preferably, The Fc region (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 85 or 86; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:85 or 86; Optionally, the Fc region contains a C220S mutation in the hinge region, for example, an Fc region containing a C220S mutation. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91 or 92; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO: 91 or 92; Optionally, the Fc region contains an LALA mutation, for example, an Fc region containing an LALA mutation. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:88; (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:88; or Optionally, the Fc region contains both LALA and C220S mutations, for example, an Fc region containing both LALA and C220S mutations. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:87; (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

87.

28. The VHH antibody of any one of claims 22-24, or the heavy chain antibody of any one of claims 25-27, wherein the antibody is a chimeric antibody or a humanized antibody.

29. An antigen-binding molecule comprising the VHH antibody of any one of claims 22-24 and 28 or the heavy chain antibody of any one of claims 25-28, for example, the antigen-binding molecule being a multispecific antibody such as a bispecific antibody.

30. A nucleic acid molecule encoding an antibody of any one of claims 1-7 and 9-28, or an antigen-binding molecule as described in claim 8 or 29.

31. An expression vector comprising the nucleic acid molecule of claim 30, preferably, said expression vector is pCDNA, such as pCDNA3.

4.

32. A host cell comprising the nucleic acid molecule of claim 30 or the expression vector of claim 31, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells.

33. A method for preparing an antibody according to any one of claims 1-7 and 9-28, or an antigen-binding molecule according to claim 8 or 29, the method comprising culturing a host cell containing a nucleic acid molecule according to claim 30 or an expression vector according to claim 31 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

34. An antibody comprising any one of claims 1-7 and 9-28, or an immunoconjugate or immunofusion of an antigen-binding molecule as described in claim 8 or 29.

35. The antibody-drug conjugate shown in formula (I): Ab-(LD) p (I) Or its pharmaceutically acceptable salts or solvates, in: Ab is an antibody according to any one of claims 1-7 and 9-28, or an antigen-binding molecule according to claim 8 or 29; L is the connector; D is a drug, such as an anti-tumor compound; and p is an integer selected from 1 to 16, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12.

36. The antibody-drug conjugate of claim 35 or a pharmaceutically acceptable salt or solvate thereof, wherein the antitumor compound is a cytotoxic agent, such as a camptothecin, aurastatin, such as eczema, Dxd, MMAE, or MMAF.

37. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 35, wherein D has the structure shown in formula (D-1a) or formula (D-1b): Where R 1a Selected from H and C1-C6 alkyl groups; R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ; R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and R 4a and R 5a Independently selected from H and C1-C4 alkyl groups; or Where R 1b R 2b R 3b R 4b R 5b and R 8b Each was independently selected from C 1-8 Alkyl groups; such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; R 6b and R 7b Each was independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy; R 9b Selected from C 1-8 Alkyl groups and COOH; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and R 10b Selected from OH and H.

38. The antibody-drug conjugate according to claim 37, or a pharmaceutically acceptable salt or solvate thereof, wherein, R 1a For H; R 2a It is a C1-C6 alkyl group; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl; R 1b R 4b and R 8b Each was independently selected from C 1-2 Alkyl; preferably methyl; R 2b R 3b and R 5b Each was independently selected from C 3-4 alkyl; R 6b and R 7b Each was independently selected from C 1-2 alkoxy groups; and R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H.

39. The antibody-drug conjugate according to claim 37, or a pharmaceutically acceptable salt or solvate thereof, wherein, D has the structure shown in equation (D-2a) or equation (D-2b): Where R 1a R 2a R 3a and R 4a As defined in claim 37 or 38; or Where R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined in claim 37 or 38.

40. The antibody-drug conjugate according to claim 37, or a pharmaceutically acceptable salt or solvate thereof, wherein, D has the structure shown in formula (D-3a) or (D-3b):

41. The antibody-drug conjugate according to claim 37, or a pharmaceutically acceptable salt or solvate thereof, wherein, D has the structure shown in formula (D-4a) or (D-4b):

42. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 35-41, wherein... -L- has the following structure: -Z-L1-L2-L3- in Z is selected from Where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non-existent, Where n1 and m1 are independently selected from 0 to 20 integers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; L2 is a single amino acid or a peptide residue selected from 2-8 amino acids; and L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6. Preferably, L2 is selected from: -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-, and -Arg-Asp-Val-Thr-.

43. The antibody-drug conjugate according to claim 41, or a pharmaceutically acceptable salt or solvate thereof, wherein... Z is selected from Where m is 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non-existent, Where n1 is an integer independently selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8; L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6.

44. The antibody-drug conjugate according to claim 43, or a pharmaceutically acceptable salt or solvate thereof, wherein... Z is selected from L1 is selected from non-existent or L2 is selected from -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, and -Arg-Asp-Val-Thr-; L3 is Among them, Su and R 1c As defined in claim 43; n2 and n5 are independently 0 or 1.

45. The antibody-drug conjugate according to claim 44, or a pharmaceutically acceptable salt or solvate thereof, wherein, L3 is Wherein Su is as defined in claim 44, Preferably, Su is selected from 46. ​​The antibody-drug conjugate according to claim 45, or a pharmaceutically acceptable salt or solvate thereof, wherein, Su selected 47. The antibody-drug conjugate according to claim 46, or a pharmaceutically acceptable salt or solvate thereof, wherein, Su 48. The antibody-drug conjugate according to claim 47, or a pharmaceutically acceptable salt or solvate thereof, wherein, Su Preferably, L3 is selected from:

49. The antibody-drug conjugate according to claim 42, or a pharmaceutically acceptable salt or solvate thereof, wherein, -Z-L1-L2-L3- is selected from the following structures 50. The antibody-drug conjugate according to claim 35, or a pharmaceutically acceptable salt or solvate thereof, wherein, The antibody-drug conjugate is selected from... Wherein Ab and p are as defined in claim 35.

51. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 35-50, wherein, The antibody-drug conjugate has an average DAR of 2-10, for example, 4-8.

52. A pharmaceutical composition comprising an antibody according to any one of claims 1-7 and 9-28, or an antigen-binding molecule according to claim 8 or 29, an immunoconjugate or immunofusion according to claim 34, an antibody-drug conjugate according to any one of claims 35-51, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.

53. Use of an antibody according to any one of claims 1-7 and 9-28, or an antigen-binding molecule according to claim 8 or 29, an immunoconjugate or immunofusion according to claim 34, an antibody-drug conjugate according to any one of claims 35-51, or a pharmaceutically acceptable salt or solvate thereof, for the preparation of a medicament.

54. The use of claim 53, wherein the drug is used to treat and / or prevent cancer in an individual, preferably said cancer being CLDN18.2 and / or CDH17 positive cancer.

55. The use of claim 53 or 54, wherein the medicament is used to treat and / or prevent cancer in an individual, and the cancer is selected from digestive system cancers such as stomach cancer, pancreatic cancer, colorectal cancer, or other tumors such as ovarian cancer, head and neck cancer, etc.

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