Binding molecule and antibody-drug conjugate and use thereof

Abstract

Provided are an immunoglobulin single variable domain (ISVD) that specifically binds to EGFR and / or cMet, and an EGFR and / or cMet binding molecule comprising the immunoglobulin single variable domain and an antibody-drug conjugate (ADC). Further provided are a nucleic acid encoding the ISVD or binding molecule, a vector comprising the nucleic acid, and the therapeutic use of the ISVD or binding molecule and the ADC.

Description

Binding molecules and antibody-drug conjugates and their uses Technical Field

[0001] The present invention relates to immunoglobulin single variable domains (ISVDs) that specifically bind to EGFR and / or cMet, as well as EGFR and / or cMet binding molecules and antibody-drug conjugates (ADCs) comprising the immunoglobulin single variable domains. The present invention also relates to nucleic acids encoding the ISVDs or binding molecules and vectors comprising the nucleic acids, as well as therapeutic uses of the ISVDs or binding molecules and the ADCs. Background Art

[0002] EGFR is a transmembrane receptor protein with tyrosine kinase activity present on the cell membrane. It can send signals by binding to exogenous growth factors (such as EGF), further activating downstream pathways related to cell division, survival, and angiogenesis, thereby affecting processes such as cell proliferation, survival, and differentiation. EGFR is found to be overexpressed or mutated in many tumor types, making it an important target for the treatment of these cancers. Currently, a variety of drugs that inhibit EGFR function, such as tyrosine kinase inhibitors and monoclonal antibodies, have been developed and are widely used in the treatment of multiple malignancies, including lung cancer, colorectal cancer, and head and neck cancer.

[0003] cMet is a receptor with tyrosine kinase (RTK) activity expressed on epithelial cells, playing a key role in cell proliferation, survival, and migration. Its binding ligand is hepatocyte growth factor (HGF). Upon binding, cMet dimerizes and activates the cMet pathway, promoting cell division, angiogenesis, and immune regulation. Because cMet is overexpressed or mutated in various cancers, it has become an important drug target, and related anticancer drugs are under research and development.

[0004] The U.S. Food and Drug Administration has approved an EGFR and cMet dual-targeting antibody: Amivantamab (also known as "JNJ-61186372") for the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion mutations, to overcome resistance to targeted therapies in patients with non-small cell lung cancer. AZD9592 is an anti-EGFR / cMet bispecific antibody ADC drug currently under clinical investigation, carrying a topoisomerase 1 inhibitor as a payload, for the treatment of advanced solid tumors (Moores, Sheri L. et al., 2016. “A Novel Bispecific Antibody Targeting EGFR and cMet Is Effective against EGFR Inhibitor-Resistant Lung Tumors.” Cancer Research 76(13):3942–53. https: / / doi.org / 10.1158 / 0008-5472.CAN-15-2833; Robert Hsu et al., “A narrative review of antibody–drug conjugates in EGFR-mutated non-small cell lung cancer”, Front Oncol. 2023 Dec 1; 13:1252652. doi:10.3389 / fonc.2023.1252652).

[0005] The anti-EGFR / cMet bispecific antibody JNJ-61186372 and the ADC drug AZD9592 have a conventional four-chain antibody structure and a large molecular weight (150 kDa), which is not conducive to penetrating deep tumor tissue. Therefore, there is still an urgent need in the art to develop new antibody formats targeting EGFR and / or cMet targets and EGFR and / or cMet binding molecules and ADC molecules with favorable properties.

[0006] Immunoglobulin single variable domains (ISVDs), such as nanobodies, are small proteins composed of a single chain of antibody molecules. They possess high antigen specificity and affinity, and are smaller, more stable, and have deeper tissue penetration than conventional four-chain antibodies. Therefore, the application of nanobodies and drug molecules based on them in the field of tumor treatment is attracting widespread attention and is expected to become one of the important means of tumor treatment in the future. Summary of the Invention

[0007] The present disclosure provides novel anti-cMet and anti-EGFR immunoglobulin single variable domains (ISVDs) based on the screening of anti-EGFR and anti-cMet phage display libraries, and utilizes the ISVDs as components to construct and produce anti-EGFR binding molecules, anti-cMet binding molecules, anti-EGFR / cMet binding molecules, and antibody-drug conjugates with excellent tumor targeting, endocytosis activity, and killing activity.

[0008] Therefore, in a first aspect, the present disclosure provides immunoglobulin single variable domains (ISVDs) that specifically bind to cMet, heavy chain antibodies comprising the ISVDs, and cMet-binding molecules. In some embodiments, the cMet-binding molecules are anti-cMet bi-epitope antibodies. In some embodiments, the cMet-binding molecules further comprise a binding domain that binds to EGFR.

[0009] In a second aspect, the present disclosure provides immunoglobulin single variable domains (ISVDs) that specifically bind to EGFR, heavy chain antibodies comprising the ISVDs, and EGFR binding molecules. In some embodiments, the EGFR binding molecule further comprises a binding domain that binds to cMet.

[0010] In a third aspect, the present disclosure provides EGFR and cMet binding molecules comprising one or more immunoglobulin single variable domains (ISVDs) according to the first and / or second aspects of the present invention that specifically bind to EGFR and cMet. In some embodiments, the binding molecule is a single-chain or multi-chain antibody. In some embodiments, the antibody is a bispecific antibody or a multispecific antibody.

[0011] In a fourth aspect, the present disclosure provides nucleic acids encoding the ISVD, EGFR binding molecules, cMet binding molecules, EGFR and cMet binding molecules according to the first to third aspects of the present disclosure, vectors (preferably, expression vectors) comprising the nucleic acids, and host cells comprising the nucleic acids or the vectors. In some embodiments, the host cells are prokaryotic or eukaryotic, for example, selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing ISVDs or binding molecules. In some embodiments, the host cells are HEK 293 cells or CHO cells. The present disclosure also provides methods for preparing the ISVD, EGFR binding molecules, cMet binding molecules, EGFR and cMet binding molecules according to the first to third aspects of the present disclosure.

[0012] In some embodiments, the binding molecules that specifically bind to EGFR and / or cMet according to the present invention have a smaller molecular weight than conventional four-chain antibodies and have better tumor tissue penetration ability.

[0013] In some embodiments, the EGFR and / or cMet binding molecules according to the present invention have low specific binding activity to EGFR antigens expressed on the cell surface, thereby reducing the "on-target / off tumor" toxicity of the EGFR binding molecules, such as significantly reducing the skin toxicity often reported for drugs containing EGFR binding molecules (Taieb, Julien et al., 2023. "Adverse Events Associated with Encorafenib Plus Cetuximab in Patients with BRAFV600E-Mutant Metastatic Colorectal Cancer: An in-Depth Analysis of the BEACON CRC Study." Clinical Colorectal Cancer, Updates in Pancreatic Cancer, 22(1): 59–66. https: / / doi.org / 10.1016 / j.clcc.2022.12.003; Robert, Caroline et al., 2005. "Cutaneous Side-Effects of Kinase Inhibitors and Blocking Antibodies." The Lancet Oncology 6(7):491–500. https: / / doi.org / 10.1016 / S1470-2045(05)70243-6).

[0014] In some embodiments, the binding molecules according to the present invention have a structure that binds to a dual cMet epitope (i.e., two different epitopes on cMet), which enables the binding molecules to have better tumor targeting. In some embodiments, the dual cMet epitope design of the binding molecules according to the present invention significantly increases the affinity and endocytosis capacity of the binding molecules to target cells compared to targeting only a single epitope on cMet.

[0015] The ISVD that specifically binds to EGFR and / or cMet and the EGFR and / or cMet binding molecules comprising the ISVD of the present invention have good tumor targeting, tumor tissue penetration ability and / or target cell endocytosis ability, etc. Therefore, the ISVD or binding molecule can be used as a targeting module of a conjugate or coupling and conjugated or coupled to a chemotherapeutic agent, toxin, drug (such as an immunotherapeutic agent), radioactive element, probe or signaling molecule, etc., to provide applications such as tumor killing, immune regulation or disease detection.

[0016] In a fifth aspect, the present disclosure provides a conjugate, fusion, and antibody-drug conjugate (ADC) comprising the ISVD, EGFR-binding molecule, cMet-binding molecule, or EGFR- and cMet-binding molecules according to the first to third aspects of the present disclosure, especially an anti-EGFR / cMet multispecific antibody-drug conjugate.

[0017] In some embodiments, the antibody drug conjugates according to the present invention have the following advantages:

[0018] (1) Binding to tumor cells expressing human EGFR or cMet and tumor cells co-expressing human EGFR and cMet;

[0019] (2) demonstrated endocytic activity in tumor cells expressing human EGFR or cMet, as well as tumor cells co-expressing human EGFR and cMet;

[0020] (3) blocking the binding of HGF to cMet on tumor cells;

[0021] (4) Has bystander effects;

[0022] (5) It has a broad spectrum of anti-tumor activity, showing significant killing activity against a variety of tumors with different EGFR and cMet expression densities;

[0023] (6) Low in vivo toxicity, such as low on-target / off-tumor toxicity.

[0024] In some embodiments, the antibody drug conjugates according to the present invention further have one or more advantages selected from the following:

[0025] (7) Because of the multispecific antibodies it contains, it can target multiple antigens simultaneously and has better targeting and reduced toxic side effects (such as reduced off-target toxicity or reduced potential dose-limiting toxicity);

[0026] (8) Compared with single-target ADCs targeting EGFR or cMet, it has higher endocytosis efficiency in tumor cells;

[0027] (9) Compared with single-target ADCs targeting EGFR or cMet, it has higher affinity on tumor cells;

[0028] (10) Compared with single-target ADCs targeting EGFR or cMet, it has a stronger killing effect on tumor cells and a stronger inhibitory effect on tumor growth.

[0029] In some embodiments, the antibody drug conjugates according to the present invention further have one or more advantages selected from the following:

[0030] (11) Good product uniformity;

[0031] (12) Having good product stability; and

[0032] (13) It has good drugability.

[0033] In a sixth aspect, the present disclosure provides pharmaceutical compositions and pharmaceutical preparations comprising the ISVD, EGFR binding molecules, cMet binding molecules, or EGFR and cMet binding molecules according to the first to third aspects of the present disclosure, or the ADC of the fifth aspect of the present disclosure and a pharmaceutically acceptable carrier, and optionally further comprising one or more additional pharmaceutically active polypeptides and / or compounds, for example, further comprising other therapeutic agents selected from oncolytic drugs, cytotoxic agents, cytokines, and inhibitors of immune checkpoint molecules. In this regard, the present disclosure also provides a combination product or kit comprising the ISVD, EGFR binding molecules, cMet binding molecules, or EGFR and cMet binding molecules according to the first to third aspects of the present disclosure, or the ADC of the fifth aspect of the present disclosure.

[0034] In the seventh aspect, the present disclosure provides the use of the ISVD, EGFR binding molecule, cMet binding molecule, or EGFR and cMet binding molecule of the first to third aspects of the present disclosure, or the ADC of the fifth aspect of the present disclosure as a drug or for the preparation of a drug, wherein the drug is used to treat cancer, for example, the cancer is selected from lung cancer (e.g., lung squamous cell carcinoma, lung adenocarcinoma, non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), gastric cancer, colon cancer or head and neck cancer (e.g., pharyngeal squamous cell carcinoma). In this aspect, the present disclosure also provides a method of treating cancer, comprising administering to a subject in need thereof an effective amount of the ISVD, EGFR binding molecule, cMet binding molecule, or EGFR and cMet binding molecule of the first to third aspects of the present disclosure, or the nucleic acid or vector or host cell of the fourth aspect of the present disclosure, or the ADC of the fifth aspect of the present disclosure, wherein the subject is a mammal; preferably, the subject is a human; wherein the cancer is, for example, lung cancer (e.g., lung squamous cell carcinoma, lung adenocarcinoma, non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), gastric cancer, colon cancer or head and neck cancer (e.g., pharyngeal squamous cell carcinoma). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0036] FIG1 shows the FACS detection of the binding activity of anti-EGFR antibodies to target cells.

[0037] FIG2 shows the FACS detection of the binding activity of anti-cMet antibodies to target cells.

[0038] FIG3 shows the blocking effect of anti-cMet VHH-Fc on the binding of the ligand HGF to the target cell EBC-1.

[0039] FIG4 shows that the anti-cMet antibodies V-n7A12 and V-n9A2 bind to different epitopes on the antigen cMet.

[0040] Figures 5A and 5B show the cellular internalization of each VHH-Fc detected by FACS.

[0041] FIG6 shows the results of ELISA testing of whether the anti-cMet antibodies V-n7A12, V-n9A2, and V-n9A10 have cross-reactivity with human cMet antigen and cynomolgus monkey cMet antigen.

[0042] FIG7 shows the FACS detection of the binding of the bi-epitope antibody to target cells.

[0043] Figure 8 shows FACS detection of the internalization of the bi-epitope antibody on target cells. Compared with the single epitope, the bi-epitope mediated synergistic endocytosis activity.

[0044] FIG9 shows a schematic diagram of the molecular structure of a single-chain multispecific EGFR / cMet antibody.

[0045] FIG10 shows a schematic diagram of the molecular structure of a double-chain multispecific EGFR / cMet antibody.

[0046] FIG11 shows the binding of trispecific antibody candidate molecules on target cells EBC-1 and NCI-H1975 cells.

[0047] Figure 12 shows the internalization of single-chain trispecific antibody candidate molecules by target cells.

[0048] Figure 13 shows the internalization of two-chain trispecific antibody candidate molecules by target cells.

[0049] FIG14 shows that trispecific anti-EGFR / cMet antibodies block the binding of EBC-1 cells to the ligand HGF.

[0050] FIG15 shows FACS detection of cooperative endocytosis of trispecific antibodies mediated by anti-EGFR ISVD and anti-cMet ISVD on target cells.

[0051] FIG16 shows FACS detection of synergistic binding of trispecific antibodies mediated by anti-EGFR ISVD and anti-cMet ISVD on target cells.

[0052] FIG17 shows the binding of ADC molecules to target cells.

[0053] Figures 18A and 18B show the killing of target cells by ADC molecules.

[0054] Figure 19 shows cell binding assays of ADC molecules with linkers of PEG and / or EVC.

[0055] FIG20 shows a cell killing assay of ADC molecules with linkers of PEG and / or EVC.

[0056] FIG21 shows the in vivo efficacy of V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE, and V-26-Fc-PEG-EVC-MMAE in the CDX model.

[0057] FIG22 shows the in vivo efficacy of V-23-Fc-VA-Exd in the CDX model.

[0058] FIG23 shows the in vivo efficacy of V-23-Fc-Glu-Exd in the CDX model. DETAILED DESCRIPTION

[0059] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0060] definition

[0061] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0062] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5%, 4%, 3%, 2% or 1% less than the specified numerical value and an upper limit that is 5%, 4%, 3%, 2% or 1% greater than the specified numerical value.

[0063] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0064] In this document, when the terms "comprising" or "including" are used, unless otherwise specified, it also covers the situation consisting of the recited elements, integers or steps. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0065] The term "on-target / off tumor toxicity" refers to the fact that in addition to tumor cells, normal tissue cells also express tumor-associated antigens targeted by antibodies, thereby binding to the antibodies and causing damage.

[0066] When used in conjunction with an antigen, the terms "binding molecule" and "antigen binding molecule" are used interchangeably (e.g., EGFR binding molecule, cMet binding molecule, EGFR and cMet binding molecule) and refer to a protein or polypeptide molecule that can specifically bind to an antigen or an epitope on an antigen. The binding molecule has "affinity" and / or "specificity" for the antigen. Herein, an EGFR binding molecule refers to a protein or polypeptide that can specifically bind to EGFR, a cMet binding molecule refers to a protein or polypeptide that can specifically bind to cMet, and an EGFR and cMet binding molecule refers to a protein or polypeptide that can specifically bind to both EGFR and cMet. Some examples of binding molecules include antibodies, antibody fragments, fusion proteins, and the like, as long as they exhibit the desired antigen binding activity.

[0067] The domain actually bound to the antigen in an antigen binding molecule is referred to herein as an "antigen binding site" or "antigen binding domain". "Domain" is a folded structure in a protein or polypeptide, generally speaking, responsible for a single function of a protein or polypeptide. For example, conventional antibodies and immunoglobulins form antigen-binding domains on the surface of a VH-VL dimer through three complementary determining regions (HCDR1-3) in their heavy chain variable region (VH) and three complementary determining regions (LCDR1-3) in their light chain variable region (VL), wherein six CDRs confer specific binding of the antibody to the antigen. However, in some cases, a single immunoglobulin variable domain (e.g., a heavy chain variable domain (VH) or a light chain variable domain (VL), a heavy chain variable domain (VHH) derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) derived from fish IgNAR) can confer antigen binding. That is, the single variable domain does not need to interact with another variable domain and can independently serve as an "antigen-binding domain" for identifying and binding to a target antigen. Typically, through engineering modifications, the "antigen-binding domain" of an antibody, including the above-mentioned single immunoglobulin variable domain and the variable domain pairs of conventional antibodies, can be added, removed or transferred to other proteins or polypeptides and still exert its antigen-binding function without losing the function of the rest of the protein or polypeptide and / or the rest of the domains.

[0068] The term "binding" or "specific binding" means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by conventional binding assays known in the art. For example, the binding ability of an antibody to an antigen can be detected by an ELISA assay as described in the Examples, or the binding ability of an antibody to cells expressing the antigen on their surface can be detected by a FACS assay as described in the Examples, or the affinity constant K can be determined by the SPR technique as described in the Examples. D .

[0069] The term "antibody" is used in the broadest sense herein to refer to a protein comprising an immunoglobulin antigen binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, single-epitope and multi-epitope antibodies (e.g., bi-epitope antibodies), monospecific and multispecific antibodies (e.g., bispecific antibodies), single-chain and multi-chain antibodies, nanobodies, single-domain antibodies, heavy-chain antibodies, chimeric antibodies, humanized antibodies, complete antibodies, and antibody fragments. In some embodiments, preferably, the antibody of the present invention is a single-domain antibody, nanobody, or heavy-chain antibody. In other embodiments, preferably, the antibody of the present invention is a bi-epitope antibody, a bispecific antibody, or a multispecific antibody.

[0070] The terms "antibody fragment" or "antigen-binding fragment" of an antibody are used interchangeably and refer to molecules that are different from intact antibodies, which comprise a portion of an intact antibody and are capable of binding to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; single-chain antibody fragments (e.g., scFv, scFab); single immunoglobulin domains; variable domain fragments of camelid heavy chain antibodies; and various monospecific, bispecific, or multispecific antibody structures formed by antibody fragments, such as linear antibody fragments, diabody fragments, and the like. In this disclosure, unless otherwise specified or clearly contradicted by the context, reference to the term "antibody" is equivalent to reference to "antibodies and antibody fragments thereof." In some embodiments according to the present invention, the antibody fragment comprises a cysteine ​​residue portion for forming an interchain disulfide bond between heavy chains, for example, a cysteine ​​residue in the hinge region of an antibody, to provide an amino acid residue site that can be used for sulfhydryl coupling chemistry. In other embodiments according to the present invention, the antibody fragment comprises a cysteine ​​residue introduced into the Fc region to provide an amino acid residue site that can be used for sulfhydryl coupling chemistry.

[0071] In the present disclosure, the term "immunoglobulin single variable domain" (abbreviated as "ISVD") is used interchangeably with the term "single variable domain" to refer to an antibody polypeptide fragment that is capable of specifically recognizing and binding to an antigen of interest through a single variable domain, such as a single VHH domain, a single VH domain, or a single VL domain, without the need for pairing with additional immunoglobulin variable domains. For an ISVD composed of a VHH domain, a VH or VL domain, its structure can be considered to be composed of four framework regions ("FR"), respectively referred to as "Framework Region 1" or "FR1", "Framework Region 2" or "FR2", "Framework Region 3" or "FR3", and "Framework Region 4" or "FR4", and three complementarity determining regions ("CDRs"); the four framework regions are interrupted by three complementarity determining regions or "CDRs", respectively referred to as "Complementarity Determining Region 1" or "CDR1", "Complementarity Determining Region 2" or "CDR2", and "Complementarity Determining Region 3" or "CDR3". From the N-terminus to the C-terminus of the ISVD polypeptide, four framework regions and three complementarity-determining regions are arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin single variable domains can comprise fully human sequences, humanized sequences, sequences optimized in other ways, or chimeric immunoglobulin sequences. Immunoglobulin single variable domains can be used alone in isolated form or as part of a larger protein to exert antigen-binding function. In this disclosure, an ISVD that specifically binds to EGFR is also referred to as an anti-EGFR ISVD; an ISVD that specifically binds to cMet is also referred to as an anti-cMet ISVD; and an ISVD that specifically binds to both EGFR and cMet is also referred to as an anti-EGFR / cMet ISVD.

[0072] In the present disclosure, the terms "single domain antibody" and "single domain antibody" are used interchangeably herein and generally refer to antibodies that recognize and bind antigens through ISVDs. Examples of single domain antibodies include those derived from Camelidae (llamas and camels) and cartilaginous fish (e.g., nurse sharks) (WO 2005 / 035572).

[0073] In the present disclosure, the term "heavy-chain antibody (hcAb)" refers to an antibody having only a heavy chain and no light chain. The heavy chain of a heavy chain antibody may, for example, comprise VH-CH2-CH3 from N-terminus to C-terminus, or may comprise VH-CH1-CH2-CH3, or may comprise VHH-CH2-CH3, etc. The heavy chain may constitute a homodimer. In some embodiments, preferably, the heavy chain antibody according to the present invention is a dimer comprising two monomers, each of which comprises a VHH domain connected to an immunoglobulin constant region (CH2 and CH3 domains) via an immunoglobulin hinge region.

[0074] The term "nanobody" is used herein to refer to an antibody comprising, consisting essentially of, or consisting of a single ISVD domain (such as a VHH domain) with a molecular weight of less than 20 kDa (typically a molecular weight of about 12-15 kDa).

[0075] The term "VHH antibody" is used herein to refer to an antibody composed of a VHH domain. A "VHH domain," also known as a VHH, VHH sequence, or VHH antibody fragment, is a single-chain antibody fragment comprising FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1 from the C-terminus to the N-terminus. The use of a VHH domain (alone or as part of a larger polypeptide) to recognize and bind a target antigen offers a number of significant advantages over the use of conventional VH and VL domains, scFv, or conventional antibody fragments (e.g., Fab or F(ab')2 fragments):

[0076] Only a single domain is required to bind the antigen with high affinity and selectivity, thus eliminating the need for two separate domains and ensuring that the two domains are in the proper spatial conformation and configuration (e.g., scFv generally requires the use of a specially designed linker);

[0077] - VHH domains can be easily engineered into multivalent and multispecific formats;

[0078] - VHH domains are highly soluble and have no tendency to aggregate;

[0079] - VHH domains are highly stable to heat, pH, protein or peptide enzymes, and other denaturing agents or conditions, and therefore can be prepared, stored, or transported without the use of refrigeration equipment, thereby achieving cost, time, and environmental savings;

[0080] - VHH domains are easy and relatively cheap to prepare, even on the scale required for production;

[0081] - VHH domains are relatively small compared to conventional tetrapeptide antibodies and their antigen-binding fragments, thus exhibiting higher tissue penetration and allowing for higher dose administration;

[0082] - VHH domains can display so-called cavity binding properties (compared to conventional VH domains, VHHs have an extended CDR3 loop, thereby being able to reach target epitopes that are inaccessible to conventional tetrapeptide antibodies and their antigen-binding fragments).

[0083] VHH includes humanized VHH, camelized VH, or VHH obtained by affinity maturation.

[0084] For further description of VHH, reference may be made to WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103. In some embodiments, the antigen binding site of the antigen binding molecules and antibodies according to the present invention is preferably provided by a VHH domain.

[0085] The term "valency" refers to the number of antigen-binding sites present in an antigen-binding molecule (e.g., an antibody). Thus, "monovalent," "bivalent," "trivalent," and "tetravalent" antibodies refer to antibodies with one, two, three, and four antigen-binding sites, respectively.

[0086] In this article, "monospecific" refers to the ability of an antigen binding molecule to bind only to a single epitope. "Multispecific" refers to the ability of an antigen binding molecule to bind to two or more different epitopes (e.g., different epitopes on the same antigen and / or different antigens). Accordingly, "bispecific" refers to the ability of an antigen binding molecule to bind to two different epitopes. Monospecific antigen binding molecules (e.g., antibodies) can be monovalent or multivalent. Multispecific and bispecific antigen binding molecules (e.g., antibodies) can be divalent, trivalent, tetravalent, or more.

[0087] The term "variable region" or "variable domain" refers to the domain of the antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of heavy and light chains typically have similar structures, comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). Since the CDR sequences are responsible for most antibody-antigen interactions, antibody variants that mimic the properties of known antibodies can be constructed by transforming the variable regions. In some cases, the CDR sequences from known antibodies can be transplanted onto the framework regions of different antibodies with different properties, and one to several residue mutations, such as back mutations, can be performed as needed to refine the desired properties of the antibody. In other cases, the variable domains of an antibody can be engineered to construct humanized, immunogenic, and / or PTM (post-translational modification) variants. The properties of the modified antibody, including but not limited to target antigen binding properties or other desired functional properties, such as internalization activity, can be determined and screened in vitro or in vivo using methods known in the art and described herein. It will be appreciated that such functional variants of any variable region (e.g., VH and / or VL region, VHH region) set forth herein are contemplated by the present invention.

[0088] The term "complementarity determining region" or "CDR region" or "CDR" refers to the region of the antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contact points"). The CDRs are primarily responsible for binding to the antigen epitope and include, numbered sequentially from the N-terminus of the variable region, CDR1, CDR2, and CDR3. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Unless otherwise indicated, in this disclosure, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same AbM or Kabat numbering position as a reference CDR sequence (e.g., the CDR sequence exemplified by the present invention). In one embodiment, the CDRs of an antibody of the present invention are defined according to Kabat or Chothia or AbM or IMGT or Contact, or any combination thereof. In one embodiment, the CDRs of an antibody of the present invention are determined according to the Kabat definition scheme.

[0089] Antibodies with different specificities (i.e., for different antigenic epitopes) have different CDRs. However, although CDRs are different between antibodies, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, IMGT and Contact methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As will be appreciated by those skilled in the art, the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present disclosure also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia or AbM or IMGT or Contact can be replaced by conservative amino acid residues.

[0090] If an amino acid sequence (e.g., an ISVD) is specific for two different antigens or antigenic determinants (e.g., EGFR from different mammalian species, such as human EGFR and cynomolgus monkey EGFR, or cMet from different mammalian species, such as human cMet and cynomolgus monkey cMet), it is said to be "cross-reactive" to these two different antigens or antigenic determinants. It is advantageous for an antibody to have human-monkey species cross-reactivity, especially to have similar human-monkey antigen binding affinities, as this property can facilitate preclinical drug development of antibodies, such as toxicological testing of ADC molecules composed of antibodies. In some embodiments, the antibodies of the present invention preferably have human-monkey species cross-reactivity.

[0091] As used herein, the term "epitope" refers to the portion of an antigen to which an antibody specifically binds. An epitope can be composed of continuous and / or discontinuous amino acids that form a conformational space unit. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are closely adjacent in three-dimensional space by the folding of the protein molecule. Epitope grouping can be performed by competitive binding assays to different antibodies that bind to the same antigen. Such competitive binding assays 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 competition assay, or by the methods described in the Examples herein.

[0092] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs in the humanized antibodies correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. Humanized antibodies optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to a humanized antibody.

[0093] As used herein, the term "half-life extension domain" or the expression "half-life increasing binding moiety" are used interchangeably to refer to a chemical structure that can confer an increased circulating half-life to a molecule (e.g., an antibody) to which it binds after administration to an animal. Such chemical structures include, for example, flexible hydrophilic molecules (e.g., carbohydrates or PEG (polyethylene glycol)), immunoglobulin Fc regions, serum albumin, serum albumin binding domains, or serum albumin binding peptides (e.g., anti-HSA ISVD). The half-life extension domain can be linked to the binding molecule or antibody of the invention by chemical conjugation or fusion, depending on its specific properties.

[0094] In some embodiments, the EGFR binding molecule of the first aspect of the present disclosure, the cMet binding molecule of the second aspect of the present disclosure, and the EGFR and cMet binding molecule of the third aspect of the present disclosure comprise an ISVD that binds to human serum albumin as a half-life extension domain. In some embodiments, the ISVD that binds to human serum albumin is selected from the serum albumin binding portion of Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10, and Alb-23. In one embodiment, the serum albumin binding portion is Alb-8 or Alb-23 or a variant thereof, as shown on pages 7-9 of WO 2012 / 175400. In some preferred embodiments, the ISVD that binds to human serum albumin comprises a CDR1 consisting of or consisting of the amino acid sequence of SEQ ID NO: 46, a CDR2 consisting of or consisting of the amino acid sequence of SEQ ID NO: 47, and a CDR3 consisting of or consisting of the amino acid sequence of SEQ ID NO: 48. In some embodiments, the ISVD that binds to human serum albumin comprises the sequence of SEQ ID NO: 45, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0095] The terms "immunoglobulin Fc region," "Fc domain," "Fc portion," or "Fc region" are used interchangeably herein to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the constant region. It is known that the heavy chain constant region of each immunoglobulin comprises four or five domains, which are named in the following order: CH1-hinge-CH2-CH3 (-CH4). CH4 is present in IgM without a hinge region. In the present disclosure, an Fc domain may comprise a CH2 domain and a CH3 domain, and optionally also comprise all or part of an immunoglobulin hinge region; but does not comprise the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, as well as the heavy chain constant region CH1 and light chain constant region CL. For example, in one embodiment, an Fc domain may comprise or consist of a CH2 domain and a CH3 domain from the N-terminus to the C-terminus. In another example, the Fc domain can comprise or consist of an immunoglobulin hinge region, a CH2 domain, and a CH3 domain from the N-terminus to the C-terminus. The Fc domain often exists in a dimerized form, and each Fc domain in the dimerized form is also referred to herein as an Fc subunit.

[0096] The term "Fc region" includes native sequence Fc regions and variant Fc regions. In certain embodiments, the antibody according to the present invention comprises a human IgG heavy chain Fc region. In some embodiments, the human IgG heavy chain Fc region extends from Glu216, Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise indicated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In some embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region comprises modifications relative to the native sequence Fc region. In some specific embodiments, the Fc region has increased or decreased effector function. In some embodiments, the Fc region has enhanced or reduced binding to FcγR. In some embodiments, the Fc region contains a heavy chain mispairing-preventing mutation, such as a Knob-into-hole (KIH) mutation.

[0097] In some cases, an immunoglobulin Fc region comprising a hinge region sequence is preferred, which can, for example, promote dimerization of antibody polypeptide chains and / or provide cysteine ​​residues for coupling to other active molecules. Such a hinge region sequence may correspond substantially or in part to the hinge region of IgG1, IgG2, IgG3 or IgG4. For example, the hinge region sequence may include all or part of the core hinge region and all or part of the lower hinge region. The core hinge region has the amino acid sequence CPPC in IgG1, IgG2 and IgG3, and has the CPSC sequence in IgG4. Preferably, the hinge region contains at least one disulfide bond connecting two Fc chains. In some embodiments, the hinge region sequence comprises a hinge region sequence from E216 to T225 of IgG1 or a hinge region sequence from D221 to T225 (according to EU numbering), or corresponding hinge region sequences from other immunoglobulin isotypes. In some embodiments, the immunoglobulin single variable domain (ISVD) of the invention is connected to the Fc region via a hinge sequence comprising, for example, EPKSS (SEQ ID NO: 49) or EPKSC (SEQ ID NO: 50).

[0098] The term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0099] In the case where effector function is not required, the Fc region may include mutations that reduce or eliminate effector function. In some cases (e.g., using the antibody of the present invention as an ADC carrier), preferably, the Fc region includes mutations that reduce or eliminate the Fc region and Fcγ receptors, such as LALA mutations in which lysine (L) at positions 234 and 235 of the Fc region is converted to alanine (A) to reduce Fcγ receptor-mediated off-target cytotoxicity. Additionally or alternatively, mutations can 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 coupling active molecules. Additionally or alternatively, the Fc region can be mutated for antibody production reasons, such as removing or replacing amino acids that may undergo post-translational modification (e.g., glycosylation) to provide improved drugability and developability of therapeutic antibodies.

[0100] The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" is one of the main mechanisms by which certain cytotoxic effector cells (e.g., natural killer (NK) cells) mediate the killing of target cells and foreign host cells. The Fc region of an antibody activates NK cells to exert ADCC by binding to the Fc receptor FcγRIIIA (i.e., CD16a) expressed on NK cells, for example.

[0101] The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its corresponding antigen. To assess complement activation, a CDC assay can be performed, for example, by the method described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0102] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the association dissociation equilibrium constant (K D Affinity can be measured by common methods known in the art, including those known in the art and described herein.

[0103] As used herein, "percent (%) identity" of an amino acid sequence refers to the percentage of positions in the candidate sequence that have the same amino acid residue at the corresponding position in the alignment as the specific amino acid sequence set forth in the present disclosure, after aligning the candidate sequence with the specific amino acid sequence set forth in the present disclosure and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity.

[0104] In some embodiments, the present disclosure contemplates variants of the ISVD, binding molecule, and antibody sequences of the invention comprising amino acid changes relative to the ISVD, binding molecule, and antibody sequences specifically disclosed herein. In some embodiments, the variants have a substantial degree of identity over the comparison window relative to the ISVD, binding molecule, and antibody sequences specifically disclosed herein, for example, at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more identity. Herein, if no comparison window (i.e., region of interest to be compared) is specified, the alignment is performed over the full length of the reference sequence.

[0105] In some embodiments of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. In preferred embodiments, the amino acid changes described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid changes described herein occur in regions outside the VHH. In some embodiments, the substitutions are conservative substitutions. Conservative substitutions refer to substitutions of one amino acid with another amino acid within the same class, such as substitutions of an acidic amino acid with another acidic amino acid, substitutions of a basic amino acid with another basic amino acid, or substitutions of a neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in Table A below:

[0106] Table A

[0107] As used herein, an "isolated" antibody or antibody fragment refers to an artificial antibody or antibody fragment, a recombinantly produced antibody or antibody fragment, and an antibody or antibody fragment that has been at least partially separated from components of the natural environment in which it is produced. In some embodiments, an antibody (e.g., an anti-EGFR / cMet antibody) or antibody fragment (e.g., an anti-EGFR ISVD or anti-cMet ISVD) according to the present invention is "isolated." In some embodiments, an isolated antibody or antibody fragment is purified to greater than 90%, 95%, or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0108] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0109] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises 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 cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which the recombinant polynucleotide is incorporated.

[0110] In this article, the terms "endocytosis" and "internalization" are used interchangeably and refer to the process in which the ligand / receptor complex is internalized and delivered to the cytosol or transferred to the appropriate intracellular compartment, triggered by the binding of the ligand to the corresponding receptor on the cell surface. In some embodiments, the antibodies of the present invention trigger EGFR and / or cMet receptor-mediated endocytosis after binding to EGFR and / or cMet expressed on the cell surface. In this article, endocytosis and endocytosis rate can be measured 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 carrying anti-tumor drugs into cancer cells in the ADC of the present invention.

[0111] The term "conjugate" or "coupled substance" as used herein refers to a molecule formed by conjugating one or more immunoglobulin-related molecules or fragments thereof to one or more other molecules. A conjugate typically comprises at least one non-proteinaceous chemical structure portion, such as a chemical linker for achieving the conjugation. In some cases, the other molecules may be immunoglobulin-related molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-related molecules or fragments thereof. The one or more additional 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 (such as immunotherapeutic agents), radioactive elements, probes or signaling molecules, etc.

[0112] "Antibody-drug conjugate (ADC)" refers to a compound formed by linking an antigen-binding molecule to a (small molecule) drug via a linker. As used herein, the term "Antibody-Drug Conjugate" or "ADC" includes pharmaceutically acceptable salts and solvates thereof, as well as other equivalent forms. The drug compound portion of an ADC may be referred to herein as the "payload" or "toxin."

[0113] The term "connexon" refers to a structural fragment that covalently links a drug (e.g., a small molecule drug) to an antigen binding molecule portion. It should be understood that the connexon has a functional group that can form a key with the functional group of the antigen binding molecule before being connected to the antigen binding molecule. In some cases, the connexon can also have a degradable portion and optionally a hydrophilicity regulating module such as a PEG segment. In some embodiments of the ADC according to the present invention, the connexon is preferably "degradable," whereby ADC can be broken and released after being delivered to the target area (e.g., target tumor tissue site). Available "degradable connexons" include, for example, acid-labile connexons, peptidase-sensitive connexons, light-labile connexons, dimethyl connexons, or connexons containing disulfide.

[0114] The term "linker-payload" refers to a compound formed by linking a payload to a linker. In some cases, a linker-payload is used as an intermediate in ADC synthesis.

[0115] The term "therapeutic agent" encompasses any substance effective in preventing or treating a disease, such as cancer, including chemotherapeutic agents, cytotoxic agents, immunomodulators (eg, immunosuppressants), other antibodies, small molecule drugs, angiogenesis inhibitors, or cytokines.

[0116] The term "drug" refers to a compound that is able to modulate biological processes, in particular to change or prevent pathological processes. In this context, the drug preferably refers to an antitumor compound.

[0117] The term "small molecule drug" refers to a low molecular weight drug that can modulate biological processes, particularly alter or prevent pathological processes. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD, more preferably less than 500 kD. Small molecule drugs include, but are not limited to, organic molecules having a molecular weight as defined above, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.

[0118] "Anti-tumor compounds" are pharmaceutically active compounds that have effects on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773 and US5658920, camptothecin compounds such as Exatecan and Dxd (Exatecan derivatives), and auristatin compounds such as monomethyl auristatin E (MMAE) and MMAF.

[0119] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0120] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0121] The term "alkyl" as used herein refers to a fully saturated branched or unbranched hydrocarbon group. The alkyl group preferably contains 1 to 16 carbon atoms, such as 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 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.

[0122] The term "alkenyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and including at least one double bond and no triple bonds. Alkenyl groups preferably contain 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0123] The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and at least one triple bond. Alkynyl groups preferably contain 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

[0124] The term "halogen" or "halo" refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0125] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogen groups as defined herein. Halogenated alkyl groups may preferably be monohalogenated alkyl, dihalogenated alkyl, or polyhalogenated alkyl (including perhalogenated alkyl). Monohalogenated alkyl groups may contain one iodine, bromine, chlorine, or fluorine in the alkyl group. Dihalogenated alkyl and polyhalogenated alkyl groups may contain two or more identical halogen atoms or a combination of different halo groups in the alkyl group. Preferably, polyhalogenated alkyl groups contain up to 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. Perhalogenated alkyl groups refer to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0126] The term "haloalkenyl" refers to an alkenyl group, as defined herein, substituted with one or more halo groups, as defined herein. The term "haloalkynyl" refers to an alkynyl group, as defined herein, substituted with one or more halo groups, as defined herein. The meaning of "halo" as defined for "haloalkyl" applies to both "haloalkenyl" and "haloalkynyl."

[0127] The terms "alkoxy" and "alkyl-O-" are used interchangeably to denote an alkyl group as defined above attached through an oxygen atom. Preferably, the alkoxy group has 1 to 8 carbon atoms (C 1-8 Alkoxy), 1-6 carbon atoms (C 1-6 Alkoxy), 1-4 carbon atoms (C 1-4 Alkoxy) or 1-3 carbon atoms (C 1-3Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy, isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexyloxy, heptyloxy, octyloxy, etc.

[0128] The term "amino acid" refers to naturally occurring and synthetic amino acids, amino acid analogs, and artificially modified forms thereof. Amino acids may be L or D isomers. In the present disclosure, the 20 natural amino acids are represented by single-letter and three-letter abbreviations known in the art, for example: phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn; N), isoleucine (Ile; I), arginine (Arg; R), proline (Pro; P), and glutamine (Gln; Q). The remaining amino acids are represented by their full names or multi-letter abbreviations known in the art, for example, citrulline can be represented by Cit; cyclobutane-1,1-dicarboxamide-citrulline is represented by cBu-Cit. Unless otherwise specified, the amino acids of the present invention refer to L-amino acids.

[0129] The term "penturonic acid" refers to a compound formed by oxidation of the primary hydroxyl group of a pentose as defined above to a carboxyl group. Examples of penturonic acids include, but are not limited to, xyluronic acid and arabinuronic acid.

[0130] The term "hexuronic acid" refers to a compound formed by oxidation of the primary hydroxyl group of a hexose as defined above to a carboxyl group. Examples of hexuronic acids include, but are not limited to, glucuronic acid, galacturonic acid, and mannuronic acid.

[0131] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, when a group or structure is "optionally substituted," the group or structure may be substituted or unsubstituted.

[0132] "Pharmaceutically acceptable" herein refers to being able to be used for administration to an individual or subject without causing any biological or other undesirable side effects, such as severe intolerable side effects. In the absence of any contradiction in the context, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably herein.

[0133] 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 is not biologically or otherwise undesirable. The ADC conjugates of the present invention may exist as pharmaceutically acceptable salts thereof, 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 between the ADC conjugates of the present invention and 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, and the like. 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 with organic bases containing an N group.

[0134] The term "solvate" refers to an association formed between one or more solvent molecules and the ADC antibody-drug conjugate of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0135] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety. In some embodiments described herein, the DAR can be determined by p in Formula I, for example, the DAR can be 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR can also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio of the drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in a product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), and this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugate of the invention is 1 to 16, e.g., 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 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, 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, and ranges having two of these values ​​as endpoints. It should be understood that when referring to an average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules comprising ADC molecules with the same and / or different DARs.

[0136] The term "median effective concentration (EC50) 50 )" refers to the concentration of a drug, antibody, ADC or toxicant that induces a response that is 50% between baseline and maximum after a specified exposure time. In the context of this application, EC 50 The unit is "nM".

[0137] The term "fluorescence activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers one cell at a time based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). Instruments for performing FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the Epics C from Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).

[0138] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0139] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), carrier, stabilizer, etc., which is administered together with the active substance.

[0140] The terms "drug combination", "combination product", "drug association" or "combination product" refer to non-fixed combination products or fixed combination products, including but not limited to kits and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antigen binding molecules or ADC molecules of the present invention, including pharmaceutically acceptable salts thereof, and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents at a preventive or therapeutically effective level in the patient. In some embodiments, the antigen binding molecules or ADC molecules of the present invention and other therapeutic agents used in the drug combination are administered at a level no greater than when they are 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. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0141] 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, the individual or subject is a human.

[0142] The terms "tumor" and "cancer" are used interchangeably herein to refer to the physiological condition in mammals in which cell growth is unregulated and encompasses both solid and liquid tumors, and encompasses both malignant and benign tumors, and all pre-cancerous and cancerous cells and tissues.

[0143] As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual being treated. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, improving or alleviating the disease state, and alleviating or improving prognosis. In the context of tumor or cancer treatment, "treatment" encompasses anti-tumor biological effects that can be induced by human intervention (e.g., by the administration of a drug), including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0144] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0145] The term "effective amount" refers to an amount or dosage of an antigen-binding molecule or ADC molecule or composition or combination of the present invention that, after administration to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. Depending on the desired effect, both "therapeutically effective amount" and "prophylactically effective amount" may be included.

[0146] The term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or ADC can vary depending on a variety of factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or ADC to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or ADC are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (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% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that is predictive of efficacy in human tumors.

[0147] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, a prophylactic dose is less than a therapeutically effective amount because a prophylactic dose is used in a subject before or at an earlier stage of the disease.

[0148] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0149] The present invention is described in detail below. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any technical features described in any of the following sections, subsections, or embodiments may be combined with any technical features described in any other sections, subsections, or embodiments, and these combinations are all within the scope of the present invention.

[0150] I. First Aspect of the Disclosure: ISVDs That Specifically Bind to cMet and cMet-Binding Molecules Comprising Such ISVDs

[0151] cMet (or c-Met) is the gene product of the proto-oncogene MET, encoded on chromosome 7. It recognizes only one known ligand, hepatocyte growth factor (HGF). The cMet protein is a receptor tyrosine kinase that is overexpressed or mutated in many tumor cell types and plays a key role in tumor cell proliferation, survival, invasion, metastasis, and tumor angiogenesis. Inhibition of cMet can induce cell death in tumor cells that overexpress or constitutively activate the cMet protein.

[0152] In a first aspect, the present disclosure provides an ISVD that specifically binds to cMet (i.e., an anti-cMet ISVD), and heavy chain antibodies and cMet-binding molecules comprising the ISVD. In some embodiments, the anti-cMet ISVD, heavy chain antibodies, and cMet-binding molecules of the present invention bind to human cMet with moderate or high affinity. In some embodiments, the anti-cMet ISVD and cMet-binding molecules of the present invention have improved tissue penetration compared to conventional four-chain antibodies.

[0153] The ISVD specifically binding to cMet of the present invention comprises three complementarity determining regions from the N-terminus to the C-terminus, namely CDR1, CDR2 and CDR3.

[0154] In some embodiments, the immunoglobulin single variable domain (ISVD) that specifically binds to cMet of the present invention comprises

[0155] (a) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 16, 39-40, and 121-130;

[0156] (b) three CDRs in the amino acid sequence shown in SEQ ID NO: 21 or one of SEQ ID NOs: 42 and 134-136; or

[0157] (c) 3 CDRs in the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the ISVD that specifically binds to cMet of the present invention comprises a variant having a single or multiple CDRs with no more than 1 to 3 amino acid changes per CDR compared to the 3 CDRs described in one of (a) to (c) above; wherein the amino acid changes are amino acid additions, deletions, or conservative amino acid substitutions. In some embodiments, the CDRs according to the present invention are defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof, preferably according to Kabat or AbM, or a combination thereof.

[0158] In some embodiments, an ISVD of the invention that specifically binds cMet comprises a

[0159] (a) CDR1 set forth in SEQ ID NO: 18 or a variant of CDR1 set forth in SEQ ID NO: 18 with no more than one amino acid change (e.g., CDR1 set forth in any one of SEQ ID NOs: 41 and 131-133), CDR2 set forth in SEQ ID NO: 19 or a variant of CDR2 set forth in SEQ ID NO: 19 with no more than two amino acid changes, and CDR3 set forth in SEQ ID NO: 20 or a variant of CDR3 set forth in SEQ ID NO: 20 with no more than two amino acid changes;

[0160] (b) CDR1 set forth in SEQ ID NO: 23 or a variant of CDR1 set forth in SEQ ID NO: 23 with no more than one amino acid change, CDR2 set forth in SEQ ID NO: 24 or a variant of CDR2 set forth in SEQ ID NO: 24 with no more than two amino acid changes, and CDR3 set forth in SEQ ID NO: 25 or a variant of CDR3 set forth in SEQ ID NO: 25 with no more than two amino acid changes; or

[0161] (c) CDR1 set forth in SEQ ID NO: 28 or a variant of CDR1 set forth in SEQ ID NO: 28 with no more than one amino acid change, CDR2 set forth in SEQ ID NO: 29 or a variant of CDR2 set forth in SEQ ID NO: 29 with no more than two amino acid changes, and CDR3 set forth in SEQ ID NO: 30 or a variant of CDR3 set forth in SEQ ID NO: 30 with no more than two amino acid changes;

[0162] The amino acid changes are amino acid additions, deletions or conservative amino acid substitutions.

[0163] In some embodiments, the present disclosure provides an immunoglobulin single variable domain (ISVD) that specifically binds cMet, wherein the ISVD comprises:

[0164] (a) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 18, 19 and 20, respectively;

[0165] (b) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 41, 19 and 20, respectively;

[0166] (c) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 131, 19 and 20, respectively;

[0167] (d) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 132, 19 and 20, respectively;

[0168] (e) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 133, 19 and 20, respectively; or

[0169] (f) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 23, 24 and 25, respectively. In some embodiments, the ISVD defined in (b) or (f) above is preferred.

[0170] In some embodiments, the ISVD that specifically binds to cMet of the present invention comprises or consists of a VHH. In some embodiments, the ISVD that specifically binds to cMet of the present invention comprises or consists of the following sequence:

[0171] (a) a sequence of one of SEQ ID NOs: 16, 39-40, and 121-130, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0172] (b) a sequence of one of SEQ ID NOs: 21, 42, and 134-136, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0173] (c) a sequence of SEQ ID NO: 26, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0174] Preferably, the amino acid changes do not occur in the CDR regions. In some embodiments, the ISVD comprising the amino acid sequence of SEQ ID NO: 39, 40 or 42 is preferred.

[0175] In some embodiments, the present disclosure provides antibodies comprising an ISVD of the invention that specifically binds to cMet, particularly anti-cMet heavy chain antibodies.

[0176] In some embodiments, the present disclosure provides a binding molecule comprising an ISVD of the present invention that specifically binds to cMet. In some embodiments, the cMet binding molecule comprises or consists of an antibody selected from the group consisting of a single domain antibody, a nanobody, a VHH antibody, or a heavy chain antibody. In other embodiments, the cMet binding molecule is selected from a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0177] In some embodiments, the cMet-binding molecules of the present invention comprise at least one ISVD that specifically binds to cMet of the present invention, for example, they comprise two, three, four or more identical or different ISVDs that specifically bind to cMet of the present invention, preferably, they comprise two, three or four different ISVDs that specifically bind to cMet of the present invention. In some embodiments, the cMet-binding molecules provided by the present invention comprise two ISVDs that bind to different epitopes of cMet.

[0178] In some embodiments, the anti-cMet ISVD contained in the cMet-binding molecules of the present invention is preferably a humanized VHH domain. Compared with camelid VHHs, humanized VHHs have reduced human anti-camelid antibody responses in humans, thereby improving the safety of antibody applications.

[0179] In some embodiments, the ISVD or cMet-binding molecule of the invention that specifically binds to cMet has one or more of the following properties:

[0180] (1) Binds to human cMet with moderate or high affinity;

[0181] (2) specifically binds to cMet expressed on the cell surface;

[0182] (3) In the presence of HGF ligand, it blocks the binding of HGF ligand to cMet on the cell surface;

[0183] (4) internalized by cells expressing cMet;

[0184] (5) Cross-reactivity with human cMet and cynomolgus monkey cMet;

[0185] (6) cooperative binding mediated by different cMet bi-epitopes;

[0186] (7) Coordinated endocytosis mediated by different cMet dual epitopes.

[0187] In some embodiments, the cMet-binding molecules of the present invention are in the form of monospecific, bispecific, or multispecific antibody molecules. For example, the multispecific antibody molecule can be a trispecific antibody molecule comprising a first binding specificity for cMet and second and third binding specificities for one or more other molecules.

[0188] In some embodiments, the cMet-binding molecules of the present invention comprise a first and a second ISVD that specifically bind to the same epitope on cMet. In some embodiments, the first and second ISVDs are each an ISVD according to the present invention that specifically binds to the same epitope on cMet. In some further embodiments, the first and second ISVDs comprise: (i) a CDR1 comprising or consisting of an amino acid sequence selected from SEQ ID NO: 18 or 41, and a CDR2 and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or (ii) a CDR1, a CDR2, and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 23-25, respectively. In some further embodiments, the first and second ISVDs comprise, consist essentially of, or consist of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in one of SEQ ID NOs: 16, 39, 40, or SEQ ID NOs: 21 or 42. In some preferred embodiments, the first and second ISVDs comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NOs: 16, 39, 40, or SEQ ID NOs: 21 or 42.

[0189] In some embodiments, the cMet-binding molecules of the present invention comprise a first and a second ISVD that specifically bind to different epitopes on cMet. In some embodiments, the first and second ISVDs are each an ISVD according to the present invention that specifically binds to different epitopes on cMet. In some embodiments, the first ISVD comprises a first anti-cMet VHH domain and the second ISVD comprises a second anti-cMet VHH domain, or vice versa, wherein the first and second cMet VHH domains are different from each other. In some embodiments, the first anti-cMet VHH domain comprises: a CDR1, a CDR2, and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NOs: 23-25, respectively; and the second anti-cMet VHH domain comprises: a CDR1 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 18 or 41, and a CDR2 and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NOs: 19 and 20, respectively. In some further embodiments, the first anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16, 39 or 40. In some further embodiments, the first anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, 39, or 40. In some preferred embodiments, the first ISVD comprises the first anti-cMet VHH domain, and the second ISVD comprises the second anti-cMet VHH domain. In some embodiments, the binding molecule is an anti-cMet bi-epitopic antibody.

[0190] In some embodiments, the cMet binding molecules of the present invention can be in single-chain or multi-chain form. In some embodiments, the binding molecules further comprise a peptide linker for connecting different domains (e.g., two or more ISVDs) located on the same polypeptide chain. In other embodiments, the binding molecules further comprise an immunoglobulin Fc region.

[0191] In some embodiments, the present disclosure provides an anti-cMet bi-epitope antibody, wherein the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from N-terminus to C-terminus,

[0192] The first polypeptide chain comprises: a first ISVD that specifically binds to cMet and an immunoglobulin Fc region;

[0193] The second polypeptide chain comprises: a second ISVD that specifically binds to cMet and an immunoglobulin Fc region,

[0194] Preferably, wherein:

[0195] - the first polypeptide chain comprises a sequence of SEQ ID NO: 88, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0196] - the second polypeptide chain comprises the sequence of SEQ ID NO: 89, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto,

[0197] More preferably, the first polypeptide chain comprises or consists of the sequence of SEQ ID NO: 88; and the second polypeptide chain comprises or consists of the sequence of SEQ ID NO: 89.

[0198] In some embodiments, the cMet-binding molecules of the present invention further comprise at least one ISVD that specifically binds to EGFR, preferably at least one (eg, 1) anti-EGFR ISVD according to the present invention.

[0199] In some embodiments, the cMet-binding molecules of the present invention are linked at their N-terminus or C-terminus via one or more peptide linkers to one or more other groups, residues or moieties, wherein the one or more other groups, residues or moieties provide for increased half-life, provide for effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), compared to the corresponding cMet-binding molecules without the one or more other groups, residues or moieties linked.

[0200] In some embodiments, the one or more other groups, residues, moieties that provide increased half-life are selected from a polyethylene glycol molecule, a serum protein or fragment thereof, a moiety that can bind to a serum protein (e.g., serum albumin (such as human serum albumin)), a moiety that binds to a serum immunoglobulin (such as IgG)), or an Fc domain.

[0201] In some embodiments, the cMet-binding molecules of the present invention are linked to an ISVD that binds to human serum albumin at their N-terminus or C-terminus via one or more peptide linkers. In some embodiments, the ISVD that binds to human serum albumin comprises a CDR1 consisting of or consisting of the amino acid sequence of SEQ ID NO: 46, a CDR2 consisting of or consisting of the amino acid sequence of SEQ ID NO: 47, and a CDR3 consisting of or consisting of the amino acid sequence of SEQ ID NO: 48. In some embodiments, the ISVD that binds to human serum albumin comprises the sequence of SEQ ID NO: 45, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0202] In certain embodiments, the cMet-binding molecules provided herein are altered to increase or decrease the degree of their glycosylation. Addition or deletion of glycosylation sites to the cMet-binding molecules can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the cMet-binding molecule comprises an Fc region, the carbohydrate attached to the Fc region can be altered. In some applications, modifications to remove unwanted glycosylation sites can be useful, such as removing fucose moieties to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, galactosylation modifications can be performed to modulate complement-dependent cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the cMet-binding molecules provided herein to generate Fc region variants in order to enhance, for example, the effectiveness of the cMet-binding molecules of the present invention in treating cancer.

[0203] II. Second Aspect of the Present Disclosure: ISVDs That Specifically Bind to EGFR and EGFR-Binding Molecules Comprising Such ISVDs

[0204] Epidermal growth factor receptor (EGFR, ErbB1 or HER1) is a type I transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. EGFR is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases (RTKs), which includes HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Increased expression or kinase activity of EGFR is associated with a variety of human cancers, making EGFR an attractive target for cancer treatment.

[0205] In this specification, "EGFR" refers to EGFR from any species, and includes EGFR isoforms, fragments, variants or homologs from any species. Human EGFR is the protein shown in UniProt P00533. Alternative splicing of the mRNA encoded by the human EGFR gene produces four isoforms: isoform 1-isoform 4. EGFR is a transmembrane protein that comprises a large extracellular region, a single transmembrane domain, an intracellular juxtamembrane domain, a tyrosine kinase domain and a C-terminal regulatory region. The binding of EGFR to a ligand induces receptor dimerization and autophosphorylation of several tyrosine residues (Y992, Y1045, Y1068, Y1148 and Y1173) in the C-terminal regulatory region of EGFR. Abnormal EGFR expression / activity is associated with many diseases (e.g., cancer).

[0206] The second aspect of the present disclosure provides an ISVD (anti-EGFR ISVD) that specifically binds to EGFR and an EGFR binding molecule comprising the ISVD. In some embodiments, the anti-EGFR ISVD and EGFR binding molecules of the present invention have improved tissue permeability compared to conventional four-chain antibodies. In some embodiments, the anti-EGFR ISVD and EGFR binding molecules of the present invention bind to human EGFR with medium or low affinity. In this article, "medium affinity" refers to the binding affinity of the antibody for the target epitope, for example, as measured by surface plasmon resonance technology (SPR). D The value is equal to or higher than 1 nM, but less than 50 nM; "low affinity" means that the antibody has a binding affinity K of 100 for the target epitope. D In some embodiments, the K D Compared to "high-affinity" anti-EGFR ISVDs or EGFR-binding molecules comprising said ISVDs having a kinase inhibitory value of less than 1 nM, in particular less than 0.1 nM, the medium- or low-affinity anti-EGFR ISVDs or EGFR-binding molecules comprising said ISVDs of the present invention have at least one of the following advantages: (i) increased tumor tissue specificity; (ii) exhibit reduced on-target toxicity in normal tissues, such as skin toxicity; (iii) have an improved safety profile; and (iv) are more effective in treating cancer.

[0207] The immunoglobulin single variable domain (ISVD) that specifically binds to EGFR of the present invention comprises three complementarity determining regions from N-terminus to C-terminus, namely CDR1, CDR2 and CDR3. In some embodiments, the immunoglobulin single variable domain (ISVD) that specifically binds to EGFR of the present invention comprises

[0208] (a) three CDRs in the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 31, and one of SEQ ID NO: 94-99;

[0209] (b) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 6, 32, and 100-102; or

[0210] (c) 3 CDRs in the amino acid sequence shown in one of SEQ ID NOs: 11, 36, 84, and 105-114.

[0211] In some embodiments, the ISVD that specifically binds to EGFR of the present invention comprises a variant having a single or multiple CDRs with no more than 1 to 3 amino acid changes in each CDR compared to the 3 CDRs described in one of (a) to (c) above, wherein the amino acid changes are additions, deletions, or conservative amino acid substitutions of amino acids. In some embodiments, the CDRs according to the present invention are defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof, preferably according to Kabat or AbM, or a combination thereof.

[0212] In some embodiments, an ISVD that specifically binds to EGFR of the invention comprises a

[0213] (a) CDR1 set forth in SEQ ID NO: 3 or a variant of CDR1 set forth in SEQ ID NO: 3 with no more than one amino acid change, CDR2 set forth in SEQ ID NO: 4 or a variant of CDR2 set forth in SEQ ID NO: 4 with no more than two amino acid changes, and CDR3 set forth in SEQ ID NO: 5 or a variant of CDR3 set forth in SEQ ID NO: 5 with no more than two amino acid changes;

[0214] (b) CDR1 set forth in SEQ ID NO: 8 or a variant of CDR1 set forth in SEQ ID NO: 8 with no more than one amino acid change, CDR2 set forth in SEQ ID NO: 9 or a variant of CDR2 set forth in SEQ ID NO: 9 with no more than two amino acid changes (e.g., CDR2 set forth in SEQ ID NO: 34 or 103), and CDR3 set forth in SEQ ID NO: 10 or a variant of CDR3 set forth in SEQ ID NO: 10 with no more than two amino acid changes (e.g., CDR3 set forth in SEQ ID NO: 35 or 104); or

[0215] (c) CDR1 set forth in SEQ ID NO: 13 or a variant of CDR1 set forth in SEQ ID NO: 13 with no more than one amino acid change, CDR2 set forth in SEQ ID NO: 14 or a variant of CDR2 set forth in SEQ ID NO: 14 with no more than two amino acid changes (e.g., CDR2 set forth in SEQ ID NO: 38 or any one of SEQ ID NOs: 85, 115-120), and CDR3 set forth in SEQ ID NO: 15 or a variant of CDR3 set forth in SEQ ID NO: 15 with no more than two amino acid changes;

[0216] The amino acid changes are amino acid additions, deletions or conservative amino acid substitutions.

[0217] In some embodiments, the present invention provides an immunoglobulin single variable domain (ISVD) that specifically binds to EGFR, wherein the ISVD comprises:

[0218] (a) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 3, 4 and 5, respectively;

[0219] (b) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 8, 9 and 10, respectively;

[0220] (c) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 8, 34 and 35, respectively;

[0221] (d) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 8, 103 and 104, respectively;

[0222] (e) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 13, 14 and 15, respectively;

[0223] (f) a CDR1 comprising or consisting of SEQ ID No: 13, a CDR2 comprising or consisting of one of SEQ ID Nos: 115-120, and a CDR3 comprising or consisting of SEQ ID No: 15;

[0224] (g) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 13, 38 and 15, respectively; or

[0225] (h) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 13, 85 and 15, respectively. In some embodiments, the ISVD defined in (a), (c) or (h) above is preferred. In some embodiments, the ISVD defined in (c) is more preferred.

[0226] In some embodiments, the ISVD that specifically binds to EGFR of the present invention comprises a VHH or consists of a VHH. In some embodiments, the ISVD that specifically binds to EGFR of the present invention comprises the following sequence or consists of the following sequence:

[0227] (a) a sequence of one of SEQ ID NOs: 1, 31, and 94-99, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0228] (b) a sequence of one of SEQ ID NOs: 6, 32, and 100-102, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0229] (c) a sequence of one of SEQ ID NOs: 11, 36, 84, and 105-114, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; preferably, the amino acid changes do not occur in the CDR regions. In some embodiments, the ISVD comprising the amino acid sequence of SEQ ID NOs: 31, 32, 84, or 36 is preferred. In other embodiments, the ISVD comprising the amino acid sequence of SEQ ID NO: 32 is more preferred.

[0230] In some embodiments, the present disclosure provides antibodies comprising an ISVD of the present invention that specifically binds to EGFR, particularly anti-EGFR heavy chain antibodies.

[0231] In some embodiments, the present disclosure provides binding molecules that specifically bind to EGFR. In some embodiments, the EGFR binding molecules comprise an ISVD that specifically binds to EGFR of the present invention. In some embodiments, the EGFR binding molecules comprise or consist of an antibody selected from the group consisting of a single domain antibody, a nanobody, a VHH antibody, or a heavy chain antibody. In other embodiments, the EGFR binding molecules are monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0232] In some embodiments, the EGFR binding molecules of the invention comprise at least one ISVD of the invention that specifically binds to EGFR, for example, they comprise two, three, four or more identical or different ISVDs of the invention that specifically bind to EGFR.

[0233] In some embodiments, the ISVD contained in the EGFR binding molecule of the present invention is preferably a humanized VHH. Compared with the VHH of camelid animals, the humanized VHH has a reduced human anti-camelid antibody response to humans, thereby improving the safety of antibody applications.

[0234] In some embodiments, the ISVD or EGFR binding molecule of the invention that specifically binds to EGFR has one or more of the following properties:

[0235] (1) Binds to human EGFR with moderate or low affinity;

[0236] (2) cross-reactivity with human EGFR and cynomolgus monkey EGFR;

[0237] (3) specifically binds to EGFR expressed on the surface of tumor cells;

[0238] (4) Internalized by tumor cells expressing EGFR.

[0239] In some embodiments, the EGFR binding molecules of the present invention are in the form of bispecific or multispecific antibody molecules. The multispecific antibody molecule can be, for example, a trispecific antibody molecule comprising a first binding specificity for EGFR and a second and third binding specificity for one or more other molecules. In some embodiments, the EGFR binding molecules of the present invention further comprise at least one ISVD that specifically binds to cMet, preferably at least one or two anti-cMet ISVDs according to the present invention.

[0240] In some embodiments, the EGFR binding molecules of the present invention are linked to one or more other groups, residues or moieties at their N-terminus or C-terminus via one or more peptide linkers, wherein the one or more other groups, residues or moieties provide increased half-life, provide effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), compared to the corresponding EGFR binding molecules without the one or more other groups, residues or moieties linked.

[0241] In some embodiments, the one or more other groups, residues, moieties that provide increased half-life are selected from a polyethylene glycol molecule, a serum protein or fragment thereof, a moiety that can bind to a serum protein (e.g., serum albumin (such as human serum albumin)), a moiety that binds to a serum immunoglobulin (such as IgG)), or an Fc domain.

[0242] In some embodiments, the EGFR-binding molecules of the present invention are linked to an ISVD that binds to human serum albumin at their N-terminus or C-terminus via one or more peptide linkers. In some embodiments, the ISVD that binds to human serum albumin comprises, for example, a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 47, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 48. In some embodiments, the ISVD that binds to human serum albumin comprises the sequence of SEQ ID NO: 45, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0243] In certain embodiments, the EGFR binding molecules provided herein are modified to increase or reduce the degree of their glycosylation. The addition or deletion of the glycosylation sites of the EGFR binding molecules can be easily achieved by changing the amino acid sequence to produce or remove one or more glycosylation sites. When the EGFR binding molecules comprise an Fc region, the carbohydrate connected to the Fc region can be changed. In some applications, it can be useful to remove the modification of unwanted glycosylation sites, such as removing the fucose module to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, galactosidation modification can be performed to regulate complement-dependent cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the EGFR binding molecules provided herein to produce Fc region variants, such as to enhance the effectiveness of the EGFR binding molecules of the present invention in treating cancer.

[0244] III. Third Aspect of the Disclosure: EGFR and cMet Binding Molecules

[0245] In a third aspect, the present disclosure provides EGFR and cMet binding molecules. Preferably, the binding molecules are multispecific antibodies capable of simultaneously binding to EGFR and cMet. In some embodiments, the multispecific antibodies according to the present invention have one or more of the following properties:

[0246] (1) Bind to EGFR, such as human EGFR, with moderate or low affinity;

[0247] (2) internalized by tumor cells expressing EGFR;

[0248] (2) specifically binds to cMet, such as human cMet;

[0249] (3) In the presence of HGF ligand, it blocks the binding of HGF ligand to cMet on the cell surface;

[0250] (4) Internalization by tumor cells expressing cMet; in particular, synergistic internalization enhancement occurs when binding to different cMet epitopes;

[0251] (5) bind to tumor cells expressing both EGFR and cMet, and preferably exhibit synergistic binding activity;

[0252] (6) being internalized by tumor cells expressing both EGFR and cMet, and preferably exhibiting synergistic endocytic activity;

[0253] (7) Cross-reactivity with human EGFR and cynomolgus monkey EGFR;

[0254] (8) cross-reactivity with human cMet and cynomolgus monkey cMet; and

[0255] (9) Reduce tumor cell proliferation and metastasis.

[0256] Monoclonal antibodies have been established as anti-tumor therapeutics over the past two decades, with multiple monoclonal antibodies targeting EGFR and cMet approved or in clinical development. However, acquired resistance in tumors limits their long-term efficacy. Because multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies) can specifically bind to different antigenic epitopes, when designed to simultaneously act on the signal transduction pathways of two or more different mediators, they can help avoid acquired resistance in tumors.

[0257] In some embodiments, the multispecific antibody of the present invention is an EGFR and cMet binding molecule, comprising at least one ISVD that specifically binds to EGFR as defined in the second aspect of the disclosure (e.g., Section II herein), for example, one or two EGFR-binding ISVDs of the present invention; and further comprising at least one ISVD that binds to cMet as defined in the first aspect of the disclosure (e.g., Section I herein), for example, one or two cMet-binding ISVDs of the present invention, optionally wherein the ISVDs located on the same polypeptide chain are connected via one or more peptide linkers.

[0258] Generally, EGFR and cMet binding molecules comprising two or more ISVDs are also referred to herein as "multivalent" EGFR and cMet binding molecules. For example, a "bivalent" EGFR and cMet binding molecule can comprise one EGFR-binding ISVD and one cMet-binding ISVD, optionally linked by one peptide linker. A "trivalent" EGFR and cMet binding molecule can comprise one EGFR-binding ISVD and two cMet-binding ISVDs, optionally linked by two peptide linkers; or can comprise two EGFR-binding ISVDs and one cMet-binding ISVD, optionally linked by two peptide linkers. A "tetravalent" EGFR and cMet binding molecule can comprise two EGFR-binding ISVDs and two cMet-binding ISVDs, optionally linked by three peptide linkers; or can comprise one EGFR-binding ISVD and three cMet-binding ISVDs, optionally linked by three peptide linkers; or can comprise three EGFR-binding ISVDs and one cMet-binding ISVD, optionally linked by three peptide linkers, etc.

[0259] In a multivalent EGFR and cMet binding molecule, the two or more ISVDs may be the same or different and may be directed against the same epitope of EGFR, the same epitope of cMet, or may be directed against different epitopes of EGFR, different epitopes of cMet; or any suitable combination thereof.

[0260] In some preferred embodiments, the multispecific EGFR and cMet binding molecules of the present invention comprise at least one (preferably one) ISVD directed against EGFR and two ISVDs directed against different epitopes on cMet. In other preferred embodiments, the multispecific EGFR and cMet binding molecules of the present invention comprise at least one (preferably one) ISVD directed against EGFR and two ISVDs directed against the same epitope of cMet.

[0261] Structural forms of the multispecific antibodies of the present invention

[0262] Multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies) can be divided into many categories according to their different components and construction methods. For example, based on the basic symmetry of the left and right sides of the multispecific antibody structure, it can be divided into symmetrical structures and asymmetrical structures; based on whether the multispecific antibody has an IgG Fc region, it can be divided into antibody styles with Fc regions and antibody styles without Fc regions; based on the number of antigen binding sites in the multispecific antibody, it can be divided into bivalent, trivalent, tetravalent or more valent antibodies, etc.; based on the number of polypeptide chains constituting the multispecific antibody, it can be divided into single-chain or multi-chain forms. See, for example, Brinkmann U. and Kontermann RE, The making of bispecific antibodies, Mabs, 2017, 9 (2): 182-212. These multispecific antibody structural forms known in the art are all considered in the present invention.

[0263] Single-chain multispecific antibodies

[0264] In some embodiments, the present disclosure provides EGFR and cMet binding molecules in single-chain form (e.g., see Figure 9), wherein at least one ISVD that specifically binds to EGFR and at least one ISVD that specifically binds to cMet are located on one polypeptide chain, wherein the ISVDs are linked via a peptide linker or directly linked.

[0265] In some embodiments, the present disclosure provides a multispecific antibody in single-chain form comprising a single polypeptide chain, wherein the polypeptide chain comprises, from N-terminus to C-terminus: (ISVD A ) n1 -(ISVD B ) n2 -(HLE) n3 -(ISVD A ) n4 -(ISVD B ) n5 -(HLE) n6 , (I)

[0266] wherein n1, n2, n3, n4, n5 and n6 are independently selected from integers of 0, 1 or 2; wherein ISVD A and ISVD B Respectively represent ISVD domains that bind to antigens A and B, wherein A and B are different from each other and are independently selected from EGFR and cMet; wherein HLE represents a serum albumin binding peptide as a half-life extension domain; wherein the symbol "-" represents connection via a peptide linker or direct connection, preferably a peptide linker of 5-15 amino acids in length. Each ISVD in formula (I) AThe ISVDs may independently target the same epitope or different epitopes on the A antigen. B They can independently target the same epitope or different epitopes on the B antigen. In some cases, it is preferred that the multispecific antibody has 2 to 6 valencies (i.e., n1+n2+n4+n5=2 to 6), more preferably no more than 4 valencies (i.e., n1+n2+n4+n5=2 to 4), for example, 2 valencies, 3 valencies, or 4 valencies.

[0267] The number of anti-EGFR ISVD domains and anti-cMet ISVD domains in the multispecific antibody may be equal or unequal. The multispecific antibody may contain or not contain an HLE domain as desired. In some embodiments, the number of anti-EGFR ISVD domains is no more than 4, preferably no more than 3, for example, 1. In some embodiments, the number of anti-cMet ISVD domains is no more than 4, preferably no more than 3, for example, 2. In some embodiments, the ratio of the number of anti-EGFR ISVD domains to anti-cMet ISVD domains is 1:1 or 1:2. In some embodiments, the antibody contains 0 or 1 HLE.

[0268] In some embodiments, the present disclosure provides a multispecific antibody comprising a single polypeptide chain, wherein the polypeptide chain comprises, from N-terminus to C-terminus:

[0269] (i)ISVD A -ISVD B ;

[0270] (ii)ISVD A -ISVD B -ISVD B , where each ISVD B each targeting the same epitope or preferably different epitopes of the B antigen;

[0271] (iii)ISVD B -ISVD A -ISVD B , where each ISVD B each targeting the same epitope or preferably different epitopes of the B antigen;

[0272] wherein preferably A represents EGFR and B represents cMet. In some embodiments, the polypeptide chain of (i)-(iii) comprises an HLE located at the N-terminus or preferably the C-terminus.

[0273] In some embodiments, the HLE is an anti-HSA ISVD that binds to human serum albumin. In some embodiments, the anti-HSA ISVD comprises a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 47, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 48. In some embodiments, the ISVD that binds to human serum albumin comprises the sequence of SEQ ID NO: 45, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0274] In some embodiments, the present invention provides a multispecific antibody comprising a single polypeptide chain, wherein the polypeptide chain comprises:

[0275] (a) an ISVD that specifically binds to EGFR, preferably selected from the ISVD that specifically binds to EGFR according to the second aspect of the present disclosure; and (b) a first ISVD that specifically binds to cMet and a second ISVD that specifically binds to cMet, preferably each selected from the ISVD that specifically binds to cMet according to the first aspect of the present disclosure, wherein the first ISVD and the second ISVD are the same or different; preferably, the first ISVD and the second ISVD specifically bind to different epitopes of cMet. In some embodiments, the polypeptide chain further comprises an ISVD that specifically binds to HSA, preferably located at the N-terminus or C-terminus of the polypeptide chain. In some embodiments, preferably, from the N-terminus to the C-terminus, the polypeptide chain comprises: an ISVD that specifically binds to EGFR, a first peptide linker, a first ISVD that specifically binds to cMet, a second peptide linker, a second ISVD that specifically binds to cMet, and optionally a third peptide linker and an ISVD that specifically binds to HSA.

[0276] Two-chain multispecific antibodies

[0277] In some embodiments, the present disclosure provides a two-chain form of an EGFR and cMet binding molecule (e.g., see Figure 10), comprising at least one ISVD that specifically binds to EGFR and at least one ISVD that specifically binds to cMet and at least one half-life extending domain, wherein the half-life extending domain is an immunoglobulin Fc region, optionally wherein the ISVDs located on the same polypeptide chain are linked via a peptide linker or directly linked.

[0278] In some embodiments, the present disclosure provides a multispecific antibody comprising a first polypeptide chain and a second polypeptide chain, wherein

[0279] The first polypeptide chain comprises from N-terminus to C-terminus: (ISVD A ) n1 -(ISVD B ) n2 -HLE-(ISVD A ) n3 -(ISVD B ) n4， (II)

[0280] The second polypeptide chain comprises from N-terminus to C-terminus: (ISVD B ) m1 -(ISVD A ) m2 -HLE-(ISVD B ) m3 -(ISVD A ) m4， (III)

[0281] wherein n1, n2, n3 and n4 and m1, m2, m3 and m4 are independently selected from an integer of 0, 1 or 2;

[0282] ISVD A and ISVD B Respectively represent ISVD domains that bind to antigens A and B, wherein A and B are different from each other and are independently selected from EGFR and cMet; wherein HLE represents an immunoglobulin Fc region as a half-life extension domain, especially a human IgG1 or IgG4 Fc region; wherein the symbol "-" represents connection via a peptide linker or direct connection, preferably a peptide linker of 5-15 amino acids in length. Each ISVD in formula (II) and formula (III) A The ISVDs in formula (II) and (III) may target the same epitope or different epitopes on the A antigen independently of each other. B They can independently target the same epitope or different epitopes on the B antigen. In some cases, it is preferred that the multispecific antibody has 2 to 6 valencies (i.e., the sum of n1, n2, n3 and n4 and m1, m2, m3 and m4 is 2-6), more preferably, no more than 4 valencies, for example, 2, 3 or 4 valencies.

[0283] The number of anti-EGFR ISVD domains and anti-cMet ISVD domains in the multispecific antibody may be equal or unequal. In some embodiments, the number of anti-EGFR ISVD domains is no more than 4, preferably no more than 3, for example, 1. In some embodiments, the number of anti-cMet ISVD domains is no more than 4, preferably no more than 3, for example, 2. In some embodiments, the ratio of the number of anti-EGFR ISVD domains to anti-cMet ISVD domains is 1:1 or 1:2.

[0284] In some preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0285] The first polypeptide chain comprises from N-terminus to C-terminus: (ISVD A )-HLE,

[0286] The second polypeptide chain comprises from N-terminus to C-terminus: (ISVD B )-HLE;

[0287] or,

[0288] The first polypeptide chain comprises from N-terminus to C-terminus: (ISVD B )-(ISVD A )-HLE,

[0289] The second polypeptide chain comprises from N-terminus to C-terminus: (ISVD B )-HLE;

[0290] Each ISVD B each targeting the same epitope or preferably different epitopes of the B antigen;

[0291] or,

[0292] The first polypeptide chain comprises from N-terminus to C-terminus: (ISVD A )-HLE;

[0293] The second polypeptide chain comprises from N-terminus to C-terminus: (ISVD B )-(ISVD B )-HLE,

[0294] Each ISVD B each targeting the same epitope or preferably different epitopes of the B antigen;

[0295] Preferably, A represents EGFR, and B represents cMet.

[0296] Due to the dimerization of the immunoglobulin Fc region, the first and second polypeptide chains of the above-mentioned multispecific antibody can associate to form a heterodimer, thereby generating a two-chain multispecific binding molecule. Preferably, in order to promote heterodimerization of the first and second polypeptide chains, a knob-into-hole mutation can be introduced into the Fc region of the first and second polypeptide chains, such as a (T366W / T366S, L368A, Y407V) mutation or a (T366Y / Y407T) mutation. Preferably, the Fc region comprises an amino acid sequence from human IgG1 or IgG4, and more preferably, the Fc region further comprises a mutation that reduces or eliminates Fcγ receptor binding, such as a LALA mutation.

[0297] In some embodiments, the EGFR and cMet binding molecules of the present invention are in a double-chain form comprising:

[0298] (a) an ISVD that specifically binds to EGFR is located on a polypeptide chain, preferably the ISVD is selected from the ISVD that specifically binds to EGFR according to the second aspect of the present disclosure, wherein an Fc region (Fc subunit) is connected to the C-terminus of the ISVD; and

[0299] (b) a first ISVD that specifically binds to cMet and a second ISVD that specifically binds to cMet are located on another chain from the N-terminus to the C-terminus, and are preferably respectively selected from the ISVD that specifically binds to cMet according to the first aspect of the present disclosure, wherein the first ISVD and the second ISVD are the same or different; preferably, the first ISVD and the second ISVD specifically bind to different epitopes of cMet; optionally, the first ISVD and the second ISVD are connected via one or more peptide linkers,

[0300] The Fc region (Fc subunit) is connected to the C-terminus of the second ISVD.

[0301] In some specific embodiments, the EGFR and cMet binding molecules of the present invention are two-chain multispecific antibodies comprising a first polypeptide chain and a second polypeptide chain, wherein, from N-terminus to C-terminus, the first polypeptide chain comprises: an ISVD that specifically binds to EGFR and an immunoglobulin Fc region, and the second polypeptide chain comprises: a first ISVD that specifically binds to cMet, a peptide linker, a second ISVD that specifically binds to cMet, and an immunoglobulin Fc region.

[0302] Example antigenic domain combinations

[0303] In some embodiments of the multispecific antibodies according to the present invention described above, preferably, the multispecific antibodies according to the present invention comprise at least one anti-cMet ISVD according to the first aspect of the disclosure (e.g., as defined in Section I) and / or at least one anti-EGFR ISVD according to the second aspect of the disclosure (e.g., as defined in Section II).

[0304] In some further preferred embodiments, the multispecific antibodies according to the present invention comprise a first and a second ISVD that specifically bind to the same epitope on cMet. In some embodiments, the first and second ISVDs are anti-cMet ISVDs according to the first disclosed aspect that specifically bind to the same epitope on cMet. In some further embodiments, the first and second ISVDs comprise: (i) a CDR1 comprising or consisting of an amino acid sequence selected from SEQ ID NO: 18 or 41, and a CDR2 and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or (ii) a CDR1, a CDR2, and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 23-25, respectively. In some further embodiments, the first and second ISVDs comprise, consist essentially of, or consist of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in one of SEQ ID NOs: 16, 39, 40, or SEQ ID NOs: 21 or 42. In some preferred embodiments, the first and second ISVDs comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NOs: 16, 39, 40, or SEQ ID NOs: 21 or 42.

[0305] In other further preferred embodiments, the multispecific antibodies according to the present invention comprise a first and a second ISVD that specifically bind to different epitopes on cMet. In some embodiments, the first and second ISVDs are anti-cMet ISVDs according to the first aspect of the present disclosure that specifically bind to different epitopes on cMet. In some embodiments, the first ISVD comprises a first anti-cMet VHH domain and the second ISVD comprises a second anti-cMet VHH domain, or vice versa, wherein the first and second cMet VHH domains are different from each other. In some embodiments, the first anti-cMet VHH domain comprises: CDR1, CDR2, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NOs: 23-25, respectively; and the second anti-cMet VHH domain comprises: CDR1 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 18 or 41, and CDR2 and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NOs: 19 and 20, respectively. In some further embodiments, the first anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16, 39 or 40. In some further embodiments, the first anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 21 or 42, and the second anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, 39, or 40. In some preferred embodiments, the first ISVD comprises the first anti-cMet VHH domain, and the second ISVD comprises the second anti-cMet VHH domain.

[0306] In some preferred embodiments of the multispecific antibodies of the present invention comprising a first and a second ISVD that bind to the same cMet epitope or different cMet epitopes, the antibodies further comprise at least one (preferably 1) anti-EGFR ISVD according to the second aspect of the present disclosure. In some embodiments, the anti-EGFR ISVD comprises:

[0307] (i) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 3, 4 and 5, respectively;

[0308] (ii) CDR1, CDR2 and CDR3 comprising, or consisting of, the amino acid sequences of SEQ ID NOs: 8, 9 and 10, or the amino acid sequences of SEQ ID NOs: 8, 34 and 35, or the amino acid sequences of SEQ ID NOs: 8, 103 and 104, respectively; or

[0309] (iii) CDR1, CDR2, and CDR3 comprising, or consisting of, the amino acid sequences of SEQ ID NOs: 13, 14, and 15, or the amino acid sequences of SEQ ID NOs: 13, 85, and 15, or the amino acid sequences of SEQ ID NOs: 13, 38, and 15, respectively;

[0310] More preferably, the anti-EGFR ISVD:

[0311] (a) comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 1, 31, and 94-99, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;

[0312] (b) comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 6, 32, 100-102, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0313] (c) comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 11, 36, 84, and 105-114, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;

[0314] More preferably, the anti-EGFR ISVD:

[0315] (a) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 31;

[0316] (b) comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 32; or

[0317] (c) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 84.

[0318] In some embodiments, the present disclosure provides single-chain multispecific antibodies comprising an anti-EGFR ISVD in combination with a first and a second anti-cMet ISVD selected from:

[0319] (a) the anti-EGFR ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 1, 6, or 13 and the first anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 21 and the second anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 16; or

[0320] (b) the anti-EGFR ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 1, 6 or 13 and the first anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 16 and the second anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 21; wherein the CDRs are preferably according to the Kabat definition,

[0321] Preferably, the antibody comprises an anti-EGFR ISVD in combination with a first and a second anti-cMet ISVD selected from the group consisting of:

[0322] (a) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 1, 6, or 13 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 21 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 16; or

[0323] (b) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 1, 6, or 13, the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 16, and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 21. In some further embodiments of this single-chain multispecific antibody, the antibody comprises, from N-terminus to C-terminus, the anti-EGFR ISVD, a peptide linker, the first anti-cMet ISVD, a peptide linker, and the second anti-cMet ISVD.

[0324] In some embodiments, the present disclosure provides a two-chain multispecific antibody comprising an anti-EGFR ISVD in combination with a first and a second anti-cMet ISVD selected from:

[0325] (a) the anti-EGFR ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 31, 32, or 84 and the first anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 39;

[0326] (b) the anti-EGFR ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 31, 32, or 84 and the first anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 39 and the second anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 39;

[0327] (c) the anti-EGFR ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 31, 32, or 84 and the first anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 42; or

[0328] (d) the anti-EGFR ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 31, 32 or 84 and the first anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 21 and the second anti-cMet ISVD comprises the three CDRs of the amino acid sequence of SEQ ID NO: 40; wherein the CDRs are preferably according to the Kabat definition,

[0329] Preferably, the antibody comprises an anti-EGFR ISVD in combination with a first and a second anti-cMet ISVD selected from the group consisting of:

[0330] (a) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 31, 32, or 84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 39; or

[0331] (b) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 31, 32, or 84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 39 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 39;

[0332] (c) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 31, 32, or 84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 42; or

[0333] (d) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 31, 32, or 84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 21 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 40;

[0334] Still more preferably, the antibody comprises an anti-EGFR ISVD in combination with a first and a second anti-cMet ISVD selected from the group consisting of:

[0335] (a) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 31 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 39;

[0336] (b) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 32 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 39;

[0337] (c) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 21 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 40;

[0338] More preferably, the antibody comprises: an anti-EGFR ISVD comprising the amino acid sequence of SEQ ID NO:31, a first anti-cMet ISVD comprising the amino acid sequence of SEQ ID NO:42, and a second anti-cMet ISVD comprising the amino acid sequence of SEQ ID NO:39.

[0339] In some further embodiments of this two-chain multispecific antibody, the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from N-terminus to C-terminus, the first polypeptide chain comprises: an ISVD that specifically binds to EGFR and an immunoglobulin Fc region, and the second polypeptide chain comprises: a first ISVD that specifically binds to cMet, a peptide linker, a second ISVD that specifically binds to cMet, and an immunoglobulin Fc region.

[0340] Exemplary peptide linkers

[0341] In some embodiments of the multispecific antibodies of the present invention, the antibodies comprise a peptide linker. As used herein, a "peptide linker" in the binding molecules and antibodies of the present invention refers to a short amino acid sequence composed of natural amino acids. There are no particular restrictions on the length or flexibility of the peptide linker used in the binding molecules and antibodies of the present invention. The peptide linker used in the binding molecules and antibodies of the present invention can be any suitable amino acid sequence, in particular an amino acid sequence of 1 to 50, preferably 1 to 30, for example 1 to 10 amino acid residues. In some embodiments, the peptide linker consists essentially of glycine (G) and serine (S) residues, preferably comprising one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 43) motif (e.g., comprising the formula (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7 or greater), for example, GGGGSGGGGS (SEQ ID NO: 44). In some embodiments, the peptide linker used in the binding molecules and antibodies of the present invention to connect two ISVDs preferably comprises the amino acid sequence of SEQ ID NO: 44. Based on the disclosure herein, one skilled in the art will be able to determine, after limited routine experimentation, the optimal peptide linker for use in a binding molecule of the invention or an antibody of the invention.

[0342] Exemplary Immunoglobulin Fc Regions

[0343] In some embodiments of the multispecific antibodies of the present invention, the antibodies comprise an Fc region (Fc subunit). The Fc region useful in the binding molecules and antibodies of the present invention can be an Fc region derived from IgG1, IgG2, IgG3 or IgG4.

[0344] In some embodiments, the Fc region of the EGFR and cMet binding molecules of the present invention utilizes the "knobs-into-holes" technology (see, e.g., John B. B. Ridgway et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, 1996. 9(7): p. 617-21; Shane Atwell et al., Stable heterodimers form remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol, 1997. 270: p. 26-35). This technology can remodel the interface between the two chains of the EGFR and cMet binding molecules of the present invention to promote the correct association of the two chains of the EGFR and cMet binding molecules of the present invention. Generally, this technology involves introducing a "knob" at the interface of one chain and a corresponding "hole" at the interface of the other chain to be paired with it, so that the knob can be placed in the hole. The preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to be paired. The protrusion can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired.

[0345] In one embodiment, the Fc region on both chains of the EGFR and cMet binding molecules of the present invention comprises a modification of the binding affinity for an Fc receptor. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In one embodiment, the modification reduces the effector function of the EGFR and cMet binding molecules of the present invention. In a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the modification is within the Fc region of the EGFR and cMet binding molecules of the present invention, particularly within its CH2 region. In one embodiment, the EGFR and cMet binding molecules of the present invention comprise an amino acid replacement at position 329 (EU numbering) of the heavy chain. In a specific embodiment, the amino acid replacement is P329G. In one embodiment, the EGFR and cMet binding molecules of the present invention comprise amino acid replacements at positions 234 and 235 (EU numbering) of the heavy chain. In a specific embodiment, the amino acid substitutions are L234A and L235A (LALA mutations) (Armour KL et al., Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol, 1999. 29(8):2613-24). In one embodiment, the EGFR and cMet binding molecules of the present invention comprise amino acid substitutions at positions 234, 235, and 329 of the heavy chain (EU numbering). In a specific embodiment, the EGFR and cMet binding molecules of the present invention comprise amino acid substitutions L234A, L235A, and P329G (EU numbering) in the heavy chain.

[0346] In one embodiment, the Fc region used in the binding molecules and antibodies of the invention has the mutation YTE, i.e., has a combination of the mutations M252Y (Met252Tyr), S254T (Ser254Thr) and T256E (Thr256Glu) numbered according to the EU index of Kabat, to provide increased half-life.

[0347] In some embodiments, the immunoglobulin Fc region used in the two-chain multispecific antibody of the invention comprises:

[0348] An Fc chain with a Hole mutation (also referred to as a clasp chain) comprising the sequence of SEQ ID NO: 137, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0349] An Fc chain with a Knob mutation (also referred to as a knob chain) comprising the sequence of SEQ ID NO: 138, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0350] Exemplary multispecific antibodies

[0351] In some embodiments, the present invention provides a multispecific antibody comprising a first polypeptide chain and a second polypeptide chain, wherein:

[0352] - the first polypeptide chain comprises a sequence selected from SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72 or SEQ ID NO: 86, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0353] - the second polypeptide chain comprises a sequence selected from SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 87, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. Preferably, in some embodiments,

[0354] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 70, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73, 75 or 74, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0355] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73, 75 or 74, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0356] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73, 75 or 74, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0357] (iv) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:86, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:87, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0358] More preferably, in some embodiments,

[0359] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0360] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0361] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 87.

[0362] In other embodiments, the present disclosure provides multispecific antibodies comprising a single polypeptide chain, wherein the polypeptide chain comprises a sequence selected from SEQ ID NOs: 51-66, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or wherein the polypeptide chain comprises a sequence selected from SEQ ID NOs: 67-68 and 76-83, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. Preferably, in some embodiments, the polypeptide chain comprises or consists of a sequence selected from SEQ ID NOs: 51-56.

[0363] Properties of the EGFR and cMet Binding Molecules of the Invention

[0364] EGFR affinity

[0365] The EGFR and cMet binding molecules of the present invention comprise ISVDs that bind to EGFR with moderate or low affinity. EGFR is expressed at low levels in normal tissues (e.g., skin). The EGFR and cMet binding molecules of the present invention that bind to EGFR with moderate or low affinity exhibit reduced on-target toxicity in normal tissues while still being able to target tumors that express high levels of EGFR, thereby resulting in an improved safety profile.

[0366] In some embodiments, the ISVD that binds to human EGFR contained in the binding molecules of the present invention can also bind to cynomolgus monkey EGFR. For example, the ISVD that binds to human EGFR can be expressed with a similar K D Binds to cynomolgus monkey EGFR. In this article, the similar K D Refers to the two K being compared D The difference between the values ​​is no more than 10-fold, preferably no more than about 5-fold, or more preferably no more than 2-fold. The EGFR and cMet binding molecules of the present invention are able to bind to both human EGFR and cynomolgus monkey EGFR, and this cross-reactivity is advantageous because it allows the administration and safety testing of the EGFR and cMet binding molecules of the present invention in cynomolgus monkeys during preclinical development.

[0367] cMet affinity

[0368] The EGFR and cMet binding molecules of the present invention comprise an ISVD that specifically binds to cMet. In some embodiments, the EGFR and cMet binding molecules of the present invention comprise at least two ISVDs that specifically bind to different epitopes on cMet, which enables the EGFR and cMet binding molecules of the present invention to have better tumor targeting and internalization.

[0369] In some embodiments, the ISVD that binds to human cMet contained in the binding molecules of the invention can be expressed as D Alternatively, the ISVD that binds to human cMet can have an affinity of less than 80 nM, 50 nM, 20 nM, 15 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2.5 nM. D Binds to human cMet with an affinity of 1 to 20 nM, 1 to 15 nM, 1 to 10 nM, 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, 1 to 3 nM, 1 to 2.5 nM, or 2 to 2.5 nM.

[0370] In some embodiments, an ISVD that binds to human cMet contained in a binding molecule of the invention may also bind to cynomolgus monkey cMet. For example, an ISVD that binds to human cMet may be expressed with a similar K D Binding to cynomolgus monkey cMet. The EGFR and cMet binding molecules of the present invention are able to bind to both human cMet and cynomolgus monkey cMet. This cross-reactivity is advantageous because it allows dosing and safety testing of the EGFR and cMet binding molecules of the present invention in cynomolgus monkeys during preclinical development.

[0371] Simultaneously and specifically binds to EGFR and cMet

[0372] The EGFR and cMet binding molecules of the present invention can bind to both EGFR and cMet targets simultaneously. Many tumors are known to co-express both EGFR and cMet, and therefore, the EGFR and cMet binding molecules of the present invention having the ability to bind to both EGFR and cMet simultaneously are expected to be advantageous.

[0373] Internalization

[0374] The EGFR and cMet binding molecules of the present invention can mediate efficient internalization. This is particularly useful for conjugates or couplings because it ensures that the conjugate or coupling is internalized into the cell and delivered to the lysosomes, where the antibody molecule is subsequently degraded and the drug is released into the cell to exert its cellular effect, such as cytotoxicity.

[0375] Internalization of the EGFR and cMet binding molecules of the invention by cells can be analyzed by contacting living cells with the EGFR and cMet binding molecules of the invention and detecting the EGFR and cMet binding molecules of the invention after a sufficient period of internalization. When the antibody molecule is retained on the cell surface (e.g., detected on the cell surface and / or not detected within the cell), it is determined that the antibody molecule has not been internalized by the cell. When the antibody molecule is detected within the cell (e.g., located in the cytoplasm or an organelle), it is determined that the antibody molecule has been internalized.

[0376] When compared to EGFR monospecific binding molecules or cMet monospecific binding molecules, the internalization mediated by the EGFR and cMet binding molecules of the present invention exhibits greater selectivity for tumor cells that co-express both targets, thereby minimizing its adverse effects in normal tissues that do not show significant levels of EGFR and cMet co-expression.

[0377] In vitro activity

[0378] The EGFR and cMet binding molecules of the present invention have cytotoxic activity in vitro. Cytotoxic activity can be measured using in vitro cell viability assays, such as (Promega) assay. In some embodiments, the cell is a cell that expresses both EGFR and cMet.

[0379] In some embodiments, the EGFR and cMet binding molecules of the present invention are capable of increasing the killing of cells, such as tumor cells, that express significant amounts of both EGFR and cMet compared to cells that express low levels of EGFR and / or cMet. Cells that express significant amounts of both EGFR and cMet can be identified by measuring the relative EGFR and cMet receptor density on the cell surface.

[0380] In vivo activity

[0381] The EGFR and cMet binding molecules of the present invention can inhibit the development or progression of cancer in vivo. In some embodiments, the cancer can be a cancer that expresses both or one of EGFR and cMet. Cancer cells can express one or both of EGFR and cMet on the cell surface. The cancer can be, for example, selected from lung cancer (e.g., squamous cell lung carcinoma, lung adenocarcinoma, non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), colon cancer, and pharyngeal squamous cell carcinoma.

[0382] IV. Fourth Aspect of the Disclosure: Nucleic Acids, Vectors, Hosts, and Production Methods

[0383] In a fourth aspect, the present disclosure provides nucleic acids encoding, vectors, host cells and production methods of the ISVDs, binding molecules and polypeptides according to the first to third aspects of the present disclosure.

[0384] In one embodiment, the present disclosure provides a method for preparing an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody of the present invention, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody, and optionally isolating the ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody. In a certain embodiment, the method further comprises recovering the ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody from the host cell (or host cell culture medium).

[0385] To recombinantly produce an ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody of the present invention, nucleic acid encoding the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody of the present invention is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to nucleic acids encoding the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody of the present invention.

[0386] The ISVDs, EGFR-binding molecules, cMet-binding molecules, or multispecific antibodies of the present invention, prepared as described herein, can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. The purity of the ISVDs, EGFR-binding molecules, cMet-binding molecules, or multispecific antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0387] V. Fifth Aspect of the Disclosure: Immunofusions, Immunoconjugates, and Antibody Drug Conjugates (ADCs)

[0388] In a fifth aspect, the present disclosure provides immunofusions, immunoconjugates, and antibody drug conjugates comprising the ISVD, antibodies, and antigen binding molecules according to the first to third aspects of the present disclosure.

[0389] Immunofusions and immunoconjugates

[0390] In one embodiment, the present disclosure provides immunofusions or immunoconjugates produced by fusing or conjugating the ISVDs, antibodies, and antigen binding molecules according to the first to third aspects of the present disclosure to a heterologous molecule.

[0391] In one embodiment, in an immunofusion, an antigen-binding molecule (e.g., an antibody) of the invention is linked to a heterologous peptide or polypeptide molecule directly or via an amino acid linker. Examples of heterologous peptides or polypeptides include, but are not limited to, proteins or polypeptides that confer another functional activity to the fusion, or tag peptides that facilitate purification or detection of the immunofusion.

[0392] In one embodiment, in immunoconjugates, the antigen binding molecules of the present invention (such as antibodies) are conjugated to therapeutic agents or diagnostic agents or detectable agents. In conjugates, chemical linkers can be used to covalently link the different entities of the conjugate. In some cases, advantageously, chemical linkers are "cleavable linkers" that are beneficial to the antigen binding molecule polypeptides and released after being delivered to the target site. For example, acid labile linkers, peptidase-sensitive linkers, light labile linkers, dimethyl linkers or disulfide-containing linkers can be used.

[0393] In embodiments where conjugated to a therapeutic agent, therapeutic agents suitable for use in the conjugate include, but are not limited to, cytotoxins (eg, cytostatic or cell-killing agents), drugs, or radioisotopes.

[0394] In embodiments conjugated to diagnostic or detectable agents, such conjugates can be used as part of a clinical test method (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 diagnosis and detection can be achieved by coupling the antibody to a detectable agent, 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 metals and non-radioactive paramagnetic metal ions used in various positron emission tomography techniques.

[0395] In some embodiments, therapeutic agents suitable for use in the conjugates include, but are not limited to, drugs (e.g., anti-tumor drugs); in other embodiments, diagnostic agents suitable for use in the conjugates include, but are not limited to, radioactive diagnostic agents, fluorescent substances, or luminescent substances.

[0396] Antibody-drug conjugates (ADCs)

[0397] In some preferred embodiments, the present disclosure provides antibody-drug conjugates (ADCs).

[0398] In some embodiments, the present disclosure provides an antibody drug conjugate (ADC) having formula (I0) or a pharmaceutically acceptable salt or solvate thereof: Ab-(LD) p (I0)

[0399] in:

[0400] Ab is a binding molecule of the present invention, such as an antibody, such as the above-mentioned antibody that specifically binds to EGFR and / or cMet, or a fragment thereof (e.g., an antigen-binding fragment);

[0401] L is a linker;

[0402] D is a drug, such as an anti-tumor compound; and

[0403] p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, Ab is an EGFR and cMet binding molecule according to the third aspect of the present disclosure, in particular a multispecific antibody according to the third aspect of the present disclosure. In some particularly preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0404] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0405] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0406] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 87.

[0407] It will be appreciated that the -LD moiety can be covalently linked to the Ab by any means known in the art. In some embodiments, the -LD moiety is covalently linked to the Ab via a sulfur (S) atom from the Ab, i.e., the -LD moiety and the Ab are linked via an -S- bond. In some embodiments, the sulfur atom originates from the opening of an interchain disulfide bond of the Ab. In some embodiments, the sulfur atom originates from a (engineered or natural) cysteine ​​in the Ab.

[0408] In some embodiments, the present invention provides an antibody-drug conjugate (ADC) having formula (I) or a pharmaceutically acceptable salt or solvate thereof: Ab-(SLD) p (I)

[0409] in:

[0410] Ab is a binding molecule of the present invention, such as an antibody, such as the above-mentioned antibody that specifically binds to EGFR and / or cMet, or a fragment thereof (e.g., an antigen-binding fragment);

[0411] L is a linker;

[0412] D is a drug, such as an anti-tumor compound; and

[0413] p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12. It should be understood that S in formula (I) is sulfur from the antibody Ab. In some embodiments, Ab is an EGFR and cMet binding molecule according to the third aspect of the present disclosure, in particular a multispecific antibody according to the third aspect of the present disclosure. In some particularly preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0414] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0415] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0416] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 87.

[0417] It is understood that p refers to the number of -LD groups attached to Ab in the antibody-drug conjugate molecule of formula (I0) or (I), which can also be called DAR.

[0418] In some embodiments, D in formula (I0) or (I) of the present invention can be any anti-tumor compound, without particular limitation, as long as it has an anti-tumor effect and a structural portion that can be connected to a linker. The anti-tumor compound can be a pharmaceutically active compound that has an effect on tumors. For anti-tumor compounds, part or all of the linker is preferably cleavable within tumor cells, freeing the anti-tumor compound portion, thereby exhibiting an anti-tumor effect.

[0419] In some embodiments, the anti-tumor compound can be, for example, a cytotoxic agent, such as a camptothecin or an auristatin.

[0420] In some embodiments, D has the structure shown in Formula (D-1a) or Formula (D-1b):

[0421] where R 1a Selected from H and C1-C6 alkyl;

[0422] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and-SR 5a ;

[0423] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0424] R 4a and R 5a Independently selected from H and C1-C4 alkyl;

[0425] In some embodiments, R 1a H; R 2a is a C1-C6 alkyl group; R 3a is halogen, preferably -F; R 4a is a C1-C4 alkyl group, preferably an ethyl group;

[0426] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 8b Each independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0427] R 6b and R 7bEach independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;

[0428] R 9b Selected from C 1-8 Alkyl and COOH; preferably C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and

[0429] R 10b Selected from OH and H.

[0430] In some embodiments, R 1b 、R 4b and R 8b Each independently selected from C 1-2 Alkyl; preferably methyl;

[0431] R 2b 、R 3b and R 5b Each independently selected from C 3-4 alkyl;

[0432] R 6b and R 7b Each independently selected from C 1-2 alkoxy; and

[0433] R 9b Selected from C 1-4 Alkyl and R 10b OH; or R 9b is COOH and R 10b For H.

[0434] It will 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 structural formula of D indicates that the valence bond is connected to L.

[0435] In some embodiments, D has the structure shown in Formula (D-2a) or Formula (D-2b):

[0436] where R 1a 、R 2a 、R 3a and R 4a as defined above; or

[0437] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b 、R 6b 、R 7b 、R 8b、R 9b and R 10b As defined above.

[0438] In some embodiments, D has the structure shown in formula (D-3a) or (D-3b):

[0439] In some embodiments, D has the structure shown in formula (D-4a) or (D-4b):

[0440] In some embodiments, in the ADC of the present disclosure, the drug is Exatecan, Dxd, SN-38, monomethyl auristatin E (MMAE) or MMAF. The structural formula is shown below:

[0441] In some embodiments, -L- has the structure: -Z-L1-L2-L3-

[0442] in

[0443] Z is selected from wherein m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0444] L1 is selected from the group consisting of: wherein n1 and m1 are each independently an integer selected from 0-20, for example an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0445] L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and

[0446] L3 is selected from: wherein X is selected from -NH-, -O- and -S-; R 1c Each independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkoxy, halogen, nitro and cyano; Su are each 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

[0447] Among them, Z is connected to S on Ab, and L3 is connected to D.

[0448] In some embodiments, -L- has the structure: -Z-L1-L2-L3-

[0449] in

[0450] Z is selected from wherein m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0451] L1 is selected from the group consisting of: wherein n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0452] L2 is a peptide residue consisting of 2-8 amino acids; and

[0453] L3 is selected from: where R 1c is selected from: H and C1-C6 alkyl; n2 is 1, 2, 3 or 4; and n3 and n4 are independently 1, 2, 3, 4, 5 or 6.

[0454] It should be understood that in the above -Z-L1-L2-L3-, Z is connected to Ab, for example, connected to S on Ab, and L3 is connected to D.

[0455] In some embodiments, Z is selected from wherein m is 1, 2, 3, 4, 5, 6, 7 or 8.

[0456] In some embodiments, Z is selected from

[0457] In some embodiments, L1 is selected from absent, wherein n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0458] In some embodiments, L1 is selected from absent,

[0459] In some embodiments, L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6, or 7 amino acids. In some embodiments, L2 is an amino acid residue. In some embodiments, L2 is a peptide residue consisting of 2, 3, 4, 5, 6, or 7 amino acids.

[0460] In some embodiments, the amino acid residue or amino acid is preferably an L-amino acid. In addition to α-amino acids, the amino acid residue or amino acid may be an amino acid residue or amino acid with a structure such as β-alanine, ε-aminocaproic acid, or γ-aminobutyric acid, or may be a non-natural amino acid, such as an N-methylated amino acid.

[0461] In some embodiments, the amino acid residues or amino acids are each independently selected from valine (Val), alanine (Ala), glycine (Gly), 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). In some embodiments, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit), phenylalanine (Phe), lysine (Lys), glutamic acid (Glu) and glutamine (Gln). In some embodiments, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit) and glutamic acid (Glu).

[0462] In some embodiments, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-.

[0463] In some embodiments, L2 is selected from -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, and -Gly-Gly-Phe-Gly-. In some embodiments, L2 is -Gly-. In some embodiments, L2 is selected from -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, and -Glu-Val-Cit-.

[0464] It should be understood that L2 is linked to L1 or Z via the amino group of the left amino acid and to L3 via the carbonyl group of the right amino acid, which is consistent with the following explanation.

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

[0466] where R 1c Each independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkoxy, halogen, nitro and cyano; Su are each independently selected from 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.

[0467] In some embodiments, L3 is selected from:

[0468] wherein the variables are as defined herein.

[0469] In some embodiments, L3 is selected from:

[0470] wherein the variables are as defined herein.

[0471] In some embodiments, L3 is selected from: wherein the variables are as defined herein.

[0472] In some embodiments, Su is selected from the group consisting of xylose, arabinose, xyluronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid, and mannuronic acid.

[0473] In some embodiments, Su is selected from

[0474] In some embodiments, Su are each independently:

[0475] In some embodiments, Su are each independently

[0476] In some embodiments, Su are each independently In some embodiments, L3 is selected from:

[0477] In some embodiments, L3 is selected from

[0478] In some embodiments, L3 is selected from:

[0479] It should be understood that L3 is connected to L2 via the amino group on the left and to D via the carbonyl group on the right, which is consistent with the following explanation.

[0480] In some embodiments, -Z-L1-L2-L3- is each independently selected from the following structures:

[0481] wherein m is each independently an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0482] n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 6 or 8; and

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

[0484] In some embodiments, -Z-L1-L2-L3- are each independently selected from the following structures

[0485] wherein n1 is independently an integer selected from 0-8, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 8.

[0486] It should be understood that, unless otherwise specified and not contradictory to the context, for the ADCs of the present invention, the left-side bond of the divalent group shown herein is connected to Ab or a group near the end of Ab, and the right-side bond of the divalent group is connected to D or a group near the end of D. For example, when L2 is When , the amino group on the left is connected to L1, and the carbonyl group on the right is connected to L3;

[0487] In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, or 2-4, or 2-6.

[0488] In some embodiments, the antibody-drug conjugate is selected from

[0489] Wherein Ab is a binding molecule of the present invention, such as a multispecific antibody, preferably V-17-Fc, V-20-Fc, V-23-Fc and V-26-Fc; p is as defined above, for example, p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Preferably, the antibody-drug conjugate has an average DAR of, for example, 2-10, 6-10, 4-8, 7-9 or 2-4 or 2-6. In some embodiments, the multispecific antibody is a multispecific antibody according to the third aspect of the present disclosure. In some particularly preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0490] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0491] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0492] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 87.

[0493] It should be understood that the S atom attached to the Ab in the above ADC is derived from the antibody Ab. Ab, under the action of a reducing agent such as TCEP, breaks disulfide bonds (e.g., interchain disulfide bonds) to generate sulfhydryl groups -SH, which are then attached to the terminal functional group of the linker, such as a maleimide moiety. In some embodiments, the S atom attached to the Ab is derived from a cysteine ​​residue in the Ab.

[0494] It should be noted that the above-mentioned and other technical solutions of the present disclosure and one or more features therein can be arbitrarily combined to constitute technical solutions not directly described in this document, and these technical solutions not directly described are also included in the scope of disclosure of this application.

[0495] Preparation of ADC molecules of the present invention

[0496] Another aspect of the present invention provides a method for preparing an ADC using the antibody of the present invention. "ADC" in the present invention is defined as an antibody coupled to a biologically and / or pharmaceutically active active substance (D, also referred to as a payload) via a linker (L). The method comprises coupling an antibody of the present invention (Ab) to one or more active substances D via one or more linkers (L) (e.g., as defined herein). Preferably, the linker-active substance is site-specifically coupled to the antibody.

[0497] In some embodiments, the method includes preparing an Ab for ADC, comprising culturing a host cell comprising a nucleic acid encoding the Ab (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid, as provided above, under conditions suitable for expression of the Ab or its chains, and optionally recovering the Ab from the host cell (or host cell culture medium).

[0498] In some embodiments, the method comprises the steps of:

[0499] (a) adding the antibody Ab to a buffer solution, adding a reducing agent, and then incubating;

[0500] (b) adding a linker-payload to the reaction solution in step (a) for coupling to obtain a crude product; and

[0501] (c) optionally purifying the crude product to obtain the antibody drug conjugate of the present invention;

[0502] wherein Ab is as defined above.

[0503] It should be understood that the linker-payload reacts with Ab to provide the -LD portion in the compound of Formula I. In the case where -LD is clearly defined, the structure of the linker-payload can be determined based on the prior art.

[0504] In some embodiments, the buffer solution in step a) is a PBS buffer, preferably, having a pH of 5.0-9.0, such as 6.0-8.0.

[0505] In some embodiments, the reducing agent of step a) is TCEP.

[0506] In some embodiments, the linker-payload has the following structure: Z'-L1-L2-L3-D, wherein L1, L2, L3, and D are as defined above, and Z' is m is as defined above, for example, 1, 2, 3, 4, 5, 6, 7 or 8.

[0507] In some embodiments, Z' is selected from

[0508] In some embodiments, for the synthesis of wherein Z is The method for producing an ADC further comprises an additional hydrolysis step to open the maleimide ring.

[0509] In some embodiments, the steps are performed under the specific reaction conditions disclosed in the Examples.

[0510] It should be noted that embodiments in which the ranges or specific values ​​of the specific reaction conditions disclosed in the examples are varied by 100%, 80%, 60%, 40%, 20% or 10% are also contemplated by the present invention.

[0511] VI. Sixth Aspect of the Disclosure: Pharmaceutical Compositions and Pharmaceutical Formulations, Combination Products, and Kits

[0512] In some embodiments, the present disclosure provides a composition comprising an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC as described herein, preferably a pharmaceutical composition or pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition comprises an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC of the present invention, and a combination of one or more other therapeutic agents (e.g., chemotherapeutic drugs, tumor vaccines, antibodies that bind to other specific antigens on tumor cells, other antibodies that deplete tumor cells).

[0513] In some embodiments, the composition of the present invention is a pharmaceutical composition or pharmaceutical preparation, which contains suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art. As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.

[0514] The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient, as required for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. When used to treat cancer, such active ingredients include, but are not limited to, anticancer agents and chemotherapeutic agents; when used to treat infectious diseases, such active ingredients include, but are not limited to, antiviral agents and antibiotics. The active ingredients are suitably combined in amounts effective for the intended use.

[0515] In some embodiments, the present disclosure also provides combination products comprising at least one ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC of the present invention, or further comprising one or more other anti-tumor agents.

[0516] In some embodiments, two or more components of the combination product may be co-administered to a subject sequentially, separately, or simultaneously.

[0517] In some embodiments, the present disclosure also provides kits comprising the ISVD, EGFR binding molecule, cMet binding molecule, multispecific antibody, ADC, pharmaceutical composition or combination product of the invention, and optionally a package insert directing administration.

[0518] In some embodiments, the present disclosure also provides pharmaceutical products comprising the ISVD, EGFR binding molecule, cMet binding molecule, multispecific antibody, ADC, pharmaceutical composition, combination product of the present invention, optionally further comprising a package insert directing administration.

[0519] VII. Seventh Aspect of the Disclosure: Uses and Methods

[0520] The ISVDs, EGFR-binding molecules, cMet-binding molecules, or multispecific antibodies comprising the same disclosed herein have in vitro and in vivo diagnostic and therapeutic uses. For example, these molecules can be administered to cultured cells in vitro or ex vivo or to subjects, e.g., human subjects, to treat and / or diagnose diseases associated with EGFR antigen and / or cMet, such as cancer.

[0521] In some embodiments, the present disclosure provides a diagnostic method for detecting the presence of relevant EGFR and / or cMet antigens in a biological sample, such as serum, semen, or urine, or a tissue biopsy sample (e.g., from a hyperproliferative or cancerous lesion) in vitro or in vivo. The diagnostic method comprises: (i) contacting the sample (and optionally a control sample) with an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody as described herein or administering the ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody to a subject under conditions that allow the interaction to occur and (ii) detecting the formation of a complex between the ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody and the sample (and optionally a control sample). The formation of a complex indicates the presence of the relevant antigen and can indicate the suitability or need for a treatment described herein.

[0522] In some embodiments, the relevant antigen is detected before treatment, for example, before the initiation of treatment or before a treatment after a treatment interval. Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR techniques (e.g., RT-PCR), or in vivo imaging techniques. Generally, the ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody used in in vivo and in vitro detection methods is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound conjugates. Suitable detectable substances include various biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances, paramagnetic (e.g., nuclear magnetic resonance active) substances, and radioactive substances.

[0523] In some embodiments, the level and / or distribution of the relevant antigen is determined in vivo, e.g., non-invasively (e.g., by detecting a detectably labeled ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody of the invention using a suitable imaging technique (e.g., positron emission tomography (PET) scan). In one embodiment, for example, by detecting a detectably labeled ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody of the invention using a PET agent (e.g., 18 The levels and / or distribution of the relevant antigens are determined in vivo using an ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody of the invention detectably labeled with F-fluorodeoxyglucose (FDG).

[0524] In one embodiment, the present invention provides a diagnostic kit comprising an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody described herein and instructions for use.

[0525] In some embodiments, the present disclosure relates to the use of an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC of the present invention to treat cancer in vivo and inhibit the growth or metastasis of a tumor expressing EGFR and / or cMet in a subject, thereby inhibiting or reducing the growth or metastasis of the cancer. The EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC of the present invention can be used alone. Alternatively, the ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC of the present invention can be administered in combination with other cancer therapeutics / preventives. When the ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC of the present invention is administered in combination with one or more other drugs, the combination can be administered in any order or simultaneously.

[0526] Thus, in one embodiment, the present invention provides a method of treating cancer, comprising administering to a subject a therapeutically effective amount of an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC described herein. In another embodiment, the present invention provides a method of preventing the emergence of cancer drug resistance in a subject, comprising administering to a subject a therapeutically effective amount of an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC described herein.

[0527] In some embodiments, cancers treated with an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody include, but are not limited to, cancers expressing EGFR and / or cMet, e.g., lung cancer (e.g., lung squamous cell carcinoma, lung adenocarcinoma, non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), colon cancer, or pharyngeal squamous cell carcinoma.

[0528] The following examples are described to assist understanding of the present invention. The examples are not intended to, and should not be interpreted in any way as, limiting the scope of protection of the present invention.

[0529] Example

[0530] Example 1 Preparation and Purification of Reference Antibodies and Reference Antibody-Drug Conjugates

[0531] The reference antibody, JNJ-61186372, is a bispecific antibody targeting EGFR and cMET, consisting of an anti-EGFR half-antibody and an anti-cMET half-antibody. JNJ-61186372 is based on the sequences and preparation methods of SEQ ID NOs: 199-202 in patent WO2014081954A1.

[0532] The reference benchmark antibody ABT-700 is a monospecific IgG antibody targeting cMet, which was prepared based on the sequence of SEQ ID NO:86-87 in patent CN109562189B and the method described therein.

[0533] The reference benchmark antibody RAA22 / B09-57 (also referred to herein as BMK-AZD, or AZD Ab) is a bispecific antibody targeting EGFR and cMet, composed of an anti-EGFR half antibody and an anti-cMET half antibody, and is prepared based on the SEQ ID NO:59-62 sequence in patent application No. US2023 / 0183358A1 and the method described therein.

[0534] The reference benchmark antibody-drug conjugate ABBV399 is a monospecific antibody (ABT-700) targeting cMet coupled to an MMAE molecule, and can be prepared based on the SEQ ID NO:86-87 sequence and conjugation method in patent CN109562189B.

[0535] The reference benchmark antibody-drug conjugate AZD9592 (also referred to herein as AZD ADC) is a bispecific antibody (RAA22 / B09-57) targeting EGFR and cMet coupled to a camptothecin molecule, and can be prepared based on the SEQ ID NO:59-62 sequence and conjugation method in patent application number US2023 / 0183358A1.

[0536] Example 2 Generation of ISVDs that specifically bind to EGFR and cMet, respectively

[0537] 2.1 Alpaca Immunity

[0538] Recombinant his-tagged human EGFR ECD protein (Aikang Biotech) and recombinant his-tagged human cMet ECD protein (Aikang Biotech) were used as target antigens for alpaca immunization. The immunization process was carried out as follows.

[0539] Two healthy adult alpacas (Aikangde Biotechnology) were selected and 0.5 mg of the target antigen (recombinant his-tagged human EGFR ECD protein or recombinant his-tagged human cMet ECD protein) was mixed with Gerbu adjuvant (GERBU biochemicals GmbH) in a 1:1 ratio. The alpacas were immunized by multiple subcutaneous injections into the cervical lymph nodes. The first immunization was 500 μg, and subsequent immunizations were 250 μg, for a total of 3-4 doses, with an immunization interval of 18-21 days. Seven days after the second and third immunizations, 5 ml of jugular vein blood was collected from the alpacas for ELISA serum titer testing. The results showed that after three rounds of immunization, the blood collection and library construction standards were successfully met, and blood collection and library construction were arranged.

[0540] 2.2 Construction and selection of phage display libraries

[0541] As described in Example 2.1, after the alpacas were immunized for the third time with either EGFR or cMet, 100 ml of jugular vein blood was collected from each alpaca and peripheral blood mononuclear cells (PBMCs) were extracted by centrifugation. Total RNA was extracted from the PBMCs, and cDNA was generated by reverse transcription using the PrimeScript Reverse Transcription Kit (Takara) using RNA as a template. Using the cDNA as a template, a first round of PCR amplification produced nucleic acid fragments of conventional IgG (VH) and pure heavy chain IgG lacking the CH1 domain (containing VHH as an ISVD). These two types of nucleic acids were separated on an agarose gel, and the nucleic acid encoding VHH was extracted and purified, followed by a second round of PCR amplification to obtain a nucleic acid fragment containing only the VHH gene fragment. The extracted and purified VHH gene fragment was inserted into a phage display vector and electroporated into competent Escherichia coli cells, and the bacterial solution was frozen at -80°C.

[0542] Escherichia coli ER2738 cells were revived and inoculated into 100 ml of 2YT-A medium (Shanghai Bioengineering Co., Ltd.). Helper phage (New England Biolabs) was added for infection. The cells were resuspended in 2×YT-AK medium (Shanghai Bioengineering Co., Ltd.) and cultured overnight at 37°C at 200 rpm. The culture supernatant was collected and recombinant phage was prepared using PEG / NaCl precipitation. Anti-EGFR and anti-cMet phage display libraries were prepared, respectively. Biotin-human EGFR protein and biotin-human cMet protein were used to enrich and pan these anti-EGFR and anti-cMet phage display libraries for subsequent selection of positive clones by ELISA.

[0543] 2.3 ELISA positive clone selection and sequencing

[0544] The expression supernatant of the E. coli ER2738 clone obtained by enrichment and panning in Example 2.2 was tested using an ELISA binding assay. The test was performed as follows.

[0545] Take the antigen (human EGFR-his antigen or human cMet-his antigen) and dilute it to 2μg / mL with PBS buffer, coat the 96-well ELISA plate, and incubate at 4°C overnight. Wash the antigen-coated plate 5 times with PBST, add PBST blocking solution containing 5% skim milk, and block at room temperature for 1 hour. Wash 6 times with PBST, add the expression supernatant of Escherichia coli ER2738 obtained in Example 2.2, and incubate at 37°C for 1 hour. Wash 6 times with PBST, add PBS-diluted anti-M13-HRP secondary antibody (Aikand Biotechnology (Suzhou) Co., Ltd.), and shake at 37°C for 45 minutes. Wash 5 times with PBST, add TMB colorimetric solution, and color for 5-15 minutes in the dark. Then add the stop solution. Read the plate with an enzyme reader and measure the OD 450nm -OD 650nm Absorbance value. Select bacterial clones with a read value greater than 1 and send them for Sanger sequencing. Select ER2738 bacterial single clones containing the corresponding positive VHH sequence, add glycerol, and freeze in a -80℃ freezer.

[0546] 2.4 Production of VHH-HIS and VHH-FC

[0547] Positive anti-EGFR VHH sequences and positive anti-cMET VHH sequences were obtained by PCR from anti-EGFR and anti-cMET positive clones, respectively. Tags (either a 6xHis tag or an Fc tag (hIgG1 isotype)) were added to their C-termini. These sequences were then inserted into the pcDNA3.4 expression vector and transiently transfected into HEK-293F cells (hereinafter referred to as "293F cells"), and the supernatants were collected. The 6xHis-tagged protein was initially purified using a Ni column and further purified using an ion exchange column; the hFc fusion protein was purified using a Protein A column. The purity of the VHH antibodies was determined by SDS-PAGE and SEC-HPLC. This yielded two tagged VHHs for anti-EGFR: anti-EGFR VHH-his and anti-EGFR VHH-Fc; and two tagged VHHs for anti-cMet: anti-cMet VHH-his and anti-cMet VHH-Fc.

[0548] 2.5 FACS detection of antibody binding to target cells

[0549] The binding of antibodies expressed in the supernatant of transiently transfected HEK-293F cells to target cells was detected by FACS.

[0550] Specifically, 100 μL of the anti-EGFR supernatant obtained by transiently transfecting HEK-293F cells in Example 2.4 and 100 μL of the anti-cMet supernatant were collected as test samples. Binding of the anti-EGFR supernatant to target cells (CHO-S-EGFR cells or CHO-S cells) was examined by FACS, and binding of the anti-cMet supernatant to target cells (CHO-S-cMet cells or CHO-S cells) was examined by FACS. CHO-S-EGFR cells is the abbreviation for the CHO-S engineered cell line expressing human EGFR. CHO-S-cMet cells is the abbreviation for the CHO-S engineered cell line expressing human cMet.

[0551] FACS assay was performed as follows. 5Cells were plated at a density of 100 cells / well in a 96-well plate and centrifuged at 300 g for 5 minutes at 4°C. The supernatant to be tested (100 μl / well) was added and incubated for one hour. After the addition of a secondary antibody, PE-anti-human IgG (Invitrogen, Cat#: 12-4998-82), and incubation at 4°C for half an hour, the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (Life Technologies) and analyzed using FlowJo. The results showed that all 23 anti-EGFR VHHs screened bound to the target CHO-S-EGFR cells to varying degrees. All 18 anti-cMet VHHs screened bound to the target CHO-S-cMet cells to varying degrees. The anti-EGFR and anti-cMet supernatants showed little binding to CHO-S cells.

[0552] Example 3 In vitro biological activity detection of each VHH as ISVD

[0553] 3.1 FACS detection of VHH binding to cells

[0554] The binding of the Fc-tagged anti-EGFR VHH and Fc-tagged anti-cMet VHH prepared in Example 2 to target cells was detected by FACS. In the anti-EGFR VHH assay, the target cells used were EGFR-expressing tumor cells NCI-H1975 (a human lung adenocarcinoma cell line); in the anti-cMet VHH assay, the target cells used were cMet-expressing tumor cells EBC-1 (a human lung squamous cell carcinoma cell line). FACS analysis was performed as follows.

[0555] The target cells were 1.5×10 5 Cells were plated at a density of 100 cells / well in a 96-well plate and centrifuged at 300 g for 5 minutes at 4°C. Anti-EGFR VHH-Fc (375 nM, 3-fold serial dilution) or anti-cMet VHH-Fc (50 nM, 4-fold serial dilution) was added, resuspended, and incubated at 4°C for 1 hour. Secondary antibody, PE-anti-human IgG (eBioscience, cat#: 12-4998-82), was added and incubated at 4°C for half an hour. The MFI of the cells was measured using a flow cytometer (Life Technologies).

[0556] The results of anti-EGFR VHH-Fc assays are shown in Figure 1. Anti-EGFR VHH candidates V-n5B10, V-n9B8, and V-n10A1 bound to target cells in a dose-dependent manner, with binding activity lower than that of the control antibody. For convenience, the anti-EGFR VHHs V-n5B10, V-n9B8, and V-n10A1 will be referred to as 5B10, 9B8, and 10A1, respectively, for the sake of description.

[0557] The results of anti-cMet VHH-Fc assays are shown in Figure 2. V-n7A12, V-n9A2, and V-n9A10, candidate anti-cMet VHHs, bound to target cells in a dose-dependent manner. For ease of description, the anti-cMet VHHs V-n7A12, V-n9A2, and V-n9A10 are sometimes referred to as 7A12, 9A2, and 9A10, respectively, throughout this article.

[0558] 3.2 Determination of the blocking effect of anti-cMet VHH-Fc on the binding of the ligand HGF to the target cell EBC-1

[0559] The target cells EBC-1 cells were cultured at a rate of 1.5 × 10 5 Cells were plated at a density of 100 cells / well in a 96-well plate and centrifuged at 300 g for 5 minutes at 4°C. A 50 μL ligand, HGF-His (1 μg / mL) (Beijing Biopsies Technology Co., Ltd.), was added and incubated for 1 hour. Then, 50 μL of anti-cMet VHH-Fc V-n7A12, V-n9A2, or V-n9A10 (100 nM, 5-fold serial dilutions) was added, mixed, and incubated for 1 hour. Secondary antibody, iF647-anti-his (GenScript), was added and incubated for half an hour at 4°C. The MFI of the cells was measured using a flow cytometer (Life Technologies).

[0560] As shown in Figure 3, V-n7A12, V-n9A2, and V-n9A10 blocked the binding of EBC-1 cells to the ligand HGF, with MFI values ​​at the highest concentrations being 40,640, 82,172, and 77,193, respectively. The MFI ratios at the highest to lowest concentrations were 2.22, 1.12, and 1.14, respectively. These results demonstrate that the candidate molecule V-n7A12 blocked the binding of EBC-1 cells to the ligand HGF in a dose-dependent manner. V-n9A2 and V-n9A10 weakly blocked the binding of EBC-1 cells to the ligand HGF.

[0561] 3.3 Epitope identification

[0562] Surface Plasmon Resonance (SPR) technology was used to detect whether the anti-cMet VHH-Fc antibodies V-n7A12 and V-n9A2 bind to the same epitope as the antigen cMet.

[0563] The V-n9A2 antibody was immobilized on a CM5 sensor chip (Cytiva). Human cMet antigen (300 nM) was injected onto the sensor chip at a flow rate of 50 μL / min, with a 200-second binding phase. The anti-cMet antibody V-n7A12 was then injected onto the sensor chip at a flow rate of 50 μL / min, with a 120-second binding phase.

[0564] The results are shown in Figure 4. V-n9A2 binds to the human cMet antigen and does not affect the binding of V-n7A12 to the human cMet antigen. This indicates that the anti-cMet antibodies V-n7A12 and V-n9A2 bind to different epitopes on the cMet antigen.

[0565] 3.4 FACS detection of cell internalization of each VHH-Fc

[0566] The internalization ability of tumor target cells to the anti-EGFR VHH-Fc and anti-cMet VHH-Fc prepared in Example 2 was detected by FACS.

[0567] Prepare tumor target cells NCI-H1975 and EBC-1, with the number of cells per well being 1-1.5×10 5 Cells were plated in 96-well plates. Anti-EGFR VHH-Fc (125 nM, 3-fold dilution) or anti-cMet VHH-Fc (50 nM, 4-fold dilution) prepared in Example 2 were added, incubated at 4°C for 30 minutes, and the supernatant containing each VHH-Fc was removed by centrifugation. The cells were divided equally into two groups and incubated at 4°C and 37°C for 4 hours, respectively. Upon completion of the incubation, ice-cold PBS was immediately added to terminate the endocytosis experiment. A secondary antibody, PE-anti-human IgG (eBioscience, Cat. No. 12-4998-82), was added and incubated at 4°C for 30 minutes. The MFI of the cells was measured using a flow cytometer (Life Technologies).

[0568] The internalization level of cell surface-bound antibody was calculated using the following formula:

[0569] Endocytosis = MFI of sample incubated at 4°C - MFI of sample incubated at 37°C.

[0570] Internalization rate (%) = 100% - (MFI of the sample incubated at 37°C / MFI of the sample incubated at 4°C) × 100%.

[0571] The results of endocytosis of candidate anti-EGFR antibody molecules in tumor target cells NCI-H1975 are shown in Figure 5A , which shows the MFI of samples incubated at 4°C and 37°C at the tested antibody concentrations. The results of endocytosis of candidate anti-cMet antibody molecules in tumor target cells EBC-1 are shown in Figure 5B , which shows the endocytic MFI determined according to the above formula at the tested antibody concentrations.

[0572] 3.5 ELISA detection of cross-reactivity of each VHH-Fc with antigens from different species

[0573] Human cMet-His antigen or cynomolgus macaque cMet-His antigen was diluted to 1 μg / mL in PBS buffer and coated onto a 96-well ELISA plate overnight at 4°C. The antigen-coated plate was washed three times with PBST (300 μL / well) and then blocked with PBS containing 5% skim milk (200 μL / well). The plate was then shaken at 37°C for 2 hours. The plate was then washed three times with PBST and a 5-fold serial dilution of each anti-cMet VHH-Fc was added. The plate was then shaken at 37°C for 1 hour. The plate was then washed three times with PBST and 100 μL / well of anti-human Fc-HRP secondary antibody (Abcam, CAT# ab97225) diluted in PBS was added to each well. The plate was then shaken at 37°C for 45 minutes. The plate was then washed three times with PBST (300 μL / well). TMB color development solution was added to each well at 100 μL / well and developed for 5-10 minutes in the dark. Then add the stop solution, the volume of the stop solution is 50uL / well. Read the plate reader and measure the OD 450nm -OD 650nm The ELISA test results are shown in Figure 6 and Table 1.

[0574] Table 1. ELISA detection of binding of anti-cMet antibodies to human cMet antigen or monkey cMet antigen Note: “-” means EC cannot be fitted 50 value.

[0575] The results showed that the anti-cMet candidate molecules V-n7A12 and V-n9A2 showed cross-reactivity with human cMet antigen and cynomolgus macaque cMet antigen, and exhibited similar binding affinities for human and monkey cMet. However, the affinity of V-n9A10 for human cMet antigen was significantly different from that for monkey cMet antigen.

[0576] Example 4. VHH sequence optimization and characterization

[0577] 4.1 VHH sequence optimization

[0578] The original VHH sequence prepared in Example 2 was humanized using the "best match method". The amino acid sequences of the VHH framework region were compared and analyzed using the human germline V gene database to select the best germline sequence. Using the Kabat CDR definition, the VHH CDR sequence was used to replace the best matching human CDR sequence to generate a humanized VHH sequence. Multiple residues in the framework region were backmutated, and post-translational modification (PTM) was removed as appropriate. The optimized sequence was reverse translated and sent to Jin Weizhi (Shanghai, China) for gene synthesis. It was then constructed into a pcDNA 3.4 expression vector to express monovalent humanized VHHs with a C-terminal fusion human His tag. Thus, a humanized VHH cloned protein was obtained.

[0579] After humanization and PTM removal, the VHH sequences were analyzed for immunogenicity. If the sequence was considered high-risk, amino acid mutations were performed on the TCE (T cell epitope) epitopes to eliminate or reduce the immunogenicity risk. The mutated sequences were sent to GeneWeiZ (Shanghai, China) for gene synthesis and then constructed into the pcDNA 3.4 expression vector to express VHHs with a C-terminal His-tag fusion. This resulted in VHH clones with low immunogenicity risk.

[0580] Sequence optimization of anti-EGFR VHH

[0581] Each parent anti-EGFR antibody and its optimized sequence are shown in Tables 2A to 2C below.

[0582] Table 2A. Anti-EGFR VHH-10A1 antibody and its optimized sequence

[0583] Table 2B. Anti-EGFR VHH 9B8 antibody and its optimized sequence

[0584] Table 2C. Anti-EGFR VHH 5B10 antibody and its optimized sequence

[0585] Sequence optimization of anti-cMET VHH

[0586] Each parent anti-cMet antibody and its optimized sequence are shown in Tables 2D to 2E below.

[0587] Table 2D Anti-cMet VHH 9A2 antibody and its optimized sequence

[0588] Table 2E. Anti-cMet VHH 7A12 antibody and its optimized sequence

[0589] 4.2 Functional characterization of optimized antibodies

[0590] Using FACS binding assays, we tested the binding of the anti-EGFR VHH-His candidate antibodies V-n10A1, V-n9B8, and V-n5B10, as well as the anti-cMet VHH-His candidate antibodies V-n9A2 and V-n7A12, and their corresponding sequence-optimized variants, to target cells: MDA-MB-468 (a triple-negative breast cancer cell line), EBC-1 (a human non-small cell lung cancer cell line), MKN45 (a human gastric cancer cell line), or CHOK1 cells overexpressing EGFR or cMet. The assays were performed as follows.

[0591] 1.5×10 5 Target cells were plated in a 96-well plate at a density of 10 cells / well and centrifuged at 300 g for 5 minutes at 4°C. Test VHH-His or reference antibodies were added, resuspended, and incubated at 4°C for 1 hour. Secondary antibodies, iF647-anti-his (Genscript, 1:1000 dilution, Catalog No. A01802-100) or APC-anti-his (BioLegend, 1:200 dilution, Catalog No. 362605), were added and incubated at 4°C for half an hour or 45 minutes. The MFI of the cells was measured using a flow cytometer (Life Technologies).

[0592] 1) Sequence optimization and modification of anti-EGFR antibodies

[0593] After sequence optimization of the parental antibodies V-n10A1, V-n9B8, and V-n5B10, the binding activity of each optimized anti-EGFR antibody to target cells was detected by FACS binding assay. The results are shown in Tables 3A and 3B.

[0594] Table 3A. Binding activity of humanized anti-EGFR antibodies to target cells Note: “-” means EC cannot be fitted 50 value.

[0595] Table 3B. Binding activity of anti-EGFR antibodies to target cells after PTM removal sequence optimization Note: “-” means EC cannot be fitted 50 value.

[0596] 2) Sequence optimization and modification of anti-cMet antibodies:

[0597] After sequence optimization of the parental antibodies V-n7A12 and V-n9A2, the binding activity of each optimized anti-cMet antibody to target cells was detected by FACS binding assay. The results are shown in Tables 3C to 3F below.

[0598] Table 3C. Binding activity of each humanized anti-cMet antibody to target cells Note: “-” means EC cannot be fitted 50 value.

[0599] Table 3D. Binding activity of each anti-cMet antibody to target cells after PTM removal sequence optimization

[0600] Table 3E. Binding activity of anti-cMet antibodies after first immunogenicity removal to target cells

[0601] Table 3F. Binding activity of anti-cMet antibodies after second immunogenicity removal to target cells

[0602] 4.4 SPR detection of anti-cMet VHH and anti-EGFR VHH

[0603] Using the SPR method, the binding affinity of anti-cMet VHH and anti-EGFR VHH to the target antigen was characterized.

[0604] For monovalent antibodies with VHH_His tags, assays were performed using Method 1. 10 μg / mL of antigen (EGFR or cMet from different species) was immobilized on a CM5 chip. The antibodies to be tested (using a gradient dilution) were then injected at a flow rate of 30 μL / min. The binding time was set to 120 s and the dissociation time to 200 s. After dissociation, the cells were regenerated with 10 mM glycine (pH 2.0). The data were analyzed using a 1:1 binding model. The results are shown in Table 4A below.

[0605] For bivalent antibodies with VHH_Fc tags, detection was performed according to Method 2. Each test antibody was captured on a Protein A chip (at a concentration of 10 μg / ml). Antigen (EGFR antigen from different species or cMet antigen) was then injected at a flow rate of 30 μL / min. The binding time was set to 120 s and the dissociation time was set to 200 s. After dissociation, the cells were regenerated with 10 mM glycine (pH 2.0). The data were analyzed using a 1:1 binding model. The results are shown in Table 4B below.

[0606] Table 4A. SPR data of VHH_His-tagged monovalent anti-cMet and anti-EGFR antibodies Note: All Vn series antibodies tested in the table are monovalent VHHs with a His tag at the C-terminus and are detected using method 1; the control antibodies in the table are detected using method 2.

[0607] Table 4B: SPR data of VHH_Fc-tagged bivalent anti-EGFR antibodies: Note: All antibodies in the table are dimers of VHH with an Fc tag at the C-terminus and are detected using method 2.

[0608] Example 5. Construction and characterization of anti-cMet bi-epitope antibodies

[0609] 5.1 Construction and Preparation of Anti-cMet Bi-epitope Antibodies

[0610] The anti-cMet dual-epitope M-1 molecule of the present invention is an asymmetric double-chain form, which has a first polypeptide chain (9A2-Fc chain) shown in SEQ ID NO: 88 and a second polypeptide chain (hu7A12 b -Fc chain).

[0611] The anti-cMet single-epitope M-2 molecule of the present invention is in a symmetrical double-chain form, comprising a first polypeptide chain and a second polypeptide chain (9A2-Fc chain) as shown in SEQ ID NO: 90.

[0612] The M-3 molecule of the present invention, which is an anti-cMet single epitope, is a symmetrical double-chain form, which has a first polypeptide chain and a second polypeptide chain (hu7A12 b -Fc chain).

[0613] The first / second polypeptide chains of M-1, M-2, and M-3 were constructed into the pcDNA 3.4 expression vector (if the antibody has a symmetrical structure, one vector is generated; if the antibody has an asymmetric structure, two vectors containing the first and second polypeptide chain encoding genes are generated, respectively) and transfected into HEK293F cells. The cells were cultured for 3 days, and the culture supernatant of the transfected cells was collected and loaded onto a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibodies were eluted with acetic acid-sodium acetate solution (pH 3.5) and immediately neutralized with 2M Tris. The antibody concentration was measured using NanoDrop. Protein purity was determined using SDS-PAGE and analytical HPLC-SEC.

[0614] 5.2 FACS detection of the binding of bi-epitope antibodies to target cells

[0615] The binding of tumor target cells EBC-1 to anti-cMet molecules was detected by FACS.

[0616] Prepare tumor target cells EBC-1, with the number of cells per well being 1.5×10 5 Cells were plated in 96-well plates. Anti-cMet M-1, M-2, and M-3 (50 nM, 4-fold dilution) were added and incubated at 4°C for 1 hour. The supernatant containing each VHH-Fc was removed by centrifugation. A secondary antibody, PE-anti-human IgG (eBioscience, catalog number 12-4998-82), was added and incubated at 4°C for 30 minutes. The MFI of the cells was measured using a flow cytometer (Life Technologies).

[0617] The results are shown in FIG7 , which indicate that compared with the anti-cMet single-epitope M-2 and M-3 molecules, the tumor target cell EBC-1 has a stronger binding to the anti-cMet dual-epitope M-1 molecule, and the binding activity of the dual-epitope molecule to the cells is also better than that of the control group ABT700.

[0618] 5.3 FACS detection of cooperative endocytosis of bi-epitope antibodies

[0619] The cooperative endocytosis of the anti-cMet dual-epitope molecule M-1 was detected by FACS.

[0620] Prepare tumor target cells EBC-1, with the number of cells per well being 1.5×10 5 Cells were plated in 96-well plates. Anti-cMet M-1, M-2, and M-3 molecules (50 nM, 4-fold dilution) were added and incubated at 4°C for 30 minutes. The supernatant containing each VHH-Fc was removed by centrifugation. The cells were divided equally into two groups and incubated at 4°C and 37°C for 4 hours, respectively. After the incubation period, ice-cold PBS was immediately added to terminate the endocytosis experiment. A secondary antibody, PE-anti-human IgG (eBioscience, catalog number 12-4998-82), was then added and incubated at 4°C for 30 minutes. The MFI of the cells was measured using a flow cytometer (Life Technologies).

[0621] The internalization level of cell surface-bound antibody was calculated using the following formula:

[0622] Endocytosis = MFI of sample incubated at 4°C - MFI of sample incubated at 37°C.

[0623] As shown in the experimental results of Figure 8, the above experimental results indicate that the tumor target cell EBC-1 has a higher endocytosis of the anti-cMet dual-epitope M-1 molecule (compared with the anti-cMet single-epitope M-2 and M-3 molecules), and the anti-cMet dual-epitope M-1 molecule has a better endocytosis effect than ABT700.

[0624] Example 6 Generation and Detection of Multispecific Anti-EGFR / CMet Antibody Molecules

[0625] 6.1 Generation of Multispecific Anti-EGFR / CMet Antibody Molecules

[0626] Based on the analysis and detection results of Examples 1 to 5 above, anti-EGFR VHH and anti-cMet VHH are combined together to form single-chain or multi-chain multispecific antibodies.

[0627] single-chain multispecific antibody

[0628] Anti-EGFR VHH sequences and anti-cMet VHH sequences were combined into a single-chain antibody format. Specifically, one anti-EGFR VHH sequence (V-n5B10, V-n9B8, V-n10A1) or a humanized VHH sequence thereof was combined with two anti-cMet VHH sequences (V-n7A12, V-n9A2) or a humanized VHH sequence thereof, targeting the same or different epitopes, to form a specific anti-EGFR / cMet antibody molecule. A GGGGS peptide linker (SEQ ID NO: 43) was added to the C-terminus of the anti-EGFR VHH, followed by an anti-cMet VHH (a first anti-cMet ISVD), followed by a GGGGSGGGGS peptide linker (SEQ ID NO: 44), and then another anti-cMet VHH (a second anti-cMet ISVD). Exemplary multispecific antibody molecules formed by this combination method and their amino acid sequences are shown in Table 5.

[0629] Table 5: Exemplary single-chain multispecific antibody molecules Note: In Table 5, the anti-EGFR VHHs V-n5B10, V-n9B8, and V-n10A1 are abbreviated as 5B10, 9B8, and 10A1, respectively; the anti-cMet VHHs V-n7A12, V-n9A2, and V-n9A10 are abbreviated as 7A12, 9A2, and 9A10, respectively.

[0630] Optionally, the specific antibody molecule is linked to an ISVD that binds to human serum albumin, such as Alb8, to extend the half-life of the specific anti-EGFR / cMet antibody molecule. For example, the C-terminus of the specific antibody molecule is linked to an ISVD that binds to human serum albumin, such as Alb8 (SEQ ID NO: 45), via a peptide linker (such as GGGGS as shown in SEQ ID NO: 43).

[0631] Specifically, taking V-17 as an example, as illustrated in FIG9 , a GGGGS peptide linker (SEQ ID NO: 43) was connected to the C-terminus of V-17, followed by an anti-HSA-Alb8 (SEQ ID NO: 45), thereby forming hu10A1-hu7A12-hu9A2-anti-HSA-Alb8 (also referred to as "V-17-anti-HSA-Alb8"). Similarly, for the other specific antibody molecules described in Table 5, a GGGGS peptide linker (SEQ ID NO: 43) was connected to the C-terminus, followed by an anti-HSA-Alb8 (SEQ ID NO: 45), to form V-1-anti-HSA-Alb8, V-3-anti-HSA-Alb8, V-7-anti-HSA-Alb8, V-9-anti-HSA-Alb8, V-13-anti-HSA-Alb8, V-15-anti-HSA-Alb8, V-16-anti-HSA-Alb8 (SEQ ID NO: 67), V-17-anti-HSA-Alb8 (SEQ ID NO: 68), V-18-anti-HSA-Alb8 (SEQ ID NO: 76), V-19-anti-HSA-Alb8 (SEQ ID NO: 77), V-20-anti-HSA-Alb8 (SEQ ID NO: 78), respectively. NO:78), V-21-anti-HSA-Alb8 (SEQ ID NO:79), V-22-anti-HSA-Alb8 (SEQ ID NO:80), V-23-anti-HSA-Alb8 (SEQ ID NO:81), V-24-anti-HSA-Alb8 (SEQ ID NO:82), V-25-anti-HSA-Alb8 (SEQ ID NO:83).

[0632] The single-chain, multispecific anti-EGFR / cMet antibody described in the present disclosure was constructed into the pcDNA 3.4 expression vector. It was transfected into HEK293F cells, cultured for 3 days, and the culture supernatant of the transfected cells was collected and loaded into Cytiva PrismA filler (Cytiva) for purification. The antibody was eluted with acetic acid-sodium acetate solution (pH 3.5) and immediately neutralized with 2M Tris. The antibody concentration was measured using a NanoDrop. The protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[0633] Multi-chain multispecific antibodies

[0634] The anti-EGFR VHH sequence and the anti-cMet VHH sequence of the same or different epitopes are respectively combined into polypeptide chains containing Fc subunits, and the polypeptide chains can further associate to form homologous or heterologous dimers to generate two-chain multispecific EGFR and cMet binding molecules.

[0635] Figure 10 shows an exemplary multi-chain multispecific antibody structure. Specifically, the anti-EGFR VHH sequence is combined with an Fc subunit to form a polypeptide chain (anti-EGFR-Fc), and optionally the Fc subunit contains a LALA mutation and / or a Knob-into-hole structure. The prepared polypeptide chain includes the sequence shown in Table 6A:

[0636] Table 6A: Exemplary sequences of anti-EGFR-Fc

[0637] The anti-cMet VHH sequence is combined with an Fc subunit to form a polypeptide chain (anti-cMet1-anti-cMet2-Fc). Specifically, the C-terminus of the first anti-cMet VHH sequence is linked to the N-terminus of the same or different second anti-cMet VHH sequence via a peptide linker (e.g., GGGGSGGGGS as shown in SEQ ID NO: 42). The C-terminus of the second anti-cMet VHH is then combined with the Fc subunit via a hinge region to form a polypeptide chain. Optionally, the Fc subunit contains a LALA mutation and / or a knob-into-hole structure. The prepared polypeptide chain comprises the sequence shown in Table 6B:

[0638] Table 6B: Exemplary sequences of anti-cMet1-anti-cMet2-Fc

[0639] Any of the above-mentioned anti-EGFR-Fc polypeptide chains can be combined with any of the anti-cMet1-anti-cMet2-Fc polypeptide chains to form a dimer, for example, to synthesize the double-chain multispecific anti-EGFR / cMet antibodies shown in Table 7:

[0640] Table 7: Examples of multispecific anti-EGFR / cMet antibodies in two-chain format

[0641] Polypeptide chains 1 and 2 of the double-chain multispecific anti-EGFR / cMet antibody described herein were separately constructed into the pcDNA 3.4 expression vector and transfected into HEK293F cells. The cells were cultured for 3 days, and the culture supernatant of the transfected cells was collected and loaded onto a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody was eluted with acetic acid-sodium acetate solution (pH 3.5) and immediately neutralized with 2M Tris. The antibody concentration was measured using a NanoDrop. The protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[0642] 6.2 FACS BINDING Detection

[0643] The binding of the above trispecific anti-EGFR / cMet antibody molecules to target cells EBC-1 and NCI-H1975 cells (cell bank of Chinese Academy of Sciences) was detected by FACS method.

[0644] Prepare target cells separately, with 1.5×10 cells per well. 5 Cells were plated onto a 96-well plate. Each of the trispecific antibodies prepared in Example 6.1 (250 nM, 3-fold dilution) was added and incubated at 4°C for 1 hour. The plates were then centrifuged at 300 g for 4 minutes and the supernatant removed. The plates were washed twice with FACS buffer (1% BSA). A secondary antibody, PE-anti-human IgG (eBioscience, catalog number 12-4998-82), was added and incubated at 4°C for 1 hour. The plates were then washed twice more with centrifugation. Finally, the cells were resuspended in FACS buffer (1% BSA) and the MFI of the cells was measured using a flow cytometer (Life Technologies).

[0645] As shown in FIG11 , the trispecific antibodies V-17-Fc, V-20-Fc, V-23-Fc and V-26-Fc were able to bind to EBC-1 and NCI-H1975 cells.

[0646] 6.3 FACS endocytosis detection

[0647] The internalization ability of the target cells EBC-1 and NCI-H1975 cells (Cell Bank of Chinese Academy of Sciences) to the above-mentioned trispecific anti-EGFR / cMet antibody molecules was detected by FACS method.

[0648] Prepare target cells separately, with 3×10 cells per well. 5 Cells were plated on a 96-well plate. Each trispecific antibody prepared in Example 6.1 (250 nM, 3-fold dilution) was added and incubated at 4°C for 30 minutes. The supernatant containing the trispecific antibodies was removed by centrifugation. The cells were divided equally into two groups and incubated at 4°C and 37°C for 4 hours, respectively. After the incubation period, ice-cold PBS was immediately added to terminate the endocytosis experiment. After adding the secondary antibody and incubating at 4°C for 30 minutes, the MFI of the cells was measured using a flow cytometer (Life Technologies). For VHH-Fc molecules, the secondary antibody used was PE-anti-human IgG (eBioscience, Catalog No.: 12-4998-82); for VHH molecules with a his tag, the secondary antibody used was APC-anti-his (BioLegend, Catalog No.: 362605).

[0649] The internalization level of cell surface-bound antibody was calculated using the following formula:

[0650] Endocytosis = MFI of samples incubated at 4°C − MFI of samples incubated at 37°C;

[0651] The results of endocytosis of trispecific antibody candidate molecules V-1, V-3, V-7, V-9, V-13, V-15, V-17-Fc, V-20-Fc, V-23-Fc and V-26-Fc by target cells MDA-MB-468, EBC-1 and NCI-H1975 are shown in Figures 12 and 13.

[0652] As shown in Figures 12 and 13, target cells were able to internalize trispecific anti-EGFR / cMet antibody molecules V-1, V-3, V-7, V-9, V-13, V-15, V-17-Fc, V-20-Fc, V-23-Fc and V-26-Fc.

[0653] 6.4 SPR Detection of Affinity of Trispecific Antibodies to Human and Cynomolgus Monkey Antigens

[0654] 6.4.1 SPR Detection of Affinity of Trispecific Antibodies for Human and Cynomolgus Monkey EGFR Antigens

[0655] The affinity of trispecific anti-EGFR / cMet antibody molecules to human and cynomolgus macaque EGFR (Beijing Sino Biological Technology Co., Ltd.) was detected by Cytiva using SPR method.

[0656] Human EGFR-his (ECD, Met1-Ser645) and cynomolgus monkey EGFR-his (ECD, Met1-Ser645) (Beijing Sino-Bio Scientific Co., Ltd.) amino-coupled antigens (20 μg / mL) were immobilized on a GLM sensor chip (Bio-Rad). Proportionally diluted trispecific anti-EGFR / cMet antibodies from Example 6.1 were injected onto the sensor chip at a flow rate of 100 μL / min. The association phase lasted 100 seconds, followed by a dissociation phase of 180 seconds. After each dissociation phase, the buffer was regenerated with 10 mM glycine (pH 1.5). The sensorgrams of the blank surface and buffer channel were subtracted from the sensorgrams tested. Langmuir analysis of the experimental data was performed using a 1:1 binding model. The affinity of the candidate molecules for human and cynomolgus monkey EGFR is shown in Table 8.

[0657] The results showed that the candidate molecules showed similar binding and dissociation rates to human EGFR antigen and cynomolgus monkey EGFR antigen, and these trispecific anti-EGFR / cMet antibody molecules cross-reacted with cynomolgus monkey EGFR.

[0658] Table 8. Affinity of trispecific antibodies for human and cynomolgus monkey EGFR antigens detected by SPR

[0659] 6.4.2 SPR Detection of Affinity of Trispecific Antibodies for Human and Cynomolgus Monkey cMet Antigens

[0660] Amino-coupled antigens human cMet-his (ECD, Met1-Thr932) and cynomolgus monkey cMet-his (ECD, Met1-Thr932) (Beijing Sino-Bio Technologies Co., Ltd.) (20 μg / mL) were immobilized on a GLM sensor chip (Bio-Rad). The trispecific anti-EGFR / cMet antibody molecule of Example 6.1 (500 nM) diluted proportionally was injected onto the sensor chip at a flow rate of 30 μL / min. The association phase was 100 seconds, followed by a dissociation phase of 180 seconds. After each dissociation phase, 10 mM glycine (pH 1.5) was used to regenerate the buffer. The sensorgrams of the blank surface and buffer channel were subtracted from the sensorgrams tested. The experimental data were analyzed using a 1:1 binding model. The affinity results of the candidate molecules to human cMet and cynomolgus monkey cMet are shown in Table 9.

[0661] The results showed that the candidate molecules displayed similar binding and dissociation rates to human cMet antigen and cynomolgus monkey cMet antigen, and these trispecific anti-EGFR / cMet antibody molecules cross-reacted with cynomolgus monkey cMet.

[0662] Table 9. Affinity of trispecific antibodies for human and cynomolgus monkey cMet antigens detected by SPR

[0663] 6.5 Determination of the blocking effect of trispecific anti-EGFR / cMet antibody molecules on the binding of the ligand HGF to the target cell EBC-1

[0664] The target cells EBC-1 cells were cultured at a rate of 1.5 × 10 5 Cells were plated in a 96-well plate at a density of 10 cells / well and centrifuged at 300 g for 5 minutes at 4°C. Trispecific anti-EGFR / cMet antibodies (200 nM, 4-fold serial dilutions) were added and incubated with the cells for 30 minutes. The ligand HGF-His (2 μg / mL) (Beijing Sino Biological Science and Technology Co., Ltd.) was then added and incubated at 4°C for 1 hour. Secondary antibodies, iF647-anti-His (GenScript), were added and incubated at 4°C for half an hour. The MFI of the cells was measured using a flow cytometer (Life Technologies).

[0665] The results showed that, as shown in FIG14 , the trispecific anti-EGFR / cMet antibody of the present invention can block the binding of EBC-1 cells to the ligand HGF, and the blocking activity is better than that of ABT700 and BMK-AZD.

[0666] 6.6 FACS detection of the coordinated endocytosis of trispecific antibodies in target cells

[0667] The M-4 molecule serves as a control molecule, which is an asymmetric two-chain Fc form, having a first polypeptide chain (hu9A2-hu7A12-Fc) shown in SEQ ID NO: 73 and a second polypeptide chain (anti-Covid-19 chain) shown in SEQ ID NO: 92, wherein the anti-Covid-19 chain does not bind to the target cell EGFR or cMET.

[0668] The M-5 molecule serves as a control molecule, which is an asymmetric two-chain Fc form, having a first polypeptide chain (anti-Covid-19 chain) shown in SEQ ID NO: 93 and a second polypeptide chain (hu5B10-Fc) shown in SEQ ID NO: 71, wherein the anti-Covid-19 chain does not bind to the target cell EGFR or cMET.

[0669] The M-6 molecule serves as a control molecule, which is an asymmetric two-chain Fc form, having a first polypeptide chain (anti-Covid-19 chain) shown in SEQ ID NO: 93 and a second polypeptide chain (hu9B8-Fc) shown in SEQ ID NO: 72, wherein the anti-Covid-19 chain does not bind to the target cell EGFR or cMET.

[0670] The M-7 molecule serves as a control molecule, which is an asymmetric two-chain Fc form, having a first polypeptide chain (anti-Covid-19 chain) shown in SEQ ID NO: 93 and a second polypeptide chain (hu10A1-Fc) shown in SEQ ID NO: 70, wherein the anti-Covid-19 chain does not bind to the target cell EGFR or cMET.

[0671] The first and second polypeptide chains of the above-mentioned M-4, M-5, M-6, and M-7 molecules were constructed into the pcDNA 3.4 expression vector (if the antibody has a symmetrical structure, one vector is generated; if the antibody has an asymmetric structure, two vectors containing the genes encoding the first and second polypeptide chains are generated respectively) and transfected into HEK293F cells. The cells were cultured for 3 days, and the culture supernatant of the transfected cells was collected and loaded onto a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibodies were eluted with acetic acid-sodium acetate solution (pH 3.5) and immediately neutralized with 2M Tris. The antibody concentration was measured using a NanoDrop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC.

[0672] The enhanced internalization ability of the trispecific anti-EGFR / cMet antibody by tumor target cell EBC-1 was detected by FACS.

[0673] Prepare tumor target cells EBC-1, with 3 × 10 cells per well. 5 Cells were plated in 96-well plates. Trispecific anti-EGFR / cMet antibodies V-20-Fc and V-23-Fc, anti-cMet M-4, and anti-EGFR M-5, M-6, and M-7 (40 nM, 5-fold dilution) were added and incubated at 4°C for 30 minutes. The supernatant containing each VHH-Fc was removed by centrifugation. The cells were divided equally into two groups and incubated at 4°C and 37°C for 4 hours, respectively. Upon completion of the incubation, the endocytosis experiment was terminated by immediately adding ice-cold PBS. After incubation at 4°C for 30 minutes with the addition of a secondary antibody, PE-anti-human IgG (eBioscience, catalog number 12-4998-82), the cells were then incubated for 30 minutes at 4°C. The MFI of the cells was determined using a flow cytometer (Life Technologies).

[0674] The internalization level of cell surface-bound antibody was calculated using the following formula:

[0675] Endocytosis = MFI of sample incubated at 4°C - MFI of sample incubated at 37°C.

[0676] As shown in FIG15 , the tumor target cell EBC-1 has a higher internalization of the trispecific antibody molecules V-20-Fc and V-23-Fc (compared to the anti-cMet M-4, and the anti-EGFR M-5, M-6, and M-7 molecules).

[0677] 6.7 FACS detection of synergistic binding of trispecific antibodies to target cells

[0678] NCI-H1975 cells were grown at a density of 1.5 × 10 cells per well. 5 Cells were plated onto a 96-well plate. A trispecific antibody of the invention (200 nM, 5-fold dilution) was added and incubated at 4°C for 1 hour. The cells were then centrifuged at 300 g for 4 minutes and the supernatant removed. The plates were washed twice with FACS buffer (1% BSA). A secondary antibody, PE-anti-human IgG (eBioscience, catalog number 12-4998-82), was added and incubated at 4°C for 1 hour. The plates were then washed twice more with centrifugation. Finally, the cells were resuspended in FACS buffer (1% BSA) and the MFI of the cells was measured using a flow cytometer (Life Technologies).

[0679] As shown in FIG16 , V-23-Fc has a stronger binding ability than its corresponding control antibodies (M4 and M6), indicating that the two targets have a synergistic binding effect.

[0680] Example 7. Preparation, characterization and experiments of antibody-drug conjugates (ADCs)

[0681] The linker-payloads used in the Examples of the present invention are known in the art and / or commercially available, or can be prepared as described herein. When the drawn structure is inconsistent with the actual situation, it should be allowed to modify or correct the structure according to the actual situation.

[0682] General Synthesis Method A

[0683] 5.45 mg / ml of the antibody of the present invention in 20 mM His-hac, 150 mM NaCl, pH 5.5 was placed in an Eppendorf tube. 6 molar equivalents of TCEP (Tris (2-carboxyethyl) phosphine hydrochloride, 5 mM concentration) were added to the antibody buffer (TCEP:antibody = 6:1). The Eppendorf tube containing the reaction mixture was placed on a shaker (x500 rpm) and reacted at 37°C for 2 hours. An additional 6 molar equivalents of TCEP (5 mM) were added to the mixture. The reaction mixture was placed on a shaker (500 rpm) and shaken at 37°C for another 2 hours. Then, ultrafiltration (MWCO 30 kd) was performed to remove TCEP, and the buffer was supplemented with 20 mM His-hac buffer. 6 molar equivalents of linker-payload (5 mg / ml in DMA) were added dropwise to the fully reduced antibody, while the DMA concentration was kept below 20% (v / v). The reaction was maintained on a shaker at room temperature for 2 hours (linker-payload:antibody = 6:1). Conversion was monitored by HIC-HLPC and purification was performed when conversion was complete. The reaction mixture was transferred to an ultrafiltration tube (MWCO 30 kd) and the sample was centrifuged at 10,000 rpm for 5 minutes to half the solution volume. The volume was then replenished with His-HAc buffer (containing 10% DMA). The flow-through was discarded. The washing step was repeated 10 times. The solution was then replaced with 10 mM His-HAc pH = 5.0, and the remaining solution was transferred and adjusted to the appropriate concentration.

[0684] When using the alternative method, Pro A, for purification (Pro A product labeled 40 g / L capacity), wash with 10 CV of 10 mM His-Hac before use, then load the sample onto a gravity column packed with Pro A resin. Wash with at least 10 CV of 10 mM His-Hac buffer containing 10% DMSO and 50 CV of 10 mM His-Hac buffer without DMSO to completely remove free payload. Elute with 50 mM acetic acid at pH 3 and immediately neutralize with 2 M Tris buffer (pH 12.0) to a pH of 5.5 to 6.0. Combine the conjugate solutions in Eppendorf tubes. Measure the concentration (Nanodrop, A280). Use 10 mM His-Hac (pH 6) as a blank. If the final concentration is lower than expected, concentrate to the desired concentration using centrifugal ultrafiltration (MWCO 30 kd ultrafiltration membrane).

[0685] Purity was determined by HIC and SEC-HPLC methods. Free linker-payload was determined by RP-HPLC method.

[0686] General methods and / or parameters for determining or detecting ADCs

[0687] Size exclusion chromatography (SEC) method (for total ADC detection)

[0688] SEC-HPLC method parameters

[0689] Reversed-phase HPLC (RP HPLC) method (for free drug detection)

[0690] RP HPLC method parameters

[0691] Elute according to the table below

[0692] ◆HIC-HPLC method (for free antibody and DAR distribution detection)

[0693] HIC-HPLC conditions.

[0694] Elute according to the table below

[0695] 7.1 Preparation and Characterization of ADC Molecules

[0696] a) Preparation of ADC molecules coupled with Mal-PEG8-VA-PAB-Exatecan

[0697] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It is understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10.

[0698] According to the above-mentioned synthesis method A, antibody V-26-Fc and linker-payload Mal-PEG8-VA-PAB-Exatecan (CAS No.: 2679821-39-5; MedChemExpress, HY-147271) were used to prepare V-26-Fc-VA-Exd with MW 96.37, average Dar 4.0, yield 63%, and purity 91.73%.

[0699] According to the preparation method of V-26-Fc-VA-Exd, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-VA-Exd, V-20-Fc-VA-Exd, and V-23-Fc-VA-Exd.

[0700] b) Preparation of ADC molecules coupled with MC-VC-PAB-MMAE

[0701] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It is understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10.

[0702] According to the above-mentioned synthesis method A, V-26-Fc-VC-MMAE was prepared using antibody V-26-Fc and linker-payload MC-VC-PAB-MMAE (CAS No.: 646502-53-6; MedChemExpress, HY-15575).

[0703] According to the V-26-Fc-VC-MMAE preparation method, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-VC-MMAE, V-20-Fc-VC-MMAE, and V-23-Fc-VC-MMAE.

[0704] c) Preparation of ADC molecules coupled with Mal-PEG8-VC-PAB-MMAE

[0705] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It is understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10.

[0706] According to the above-mentioned synthesis method A, V-26-Fc-PEG-VC-MMAE was prepared using antibody V-26-Fc and linker-payload Mal-PEG8-VC-PAB-MMAE (CAS No.: 2353409-69-3; MedChemExpress, HY-141156).

[0707] According to the V-26-Fc-PEG-VC-MMAE preparation method, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-PEG-VC-MMAE, V-20-Fc-PEG-VC-MMAE, and V-23-Fc-PEG-VC-MMAE.

[0708] d) Preparation of ADC molecules coupled with Mal-PEG8-EVC-PAB-MMAE

[0709] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It is understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10.

[0710] According to the above-mentioned synthesis method A, V-26-Fc-PEG-EVC-MMAE was prepared using antibody V-26-Fc and linker-payload Mal-PEG8-EVC-PAB-MMAE (prepared according to the preparation method of this application).

[0711] According to the V-26-Fc-PEG-EVC-MMAE preparation method, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-PEG-EVC-MMAE, V-20-Fc-PEG-EVC-MMAE, and V-23-Fc-PEG-EVC-MMAE.

[0712] e) Preparation of ADC molecules coupled with MC-EVC-PAB-MMAE

[0713] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It is understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10.

[0714] According to the above-mentioned synthesis method A, V-26-Fc-EVC-MMAE was prepared using antibody V-26-Fc and linker-payload MC-EVC-PAB-MMAE (CAS No.: 2873452-49-2; MedChemExpress, HY-154915).

[0715] According to the V-26-Fc-EVC-MMAE preparation method, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-EVC-MMAE, V-20-Fc-EVC-MMAE, and V-23-Fc-EVC-MMAE.

[0716] f) Preparation of ADC molecules coupled to Mal-Gly-Exatecan-D-glucuronic acid:

[0717] Ab is the antibody V-23-Fc prepared in the present application, and p is mainly 4. It is understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10.

[0718] According to the above-mentioned synthesis method A, V-23-Fc-Gluc-Exd (or V-23-Fc-Glu-Exd) was prepared using the antibody V-23-Fc and the linker-payload Mal-Gly-Exatecan-D-glucuronic acid (CA No.: 2763252-25-9; MedChemExpress, HY-153179) with a MW of 96, an average Dar of 4.0, a yield of 50%, and a purity of 84.31%.

[0719] According to the preparation method of V-23-Fc-Gluc-Exd, the antibody V-23-Fc was replaced with V-26-Fc, V-17-Fc, and V-20-Fc, respectively, to prepare V-26-Fc-Gluc-Exd, V-17-Fc-Gluc-Exd, and V-23-Fc-Gluc-Exd.

[0720] The characterization data of the exemplary ADCs prepared in this application are as follows:

[0721] Preparation of the reference benchmark antibody-drug conjugate AZD9592

[0722] To the antibody (RAA22 / B09-57, also referred to herein as BMK-AZD or AZD Ab) solution, 15 equivalents of TCEP solution were added, mixed thoroughly, and reacted at 37°C for 2 hours. DMA and 16 equivalents of the linker-payload (Mal-PEG8-amide-Val-Ala-(4-NH2)-Exatecan) dissolved in DMA to a 10 mM stock solution were added to the reaction, resulting in a final organic solvent content of 10%. The reaction was mixed and incubated on a 22°C incubator for 1 hour, followed by quenching with N-acetylcysteine. The conjugated sample was purified using an ultrafiltration concentrator and exchanged into the final ADC storage buffer (30 mM histidine, 30 mM arginine-HCl, containing 0.02% PS 80, pH 6.8). The sample was sterilized by filtration using a 0.22 μm syringe filter.

[0723] The characterization data of the reference antibody drug conjugate involved in this application are as follows:

[0724] Preparation of Mal-PEG8-EVC-PAB-MMAE in this application

[0725] Step 1:

[0726] (1) Compound HM-2039_1 (CAS: 13726-84-6) (100 mg, 0.33 mmol, 1 eq) and HN-078A_7 (Catalog: HY-100374, CAS: 644981-35-1) (388.86 mg, 0.346, 1.05 eq) were added to DMF (6 mL), and HATU (188 mg) and triethylamine (66.7 mg) were added, and the mixture was reacted under N2 protection for 4 h.

[0727] (2) After the reaction was completed by LCMS monitoring, the reaction solution was dropped into water and extracted twice with EA (30 mL*2). The organic phases were combined and washed three times with saturated brine (30 mL*3), dried over anhydrous sodium sulfate, and concentrated to obtain 410 mg (88.29% yield) of a white solid.

[0728] Step 2:

[0729] (1) Compound HM-2039_2 (410 mg, 0.291 mmol, 1 eq) was added to DCM (4 mL), and TFA (1 mL) was added. The mixture was reacted under nitrogen protection (0-10°C) for 1 h.

[0730] (2) After the reaction was completed by LCMS monitoring, the crude product was concentrated at low temperature to remove DCM, and the crude product was passed through a reverse phase column to obtain the product: 200 mg, 54.86% yield, as a white solid.

[0731] Step 3:

[0732] (1) HM-2039_3 (0.1 g, 79.84 μmol, 1 eq) and HM-2039_4 (CAS: 1818294-46-0) (51.86 mg, 83.83 μmol, 1.05 eq) were dissolved in DMF, and the reaction mixture was stirred for 2 h.

[0733] (2) After the reaction was completed as monitored by LCMS, the reaction solution was directly prepared (TFA in water, ACN) to obtain 30 mg (23.54% yield) of the target product as a white solid with a MW of 1756.08 and a purity of 99.18%.

[0734] 1 H NMR (400MHz, DMSO) δ10.04(s,1H),8.35-8.25(m,0.28H),8.25-8.15(m,1H),8.13-8.03(m,1.5H),7.94-7.87(m,0.4H),7.76-7.70(m,1H), 7.67-7.63(m,0.4H),7.62-7.57(m,1.7H),7.38-7.25(m,7H),7.22-7 .14(m,1H),7.04(s,2H),5.99(s,1H),5.66–5.20(m,2H),5.15-4.95( s,2H),4.80–4.15(m,8H),4.10-3.90(m,2H),3.63-3.56(m,5H),3.52 -3.48(m,28H),3.28-3.24(m,4H),3.22-3.18(m,5H),3.16-3.10(m,2 H),3.10–2.84(m,9H),2.50-2.40(m,2H),2.30-2.20(m,3H),2.20-1. 69(m,10H),1.67-1.30(m,7H),1.08–0.98(m,7H),0.89–0.76(m,26H).

[0735] 7.2 FACS Binding Assays of ADCs of the Invention

[0736] The FACS method was used to detect the binding of the above-mentioned trispecific anti-EGFR / cMet antibody molecules to the target cells MDA-MB-468, EBC-1 and NCI-H1975 cells (cell bank of the Chinese Academy of Sciences) after they were coupled with toxin molecules (i.e., payloads) to form ADC molecules.

[0737] The test method is as described in Example 6.2. Prepare target cells separately, with 1.5×10 cells per well. 5 Cells were plated in a 96-well plate. Test ADCs (200 nM or 40 nM, 5-fold dilution) were added and incubated at 4°C for 1 hour. The cells were then centrifuged at 300 g for 4 minutes and the supernatant removed. The plates were washed twice with FACS buffer (1% BSA). Secondary antibody (PE-anti-human IgG (eBioscience, Cat. No. 12-4998-82)) was added and incubated at 4°C for 1 hour. The plates were then washed twice more by centrifugation. Finally, the cells were resuspended in FACS buffer (1% BSA) and the MFI of the cells was measured using a flow cytometer (Life Technologies).

[0738] As shown in Figure 17, V-26-Fc-PEG-EVC-MMAE, V-20-Fc-PEG-EVC-MMAE, and V-23-Fc-PEG-EVC-MMAE were able to bind to EBC-1 and NCI-H1975 cells. Similarly, V-23-Fc-VA-Exd and V-23-Fc-Gluc-Exd were able to bind to MDA-MB-468 and NCI-H1975 cells, with binding activity superior to that of the control groups AZD9592 and ABBV399.

[0739] 7.3 Cell Killing Assays of ADCs of the Invention

[0740] The FACS method was used to detect the killing effect of the above-mentioned trispecific anti-EGFR / cMet antibody molecules coupled with toxins to form ADC molecules on target cells MDA-MB-468, EBC-1 and NCI-H1975 cells (cell bank of the Chinese Academy of Sciences).

[0741] 100ul of cells (1x10 4) were inoculated into each well of a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. The next day, 100 μL of serially diluted V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE, and V-26-Fc-PEG-EVC-MMAE, as well as positive control ADCs ABBV399 and AZD9592, were added to the corresponding wells. Cells were cultured for another 3-5 days. Then, 20 μL of CCK8 (Shanghai Life iLab Biotech, Cat. No. AC11L054) was added to each well and incubated in a 37°C, 5% CO2 incubator until color developed. The absorbance at 450 nm was recorded using a microplate reader (Molecular Device, SpectraMax M5).

[0742] Cell viability was calculated as follows: Percent cell viability = [(As-Ab) / (Ac-Ab)] × 100. As = absorbance of the experimental well (cells, culture medium, CCK8, and ADC); Ab = absorbance of the blank well (culture medium and CCK8); Ac = absorbance of the control well (cells, culture medium, and CCK8).

[0743] The cytotoxicity results of V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE, and V-26-Fc-PEG-EVC-MMAE against EBC-1, NCI-H1975, and MDA-MB-468 are shown in Table 10A and Figure 18A below. The cytotoxicity results of V-23-Fc-VA-Exd and V-23-Fc-Gluc-Exd against EBC-1 and NCI-H1975 are shown in Table 10B and Figure 18B below. These results demonstrate that the cytotoxicity of each ADC against target cells is substantially equal to or superior to that of the positive control drugs (ABBV399 and AZD9592).

[0744] Table 10A. Killing of target cells by V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE and V-26-Fc-PEG-EVC-MMAE Note: “-” means IC cannot be fitted 50 value.

[0745] Table 10B. Killing of target cells by V-23-Fc-VA-Exd and V-23-Fc-Gluc-Exd Note: “-” means IC cannot be fitted 50 value.

[0746] 7.4 Cell Binding and Cell Killing Assays of PEGylated and / or EVC-Modified ADCs

[0747] Using V-26-Fc as a representative antibody moiety, Mal-PEG8-EVC-PAB-MMAE (V-26-Fc-PEG-EVC-MMAE), Mal-PEG8-VC-PAB-MMAE (V-26-Fc-PEG-VC-MMAE), MC-VC-PAB-MMAE (V-26-Fc-VC-MMAE), and Mal-PEG8-VA-PAB-Exatecan (V-26-Fc-VA-Exd) were conjugated. The FACS binding assay method was as described above (e.g., Example 6.2 or 7.2), and the test results are shown in Figure 19. The cell killing assay steps were as described in Section 7.3, and the test results are shown in Figure 20.

[0748] Example 8. In vivo efficacy evaluation of multispecific anti-EGFR / cMet antibody drug conjugates (ADCs)

[0749] The EBC-1, NCI-H1975, NCI-H441 (lung adenocarcinoma cells), MDA-MB-468, FADU (head and neck cancer cells), and SW48 (colon adenocarcinoma cells) CDX mouse models were used to evaluate the in vivo efficacy of trispecific anti-EGFR / cMet antibody molecules coupled to toxin molecules to form ADC molecules. Fresh culture medium was used to revive each tumor cell. Cells in the logarithmic growth phase were collected. During the cell collection process, the culture medium was removed and washed twice, and then resuspended in DPBS. The cells were then subcutaneously injected into BALB / c nu / nu mice. The inoculum size for the EBC-1, NCI-H1975, NCI-H441, MDA-MB-468, FADU, and SW48 CDX models was 3×10 6 / 100μL、3×10 6 / 100μL, 1×10 7 / 100μL, 1×10 7 / 100μL, 5×10 6 / 100μL and 5×10 6 / 100μL. When the average tumor volume of mice reaches 110-250mm 3 At the beginning of the study, the mice were randomly divided into groups according to the tumor volume. The day of grouping was defined as day 0. Each CDX mouse model was administered with a single intravenous push, and the test article was administered intravenously on D0. After the start of administration, the mice were weighed 1-2 times a week, the tumor volume was measured 1-2 times, and the animals were observed twice a day until the end of the study. The formula for calculating tumor volume is: tumor volume (cubic millimeters) = 0.5 * tumor length * tumor width 2The change in tumor size (based on the baseline) is used to reflect the tumor inhibition and to evaluate the anti-tumor efficacy of the test article. The calculation formula for the change in tumor size (based on the baseline) is as follows: Tumor volume change = (V t -V0) / V0×100%

[0750] V t : Average tumor volume of mice in the test article-administered group on day t;

[0751] V0: the average tumor volume of mice in the test article-administered group on day 0;

[0752] 8.1 In vivo efficacy of V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE, and V-26-PEG-EVC-MMAE in the CDX model

[0753] The experimental results are shown in Figure 21. V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE and V-26-Fc-PEG-EVC-MMAE all have strong tumor inhibitory effects. After intravenous injection of the drugs, the tumor volume was significantly reduced, and the efficacy was better than that of the positive control drugs ABBV399 and AZD9592.

[0754] 8.2 In vivo efficacy of V-23-Fc-VA-Exd in the CDX model

[0755] The in vivo efficacy of V-23-Fc-VA-Exd was evaluated in EBC-1, NCI-H1975, NCI-H441, and MDA-MB-468CDX mouse models. As shown in Figure 22, V-23-Fc-VA-Exd demonstrated superior in vivo tumor inhibition compared to the positive control drug AZD9592.

[0756] In the EBC-1 model, tumors were completely eliminated at high doses of V-23-Fc-VA-Exd and AZD9592 on day 54 after administration; at low doses, V-23-Fc-VA-Exd was more effective than AZD9592.

[0757] In the NCI-H1975 model, on day 29 after administration, the tumor inhibitory effect of V-23-Fc-VA-Exd at 2 mg / kg was comparable to that of AZD9592 at 4 mg / kg; at the same dose, V-23-Fc-VA-Exd was more effective than AZD9592.

[0758] In the NCI-H441 model, at 45 days after administration, at the same dose, V-23-Fc-VA-Exd was more effective than AZD9592; the tumor inhibitory effect of V-23-Fc-VA-Exd at 2 mg / kg was equivalent to that of AZD9592 at 8 mg / kg.

[0759] In the MDA-MB-468 model, tumors were completely eliminated at day 42 after administration with V-23-Fc-VA-Exd and AZD9592 at 4 mg / kg and 8 mg / kg, respectively; the tumor volume change rate of V-23-Fc-VA-Exd at 2 mg / kg was -72%.

[0760] 8.3 In vivo efficacy of V-23-Fc-Gluc-Exd in the CDX model

[0761] SW48 CDX (colorectal cancer model, EGFR ++ &cMet + ), FADU CDX (head and neck cancer model, EGFR +++ &cMet + / - ), EBC-1 CDX (non-small cell lung cancer model, EGFR ++ &cMet +++ ) and NCI-H1975 CDX (non-small cell lung cancer model, EGFR + &cMet + The in vivo efficacy of V-23-Fc-Gluc-Exd was evaluated in a 1:1 mouse model. The results, shown in Figure 23, demonstrate that V-23-Fc-Gluc-Exd exhibits superior in vivo tumor inhibition compared to the positive control drug.

[0762] In the SW48 model, FADU model, and NCI-H1975 model, V-23-Fc-Gluc-Exd was more effective than positive control drugs, including AZD9592 and ABBV399, at the same dose.

[0763] In the EBC-1 CDX model, V-23-Fc-Gluc-Exd was more effective than AZD9592 at a low dose (1 mg / kg) on ​​day 36 after administration, and tumors were completely eliminated in both groups of animals at a dose of 3 mg / kg.

[0764] While the exemplary embodiments of the present invention have been described above, it should be understood by those skilled in the art that these disclosures are merely exemplary and that various other substitutions, adaptations, and modifications may be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0765] Sequence Listing Overview:

Claims

1. An immunoglobulin single variable domain (ISVD) that specifically binds to cMet, wherein the ISVD comprises or consists of a VHH domain, wherein the VHH domain comprises (a) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 16, 39-40 and 121-130; (b) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 21, 42 and 134-136; or (c) three CDRs in the amino acid sequence shown in SEQ ID NO:26; Preferably, the VHH domain comprises: (i) a CDR1 comprising or consisting of an amino acid sequence selected from one of SEQ ID NOs: 18, 41, and 131-133, and a CDR2 and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NOs: 19 and 20, respectively; in particular, a CDR1, a CDR2, and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NOs: 41, 19, and 20, respectively; (ii) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 23-25, respectively; or (iii) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 28-30, respectively; More preferably, the VHH domain comprises: (a) a sequence of one of SEQ ID NOs: 16, 39-40, and 121-130, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (b) a sequence of one of SEQ ID NOs: 21, 42, and 134-136, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (c) a sequence of SEQ ID NO:26, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; More preferably, the VHH domain comprises: (a) the amino acid sequence shown in one of SEQ ID NOs: 16, 39-40 and 121-130; (b) the amino acid sequence shown in one of SEQ ID NOs: 21, 42 and 134-136; or (c) the amino acid sequence shown in SEQ ID NO: 26, In particular, the VHH domain comprises the amino acid sequence of SEQ ID NO: 39 or 40, or comprises the amino acid sequence of SEQ ID NO:

42.

2. An immunoglobulin single variable domain (ISVD) that specifically binds to EGFR, comprising or consisting of a VHH domain, wherein the VHH domain comprises (a) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 1, 31 and 94-99; (b) three CDRs in the amino acid sequence of one of SEQ ID NOs: 6, 32, 100-102; or (c) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 11, 36, 84 and 105-114; Preferably, the VHH domain comprises: (i) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 3-5, respectively; (ii) a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 9, 34 or 103, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 10, 35 or 104; in particular, CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NO: 8, 34 and 35, respectively; or (iii) a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising or consisting of the amino acid sequence of one of SEQ ID NOs: 14, 38, 85, 115-120, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 15; in particular, a CDR1, a CDR2 and a CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 13, 85 and 15, or SEQ ID NOs: 13, 38 and 15, respectively; More preferably, the VHH domain comprises: (a) a sequence of one of SEQ ID NOs: 1, 31, and 94-99, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (b) a sequence of one of SEQ ID NOs: 6, 32, 100-102, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (c) a sequence of one of SEQ ID NOs: 11, 36, 84, and 105-114, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; More preferably, the VHH domain comprises: (a) the amino acid sequence shown in any one of SEQ ID NOs: 1, 31 and 94-99; (b) the amino acid sequence shown in any one of SEQ ID NOs: 6, 32, 100-102; or (c) the amino acid sequence shown in one of SEQ ID NOs: 11, 36, 84 and 105-114; In particular, the VHH domain comprises the amino acid sequence of SEQ ID NO:31, or comprises the amino acid sequence of SEQ ID NO:32, or comprises the amino acid sequence of SEQ ID NO:36, or comprises the amino acid sequence of SEQ ID NO:

84.

3. A binding molecule comprising the ISVD according to claim 1 or 2.

4. A binding molecule according to claim 3, comprising or consisting of an antibody selected from the group consisting of: Single domain antibodies, nanobodies, heavy chain antibodies, monospecific antibodies or multispecific antibodies.

5. An antibody comprising at least one (eg, 1, 2, 3, 4 or more) ISVD that specifically binds to cMet according to claim 1.

6. The antibody according to claim 5, wherein the antibody further comprises at least one (eg, 1, 2, 3, 4 or more) ISVD that specifically binds to EGFR, preferably the ISVD is the ISVD according to claim 2.

7. The antibody according to any one of claims 5-6, wherein: (a) the antibody specifically binds to cMet and comprises two ISVDs that specifically bind to the same epitope on cMet; or (b) The antibody specifically binds to cMet and comprises two ISVDs that specifically bind to different epitopes on cMet.

8. An antibody according to any one of claims 5 to 7, wherein: (a) the antibody specifically binds to EGFR and cMet, and comprises at least one (preferably one) ISVD that specifically binds to EGFR and two ISVDs that specifically bind to the same epitope on cMet, or (b) The antibody specifically binds to EGFR and cMet, and comprises at least one (preferably one) ISVD that specifically binds to EGFR and two ISVDs that specifically bind to different epitopes on cMet.

9. The antibody according to any one of claims 5 to 8, wherein the antibody comprises a first and a second ISVD that specifically bind to cMet, wherein the first and the second ISVD are respectively the ISVD according to claim 1 that specifically bind to the same epitope on cMet, Preferably, the first and second ISVDs comprise: (i) a CDR1 comprising or consisting of an amino acid sequence selected from SEQ ID NO: 18 or 41, and a CDR2 and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or (ii) a CDR1, a CDR2 and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 23-25, respectively; Still more preferably, the first and second ISVDs comprise, consist essentially of, or consist of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in one of SEQ ID NOs: 16, 39, 40 or SEQ ID NOs: 21 or 42; Still more preferably, the first and second ISVDs comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 16, 39, 40 or SEQ ID NO: 21 or 42.

10. The antibody according to any one of claims 5 to 9, wherein the antibody comprises a first and a second ISVD that specifically bind to cMet, wherein the first and the second ISVD are the ISVD according to claim 1 that specifically bind to different epitopes on cMet, Preferably, wherein: The first ISVD comprises a first anti-cMet VHH domain and the second ISVD comprises a second anti-cMet VHH domain; or the first ISVD comprises a second anti-cMet VHH domain and the second ISVD comprises a first anti-cMet VHH domain, wherein the first anti-cMet VHH domain comprises: CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 23-25, respectively; and the second anti-cMet VHH domain comprises: CDR1 comprising or consisting of the amino acid sequence selected from SEQ ID NOs: 18 or 41, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 19 and SEQ ID NOs: 20, respectively; Preferably, the first anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16, 39 or 40; More preferably, the first anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, 39 or 40. Preferably, the first ISVD comprises a first anti-cMet VHH domain and the second ISVD comprises a second anti-cMet VHH domain.

11. The antibody according to any one of claims 5 to 10, wherein the antibody further comprises an anti-EGFR ISVD that specifically binds to EGFR, wherein the anti-EGFR ISVD is the ISVD according to claim 2, Preferably, the anti-EGFR ISVD comprises: (i) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 3, 4 and 5, respectively; (ii) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 8, 9 and 10, or the amino acid sequences of SEQ ID NOs: 8, 34 and 35, or the amino acid sequences of SEQ ID NOs: 8, 103 and 104, respectively; or (iii) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 13, 14 and 15, or the amino acid sequences of SEQ ID NOs: 13, 85 and 15, or the amino acid sequences of SEQ ID NOs: 13, 38 and 15, respectively; More preferably, the anti-EGFR ISVD: (a) comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 1, 31, and 94-99, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (b) comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 6, 32, 100-102, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; or (c) comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 11, 36, 84, and 105-114, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; More preferably, the anti-EGFR ISVD: (a) comprising, or consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 31; (b) comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 32; or (c) comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:

84.

12. The antibody according to any one of claims 5 to 11, wherein the antibody is in single-chain form or in double-chain form.

13. The antibody according to any one of claims 5 to 12, wherein the ISVDs on the same polypeptide chain are connected via one or more peptide linkers, preferably, the peptide linker comprises (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6 or 7, for example, GGGGS (SEQ ID NO: 43) or GGGGSGGGGS (SEQ ID NO: 44).

14. The antibody according to any one of claims 5 to 13, wherein the antibody further comprises a half-life increasing moiety, preferably an immunoglobulin Fc region or an ISVD that binds to human serum albumin, optionally wherein: - the immunoglobulin Fc region is an Fc region of human IgG1, IgG2, IgG3 or IgG4 isotype; - The ISVD that binds to human serum albumin is an anti-HSA ISVD comprising or consisting of a VHH domain, wherein the VHH domain comprises CDR1-3 of SEQ ID NOs: 46-48.

15. The antibody according to any one of claims 5 to 14, wherein the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein: From N-terminus to C-terminus, The first polypeptide chain comprises: a first ISVD that specifically binds to cMET and an immunoglobulin Fc region; The second polypeptide chain comprises: a second ISVD that specifically binds to cMET and an immunoglobulin Fc region, Preferably, wherein: - the first polypeptide chain comprises the sequence of SEQ ID NO: 88, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and - the second polypeptide chain comprises the sequence of SEQ ID NO: 89, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, More preferably, the first polypeptide chain comprises or consists of the sequence of SEQ ID NO: 88; and the second polypeptide chain comprises or consists of the sequence of SEQ ID NO:

89.

16. The antibody according to any one of claims 5 to 14, wherein the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein: From N-terminus to C-terminus The first polypeptide chain comprises: an ISVD that specifically binds to EGFR and an immunoglobulin Fc region, The second polypeptide chain comprises: a first ISVD that specifically binds to cMET, a peptide linker, a second ISVD that specifically binds to cMET, and an immunoglobulin Fc region.

17. The antibody according to any one of claims 5 to 14, wherein the antibody comprises a single polypeptide chain, in, The polypeptide chain comprises: a first ISVD that specifically binds to cMET, a second ISVD that specifically binds to cMET, and an ISVD that specifically binds to EGFR, and optionally an ISVD that specifically binds to HSA, Preferably, from N-terminus to C-terminus, the polypeptide chain comprises: An ISVD that specifically binds to EGFR, a first peptide linker, a first ISVD that specifically binds to cMET, a second peptide linker, a second ISVD that specifically binds to cMET, and optionally a third peptide linker and an ISVD that specifically binds to HSA.

18. The antibody according to claim 16, wherein the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein: - the first polypeptide chain comprises a sequence selected from SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:86, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, - the second polypeptide chain comprises a sequence selected from SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, or SEQ ID NO:87, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and Preferably, wherein: (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:70, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:73, 75 or 74, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:71, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:73, 75 or 74, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:72, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:73, 75 or 74, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iv) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:86, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:87, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; More preferably, wherein: (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73; (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73; (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain comprises, or consists of the amino acid sequence of SEQ ID NO:

87.

19. The antibody according to claim 17, wherein the antibody comprises a single polypeptide chain, wherein the polypeptide chain comprises a sequence selected from the group consisting of SEQ ID NOs: 51-66, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or wherein the polypeptide chain comprises a sequence selected from the group consisting of SEQ ID NOs: 67-68 and 76-83, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; Preferably wherein the polypeptide chain comprises or consists of a sequence selected from SEQ ID NOs: 51-56.

20. The antibody according to any one of claims 5-19, which is a multispecific antibody that specifically binds to EGFR and cMet and has one or more of the following properties: (1) binds to EGFR, such as human EGFR, with moderate or low affinity; and specifically binds to cMet, such as human cMet; (2) Binds to EGFR expressed on the cell surface at a low level and specifically binds to cMet expressed on the cell surface; (3) In the presence of HGF ligand, blocking the binding of HGF ligand to cMet on the cell surface; (4) internalized by cells expressing cMet; (5) internalized by cells expressing EGFR; (6) It has cross-reactivity with human EGFR and cynomolgus monkey EGFR; and cross-reactivity with human cMet and cynomolgus monkey cMet.

21. An antibody comprising at least one ISVD that specifically binds to EGFR according to claim 2, and optionally further comprising at least one ISVD that specifically binds to cMet, preferably the ISVD that specifically binds to cMet is the ISVD according to claim 1. Preferably, the antibody has one or more of the following properties: (1) Bind to EGFR, such as human EGFR, with low affinity; (2) It has cross-reactivity with human EGFR and cynomolgus monkey EGFR.

22. An isolated nucleic acid encoding the ISVD of claim 1 or 2, the binding molecule of claim 3 or 4, or the antibody of any one of claims 5-21.

23. A vector comprising the nucleic acid of claim 22, preferably said vector is an expression vector.

24. A host cell comprising the nucleic acid of claim 22 or the vector of claim 23, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing ISVDs, most preferably, the host cell is HEK 293 cells or CHO cells.

25. A method of preparation, the method comprising culturing the host cell of claim 24, and optionally recovering the ISVD of claim 1 or 2, the binding molecule of claim 3 or 4, or the antibody of any one of claims 5-21 from the host cell or from the culture medium.

26. An immunoconjugate or immunofusion comprising the ISVD of claim 1 or 2, the binding molecule of claim 3 or 4, or the antibody of any one of claims 5-21.

27. An antibody-drug conjugate having the formula (I0) or a pharmaceutically acceptable salt or solvate thereof: Ab-(LD) p (I0) in: Ab is the ISVD of claim 1 or 2, the binding molecule of claim 3 or 4, or the antibody of any one of claims 5 to 21; L is a linker; D is a drug, such as an anti-tumor compound; p is an integer selected from 1 to 16, for example an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

28. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 27, wherein the antibody drug conjugate has formula (I): Ab-(SLD) p (I) in: Ab is the ISVD of claim 1 or 2, the binding molecule of claim 3 or 4, or the antibody of any one of claims 5 to 21; L is a linker; D is a drug, such as an anti-tumor compound; p is an integer selected from 1 to 16, for example an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12; and S in formula (I) is sulfur from Ab.

29. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 27 or 28, wherein the drug is a cytotoxic agent, such as a camptothecin compound or an auristatin compound.

30. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 27 to 29, wherein D has a structure represented by formula (D-1a) or formula (D-1b): Where R 1a Selected from H and C1-C6 alkyl; R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and-SR 5a ; R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and R 4a and R 5a Independently selected from H and C1-C4 alkyl; or Where R 1b , R 2b , R 3b , R 4b , R 5b and R 8b Each independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; R 6b and R 7b Each independently selected from C 1-8 Alkoxy, such as methoxy, ethoxy or propoxy; R 9b Selected from C 1-8 Alkyl and COOH; preferably C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and R 10b Selected from OH and H; The wavy line in the D structure indicates that the bond is connected to L.

31. The antibody drug conjugate according to claim 30, or a pharmaceutically acceptable salt or solvate thereof, wherein D has a structure of formula (D-1a), and wherein R 1a H; R 2a is a C1-C6 alkyl group; R 3a is halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably an ethyl group.

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

33. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 27 to 32, wherein D has a structure represented by formula (D-2a) or formula (D-2b): Where R 1a , R 2a , R 3a and R 4a As defined in formula (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 formula (D-1b).

34. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 27 to 29, wherein D has a structure represented by formula (D-3a) or (D-3b): Preferably, D has a structure shown in formula (D-4a) or (D-4b):

35. The antibody drug conjugate according to any one of claims 27 to 29, or a pharmaceutically acceptable salt or solvate thereof, wherein the drug is Exatecan, Dxd, SN-38, monomethyl auristatin E (MMAE) or MMAF.

36. The antibody drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 27 to 35, wherein -L- has the following structure: -Z-L1-L2-L3- in Z is selected from wherein m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from the group consisting of: wherein n1 and m1 are each independently an integer selected from 0-20, such as an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8; L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and L3 is selected from: wherein X is selected from -NH-, -O- and -S-; R 1c Each independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 alkoxy, halogen, nitro and cyano; Su is each independently selected from pentose, pentose uronic acid, hexose and hexose uronic 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 S on Ab, and L3 is connected to D.

37. The antibody drug conjugate according to claim 36 or a pharmaceutically acceptable salt or solvate thereof, wherein Z is selected from Wherein m is 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, Z is selected from 38. The antibody drug conjugate according to claim 36 or 37, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is selected from the group consisting of: wherein n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, L1 is selected from the group consisting of:

39. The antibody drug conjugate according to any one of claims 36 to 38, or a pharmaceutically acceptable salt or solvate thereof, wherein L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids; preferably, wherein the amino acid residue or amino acid is each independently selected from valine (Val), alanine (Ala), glycine (Gly), 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); More preferably, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit), phenylalanine (Phe), lysine (Lys), glutamic acid (Glu) and glutamine (Gln); Still more preferably, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit) and glutamic acid (Glu).

40. The antibody drug conjugate according to any one of claims 36 to 38, or a pharmaceutically acceptable salt or solvate thereof, wherein L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-; Preferably, L2 is selected from -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit- and -Gly-Gly-Phe-Gly-.

41. The antibody drug conjugate according to any one of claims 36 to 40, or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: Where R 1c Each independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkoxy, halogen, nitro and cyano; Su are each independently selected from 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 according to any one of claims 36 to 41, or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: Preferably, L3 is selected from: More preferably, L3 is selected from:

43. The antibody drug conjugate according to any one of claims 36 to 42, or a pharmaceutically acceptable salt or solvate thereof, wherein Su is each independently: Preferably, Su are each independently Also preferably, Su are each independently 44. The antibody drug conjugate according to any one of claims 36 to 43, or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: Preferably, L3 is selected from 45. The antibody drug conjugate according to claim 36 or a pharmaceutically acceptable salt or solvate thereof, wherein -Z-L1-L2-L3- is each independently selected from the following structures: in, m is each independently an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 6 or 8; and The left side of the group is connected to S on Ab, and the right side is connected to D.

46. ​​The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 27 or 28, wherein the antibody drug conjugate is an antibody drug conjugate having a structure selected from the following: wherein Ab is the ISVD of claim 1 or 2, the binding molecule of claim 3 or 4, or the antibody of any one of claims 5 to 21; and p is an integer selected from 1 to 16, for example an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

47. The antibody drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 27 to 46, wherein the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, or 2-4 or 2-6.

48. A pharmaceutical composition comprising the ISVD of claim 1 or 2, the binding molecule of claim 3 or 4, the antibody of any one of claims 5-21, the immunoconjugate or immunofusion of claim 26, or the antibody-drug conjugate of any one of claims 27-47, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, and optionally further comprising one or more additional pharmaceutically active polypeptides and / or compounds, for example, further comprising other therapeutic agents selected from oncolytic drugs, cytotoxic agents, cytokines, and inhibitors of immune checkpoint molecules.

49. Use of the ISVD of claim 1 or 2, the binding molecule of claim 3 or 4, the antibody of any one of claims 5-21, or the immunoconjugate or immunofusion of claim 26, or the antibody drug conjugate of any one of claims 27-47, or a pharmaceutically acceptable salt or solvate thereof, as a medicament or for the preparation of a medicament, wherein preferably the medicament is used to treat cancer, for example, selected from lung cancer (e.g., lung squamous cell carcinoma, lung adenocarcinoma, non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), gastric cancer, colon cancer, and head and neck cancer (e.g., pharyngeal squamous cell carcinoma).