Antibody, antibody-drug conjugate, and use thereof

Abstract

The present invention relates to an antigen binding molecule and an antibody-drug conjugate (ADC), and in particular to an antibody and ADC targeting CDH17 and / or EGFR, and a composition containing the antigen binding molecule or the ADC and a therapeutic use of the antigen binding molecule or the ADC.

Description

Antibodies and their drug conjugates and their uses

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to PCT International Patent Application No. PCT / CN2025 / 071545, filed on January 9, 2025, 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 EGFR and / or CDH17, as well as compositions containing said antibodies or ADCs and their therapeutic applications. Background Technology

[0004] Epidermal growth factor receptor (EGFR, ErbB-1, or HER1) is a cell surface receptor for extracellular protein ligands belonging to the epidermal growth factor family (EGF-family). EGFR is a member of the ErbB receptor family, a subfamily of tightly linked proteins consisting of four receptor tyrosine kinases: EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). Mutations affecting EGFR expression or activity may lead to cancer. EGFR is a transmembrane protein widely expressed on the surface of various normal and cancerous cells. EGFR plays a crucial role in cell proliferation, differentiation, and survival; its overexpression or mutation is often associated with the occurrence and progression of various cancers. Targeted therapy against EGFR has become an important strategy in cancer treatment. Antibody-drug conjugates (ADCs) are a new class of targeted therapies that selectively kill cancer cells while reducing damage to normal cells by conjugating cytotoxic drugs with specific antibodies.

[0005] 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.

[0006] 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).

[0007] 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 EGFR and CDH17. Intratumoral and intertumoral heterogeneity leads to resistance to these treatments, limiting their efficacy. Furthermore, single-target strategies cannot adequately address the complex biological characteristics and microenvironment of tumors, resulting in poor therapeutic outcomes. By targeting two different biomarkers, different tumor subpopulations can be effectively attacked, reducing or delaying the development of treatment resistance. Therefore, developing a novel bispecific ADC not only holds promise for providing a more effective treatment for specific tumor types but also brings new strategies and hope to cancer therapy.

[0008] Invention Overview

[0009] Analysis of immunohistochemical and bioinformatics data from clinicopathological samples (see, e.g., DOI:10.1038 / s41421-021-00312-y) revealed that EGFR and CDH17 targets are co-expressed in many tumors, especially colorectal cancer tissue samples, while their co-expression rate is low in normal tissues. Therefore, the inventors first obtained single-domain antibodies targeting EGFR and CDH17, and based on these single-domain antibodies, proposed and designed antibody molecules that simultaneously target EGFR and CDH17 to cover tumor cells expressing EGFR and / or CDH17 targets, thereby overcoming intratumoral heterogeneity and improving therapeutic efficacy. Furthermore, the inventors proposed an innovative antibody model based on nanobodies, resulting in a final antibody molecular weight that is only about 70% of the molecular weight of conventional four-chain antibodies. This achieves a higher enrichment rate of antibody molecules in tumor tissues, further improving therapeutic efficacy. Based on these designs and discoveries, the inventors have developed the single-domain antibody of the present invention, as well as multispecific antibodies constructed therefrom, and drug conjugates constructed based on said multispecific antibodies and their uses, particularly in cancer treatment.

[0010] Therefore, in a first aspect, the present invention provides a multispecific antibody that binds EGFR and CDH17, a pharmaceutical composition thereof, and use thereof, wherein the antibody comprises at least one antigen-binding domain that specifically binds EGFR and at least one antigen-binding domain that specifically binds CDH17.

[0011] In some specific embodiments, the present invention provides a bispecific antibody that specifically binds to EGFR and CDH17, wherein the bispecific antibody comprises one or two antigen-binding domains, such as VHH, that specifically bind to EGFR and two antigen-binding domains, such as VHH, that specifically bind to CDH17.

[0012] In some specific embodiments, the present invention provides a bispecific antibody, wherein the bispecific antibody comprises an antigen-binding domain VHH that specifically binds to EGFR. EGFR And two antigen-binding domains that specifically bind to CDH17, VHH CDH17 The bispecific antibody comprises two polypeptide chains, one polypeptide chain comprising two tandem VHHs and optionally an Fc region, and the other polypeptide chain comprising one VHH and optionally an Fc region.

[0013] In some specific embodiments, the present invention provides a bispecific antibody, wherein the bispecific antibody comprises two antigen-binding domains VHH that specifically bind to EGFR. EGFR And two antigen-binding domains that specifically bind to CDH17, VHHCDH17 The bispecific antibody comprises two polypeptide chains, one of which contains two tandem VHH molecules. CDH17 and VHH EGFR And optionally the Fc region, and another polypeptide chain contains two tandem VHHs CDH17 and VHH EGFR , and optionally the Fc region; preferably, the two polypeptide chains are identical.

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding the multispecific antibody of the present invention, an expression vector comprising the nucleic acid molecule, a host cell comprising the nucleic acid molecule or the expression vector, or a method for preparing the multispecific antibody.

[0015] In a third aspect, the present invention provides an antibody-drug conjugate (ADC) comprising the antibody of the present invention, a pharmaceutical composition thereof, and its use.

[0016] The antibody-drug conjugate of the present invention has the following advantages:

[0017] (1) It specifically binds to target cells expressing human CDH17 and / or EGFR; (2) It has a significant bystander effect;

[0018] (3) It has high anti-tumor efficacy, stronger killing effect on tumor cells and / or stronger inhibitory effect on tumor growth, especially stronger killing effect on tumors with high expression of CDH17 and / or EGFR and / or stronger inhibitory effect on tumor growth.

[0019] (4) It has significantly enhanced and even unexpectedly enhanced antitumor activity;

[0020] (5) It has better product uniformity.

[0021] (6) It has low toxicity;

[0022] (7) It has good physicochemical stability and;

[0023] (8) It has good drug-like properties.

[0024] 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.

[0025] Invention Details

[0026] 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.

[0027] 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.

[0028] I. Definition

[0029] 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.

[0030] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0031] 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.

[0032] When referring to “molecules of the present invention” or “molecules of this document”, it encompasses the various molecules as defined herein, including antigen-binding molecules such as antibodies, immunoconjugates, immunofusions, antibody-drug conjugates, or pharmaceutically acceptable salts or solvates thereof.

[0033] In this document, the term "antigen-binding molecule" refers to a molecule, such as a protein or polypeptide, 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 EGFR and / or CDH17, antigen-binding molecules binding EGFR and / or CDH17 are also referred to as EGFR-binding molecules, CDH17-binding molecules, or EGFR / 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".

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In this document, unless otherwise stated, the term "EGFR" includes any variant of human EGFR, 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 EGFR expressed naturally or recombinantly in cells or expressed on natural or recombinant cells, but also recombinantly expressed fusion proteins containing EGFR or fragments thereof. An example of EGFR is the human EGFR protein containing the amino acid sequence under UniProt_P00533. In this document, unless specifically indicated, the term "EGFR" refers to EGFR derived from humans. In the antibodies of this invention, "antigen-binding specificity against EGFR," i.e., "antigen-binding domain that specifically binds to EGFR," is provided by the VHH domain.

[0042] In this document, the term "EGFR-positive" cell refers to a cell that expresses EGFR positively on its cell surface, such as cancer cells, modified cancer cells, or modified non-tumor cells. The EGFR expression level on the cell surface can be determined using any conventional method known in the art for determining cell surface antigen expression levels, such as FACS detection or immunofluorescence staining. EGFR is expressed at significantly higher levels on a variety of tumor cells than on normal tissues / cells, for example, AsPc-1 (pancreatic cancer cells). Preferably, in this document, EGFR-positive cells are EGFR-positive tumor cells.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] In this document, the term "native 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 Pro230 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 native immunoglobulin, such as the sequence E216 to P230 or the sequence D221 to P230 according to EU designations.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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 .

[0060] 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., EGFR 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., EGFR 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.

[0061] 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.

[0062] In this document, if an amino acid sequence (e.g., VHH) is specific for two different antigens or antigenic determinants (e.g., EGFR or CDH17 from different mammalian species, such as human EGFR or human CDH17, cynomolgus monkey EGFR or cynomolgus monkey CDH17), then it is said to be "cross-reactive" to these two different antigens or antigenic determinants. Antibodies possessing 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 ADC molecules composed of antibodies. In some embodiments, the antibodies of the present invention, such as anti-EGFR antibodies or anti-CDH17 antibodies or bispecific antibodies, preferably possess human-monkey species cross-reactivity.

[0063] 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 initiate endocytosis mediated by EGFR and / or CDH17 receptors upon binding to EGFR and / 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.

[0064] 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.

[0065] 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.

[0066] In this document, the terms "immunoconjugate," "immunoconjugate fusion," or "immunoconjugate" 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.

[0067] The term "antibody-drug conjugate (ADC)" refers to a compound entity obtained by linking an antigen-binding molecule to a (small molecule) drug via a linker. In this document, the term "antibody-drug conjugate" or "ADC" includes its pharmaceutically acceptable salts and solvent compounds, as well as other equivalents. The drug compound portion of an ADC may be referred to herein as the "payload" or "toxin."

[0068] The term "linker" refers to a structural segment that covalently links a drug (e.g., a small molecule drug) to an antigen-binding molecule. It should be understood that a linker has functional groups that can form bonds with functional groups of the antigen-binding molecule before being linked. In some cases, the linker may also have a degradable portion and optionally a hydrophilicity modulating module, such as a PEG segment. In some embodiments of the ADC according to the invention, the linker is preferably "degradable," thereby enabling it to break and release the payload after the ADC is delivered to the target region (e.g., a target tumor tissue site). Such "degradable linkers" available include, for example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers. Non-limiting examples of linkers include those involved in the embodiments and examples of the invention.

[0069] The term "linker-payload" refers to a compound formed by the connection of a payload and a linker. In some cases, linker-payloads are used as intermediates in ADC synthesis. Non-limiting examples of linker-payloads include those involved in embodiments and examples of the present invention.

[0070] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 1-16 carbon atoms, for example, 1-12 carbon atoms, 1-10 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. 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.

[0071] 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. The alkenyl group preferably contains 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl groups.

[0072] 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, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentyynyl, and hexynyl.

[0073] The term “halogen” or “halogenated” refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0074] The term "haloalkyl" refers to an alkyl group as defined herein, which is substituted with one or more halogen groups. Haloalkyl groups may preferably be monohaloalkyl, dihaloalkyl, or polyhaloalkyl (including perhaloalkyl). Monohaloalkyl groups may contain one iodine, bromine, chlorine, or fluorine group in the alkyl group. Dihaloalkyl and polyhaloalkyl groups may contain two or more identical halogen atoms or combinations of different halogen groups in the alkyl group. Preferably, polyhaloalkyl groups contain at most 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhaloalkyl refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0075] The term “haloalkenyl” refers to an alkenyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The term “haloynyl” refers to an ynyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The meaning of “halogenated” as defined for “haloalkyl” may apply to both “haloalkenyl” and “haloynyl”.

[0076] The terms "alkoxy" and "alkyl-O-" are used interchangeably to refer to an alkyl group as defined above, linked by an oxygen atom. Preferably, the alkoxy group has 1-8 carbon atoms (C... 1-8 alkoxy group), 1-6 carbon atoms (C 1-6 alkoxy group), 1-4 carbon atoms (C 1-4 alkoxy group or 1-3 carbon atoms (C 1-3 Alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexoxy, heptoxy, octoxy, etc.

[0077] The term "penturonic acid" refers to compounds formed by oxidizing the primary hydroxyl group of a pentose sugar to a carboxyl group. Examples of penturonic acids include, but are not limited to, xyuronic acid and arabinuronic acid.

[0078] The term "hexuronic acid" refers to compounds formed by oxidizing the primary hydroxyl group of a hexose to a carboxyl group. Examples of hexuronic acids include, but are not limited to, glucuronic acid, galacturonic acid, and mannuronic acid.

[0079] 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:

[0080] 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.

[0081] Unless otherwise specified, the amino acids in this invention refer to L-amino acids.

[0082] 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.

[0083] 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, intolerable side effects. Where there is no contradiction in the context, "pharmaceutically acceptable" and "medicinal" are used interchangeably.

[0084] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention, 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 conjugates 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.

[0085] The term "solvent" refers to an association formed by one or more solvent molecules with the ADC antibody-drug conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0086] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug portion (D) coupled to the Ab portion described herein to the Ab portion. In some embodiments described herein, the DAR may be determined by p in Formula I, for example, the DAR may be 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR may also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug portion (D) coupled to the Ab portion described herein to the Ab portion in the product as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 16, for example 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, for example 1.0-8.0, 2.0-6.0, for example 0.5, 0.6, 0.7, 0.8, 0.9, 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, 0, 7.9, 8, 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. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population or mixture of ADC molecules that contains ADC molecules having the same and / or different DAR values.

[0087] 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.

[0088] 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.

[0089] The term "therapeutic agent" encompasses any substance that is effective in preventing or treating diseases such as cancer, including chemotherapeutic agents, cytotoxic agents, immunomodulators (such as immunosuppressants), other antibodies, small molecule drugs, angiogenesis inhibitors, or cytokines.

[0090] "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 such as eczetidine (a topoisomerase I inhibitor Exatecan) and Dxd (a novel topoisomerase I inhibitor Exatecan derivative), and auristatin compounds such as monomethyl auristatin E (MMAE) and MMAF.

[0091] 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.

[0092] The term "chemotherapeutic agents" includes chemical compounds that are useful in treating cancer or immune system diseases.

[0093] The term "immunomodulator" refers to natural or synthetic active agents or drugs that inhibit or modulate (e.g., activate) 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.

[0094] 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 liquid tumors. In some embodiments, cancers suitable for treatment by the antibodies or immune conjugates or immune fusions of the present invention include CDH17-positive and / or EGFR-positive tumors / cancers, including their metastatic forms. Examples of cancer include the specific cancers mentioned in the embodiments section herein.

[0095] 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, symptom relief, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or mitigation of the disease state, and relief or improvement of 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.

[0096] In this document, "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 programs. 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.

[0097] 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".

[0098] The term "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 doses of antibodies or ADCs can vary depending on various factors such as disease state, individual age, sex, weight, and the ability of the antibody or ADC to elicit the desired response in the individual. A therapeutic effective dose is also a dose in which any toxic or harmful effects of the antibody or ADC are less than the beneficial therapeutic effect. Relative to untreated subjects, a "therapeutic effective dose" preferably inhibits measurable parameters (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit measurable parameters (e.g., cancer) can be evaluated in animal model systems that predict efficacy in human tumors.

[0099] The term "preventive 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 preventive effective dose is less than the therapeutic effective dose.

[0100] 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.

[0101] The terms “individual” or “subject” are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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 or subject is a human being.

[0102] 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.

[0103] 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.

[0104] The terms “drug combination,” “combination product,” “drug conjugate,” or “combination product” refer to non-fixed or fixed combinations, 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 separate formulations, and their formulations may be the same or different.

[0105] 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.

[0106] 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).

[0107] II. Antigen-binding molecules, nucleic acids, and their preparation methods

[0108] In some embodiments, the present invention relates to antigen-binding molecules that specifically bind to CDH17 and / or EGFR, such as VHH antibodies, heavy chain antibodies, full-length antibodies, or multispecific antibodies.

[0109] I. CDH17 single-domain antibody and its heavy chain antibody

[0110] In one aspect, this 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 this invention specifically binds to mammalian CDH17, such as human CDH17 or cynomolgus monkey CDH17.

[0111] 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 .

[0112] 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:20-32; preferably, the CDR sequences are defined according to ABM.

[0113] 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:20-32; preferably, said CDR sequence is defined according to ABM.

[0114] 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

[0115] The VHH CDR1 comprises, or is composed of, an amino acid sequence selected from SEQ ID NO:14, 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:14; or

[0116] The VHH CDR2 comprises, or is composed of, the amino acid sequence of SEQ ID NO:15, 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:15; or

[0117] The VHH CDR3 comprises or is composed of an amino acid sequence selected from any one of SEQ ID NO:16-19, 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:16-19.

[0118] 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 VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:14, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15, and VHH CDR3 comprises or is composed of the amino acid sequence shown in any one of SEQ ID NO:16-19.

[0119] 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

[0120] (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: 20-32; or

[0121] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:20-32; or

[0122] (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:20-32, preferably, the amino acid changes do not occur in the CDR region.

[0123] 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:20-32.

[0124] 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.

[0125] 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...

[0126] (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;

[0127] (ii) Replace the best-matching human CDR sequence with a VHH CDR sequence;

[0128] (iii) Optionally, humanized VHH sequences can be generated by reverting multiple residues in the mutant framework region and removing them by post-translational modification (PTM);

[0129] (iv) 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;

[0130] (v) Optional sequencing of VHH antibodies.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] In some embodiments, the anti-CDH17 heavy chain antibody of the present invention comprises VHH as defined herein. CDH17 Or the heavy chain variable region therein, and the heavy chain constant region or the Fc region of the heavy chain constant region.

[0135] 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.

[0136] 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.

[0137] 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:40. 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:41. 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:57. In some embodiments, the Fc region linked to the VHH antibody retains cysteine ​​C at position 220.

[0138] In some embodiments, the Fc region includes a mutation that reduces or eliminates the Fc region from the Fcγ receptor, such as the LALA mutation. For example, an 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:35, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:35.

[0139] In some embodiments, the Fc region includes mutations that reduce or eliminate the Fc region and the Fcγ receptor, such as the LALA mutation, and a complete hinge region containing the C220S mutation. For example, the Fc region containing the LALA mutation and C220S 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:56, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:56.

[0140] 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.

[0141] The Fc region described in this context applies to the heavy chain antibody of the present invention that specifically binds to CDH17.

[0142] In some embodiments, the Fc region is derived from IgG1 and comprises the entire hinge region, which contains 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: 33, 34, 35, or 56.

[0143] 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:

[0144] (i) It binds to CDH17-expressing cells, such as tumor cells, with high affinity;

[0145] (ii) exhibits species cross-reactivity with cynomolgus monkey CDH17; and / or

[0146] (iii) Having endocytic activity on CDH17-positive cells, for example, antibodies with higher levels of known CDH17.

[0147] 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.

[0148] In some aspects, the CDH17-specific single-domain antibody VHH of the present invention, or heavy-chain antibodies containing it such as VHH-Fc, or multispecific antibodies, exhibit 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).

[0149] In some embodiments, the single-domain antibody VHH that specifically binds to CDH17 of the present invention, or heavy-chain antibodies containing it such as VHH-Fc, or multispecific antibodies, exhibit cross-reactivity with human and monkey CDH17.

[0150] In some embodiments, the CDH17-specific single-domain antibody VHH of the present invention, or a heavy-chain antibody containing it such as VHH-Fc, or a multispecific antibody, has 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.

[0151] In some embodiments, after one-step purification from antibody products generated from recombinant mammalian cells using protein A affinity chromatography, the single-domain antibody VHH that specifically binds to CDH17 of the present invention, or heavy-chain antibodies containing it such as VHH-Fc, or multispecific antibodies, can achieve a purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher, as determined by SEC-HPLC.

[0152] II. EGFR single-domain antibodies and their heavy chain antibodies

[0153] In another aspect, the present invention relates to an antigen-binding molecule that specifically binds to EGFR. In some embodiments, the antigen-binding molecule is a single-domain antibody that binds to EGFR, or a heavy-chain antibody or multispecific antibody comprising said single-domain antibody. In some embodiments, the antigen-binding molecule of the present invention binds to mammalian EGFR, such as human EGFR or cynomolgus monkey EGFR.

[0154] In some embodiments, the anti-EGFR 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. However, it should be understood that the CDR can also be defined in any other manner known in the art. In this document, the anti-EGFR single-domain antibody or VHH antibody will also be referred to as VHH. EGFR .

[0155] In some embodiments, the anti-EGFR 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:8-13; preferably, the CDR sequences are defined according to ABM.

[0156] In some embodiments, the anti-EGFR 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:8-13; preferably, said CDR sequence is defined according to AbM.

[0157] In some embodiments, the anti-EGFR VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-EGFR 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

[0158] The VHH CDR1 comprises, or is composed of, an amino acid sequence selected from SEQ ID NO:1, 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:1; or

[0159] The VHH CDR2 comprises, or is composed of, any of the amino acid sequences in SEQ ID NO:2-4, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to any of the amino acid sequences in SEQ ID NO:2-4; or

[0160] The VHH CDR3 comprises or is composed of an amino acid sequence selected from any one of SEQ ID NO:5-7, 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:5-7.

[0161] In some embodiments, the anti-EGFR VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-EGFR 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

[0162] (i) The VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1, the VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:2, and the VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:5.

[0163] (ii) The VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:1, the VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:3, and the VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:6; or

[0164] (iii) The VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1, the VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:4, and the VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:7.

[0165] In some embodiments, the anti-EGFR VHH antibody of the present invention comprises or is composed of a heavy chain variable region, said heavy chain variable region

[0166] (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: 8-13; or

[0167] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:8-13; or

[0168] (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 shown in any of SEQ ID NO:8-13, preferably, the amino acid changes do not occur in the CDR region.

[0169] In some embodiments, the anti-EGFR 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:8-13.

[0170] In some embodiments, the anti-EGFR 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.

[0171] In one embodiment, the anti-EGFR 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...

[0172] (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;

[0173] (ii) Replace the best-matching human CDR sequence with a VHH CDR sequence;

[0174] (iii) and optionally by reverting multiple residues in the mutant framework region and removing the mutation by post-translational modification (PTM), a humanized VHH sequence is generated;

[0175] (iv) 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;

[0176] (iv) Optional sequencing of VHH antibodies.

[0177] 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.

[0178] In another aspect of the invention, the invention also provides a heavy chain antibody comprising the heavy chain variable region of the anti-EGFR VHH antibody of the invention.

[0179] In some embodiments, the anti-EGFR 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, a VHH-CH1-Fc region, or a VHH-Fc region. In one embodiment, the heavy chain antibody comprises the VHH antibody of the present invention and an Fc region located at its C-terminus.

[0180] In some embodiments, the anti-EGFR heavy chain antibody of the present invention comprises VHH as defined herein. EGFR Or the heavy chain variable region therein, and the heavy chain constant region or the Fc region of the heavy chain constant region.

[0181] In one embodiment, the anti-EGFR heavy chain antibody comprises the Fc portion 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.

[0182] In some embodiments, the anti-EGFR 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.

[0183] In some embodiments, the anti-EGFR 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:40. 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:41. 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:57. In some embodiments, the Fc region linked to the VHH antibody retains cysteine ​​C at position 220.

[0184] In some embodiments, the Fc region includes a mutation that reduces or eliminates the Fc region from the Fcγ receptor, such as the LALA mutation. For example, an 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:35, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:35.

[0185] In some embodiments, the Fc region includes mutations that reduce or eliminate the Fc region and the Fcγ receptor, such as the LALA mutation, and the entire hinge region containing the C220S mutation. For example, the Fc region containing the LALA mutation and C220S 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:56, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:56.

[0186] In one embodiment, the heavy chain antibody that specifically binds to EGFR 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.

[0187] The Fc region described in this context applies to the heavy chain antibodies of this invention that specifically bind to EGFR.

[0188] In some embodiments, the Fc region is derived from IgG1 and comprises the entire hinge region, which contains 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: 33, 34, 35, or 56.

[0189] The single-domain antibody VHH that specifically binds to EGFR, or a heavy-chain antibody containing it, such as VHH-Fc, may have one or more of the following properties:

[0190] (i) Binds to EGFR-expressing cells, such as tumor cells, with appropriate affinity;

[0191] (ii) exhibiting species cross-reactivity with EGFR in cynomolgus monkeys; and / or

[0192] (iii) It has endocytic activity on EGFR-positive cells.

[0193] In some embodiments, the single-domain antibody VHH that specifically binds to EGFR of the present invention, or a heavy-chain antibody containing it, such as VHH-Fc, or a multispecific antibody, specifically binds to human EGFR or cynomolgus monkey EGFR.

[0194] In some aspects, the EGFR-specific single-domain antibody VHH of the present invention, or heavy-chain antibodies containing it such as VHH-Fc, or multispecific antibodies, exhibit appropriate binding affinity to EGFR-expressing tumor cells. In some embodiments, the EGFR-specific single-domain antibody VHH of the present invention, or heavy-chain antibodies containing it such as VHH-Fc, or multispecific antibodies, exhibit appropriate affinity to EGFR-expressing tumor cells and therefore lower EGFR-related toxicity. The cell-binding affinity of the antibody to EGFR-positive tumor cells can be reflected by FACS or ELISA assays (e.g., the assays described in the examples).

[0195] In some embodiments, the single-domain antibody VHH that specifically binds to EGFR, or heavy-chain antibodies containing it such as VHH-Fc, or multispecific antibodies, exhibit cross-reactivity with human and monkey EGFR.

[0196] In some embodiments, the multispecific EGFR-binding single-domain antibody VHH or a heavy-chain antibody containing it, such as VHH-Fc, or a multispecific antibody of the present invention, possesses EGFR-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.

[0197] In some embodiments, after one-step purification from antibody products generated from recombinant mammalian cells using protein A affinity chromatography, the purity of the EGFR-specific single-domain antibody VHH or heavy-chain antibodies containing it, such as VHH-Fc or multispecific antibodies, can reach 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher, as determined by SEC-HPLC.

[0198] III. The multispecific antibody of the present invention

[0199] Through in-depth research, the inventors discovered that by designing antibody molecules that simultaneously target EGFR and CDH17, it is possible to target more tumor cells, overcome intratumoral heterogeneity, and improve anti-tumor efficacy.

[0200] Therefore, in a third aspect, the antigen-binding molecule of the present invention is a multispecific antibody that specifically binds to EGFR and CDH17, wherein the antibody comprises at least one antigen-binding domain specifically binding to EGFR and at least one antigen-binding domain specifically binding to CDH17. The multispecific antibody of the present invention can be a bispecific antibody. In some aspects, to increase the half-life of the antibody of the present invention in animal circulation, 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 polypeptide chains, wherein the domains located on the same chain are linked by a linker or directly as needed.

[0201] In some embodiments, the present invention relates to a multispecific antibody comprising

[0202] (i) Specifically binds to the antigen-binding domain of EGFR; and

[0203] (ii) Specifically binds to the antigen-binding domain of CDH17.

[0204] In some embodiments, the multispecific antibody of the present invention is a bispecific antibody that specifically binds to EGFR and specifically binds to CDH17.

[0205] In some embodiments, the valence (i.e., the total number of antigen-binding domains) of the multispecific antibody according to the invention is 2-6 valence, preferably 3-4 valence, and most preferably 3 valence.

[0206] In some embodiments, the multispecific antibody comprises at least one (preferably one or two) antigen-binding domains that specifically bind to EGFR, such as VHH, and at least one (preferably two) antigen-binding domains that specifically bind to CDH17, such as VHH.

[0207] In some embodiments, the multispecific antibody comprises one or two antigen-binding domains that specifically bind to EGFR, such as VHH, and two antigen-binding domains that specifically bind to CDH17, such as VHH. Preferably, the two antigen-binding domains that specifically bind to EGFR are identical, and / or the two antigen-binding domains that specifically bind to CDH17 are identical.

[0208] In some preferred embodiments, the multispecific antibody comprises one antigen-binding domain, such as VHH, that specifically binds to EGFR and two antigen-binding domains, such as VHH, that specifically bind to CDH17. Preferably, the two antigen-binding domains that specifically bind to CDH17 are identical.

[0209] In some implementations, the multispecific antibody is a bispecific antibody.

[0210] 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.

[0211] Specifically binds to the antigen-binding domain of EGFR

[0212] In some embodiments of the multispecific antibody according to the present invention, preferably, the antigen-binding domain that specifically binds to EGFR comprises or is composed of an anti-EGFR antibody or its antigen-binding fragment, as long as it can specifically bind to EGFR, including but not limited to, for example, full-length antibodies that specifically bind to EGFR, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy chain antibodies, etc.

[0213] In some implementations, the antigen-binding domain that specifically binds to EGFR is an anti-EGFR VHH (VHH) as defined herein (e.g., in Part II). EGFR ).

[0214] Specifically binds to the CDH17 antigen-binding domain

[0215] 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.

[0216] In some implementations, the antigen-binding domain that specifically binds to CDH17 is the anti-CDH17 VHH (VHH) as defined herein (e.g., Part I). CDH17 ).

[0217] Immunoglobulin Fc region

[0218] 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.

[0219] 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 comprises at least an immunoglobulin CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin Fc region further comprises all or part of a hinge region, for example, a partial hinge region being the amino acid sequence shown in SEQ ID NO:41, or a complete hinge region being the amino acid sequence shown in SEQ ID NO:40. In some embodiments, the immunoglobulin Fc region comprises, or consists of, an immunoglobulin hinge region or a partial hinge region, a CH2 domain and a CH3 domain, or both, from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region comprises, or consists of, a CH2 domain and a CH3 domain, from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, or IgG4, or a subtype thereof. Preferably, the immunoglobulin Fc region comprises an Fc region sequence derived from humans.

[0220] Immunoglobulin Fc regions can fuse to other domains (i.e., EGFR or CDH17 binding domains, such as VHH). EGFR 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.

[0221] 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 include 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 (SEQ ID NO:74) in IgG1, IgG2, and IgG3, and the CPSC sequence (SEQ ID NO:76) in IgG4. Preferably, the hinge region contains at least one disulfide bond connecting two Fc chains. In some embodiments, the hinge region sequence comprises a hinge region sequence from E216 to P230 of IgG1 (e.g., the amino acid sequence shown in SEQ ID NO:40) or a hinge region sequence from D221 to P230 (e.g., the amino acid sequence shown in SEQ ID NO:41) (according to EU designations), or a corresponding hinge region sequence from other immunoglobulin isotypes. In this document, when referring to a complete or all-encompassing hinge region, it generally refers to the hinge region sequence corresponding to E216 to P230 of IgG1 (according to EU designations), for example, the complete or all-encompassing hinge region of the IgG1 Fc region is the amino acid sequence shown in SEQ ID NO:40. 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 designations), for example, the partial hinge region of IgG1 Fc is the amino acid sequence shown in SEQ ID NO:41. In some embodiments, when the Fc region of an immunoglobulin is connected to the VHH domain, such as its C-terminus, via its N-terminus, the Fc region preferably contains the entire hinge region sequence, such as the hinge region sequence from E216 to P230 of IgG1 (e.g., the amino acid sequence shown in SEQ ID NO:40).

[0222] 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:33 or SEQ ID NO:34 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.

[0223] 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.

[0224] In some embodiments, the Fc region may have a cysteine ​​residue mutated at position 220, for example, to serine. For example, the amino acid sequence of the hinge region of the Fc region may retain native cysteine ​​residues, such as the native cysteine ​​residue at position 220, for use in constructing disulfide bonds or for adding cysteine ​​coupling sites to improve the DAR of the ADC using the antibody. In some embodiments, the entire hinge region containing the C220S mutation comprises, for example, the amino acid sequence shown in SEQ ID NO:56. In some embodiments, the amino acid at position 220 of the hinge region is cysteine.

[0225] In some embodiments, if the multispecific antibody comprises dissimilar chains, such as an asymmetric structure, a Knob-into-Hole (KiH) mutation may be introduced into the CH3 domain to facilitate 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. Exemplary KiH mutations include, but are not limited to, combinations of Knob mutation T366W and Hole mutations T366S, L368A, and Y407V; or combinations of Knob mutation T366Y and Hole mutation Y407T. In some embodiments, the Fc regions may also contain cysteine ​​residue substitutions to obtain non-natural disulfide bond linkages. In some embodiments, one Fc region contains S354C or E356C, and the other Fc region contains Y349C.

[0226] In one specific implementation, the Fc region containing the Knob mutation contains the amino acid substitution T366W, and the Fc region containing the hole mutation contains the amino acid substitutions T366S, L368A, and Y407V (numbered according to the EU index).

[0227] In one specific implementation, the Fc region containing the Knob mutation contains amino acid substitutions S354C and T366W, and the Fc region containing the hole mutation contains amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the EU index).

[0228] When the multispecific antibody of the present invention contains an asymmetric double-stranded structure, the Fc region preferably contains a KiH mutation that promotes the correct heterodimerization of the antibody polypeptide chain.

[0229] 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.

[0230] 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. 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:35, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:35.

[0231] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two Fc regions heterodimerized, one Fc-region polypeptide comprising mutants S354C and T366W, and the other Fc-region polypeptide comprising mutants Y349C, T366S, L368A and Y407V.

[0232] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein the Fc regions respectively contain KiH mutations and mutations such as LALA mutations that reduce or eliminate the Fc region and the Fcγ receptor.

[0233] Therefore, in one specific embodiment, the multispecific antibody of the present invention contains two Fc regions with C220S mutations in the hinge region.

[0234] Therefore, in one specific embodiment, the multispecific antibody of the present invention contains two Fc regions that retain cysteine ​​at position 220.

[0235] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:36 and comprises the mutations S354C and T366W, and optionally the L234A / L235A mutation, and the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:37 and comprises the mutations Y349C, T366S, L368A, and Y407V, and optionally the L234A / L235A mutation.

[0236] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:36, and the other Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:37.

[0237] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:54 and comprises mutants C220S, S354C, and T366W, and optionally L234A / L235A mutations, and the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:55 and comprises mutants C220S, Y349C, T366S, L368A, and Y407V, and optionally L234A / L235A mutations.

[0238] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:54, and the other Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:55.

[0239] connector

[0240] In the multispecific antibody according to the invention, antibody components (e.g., antigen-binding domain and Fc region) can be connected by adapters.

[0241] 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.

[0242] 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.

[0243] Suitable connector sequences include, but are not limited to, G4S (SEQ ID NO: 58); (G4S)2 (SEQ ID NO: 59); (G4S)3 (SEQ ID NO: 60); GGGSG (SEQ ID NO: 61); GGSGG (SEQ ID NO: 62); GSGGG (SEQ ID NO: 63); GSGGGP (SEQ ID NO: 64); GGEPS (SEQ ID NO: 65); GGEGGGP (SEQ ID NO: 66) and GGEGGGSEGGGS (SEQ ID NO: 67); and (G4S)n (SEQ ID NO: 68), where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; TS(G4S)n (SEQ ID NO: 69), where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; G(G4S)n (SEQ ID NO: 68); G4S)n (SEQ ID NO: 69 ... NO:70), where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; (G4)n(SEQ ID NO:71), where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; (GRPGS)n(SEQ ID NO:72), 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 the present invention may also be, for example, but not limited to, the following amino acid sequences: (G3S)2 (SEQ ID NO:73), (G4S)2 (SEQ ID NO:59), (G3S)3 (SEQ ID NO:75), (G4S)3 (SEQ ID NO:60), (G3S)4 (SEQ ID NO:77), (G4S)4 (SEQ ID NO:78), (G3S)5 (SEQ ID NO:79), (G4S)5 (SEQ ID NO:80), (G3S)6 (SEQ ID NO:81), (G4S)(SEQ ID NO:82), GGG, DGGGS (SEQ ID NO:83), TGEKP (SEQ ID NO:84), GGRR (SEQ ID NO:85), EGKSSGSGSESKVD (SEQ ID NO:86), KESGSVSSEQLAQFRSLD (SEQ ID NO:87), GGRRGGGS (SEQ ID NO:88). (SEQ ID NO:88), LRQRDGERP (SEQ ID NO:89), LRQKDGGGSERP (SEQ ID NO:90), and GSTGSGSGKPGSGEGSTKG (SEQ ID NO:91). Alternatively, suitable flexible linker peptides can be rationally designed by using computer programs to simulate the three-dimensional structure of proteins and peptides, or by using phage display methods.

[0244] 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 (SEQ ID NO:92), where n is an integer equal to or greater than 1, for example, n is 1, 2, 3, 4, 5, 6 or 7.

[0245] 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.

[0246] Structure and examples of multispecific antibodies

[0247] The multispecific antibody according to the invention can take any suitable form, for example, it comprises two polypeptide chains, wherein the two polypeptide chains together contain three or four VHHs as described herein, wherein one polypeptide chain contains one or two tandem VHHs, and the other polypeptide chain contains the remaining one or two tandem VHHs. In some embodiments, the multispecific antibody contains the same VHHs that bind to the same antigen. In some specific embodiments, the two tandem VHHs are linked via a linker, for example, the C-terminus of the first VHH is linked to the N-terminus of the second VHH.

[0248] When this article refers to two VHHs connected in series, it means that the C end of the first VHH and the N end of the second VHH are connected via or without a connector, preferably via a connector.

[0249] In some embodiments, the multispecific antibody comprises two VHHs. CDH17 and 2 VHH EGFR One of the chains contains two cascaded VHHs CDH17 and VHH EGFR and Fc region, and another chain contains two tandem VHH CDH17 and VHH EGFR and the Fc region. In some embodiments, the multispecific antibody comprises two identical chains.

[0250] In some implementations, the multispecific antibody is a bispecific antibody with a “2+2” symmetrical structure, such as the structure corresponding to the format schematic diagrams in Figures 20A and 20B.

[0251] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to EGFR and CDH17, and comprises or consists of two identical polypeptide chains, each of which comprises, or consists of, the following structure from its N-terminus to its C-terminus:

[0252] VHH A -VHH B -Fc, where VHH A and VHH B These represent the VHH domains that bind antigens A and B, respectively, where A and B are distinct from each other and independently selected from EGFR and CDH17; the symbol "-" indicates linkage via a linker or direct linkage, preferably representing a linker of 5-15 amino acids in length.

[0253] Preferably, VHH A -VHH B There is a connector between them, and VHH B It connects directly to the Fc area, which contains the hinge area (e.g., the entire hinge area);

[0254] Optionally, the Fc region contains an LALA mutation;

[0255] Optionally, VHH A The C end connects to VHH via a connector. B N-terminal connection;

[0256] Optionally, VHH B The C terminal is connected to the N terminal of the Fc region.

[0257] In the above-mentioned multispecific antibodies, preferably, each antigen-binding domain on the polypeptide chain (e.g., VHH) CDH17 Domain and VHH EGFR The Fc region is connected to the other Fc region via a connector, preferably having a length of 5-15 amino acids, for example (G4S)3. Preferably, in the above-mentioned multispecific antibody, the Fc region includes all hinge regions, for example, including the hinge region shown in SEQ ID NO:40.

[0258] When the two chains of a multispecific antibody are identical, the two immunoglobulin Fc regions can associate to form a homodimer through dimerization. In some embodiments, the Fc region contains an amino acid sequence derived from human IgG1. In some embodiments, the Fc region contains a mutation that reduces or eliminates Fcγ receptor binding, such as the LALA mutation.

[0259] In some embodiments, the bispecific antibody of the present invention specifically binds to EGFR and CDH17, and comprises or consists of two identical polypeptide chains, wherein each chain comprises, or consists of, the following structure from the N-terminus to the C-terminus:

[0260] VHH CDH17 -VHH EGFR -Fc, where the symbol "-" indicates a connection via a linker or a direct connection, preferably a linker with a length of 5-15 amino acids;

[0261] Preferably, VHH CDH17 -VHH EGFR There is a connector between them, and VHH EGFR It connects directly to the Fc area, which contains the entire hinge area;

[0262] Optionally, the Fc region contains an LALA mutation;

[0263] Optionally, VHH CDH17 The C end connects to VHH via a connector. EGFR N-terminal connection;

[0264] Optionally, VHH EGFR The C terminal is connected to the N terminal of the Fc region.

[0265] In some embodiments of the multispecific antibody of the present invention having the above-described structural form, the VHH CDH17 It is a CDH17-resistant VHH (VHH) as defined herein (e.g., Part I). CDH17 In some further implementations, the VHH... EGFR It is an EGFR-resistant VHH (VHH) as defined herein (e.g., Part II). EGFR ).

[0266] In some preferred embodiments, the VHH CDH17 The VHH includes the three complementary determination regions (CDRs) contained in the VH shown in SEQ ID NO: 20-32, and in particular, includes the three complementary determination regions (CDRs) contained in the VH shown in SEQ ID NO: 31, preferably, the CDRs are defined according to the AbM scheme. More preferably, the VHH... CDH17 It comprises complement-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprises or is composed of heavy chain variable regions, wherein the heavy chain variable regions comprise complement-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:14, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:18. More preferably, the VHH... CDH17Contains the amino acid sequence shown in SEQ ID NO: 20-32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. More preferably, the VHH CDH17 It contains the amino acid sequence shown in SEQ ID NO: 31.

[0267] In some preferred embodiments, the VHH EGFR The VHH includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NOs: 8-13, and in particular, includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO: 12, preferably, the CDRs are defined according to the AbM scheme. More preferably, the VHH... EGFR 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:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:6. More preferably, the VHH... EGFR Contains the amino acid sequence shown in SEQ ID NO: 8-13 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. More preferably, the VHH EGFR It contains the amino acid sequence shown in SEQ ID NO: 12.

[0268] In some preferred embodiments, the bispecific antibody of the present invention comprises or is composed of two identical polypeptide chains, wherein each polypeptide chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence. In some preferred embodiments, the bispecific antibody of the present invention comprises or is composed of two identical polypeptide chains, wherein each polypeptide chain is composed of the amino acid sequence shown in SEQ ID NO: 42.

[0269] In some embodiments, the bispecific antibody of the present invention specifically binds to EGFR and CDH17, and comprises or consists of two identical polypeptide chains, wherein each chain comprises, or consists of, the following structure from the N-terminus to the C-terminus:

[0270] VHH EGFR -VHH CDH17 -Fc, where the symbol "-" indicates a connection via a linker or a direct connection, preferably a linker with a length of 5-15 amino acids;

[0271] Preferably, VHH EGFR -VHH CDH17 There is a connector between them, and VHH CDH17 It connects directly to the Fc area, which contains the entire hinge area;

[0272] Optionally, the Fc region contains an LALA mutation;

[0273] Optionally, VHH EGFR The C end connects to VHH via a connector. CDH17 N-terminal connection;

[0274] Optionally, VHH CDH17 The C terminal is connected to the N terminal of the Fc region.

[0275] In some embodiments of the multispecific antibody of the present invention having the above-described structural form, the VHH CDH17 It is a CDH17-resistant VHH (VHH) as defined herein (e.g., Part I). CDH17 In some further implementations, the VHH... EGFR It is an EGFR-resistant VHH (VHH) as defined herein (e.g., Part II). EGFR ).

[0276] In some preferred embodiments, the VHH CDH17 The VHH includes the three complementary determination regions (CDRs) contained in the VH shown in SEQ ID NO: 20-32, and in particular, includes the three complementary determination regions (CDRs) contained in the VH shown in SEQ ID NO: 31, preferably, the CDRs are defined according to the AbM scheme. More preferably, the VHH... CDH17It comprises complement-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprises or is composed of heavy chain variable regions, wherein the heavy chain variable regions comprise complement-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:14, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:18. More preferably, the VHH... CDH17 Contains the amino acid sequence shown in SEQ ID NO: 20-32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. More preferably, the VHH CDH17 It contains the amino acid sequence shown in SEQ ID NO: 31.

[0277] In some preferred embodiments, the VHH EGFR The VHH includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NOs: 8-13, and in particular, includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO: 12, preferably, the CDRs are defined according to the AbM scheme. More preferably, the VHH... EGFR 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:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:6. More preferably, the VHH... EGFR Contains the amino acid sequence shown in SEQ ID NO: 8-13 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. More preferably, the VHH EGFR It contains the amino acid sequence shown in SEQ ID NO: 12.

[0278] In some preferred embodiments, the bispecific antibody of the present invention comprises or is composed of two identical polypeptide chains, wherein each polypeptide chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence. In some preferred embodiments, the bispecific antibody of the present invention comprises or is composed of two identical polypeptide chains, wherein each polypeptide chain is composed of the amino acid sequence shown in SEQ ID NO: 43.

[0279] In some embodiments, the multispecific antibody of the present invention, such as a bispecific antibody, comprises two CDH17-specific antigen-binding domains and one EGFR-specific antigen-binding domain. In some embodiments, the CDH17-specific antigen-binding domain is a VHH, and / or the EGFR-specific antigen-binding domain is a VHH. In some specific embodiments, the multispecific antibody of the present invention, such as a bispecific antibody, comprises two CDH17-specific VHHs and one EGFR-specific VHH. In some specific embodiments, the multispecific antibody of the present invention is a bispecific antibody. In some specific embodiments, the multispecific antibody of the present invention, such as a bispecific antibody, comprises two polypeptide chains, one polypeptide chain comprising two tandem VHHs and optionally an Fc region, and the other polypeptide chain comprising one VHH and optionally an Fc region. In some specific embodiments, the two tandem VHHs are two CDH17-specific VHHs, and the other VHH is an EGFR-specific VHH. In some specific implementations, the two tandem VHHs are a VHH that specifically binds to CDH17 and a VHH that specifically binds to EGFR (e.g., VHH). EGFR -VHH CDH17 , or VHH CDH17 -VHH EGFR One VHH is a CDH17-specific VHH. In some specific embodiments, the two CDH17-specific VHHs are identical. In some specific embodiments, the two tandem VHHs are connected via a connector, for example, the C-terminus of the first VHH is connected to the N-terminus of the second VHH. In some specific embodiments, when a VHH is connected to an Fc region, its C-terminus is directly connected to the N-terminus of the Fc region.

[0280] In other embodiments, the multispecific antibody is a bispecific antibody with a “2+1” asymmetric structure, such as the structure corresponding to the format schematic diagram of any one of Figures 20C-E.

[0281] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to EGFR and CDH17, and the bispecific antibody contains a VHH. EGFR and two VHH CDH17 In some preferred embodiments, the multispecific antibody comprises a VHH EGFR and two VHH CDH17 Furthermore, it is a trivalent bispecific antibody. In some embodiments, the multispecific antibody comprises two VHHs. CDH17 and 1 VHH EGFR One of the polypeptide chains contains two tandem VHH CDH17 and VHH EGFR And the Fc region, and another polypeptide chain contains one VHH. CDH17 and the Fc region; or one of the polypeptide chains contains two tandem VHHs. CDH17 And the Fc region, and another polypeptide chain contains one VHH. EGFR and Fc area.

[0282] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to EGFR and CDH17, and comprises or consists of two polypeptide chains, wherein the polypeptide chains comprise or consist of the following structures from the N-terminus to the C-terminus:

[0283] First polypeptide chain: VHH A -VHH B -Fc 1 ,

[0284] Second polypeptide chain: VHH A -Fc 2 or VHH B -Fc 2

[0285] VHH A and VHH B These represent the VHH domains that bind antigens A and B, respectively, where A and B are distinct from each other and independently selected from EGFR and CDH17; the symbol "-" indicates linkage via a linker or direct linkage, preferably representing a linker of 5-15 amino acids in length; Fc 1 It refers to the Fc region in the first polypeptide chain. 2 It refers to the Fc region in the second polypeptide chain;

[0286] Preferably, VHH A -VHH B There is a connector between them, and VHH A and / or VHH BIt connects directly to the Fc area, which contains the hinge area;

[0287] Optionally, the one or two Fc regions preferably contain the LALA mutation, and optionally the one or two Fc regions also each contain the C220S mutation;

[0288] Optionally, the Fc of the first polypeptide chain 1 Containing the Knob mutation and the Fc of the second polypeptide chain 2 Includes Hole mutations, or vice versa;

[0289] Optionally, VHH in the first polypeptide chain A The C end connects to VHH via a connector. B N-terminal connection;

[0290] Optionally, VHH in the first polypeptide chain B C-end and Fc 1 The N-terminal connection of the area;

[0291] Optionally, VHH in the second polypeptide chain A C-end or VHH B C-end and Fc 2 The N-terminal connection of the area.

[0292] In some preferred embodiments, the multispecific antibody is a bispecific antibody that specifically binds to EGFR and CDH17, comprising or consisting of two polypeptide chains, wherein the polypeptide chains, from the N-terminus to the C-terminus, comprise or consist of the following structures:

[0293] First polypeptide chain: VHH EGFR -VHH CDH17 -Fc 1 ,

[0294] Second polypeptide chain: VHH CDH17 -Fc 2 ,

[0295] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker with a length of 5-15 amino acids; Fc 1 It refers to the Fc region in the first polypeptide chain. 2 It refers to the Fc region in the second polypeptide chain;

[0296] Preferably, VHH EGFR -VHH CDH17 There is a connector between them, and VHH CDH17 It connects directly to the Fc area, which contains the entire hinge area;

[0297] Optionally, the one or two Fc regions preferably contain the LALA mutation, and optionally the one or two Fc regions also each contain the C220S mutation;

[0298] Optionally, the Fc of the first polypeptide chain 1 Containing the Knob mutation and the Fc of the second polypeptide chain 2 Includes Hole mutations, or vice versa;

[0299] Optionally, in the first polypeptide chain, VHH EGFR The C end connects to VHH via a connector. CDH17 N-terminal connection;

[0300] Optionally, in the first polypeptide chain, VHH CDH17 C-end and Fc 1 The N-terminal connection of the area;

[0301] Optionally, in the second polypeptide chain, VHH CDH17 C-end and Fc 2 The N-terminal connection of the area.

[0302] In some preferred embodiments, the multispecific antibody is a bispecific antibody that specifically binds to EGFR and CDH17, comprising or consisting of two polypeptide chains, wherein the polypeptide chains, from the N-terminus to the C-terminus, comprise or consist of the following structures:

[0303] First polypeptide chain: VHH CDH17 -VHH EGFR -Fc 1 ,

[0304] Second polypeptide chain: VHH CDH17 -Fc 2 ,

[0305] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker with a length of 5-15 amino acids; Fc 1 It refers to the Fc region in the first polypeptide chain. 2 It refers to the Fc region in the second polypeptide chain;

[0306] Preferably, VHH CDH17 -VHH EGFR The linker is present between them, and VHH is present in the first polypeptide chain. EGFR The VHH in the second polypeptide chain is directly connected to the Fc region, which contains the entire hinge region. CDH17 It connects directly to the Fc area, which contains the entire hinge area;

[0307] Optionally, the one or two Fc regions preferably contain the LALA mutation, and optionally the one or two Fc regions also each contain the C220S mutation;

[0308] Optionally, the Fc of the first polypeptide chain 1 Containing the Knob mutation and the Fc of the second polypeptide chain 2 Includes Hole mutations, or vice versa;

[0309] Optionally, in the first polypeptide chain, VHH CDH17 The C end connects to VHH via a connector. EGFR N-terminal connection;

[0310] Optionally, in the first polypeptide chain, VHH EGFR C-end and Fc 1 The N-terminal connection of the area;

[0311] Optionally, in the second polypeptide chain, VHH CDH17 C-end and Fc 2 The N-terminal connection of the area.

[0312] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to EGFR and CDH17, and comprises or consists of two polypeptide chains, wherein the polypeptide chains comprise or consist of the following structures from the N-terminus to the C-terminus:

[0313] First polypeptide chain: VHH A -VHH A -Fc 1 ,

[0314] Second polypeptide chain: VHH B -Fc 2

[0315] VHH A and VHH B These represent the VHH domains that bind antigens A and B, respectively, where A and B are distinct from each other and independently selected from EGFR and CDH17; the symbol "-" indicates linkage via a linker or direct linkage, preferably representing a linker of 5-15 amino acids in length; Fc 1 It refers to the Fc region in the first polypeptide chain. 2 It refers to the Fc region in the second polypeptide chain;

[0316] Preferably, VHH A -VHH A The linker is present between them, and the second VHH in the first polypeptide chain A VHH in the second polypeptide chain B It connects directly to the Fc area, which contains the hinge area;

[0317] Optionally, the one or two Fc regions preferably contain the LALA mutation, and optionally the one or two Fc regions also each contain the C220S mutation;

[0318] Optionally, the Fc of the first polypeptide chain 1 Containing the Knob mutation and the Fc of the second polypeptide chain 2 Includes Hole mutations, or vice versa;

[0319] Optionally, the first VHH in the first polypeptide chain A The C end is connected to the second VHH via a connector. A N-terminal connection;

[0320] Optionally, the second VHH in the first polypeptide chain A C-end and Fc 1 The N-terminal connection of the area;

[0321] Optionally, VHH in the second polypeptide chain B C-end and Fc 2 The N-terminal connection of the area.

[0322] In some preferred embodiments, the multispecific antibody is a bispecific antibody that specifically binds to EGFR and CDH17, comprising or consisting of two polypeptide chains, wherein the polypeptide chains, from the N-terminus to the C-terminus, comprise or consist of the following structures:

[0323] First polypeptide chain: VHH CDH17 -VHH CDH17 -Fc 1 ,

[0324] Second polypeptide chain: VHH EGFR -Fc 2 ,

[0325] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker with a length of 5-15 amino acids; Fc 1 It refers to the Fc region in the first polypeptide chain. 2 It refers to the Fc region in the second polypeptide chain;

[0326] Preferably, VHH CDH17 -VHH CDH17 The linker is present between them, and the second VHH in the first polypeptide chain CDH17 Directly connected to the Fc region containing the entire hinge region, and the VHH in the second polypeptide chain. EGFR It connects directly to the Fc area, which contains the entire hinge area;

[0327] Optionally, the one or two Fc regions preferably contain the LALA mutation, and optionally the one or two Fc regions also each contain the C220S mutation;

[0328] Optionally, the Fc of the first polypeptide chain 1 Containing the Knob mutation and the Fc of the second polypeptide chain 2 Includes Hole mutations, or vice versa;

[0329] Optionally, in the first polypeptide chain, the first VHH CDH17 The C end is connected to the second VHH via a connector. CDH17 N-terminal connection;

[0330] Optionally, in the first polypeptide chain, the second VHH CDH17 C-end and Fc 1 The N-terminal connection of the area;

[0331] Optionally, in the second polypeptide chain, VHH EGFR C-end and Fc 2 The N-terminal connection of the area.

[0332] In the above-mentioned multispecific antibodies, preferably, each antigen-binding domain on the polypeptide chain (e.g., VHH) CDH17 Domain and VHH EGFR The domains are connected by a linker, preferably having a length of 5-15 amino acids, for example (G4S)3. Preferably, in the above-mentioned multispecific antibody, the Fc region includes all hinge regions, for example, the hinge region shown in SEQ ID NO:40. Preferably, the Fc region includes all hinge regions containing the C220S mutation, for example, the hinge region shown in SEQ ID NO:57.

[0333] In the above-mentioned multispecific antibodies, preferably, the antigen-binding domains or Fc regions on the polypeptide chain are as defined herein.

[0334] When the two polypeptide chains of a multispecific antibody are different, the first and second polypeptide chains of the aforementioned multispecific antibody can associate to form a heterodimer due to the dimerization effect of the Fc region of the immunoglobulin. Preferably, to promote the heterodimerization of the first and second polypeptide chains, a Knob-into-hole mutation, such as (T366W / T366S, L368A, Y407V) mutation or (T366Y / Y407T) mutation, and optionally S354C / Y349C, can be introduced into the Fc region of the first and second polypeptide chains. Preferably, the Fc region contains an amino acid sequence from human IgG1 or IgG4, and preferably, the Fc region also contains a mutation that reduces or eliminates Fcγ receptor binding, such as the LALA mutation.

[0335] In some embodiments of the multispecific antibody of the present invention having the above-described structural form, the VHH CDH17 It is a CDH17-resistant VHH (VHH) as defined herein (e.g., Part I). CDH17 In some further implementations, the VHH... EGFR It is an EGFR-resistant VHH (VHH) as defined herein (e.g., Part II). EGFR ).

[0336] In some preferred embodiments, the VHH CDH17 The VHH includes the three complementary determination regions (CDRs) contained in the VH shown in SEQ ID NO: 20-32, and in particular, includes the three complementary determination regions (CDRs) contained in the VH shown in SEQ ID NO: 31, preferably, the CDRs are defined according to the AbM scheme. More preferably, the VHH... CDH17 It comprises complement-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprises or is composed of heavy chain variable regions, wherein the heavy chain variable regions comprise complement-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:14, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:18. More preferably, the VHH... CDH17 Contains the amino acid sequence shown in SEQ ID NO: 20-32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. More preferably, the VHH CDH17 It contains the amino acid sequence shown in SEQ ID NO: 31.

[0337] In some preferred embodiments, the VHH EGFR The VHH includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NOs: 8-13, and in particular, includes the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO: 12, preferably, the CDRs are defined according to the AbM scheme. More preferably, the VHH... EGFRThe 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:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:6. More preferably, the VHH... EGFR Contains the amino acid sequence shown in SEQ ID NO: 8-13 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. More preferably, the VHH EGFR It contains the amino acid sequence shown in SEQ ID NO: 12.

[0338] In some embodiments, the bispecific antibody comprises or consists of a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of:

[0339] (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 44 and 45, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences,

[0340] (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 46 and 47, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences,

[0341] (iii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 48 and 49, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences.

[0342] (iv) Each of the amino acid sequences shown in SEQ ID NOs: 52 and 53, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences.

[0343] In some embodiments, the bispecific antibody comprises or is composed of a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are respectively as shown in the following amino acid sequences:

[0344] SEQ ID NO:44 and SEQ ID NO:45;

[0345] SEQ ID NO:46 and SEQ ID NO:47;

[0346] SEQ ID NO:48 and SEQ ID NO:49;

[0347] SEQ ID NO:52 and SEQ ID NO:53.

[0348] In some more specific embodiments, the bispecific antibody is V-F1, V-F2, V-F3, V-F7, V-F8, or V-F9, wherein V-F1, V-F2, V-F3, V-F7, V-F8, or V-F9 is composed of the following chains:

[0349] V-F1: Two chains as shown in SEQ ID NO:42;

[0350] V-F2: Two chains as shown in SEQ ID NO:43;

[0351] V-F3: the first strand as shown in SEQ ID NO:44, and the second strand as shown in SEQ ID NO:45;

[0352] V-F7: the first chain as shown in SEQ ID NO:46, and the second chain as shown in SEQ ID NO:47;

[0353] V-F8: the first chain as shown in SEQ ID NO:48, and the second chain as shown in SEQ ID NO:49;

[0354] V-F9: the first chain as shown in SEQ ID NO:52, and the second chain as shown in SEQ ID NO:53.

[0355] Characteristics of the multispecific antibody of this invention

[0356] The multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, may have one or more of the following properties:

[0357] (i) Specific binding to tumor cells expressing EGFR and / or CDH17 antigens, preferably specific binding to tumor cells co-expressing EGFR and CDH17 antigens;

[0358] (ii) It exhibits species cross-reactivity with cynomolgus monkey EGFR and CDH17;

[0359] (iii) It has EGFR and / or CDH17-mediated endocytic activity, particularly in tumor cells expressing EGFR and / or CDH17, and preferably has synergistic endocytic activity in tumor cells that specifically bind to co-expressing EGFR and CDH17 antigens.

[0360] (iv) Having killing activity against tumor cells expressing EGFR and / or CDH17, preferably specifically binding to tumor cells co-expressing EGFR and CDH17 antigens, and preferably synergistic killing activity.

[0361] In some aspects, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, target cells expressing EGFR and / or CDH17 antigens. The EC50 value and / or maximum binding amount of the antibodies of the present invention against EGFR and / or CDH17-positive tumor cells can be determined by FACS or ELISA assays (e.g., the assays described in the examples) and optionally compared with a reference antibody to reflect the cell-binding affinity of the antibodies. Exemplary affinity assay methods are described in the examples, particularly in the materials and methods section.

[0362] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, are capable of specifically binding to EGFR and CDH17, for example, human or cynomolgus monkey EGFR 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. In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, are...

[0363] (i) K binding affinity with human CDH17 D The value is less than or equal to about 100 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM or, for example, about 10 nM, for example less than about 5 nM or 4 nM, preferably between 0.5 and 10 nM, for example between 1 and 5 nM, as determined by the SPR method.

[0364] (ii) K binding affinity with CDH17 of cynomolgus monkeys D The value is less than or equal to about 100 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM or, for example, about 10 nM, for example, less than about 8 nM, 7 nM or 6 nM, preferably between 1 and 10 nM, for example between 1 and 7 nM or between 2 and 6 nM, for example, as determined by the SPR method;

[0365] (iii) K binding affinity with human EGFR D Values ​​less than or equal to about 100 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, or for example about 15 nM, for example less than about 12 nM, 11 nM, 7 nM, or 5 nM, preferably between 1 and 15 nM, for example between 2 and 12 nM or between 3 and 11 nM, as determined by the SPR method; and / or

[0366] (iv) K binding affinity to EGFR in cynomolgus monkeys D The value is less than or equal to about 100 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM or, for example, about 15 nM, for example less than about 12 nM, 11 nM or 10 nM, preferably between 1 and 15 nM, for example between 2 and 12 nM or between 2 and 10 nM, for example as determined by the SPR method.

[0367] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, exhibit cross-reactivity with human and / or monkey EGFR 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 their KD values ​​is between 1 and 10, for example, between 1 and 5, approximately between 1 and 4, approximately between 1 and 3, or approximately between 1 and 2.

[0368] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17 (especially synergistic antibodies), specifically bind to EGFR-positive and / or CDH17-positive cells, particularly EGFR-positive and CDH17-positive tumor cells.

[0369] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, have a synergistic effect with the binding of EGFR and CDH17 targets, i.e., higher binding activity with EGFR and / or CDH17 positive, preferably EGFR positive and CDH17 positive tumor cells, for example, compared with the binding of EGFR monospecific antibodies (e.g., EGFR parental antibodies) to EGFR positive tumor cells, or compared with the binding of CDH17 monospecific antibodies (e.g., CDH17 parental antibodies) to CDH17 positive tumor cells.

[0370] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, bind more strongly to EGFR-positive cells (optionally also CDH17-positive) than EGFR monospecific antibodies (e.g., parental EGFR antibodies). In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, bind more strongly to CDH17-positive cells (optionally also EGFR-positive) than CDH17 monospecific antibodies (e.g., parental CDH17 antibodies).

[0371] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, specifically kill EGFR-positive and / or CDH17-positive cells, particularly EGFR-positive and CDH17-positive tumor cells, for example, specifically killing EGFR-positive and / or CDH17-positive cells, particularly EGFR-positive and CDH17-positive tumor cells, with a synergistic effect. In particular, the multispecific antibodies of the present invention exhibit greater killing activity against EGFR-positive and / or CDH17-positive tumor cells, such as EGFR-positive and CDH17-positive tumor cells, for example, synergistic killing activity, for example, greater killing activity against EGFR-positive tumor cells compared to EGFR monospecific antibodies (e.g., EGFR parental antibodies), or greater killing activity against CDH17-positive tumor cells compared to CDH17 monospecific antibodies (e.g., CDH17 parental antibodies).

[0372] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, exhibit stronger cytotoxic activity against CDH17-positive (optionally also EGFR-positive) tumor cells than CDH17 monospecific antibodies (e.g., CDH17 parental antibodies). In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, exhibit stronger cytotoxic activity against EGFR-positive (optionally also CDH17-positive) tumor cells than EGFR monospecific antibodies (e.g., EGFR parental antibodies).

[0373] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, can be internalized by cells expressing EGFR and / or CDH17. In particular, the multispecific antibodies of the present invention have a greater degree of internalization activity in EGFR-positive and / or CDH17-positive tumor cells, such as EGFR-positive and CDH17-positive tumor cells, for example, co-endocytic activity, for example, greater internalization activity compared to EGFR monospecific antibodies (e.g., EGFR parental antibodies) in EGFR-positive tumor cells, or greater internalization activity compared to CDH17 monospecific antibodies (e.g., CDH17 parental antibodies) in CDH17-positive tumor cells.

[0374] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, exhibit stronger endocytosis activity with CDH17-positive (optionally also EGFR-positive) tumor cells than CDH17 monospecific antibodies (e.g., CDH17 parental antibodies). In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies that specifically bind to EGFR and CDH17, exhibit stronger endocytosis activity with EGFR-positive (optionally also CDH17-positive) tumor cells than EGFR monospecific antibodies (e.g., EGFR parental antibodies).

[0375] The endocytic activity of antibodies can be evaluated in cell-based assays, such as those described in the examples. Exemplary methods are described in the examples, particularly in the materials and methods.

[0376] 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% or 95% or higher as determined by SEC-HPLC.

[0377] IV. Other antigen-binding molecules

[0378] 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.

[0379] V. Production and purification of antibody or antigen-binding molecules of the present invention

[0380] 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.

[0381] 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 of the present invention, such as an antibody, 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 of the present invention, such as an antibody, the method comprising: culturing host cells containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the antigen-binding molecule, such as an antibody; and assembling the polypeptide chain to produce the antibody under conditions suitable for assembling the polypeptide chain into the antigen-binding molecule, such as an antibody.

[0382] 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.

[0383] 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.

[0384] 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 antigen-binding molecules, such as antibodies, provided herein can be identified, screened, or characterized using a variety of assays known in the art.

[0385] 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.

[0386] III. Immunoconjugates

[0387] In another aspect, the present invention provides immunofusions, immunoconjugates, and antibody-drug conjugates comprising antigen-binding molecules of the present invention, such as antibodies.

[0388] Immunofusions and Immunoconjugates

[0389] In one embodiment, the present invention provides an immunofusion or immunoconjugate produced by fusing or conjugating an antigen-binding molecule of the present invention, such as an antibody, to a heterologous molecule.

[0390] In one embodiment, in the immunofusion, the antigen-binding molecule (such as an antibody) of the present invention 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.

[0391] In one embodiment, in the immunoconjugate, the antigen-binding molecule (such as an antibody) of the present invention is conjugated to a therapeutic agent, diagnostic agent, or detectable agent. In the conjugate, chemical linkers can be used to covalently link different entities of the conjugate. In some cases, it is advantageous that the chemical linker is a "cleavable linker" that facilitates the release of the antigen-binding molecule 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.

[0392] In embodiments where a therapeutic agent is conjugated, the therapeutic agent suitable for the conjugation includes, but is not limited to, drugs (e.g., antitumor drugs, such as cytotoxins) or diagnostic agents (e.g., radioisotopes, fluorescent substances, or luminescent substances).

[0393] 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.

[0394] In some embodiments, therapeutic agents suitable for the conjugate include, but are not limited to, drugs (e.g., antitumor drugs); in other embodiments, diagnostic agents suitable for the conjugate include, but are not limited to, radiodiagnostic agents, fluorescent substances, or luminescent substances.

[0395] Antibody-drug conjugates (ADCs)

[0396] In some implementations, this disclosure provides antibody-drug conjugates (ADCs).

[0397] In some embodiments, this disclosure provides an antibody-drug conjugate having formula (I) or a pharmaceutically acceptable salt or solvation thereof: Ab-(LD) p (I)

[0398] in:

[0399] Ab is the antigen-binding molecule of the present invention, and in particular the antigen-binding molecule defined in the second paragraph of the detailed description of the invention herein;

[0400] L is the connector;

[0401] D represents a drug, such as an anti-tumor compound;

[0402] p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, for example, an integer selected from 4 to 6.

[0403] It can be understood that p refers to the number of -LDs that Ab is linked to in the antibody-drug conjugate molecule of formula (I), which can also be called the DAR of the molecule.

[0404] In some embodiments, D in formula (I) of the present invention can be any antitumor compound, as long as it has antitumor effects and a structural portion that can be attached to the linker, without particular limitation. The antitumor compound can be a pharmaceutically active compound that acts on tumors. Preferably, part or all of the linker can be cleaved within tumor cells to release the antitumor compound portion, thereby exhibiting an antitumor effect.

[0405] In some embodiments, the drug is an antitumor compound, such as a cytotoxic agent, like camptothecin or auroretamine.

[0406] In some implementations, D has the structure shown in formula (D-1a) or formula (D-1b):

[0407] Where R 1a Selected from H and C1-C6 alkyl groups;

[0408] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ;

[0409] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0410] R 4a and R 5a Each is independently selected from H and C1-C4 alkyl groups;

[0411] or

[0412] Where R 1b R 2b R 3b R 4b R 5b and R 8b Each is C independently 1-8 Alkyl, preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl;

[0413] R 6b and R 7b Each is independently selected from C 1-8 Alkoxy, preferably C 1-4 Alkyl groups, such as methoxy, ethoxy, or propoxy;

[0414] R 9b Selected from C 1-8Alkyl groups and COOH, preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and

[0415] R 10b Selected from OH and H;

[0416] In the D structure, the wavy line indicates that the valence bond is connected to L.

[0417] It is understandable that in the structural formula, the chiral centers whose configurations are not specified are each independently of the R or S configuration.

[0418] In some implementations, D has the structure of formula (D-1a), and R 1a For H; R 2a It is a C1-C4 alkyl group, preferably methyl; R 3a For halogens, F and R are preferred. 4a It is a C1-C4 alkyl group, preferably ethyl.

[0419] In some implementations, D has the structure of formula (D-1b), and wherein

[0420] R 1b R 4b and R 8b Each is independently selected from C 1-2 Alkyl groups, preferably methyl groups;

[0421] R 2b R 3b and R 5b Each is independently selected from C 3-4 alkyl;

[0422] R 6b and R 7b Each is independently selected from C 1-2 alkoxy groups; and

[0423] R 9b Selected from C 1-4 Alkyl and R 10b It is OH or H; or R 9b It is COOH and R 10b For H.

[0424] In some implementations, D has the structure shown in formula (D-2a) or formula (D-2b):

[0425] Where R 1a R 2a R 3a and R 4a As defined in equation (D-1a); or

[0426] 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 equation (D-1b).

[0427] In some implementations, D has the structure shown in formula (D-3a) or (D-3b):

[0428] In some implementations, the unspecified chiral centers are each independently of the R or S configuration.

[0429] In some implementations, D has the structure shown in formula (D-4a) or (D-4b):

[0430] It can be understood that the wavy line in the structural formula indicates that the valence bond is connected to the rest of the molecule. For example, the wavy line in the D structural formula indicates that the valence bond is connected to L.

[0431] In some embodiments, in the ADC of the present invention, the drug is: Exatecan, Dxd, SN-38, monomethylaurestatin E (MMAE), or MMAF. The structural formula is shown below:

[0432] In some implementations, -L- has the following structure: -Z-L1-L2-L3-

[0433] in

[0434] 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;

[0435] L1 is selected from non-existent, Where n1 and m1 are each an integer selected from 0 to 20, for example, an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0436] L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and

[0437] L3 is Where X is selected from -NH-, -O-, and -S-; R 1c Each is independently selected from C 1-8 Alkyl, C1-8 Haloalkyl, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is independently 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; and

[0438] Among them, Z is connected to Ab, preferably connected to S on Ab, and L3 is connected to D.

[0439] In some implementations, -L- has the following structure: -Z-L1-L2-L3-

[0440] in

[0441] 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;

[0442] L1 is selected from non-existent, Where n1 and m1 are each an integer selected from 0 to 20, for example, an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0443] L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and

[0444] L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; n3 and n4 are independently 1, 2, 3, 4, 5, or 6; and

[0445] Among them, Z is connected to Ab, preferably connected to S on Ab, and L3 is connected to D.

[0446] In this document, unless otherwise specified, the divalent groups are connected in the directions shown. It is understood that when the variable to be connected indicates absence, it is connected to the next variable. For example, for the Z group, its left side (e.g., the maleimide moiety) is connected to Ab, and its right side (e.g., the carbonyl group) is connected to L1, which covers the following cases: when L1 indicates absence, the right side is actually connected to L2; when both L1 and L2 indicate absence, the right side is actually connected to L3; and when L1, L2, and L3 all indicate absence, the right side is actually connected to D.

[0447] In some implementation schemes, Z is selected from Preferred is Where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, m is 2, 3, 4, or 5, especially 2 or 5.

[0448] In some implementation schemes, Z is selected from

[0449] In some implementation schemes, Z is

[0450] In some implementations, L1 is selected from non-existent, Where n1 is an integer independently selected from 0-12, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some implementations, L1 is non-existent. In some implementations, L1 is... In some implementations, L1 is

[0451] In some implementations, L1 is selected from non-existent,

[0452] In some implementations, L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6, 7 or 8 amino acids.

[0453] In some embodiments, the amino acid residue or amino acid is preferably an L-amino acid. Moreover, in addition to α-amino acids, the amino acid residue or amino acid can be an amino acid residue or amino acid with structures such as β-alanine, ε-aminohexanoic acid, γ-aminobutyric acid, etc., and can also be a non-natural amino acid, such as an N-methylated amino acid.

[0454] In some embodiments, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine ​​(Cys), histidine (His), and threonine (Thr), wherein the amino acid residues or amino acids are optionally converted by one or more C 1-6 Alkyl substitution.

[0455] In some embodiments, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), aspartic acid (Asp), asparagine (Asn), arginine (Arg), and threonine (Thr).

[0456] In some embodiments, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), aspartic acid (Asp), arginine (Arg), and threonine (Thr).

[0457] In some implementations, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Arg-Asp-Val-Thr-, and -Gly-Gly-Phe-Gly-. In some implementations, L2 is selected from -Gly- and -Arg-Asp-Val-Thr-. In some implementations, L2 is -Gly-. In some implementations, L2 is -Arg-Asp-Val-Thr-.

[0458] It should be understood that L2 is connected to L1 via the amino group of the amino acid on the left, or to Z when L1 is absent, and to L3 via the carbonyl group of the amino acid on the right.

[0459] In some implementations, L3 is:

[0460] Where R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n3 and n4 are independently 1, 2, 3, 4, 5, or 6; and n5 is 0, 1, 2, or 3.

[0461] In some implementations, L3 is selected from:

[0462] Where R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is selected independently from each of the following groups: 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.

[0463] In some implementations, L3 is selected from:

[0464] The variables are as defined in this paper.

[0465] In some implementations, L3 is selected from:

[0466] The variables are as defined in this paper.

[0467] In some implementations, L3 is selected from: For example is The variables are as defined in this paper.

[0468] In some implementations, L3 is In some implementations, L3 is Where Su is as defined herein. In some implementations, L3 is... Where Su is as defined in this article.

[0469] In some embodiments, Su is selected from xylose, arabinose, xyuronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid, and mannuronic acid.

[0470] In some implementation schemes, Su is selected from

[0471] In some implementations, Su is independently:

[0472] In some implementation schemes, Su is independently

[0473] In some implementation schemes, Su is independently

[0474] In some implementations, L3 is selected from:

[0475] In some implementations, L3 is selected from:

[0476] In some implementations, L3 is In some implementations, L3 is

[0477] It should be understood that L3 is connected to L2 through the amino group on the left and to D through the carbonyl group on the right.

[0478] In some implementations, -L- or -Z-L1-L2-L3- are each independently selected from the following structures:

[0479] Where each m is an independent integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

[0480] The group is connected to Ab on the left and to D on the right.

[0481] In some implementations, -L- or -Z-L1-L2-L3- are each independently selected from the following structures:

[0482] Where each m is an independent integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

[0483] The group is connected to Ab on the left and to D on the right.

[0484] In some implementations, -L- or -Z-L1-L2-L3- are independently:

[0485] The group is connected to Ab on the left and to D on the right.

[0486] In some embodiments, the antibody-drug conjugate is selected from:

[0487] Where Ab is an antigen-binding molecule described herein, for example, particularly as defined in the second paragraph of the detailed description of the invention herein, such as the multispecific antibody of the present invention, such as a bispecific antibody; and

[0488] m are each an independent integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

[0489] p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 and 16, for example, an integer selected from 4 to 6.

[0490] In some embodiments, the antibody-drug conjugate is selected from:

[0491] Wherein, Ab is an antigen-binding molecule described herein, for example, particularly the antigen-binding molecule defined in the second detailed description of the invention herein, such as the multispecific antibody of the present invention, such as a bispecific antibody; and

[0492] p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 4 to 6, mainly 4 or 6;

[0493] More preferably, Ab is V-F1, V-F2, V-F3, V-F7 or V-F8 and p is 6, or Ab is V-F9 and p is 4, wherein V-F1, V-F2, V-F3, V-F7, V-F8 or V-F9 are each composed of the following chains:

[0494] V-F1: Two chains as shown in SEQ ID NO:42;

[0495] V-F2: Two chains as shown in SEQ ID NO:43;

[0496] V-F3: the first strand as shown in SEQ ID NO:44, and the second strand as shown in SEQ ID NO:45;

[0497] V-F7: the first chain as shown in SEQ ID NO:46, and the second chain as shown in SEQ ID NO:47;

[0498] V-F8: the first chain as shown in SEQ ID NO:48, and the second chain as shown in SEQ ID NO:49;

[0499] V-F9: the first chain as shown in SEQ ID NO:52, and the second chain as shown in SEQ ID NO:53.

[0500] It is understood that, unless otherwise specified and without contradiction in the context, for the ADCs of this invention, the left-hand bond of the divalent group shown herein is connected to an Ab group or a group near the Ab end, and the right-hand bond of the divalent group is connected to a D group or a group near the D end. For example, when L2 is In this case, the amino group on the left is connected to L1, and the carbonyl group on the right is connected to L3.

[0501] It is also understood that the -LD portion can be covalently linked to the Ab in any suitable manner known in the art. In some embodiments, the -LD portion is covalently linked to the Ab via a sulfur (S) atom from the Ab, i.e., the -LD portion and the Ab are linked via -S-. In some embodiments, the sulfur atom originates from the opening of interchain disulfide bonds in the Ab. For example, the interchain disulfide bonds in the Ab are opened by a reducing agent such as TCEP, generating a thiol group (-SH), which is then linked to a terminal functional group of the linker, such as a maleimide moiety. In some embodiments, the sulfur atom originates from (engineered or unengineered) cysteine ​​residues in the Ab.

[0502] In some embodiments, the antibody-drug conjugate of the present invention has an average DAR of 2-10, for example 4-8, or for example 4-6, such as about 4 or about 6. In some embodiments, the antibody-drug conjugate of the present invention has an average DAR of about 4. In some embodiments, the antibody-drug conjugate of the present invention has an average DAR of about 6.

[0503] It should be noted that the above and other technical solutions of the present invention and one or more features thereof can be arbitrarily combined to constitute technical solutions not directly described herein, and these undescribed technical solutions are also covered within the scope of this application.

[0504] Preparation of ADCs molecules of the present invention

[0505] Another aspect of the invention provides a method for preparing an antibody-drug conjugate (ADC) using the antigen-binding molecule of the invention, formula (I). The method includes conjugating an antibody (Ab) of the invention to a drug moiety (D) via a linker (L).

[0506] In some implementations, the method includes the following steps:

[0507] (a) Add a reducing agent to antibody Ab in buffer solution and incubate;

[0508] (b) Couple the reaction solution from step (a) by adding a linker-load to the reaction solution to obtain the crude product; and

[0509] (c) Optionally, the crude product is purified to obtain the antibody-drug conjugate of the present invention;

[0510] Ab is defined as in this article.

[0511] It should be understood that the connector-payload and Ab response provide the -LD portion in equation (I). Given a clear definition of -LD, the structure of the connector-payload can be determined according to existing technology.

[0512] In some implementations, the reducing agent in step a) is TCEP.

[0513] In some embodiments, the buffer in step a) is a NaOAC-HOAc buffer, a HIS-HAC buffer, or a PBS buffer. Preferably, the buffer has a pH of 5.0-9.0, for example, 6.0-8.0.

[0514] In some implementations, the connector-payload has the following structure: Z'-L1-L2-L3-D, where L1, L2, L3, and D are as defined above, and Z' is... m is defined above, for example, as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0515] In some implementations, Z' is selected from

[0516] In some implementation schemes, for the preparation of Z as The method for antibody-drug conjugates (ADCs) further includes a hydrolysis step that opens the ring of maleimide.

[0517] In some implementations, the methods of this application are carried out under the specific reaction conditions disclosed in the embodiments.

[0518] 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.

[0519] 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.

[0520] IV. Pharmaceutical Combinations, Drug Conjugates, and Reagent Kits

[0521] In one aspect, the present invention provides compositions, such as pharmaceutical compositions, comprising molecules described herein (encompassing antigen-binding molecules such as antibodies, immunoconjugates, immunofusions, antibody-drug conjugates, or pharmaceutically acceptable salts or solvates thereof) formulated together with pharmaceutically acceptable excipients.

[0522] As used herein, "pharmaceutical excipients" include any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. 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 (covering antigen-binding molecules described herein such as antibodies, immunoconjugates, immunofusions, antibody-drug conjugates, or pharmaceutically acceptable salts or solvates thereof) is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise the molecule of the present invention (covering antigen-binding molecules described herein such as antibodies, immunoconjugates, immunofusions, antibody-drug conjugates, or pharmaceutically acceptable salts or solvates thereof) with one or more therapeutic agents.

[0523] In another aspect, the present invention also provides a pharmaceutical combination comprising the molecules of the present invention with one or more therapeutic agents.

[0524] 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.

[0525] 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.

[0526] 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.

[0527] V. Uses and Methods

[0528] Based on the excellent targeting properties of the molecules of the present invention (including antigen-binding molecules described herein such as antibodies, immunoconjugates, immunofusions, antibody-drug conjugates, or pharmaceutically acceptable salts or solvates thereof) to tumor cells expressing CDH17 and / or EGFR, as well as the other superior properties described above, the present invention also provides the molecules of the present invention and their applications and methods in the treatment and prevention of CDH17 and / or EGFR-related diseases.

[0529] EGFR and CDH17 are overexpressed in cancer tissues from various sources (e.g., on cell surfaces), and are therefore suitable targets for developing cancer therapies. In one aspect, the present invention provides the use of the molecules of the invention for the prevention and / or treatment of EGFR and / or CDH17-positive tumors in a subject. In said application, 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; drugs that target immune checkpoints to activate the subject's immune system, prompting it to spontaneously eliminate tumors; drugs that target T cell engagers to activate the immune system to eliminate tumor cells by recruiting T cells; or small molecule tyrosine gammidine inhibitors that inhibit tumor cell division and proliferation by binding to tyrosine kinases and blocking tumor cell signaling.

[0530] In another aspect, the present invention also provides a method for preventing or treating EGFR and / or CDH17-related diseases in subjects, comprising administering the molecules of the present invention to subjects in need. In some embodiments, the EGFR and / or CDH17-related disease is a tumor, such as cancer, for example, an EGFR- and / or CDH17-positive tumor or cancer, such as an EGFR-positive tumor or cancer, or a CDH17-positive tumor or cancer, or an EGFR-positive and CDH17-positive tumor or cancer.

[0531] The tumors suitable for the methods and applications of this invention can be selected from various solid tumors, such as gastrointestinal tumors or pancreatic cancer, for example: colon cancer, colorectal cancer (e.g., colorectal adenocarcinoma), lung cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung, pancreatic cancer, gastric cancer (e.g., gastric adenocarcinoma), gastroesophageal adenocarcinoma, cervical cancer, ovarian cancer, or neuroendocrine tumors, etc. In some embodiments, the solid tumor is an EGFR and / or CDH17 positive tumor, particularly an EGFR and CDH17 positive tumor.

[0532] The EGFR and / or CDH17-positive tumors suitable for the methods and applications of this invention can be early, intermediate, or late-stage cancers, or metastatic cancers. Furthermore, the EGFR 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.

[0533] In some embodiments, EGFR and / or CDH17 positive tumors suitable for prevention or treatment according to the method of the present invention have overexpression of EGFR and / or CDH17, for example, overexpression of EGFR 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 the adjacent healthy tissues or healthy cells of the same subject's tumor tissues or cells.

[0534] In some embodiments, the EGFR 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% EGFR and / or CDH17-positive cells. In some embodiments, the EGFR-positive tumors have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% EGFR-positive cells. In some embodiments, the 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 implementations, EGFR-positive and CDH17-positive tumors have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% EGFR and at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% CDH17 double-positive cells. EGFR and / or CDH17 expression levels on tumor biopsies can be assessed by immunohistochemistry. High percentages of EGFR and / or CDH17-positive cells have been detected in biopsies from various cancers, such as the aforementioned tumors or cancers.

[0535] Preferably, in some embodiments, the method according to the invention is used to treat tumors having a high percentage of EGFR and / or CDH17 positive cells, for example, tumors having at least 25%, 50%, 75%, or 100% EGFR and / or CDH17 positive cells. In some embodiments, tumors having a high percentage of EGFR positive cells have at least 25%, 50%, 75%, or 100% EGFR positive cells. In some embodiments, tumors having a high percentage of CDH17 positive cells have at least 25%, 50%, 75%, or 100% CDH17 positive cells. In some embodiments, tumors having a high percentage of both EGFR and CDH17 positive cells have at least 25%, 50%, 75%, or 100% EGFR and at least 25%, 50%, 75%, or 100% CDH17 double positive cells.

[0536] In some embodiments, the method according to the invention can also be used to treat cancers with less than 25% or 20% of EGFR and / or CDH17 positive cells, preferably, the tumor has less than 25% or 20% of EGFR positive cells but is a tumor with a high percentage of CDH17 positive cells; or the tumor has less than 25% or 20% of CDH17 positive cells but is a tumor with a high percentage of EGFR positive cells. In some embodiments, the tumor has less than 25% or 20% of both EGFR positive cells and CDH17 positive cells.

[0537] In some embodiments, the application of the method of the present invention in said cancer results in tumor growth inhibition.

[0538] In some embodiments, the application of the method of the present invention in said cancer induces tumor regression.

[0539] 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.

[0540] 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.

[0541] 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.

[0542] On the other hand, the present invention provides molecules of the invention for therapeutic purposes, such as for the prevention and / or treatment of EGFR and / or CDH17-related diseases as defined herein.

[0543] On the other hand, the present invention provides molecules of the present invention that are used as medicines, such as medicines for preventive or therapeutic purposes of the present invention, for example, medicines for the prevention and / or treatment of EGFR and / or CDH17-related diseases as defined herein.

[0544] 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 prevention and / or treatment of EGFR and / or CDH17-related diseases as defined herein. VI. Specific Implementation Plan

[0545] Some aspects of this invention relate to the following specific embodiments:

[0546] 1. A multispecific antibody that specifically binds to EGFR and CDH17, wherein the multispecific antibody comprises at least one (preferably one or two) antigen-binding domains specifically binding to EGFR, such as VHH, and at least one (preferably two) antigen-binding domains specifically binding to CDH17, such as VHH.

[0547] Preferably, the multispecific antibody comprises one or two antigen-binding domains that specifically bind to EGFR, such as VHH, and two antigen-binding domains that specifically bind to CDH17, such as VHH.

[0548] Preferably, the multispecific antibody is a bispecific antibody.

[0549] 2. The multispecific antibody of implementation scheme 1, wherein the multispecific antibody comprises an antigen-binding domain VHH that specifically binds to EGFR. EGFR And two antigen-binding domains that specifically bind to CDH17, VHH CDH17 Preferably, the multispecific antibody comprises two polypeptide chains, one polypeptide chain comprising two tandem VHHs and optionally an Fc region, and the other polypeptide chain comprising one VHH and optionally an Fc region.

[0550] Optionally, the multispecific antibody is a bispecific antibody, and the bispecific antibody contains an antigen-binding domain VHH that specifically binds to EGFR. EGFR And two antigen-binding domains that specifically bind to CDH17, VHH CDH17Preferably, the bispecific antibody comprises two polypeptide chains, one polypeptide chain comprising two tandem VHHs and optionally an Fc region, and the other polypeptide chain comprising one VHH and optionally an Fc region.

[0551] 3. The multispecific antibody of embodiment 2, which is a bispecific antibody, wherein the bispecific antibody comprises or is composed of two polypeptide chains, wherein the polypeptide chains comprise or are composed of the following structure from the N-terminus to the C-terminus:

[0552] First polypeptide chain: VHH CDH17 -VHH CDH17 -Fc 1 ,

[0553] Second polypeptide chain: VHH EGFR -Fc 2 ,

[0554] or

[0555] First polypeptide chain: VHH EGFR -VHH CDH17 -Fc 1 ,

[0556] Second polypeptide chain: VHH CDH17 -Fc 2 ,

[0557] or

[0558] First polypeptide chain: VHH CDH17 -VHH EGFR -Fc 1 ,

[0559] Second polypeptide chain: VHH CDH17 -Fc 2 ,

[0560] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length; preferably, the two tandem VHHs of the first polypeptide chain are connected via a linker.

[0561] Optionally, the C-terminus of the second VHH of the first polypeptide chain is connected to the Fc... 1 N-terminal connection;

[0562] Optionally, the C-terminus of the first VHH of the first polypeptide chain is connected to the N-terminus of the second VHH via a connector;

[0563] Optionally, the C-terminus of the VHH of the second polypeptide chain is connected to the Fc... 2 The N-terminal connection.

[0564] 4. The multispecific antibody according to embodiment 2 or 3, wherein the Fc region is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc, for example, the Fc region comprises an amino acid sequence SEQ ID NO:33 or SEQ ID NO:34 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity with the amino acid sequence, or is composed of the amino acid sequence.

[0565] Optionally, the Fc region contains a mutation that reduces or eliminates the connection between the Fc region and the Fcγ receptor, such as the LALA mutation. For example, an 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:35, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:35.

[0566] Optionally, the Fc region contains a C220S mutation;

[0567] Optionally, the Fc region containing the LALA mutation and the C220S mutation 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:56, for example, containing or consisting of the amino acid sequence shown in SEQ ID NO:56.

[0568] 5. A multispecific antibody according to any one of embodiments 2-4, wherein the two Fc regions respectively contain a Knob mutation and a Hole mutation, for example, the Knob mutation T366W, and the Hole mutations T366S, L368A, and Y407V combined; or the Knob mutation is T366Y, and the Hole mutation is Y407T; optionally, the Fc region containing the Knob mutation further contains the S354C mutation, and the Fc region containing the Hole mutation further contains the Y349C mutation.

[0569] For example, the Fc region containing the Knob mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:36, while the Fc region containing the Hole mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:37; or

[0570] The Fc region containing the Knob mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:54, while the Fc region containing the Hole mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:55.

[0571] 6. The multispecific antibody of implementation scheme 1, which contains two antigen-binding domains VHH that specifically bind to EGFR. EGFR And two antigen-binding domains that specifically bind to CDH17, VHH CDH17 Preferably, the multispecific antibody comprises two polypeptide chains, wherein one polypeptide chain contains two tandem VHHs. CDH17 and VHH EGFR And optionally the Fc region, and another polypeptide chain contains two tandem VHHs CDH17 and VHH EGFR , and optionally the Fc region; preferably, the two polypeptide chains are identical;

[0572] Optionally, the multispecific antibody is a bispecific antibody and contains two antigen-binding domains VHH that specifically bind to EGFR. EGFR And two antigen-binding domains that specifically bind to CDH17, VHH CDH17 Preferably, the bispecific antibody comprises two polypeptide chains, one of which contains two tandem VHH molecules. CDH17 and VHH EGFR And optionally the Fc region, and another polypeptide chain contains two tandem VHHs CDH17 and VHH EGFR , and optionally the Fc region; preferably, the two polypeptide chains are identical.

[0573] 7. The multispecific antibody of embodiment 6, which is a bispecific antibody, wherein the bispecific antibody comprises or is composed of two identical polypeptide chains, wherein each polypeptide chain comprises, or is composed of, the following structure from the N-terminus to the C-terminus:

[0574] VHH CDH17 -VHH EGFR -Fc or VHH EGFR -VHH CDH17 -Fc

[0575] The symbol "-" indicates a connection via a connector or a direct connection, preferably a connector with a length of 5-15 amino acids;

[0576] Optionally, VHH CDH17 With VHH EGFR They are connected via a connector;

[0577] Optionally, the C terminal of the first VHH is connected to the N terminal of the second VHH via a connector; the C terminal of the second VHH is connected to the N terminal of Fc.

[0578] 8. The multispecific antibody of embodiment 6 or 7, wherein the Fc region is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc, for example, the Fc region comprises an amino acid sequence SEQ ID NO:33 or SEQ ID NO:34 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity with the amino acid sequence, or is composed of the amino acid sequence, optionally, the Fc region comprises a mutation that reduces or eliminates the Fc region from the Fcγ receptor, such as the LALA mutation, for example, the Fc region containing the LALA mutation comprises 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:35, for example, comprising or composed of the amino acid sequence shown in SEQ ID NO:35.

[0579] 9. A multispecific antibody according to any one of embodiments 1-8, wherein the multispecific antibody comprises a linker and the linker comprises (G4S)n, wherein n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5.

[0580] 10. The multispecific antibody according to any one of embodiments 1-9, comprising VHH (VHH) that specifically binds to CDH17. CDH17 ), and the VHH CDH17 It contains or is composed of a heavy chain variable region, the heavy chain variable region comprising the three complementary determinant regions (CDRs) contained in the VH shown in any one of SEQ ID NO:31, 20-30 and 32; preferably, the CDR sequence is defined according to ABM.

[0581] 11. The multispecific antibody described in Implementation Scheme 10, wherein the VHH CDH17 It includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or it includes or is composed of heavy chain variable regions, wherein the heavy chain variable regions include complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0582] VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:14, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:15, and VHH CDR3 contains or is composed of the amino acid sequence shown in any one of SEQ ID NO:18, 16, 17, and 19.

[0583] 12. The multispecific antibody according to embodiment 10 or 11, wherein the VHH CDH17 Contains or is composed of heavy chain variable regions, said heavy chain variable regions

[0584] (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: 31, 20-30, and 32; or

[0585] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:31, 20-30 and 32.

[0586] 13. A multispecific antibody according to any one of implementation schemes 1-12, comprising VHH (VHH) that specifically binds to EGFR. EGFR ), and the VHH EGFR It contains or is composed of a heavy chain variable region, the heavy chain variable region comprising the three complementary determinant regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 12, 8-10 and 13; preferably, the CDR sequence is defined according to ABM.

[0587] 14. The multispecific antibody described in Implementation Scheme 13, wherein the VHH EGFR It includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or it includes or is composed of heavy chain variable regions, wherein the heavy chain variable regions include complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0588] (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:6.

[0589] (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:2, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:5; or

[0590] (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:4, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:7.

[0591] 15. The multispecific antibody according to embodiment 13 or 14, wherein the VHH EGFR Contains or is composed of heavy chain variable regions, said heavy chain variable regions

[0592] (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: 12, 8-10, and 13; or

[0593] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:12, 8-10 and 13.

[0594] 16. The multispecific antibody according to any one of embodiments 1-15, wherein the antigen-binding domain that specifically binds to CDH17 is VHH CDH17 It contains or is composed of a heavy chain variable region, the heavy chain variable region comprising the three complementarity-determining regions (CDRs) contained in VH shown in SEQ ID NO:31; preferably, the CDR sequence is defined according to ABM; and the antigen-binding domain that specifically binds to EGFR is VHH. EGFR It contains or is composed of a heavy chain variable region, the heavy chain variable region containing the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:12; preferably, the CDR sequence is defined according to ABM.

[0595] 17. The multispecific antibody according to any one of embodiments 1-16, wherein the antigen-binding domain that specifically binds to CDH17 is VHH CDH17The EGFR-specific antigen-binding domain 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:14, VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15, and VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:18; and the EGFR-specific antigen-binding domain is VHH. EGFR It contains complementarity-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 complementarity-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:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:6.

[0596] 18. The multispecific antibody according to any one of embodiments 1-17, wherein the antigen-binding domain that specifically binds to CDH17 is VHH CDH17 It contains or is composed of a heavy chain variable region, the heavy chain variable region containing or being composed of the amino acid sequence shown in SEQ ID NO:31; and the antigen-binding domain that specifically binds to EGFR is VHH. EGFR It contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0597] 19. The multispecific antibody according to any one of embodiments 1-18, wherein it is a bispecific antibody, and wherein the bispecific antibody comprises or is composed of two polypeptide chains, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of:

[0598] (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 44 and 45, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences,

[0599] (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 46 and 47, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences,

[0600] (iii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 48 and 49, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences; or

[0601] (iv) Each of the amino acid sequences shown in SEQ ID NOs: 52 and 53, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences.

[0602] 20. The multispecific antibody according to any one of embodiments 1-18, which is a bispecific antibody, wherein the bispecific antibody comprises or is composed of two polypeptide chains, wherein the first polypeptide chain and the second polypeptide chain respectively comprise or are composed of the amino acid sequences shown in SEQ ID NOs: 44 and 45.

[0603] 21. The multispecific antibody according to any one of embodiments 1-18, which is a bispecific antibody, and wherein the bispecific antibody comprises or is composed of two identical polypeptide chains, wherein each polypeptide chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 42 or 43 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

[0604] 22. The multispecific antibody according to any one of embodiments 1-21, wherein it is a bispecific antibody, and wherein the bispecific antibody has one or more of the following properties:

[0605] (i) K binding affinity with human CDH17 D The value is less than about 10 nM, for example less than about 5 nM or 4 nM, preferably between 0.5 and 10 nM, for example between 1 and 5 nM, as determined by the SPR method;

[0606] (ii) K binding affinity with CDH17 of cynomolgus monkeys DThe value is less than about 10 nM, for example less than about 8 nM, 7 nM or 6 nM, preferably between 1 and 10 nM, for example between 1 and 7 nM or between 2 and 6 nM, as determined by the SPR method;

[0607] (iii) K binding affinity with human EGFR D The value is less than about 50 nM, for example less than about 40 nM, 30 nM, 20 nM or 15 nM, for example less than about 12 nM or 11 nM or 7 nM or 5 nM, preferably between 1 and 15 nM, for example between 2 and 12 nM or between 3 and 11 nM, for example as determined by the SPR method;

[0608] (iv) K binding affinity to EGFR in cynomolgus monkeys D The value is less than about 50 nM, for example less than about 40 nM, 30 nM, 20 nM or 15 nM, for example less than about 12 nM, 11 nM or 10 nM, preferably between 1 and 15 nM, for example between 2 and 12 nM or between 2 and 10 nM, for example as determined by the SPR method;

[0609] (v) It exhibits cross-reactivity to human and monkey EGFR and / or CDH17;

[0610] (vi) It has a synergistic effect with the binding of EGFR and CDH17 targets, that is, it has higher binding activity with EGFR and / or CDH17 positive, preferably EGFR positive and CDH17 positive tumor cells, for example, compared with the binding of EGFR monospecific antibody (e.g., EGFR parent antibody) to EGFR positive tumor cells, or compared with the binding of CDH17 monospecific antibody (e.g., CDH17 parent antibody) to CDH17 positive tumor cells;

[0611] (vii) Capable of being internalized by cells expressing EGFR and / or CDH17, particularly, the bispecific antibody exhibits greater endocytic activity in EGFR-positive and / or CDH17-positive tumor cells, such as EGFR-positive and CDH17-positive tumor cells, for example, co-endocytic activity, for example, compared to the endocytic activity of EGFR monospecific antibodies (e.g., EGFR parental antibodies) in EGFR-positive tumor cells, or compared to the endocytic activity of CDH17 monospecific antibodies (e.g., CDH17 parental antibodies) in CDH17-positive tumor cells; or

[0612] (viii) Capable of killing cells expressing EGFR and / or CDH17, particularly, the bispecific antibody exhibits greater killing activity against EGFR-positive and / or CDH17-positive tumor cells, such as EGFR-positive and CDH17-positive tumor cells, for example, synergistic killing activity, for example, compared to the killing activity of EGFR-positive tumor cells by EGFR monospecific antibodies (e.g., EGFR parental antibodies), or compared to the killing activity of CDH17-positive tumor cells by CDH17 monospecific antibodies (e.g., CDH17 parental antibodies).

[0613] 23. A nucleic acid molecule, which encodes a multispecific antibody or any polypeptide chain thereof, according to any one of the implementation schemes 1-22.

[0614] 24. An expression vector comprising the nucleic acid molecule of embodiment 23, preferably, the expression vector being pCDNA, such as pCDNA3.4.

[0615] 25. A host cell comprising the nucleic acid molecule described in embodiment 23 or the expression vector described in embodiment 24, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells.

[0616] 26. A method for preparing a multispecific antibody according to any one of embodiments 1-22, the method comprising culturing a host cell containing the nucleic acid molecule of embodiment 23 or the expression vector of embodiment 24 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0617] 27. An immunoconjugate or immunofusion containing any of the multispecific antibodies in embodiments 1-22.

[0618] 28. Antibody-drug conjugates having formula (I) or pharmaceutically acceptable salts or solvates thereof: Ab-(LD) p (I)

[0619] in:

[0620] Ab is a multispecific antibody according to any one of implementation schemes 1-22;

[0621] L is the connector;

[0622] D represents a drug, such as an anti-tumor compound;

[0623] p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, for example, an integer selected from 4 to 6.

[0624] 29. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 28, wherein the drug is a cytotoxic agent, such as a camptothecin or auratestatin.

[0625] 30. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 28, wherein D has the structure shown in formula (D-1a) or formula (D-1b):

[0626] Where R 1a Selected from H and C1-C6 alkyl groups;

[0627] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ;

[0628] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0629] R 4a and R 5a Each is independently selected from H and C1-C4 alkyl groups;

[0630] or

[0631] Where R 1b R 2b R 3b R 4b R 5b and R 8b Each is C independently 1-8 Alkyl, preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl;

[0632] R 6b and R 7b Each is independently selected from C 1-8 Alkoxy, preferably C 1-4 Alkyl groups, such as methoxy, ethoxy, or propoxy;

[0633] 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

[0634] R 10b Selected from OH and H;

[0635] In the D structure, the wavy line indicates that the valence bond is connected to L.

[0636] 31. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 30, wherein D has the structure of formula (D-1a), and wherein R 1a For H; R 2a It is a C1-C4 alkyl group, preferably methyl; R 3a For halogens, F and R are preferred. 4a It is a C1-C4 alkyl group, preferably ethyl.

[0637] 32. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 30, wherein D has the structure of formula (D-1b), and wherein

[0638] R 1b R 4b and R 8b Each is independently selected from C 1-2 Alkyl groups, preferably methyl groups;

[0639] R 2b R 3b and R 5b Each is independently selected from C 3-4 alkyl;

[0640] R 6b and R 7b Each is independently selected from C 1-2 alkoxy groups; and

[0641] R 9b Selected from C 1-4 Alkyl and R 10b It is OH or H; or R 9b It is COOH and R 10b For H.

[0642] 33. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 30-32, wherein D has the structure shown in formula (D-2a) or formula (D-2b):

[0643] Where R 1a R 2a R 3a and R 4a As defined in equation (D-1a); or

[0644] Where R 1b R 2b R 3b R4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined in equation (D-1b).

[0645] 34. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 30, wherein D has the structure shown in formula (D-3a) or (D-3b):

[0646] Preferably, D has the structure shown in formula (D-4a) or (D-4b):

[0647] 35. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 28, wherein the drug is Exatecan, Dxd, SN-38, monomethylaurestatin E (MMAE) or MMAF.

[0648] 36. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 28-35, wherein -L- has the following structure: -Z-L1-L2-L3-

[0649] in

[0650] Z is selected from Where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0651] L1 is selected from non-existent, Where n1 and m1 are each an independent integer selected from 0 to 20, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

[0652] L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and

[0653] L3 is Where X is selected from -NH-, -O-, and -S-; R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8Alkyl, halogen, nitro, and cyano groups; Su is independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; n3 and n4 are independently 1, 2, 3, 4, 5, or 6; and

[0654] Among them, Z is connected to Ab, preferably connected to S on Ab, and L3 is connected to D.

[0655] 37. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in embodiment 36, wherein...

[0656] Z is selected from Preferred is Where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0657] Preferably, Z is selected from

[0658] More preferably, Z is

[0659] 38. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36 or 37, wherein L1 is selected from those that do not exist. Where n1 is an integer independently selected from 0 to 12, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0660] Preferably, L1 is selected from non-existent,

[0661] 39. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 36-38, wherein L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6, 7 or 8 amino acids; preferably, wherein each amino acid residue or amino acid is independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine ​​(Cys), histidine (His) and threonine (Thr), wherein the amino acid residue or amino acid is optionally divided by one or more C 1-6 Alkyl substitution;

[0662] More preferably, the amino acid residues or each amino acid is independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), aspartic acid (Asp), asparagine (Asn), arginine (Arg), and threonine (Thr);

[0663] More preferably, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), aspartic acid (Asp), arginine (Arg) and threonine (Thr).

[0664] 40. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-39, wherein L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Arg-Asp-Val-Thr-, and -Gly-Gly-Phe-Gly-, preferably selected from -Gly- and -Arg-Asp-Val-Thr-.

[0665] 41. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-40, wherein L3 is selected from:

[0666] Optionally, L3 is

[0667] Optional, L3 is

[0668] Where R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is selected independently from each of the following groups: 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.

[0669] 42. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-41, wherein L3 is selected from:

[0670] Preferably, L3 is selected from:

[0671] More preferably, L3 is selected from: For example is

[0672] 43. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-42, wherein each of Su is independently:

[0673] Preferably, Su is independently

[0674] Alternatively, preferably, each Su is independently...

[0675] 44. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-43, wherein L3 is selected from:

[0676] Preferably, L3 is selected from

[0677] 45. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 30-35, wherein -L- are each independently selected from the following structures:

[0678] Preferred is

[0679] Where each m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0680] Optionally, -L- independently are:

[0681] The group is connected to Ab on the left and to D on the right.

[0682] 46. ​​The antibody-drug conjugate according to embodiment 28 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is selected from:

[0683] Where Ab is a multispecific antibody of any one of the implementation schemes 1-22;

[0684] m are each an independent integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

[0685] p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 and 16, for example, an integer selected from 4 to 6.

[0686] 47. The antibody-drug conjugate according to embodiment 28, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is:

[0687] Wherein, Ab is a multispecific antibody of any one of implementation schemes 1-22; and

[0688] p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 4 to 6, mainly 4 or 6.

[0689] 48. The antibody-drug conjugate according to embodiment 28, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is:

[0690] Wherein, Ab is a multispecific antibody of any one of implementation schemes 1-22; and

[0691] p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 4 to 6, mainly 4 or 6.

[0692] 49. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 47 or 48, wherein the Ab is V-F1, V-F2, V-F3, V-F7 or V-F8 and p is 6, or the Ab is V-F9 and p is 4, wherein V-F1, V-F2, V-F3, V-F7, V-F8 or V-F9 are each composed of the following chains:

[0693] V-F1: Two chains as shown in SEQ ID NO:42;

[0694] V-F2: Two chains as shown in SEQ ID NO:43;

[0695] V-F3: the first strand as shown in SEQ ID NO:44, and the second strand as shown in SEQ ID NO:45;

[0696] V-F7: the first chain as shown in SEQ ID NO:46, and the second chain as shown in SEQ ID NO:47;

[0697] V-F8: the first chain as shown in SEQ ID NO:48, and the second chain as shown in SEQ ID NO:49;

[0698] V-F9: the first chain as shown in SEQ ID NO:52, and the second chain as shown in SEQ ID NO:53.

[0699] 50. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 28-49, wherein the LD portion is covalently linked to the Ab via a sulfur atom from the Ab.

[0700] 51. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 28-50, wherein the antibody-drug conjugate has an average DAR of 2-10, for example 4-8, for example about 4-6, for example about 4 or about 6.

[0701] 52. A pharmaceutical composition comprising a multispecific antibody of any one of embodiments 1-22, a nucleic acid molecule of embodiment 23, an immunoconjugate or immunofusion of embodiment 27, an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of embodiments 28-51, and a pharmaceutically acceptable excipient, and optionally further comprising one or more additional therapeutically active substances.

[0702] 53. The use of any multispecific antibody of embodiments 1-22, the nucleic acid molecule of embodiment 23, the immunoconjugate or immunofusion of embodiment 27, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate of any embodiment 28-51 as a drug or for the preparation of a drug.

[0703] 54. Use of embodiment 53, wherein the drug is used to treat and / or prevent cancer in an individual, preferably said cancer being CDH17 and / or EGFR positive cancer.

[0704] 55. The use of embodiment 54, wherein the cancer is selected from, for example, gastrointestinal tumors or pancreatic cancer, such as: colon cancer, colorectal cancer (e.g., colorectal adenocarcinoma), lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, gastric cancer (e.g., gastric adenocarcinoma), gastroesophageal adenocarcinoma, cervical cancer, ovarian cancer, or neuroendocrine tumor.

[0705] 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.

[0706] Brief description of the attached diagram

[0707] Figure 1. FACS binding curve of an exemplary antibody EGFR VHH-Fc

[0708] Figure 2. Exemplary FACS binding curves for anti-CDH17 VHH-Fc.

[0709] Figure 3. Exemplary detection of anti-EGFR VHH-Fc endocytosis in NCI-H1975 cells.

[0710] Figure 4. Exemplary detection of anti-CDH17 VHH-Fc endocytosis in SNU5 and SNU620 cells.

[0711] Figure 5. Expression of EGFR and CDH17 in different tumor cell lines

[0712] Figure 6. Exemplary binding of bispecific antibodies to tumor cells with different antigen expression levels.

[0713] Figure 7. Endocytosis results of exemplary bispecific antibody molecules by different tumor cells.

[0714] Figure 8 shows the in vitro killing activity of ADCs.

[0715] Figure 9 shows the antitumor effect (A) and weight change (B) of ADCs in a mouse SW48 subcutaneous transplantation model.

[0716] Figure 10 shows the antitumor effect (A) and weight change (B) of ADCs in a mouse SW48 subcutaneous transplantation model.

[0717] Figure 11 shows the antitumor effect (A) and weight change (B) of ADCs in a mouse SNU5 subcutaneous transplantation model.

[0718] Figure 12 shows the antitumor effect (A) and weight change (B) of ADCs in a mouse SNU620 subcutaneous transplantation model.

[0719] Figure 13 shows the antitumor effect (A) and weight change (B) of ADCs in a mouse Colo205 subcutaneous transplantation model.

[0720] Figure 14 shows the antitumor effect (A) and weight change (B) of ADCs in a mouse AsPc-1 subcutaneous transplantation model.

[0721] Figure 15 shows the antitumor effect (A) and weight change (B) of ADCs in the mouse SNU-16 subcutaneous transplantation model.

[0722] Figure 16 shows the antitumor effect (A) and weight change (B) of ADCs in a mouse EBC-1 subcutaneous transplantation model.

[0723] Figure 17 shows the antitumor effect (A) and weight change (B) of ADCs in a mouse T84 subcutaneous transplantation model.

[0724] Figure 18 shows the antitumor effect of ADCs in the human ovarian cancer xenograft LD1-0032-411098 model.

[0725] Figure 19 shows the antitumor effect of ADCs in a mouse model of subcutaneous transplantation of human pancreatic cancer Aspc-1.

[0726] Figure 20 shows the configurations of the bispecific antibodies V-F1, V-F2, V-F3 / V-F9, V-F7, and V-F8 of the present invention. Example

[0727] The following embodiments are described to aid in understanding the invention. These embodiments are not intended and should not be construed as limiting the scope of the invention in any way. In this application, when chemical names and structural formulas differ, the structural formula shall prevail unless the chemical name, rather than the structural formula, is correct based on the context or by reason of a person skilled in the art. For simplicity, not all hydrogen atoms are explicitly indicated in the structural formulas of some compounds given in this application. The presence of a vacant valence in a compound indicates the presence of unindicated hydrogen atoms. Element symbols include their isotopic equivalents. For example, H (hydrogen) includes H0. 1 H 2 or H 3 .

[0728] Unless otherwise specified, all experimental materials and reagents used in the examples are commercially available or can be readily prepared using methods known to those skilled in the art. Solvent-to-solvent ratios are generally expressed as volume ratios unless otherwise indicated.

[0729] The compounds, reagents, raw materials, and linkers-payloads used in some embodiments of this invention are known in the prior art and / or commercially available. When the drawn structure is inconsistent with the actual situation, modifications to the structure should be allowed according to the actual situation.

[0730] The abbreviations used herein generally have the meanings commonly understood in the art. In particular, unless otherwise specified and without contradiction of the context, the abbreviations used have the following meanings:

[0731] Materials and methods

[0732] Reference antibody and its preparation

[0733] In this embodiment, the reference antibody JNJ-61186372 was used. JNJ-61186372 is a bispecific antibody targeting EGFR and cMet. JNJ-61186372 is generated based on the SEQ ID NO:199-202 sequence and preparation method in patent WO2014081954A1.

[0734] In addition, the reference antibody PTA001_A4 (also known as V-BMK2) is used. V-BMK2 is a monospecific bivalent antibody disclosed in US20160039933A1. It is an anti-CDH17 antibody constructed from the amino acid sequences of the heavy and light chain variable regions of PTA001_A4 (SEQ ID NO 38 and 49 in US20160039933A1). In the context of the reference antibody V-BMK2, 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: 39) and a kappa light chain (SEQ ID NO: 38).

[0735] The reference antibody was 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.

[0736] Antigens and their preparation

[0737] Extracellular sequence information for human EGFR (UniProt_P00533), CDH17 (UniProt_Q12864), and extracellular sequence information for EGFR (Leu25-Ser645), CDH17 (Gln23-Met787), and cynomolgus monkey CDH17 (NCBI_XP_005563762.1) (CDH17-Glu23-Thr784) were retrieved from the database. His tags were 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 Sanger sequencing verification, plasmids were extracted for later use. These constructed eukaryotic expression vectors were transiently transfected into Expi293F cells. 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 activity of the prepared human CDH17-His and cynomolgus monkey CDH17-His recombinant antigen proteins was confirmed by ELISA.

[0738] Recombinant engineered cell lines expressing antigens and their preparation

[0739] Prepare CHO-K1 engineered cell lines expressing human and monkey EGFR as described below.

[0740] The full-length genes of human EGFR (UniProt_P00533) and monkey EGFR (UniProt: A0A2K5WKD8) were inserted into the lentiviral expression vector pLVX-puro, respectively. Lentiviral viruses were packaged in 293T cells and used to infect CHO-K1 cells. Cells were cultured in medium containing 8 μg / ml puromycin selection pressure to obtain polyclonal cell lines. Stable transgenic CHO-K1-human EGFR cell lines (CHO-K1-huEGFR) and CHO-K1-monkey EGFR cell lines (CHO-K1-cynoEGFR) expressing high levels of the target antigen were selected through limiting dilution. FACS analysis using the reference antibody JNJ-61186372 showed that the antigen expression positivity rate of the stable transgenic cell lines was >90%.

[0741] HEK293 engineered cell lines expressing human and monkey CDH17 were prepared as follows.

[0742] 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%.

[0743] ELISA combination assay

[0744] 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- OD650 .

[0745] FACS Combined Test

[0746] Target cells at 1-5 × 10⁶ cells per well 5 Seed cells at a density of 100 μL / well in 96-well plates and centrifuge at 300 g for 5 min at 4 °C. Add the test sample diluted to the appropriate concentration and incubate at 2-8 °C for approximately 1 h. 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.

[0747] endocytosis test

[0748] Target cells at 1-5 × 10⁶ cells per well 5 Seed cells onto plates, add the test sample diluted to the appropriate concentration, and incubate at 2-8°C for approximately 0.5 hours to allow the test sample to bind to the cells. Centrifuge at 400xg for 4 minutes at 4°C, 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°C and 37°C for 4 hours, respectively. After incubation, immediately add ice-cold FACS buffer to terminate the endocytosis experiment. Centrifuge at 400xg for 4 minutes at 4°C, 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°C in the dark for approximately 30 minutes to 1 hour. 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:

[0749] MFI of samples incubated at 4°C - MFI of samples incubated at 37°C.

[0750] Internalization rate % = 100% - (MFI of samples incubated at 37℃ / MFI of samples incubated at 4℃) × 100%.

[0751] SPR measurement

[0752] The binding affinity between antibodies and antigens is detected using surface plasmon resonance (SPR) technology.

[0753] 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.

[0754] 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.

[0755] SEC-HPLC

[0756] 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.

[0757] HIC-HPLC

[0758] A suitable amount of protein sample was loaded onto a MAbPac™ HIC-Butyl column (Thermo SCIENTIFIC) at a column temperature of 25 °C. Using an Agilent 1260 HPLC system, the sample was eluted for 25 min at a gradient elution rate of 0.8 mL / 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 20% isopropanol, pH 6.0 ± 0.1. The eluted protein was detected using UV absorbance at 280 nm.

[0759] Example 1: Screening, construction, and validation of VHH antibodies and multispecific antibodies

[0760] Example 1.1 VHH Screening

[0761] Using alpaca immunoassay and magnetic sorting techniques, candidate VHH sequences binding EGFR and CDH17 were screened through preliminary property characterization.

[0762] In short, recombinant his-tagged human EGFR ECD protein (Aikon Biotechnology Co., Ltd.) was used as the target antigen for alpaca immunization. The immunization process was as follows: Two healthy adult alpacas (Aikon Biotechnology Co., Ltd.) were selected, and 0.5 mg of the target antigen (recombinant his-tagged human EGFR ECD protein) was mixed with Gerbu adjuvant (GERBU biochemicals GmbH) at a 1:1 ratio and immunized by multiple subcutaneous injections into the cervical lymph nodes. The initial immunization was 500 μg, followed by 250 μg, for a total of 3-4 doses, with immunization intervals of 18-21 days. Seven days after the second and third immunizations, 5 ml of jugular venous blood was collected from the alpacas for ELISA serum titer detection. The results showed that the blood collection and bank establishment criteria were successfully met after three rounds of immunization, and blood collection and bank establishment were arranged.

[0763] Alpacas were immunized with Hu-CDH17-ECD-Fc (Gln23-Met787; UniProt_Q12864; Sanyou Biopharmaceutical (Shanghai) Co., Ltd.) and Hu-CDH17(EC1-2)-His antigen (Pro30-Pro244; UniProt_Q12864; Sanyou Biopharmaceutical (Shanghai) Co., Ltd.) for a total of 6 immunizations. One week after the immunization period, blood was collected for serum titer testing. Blood was collected for blood bank establishment only after the titer test was passed.

[0764] RNA was extracted from immunized alpaca PBMCs and further reverse transcribed into cDNA to obtain all antibody nucleotide sequences. These sequences were then constructed into phage display vectors using molecular cloning technology. The constructed vectors were then electroporated into *E. coli* to obtain an alpaca immunized phage display library. The phage display library was screened using solid-phase, solid-liquid-phase cross-conversion, and cell screening methods. Positive samples were sequenced. 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 a human IgG1Fc sequence. 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. The anti-EGFR VHH antibody and anti-CDH17 VHH antibody, as shown in Table 1 below, were finally obtained.

[0765] Table 1. VHH antibodies and their variable region sequences

[0766] Example 1.2. In vitro characterization of candidate VHH

[0767] Candidate VHH-Fc antibody expression and purification

[0768] The encoding gene for the VHH antibody sequence described above was synthesized and inserted into the expression vector pcDNA3.4, fusing the hIgG1 Fc sequence (SEQ ID NO:33) 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. 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. Double-chain VHH-hIgG1 Fc antibody was obtained.

[0769] FACS antigen binding property detection

[0770] The binding of the anti-EGFR VHH-Fc candidate antibody molecule n9B8 to target cells was detected using a FACS binding assay. The assay was performed under the following conditions: NCI-H1975 cells (human non-small cell lung cancer cell line) (1.5 × 10⁻⁶ cells). 5 ( / well) + VHH-Fc + reference antibody (50nM, 4× dilution, 4℃ 1h) + anti-hu IgG Fc-PE secondary antibody (1:500, 4℃ 0.5h). The FACS binding results are shown in Figure 1. The binding affinity of the n9B8-Fc antibody is weaker than that of the reference antibody, thus exhibiting reduced EGFR-related toxicity.

[0771] The binding of the anti-CDH17 VHH-Fc candidate antibody molecule A004 to target cells was detected using a FACS binding assay. The assay was performed under the following conditions: SNU5 (human gastric cancer cell line, CBP60505, Nanjing Kebai Biotechnology Co., Ltd.) or SNU620 target cells (human gastric adenocarcinoma cell line, CBP60508, Nanjing Kebai Biomedical Technology Co., Ltd.) (3 × 10⁻⁶ cells). 5 / well) + D 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 the candidate antibody A004-Fc exhibited good target cell binding properties, comparable to or better than the control antibody V-BMK2.

[0772] endocytosis detection

[0773] The internalization capacity of tumor target cells for the anti-EGFR VHH-Fc prepared in Example 1.2 was detected using the FACS method.

[0774] Prepare tumor target cells NCI-H1975 (human lung adenocarcinoma cell line; SCSP-597; Cell Bank of the Chinese Academy of Sciences), with 1–1.5 × 10⁶ cells per well. 5 Cells were seeded into 96-well plates. Anti-EGFR VHH-Fc (125 nM, 3-fold dilution) prepared in Example 1.2 was added, and the cells were incubated at 4°C for 30 min. The supernatant containing each VHH-Fc cell was removed by centrifugation. The cells were divided into two groups and incubated at 4°C and 37°C for 4 hours, respectively. After incubation, PBS was immediately added to ice to terminate the endocytosis experiment. Anti-hIgG Fc-PE secondary antibody (1:500 dilution) (12-4998-82, Invitrogen) was added, and the cells were incubated at 4°C for 30 min. The MFI of the cells was measured using flow cytometry (Life Technologies).

[0775] Incubation was performed at two temperatures, 4°C and 37°C, to promote antibody internalization within cells through temperature changes.

[0776] Figure 3 shows the endocytosis of n9B8, a candidate molecule for anti-EGFR antibody, in tumor target cells NCI-H1975.

[0777] 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 the anti-CDH17VHH-Fc antibody prepared in Example 2, serially diluted 4x, 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 using anti-hIgG-Fc-PE (1:500 dilution) (12-4998-82, Invitrogen). The cells were then fixed with 1% paraformaldehyde (PFA) and subjected to FACS detection.

[0778] As shown in Figure 4, for the candidate anti-CDH17 VHH-Fc antibody A004-Fc, the endocytosis rate at low concentrations was better than that of V-BMK2, regardless of whether it was in SNU5 or SNU620, while the endocytosis rate at saturation concentration was comparable to that of V-BMK2.

[0779] Example 1.3. Sequence optimization and characterization of VHH components

[0780] Sequence optimization of anti-EGFR VHH components

[0781] The anti-EGFR VHH sequence 9B8 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 aligned 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. This sequence was synthesized by Genewiz (Shanghai, China). It was then constructed into the pcDNA 3.4 expression vector, with a human HIS tag (6*HIS) fused to the C-terminus to express humanized VHHs, thereby obtaining the VHH antibody protein.

[0782] Tables 2 and 3 below show the humanized and PTM-removed VHH sequences and their corresponding original VHH sequences.

[0783] Table 2. Humanized sequence of anti-EGFR VHH 9B8

[0784] Table 3. Anti-EGFR VHH zn9B8.m2 PTM removal sequence

[0785] The obtained antibodies zn9B8.m2 and zn9B8.m4 were subjected to FACS detection. The binding of zn9B8.m2 to target cells MDA-MB-468 (human breast cancer cell line; TCHU136, Chinese Academy of Sciences Cell Bank) was similar to that of the parent 9B8 antibody (Table 4). Table 5 shows that pzn9B8.m2.m11 bound to target cells MDA-MB-468, CHO-K1-huEGFR, and CHO-K1-cynoEGFR, respectively, and their binding was similar to that of the parent 9B8 antibody. The FACS binding assay conditions were as follows: target cells MDA-MB-468 (1×10⁻⁶ cells). 5 / well) + test antibody (1333nM, 4X dilution, incubate at 4℃ for 1h) + anti-his (BioLegend, catalog number: 362605. 1:200, incubate at 4℃ for 0.5h) + 1% Paraformaldehyde or target cells CHO-K1-huEGFR or CHO-K1-cynoEGFR + test antibody (200nM, 4X dilution, incubate at 4℃ for 1h) + iF647-anti-his (Genscript, catalog number: A01802-100) (1:1000, incubate at 4℃ for 45min).

[0786] Table 4.9 Binding activity of humanized antibody B8 to target cells

[0787] Table 5. Binding activity of zn9B8.m2 PTM removal antibody to target cells.

[0788] Sequence optimization of CDH17 VHH-resistant components

[0789] The anti-CDH17 VHH sequence A004 (hereinafter referred to as "A004") was selected and its sequence optimized. The original VHH sequence was humanized using the "best-matching method". The amino acid sequence of the VHH framework region was 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 a humanized VHH sequence. Multiple residues in the reverse mutant framework region were removed by post-translational modification (PTM). The humanized sequence was then sent to Genewiz (Shanghai, China) for gene synthesis. The base sequence was optimized, and the optimized sequence was synthesized into the pcDNA 3.4 expression vector. Human IgG1 sequence (SEQ ID NO:33) was fused to the C-terminus to generate human IgG1 and express humanized VHHs, thereby obtaining the VHH antibody protein.

[0790] Table 6 below shows the humanized and PTM-removed VHH sequences and their corresponding original VHH sequences.

[0791] Table 6. Parent Stock and Optimized Sequence of A004 VHH

[0792] 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 7 and 8).

[0793] Table 7. Binding affinity kinetics of A004 and humanized and PTM-removed antibodies with human CDH17 antigen.

[0794] Table 8. Binding affinity kinetics of A004 and humanized and PTM-removed antibodies with human CDH17 antigen.

[0795] The sequence-optimized antibodies were subjected to FACS binding assays. When binding to target cells SNU5 or SNU620, the humanized A004.m6-Fc and the PTM-removed A004.m11-Fc still showed similar binding to A004-Fc (Table 9). The FACS assays used the following conditions: SUN5 or SNU620 target cells (2 × 10⁻⁶). 5 / well) + antibody sample (200nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (1:500, 4℃ 1h).

[0796] Based on the above data, A004.m6-Fc and A004.m11-Fc were combined to obtain the final optimized sequence A004.m6m11. The bivalent antibody in Fc form constructed from the A004.m6m11 sequence is A004.m6m11-Fc. The FACS binding experimental results are shown in Table 10, exhibiting characteristics similar to the parent antibody.

[0797] Table 9. Binding activity of A004 humanized and PTM-depleted antibodies to target cells.

[0798] Table 10. Binding activity of A004 humanized and PTM-removed antibodies to target cells.

[0799] Example 1.4 Generation of multispecific anti-EGFR / CDH17 antibody molecules

[0800] This embodiment describes the structure of an exemplary anti-EGFR / CDH17 bispecific antibody (BsAb) and the design and construction of its expression vector.

[0801] Multispecific antibody molecule design

[0802] Six bispecific antibody molecule constructs were designed as shown in Figure 20 (where V-F3 and V-F9 have the same bispecific antibody knot structure). They are either symmetrical double-stranded or asymmetrical double-stranded. The symmetrical double-stranded form consists of two identical polypeptide chains, each of which contains the following structure from the N-terminus to the C-terminus, or is composed of the following structure:

[0803] VHH A -VHH B -Fc, where VHH A and VHH B The symbols represent the VHH domains that bind to antigens A and B, respectively, wherein A and B are distinct from each other and independently selected from EGFR and CDH17; the symbol "-" indicates linkage via a linker or direct linkage, preferably representing a linker of 5-15 amino acids in length.

[0804] The asymmetric double-stranded form consists of two distinct polypeptide chains, each containing, or composed of, the following structures from its N-terminus to its C-terminus:

[0805] First polypeptide chain: VHH A -VHH A -Fc 1 ,

[0806] Second polypeptide chain: VHH B -Fc 2 ,

[0807] VHH A and VHH B These represent the VHH domains that bind antigens A and B, respectively, where A and B are distinct from each other and independently selected from EGFR and CDH17; the symbol "-" indicates linkage via a linker or direct linkage, preferably representing a linker of 5-15 amino acids in length; Fc 1 Fc represents the Fc region in the first polypeptide chain. 2 This represents the Fc region in the second polypeptide chain.

[0808] Alternatively, the asymmetric double-stranded form consists of two distinct polypeptide chains, each containing the following structure from the N-terminus to the C-terminus, or composed of the following structures:

[0809] First polypeptide chain: VHH A -VHH B -Fc 1 ,

[0810] Second polypeptide chain: VHH A -Fc 2 or VHH B -Fc 2 ,

[0811] VHH A and VHH B These represent the VHH domains that bind antigens A and B, respectively, where A and B are distinct from each other and independently selected from EGFR and CDH17; the symbol "-" indicates linkage via a linker or direct linkage, preferably representing a linker of 5-15 amino acids in length; Fc 1 Fc represents the Fc region in the first polypeptide chain. 2 This represents the Fc region in the second polypeptide chain.

[0812] Specifically, VHH A and VHH B The two polypeptide chains are linked by a linker (G4S)3, with a direct link between the VHH domain and the Fc domain. Due to the dimerization of the Fc region of immunoglobulins, the two polypeptide chains can associate to form homodimers or heterodimers, thereby producing a double-stranded, multispecific binding molecule.

[0813] When two polypeptide chains associate to form a heterodimer, the Fc region of the first polypeptide chain and the Fc region of the second polypeptide chain can be different, for example, containing Knob and Hole mutations respectively.

[0814] Construction of multispecific antibody molecules

[0815] The exemplary multi-chain form of bispecific antibodies constructed as shown in Table 11 below is illustrated in 20A-20E, and the corresponding amino acid sequences are provided in the sequence listing.

[0816] In Table 11, the optimized VHH sequence is used as the building block, where VHH EGFR The amino acid sequence is from pzn9B8.m2.m11, and its amino acid sequence is shown in SEQ ID NO:12; VHH DH17 The amino acid sequence is from pznA004.m6m11, and its amino acid sequence is shown in SEQ ID NO:31; the Fc region of the immunoprotein, which is the half-life extension domain (HLE), is from human IgG1 (containing only the Fc region with LALA mutation: SEQ ID NO:35); the symbol "-" indicates that the two domains are connected by a linker or directly.

[0817] Table 11. Exemplary Multispecific Antibodies

[0818] Note: The two VHH domains are connected by a linker (G4S)3. The VHH domain and the Fc domain are directly connected or connected via G4S. When the first and second strands are the same, the Fc region contains the LALA mutation. When the first and second strands are different, the first strand contains the S354C / T366W and LALA mutation (SEQ ID NO:36); the second strand contains the Y349C / T366S / L368A / Y407V and LALA mutation (SEQ ID NO:37); or the first strand contains the C220S, S354C / T366W and LALA mutation (SEQ ID NO:54); the second strand contains the C220S, Y349C / T366S / L368A / Y407V and LALA mutation (SEQ ID NO:55).

[0819] The first / second polypeptide chains of the Fc dimer double-stranded antibodies shown in Table 11 were constructed into pcDNA 3.4 expression vectors (one vector was generated if the antibody had a symmetrical structure; two vectors containing the encoding genes of the first and second polypeptide chains were generated if the antibody had an asymmetrical structure), and transfected into HEK293F cells. Cells were cultured for 3 days, and the culture supernatant of the transfected cells 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.

[0820] Example 1.5 Characterization of multispecific anti-EGFR / CDH17 antibody molecules

[0821] Analysis of target antigen expression levels on tumor cells

[0822] Using the reference antibody Anti-EGFR parental and V-BMK2, the expression of EGFR and CDH17 antigens on various target tumor cell lines, including AsPC-1 (pancreatic cancer cell line, CBP60546, Nanjing Kebai Biotechnology Co., Ltd.), SNU5 (gastric cancer cell line, CBP60505, Nanjing Kebai Biotechnology Co., Ltd.), SUN620 (gastric cancer cell line, CBP60508, Nanjing Kebai Biotechnology Co., Ltd.), Colo205 (human colon cancer cell line, CBP60026, Nanjing Kebai Biotechnology Co., Ltd.), SW48 (human colon adenocarcinoma cell line, CBP60018, Nanjing Kebai Biotechnology Co., Ltd.), and NCI-H508 (human colorectal adenocarcinoma cell line, CBP60795, Nanjing Kebai Biotechnology Co., Ltd.), was detected by FACS binding assay.

[0823] The test results are shown in Figure 5. In the tested human pancreatic cancer cells AsPC-1, EGFR antigen showed high expression levels, while CDH17 antigen showed low expression levels, classifying them as EGFR and CDH17 double-positive cells. In colorectal cancer cells SW48, EGFR antigen showed moderate expression levels, with no CDH17 antigen expression, classifying them as EGFR single-positive cells. In gastric cancer cells SNU-5, EGFR antigen showed moderate expression levels, while CDH17 antigen showed high expression levels, classifying them as EGFR and CDH17 double-positive cells. In colorectal cancer cells Colo205, both EGFR antigen and CDH17 showed low expression levels, classifying them as EGFR and CDH17 double-positive cells. In gastric cancer cells SNU620, only CDH17 showed moderate expression levels, classifying them as CDH17 single-positive cells. In colorectal cancer cells NCI-H508, both EGFR antigen and CDH17 showed low expression levels, classifying them as EGFR and CDH17 double-positive cells.

[0824] Tumor cell binding activity

[0825] Based on the above analysis of antigen expression on tumor cells, cell lines with different target antigen expression levels were selected and 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.

[0826] FACS was performed in conjunction with experimental conditions: target cells (2 × 10⁶). 5 ( / well) + sample (200nM, 3× dilution, 4℃ 1h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5h).

[0827] As shown in Figure 6, compared to other cell lines with low or no CDH17 expression, such as AsPc-1 (A), SW48 (B), and NCI-H508 (C), V-F1 showed a lower EC50 binding value to tumor cells, while V-F3 showed a higher Bmax binding value. In SNU5 (D) tumor cells with high CDH17 expression, the binding abilities of the three bispecific molecules were similar. In EGFR-positive cells (SW48) (E), V-F7 and V-F8 showed non-inferior binding to V-F3. In the AsPc-1 (F) cell line, V-F9 and V-F3 showed comparable binding abilities.

[0828] Internalization assay

[0829] The internalization ability of the bispecific antibody molecule V BsAb of this invention on different target cells was detected using an endocytosis assay. As shown in Figure 7, in AsPc-1 (PC; EGFR) Positive CDH17 Positive ), SW48 (CRC; EGFR Positive CDH17 Negative SNU5(GC; EGFR) Positive CDH17 Positive ), NCI-H508 (CRC; EGFR Positive CDH17 Positive On SW48 (CRC; EGFR), V-F3 showed the best endocytic activity, while there was no significant difference in endocytic activity between V-F1 and V-F2. Positive CDH17 Negative On the cytotoxicity level, V-F7, V-F8 and V-F3 have comparable endocytic activity (MFI 4°C - 37°C).

[0830] Combined with kinetic SPR measurement

[0831] The affinity of bispecific antibody molecules V-F3 and V-F8 for human and cynomolgus monkey EGFR or 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.

[0832] The affinity results of candidate molecules V-F3 and V-F8 for human and cynomolgus monkey EGFR antigens are shown in Table 12. The results indicate that candidate molecules V-F3 and V-F8 exhibit cross-reactivity with cynomolgus monkey EGFR, and show similar binding and dissociation rates with human EGFR antigen and cynomolgus monkey CDH17 antigen.

[0833] Table 12. Affinity to human and cynomolgus monkey EGFR antigens

[0834] In addition, the affinity of bispecific antibody molecules V-F3 and V-F8 for human and cynomolgus monkey CDH17 antigens was also detected by the SPR method, and the results are shown in Table 13 below.

[0835] Table 13. Affinity to human and cynomolgus monkey CDH17 antigens

[0836] Example 2. Synthesis of Connector-Payload (MC-RDVT-PAB-EXD)

[0837] Example 2.1 Preparation of HM-2100D_2

[0838] In this document, including Example 2.1 and the following examples, It refers to resin.

[0839] Weigh 10 g (15 mmol, ~1.5 mmol / g) of HM-2100C_2 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 15 mmol of HM-2100D_2. Treat a small sample with HFIP / DCM (1 / 4, v / v), remove the resin, and send to LC-MS to confirm resin incorporation. LC-MS (ESI) m / z: 342.1 [M-56+H] + .

[0840] Example 2.2 Preparation of HM-2100D_3

[0841] Add 20% piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100D_2 (6.67 mmol), shake on a shaker for 30 minutes, then dry. Add another 20% piperidine / DMF (150 mL), shake on a shaker for 30 minutes, then dry. Wash the resin sequentially with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3). 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. A small sample was added to ninhydrin hydrate and heated to 110°C. No blue color was observed. The solution was dried under vacuum. The resin was washed 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. A small sample was treated with HFIP / DCM (1 / 4, v / v), the resin was removed, and the solution was sent to LC-MS. HM-297D_1 was confirmed to have been attached. LC-MS (ESI) m / z: 497.3 [M+H] + .

[0842] Example 2.3 Preparation of HM-2100D_4

[0843] 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.

[0844] Add a 150 mL solution of DMF containing 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. Treat a small sample with HFIP / DCM (1 / 4, v / v), remove the resin, and send to LC-MS to confirm the attachment of HM-588_3A. LC-MS (ESI) m / z: 668.3 [M+H] + .

[0845] Example 2.4 Preparation of HM-2100D_5

[0846] 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.

[0847] 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 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_5. Treat a small sample with HFIP / DCM (1 / 4, v / v), remove the resin, and send to LCMS to confirm the attachment of HM-2100C_7.

[0848] LC-MS (ESI) m / z: 954.5 [M+H] + .

[0849] Example 2.5 Preparation of HM-2100D_6

[0850] HFIP / DCM (1 / 4, v / v) (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, and washed with DCM (200 mL x 4). The filtrate was concentrated, and residual HFIP was removed with DCM (400 mL x 4) to obtain the target product HM-2100D_6 (6.67 mmol, light pink solid). LC-MS (ESI) m / z: 954.5 [M+H] + .

[0851] Example 2.6 Preparation of HM-2100D_7

[0852] Under ice bath conditions (5°C), COMU (7.41 g, 17.3 mmol) and 2,6-dimethylpyridine (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, the solid was 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] + .

[0853] Example 2.7 Preparation of HM-2100D_8

[0854] 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). The mixture was stirred at room temperature (15°C) for 16 hours. The reaction solution was poured into water (1 L), extracted with ethyl acetate, concentrated, and the residue was dissolved in a small amount of ethyl acetate. This residue was 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] + .

[0855] Example 2.8 Preparation of HM-2100D_9

[0856] At room temperature (15℃), 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), pyridine (4.5 mL), and HOAt (115 mg, 0.8475 mmol) in DMF (18 mL). The mixture was stirred at room temperature (15℃) 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). LC-MS (ESI) m / z: 1520.6 [M+H] + .

[0857] Example 2.9 Preparation of HM-2100D_10

[0858] 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, containing 0.1% TFA), lyophilized, and the target product HM-2100D_10 (360 mg, yield: 93%, yellow solid, TFA salt) was obtained. LC-MS (ESI) m / z: 1056.6 [M+H] + .

[0859] Example 2.10 Preparation of HM-2100D_11

[0860] At room temperature (15°C), DIEA (50 mg, 0.384 mmol) was added to a 3 mL solution of HM-2100D_10 (150 mg, 0.128 mmol) and HM-297Q_6 (60 mg, 0.193 mmol) in DMF. 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] + .

[0861] 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).

[0862] MS(ES-API): 1249.4(M+H) +

[0863] Example 3. Preparation and characterization of antibody-drug conjugates (ADCs)

[0864] Materials and methods

[0865] The connectors-payloads used in some embodiments of this invention are known and / or commercially available in the prior art, or can be prepared according to this application. When the drawn structure differs from the actual situation, modifications or corrections to the structure should be permitted based on the actual situation. Specifically, the connector-payload Mal-Gly-Exatecan-D-glucuronic acid is available from MedChemExpress (CAS No.: 2763252-25-9; HY-153179), and the connector-payload MC-RDVT-PAB-EXD is prepared according to the description in Example 2.

[0866] General Coupling and Purification Method A:

[0867] Antibody reduction: A certain amount of antibody solution (e.g., the corresponding bispecific antibody solution prepared according to the antibody preparation examples above, or by replacing the solvent with sodium acetate buffer) was placed in a Biofil tube, and 6 molar equivalents of TCEP (5 mM concentration) were added (TCEP:Ab = 6:1). The Biofil tube containing the reaction mixture was placed on a shaker (x 500 rpm) and reacted at 37°C for 2 hours. Then, another 6 molar equivalents of TCEP (5 mM) were added to the mixture, and the reaction mixture was placed on a shaker (x 500 rpm) and reacted at 37°C for another 2 hours.

[0868] Purification of the reduced intermediate: Ultrafiltration was performed using Amicon Utral-15 (MWCO 30kd) to remove excess TCEP and the buffer was changed. The buffer used was 20mM NaOAc-HOAc (pH 5.5), and the ultrafiltration was performed 10 times the volume.

[0869] Linker-load coupling: 8 molar equivalents of linker-load (5 mg / ml DMA, linker-load:Ab = 8:1) were added dropwise to the ultrafiltered, fully reduced antibody, while maintaining the DMA concentration below 20% (v / v). After the addition was complete, the reaction mixture was incubated at room temperature for 2 hours on a shaker (x500 rpm).

[0870] ADC purification: Excess linkers, payloads, and DMA were removed using Amicon Ultraral-15 (MWCO 30kd) ultrafiltration. The first 8 volumes were purified using Buffer 1 (20mM NaOAc-HOAc containing 10% DMA, 8% (w / v) trehalose buffer, pH 5.5), and the next 10 volumes were purified using Buffer 2 (20mM NaOAc-HOAc, 8% trehalose buffer, pH 5.5). After ultrafiltration, the ADC was concentrated to the desired concentration.

[0871] Purity was determined by HIC and SEC-HPLC methods. Free linker-load was determined by RP-HPLC.

[0872] General methods and / or parameters for determining or detecting ADCs

[0873] ◆Size exclusion chromatography-HPLC method (for total ADC detection) parameters:

[0874] ◆ Reversed-phase HPLC (RP-HPLC) method (for the detection of free drugs) parameters

[0875] Perform elution according to the table below.

[0876] ◆HIC-HPLC method (for the detection of free antibodies and DAR distribution) parameters

[0877] Perform elution according to the table below.

[0878] Example 3.1 Preparation of V-F1-Gluc-Exd

[0879] Wherein, Ab is the bispecific antibody V-F1, and p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer from 4 to 8, for example, an integer from 4 to 6, mainly 6.

[0880] Following general method A, V-F1-Gluc-Exd was prepared using the bispecific antibody V-F1 prepared according to the above examples and the linker-loaded Mal-Gly-Exatecan-D-glucuronic acid (CAS No.: 2763252-25-9; MCE, HY-153179). The product V-F1-Gluc-Exd had an average DAR value of approximately 5.9 and a SEC-HPLC purity of 97.8%.

[0881] Example 3.2 Preparation of V-F9-Gluc-Exd

[0882] Wherein, Ab is the bispecific antibody V-F9, and p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer from 4 to 8, for example, an integer from 4 to 6, mainly 4.

[0883] Following general method A, V-F9-Gluc-Exd was prepared using the bispecific antibody V-F9 prepared according to the above examples and the linker-loaded Mal-Gly-Exatecan-D-glucuronic acid (CAS No.: 2763252-25-9; MCE, HY-153179). The product V-F9-Gluc-Exd had an average DAR value of approximately 3.95 and a SEC-HPLC purity of 94.7%.

[0884] Example 3.3 Preparation of V-F7-RDVT-Exd

[0885] Wherein, Ab is the bispecific antibody V-F7, and p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer from 4 to 8, for example, an integer from 4 to 6, mainly 6.

[0886] Following general method A, V-F7-RDVT-Exd was prepared using the bispecific antibody V-F7 prepared according to the above examples and the linker-loaded MC-RDVT-PAB-EXD (prepared as in Example 2, MCE, HY-X3661). The product V-F7-RDVT-Exd was found to have an average DAR value of approximately 5.9 and a SEC-HPLC purity of 95.9%.

[0887] Example 3.4 Preparation of other ADCs

[0888] Following the method described in Example 3.1, but using different antibodies (V-F2, V-F3, V-F7, V-F8) instead of V-F1, V-F2-Gluc-Exd, V-F3-Gluc-Exd, V-F7-Gluc-Exd, and V-F8-Gluc-Exd were prepared, wherein in each ADC, p is an integer selected from 1 to 16, mainly 6.

[0889] V-F8-RDVT-Exd was prepared by following the method described in Example 3.3, but using a different antibody V-F8 instead of V-F7, where p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, 4 to 8, and mainly 6.

[0890] Table 14 provides some relevant information about the ADCs prepared in the embodiments of this application and the control ADC (prepared by a method similar to that in Example 3.1).

[0891] Table 14. Basic Information of ADCs

[0892] The “Isotype control antibody” is constructed based on the 2B10 sequence of the single-domain antibody against Anti-Rotavirus disclosed 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:35) to form a bivalent fusion antibody protein.

[0893] Example 4. In vitro experiments of ADC

[0894] Target cells NCI-H508 (human colorectal adenocarcinoma cells, CBP60795, Nanjing Kebai Biotechnology Co., Ltd.), AsPc-1 (human pancreatic cancer cells, CBP60546, Nanjing Kebai Biotechnology Co., Ltd.), SW48 (human colon cancer cells, CBP60018, Nanjing Kebai Biotechnology Co., Ltd.), SNU5 (human gastric cancer cells, CBP60505, Nanjing Kebai Biotechnology Co., Ltd.), or SW403 (human colon cancer cells, CBP60017, Nanjing Kebai Biotechnology Co., Ltd.) were seeded at 3000 cells / well, 100 μL / well, and incubated overnight at 37°C in a 5% CO2 incubator. ADCs samples were diluted to 400 nM with complete culture medium, serially diluted 2.5-fold, and 100 μL / well was added to each well of the seeded 96-well culture plate, and incubated at 37°C in a 5% CO2 incubator for 6 days. Add 20 μL / well to CCK-8 (Cell Counting Kit-8; Life-iLab; Cat#AC11L054) and incubate at 37℃ in a 5% CO2 incubator for 1–5 h. OD values ​​are read by spectroscopic analysis. 450 The results are shown in Figure 8.

[0895] Example 5. In vivo effects experiment of ADC

[0896] Example 5.1: Antitumor effect of ADCs in mouse SW48 subcutaneous transplantation model

[0897] This experiment was used to evaluate the efficacy of test products (including the ADCs of this invention) in a mouse nu / nu human colon cancer cell subcutaneous transplantation model of SW48.

[0898] Experiment A

[0899] Model Construction and Grouping: 7-8 week old female nu / nu mice (nu / nu, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 1 week, and then injected into the right scapula with SW48 (CBP60018, Nanjing Kebai Biotechnology Co., Ltd.) and PBS resuspended cell solution (5×10⁻⁶). 6 (cells / animals), until the tumor volume grows to 100-220 mm 3 Patients were randomly assigned to groups (n=5) based on mean tumor volume. The day of group assignment was defined as D0, and the test drug was administered intravenously on D1 as a single dose.

[0900] Dosage volume: Adjusted according to mouse body weight (dosage volume for mice = 10 μL / g × mouse body weight (g))

[0901] 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.

[0902] 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 photographed.

[0903] Endpoint analysis: At the end of the trial, the following indicators were analyzed: tumor volume change (TGI) TV and weight changes. TGI TV The calculation formula is as follows: TGI TV ={1-[(V t -V0) / (C t -C0)]}×100%

[0904] Among them, V t : The average tumor volume of mice in the test drug administration group on day t;

[0905] V0: The average tumor volume of mice in the test drug administration group on day 0;

[0906] C t : Average tumor volume of the solvent group mice on day t;

[0907] C0: Average tumor volume of the solvent group mice on day 0.

[0908] When TGI is calculated as described above TV When the value is >100%, the TGI calculation formula is as follows: TGI TV =100%-[(Vt-V0) / V0)]×100%.

[0909] Statistical Analysis: Analysis, processing, and reporting were performed. Quantitative indicators were described using mean ± standard error (Mean ± SEM / SD). One-way ANOVA or two-way ANOVA was used for quantitative analysis. For inter-group comparisons, the t-test was used, and p < 0.05 was considered statistically significant. Both statistical and biological significance were considered in the analysis of results.

[0910] The results are shown in Figures 9 (A and B). It can be seen that at equal doses, V-F3-Gluc-Exd was superior in efficacy regardless of the dosage (low or high), while the animals showed steady weight gain and good drug safety. V-F1-Gluc-Exd and V-F2-Gluc-Exd had comparable efficacy.

[0911] Experiment B

[0912] Mice inoculated with SW48 were randomly divided into 7 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 10(A).

[0913] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 10 (A and B). It can be seen that the ADCs of the present invention all exhibit significant tumor growth inhibition effects, and the weight changes in animals treated with the ADCs of the present invention are recoverable, demonstrating good drug safety and tolerability.

[0914] Example 5.2: Antitumor effects of ADCs in the SNU-5 model

[0915] This experiment was used to evaluate the efficacy of test products (including the ADCs of this invention) in a nu / nu mouse model of human gastric cancer SNU5 subcutaneous transplantation.

[0916] Model construction and grouping: 7-8 week old female mice (nu / nu, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 1 week and then injected with SNU5 (CBP60505, Nanjing Kebai Biomedical Technology Co., Ltd.) and PBS-resuspended cell solution (5×10⁻⁶) into the right scapula. 6 (cells / animals), until the tumor volume grows to 100-200 mm 3 The tumors were grouped according to their average volume (n=4), and the day of grouping was defined as D0. The test drug was administered intravenously on D0 as a single dose. The dosage for each group is shown in Figure 11.

[0917] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)).

[0918] 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.

[0919] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 11 (A and B). It can be seen that the ADCs of the present invention all exhibit significant anti-tumor growth effects, and the animals treated with the ADCs of the present invention maintained weight gain, demonstrating good drug safety and tolerability. Specifically, at a low dose of 3 mg / kg, V-F2-Gluc-Exd and V-F3-Gluc-Exd were comparable in efficacy, both superior to V-F1-Gluc-Exd. At a high dose of 8 mg / kg, V-F3-Gluc-Exd showed superior tumor suppression compared to V-F2-Gluc-Exd, which in turn was superior to V-F1-Gluc-Exd, achieving a 50% (2 / 4) CR (complete remission) rate.

[0920] Example 5.3: Antitumor effects of ADCs in the SNU620 model

[0921] This experiment was used to evaluate the efficacy of test products (including the ADCs of this invention) in a subcutaneous transplantation model of human gastric cancer cells SNU620 (CBP60508, Nanjing Kebai Biomedical Technology Co., Ltd.) in NSG mice.

[0922] Mice inoculated with SNU620 were randomly divided into 11 groups (n=5), with the day of grouping defined as D0. The test drug was administered intravenously on D0 and D7, once a week for a total of 2 weeks. The dosage for each group is shown in Figure 12(A).

[0923] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 12 (A and B). It can be seen that the ADCs of the present invention all exhibit significant tumor growth inhibition effects, and the animals treated with the ADCs maintained weight gain, demonstrating good drug safety and tolerability. In particular, all ADCs demonstrated significant tumor inhibition effects.

[0924] Example 5.4 Antitumor effect of ADCs in the colo205 model

[0925] 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 colorectal cancer colo205.

[0926] Model Construction and Grouping: 7-8 week old female BALB / c nude mice (BALB / c nude, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 1 week, and then injected into the right scapula with colo205 (CL-0052, Wuhan Pronosei Life Sciences Co., Ltd.) and PBS resuspended cell solution (5×10⁻⁶). 6 (cells / animals), until the tumor volume grows to 220-350 mm 3 The tumors were grouped according to their average volume (n=4). The day of grouping was defined as D0. The test drug was administered intravenously on D0 and D7, once a week for a total of 2 weeks. The dosage details are shown in Figure 13.

[0927] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)).

[0928] 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.

[0929] Data processing was performed according to Example 5.1, and the experimental results are shown in Figures 13 (A and B). It can be seen that the ADCs of the present invention all exhibit significant anti-tumor growth effects, and the animals treated with the ADCs of the present invention maintained weight gain, demonstrating good drug safety and tolerability. In particular, regarding tumor inhibition, V-F3-Gluc-Exd showed the best efficacy at a dose of 8 mg / kg, while V-F1-Gluc-Exd and V-F2-Gluc-Exd showed comparable efficacy.

[0930] Example 5.5 Antitumor effect of ADCs in the AsPc-1 model

[0931] This experiment was used to evaluate the efficacy of test products (including the ADCs of this invention) in the BALB / c nude mouse model of subcutaneous transplantation of human pancreatic cancer Aspc-1.

[0932] Experiment A

[0933] Model Construction and Grouping: 7-8 week old female BALB / c nude mice (BALB / c nude, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 1 week, and then injected with Aspc-1 (CBP60546, Nanjing Kebai Biomedical Technology Co., Ltd.) and PBS-resuspended cell solution (5×10⁻⁶) into the right scapula. 6 (cells / animals), until the tumor volume grows to 100-200 mm 3 The tumors were grouped according to their average volume (n=5). The day of grouping was defined as D0. The test drug was administered intravenously on D0 and D7, once a week for a total of 2 weeks. The dosage details are shown in Figure 14.

[0934] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)).

[0935] 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.

[0936] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 14 (A and B). It can be seen that the ADCs of the present invention all showed significant anti-tumor growth effects, and the weight of animals treated with the ADCs of the present invention maintained growth, with good drug safety and tolerability.

[0937] Experiment B

[0938] Model Construction and Grouping: 7-8 week old female BALB / c nude mice (BALB / c nude, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 1 week, and then injected with AsPc-1 (CBP60546, Nanjing Kebai Biomedical Technology Co., Ltd.) and PBS-resuspended cell solution (5×10⁻⁶) into the right scapula. 6 (cells / animals), until the tumor volume grows to 100-200 mm 3 The tumors were grouped according to their average volume (n=5). The day of grouping was defined as D0. The test drug was administered intravenously on D0 and D7, once a week for a total of 2 weeks. The dosage details are shown in Figure 19.

[0939] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)).

[0940] 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.

[0941] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 19 (A and B). It can be seen that the ADCs of the present invention all exhibit significant anti-tumor growth effects, and the animals treated with the ADCs of the present invention maintained weight gain, demonstrating good drug safety and tolerability.

[0942] Example 5.6 Antitumor effects of ADCs in the SNU-16 model

[0943] 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 SNU-16.

[0944] Model Construction and Grouping: 7-8 week old female BALB / c nude mice (BALB / c nude, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 1 week, and then injected with SNU-16 (CBP60502, Nanjing Kebai Biotechnology Co., Ltd.) and PBS resuspended cell solution (5×10⁻⁶) into the right scapula. 6 (cells / animals), until the tumor volume grows to 120-205 mm 3 The tumors were grouped according to their average volume (n=5). The day of grouping was defined as D0, and the test drug was administered intravenously on D0, for a total of one dose. The dosage details are shown in Figure 15.

[0945] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)).

[0946] 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.

[0947] Data processing was performed according to Example 5.1, and the experimental results are shown in Figures 15 (A and B). It can be seen that the ADCs of the present invention all exhibit significant anti-tumor growth effects, and the animals treated with the ADCs of the present invention maintained weight gain, demonstrating good drug safety and tolerability. In particular, V-F3-Gluc-Exd showed the best efficacy for tumor inhibition at a dose of 3 mg / kg. At a dose of 8 mg / kg, the efficacy of each ADC was comparable.

[0948] Example 5.7 Antitumor effect of ADCs in EBC-1 model

[0949] This experiment was used to evaluate the efficacy of test products (including the ADCs of this invention) in a subcutaneous transplantation model of EBC-1 human lung squamous cell carcinoma cells in BALB / c nude mice.

[0950] Model Construction and Grouping: 7-8 week old female BALB / c nude mice (BALB / c nude, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 1 week, and then injected with EBC-1 (CBP60091, Nanjing Kebai Biotechnology Co., Ltd.) and PBS-resuspended cell solution (5×10⁻⁶) into the right scapula. 6 (cells / animals), until the tumor volume grows to 120-220 mm 3 The tumors were grouped according to their average volume (n=4). The day of grouping was defined as D0, and the test drug was administered intravenously on D0, for a total of one dose. The dosage details are shown in Figure 16.

[0951] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)).

[0952] 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.

[0953] Data processing was performed according to Example 5.1, and the experimental results are shown in Figures 16 (A and B). It can be seen that the ADCs of the present invention all exhibited significant anti-tumor growth effects, and the animals treated with the ADCs maintained weight gain, demonstrating good drug safety and tolerability. Specifically, regarding tumor inhibition effects, on day 23 after administration, V-F3-Gluc-Exd showed the best effect (TGI: 200%), achieving a 100% CR; V-F1-Gluc-Exd was the second most effective (TGI: 170%), achieving a 40% CR.

[0954] Example 5.8 Antitumor effect of ADCs in T84 model

[0955] 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 colorectal cancer T84.

[0956] Model Construction and Grouping: 7-8 week old female BALB / c nude mice (BALB / c nude, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimatized for 1 week, and then injected into the right scapula with T84 containing 30% matrix gel (CBP60021, Nanjing Kebai Biotechnology Co., Ltd.) and PBS resuspended in 5×10⁻⁶ cells. 6 (cells / animals), until the tumor volume grows to 120-230 mm 3 The tumors were grouped according to their average volume (n=5). The day of grouping was defined as D0, and the test drug and control drug were administered on D0. The administration method, dosage, and frequency are detailed in Figure 17.

[0957] Dosage volume: Adjusted according to mouse body weight (mouse dosage volume = 10 μL / g × mouse body weight (g)).

[0958] 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.

[0959] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 17 (A and B). It can be seen that the ADCs of the present invention all exhibit significant anti-tumor growth effects, and the animals treated with the ADCs of the present invention maintained weight gain, demonstrating good drug safety and tolerability. In particular, regarding tumor suppression, on day 28 after administration, compared with the current standard treatments for colorectal cancer, the combination of Cetuximab (HY-P9905; MedChemExpress LLC) and Irinotecan (HY-13631, MedChemExpress LLC) (TGI: 94%) and the potential standard treatments Amivantamab (HY-P9977; MedChemExpress LLC) and Irinotecan (TGI: 97%), 3mpk V-F3-Gluc-Exd (TGI: 108%), V-F8-Gluc-Exd (TGI: 105%), and 8mpk V-F3-Gluc-Exd (TGI: 200%) all showed stronger therapeutic effects, with 8mpk V-F3-Gluc-Exd achieving 100% CR.

[0960] Example 5.9 Antitumor effect of ADCs in human ovarian cancer xenograft model LD1-0032-411098

[0961] 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 (Mean mean 120±30 mm3) Tumor-bearing mice were randomly divided into two groups (n=5). The day of grouping was defined as D0. The test product was administered intravenously on D0, and the administration was repeated every 3 weeks. The dosage for each group is shown in Figure 18.

[0962] Data processing was performed according to Example 5.1, and the experimental results are shown in Figure 18. It can be seen that on day 28 after administration, at a dose of 8 mg / kg, V-F3-Gluc-Exd had a significant tumor-suppressive effect on human ovarian cancer xenografts, achieving a CR rate of 40% (2 / 5).

[0963] Sequence List Overview

Claims

1. A bispecific antibody that specifically binds to EGFR and CDH17, wherein the bispecific antibody comprises one or two antigen-binding domains specifically binding to EGFR, such as VHH, and two antigen-binding domains specifically binding to CDH17, such as VHH.

2. The bispecific antibody of claim 1, wherein the bispecific antibody comprises an antigen-binding domain VHH that specifically binds to EGFR. EGFR And two antigen-binding domains that specifically bind to CDH17, VHH CDH17 , Preferably, the bispecific antibody comprises two polypeptide chains, one polypeptide chain comprising two tandem VHHs and optionally an Fc region, and the other polypeptide chain comprising one VHH and optionally an Fc region.

3. The bispecific antibody of claim 2, wherein the bispecific antibody comprises or is composed of two polypeptide chains, wherein the polypeptide chains comprise or are composed of the following structure from the N-terminus to the C-terminus: First polypeptide chain: VHH CDH17 -VHH CDH17 -Fc 1 , Second polypeptide chain: VHH EGFR -Fc 2 , or First polypeptide chain: VHH EGFR -VHH CDH17 -Fc 1 , Second polypeptide chain: VHH CDH17 -Fc 2 , or First polypeptide chain: VHH CDH17 -VHH EGFR -Fc 1 , Second polypeptide chain: VHH CDH17 -Fc 2 , The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length; preferably, the two tandem VHHs of the first polypeptide chain are connected via a linker. Optionally, the C-terminus of the second VHH of the first polypeptide chain is connected to the Fc... 1 N-terminal connection; Optionally, the C-terminus of the first VHH of the first polypeptide chain is connected to the N-terminus of the second VHH via a connector; Optionally, the C-terminus of the VHH of the second polypeptide chain is connected to the Fc... 2 The N-terminal connection.

4. The bispecific antibody of claim 2 or 3, wherein the Fc region is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, and for example, the Fc region comprises an amino acid sequence SEQ ID NO:33 or SEQ ID NO:34 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with the amino acid sequence, or is composed of the amino acid sequence. Optionally, the Fc region contains a mutation that reduces or eliminates the connection between the Fc region and the Fcγ receptor, such as the LALA mutation. For example, an 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:35, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:

35. Optionally, the Fc region contains a C220S mutation; Optionally, the Fc region containing the LALA mutation and the C220S mutation 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:56, for example, containing or consisting of the amino acid sequence shown in SEQ ID NO:

56.

5. The bispecific antibody of any one of claims 2-4, wherein the two Fc regions respectively contain a Knob mutation and a Hole mutation, for example, the Knob mutation T366W, and the Hole mutations T366S, L368A, and Y407V; or the Knob mutation is T366Y, and the Hole mutation is Y407T; optionally, the Fc region containing the Knob mutation further contains the S354C mutation, and the Fc region containing the Hole mutation further contains the Y349C mutation. For example, the Fc region containing the Knob mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:36, while the Fc region containing the Hole mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:37; or The Fc region containing the Knob mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:54, while the Fc region containing the Hole mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:

55.

6. The bispecific antibody of claim 1, wherein the bispecific antibody comprises two antigen-binding domains VHH that specifically bind to EGFR. EGFR And two antigen-binding domains that specifically bind to CDH17, VHH CDH17 ; Preferably, the bispecific antibody comprises two polypeptide chains, one of which contains two tandem VHHs. CDH17 and VHH EGFR And optionally the Fc region, and another polypeptide chain contains two tandem VHHs CDH17 and VHH EGFR , and optionally the Fc region; preferably, the two polypeptide chains are identical.

7. The bispecific antibody of claim 6, wherein the bispecific antibody comprises or is composed of two identical polypeptide chains, wherein each polypeptide chain comprises, or is composed of, the following structure from the N-terminus to the C-terminus: VHH CDH17 -VHH EGFR -Fc or VHH EGFR -VHH CDH17 -Fc The symbol "-" indicates a connection via a connector or a direct connection, preferably a connector with a length of 5-15 amino acids; Optionally, VHH CDH17 With VHH EGFR They are connected via a connector; Optionally, the C terminal of the first VHH is connected to the N terminal of the second VHH via a connector; the C terminal of the second VHH is connected to the N terminal of Fc.

8. The bispecific antibody of claim 6 or 7, wherein the Fc region is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, for example, the Fc region comprises an amino acid sequence SEQ ID NO:33 or SEQ ID NO:34 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with the amino acid sequence, or is composed of the amino acid sequence, optionally, the Fc region comprises a mutation that reduces or eliminates the Fc region from the Fcγ receptor, such as the LALA mutation, for example, an Fc region containing the LALA mutation comprises 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:35, for example, comprising or composed of the amino acid sequence shown in SEQ ID NO:

35.

9. The bispecific antibody of any one of claims 1-8, wherein the bispecific antibody comprises a linker and the linker comprises (G4S)n, wherein n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5.

10. The bispecific antibody according to any one of claims 1-9, wherein the bispecific antibody comprises VHH (VHH) that specifically binds to CDH17. CDH17 ), and the VHH CDH17 It contains or is composed of a heavy chain variable region, the heavy chain variable region comprising the three complementary determinant regions (CDRs) contained in the VH shown in any one of SEQ ID NO:31, 20-30 and 32; preferably, the CDR sequence is defined according to ABM.

11. The bispecific antibody of claim 10, wherein the VHH CDH17 It includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or it includes or is composed of heavy chain variable regions, wherein the heavy chain variable regions include complementarity-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:14, and VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:

15. CDR3 contains or consists of the amino acid sequence shown in any one of SEQ ID NO:18, 16, 17 and 19.

12. The bispecific antibody of claim 10 or 11, wherein the VHH CDH17 Contains or is composed of heavy chain variable regions, said heavy chain variable regions (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: 31, 20-30, and 32; or (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:31, 20-30 and 32.

13. The bispecific antibody of any one of claims 1-12, wherein the bispecific antibody comprises VHH (VHH) that specifically binds to EGFR. EGFR ), and the VHH EGFR It contains or is composed of a heavy chain variable region, the heavy chain variable region comprising the three complementary determinant regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 12, 8-10 and 13; preferably, the CDR sequence is defined according to ABM.

14. The bispecific antibody of claim 13, wherein the VHH EGFR The CDRs include complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or they contain or are composed of heavy chain variable regions. 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:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

6. (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:2, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:5; or (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:4, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

7.

15. The bispecific antibody of claim 13 or 14, wherein the VHH EGFR Contains or is composed of heavy chain variable regions, said heavy chain variable regions (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: 12, 8-10, and 13; or (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:12, 8-10 and 13.

16. The bispecific antibody according to any one of claims 1-15, wherein the antigen-binding domain that specifically binds to CDH17 is VHH. CDH17 It contains or is composed of a heavy chain variable region, the heavy chain variable region comprising the three complementarity-determining regions (CDRs) contained in VH shown in SEQ ID NO:31; preferably, the CDR sequence is defined according to ABM; and the antigen-binding domain that specifically binds to EGFR is VHH. EGFR And containing or consisting of a heavy chain variable region, the The heavy chain variable region contains the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:12; preferably, the CDR sequence is defined according to ABM.

17. The bispecific antibody according to any one of claims 1-16, wherein the antigen-binding domain that specifically binds to CDH17 is VHH. CDH17 The heavy chain variable region contains complementarity-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 complementarity-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:14, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:15, and VHH... CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:18; and the antigen-binding domain that specifically binds to EGFR is VHH. EGFR It contains complementarity-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 complementarity-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:1, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

6.

18. The bispecific antibody according to any one of claims 1-17, wherein the antigen-binding domain that specifically binds to CDH17 is VHH. CDH17 It contains or is composed of a heavy chain variable region, the heavy chain variable region containing or being composed of the amino acid sequence shown in SEQ ID NO:31; and the antigen-binding domain that specifically binds to EGFR is VHH. EGFR And containing or consisting of a heavy chain variable region, the The heavy chain variable region contains or consists of the amino acid sequence shown in SEQ ID NO:

12.

19. The bispecific antibody of any one of claims 1-18, wherein the bispecific antibody comprises or is composed of two polypeptide chains, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of: (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 44 and 45, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences, (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 46 and 47, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences, (iii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 48 and 49, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences; or (iv) Each of the amino acid sequences shown in SEQ ID NOs: 52 and 53, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences.

20. The bispecific antibody of any one of claims 1-18, wherein the bispecific antibody comprises or is composed of two polypeptide chains, wherein the first polypeptide chain and the second polypeptide chain respectively comprise or are composed of the amino acid sequences shown in SEQ ID NOs: 44 and 45.

21. The bispecific antibody of any one of claims 1-18, wherein the bispecific antibody comprises or is composed of two identical polypeptide chains, wherein each polypeptide chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 42 or 43 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

22. The bispecific antibody according to any one of claims 1-21, wherein the bispecific antibody has one or more of the following properties: (i) K binding affinity with human CDH17 D The value is less than about 10 nM, for example less than about 5 nM or 4 nM, preferably between 0.5 and 10 nM, for example between 1 and 5 nM, as determined by the SPR method; (ii) K binding affinity with CDH17 of cynomolgus monkeys D The value is less than about 10 nM, for example less than about 8 nM, 7 nM or 6 nM, preferably between 1 and 10 nM, for example between 1 and 7 nM or between 2 and 6 nM, as determined by the SPR method; (iii) K binding affinity with human EGFR D The value is less than about 50 nM, for example less than about 40 nM, 30 nM, 20 nM or 15 nM, for example less than about 12 nM or 11 nM or 7 nM or 5 nM, preferably between 1 and 15 nM, for example between 2 and 12 nM or between 3 and 11 nM, for example as determined by the SPR method; (iv) K binding affinity to EGFR in cynomolgus monkeys D The value is less than about 50 nM, for example less than about 40 nM, 30 nM, 20 nM or 15 nM, for example less than about 12 nM, 11 nM or 10 nM, preferably between 1 and 15 nM, for example between 2 and 12 nM or between 2 and 10 nM, for example as determined by the SPR method; (v) It exhibits cross-reactivity to human and monkey EGFR and / or CDH17; (vi) It has a synergistic effect with the binding of EGFR and CDH17 targets, that is, it has higher binding activity with EGFR and / or CDH17 positive, preferably EGFR positive and CDH17 positive tumor cells, for example, compared with the binding of EGFR monospecific antibody (e.g., EGFR parent antibody) to EGFR positive tumor cells, or compared with the binding of CDH17 monospecific antibody (e.g., CDH17 parent antibody) to CDH17 positive tumor cells; (vii) Capable of being internalized by cells expressing EGFR and / or CDH17, particularly, the bispecific antibody exhibits greater endocytic activity in EGFR-positive and / or CDH17-positive tumor cells, such as EGFR-positive and CDH17-positive tumor cells, for example, co-endocytic activity, for example, compared to the endocytic activity of EGFR monospecific antibodies (e.g., EGFR parental antibodies) in EGFR-positive tumor cells, or compared to the endocytic activity of CDH17 monospecific antibodies (e.g., CDH17 parental antibodies) in CDH17-positive tumor cells; or (viii) Capable of killing cells expressing EGFR and / or CDH17, particularly, the bispecific antibody exhibits greater killing activity against EGFR-positive and / or CDH17-positive tumor cells, such as EGFR-positive and CDH17-positive tumor cells, for example, synergistic killing activity, for example, compared to the killing activity of EGFR-positive tumor cells by EGFR monospecific antibodies (e.g., EGFR parental antibodies), or compared to the killing activity of CDH17-positive tumor cells by CDH17 monospecific antibodies (e.g., CDH17 parental antibodies).

23. A nucleic acid molecule encoding a bispecific antibody or any polypeptide chain thereof as claimed in any one of claims 1-22.

24. An expression vector comprising the nucleic acid molecule of claim 23, preferably, said expression vector is pCDNA, such as pCDNA3.

4.

25. A host cell comprising the nucleic acid molecule of claim 23 or the expression vector of claim 24, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells.

26. A method for preparing a bispecific antibody according to any one of claims 1-22, the method comprising culturing a host cell containing a nucleic acid molecule according to claim 23 or an expression vector according to claim 24 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

27. An immunoconjugate or immunofusion comprising the bispecific antibody of any one of claims 1-22.

28. Antibody-drug conjugates having formula (I) or pharmaceutically acceptable salts or solvates thereof: Ab-(L-D) p (I) in: Ab is a bispecific antibody according to any one of claims 1-22; L is the connector; D represents a drug, such as an anti-tumor compound; p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, for example, an integer selected from 4 to 6.

29. The antibody-drug conjugate of claim 28 or a pharmaceutically acceptable salt or solvate thereof, wherein the drug is a cytotoxic agent, such as a camptothecin or auratestatin.

30. The antibody-drug conjugate of claim 28 or a pharmaceutically acceptable salt or solvation thereof, 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 Each is 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 is C independently 1-8 Alkyl, preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; R 6b and R 7b Each is independently selected from C 1-8 Alkoxy, preferably C 1-4 Alkyl groups, such as methoxy, ethoxy, or propoxy; 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 R 10b Selected from OH and H; In the D structure, the wavy line indicates that the valence bond is connected to L.

31. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 30, wherein D has the structure of formula (D-1a), and wherein R 1a For H; R 2a It is a C1-C4 alkyl group, preferably methyl; R 3a For halogens, F and R are preferred. 4a It is a C1-C4 alkyl group, preferably ethyl.

32. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 30, wherein D has the structure of formula (D-1b), and wherein R 1b R 4b and R 8b Each is independently selected from C 1-2 Alkyl groups, preferably methyl groups; R 2b R 3b and R 5b Each is independently selected from C 3-4 alkyl; R 6b and R 7b Each is independently selected from C 1-2 alkoxy groups; and R 9b Selected from C 1-4 Alkyl and R 10b It is OH or H; or R 9b It is COOH and R 10b For H.

33. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 30-32, wherein D has the structure shown in formula (D-2a) or formula (D-2b): Where R 1a R 2a R 3a and R 4a As defined in equation (D-1a); 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 equation (D-1b).

34. The antibody-drug conjugate of claim 30 or a pharmaceutically acceptable salt or solvation thereof, wherein D has the structure shown in formula (D-3a) or (D-3b): Preferably, D has the structure shown in formula (D-4a) or (D-4b):

35. The antibody-drug conjugate of claim 28 or a pharmaceutically acceptable salt or solvate thereof, wherein the drug is Exatecan, Dxd, SN-38, monomethylaurestatin E (MMAE), or MMAF.

36. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 28-35, 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, 8, 9 or 10; L1 is selected from non-existent, Where n1 and m1 are each an independent integer selected from 0 to 20, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and L3 is Where X is selected from -NH-, -O-, and -S-; R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; n3 and n4 are independently 1, 2, 3, 4, 5, or 6; and in, Z is connected to Ab, preferably connected to S on Ab, and L3 is connected to D.

37. The antibody-drug conjugate of claim 36 or a pharmaceutically acceptable salt or solvate thereof, wherein... Z is selected from Preferred is Where m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, Z is selected from More preferably, Z is 38. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 36 or 37, wherein L1 is selected from the absence of... Where n1 is an integer independently selected from 0 to 12, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Preferably, L1 is selected from non-existent, 39. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 36-38, wherein L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6, 7 or 8 amino acids; preferably, wherein each of the amino acid residues or amino acids is independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine ​​(Cys), histidine (His), and threonine (Thr), wherein the amino acid residues or amino acids are optionally converted by one or more C 1-6 Alkyl substitution; More preferably, the amino acid residues or each amino acid is independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), aspartic acid (Asp), asparagine (Asn), arginine (Arg), and threonine (Thr); More preferably, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), aspartic acid (Asp), arginine (Arg) and threonine (Thr).

40. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 36-39, wherein L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Arg-Asp-Val-Thr-, and -Gly-Gly-Phe-Gly-, preferably selected from -Gly- and -Arg-Asp-Val-Thr-.

41. The antibody-drug conjugate according to any one of claims 36-40, or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: Optional, L3 is Optional, L3 is Where R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is selected independently from each of the following groups: 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.

42. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 36-41, wherein L3 is selected from: Preferably, L3 is selected from: More preferably, L3 is selected from: For example is 43. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 36-42, wherein each of Su is independently: Preferably, Su is independently Alternatively, preferably, each Su is independently...

44. The antibody-drug conjugate according to any one of claims 36-43, or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: Preferably, L3 is selected from 45. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 30-35, wherein -L- are each independently selected from the following structures: Preferred is in, Each m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Optionally, -L- independently are: The group is connected to Ab on the left and to D on the right.

46. ​​The antibody-drug conjugate of claim 28 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is selected from: Wherein Ab is a bispecific antibody according to any one of claims 1-22; m are each an independent integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 and 16, for example, an integer selected from 4 to 6.

47. The antibody-drug conjugate of claim 28 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is: in, Ab is a bispecific antibody according to any one of claims 1-22; and p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 4 to 6, mainly 4 or 6.

48. The antibody-drug conjugate of claim 28 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is: in, Ab is a bispecific antibody according to any one of claims 1-22; and p is an integer selected from 1 to 16, for example, an integer selected from 2 to 10, for example, an integer selected from 4 to 8, for example, an integer selected from 4 to 6, mainly 4 or 6.

49. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 47 or 48, wherein the Ab is V-F1, V-F2, V-F3, V-F7 or V-F8 and p is 6, or the Ab is V-F9 and p is 4, wherein V-F1, V-F2, V-F3, V-F7, V-F8 or V-F9 are each composed of the following chains: V-F1: Two chains as shown in SEQ ID NO:42; V-F2: Two chains as shown in SEQ ID NO:43; V-F3: the first strand as shown in SEQ ID NO:44, and the second strand as shown in SEQ ID NO:45; V-F7: the first chain as shown in SEQ ID NO:46, and the second chain as shown in SEQ ID NO:47; V-F8: the first chain as shown in SEQ ID NO:48, and the second chain as shown in SEQ ID NO:49; V-F9: the first chain as shown in SEQ ID NO:52, and the second chain as shown in SEQ ID NO:

53.

50. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 28-49, wherein the LD portion is covalently linked to the Ab via a sulfur atom from the Ab.

51. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 28-50, wherein the antibody-drug conjugate has an average DAR of 2-10, for example 4-8, for example about 4-6, for example about 4 or about 6.

52. A pharmaceutical composition comprising a bispecific antibody of any one of claims 1-22, a nucleic acid molecule of claim 23, an immunoconjugate or immunofusion of claim 27, an antibody-drug conjugate of any one of claims 28-51 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient, and optionally further comprising one or more additional therapeutically active substances.

53. The use of the bispecific antibody of any one of claims 1-22, the nucleic acid molecule of claim 23, the immunoconjugate or immunofusion of claim 27, the antibody-drug conjugate of any one of claims 28-51, or a pharmaceutically acceptable salt or solvate thereof as a drug or for the preparation of a drug.

54. The use of claim 53, wherein the drug is used to treat and / or prevent cancer in an individual, preferably said cancer being CDH17 and / or EGFR positive cancer.

55. The use of claim 54, wherein the cancer is selected from, for example, gastrointestinal tumors or pancreatic cancer, such as: colon cancer, colorectal cancer (e.g., colorectal adenocarcinoma), lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, gastric cancer (e.g., gastric adenocarcinoma), gastroesophageal adenocarcinoma, cervical cancer, ovarian cancer, or neuroendocrine tumor.

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