Binding molecules and antibody drug conjugates and uses

Abstract

The present disclosure provides 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 said immunoglobulin single variable domains. The present disclosure also provides nucleic acids encoding said ISVDs or binding molecules and vectors comprising said nucleic acids, as well as therapeutic applications of said ISVDs or binding molecules and said ADCs.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202411679744.0, filed on November 22, 2024, entitled "Conjugation Molecule and Antibody-Drug Conjugate and Use". Technical Field

[0002] This 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 said immunoglobulin single variable domains. The invention also relates to nucleic acids encoding said ISVDs or binding molecules and vectors comprising said nucleic acids, and the therapeutic applications of said ISVDs or binding molecules and said ADCs. Background Technology

[0003] EGFR is a transmembrane receptor protein with tyrosine kinase activity located 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 cell proliferation, survival, and differentiation. EGFR is highly expressed or mutated in many tumor types, making it an important therapeutic target for these cancers. Currently, various drugs that inhibit EGFR function have been developed, such as tyrosine kinase inhibitors and monoclonal antibodies, and these drugs have been widely used in the treatment of multiple malignant tumors, including lung cancer, colorectal cancer, and head and neck cancer.

[0004] cMet is a receptor with tyrosine kinase (RTK) activity expressed on epithelial cells, playing a crucial role in cell proliferation, survival, and migration. The binding ligand for cMet is hepatocyte growth factor (HGF). Upon binding to HGF, cMet dimers and activates the cMet pathway, promoting cell division, angiogenesis, and immune regulation. Due to its high expression or mutations in various cancers, cMet has become an important drug target, and related anticancer drugs are under research and development.

[0005] The U.S. Food and Drug Administration has approved a dual-targeting antibody against EGFR and cMet, 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 NSCLC patients. AZD9592 is an anti-EGFR / cMet bispecific antibody ADC drug currently under clinical investigation, carrying a topoisomerase 1 inhibitor as its 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., “Anarrative review of antibody–drug conjugates in EGFR-mutated non-small celllung cancer”, Front Oncol. 2023 Dec 1;13:1252652. doi: 10.3389 / fonc.2023.1252652).

[0006] The anti-EGFR / cMet bispecific antibody JNJ-61186372 and the ADC drug AZD9592 have conventional antibody quadruple chains and large molecular weights (150 kDa), which are not conducive to penetrating deep tumor tissues. Therefore, there is still an urgent need in the field to develop new antibody formats targeting EGFR and / or cMet, as well as EGFR and / or cMet binding molecules and ADC molecules with advantageous properties.

[0007] Immunoglobulin single variable domain (ISVD) nanobodies, for example, are small proteins composed of single-chain antibody molecules. They possess high antigen specificity and affinity, and compared to conventional four-chain antibodies, they exhibit smaller size, higher stability, and deeper tissue penetration. Therefore, the application of nanobodies and drug molecules constructed based on them in the field of cancer therapy is receiving widespread attention and is expected to become one of the important means of future cancer treatment. Summary of the Invention

[0008] Based on the screening of anti-EGFR and anti-cMet phage display libraries, this disclosure provides novel anti-cMet and anti-EGFR immunoglobulin single variable domains (ISVDs), and uses the ISVDs as components to construct anti-EGFR binding molecules, anti-cMet binding molecules, and anti-EGFR / cMet binding molecules and antibody-drug conjugates with excellent tumor targeting, endocytic activity and killing activity.

[0009] Therefore, in a first aspect, this disclosure provides an immunoglobulin single variable domain (ISVD) that specifically binds to cMet, a heavy chain antibody comprising said ISVD, and a cMet-binding molecule. In some embodiments, said cMet-binding molecule is an anti-cMet biepitaxy antibody. In some embodiments, said cMet-binding molecule further comprises a binding domain that binds to EGFR.

[0010] In a second aspect, this disclosure provides an immunoglobulin single variable domain (ISVD) that specifically binds to EGFR, a heavy chain antibody comprising said ISVD, and an EGFR-binding molecule. In some embodiments, said EGFR-binding molecule further comprises a cMet-binding domain.

[0011] In a third aspect, this disclosure provides an EGFR and cMet binding molecule comprising one or more immunoglobulin single variable domains (ISVDs) that specifically bind EGFR and cMet according to the first and / or second aspects of the invention. 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.

[0012] In a fourth aspect, this disclosure provides nucleic acids encoding the ISVD, EGFR-binding molecules, cMet-binding molecules, and EGFR and cMet-binding molecules described in the first to third aspects of this disclosure, vectors (preferably expression vectors) containing said nucleic acids, and host cells containing said nucleic acids or said vectors. In some embodiments, the host cell is prokaryotic or eukaryotic, for example, selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing ISVD or binding molecules. In some embodiments, the host cell is HEK 293 cells or CHO cells. This disclosure also provides methods for preparing the ISVD, EGFR-binding molecules, cMet-binding molecules, and EGFR and cMet-binding molecules described in the first to third aspects of this disclosure.

[0013] 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, thus exhibiting better tumor tissue penetration ability.

[0014] In some embodiments, the EGFR and / or cMet binding molecules according to the invention exhibit 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 commonly reported with drugs containing EGFR binding molecules (Taieb, Julien et al., 2023. "Adverse Events Associated with Encorafenib Plus Cetuximabin Patients with BRAFV600E-Mutant Metastatic Colorectal Cancer: An in-Depth Analysis of the BEACON CRC Study."). Clinical Colorectal Cancer , Updates inPancreatic 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 andBlocking Antibodies.” The Lancet Oncology 6 (7): 491–500. https: / / doi.org / 10.1016 / S1470-2045(05)70243-6 ).

[0015] In some embodiments, the binding molecule according to the invention has a structure that binds to two cMet epitopes (i.e., two different epitopes on cMet), which gives the binding molecule better tumor targeting. In some embodiments, the cMet dual-epitope design of the binding molecule according to the invention significantly increases the affinity and endocytic capacity of the binding molecule for target cells compared to targeting only a single epitope on cMet.

[0016] The ISVDs of this invention that specifically bind to EGFR and / or cMet, as well as EGFR and / or cMet binding molecules containing said ISVDs, have good tumor targeting, tumor tissue penetration and / or target cell endocytosis capabilities. Therefore, said ISVDs or binding molecules can serve as targeting modules of conjugates or couplings, and can be conjugated or coupled to chemotherapeutic agents, toxins, drugs (such as immunotherapeutic agents), radioactive elements, probes or signaling molecules, etc., to provide applications such as tumor killing, immune regulation or disease detection.

[0017] In a fifth aspect, this disclosure provides conjugates, fusions, and antibody-drug conjugates (ADCs) comprising the ISVD, EGFR-binding molecules, cMet-binding molecules, or EGFR and cMet-binding molecules described in the first to third aspects of this disclosure, particularly anti-EGFR / cMet multispecific antibody-drug conjugates.

[0018] In some embodiments, the antibody-drug conjugate according to the present invention has the following advantages: (1) Combine tumor cells expressing human EGFR or cMet and tumor cells co-expressing human EGFR and cMet; (2) Endocytotic activity was observed in tumor cells expressing human EGFR or cMet and tumor cells co-expressing human EGFR and cMet; (3) Blocking the binding of HGF to cMet on tumor cells; (4) It has a lateral killing effect; (5) It has a broad spectrum of antitumor activity and exhibits significant killing activity against various tumors with different EGFR and cMet expression densities; (6) Low in vivo toxicity, such as low on-target / detumescent toxicity.

[0019] In some embodiments, the antibody-drug conjugate according to the present invention also has one or more advantages selected from the following: (7) Because of the multispecific antibodies it contains, it can target multiple antigens at the same time and has better targeting and reduced toxic side effects (e.g., reduced off-target toxicity or reduced potential dose-limiting toxicity). (8) Compared with single-target ADCs targeting EGFR or cMet, it has higher endocytosis efficiency on tumor cells; (9) Compared with single-target ADCs targeting EGFR or cMet, it has higher affinity on tumor cells; (10) Compared with single-target ADCs that target EGFR or cMet, it has a stronger killing effect on tumor cells and a stronger inhibitory effect on tumor growth.

[0020] In some embodiments, the antibody-drug conjugate according to the present invention also has one or more advantages selected from the following: (11) It has good product uniformity; (12) It has good product stability; and (13) It has good drug-like properties.

[0021] In a sixth aspect, this disclosure provides pharmaceutical compositions and formulations comprising the ISVD, EGFR-binding molecule, cMet-binding molecule, or EGFR and cMet-binding molecule as described in the first to third aspects of this disclosure, or the ADC of the fifth aspect of this disclosure, and a pharmaceutically acceptable carrier, and optionally further comprising one or more other pharmaceutically active peptides and / or compounds, such as other therapeutic agents selected from inhibitors of oncolytic agents, cytotoxic agents, cytokines, and immune checkpoint molecules. In this aspect, this disclosure also provides combination products or kits comprising the ISVD, EGFR-binding molecule, cMet-binding molecule, or EGFR and cMet-binding molecule as described in the first to third aspects of this disclosure, or the ADC of the fifth aspect of this disclosure.

[0022] In a seventh aspect, this disclosure provides the use of the ISVD, EGFR-binding molecule, cMet-binding molecule, or EGFR and cMet-binding molecule described in the first to third aspects of the invention, or the ADC of the fifth aspect of the disclosure, as a medicament or for the preparation of a medicament, wherein the medicament is for treating cancer, for example, cancer selected from lung cancer (e.g., squamous cell carcinoma of the lung, adenocarcinoma of the lung, 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, this disclosure also provides a method of treating cancer, the method comprising administering to a subject in need an effective amount of the ISVD, EGFR-binding molecule, cMet-binding molecule, or EGFR and cMet-binding molecule described in the first to third aspects of the disclosure, or a nucleic acid or vector or host cell of the fourth aspect of the disclosure, or the ADC of the fifth aspect of the disclosure, wherein the subject is a mammal; preferably, the subject is a human; wherein the cancer is, for example, lung cancer (e.g., squamous cell carcinoma of the lung, adenocarcinoma of the lung, 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). Attached Figure Description

[0023] Combined with the following appendix Figure 1 Reading this description will provide a better understanding of the preferred embodiments of the invention as detailed below. For illustrative purposes, the figures show presently preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the figures.

[0024] Figure 1 This demonstrates the binding activity of anti-EGFR antibodies to target cells as detected by FACS.

[0025] Figure 2 This demonstrates the binding activity of anti-cMet antibodies to target cells as detected by FACS.

[0026] Figure 3 This demonstrates the blocking effect of anti-cMet VHH-Fc on the binding of ligand HGF to target cell EBC-1.

[0027] Figure 4 This shows that anti-cMet antibodies V-n7A12 and V-n9A2 bind to different epitopes on the cMet antigen.

[0028] Figure 5A and 5B The FACS assay shows the endocytosis of each VHH-Fc by cells.

[0029] Figure 6 The results show the cross-reactivity of anti-cMet antibodies V-n7A12, V-n9A2, and V-n9A10 with human cMet antigen and cynomolgus monkey cMet antigen detected by ELISA.

[0030] Figure 7 This demonstrates the binding of dual epitope antibodies to target cells as detected by FACS.

[0031] Figure 8 This study demonstrates the endocytosis of biepitaxy antibodies on target cells using FACS. Compared to monoepitaxy, biepitaxy mediates co-endocytic activity.

[0032] Figure 9 This diagram shows the molecular structure of a single-chain multispecific EGFR / cMet antibody.

[0033] Figure 10 This diagram shows the molecular structure of a double-stranded, multispecific EGFR / cMet antibody.

[0034] Figure 11 The binding of trispecific antibody candidate molecules was demonstrated on target cells EBC-1 and NCI-H1975 cells.

[0035] Figure 12 This demonstrates the endocytosis of target cells with single-chain trispecific antibody candidate molecules.

[0036] Figure 13 This demonstrates the endocytosis of target cells with double-stranded trispecific antibody candidate molecules.

[0037] Figure 14 The trispecific anti-EGFR / cMet antibody showed that it blocked the binding of EBC-1 cells to the ligand HGF.

[0038] Figure 15 The results show that FACS detection of trispecific antibodies on target cells involves synergistic endocytosis mediated by anti-EGFR ISVD and anti-cMet ISVD.

[0039] Figure 16 The results show that FACS detection of trispecific antibodies on target cells involves synergistic binding mediated by anti-EGFR ISVD and anti-cMet ISVD.

[0040] Figure 17 This demonstrates the binding of ADC molecules to target cells.

[0041] Figure 18A and 18B This demonstrates the killing effect of ADC molecules on target cells.

[0042] Figure 19 Cell binding detection of ADC molecules with PEG and / or EVC linkers.

[0043] Figure 20 Cell killing detection of ADC molecules with PEG and / or EVC linkers.

[0044] Figure 21 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 is shown.

[0045] Figure 22 This demonstrates the in vivo efficacy of V-23-Fc-VA-Exd in the CDX model.

[0046] Figure 23 This demonstrates the in vivo efficacy of V-23-Fc-Glu-Exd in the CDX model. Detailed Implementation

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0048] definition

[0049] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0050] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​that have a lower limit of 5%, 4%, 3%, 2%, or 1% smaller than the specified numeric value and an upper limit of 5%, 4%, 3%, 2%, or 1% larger than the specified numeric value.

[0051] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0052] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the variable region consists of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region that “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0053] 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 that are targeted by antibodies, thereby binding to the antibodies and causing damage.

[0054] When used with antigens, the terms "binding molecule" and "antigen-binding molecule" are used interchangeably (e.g., EGFR-binding molecule, cMet-binding molecule, EGFR and cMet-binding molecule), referring to a protein or polypeptide molecule that can specifically bind to an antigen or an epitope on the antigen. A binding molecule has "affinity" and / or "specificity" towards the antigen. In this document, an EGFR-binding molecule refers to a protein or polypeptide that specifically binds to EGFR, a cMet-binding molecule refers to a protein or polypeptide that specifically binds to cMet, and an EGFR and cMet-binding molecule refers to a protein or polypeptide that specifically binds to both EGFR and cMet. Some examples of binding molecules include antibodies, antibody fragments, fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.

[0055] The domain in an antigen-binding molecule that actually binds to the antigen is referred to in this paper as the "antigen-binding site" or "antigen-binding domain". A domain, as a folded structure in a protein or polypeptide, generally governs a single function of the protein or polypeptide. For example, conventional antibodies and immunoglobulins typically form an antigen-binding domain on the surface of a VH-VL dimer through three complementarity-determining regions (HCDR1-3) in their heavy chain variable region (VH) and three complementarity-determining regions (LCDR1-3) in their light chain variable region (VL), where six CDRs confer specific binding between the antibody and 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 camel heavy chain antibodies, or a VH-like single domain (v-NAR) derived from fish IgNARs) can confer antigen binding. That is, the single variable domain does not need to interact with another variable domain and can independently function as an "antigen-binding domain" for recognizing and binding target antigens. Typically, through engineering modifications, the "antigen-binding domains" of antibodies, including the aforementioned monoimmunoglobulin variable domains and the variable domain pairs of conventional antibodies, can be added, removed, or transferred to other proteins or peptides while still maintaining their antigen-binding function without losing the function of the remaining parts and / or remaining domains of the protein or peptide.

[0056] The terms "binding" or "specific binding" mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined using conventional binding assays known in the art. For example, the binding ability of an antibody to an antigen is detected by the ELISA assay described in the examples, or the binding ability of an antibody to cells expressing an antigen on their surface is detected by the FACS assay described in the examples, or the affinity constant K is detected by the SPR technique described in the examples. D .

[0057] The term "antibody" is used in the broadest sense herein to refer to a protein containing an antigen-binding site of an immunoglobulin, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monoepitope and polyepitope antibodies (e.g., biepitope 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, intact antibodies, and antibody fragments. In some embodiments, preferably, the antibodies of the present invention are single-domain antibodies, nanobodies, or heavy-chain antibodies. In other embodiments, preferably, the antibodies of the present invention are biepitope antibodies, bispecific antibodies, or multispecific antibodies.

[0058] The terms "antibody fragment" or "antigen-binding fragment" of an antibody are used interchangeably and refer to a molecule distinct from the intact antibody that contains a portion of the intact antibody and is capable of binding the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; single-chain antibody fragments (e.g., scFv, scFab); monoimmunoglobulin domains; variable domain fragments of camel heavy chain antibodies; and various monospecific, bispecific, or multispecific antibody structures formed from antibody fragments, such as linear antibody fragments, diabody fragments, etc. In this disclosure, unless otherwise stated or explicitly contradicted by the context, reference to the term "antibody" is equivalent to reference to "antibody and antibody fragments thereof." In some embodiments of the invention, the antibody fragment includes cysteine ​​residue portions for forming interchain disulfide bonds between heavy chains, such as cysteine ​​residues in the antibody hinge region, to provide amino acid residue sites usable for thiol coupling chemistry. In other embodiments of the invention, the antibody fragment includes cysteine ​​residues introduced into the Fc region to provide amino acid residue sites usable for thiol coupling chemistry.

[0059] In this 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 can specifically recognize and bind to a target antigen through a single variable domain, such as a single VHH domain or a single VH domain or a single VL domain, without pairing with an additional immunoglobulin variable domain. For an ISVD composed of a VHH domain or a VH or VL domain, its structure can be considered to consist of four frame regions ("FR") and three complementarity-determining regions ("CDR"), referred to as "frame region 1" or "FR1", "frame region 2" or "FR2", "frame region 3" or "FR3", and "frame region 4" or "FR4", respectively; the four frame regions are interrupted by three complementarity-determining regions or "CDRs", referred to as "complementarity-determining region 1" or "CDR1", "complementarity-determining region 2" or "CDR2", and "complementarity-determining region 3" or "CDR3", respectively. From the N-terminus to the C-terminus of the ISVD polypeptide, the four frame regions and three complementarity-determining regions are arranged in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin single variable domain may include a fully human sequence, a humanized sequence, a sequence optimized in other ways, or a chimeric immunoglobulin sequence. The immunoglobulin single variable domain can be used alone in a separate form or as part of a larger protein to perform antigen-binding functions. 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.

[0060] In this disclosure, the terms "single-domain antibody" and "single-domain antibody" are used interchangeably herein and generally refer to antibodies that recognize and bind to antigens via ISVD. Examples of single-domain antibodies include single-domain antibodies derived from camelids (lambs and camels) and cartilaginous fish (e.g., nurse sharks) (WO 2005 / 035572).

[0061] In this disclosure, the term "heavy-chain antibody (hcAb)" refers to an antibody that has only a heavy chain and no light chain. The heavy chain of a heavy-chain antibody, from the N-terminus to the C-terminus, may, for example, contain VH-CH2-CH3, or may contain VH-CH1-CH2-CH3, or may contain VHH-CH2-CH3, etc. The heavy chain can form a homodimer. In some embodiments, preferably, the heavy-chain antibody according to the invention is a dimer comprising two monomers, wherein each monomer contains a VHH domain linked to the immunoglobulin constant regions (CH2 and CH3 domains) via an immunoglobulin hinge region.

[0062] The term “nanobody” is used in this document to refer to an antibody containing, or substantially composed of, a single ISVD domain (such as a VHH domain) and having a molecular weight of less than 20 kDa (typically about 12–15 kDa).

[0063] The term "VHH antibody" is used in this document to refer to an antibody composed of a VHH domain. A "VHH domain," also known as VHH, VHH sequence, or VHH antibody fragment, is a single-chain antibody fragment containing FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1 from the C-terminus to the N-terminus. Using the VHH domain (alone, or as part of a larger polypeptide) to recognize and bind to target antigens offers many significant advantages over using conventional VH and VL domains, scFv, or conventional antibody fragments (such as Fab or F(ab')2 fragments): - Only a single domain is needed to bind to the antigen with high affinity and high selectivity, so that there is no need for two separate domains, nor is it necessary to ensure that the two domains exist in the appropriate spatial conformation and configuration (for example, scFv generally requires the use of specially designed adapters). -VHH domains can be easily modified into multivalent and multispecific formats; - The VHH domain is highly soluble and has no tendency to aggregate; The -VHH domain is highly stable to heat, pH, protein or peptidoglycan enzymes and other denaturants or conditions, and therefore can be prepared, stored or transported without the use of refrigeration equipment, thus saving costs, time and the environment; -VHH domains are easy to prepare and relatively inexpensive, even at the scale required for production; The -VHH domain is relatively small compared to conventional tetrapeptide chain antibodies and their antigen-binding fragments, thus exhibiting higher tissue penetration and allowing for higher dose administration. - The VHH domain can exhibit so-called cavity binding properties (compared to the conventional VH domain, the VHH has an extended CDR3 loop, thus reaching target epitopes that are inaccessible to conventional tetrapeptide antibodies and their antigen-binding fragments).

[0064] VHHs include humanized VHHs, camel-derived VHHs, or VHHs acquired through affinity maturation.

[0065] Further description of VHH can be found in WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103. In some embodiments, the antigen-binding site of the antigen-binding molecule and antibody according to the invention is preferably provided by the VHH domain.

[0066] The term "valence" refers to the number of antigen-binding sites present in an antigen-binding molecule (such as an antibody). Therefore, "monovalent," "bivalent," "trivalent," and "quadrivalent" antibodies refer to antibody molecules containing 1, 2, 3, and 4 antigen-binding sites, respectively.

[0067] In this article, "monospecific" means that an antigen-binding molecule has the ability to bind only a single epitope. "Multispecific" means that an antigen-binding molecule has the ability to bind two or more different epitopes (e.g., different epitopes on the same antigen and / or different antigens). Correspondingly, "bispecific" means that an antigen-binding molecule can bind 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 bivalent, trivalent, tetravalent, or more.

[0068] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. Variable domains of the heavy and light chains typically have similar structures, containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). Since the CDR sequence is responsible for most antibody-antigen interactions, antibody variants mimicking the properties of known antibodies can be constructed by modifying the variable region. In some cases, CDR sequences from known antibodies can be grafted onto the frame regions of different antibodies with different properties, and one to several residues can be mutated as needed, such as reversion mutations, to refine the desired properties of the antibody. In other cases, the variable domains of antibodies can be engineered to construct humanized, deimmunogenic, and / or PTM (post-translational modification)-removed variants. The properties of the modified antibodies, 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 should be understood that any such functional variations of any variable region (e.g., VH and / or VL regions, VHH regions) given herein are within the scope of this invention.

[0069] The term "complementarity-determining region" or "CDR region" or "CDR" refers to a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes and are sequentially numbered from the N-terminus of the variable region as 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 of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (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)) based on antibody sequence variability; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., Department of Health and Human Services, National Institutes of Health (1987)); AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics database (IMGT). (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this disclosure, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing methods. A CDR may also be determined based on having the same AbM or Kabat numbering position as a reference CDR sequence (e.g., the CDR sequence of the example of this invention). In one embodiment, the CDR of the antibody of the present invention is defined according to Kabat or Chothia or AbM or IMGT or Contact, or any combination thereof. In one embodiment, the CDR of the antibody of the present invention is determined according to the Kabat definition scheme.

[0070] Antibodies with different specificities (i.e., targeting different antigenic epitopes) have different core-response distances (CDRs). However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, IMGT, and Contact methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining portion of the CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, this disclosure also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat, Chothia, AbM, IMGT, or Contact may be substituted with conserved amino acid residues.

[0071] If an amino acid sequence (e.g., 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), then it is said to be "cross-reactive" to these two different antigens or antigenic determinants. Antibodies exhibiting human-monkey species cross-reactivity, particularly having similar human-monkey antigen binding affinity, is advantageous, as this property can facilitate preclinical drug development of the antibody, such as toxicological assays of ADC molecules composed of antibodies. In some embodiments, the antibodies of the present invention preferably exhibit human-monkey species cross-reactivity.

[0072] As used herein, the term "epitope" refers to the portion of an antigen that an antibody specifically binds to. An epitope can consist of continuous and / or discontinuous amino acids forming a conformational spatial unit. For discontinuous epitopes, the amino acids of different portions of the linear sequence of the antigen are closely spaced in three-dimensional space through the folding of the protein molecule. Epitopes can be grouped by competitive binding assays of different antibodies binding 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 competitive assays, or the methods described in the examples herein.

[0073] "Humanized" antibodies are chimeric antibodies comprising amino acid residues from nonhuman CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs in a humanized antibody correspond to those in nonhuman antibodies, and all or substantially all of the FRs correspond to those in human antibodies. Humanized antibodies may optionally contain at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a nonhuman antibody) refers to an antibody that has been humanized.

[0074] In this document, the term "half-life extension domain" or the expression "binding portion for increasing half-life" are used interchangeably and refer to a chemical structure that endows a bound molecule (e.g., an antibody) with an increased circulating half-life 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-HSAISVD). The half-life extension domain can be linked to the binding molecule or antibody of the present invention via chemical conjugation or fusion, depending on its specific properties.

[0075] In some embodiments, the EGFR-binding molecule described in the first aspect of this disclosure, the cMet-binding molecule described in the second aspect of this disclosure, and the EGFR and cMet-binding molecule described in the third aspect of this disclosure include 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 moieties 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 moieties are Alb-8 or Alb-23 or variants thereof, as shown on pages 7-9 of WO2012 / 175400. In some preferred embodiments, the ISVD binding to human serum albumin comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 46 or composed of therefrom, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 47 or composed of therefrom, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 48 or composed of therefrom. In some embodiments, the ISVD binding 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 with it.

[0076] 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 that comprises at least a portion of a constant region. It is known that the constant region of the heavy chain of each immunoglobulin comprises four or five domains, named in the following order: CH1-hinge-CH2-CH3(-CH4). CH4 is present in IgM without the hinge region. In this disclosure, the Fc domain may comprise the CH2 and CH3 domains, and optionally also comprise all or part of the immunoglobulin hinge region; but does not comprise the immunoglobulin heavy chain variable region VH and light chain variable region VL, or the heavy chain constant region CH1 and light chain constant region CL. For example, in one instance, the Fc domain may comprise or consist of the CH2 and CH3 domains from the N-terminus to the C-terminus. In another example, the Fc domain may comprise, or be composed 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.

[0077] The term "Fc region" includes both the native sequence Fc region and variant Fc regions. In some embodiments, the antibody according to the 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) of the Fc region may or may not be present. Unless otherwise stated, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In some embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region contains modifications relative to the native sequence Fc region. In some specific embodiments, the Fc region has an increase or decrease in effector function. In some specific embodiments, the Fc region is enhanced or weakened by binding to FcγR. In some specific embodiments, the Fc region contains heavy chain mismatch prevention mutations, such as Knob-into-hole (KIH) mutations.

[0078] In some cases, immunoglobulin Fc regions containing hinge region sequences are preferred, which can, for example, promote the dimerization of antibody polypeptide chains and / or provide cysteine ​​residues for coupling with other active molecules. Such hinge sequences may substantially or partially correspond to the hinge regions of IgG1, IgG2, IgG3, or IgG4. For example, the hinge region sequence may comprise all or part of a core hinge region and all or part of a lower hinge region. The core hinge region has the amino acid sequence CPPC in IgG1, IgG2, and IgG3, and the CPSC sequence in IgG4. Preferably, the hinge region contains at least one disulfide bond connecting two Fc chains. In some embodiments, the hinge region sequence comprises hinge region sequences from E216 to T225 of IgG1 or hinge region sequences from D221 to T225 (according to EU numbers), or corresponding hinge region sequences from other immunoglobulin isotypes. In some embodiments, the immunoglobulin single variable domain (ISVD) of the present invention is connected to the Fc region by a hinge sequence comprising, for example, EPKSS (SEQ ID NO: 49) or EPKSC (SEQ ID NO: 50).

[0079] The term "effective function" refers to those biological activities attributable to the Fc region of immunoglobulins that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen-presenting cell uptake of antigens, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0080] Where effector function is not required, the Fc region may contain mutations that reduce or eliminate effector function. In some cases (e.g., when the antibody of the present invention is used as an ADC vector), preferably, the Fc region contains mutations that reduce or eliminate the interaction between the Fc region and the Fcγ receptor, such as the LALA mutation that changes lysine (L) at positions 234 and 235 of the Fc region to alanine (A), to reduce Fcγ receptor-mediated off-target cytotoxicity. Alternatively or additionally, mutations may be introduced into the Fc region to increase binding to FcRn and / or remove protease sites, and / or introduce amino acid modifications that can be used for conjugation of active molecules. Alternatively or additionally, the Fc region may be mutated for antibody production purposes, for example, by removing or replacing amino acids that may undergo post-translational modifications (e.g., glycosylation), to provide improved drugability and developability of the therapeutic antibody.

[0081] The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to one of the main mechanisms by which certain cytotoxic effector cells (such as 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, for example, NK cells.

[0082] 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 (C1q) of the complement system to an antibody (appropriate subclass) that binds to its corresponding antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0083] "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 stated, when used herein, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be expressed by the binding dissociation equilibrium constant (K0). D Affinity can be expressed as a definite affinity. It can be measured using methods commonly known in the art, including those known in the prior art and those described herein.

[0084] In this document, the “identity percentage (%)” of an amino acid sequence refers to the percentage of positions in the candidate sequence that have the same amino acid residues at the corresponding positions in the alignment with the specific amino acid sequence shown in this disclosure, after comparing the candidate sequence with the specific amino acid sequence shown in this disclosure and, if necessary, introducing vacancies to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of sequence identity.

[0085] In some embodiments, this disclosure contemplates variations of the ISVD, binding molecule, and antibody sequences of the present invention, which contain amino acid changes relative to the ISVD, binding molecule, and antibody sequences specifically disclosed herein. In some embodiments, the variations have a considerable degree of identity with respect to the ISVD, binding molecule, and antibody sequences specifically disclosed herein within a comparison window, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. Where no comparison window is specified herein (i.e., the region of interest to be compared), the alignment is performed over the full length of the reference sequence.

[0086] In some embodiments of the invention, the amino acid changes described herein include substitution, insertion, or deletion of amino acids. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. In a preferred embodiment, 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 substitution is a conservative substitution. A conservative substitution means that an amino acid is substituted by another amino acid within the same class, for example, an acidic amino acid is substituted by another acidic amino acid, a basic amino acid is substituted by another basic amino acid, or a neutral amino acid is substituted by another neutral amino acid. Exemplary substitutions are shown in Table A below: Table A

[0087] In this document, "isolated" antibodies or antibody fragments refer to artificial antibodies or antibody fragments, recombinant antibodies or antibody fragments, and antibodies or antibody fragments that have been at least partially separated from components in the natural environment in which they originated. In some embodiments, antibodies (e.g., anti-EGFR / cMet antibodies) or antibody fragments (e.g., anti-EGFR ISVD or anti-cMet ISVD) according to the invention are "isolated". In some embodiments, the isolated antibodies or antibody fragments are purified to a purity of more than 90%, 95%, or 99%, 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).

[0088] In this document, the term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0089] In this document, the term "expression vector" refers to a vector containing a recombinant polynucleotide and an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including clomids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0090] In this document, the terms "endocytosis" and "internalization" are used interchangeably, referring to the process by which a ligand / receptor complex is internalized and delivered into the cytosol or translocated to a suitable intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface. In some embodiments, the antibodies of the present invention initiate endocytosis mediated by EGFR and / or cMet receptors upon binding to EGFR and / or cMet expressed on the cell surface. In this document, endocytosis and endocytosis rate can be determined, for example, by the methods described in the examples, to characterize the endocytic activity of the antibody. In some embodiments, the antibodies of the present invention having endocytic activity can be used as a tool for delivering antitumor drugs into cancer cells in the ADCs of the present invention.

[0091] The term "conjugate" or "coupler" herein refers to a molecule formed by conjugating one or more immunoglobulin-associated molecules or fragments thereof with one or more other molecules. A conjugate typically contains at least one non-protein chemical structural part, such as a chemical linker for achieving the conjugation. In some cases, the other molecules may be the same as immunoglobulin-associated molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-associated molecules or fragments thereof. The one or more additional molecules may be the same as or different from each other. For example, the other molecules may be target-binding elements and / or effector elements, such as chemotherapeutic agents, toxins, drugs (e.g., immunotherapeutic agents), radioactive elements, probes, or signaling molecules, etc.

[0092] "Antibody-drug conjugate (ADC)" refers to a compound obtained by linking an antigen-binding molecule to a (small molecule) drug via a linker. In this document, the term "antibody-drug conjugate" or "ADC" includes its pharmaceutically acceptable salts and solvent compounds, as well as other equivalents. The drug compound portion of an ADC may be referred to herein as the "payload" or "toxin."

[0093] The term "linker" refers to a structural segment that covalently links a drug (e.g., a small molecule drug) to an antigen-binding molecule. It should be understood that a linker has functional groups that can form bonds with the functional groups of the antigen-binding molecule before being linked. In some cases, the linker may also have a degradable portion and optionally a hydrophilicity modulating module, such as a PEG segment. In some embodiments of the ADC according to the invention, the linker is preferably "degradable," thereby enabling it to break and release the payload after the ADC is delivered to the target region (e.g., the target tumor tissue site). Such "degradable linkers" available include, for example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers.

[0094] The term "linker-payload" refers to a compound formed by the connection of a payload and a linker. In some cases, linker-payloads are used as intermediates in ADC synthesis.

[0095] The term "therapeutic agent" encompasses any substance that is effective in preventing or treating diseases such as cancer, including chemotherapeutic agents, cytotoxic agents, immunomodulators (such as immunosuppressants), other antibodies, small molecule drugs, angiogenesis inhibitors, or cytokines.

[0096] The term "drug" refers to a compound that can regulate biological processes, particularly altering or preventing pathological processes. In this article, "drug" preferably refers to antitumor compounds.

[0097] The term "small molecule drug" refers to a low molecular weight drug that can regulate biological processes, particularly altering or preventing pathological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa, more preferably less than 500 kDa. Small molecule drugs include, but are not limited to, organic molecules having the molecular weights defined above, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0098] "Antitumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773 and US5658920, camptothecin compounds such as eczetidine and Dxd (Exatecan derivatives), and auristatin compounds such as monomethyl auristatin E (MMAE) and MMAF.

[0099] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0100] "Chemotherapy agents" include chemical compounds that are useful in treating cancer or immune system diseases.

[0101] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 1-16 carbon atoms, for example, 1-12 carbon atoms, 1-10 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0102] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing 2-16 carbon atoms and at least one double bond but no triple bonds. The alkenyl group preferably contains 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl groups.

[0103] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing 2-16 carbon atoms and at least one triple bond. The alkynyl group preferably contains 2-12 carbon atoms, 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentyynyl, and hexynyl.

[0104] The term “halogen” or “halogenated” refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0105] The term "haloalkyl" refers to an alkyl group as defined herein, which is substituted with one or more halogen groups. Haloalkyl groups may preferably be monohaloalkyl, dihaloalkyl, or polyhaloalkyl (including perhaloalkyl). Monohaloalkyl groups may contain one iodine, bromine, chlorine, or fluorine group in the alkyl group. Dihaloalkyl and polyhaloalkyl groups may contain two or more identical halogen atoms or combinations of different halogen groups in the alkyl group. Preferably, polyhaloalkyl groups contain at most 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhaloalkyl refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0106] The term “haloalkenyl” refers to an alkenyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The term “haloynyl” refers to an ynyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The meaning of “halogenated” as defined for “haloalkyl” may apply to both “haloalkenyl” and “haloynyl”.

[0107] The terms "alkoxy" and "alkyl-O-" are used interchangeably to refer to an alkyl group as defined above, linked by an oxygen atom. Preferably, the alkoxy group has 1-8 carbon atoms (C... 1-8 alkoxy group), 1-6 carbon atoms (C 1-6 alkoxy group), 1-4 carbon atoms (C 1-4 alkoxy group or 1-3 carbon atoms (C 1-3Alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexoxy, heptoxy, octoxy, etc.

[0108] The term "amino acid" refers to naturally occurring and synthetic amino acids, amino acid analogs, and their artificially modified forms. Amino acids can be L- or D-isomers. In this disclosure, 20 naturally occurring amino acids are represented by single-letter and three-letter abbreviations known in the art, such as: 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 this invention refer to L-amino acids.

[0109] The term "penturonic acid" refers to compounds formed by oxidizing the primary hydroxyl group of a pentose sugar as defined above to a carboxyl group. Examples of penturonic acids include, but are not limited to, xyuronic acid and arabinuronic acid.

[0110] The term "hexuronic acid" refers to compounds formed by oxidizing 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.

[0111] The term "optional" or "optionally" means that the event or condition described below either occurs or does not occur, and the description includes instances where the event or condition occurs as well as instances where the event or condition does not occur. For example, when a group or structure is "optionally substituted," the group or structure may or may not be substituted.

[0112] In this article, "pharmaceutically acceptable" means that it can be administered to an individual or subject without producing biologically or otherwise undesirable side effects, such as serious, intolerable side effects. Where there is no contradiction in the context, "pharmaceutically acceptable" and "medicinal" are used interchangeably.

[0113] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention, and that such salt is not biologically or otherwise undesirable. The ADC conjugates of the present invention can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugates of the present invention with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N group.

[0114] The term "solvent" refers to an association formed by one or more solvent molecules with the ADC antibody-drug conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0115] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug portion (D) coupled to the Ab portion described herein to the Ab portion. In some embodiments described herein, the DAR may be determined by p in Formula I, for example, the DAR may be 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR may also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug portion (D) coupled to the Ab portion described herein to the Ab portion in the product as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 16, for example 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, for example 1.0-8.0, 2.0-6.0, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, a range with two of these values ​​as endpoints. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population or mixture of ADC molecules that contains ADC molecules having the same and / or different DAR values.

[0116] The term "half-maximum effective concentration (EC50)" 50 "EC" refers to the concentration of a drug, antibody, ADC, or toxicant that induces a 50% response between baseline and maximum after a specific exposure time. In the context of this application, EC 50 The unit is “nM”.

[0117] The term "fluorescence-activated cell sorting" or "FACS" refers to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). Instruments used to perform FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACS StarPlus, 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).

[0118] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0119] The term "pharmaceutical excipient" refers to diluents, adjuvants (such as Freund's adjuvants (complete and incomplete)), carriers, or stabilizers that are applied together with the active substance.

[0120] The terms "drug combination," "combination product," "drug conjugate," or "combination product" refer to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antigen-binding molecule or ADC molecule of the present invention, including its pharmaceutically acceptable salt, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of two or more active agents. In some embodiments, the antigen-binding molecule or ADC molecule of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in a separate formulation, and their formulations may be the same or different.

[0121] The terms “individual” or “subject” are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual or subject is a human being.

[0122] The terms “tumor” and “cancer” are used interchangeably in this document to refer to a physiological disorder in mammals in which cell growth is not regulated, encompassing both solid and liquid tumors, as well as malignant and benign tumors, and all precancerous and cancerous cells and tissues.

[0123] In this article, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In cases involving tumor or cancer treatment, "treatment" encompasses antitumor biological effects that can be induced by artificial intervention (e.g., through the administration of drugs), including but not limited to, reductions in tumor volume, number of tumor cells, proliferation, or survival.

[0124] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0125] The term "effective amount" refers to such an amount or dose of the antigen-binding molecule or ADC molecule or composition or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. Depending on the intended effect, it may include "therapeutic effective amount" and "preventive effective amount".

[0126] The term "therapeutic effective amount" refers to the amount that, at the required dose and sustained for the required period of time, effectively achieves the desired therapeutic outcome. Therapeutic effective amounts of antibodies or ADCs can vary depending on various factors such as disease state, individual age, sex, weight, and the ability of the antibody or ADC to elicit the desired response in the individual. A therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody or ADC are less than the beneficial therapeutic effect. Relative to untreated subjects, a "therapeutic effective amount" preferably inhibits measurable parameters (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit measurable parameters (e.g., cancer) can be evaluated in animal model systems that predict efficacy in human tumors.

[0127] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered in subjects before or at an early stage of the disease, the prophylactic effective dose is less than the therapeutic effective dose.

[0128] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0129] The present invention will now be described in detail. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any technical feature described in any of the following sections, subsections, or embodiments may be combined with any technical feature described in any other section, subsection, or embodiment, and such combinations are all within the scope of this invention.

[0130] I. First aspect of this disclosure: ISVDs that specifically bind cMet and cMet-binding molecules containing the ISVDs.

[0131] 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). cMet protein is a receptor tyrosine kinase that is overexpressed or mutated in many tumor cell types, playing a crucial role in tumor cell proliferation, survival, invasion, metastasis, and tumor angiogenesis. Inhibition of cMet can induce cell death in tumor cells that overexpress cMet protein or express constitutively activated cMet protein.

[0132] This disclosure provides, in a first aspect, an ISVD that specifically binds to cMet (i.e., an anti-cMet ISVD) and a heavy-chain antibody and cMet-binding molecule comprising the ISVD. In some embodiments, the anti-cMet ISVD, heavy-chain antibody, and cMet-binding molecule of the present invention bind human cMet with intermediate or high affinity. In some embodiments, the anti-cMet ISVD and cMet-binding molecule of the present invention have improved tissue penetration compared to conventional four-chain antibodies.

[0133] The ISVD of the present invention, which specifically binds to cMet, contains three complementary determination regions from the N end to the C end, namely CDR1, CDR2 and CDR3.

[0134] In some embodiments, the immunoglobulin single variable domain (ISVD) that specifically binds to cMet of the present invention comprises

[0135] (a) Three CDRs in the amino acid sequence shown in one of SEQ ID NO: 16, 39-40 and 121-130; (b) Three CDRs in the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 42 and 134-136; or (c) Three CDRs in the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the ISVD of the present invention specifically binding to cMet comprises a variant having, compared to the three CDRs described in any one of (a)-(c) above, a single or multiple CDRs having no more than one to three amino acid variations in each CDR; wherein said amino acid variation is the addition, deletion, or conserved amino acid substitution. 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.

[0136] In some embodiments, the ISVD of the present invention specifically binding to cMet includes a number according to the Kabat number.

[0137] (a) CDR1 shown in SEQ ID NO: 18 or a variant of CDR1 shown in SEQ ID NO: 18 with no more than one amino acid change (e.g., CDR1 shown in one of SEQ ID NO: 41 and 131-133), CDR2 shown in SEQ ID NO: 19 or a variant of CDR2 shown in SEQ ID NO: 19 with no more than two amino acid changes, and CDR3 shown in SEQ ID NO: 20 or a variant of CDR3 shown in SEQ ID NO: 20 with no more than two amino acid changes; (b) CDR1 shown in SEQ ID NO: 23 or a variant of CDR1 shown in SEQ ID NO: 23 with no more than one amino acid change, CDR2 shown in SEQ ID NO: 24 or a variant of CDR2 shown in SEQ ID NO: 24 with no more than two amino acid changes, and CDR3 shown in SEQ ID NO: 25 or a variant of CDR3 shown in SEQ ID NO: 25 with no more than two amino acid changes; or (c) CDR1 shown in SEQ ID NO: 28 or a variant of CDR1 shown in SEQ ID NO: 28 with no more than one amino acid change, CDR2 shown in SEQ ID NO: 29 or a variant of CDR2 shown in SEQ ID NO: 29 with no more than two amino acid changes, and CDR3 shown in SEQ ID NO: 30 or a variant of CDR3 shown in SEQ ID NO: 30 with no more than two amino acid changes; The amino acid changes mentioned therein are the addition, deletion, or substitution of conserved amino acids.

[0138] In some embodiments, this disclosure provides an immunoglobulin single variable domain (ISVD) that specifically binds to cMet, wherein said ISVD comprises: (a) Each of the following contains SEQ ID Nos: 18, 19 and 20 or CDR1, CDR2 and CDR3 thereof; (b) Containing SEQ ID Nos: 41, 19 and 20 or CDR1, CDR2 and CDR3 thereof respectively; (c) Each of the following contains SEQ ID Nos: 131, 19 and 20 or CDR1, CDR2 and CDR3 composed thereof; (d) Containing SEQ ID Nos: 132, 19 and 20 or CDR1, CDR2 and CDR3 composed of them respectively; (e) Containing SEQ ID Nos: 133, 19, and 20, or CDR1, CDR2, and CDR3 thereof; or (f) Each of the following comprises SEQ ID Nos: 23, 24, and 25, or CDR1, CDR2, and CDR3 thereof. In some embodiments, the ISVD defined in (b) or (f) above is preferred.

[0139] In some embodiments, the ISVD of the present invention that specifically binds to cMet comprises or consists of VHH. In some embodiments, the ISVD of the present invention that specifically binds to cMet comprises or consists of the following sequences: (a) A sequence of one of SEQ ID NO: 16, 39-40 and 121-130 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (b) A sequence of one of SEQ ID NO: 21, 42, and 134-136, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or (c) A sequence of SEQ ID NO: 26 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; Preferably, the amino acid change does not occur in the CDR region. In some embodiments, the ISVD containing the amino acid sequence of SEQ ID NO:39, 40, or 42 is preferred.

[0140] In some embodiments, this disclosure provides antibodies comprising the ISVD that specifically binds to cMet according to the present invention, particularly anti-cMet heavy chain antibodies.

[0141] In some embodiments, this disclosure provides a binding molecule comprising an ISVD that specifically binds to cMet according to the present invention. In some embodiments, the cMet binding molecule comprises or consists of an antibody selected from: single-domain antibodies, nanobodies, VHH antibodies, or heavy chain antibodies. In other embodiments, the cMet binding molecule is selected from monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0142] In some embodiments, the cMet-binding molecule of the present invention comprises at least one cMet-specific ISVD of the present invention, for example, comprising two, three, four or more identical or different cMet-specific ISVDs of the present invention, preferably comprising two, three or four different cMet-specific ISVDs of the present invention. In some embodiments, the cMet-binding molecule provided by the present invention comprises two ISVDs binding to different epitopes of cMet.

[0143] In some embodiments, the anti-cMet ISVD contained in the cMet binding molecule of the present invention is preferably a humanized VHH domain. Compared with the camel-derived VHH, the humanized VHH exhibits a reduced human anti-camel antibody response in humans, thus improving the safety of antibody application.

[0144] In some embodiments, the ISVD or cMet-binding molecule of the present invention that specifically binds to cMet has one or more of the following properties: (1) Binds to human cMet with moderate or high affinity; (2) Specifically binds to cMet expressed on the cell surface; (3) In the presence of ligand HGF, block the binding of ligand HGF to cMet on the cell surface; (4) Internalization of cMet-expressing cells; (5) It exhibits cross-reactivity with human cMet and cynomolgus monkey cMet; (6) Synergistic binding mediated by different cMet double epitopes; (7) Cooperative endocytosis mediated by different cMet double epitopes.

[0145] In some embodiments, the cMet binding molecule of the present invention is in the form of a monospecific, bispecific, or multispecific antibody molecule. A multispecific antibody molecule may, for example, be a trispecific antibody molecule, which includes a first binding specificity against cMet and a second and third binding specificity against one or more other molecules.

[0146] In some embodiments, the cMet-binding molecule of the present invention comprises first and second ISVDs that specifically bind to the same epitope on cMet. In some embodiments, the first and second ISVDs are, respectively, ISVDs according to the present invention that specifically bind to the same epitope on cMet. In some further embodiments, the first and second ISVDs comprise: (i) a CDR1 comprising an amino acid sequence selected from SEQ ID NO: 18 or 41, or CDR2 and CDR3 comprising amino acid sequences selected from SEQ ID NO: 19 and SEQ ID NO: 20, or CDR1, CDR2 and CDR3, respectively; or (ii) amino acid sequences selected from SEQ ID NO: 23-25, or CDR1, CDR2 and CDR3, respectively. In some further embodiments, the first and second ISVDs comprise, or are substantially composed of, amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with, the amino acid sequences shown in one of SEQ ID NO: 16, 39, 40, or SEQ ID NO: 21 or 42. In some preferred embodiments, the first and second ISVDs comprise, or are substantially composed of, the amino acid sequences of SEQ ID NO: 16, 39, 40, or SEQ ID NO: 21 or 42.

[0147] In some embodiments, the cMet binding molecule of the present invention comprises first and second ISVDs that specifically bind to different epitopes on cMet. In some embodiments, the first and second ISVDs are, respectively, ISVDs according to the present invention 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 composed of amino acid sequences selected from SEQ ID NO: 23-25, respectively; and the second anti-cMet VHH domain comprises: CDR1 comprising or composed of amino acid sequences selected from SEQ ID NO: 18 or 41, and CDR2 and CDR3 comprising or composed of amino acid sequences selected from SEQ ID NO: 19 and SEQ ID NO: 20, respectively. In some further embodiments, the first anti-cMet VHH domain comprises, or is substantially composed of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, or is substantially composed of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16, 39, or 40. In some further embodiments, the first anti-cMet VHH domain comprises, or is substantially composed of, the amino acid sequence shown in SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, or is substantially composed of, the amino acid sequence shown in SEQ ID NO: 16, 39, or 40. In some preferred embodiments, the first ISVD contains the first anti-cMet VHH domain, and the second ISVD contains the second anti-cMet VHH domain. In some embodiments, the binding molecule is an anti-cMet biepisode antibody.

[0148] In some embodiments, the cMet binding molecule of the present invention may be in single-chain or multi-chain form. In some embodiments, the binding molecule further comprises a peptide linker for connecting different domains (e.g., two or more ISVDs) located on the same polypeptide chain. In other embodiments, the binding molecule further comprises an immunoglobulin Fc region.

[0149] In some embodiments, this disclosure provides anti-cMet dual epitope antibodies, wherein the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus, The first polypeptide chain contains: a first ISVD that specifically binds to cMet and an immunoglobulin Fc region; The second polypeptide chain contains: a second ISVD that specifically binds to cMet and the Fc region of immunoglobulin. Preferably, wherein: - The first polypeptide chain contains the sequence of SEQ ID NO: 88 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and - The second polypeptide chain contains the sequence of SEQ ID NO: 89 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. More preferably, the first polypeptide chain comprises or is composed of the sequence of SEQ ID NO: 88; and the second polypeptide chain comprises or is composed of the sequence of SEQ ID NO: 89.

[0150] In some embodiments, the cMet binding molecule of the present invention further comprises at least one ISVD that specifically binds to EGFR, preferably at least one (e.g., one) anti-EGFR ISVD according to the present invention.

[0151] In some embodiments, the cMet binding molecule of the present invention is linked to one or more other groups, residues or portions at its N-terminus or C-terminus via one or more peptide linkers, wherein the one or more other groups, residues or portions provide an increased half-life or provide effector functions such as antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) compared to a corresponding cMet binding molecule without said one or more other groups, residues or portions.

[0152] In some embodiments, the one or more other groups, residues, portions thereof that provide an increased half-life are selected from polyethylene glycol molecules, serum proteins or fragments thereof, portions that can bind to serum proteins (e.g., portions of serum albumin (such as human serum albumin), portions that can bind to serum immunoglobulins (such as IgG)), or Fc domains.

[0153] In some embodiments, the cMet-binding molecule of the present invention is linked to an ISVD that binds to human serum albumin via one or more peptide linkers at its N-terminus or C-terminus. In some embodiments, the ISVD that binds to human serum albumin comprises CDR1, which contains the amino acid sequence shown in SEQ ID NO: 46 or is composed thereof; CDR2, which contains the amino acid sequence shown in SEQ ID NO: 47 or is composed thereof; and CDR3, which contains the amino acid sequence shown in SEQ ID NO: 48 or is composed thereof. 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 with it.

[0154] In some embodiments, the cMet binding molecules provided herein are modified to increase or decrease their degree of glycosylation. The addition or deletion of glycosylation sites in the cMet binding molecule can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the cMet binding molecule contains an Fc region, the sugars linked to the Fc region can be altered. In some applications, modifications to remove unwanted glycosylation sites can be useful, for example, removing the fucose module to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, galactosylation modifications can be performed to modulate complement-dependent cytotoxicity (CDC). In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the cMet binding molecule provided herein to create Fc region variants to enhance the efficacy of, for example, the cMet binding molecules of the present invention in treating cancer.

[0155] II. The second aspect of this disclosure: ISVDs that specifically bind to EGFR and EGFR-binding molecules containing such ISVDs.

[0156] Epidermal growth factor receptor (EGFR, ErbB1, or HER1) is a type I transmembrane glycoprotein encoded by the c-erbBl 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 various human cancers; therefore, EGFR is an attractive target for cancer therapy.

[0157] In this specification, “EGFR” refers to EGFR from any species and includes EGFR isotypes, 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 isotypes: isotype 1 through isotype 4. EGFR is a transmembrane protein containing a large extracellular region, a single transmembrane domain, an intracellular juxtamembrane domain, a tyrosine kinase domain, and a C-terminal regulatory region. EGFR binding to its 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, such as cancer.

[0158] A second aspect of this disclosure provides an EGFR-specific ISVD (anti-EGFR ISVD) and an EGFR-binding molecule comprising said ISVD. In some embodiments, the anti-EGFR ISVD and EGFR-binding molecule of the present invention have improved tissue penetration compared to conventional four-chain antibodies. In some embodiments, the anti-EGFR ISVD and EGFR-binding molecule of the present invention bind to human EGFR with intermediate or low affinity. Herein, "intermediate affinity" refers to the binding affinity K of the antibody to the target epitope, as determined by, for example, surface plasmon resonance (SPR) technology. D Values ​​equal to or higher than 1 nM, but less than 50 nM; "low affinity" refers to the antibody's binding affinity K for the target epitope. D The value is greater than 50 nM. In some implementations, it is related to K. D Compared to "high affinity" anti-EGFR ISVD or EGFR-binding molecules containing said ISVD with values ​​below 1 nM, especially below 0.1 nM, the intermediate or low affinity anti-EGFR ISVD or EGFR-binding molecules containing said ISVD of the present invention have at least one of the following advantages: (i) increased tumor tissue specificity; (ii) reduced targeting toxicity, such as skin toxicity, in normal tissues; (iii) improved safety; and (iv) greater effectiveness in treating cancer.

[0159] The EGFR-specific immunoglobulin single variable domain (ISVD) of this invention comprises three complementarity-determining regions from the N-terminus to the C-terminus: CDR1, CDR2, and CDR3. In some embodiments, the EGFR-specific immunoglobulin single variable domain (ISVD) of this invention includes...

[0160] (a) Three CDRs in the amino acid sequence shown in one of SEQ ID NO: 1, SEQ ID NO: 31, and 94-99; (b) Three CDRs in one of the amino acid sequences shown in SEQ ID NO: 6, 32, and 100-102; or (c) Three CDRs in the amino acid sequence shown in one of SEQ ID NO: 11, 36, 84 and 105-114.

[0161] In some embodiments, the EGFR-specific ISVD of the present invention comprises a variant having, compared to the three CDRs described in any one of (a)-(c) above, a single or multiple CDRs having no more than one to three amino acid changes in each CDR, wherein said amino acid changes are the addition, deletion, or conserved amino acid substitution of an amino acid. 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.

[0162] In some embodiments, the EGFR-specific ISVD of the present invention comprises a number according to the Kabat number.

[0163] (a) CDR1 shown in SEQ ID NO: 3 or a variant of CDR1 shown in SEQ ID NO: 3 with no more than one amino acid change, CDR2 shown in SEQ ID NO: 4 or a variant of CDR2 shown in SEQ ID NO: 4 with no more than two amino acid changes, and CDR3 shown in SEQ ID NO: 5 or a variant of CDR3 shown in SEQ ID NO: 5 with no more than two amino acid changes; (b) CDR1 shown in SEQ ID NO: 8 or a variant of CDR1 shown in SEQ ID NO: 8 with no more than one amino acid change, CDR2 shown in SEQ ID NO: 9 or a variant of CDR2 shown in SEQ ID NO: 9 with no more than two amino acid changes (e.g., CDR2 shown in SEQ ID NO: 34 or 103), and CDR3 shown in SEQ ID NO: 10 or a variant of CDR3 shown in SEQ ID NO: 10 with no more than two amino acid changes (e.g., CDR3 shown in SEQ ID NO: 35 or 104); or (c) CDR1 shown in SEQ ID NO: 13 or a variant of CDR1 shown in SEQ ID NO: 13 with no more than one amino acid change, CDR2 shown in SEQ ID NO: 14 or a variant of CDR2 shown in SEQ ID NO: 14 with no more than two amino acid changes (e.g., CDR2 shown in SEQ ID NO: 38 or one of SEQ ID NO: 85, 115-120), and CDR3 shown in SEQ ID NO: 15 or a variant of CDR3 shown in SEQ ID NO: 15 with no more than two amino acid changes; The amino acid changes mentioned therein are the addition, deletion, or substitution of conserved amino acids.

[0164] In some embodiments, the present invention provides an immunoglobulin single variable domain (ISVD) that specifically binds to EGFR, wherein the ISVD comprises: (a) Each of the following contains SEQ ID Nos: 3, 4 and 5 or CDR1, CDR2 and CDR3 composed of them; (b) Each of the following contains SEQ ID Nos: 8, 9 and 10 or CDR1, CDR2 and CDR3 composed of SEQ ID Nos: 8, 9 and 10 respectively; (c) Each of the following contains SEQ ID Nos: 8, 34 and 35 or CDR1, CDR2 and CDR3 composed of them; (d) Each of the following contains SEQ ID Nos: 8, 103 and 104 or CDR1, CDR2 and CDR3 composed of them; (e) Each of the following contains SEQ ID Nos: 13, 14 and 15 or CDR1, CDR2 and CDR3 composed of them; (f) CDR1 containing SEQ ID No: 13 or composed thereof, CDR2 containing one of SEQ ID Nos: 115-120 or composed thereof, and CDR3 containing SEQ ID No: 15 or composed thereof; (g) Containing SEQ ID Nos: 13, 38, and 15, or CDR1, CDR2, and CDR3 thereof; or (h) comprises SEQ ID Nos: 13, 85, and 15, or CDR1, CDR2, and CDR3 thereof. 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.

[0165] In some embodiments, the EGFR-specific ISVD of the present invention comprises or consists of VHH. In some embodiments, the EGFR-specific ISVD of the present invention comprises or consists of the following sequences: (a) A sequence of one of SEQ ID NO: 1, 31 and 94-99 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (b) A sequence of one of SEQ ID NO: 6, 32, and 100-102, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or (c) A sequence of one of SEQ ID NO: 11, 36, 84, and 105-114, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; preferably, the amino acid variation does not occur in the CDR region. In some embodiments, the ISVD containing the amino acid sequence of SEQ ID NO: 31, 32, 84, or 36 is preferred. In other embodiments, the ISVD containing the amino acid sequence of SEQ ID NO: 32 is more preferred.

[0166] In some embodiments, this disclosure provides antibodies comprising the ISVD of the present invention that specifically binds to EGFR, particularly anti-EGFR heavy chain antibodies.

[0167] In some embodiments, this disclosure provides binding molecules that specifically bind to EGFR. In some embodiments, the EGFR-binding molecule comprises the EGFR-specific ISVD of the present invention. In some embodiments, the EGFR-binding molecule comprises or consists of an antibody selected from: single-domain antibodies, nanobodies, VHH antibodies, or heavy chain antibodies. In other embodiments, the EGFR-binding molecule is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0168] In some embodiments, the EGFR-binding molecule of the present invention comprises at least one EGFR-specific ISVD of the present invention, for example, comprising two, three, four or more identical or different EGFR-specific ISVDs of the present invention.

[0169] In some embodiments, the ISVD contained in the EGFR-binding molecule of the present invention is preferably a humanized VHH. Compared with camel VHH, humanized VHH has a reduced human anti-camel antibody response in humans, thus improving the safety of antibody application.

[0170] In some embodiments, the ISVD or EGFR-binding molecule of the present invention that specifically binds to EGFR has one or more of the following properties: (1) Binds to human EGFR with moderate or low affinity; (2) It exhibits cross-reactivity with human EGFR and cynomolgus monkey EGFR; (3) It specifically binds to EGFR expressed on the surface of tumor cells; (4) Internalization of tumor cells expressing EGFR.

[0171] In some embodiments, the EGFR-binding molecule of the present invention is in the form of a bispecific or multispecific antibody molecule. A multispecific antibody molecule may be, for example, a trispecific antibody molecule, comprising a first binding specificity against EGFR and a second and third binding specificity against one or more other molecules. In some embodiments, the EGFR-binding molecule of the present invention further comprises at least one ISVD that specifically binds to cMet, preferably at least one or two anti-cMet ISVDs according to the present invention.

[0172] In some embodiments, the EGFR-binding molecule of the present invention is linked to one or more other groups, residues or portions at its N-terminus or C-terminus via one or more peptide linkers, wherein the one or more other groups, residues or portions provide an increased half-life or provide effector functions such as antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) compared to a corresponding EGFR-binding molecule without said one or more other groups, residues or portions.

[0173] In some embodiments, the one or more other groups, residues, portions thereof that provide an increased half-life are selected from polyethylene glycol molecules, serum proteins or fragments thereof, portions that can bind to serum proteins (e.g., portions of serum albumin (such as human serum albumin), portions that can bind to serum immunoglobulins (such as IgG)), or Fc domains.

[0174] In some embodiments, the EGFR-binding molecule of the present invention is linked to an ISVD that binds to human serum albumin via one or more peptide linkers at its N-terminus or C-terminus. In some embodiments, the ISVD that binds to human serum albumin comprises, for example, CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 46 or composed thereof, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 47 or composed thereof, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 48 or composed thereof. 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 with it.

[0175] In some embodiments, the EGFR-binding molecules provided herein are modified to increase or decrease their degree of glycosylation. The addition or deletion of glycosylation sites in the EGFR-binding molecule can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the EGFR-binding molecule contains an Fc region, the sugars linked to the Fc region can be altered. In some applications, modifications to remove unwanted glycosylation sites can be useful, for example, removing the fucose module to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, galactosylation modifications can be performed to modulate complement-dependent cytotoxicity (CDC). In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the EGFR-binding molecule provided herein to create Fc region variants to enhance the efficacy of, for example, the EGFR-binding molecules of the present invention in treating cancer.

[0176] III. The third aspect of this disclosure: EGFR and cMet binding molecules

[0177] In a third aspect, this disclosure provides an EGFR and cMet binding molecule. Preferably, the binding molecule is a multispecific antibody capable of simultaneously binding EGFR and cMet. In some embodiments, the multispecific antibody according to the invention has one or more of the following properties:

[0178] (1) Binds to EGFR with moderate or low affinity, such as human EGFR; (2) Internalization of tumor cells expressing EGFR; (2) Specific binding to cMet, such as human cMet; (3) In the presence of ligand HGF, block the binding of ligand HGF to cMet on the cell surface; (4) Internalization of tumor cells expressing cMet; especially when binding to different epitopes of cMet, it produces a synergistic internalization enhancement effect; (5) It binds to tumor cells expressing both EGFR and cMet, and preferably exhibits synergistic binding activity; (6) Endocytosis by tumor cells expressing both EGFR and cMet, preferably exhibiting synergistic endocytic activity; (7) It exhibits cross-reactivity with human EGFR and cynomolgus monkey EGFR; (8) It exhibits cross-reactivity with human cMet and cynomolgus monkey cMet; and (9) Reduce tumor cell proliferation and metastasis.

[0179] Over the past two decades, monoclonal antibodies have been established as anti-tumor therapeutic agents, with several monoclonal antibodies targeting EGFR and cMet approved or in clinical development. However, acquired resistance developed by tumors limits their long-term efficacy. Since multispecific antibodies (e.g., bispecific and trispecific antibodies) can specifically bind to different antigenic epitopes, when designed to act simultaneously on two or more different signal transduction pathways, it is beneficial to avoid acquired resistance in tumors.

[0180] In some embodiments, the multispecific antibody of the present invention is an EGFR and cMet binding molecule comprising at least one EGFR-specific ISVD as defined in the second aspect of this disclosure (e.g., Part II herein), such as one or two EGFR-binding ISVDs of the present invention; and further comprising at least one cMet-binding ISVD as defined in the first aspect of this disclosure (e.g., Part I herein), such as one or two cMet-binding ISVDs of the present invention, optionally wherein ISVDs located on the same polypeptide chain are linked via one or more peptide linkers.

[0181] Generally, EGFR and cMet binding molecules containing 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 may contain one EGFR-binding ISVD and one cMet-binding ISVD, optionally linked by one peptide linker. A “trivalent” EGFR and cMet binding molecule may contain one EGFR-binding ISVD and two cMet-binding ISVDs, optionally linked by two peptide linkers; or it may contain two EGFR-binding ISVDs and one cMet-binding ISVD, optionally linked by two peptide linkers. A “tetravalent” EGFR and cMet binding molecule may contain two EGFR-binding ISVDs and two cMet-binding ISVDs, optionally linked by three peptide linkers; or it may contain one EGFR-binding ISVD and three cMet-binding ISVDs, optionally linked by three peptide linkers; or it may contain three EGFR-binding ISVDs and one cMet-binding ISVD, optionally linked by three peptide linkers, etc.

[0182] In a multivalent EGFR and cMet binding molecule, two or more ISVDs may be the same or different and may target the same antigenic epitope of EGFR, the same antigenic epitope of cMet, or different antigenic epitopes of EGFR and different antigenic epitopes of cMet; or any suitable combination thereof.

[0183] In some preferred embodiments, the multispecific EGFR and cMet binding molecule of the present invention comprises at least one (preferably one) ISVD targeting EGFR and two ISVDs targeting different epitopes on cMet. In other preferred embodiments, the multispecific EGFR and cMet binding molecule of the present invention comprises at least one (preferably one) ISVD targeting EGFR and two ISVDs targeting the same epitope on cMet.

[0184] The structural form of the multispecific antibody of this invention

[0185] Multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies) can be classified into many types based on their different components and construction methods. For example, based on the substantially symmetrical structure of the multispecific antibody, they can be divided into symmetrical and asymmetrical structures; based on the presence or absence of the Fc region of IgG, they can be divided into antibody patterns with and without the Fc region; based on the number of antigen-binding sites in the multispecific antibody, they can be divided into bivalent, trivalent, tetravalent, or more valent antibodies; based on the number of polypeptide chains constituting the multispecific antibody, they 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 known multispecific antibody structures are all within the scope of consideration of this invention.

[0186] Single-chain form of multispecific antibodies

[0187] In some embodiments, this 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 a polypeptide chain, wherein the ISVDs are linked to each other via peptide linkers or directly.

[0188] In some embodiments, this disclosure provides a multispecific antibody in single-chain form comprising a single polypeptide chain, wherein the polypeptide chain comprises, from the N-terminus to the C-terminus: (ISVD A ) n1 -(ISVD B ) n2 -(HLE) n3 -(ISVD A ) n4 -(ISVD B ) n5 -(HLE) n6， (I) Where n1, n2, n3, n4, n5, and n6 are independently selected from integers of 0, 1, or 2; where ISVD A and ISVD B These represent the ISVD domains that bind antigens A and B, respectively, where A and B are distinct from each other and independently selected from EGFR and cMet; where HLE represents a serum albumin-binding peptide as a half-life extension domain; where the symbol "-" indicates linkage via a peptide linker or direct linkage, preferably representing a peptide linker of 5-15 amino acids in length. Each ISVD in formula (I)A They can independently target the same or different epitopes on the A antigen. Each ISVD in formula (I) B They can independently target the same or different epitopes on the B antigen. In some cases, the multispecific antibodies are preferably divalent to hexavalent (i.e., n1+n2+n4+n5=2 to 6), more preferably not exceeding quadrivalent (i.e., n1+n2+n4+n5=2 to 4), for example divalent, trivalent or tetravalent.

[0189] 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 or may not include an HLE domain, depending on the requirements. In some embodiments, anti-EGFR ISVD... The number of domains does not exceed four, preferably not more than three, for example, one. In some embodiments, the number of anti-cMet ISVD domains does not exceed four, preferably not more than three, for example, two. In some embodiments, the ratio 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.

[0190] In some embodiments, this disclosure provides a multispecific antibody comprising a single polypeptide chain, wherein the polypeptide chain comprises, from the N-terminus to the C-terminus: (i) ISVD A -ISVD B ; (ii) ISVD A -ISVD B -ISVD B Each ISVD B Each targets the same epitope of the B antigen, or preferably different epitopes; (iii) ISVD B -ISVD A -ISVD B Each ISVD B Each targets the same epitope of the B antigen, or preferably different epitopes; Preferably, A represents EGFR and B represents cMet. In some embodiments, the polypeptide chains of (i)-(iii) contain an HLE located at the N-terminus or preferably the C-terminus.

[0191] In some embodiments, the HLE is an anti-HSA ISVD that binds to human serum albumin. In some embodiments, the anti-HSA ISVD comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 46 or composed thereof, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 47 or composed thereof, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 48 or composed thereof. 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 with it.

[0192] In some specific embodiments, the present invention provides a multispecific antibody comprising a single polypeptide chain, wherein the polypeptide chain comprises: (a) an EGFR-specific ISVD, preferably selected from EGFR-specific ISVDs according to the second aspect of this disclosure; and (b) a first ISVD and a second ISVD specifically binding cMet, preferably selected from cMet-specific ISVDs according to the first aspect of this disclosure, wherein the first ISVD and the second ISVD are the same or different; preferably, the first ISVD and the second ISVD specifically bind different epitopes of cMet. In some embodiments, the polypeptide chain further comprises an HSA-specific ISVD, 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 EGFR-specific ISVD, a first peptide linker, a cMet-specific first ISVD, a second peptide linker, a cMet-specific second ISVD, and optionally a third peptide linker and an HSA-specific ISVD.

[0193] Double-stranded multispecific antibodies

[0194] In some embodiments, this disclosure provides a double-stranded EGFR and cMet binding molecule (e.g., see [link to relevant documentation]). Figure 10 It comprises 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 extension domain, wherein the half-life extension domain is an immunoglobulin Fc region, and optionally the ISVDs located on the same polypeptide chain are linked by a peptide linker or directly linked.

[0195] In some embodiments, this disclosure provides a multispecific antibody comprising a first polypeptide chain and a second polypeptide chain, wherein

[0196] The first polypeptide chain, from the N-terminus to the C-terminus, includes: (ISVD) A ) n1 -(ISVD B ) n2 -HLE-(ISVD A ) n3 -(ISVD B ) n4， (II)

[0197] The second polypeptide chain, from the N-terminus to the C-terminus, includes: (ISVD) B ) m1 -(ISVD A ) m2 -HLE-(ISVD B ) m3 -(ISVD A ) m4， (III)

[0198] Where n1, n2, n3, and n4, and m1, m2, m3, and m4 are each selected independently from integers of 0, 1, or 2; Among them, ISVD A and ISVD B These represent the ISVD domains that bind antigens A and B, respectively, where A and B are distinct from each other and independently selected from EGFR and cMet; where HLE represents the immunoglobulin Fc region as a half-life extension domain, especially the human IgG1 or IgG4 Fc region; where the symbol "-" indicates linkage via a peptide linker or direct linkage, preferably representing a peptide linker of 5-15 amino acids in length. The ISVDs in formulas (II) and (III) A They can independently target the same or different epitopes on the A antigen. (II) and (III) are each ISVD B They can independently target the same or different epitopes on the B antigen. In some cases, the multispecific antibody preferably has a 2 to 6 valence (i.e., the sum of n1, n2, n3 and n4 and m1, m2, m3 and m4 is 2-6), more preferably, not exceeding 4 valence, for example, 2, 3 or 4 valence.

[0199] 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 anti-EGFR ISVD domain is... The number of domains does not exceed four, preferably not more than three, for example, one. In some embodiments, the number of cMet ISVD-resistant domains does not exceed four, preferably not more than three, for example, two. In some embodiments, the ratio of EGFR ISVD-resistant domains to cMet ISVD-resistant domains is 1:1 or 1:2.

[0200] In some preferred embodiments, the multispecific antibody comprises first and second polypeptide chains, wherein: The first polypeptide chain, from the N-terminus to the C-terminus, includes: (ISVD) A )-HLE ， The second polypeptide chain, from the N-terminus to the C-terminus, includes: (ISVD) B )-HLE ； or, The first polypeptide chain, from the N-terminus to the C-terminus, includes: (ISVD) B )-(ISVD A )-HLE ， The second polypeptide chain, from the N-terminus to the C-terminus, includes: (ISVD) B )-HLE ； Each ISVD B Each targets the same epitope of the B antigen, or preferably different epitopes; or, The first polypeptide chain, from the N-terminus to the C-terminus, includes: (ISVD A )-HLE ； The second polypeptide chain, from the N-terminus to the C-terminus, includes: (ISVD) B )-(ISVD B )-HLE ， Each ISVD B Each targets the same epitope of the B antigen, or preferably different epitopes; Preferably, A represents EGFR, and B represents cMet.

[0201] Due to the dimerization of the Fc region of immunoglobulins, the first and second polypeptide chains of the aforementioned multispecific antibody can associate to form heterodimers, thereby producing a double-stranded multispecific binding molecule. Preferably, to promote heterodimerization of the first and second polypeptide chains, a Knob-into-hole mutation, such as (T366W / T366S, L368A, Y407V) or (T366Y / Y407T) mutation, can be introduced into the Fc region of the first and second polypeptide chains. Preferably, the Fc region contains an amino acid sequence from human IgG1 or IgG4; more preferably, the Fc region also contains a mutation that reduces or eliminates Fcγ receptor binding, such as the LALA mutation.

[0202] In some specific embodiments, the EGFR and cMet binding molecule of the present invention is in a double-stranded form, comprising: (a) An EGFR-specific ISVD is located on a polypeptide chain, preferably selected from EGFR-specific ISVDs according to a second aspect of this disclosure, wherein an Fc region (Fc subunit) is attached to the C-terminus of the ISVD; and (b) A first ISVD and a second ISVD that specifically bind cMet are located on another chain from the N-terminus to the C-terminus, preferably selected from specific cMet-binding ISVDs according to a first aspect of this 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 linked via one or more peptide linkers. The Fc region (Fc subunit) is connected to the C end of the second ISVD.

[0203] In some specific embodiments, the EGFR and cMet binding molecule of the present invention is a double-stranded multispecific antibody comprising a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus, the first polypeptide chain comprises: an ISVD specifically binding to EGFR and an immunoglobulin Fc region, and the second polypeptide chain comprises: a first ISVD specifically binding to cMet, a peptide linker, a second ISVD specifically binding to cMet, and an immunoglobulin Fc region.

[0204] Example antigen domain combination

[0205] In some embodiments of the multispecific antibody according to the present invention, preferably, the multispecific antibody according to the present invention comprises at least one anti-cMet ISVD according to a first aspect of the present disclosure (e.g., as defined in Part I) and / or at least one anti-EGFR ISVD according to a second aspect of the present disclosure (e.g., as defined in Part II).

[0206] In some further preferred embodiments, the multispecific antibody according to the invention comprises first and second ISVDs that specifically bind to the same epitope on cMet. In some embodiments, the first and second ISVDs are respectively anti-cMet ISVDs that specifically bind to the same epitope on cMet according to the first aspect of disclosure. In some further embodiments, the first and second ISVDs comprise: (i) CDR1 comprising an amino acid sequence selected from SEQ ID NO: 18 or 41, or CDR2 and CDR3 comprising amino acid sequences selected from SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or (ii) CDR1, CDR2 and CDR3 comprising amino acid sequences selected from SEQ ID NO: 23-25, respectively. In some further embodiments, the first and second ISVDs comprise, or are substantially composed of, amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with, the amino acid sequences shown in one of SEQ ID NO: 16, 39, 40, or SEQ ID NO: 21 or 42. In some preferred embodiments, the first and second ISVDs comprise, or are substantially composed of, the amino acid sequences of SEQ ID NO: 16, 39, 40, or SEQ ID NO: 21 or 42.

[0207] In some further preferred embodiments, the multispecific antibody according to the invention comprises first and second ISVDs that specifically bind to different epitopes on cMet. In some embodiments, the first and second ISVDs are respectively anti-cMet ISVDs that specifically bind to different epitopes on cMet according to the first aspect of the present disclosure. 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 composed of amino acid sequences selected from SEQ ID NO: 23-25, respectively; and the second anti-cMet VHH domain comprises: CDR1 comprising or composed of amino acid sequences selected from SEQ ID NO: 18 or 41, and CDR2 and CDR3 comprising or composed of amino acid sequences selected from SEQ ID NO: 19 and SEQ ID NO: 20, respectively. In some further embodiments, the first anti-cMet VHH domain comprises, or is substantially composed of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, or is substantially composed of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16, 39, or 40. In some further embodiments, the first anti-cMet VHH domain comprises, or is substantially composed of, the amino acid sequence shown in SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, or is substantially composed of, the amino acid sequence shown in SEQ ID NO: 16, 39, or 40. In some preferred embodiments, the first ISVD includes the first anti-cMet VHH domain, and the second ISVD includes the second anti-cMet VHH domain.

[0208] In some preferred embodiments of the multispecific antibodies of the present invention comprising first and second ISVDs binding to the same or different cMet epitopes, the antibodies further comprise at least one (preferably one) anti-EGFR ISVD according to the second aspect of this disclosure. In some embodiments, the anti-EGFR ISVD comprises: (i) Containing amino acid sequences of SEQ ID NOs: 3, 4 and 5, or CDR1, CDR2 and CDR3 composed thereof; (ii) Containing amino acid sequences of SEQ ID NOs: 8, 9, and 10, or amino acid sequences of SEQ ID NOs: 8, 34, and 35, or amino acid sequences of SEQ ID NOs: 8, 103, and 104, or CDR1, CDR2, and CDR3 composed thereof; or (iii) Each of the amino acid sequences comprising SEQ ID NOs: 13, 14 and 15, or SEQ ID NOs: 13, 85 and 15, or SEQ ID NOs: 13, 38 and 15, or CDR1, CDR2 and CDR3 thereof. More preferably, the anti-EGFR ISVD: (a) Contains an amino acid sequence shown in one of SEQ ID NO: 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 with it, or is substantially composed of it, or is composed of it. (b) Contains, or is substantially composed of, an amino acid sequence shown in one of SEQ ID NO: 6, 32, 100-102, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such sequence; or (c) Contains an amino acid sequence shown in one of SEQ ID NO: 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 with it, or is substantially composed of it, or is composed of it. More preferably, the anti-EGFR ISVD: (a) Contains, is substantially composed of, or is composed of the amino acid sequence shown in SEQ ID NO: 31; (b) Contains, is substantially composed of, or is composed of the amino acid sequence shown in SEQ ID NO: 32; or (c) Contains, or is substantially composed of, or is composed of the amino acid sequence shown in SEQ ID NO: 84.

[0209] In some embodiments, this disclosure provides a single-chain multispecific antibody comprising a combination of anti-EGFRISVD and first and second anti-cMet ISVD: (a) An anti-EGFR ISVD containing three CDRs of the amino acid sequence of SEQ ID NO: 1, 6, or 13, and a first anti-cMet ISVD containing three CDRs of the amino acid sequence of SEQ ID NO: 21, and a second anti-cMet ISVD containing three CDRs of the amino acid sequence of SEQ ID NO: 16; or (b) The anti-EGFR ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 1, 6, or 13, and the first anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 16, and the second anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 21; wherein the CDRs are preferably defined according to Kabat. Preferably, the antibody comprises a combination of an anti-EGFR ISVD selected from the following and a first and a second anti-cMet ISVD: (a) An anti-EGFR ISVD containing the amino acid sequence of SEQ ID NO: 1, 6, or 13, and a first anti-cMet ISVD containing the amino acid sequence of SEQ ID NO: 21, and a second anti-cMet ISVD containing the amino acid sequence of SEQ ID NO: 16; or (b) 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: 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 the N-terminus to the C-terminus, the anti-EGFR ISVD, a peptide linker, the first anti-cMet ISVD, a peptide linker, and the second anti-cMet ISVD.

[0210] In some embodiments, this disclosure provides a double-chain multispecific antibody comprising a combination of anti-EGFRISVD selected from the group consisting of first and second anti-cMet ISVD: (a) The anti-EGFR ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 31, 32 or 84, and the first anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 39; (b) The anti-EGFR ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 31, 32 or 84, and the first anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 39 and the second anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 39; (c) The anti-EGFR ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 31, 32, or 84, and the first anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 42, and the second anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 42; or (d) The anti-EGFR ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 31, 32, or 84, and the first anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 21, and the second anti-cMet ISVD contains three CDRs in the amino acid sequence of SEQ ID NO: 40; wherein the CDRs are preferably defined according to Kabat. Preferably, the antibody comprises a combination of an anti-EGFR ISVD selected from the following and a first and a second anti-cMet ISVD: (a) An anti-EGFR ISVD containing the amino acid sequence of SEQ ID NO: 31, 32, or 84, and a first anti-cMet ISVD containing the amino acid sequence of SEQ ID NO: 42, and a second anti-cMet ISVD containing the amino acid sequence of SEQ ID NO: 39; or (b) The anti-EGFR ISVD contains the amino acid sequence of SEQ ID NO: 31, 32 or 84, and the first anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 39 and the second anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 39; (c) The anti-EGFR ISVD contains the amino acid sequence of SEQ ID NO: 31, 32, or 84, and the first anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 42, and the second anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 42; or (d) The anti-EGFR ISVD contains the amino acid sequence of SEQ ID NO: 31, 32 or 84, and the first anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 21 and the second anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 40; More preferably, the antibody comprises a combination of anti-EGFR ISVD and first and second anti-cMet ISVD: (a) The anti-EGFR ISVD contains the amino acid sequence of SEQ ID NO: 31, the first anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 42, and the second anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 39; (b) The anti-EGFR ISVD contains the amino acid sequence of SEQ ID NO: 32, the first anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 42, and the second anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 39; (c) The anti-EGFR ISVD contains the amino acid sequence of SEQ ID NO: 84, the first anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 21, and the second anti-cMet ISVD contains the amino acid sequence of SEQ ID NO: 40; More preferably, the antibody comprises: an anti-EGFR ISVD containing the amino acid sequence of SEQ ID NO: 31, a first anti-cMet ISVD containing the amino acid sequence of SEQ ID NO: 42, and a second anti-cMet ISVD containing the amino acid sequence of SEQ ID NO: 39.

[0211] In some further embodiments of this double-stranded multispecific antibody, the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the 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.

[0212] Exemplary peptide linkers

[0213] In some embodiments of the multispecific antibodies of the present invention, the antibodies comprise peptide linkers. Hereinafter, the term "peptide linker" used in the binding molecules and antibodies of the present invention refers to a short amino acid sequence consisting of natural amino acids. There are no particular limitations on the length or flexibility of the peptide linkers used in the binding molecules and antibodies of the present invention. The peptide linkers used in the binding molecules and antibodies of the present invention can be any suitable amino acid sequence, particularly 1 to 50, preferably 1 to 30, for example, an amino acid sequence of 1 to 10 amino acid residues. In some embodiments, the peptide linker is substantially composed of glycine (G) and serine (S) residues, preferably comprising one or more repeats of a peptide motif such as 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 connecting two ISVDs in the binding molecules and antibodies of the present invention preferably comprises the amino acid sequence of SEQ ID NO: 44. Based on the disclosure herein, those skilled in the art will be able to determine the optimal peptide linker for use in the binding molecule or antibody of the present invention after a limited set of routine experiments.

[0214] Exemplary immunoglobulin Fc region

[0215] In some embodiments of the multispecific antibody of the present invention, the antibody comprises an Fc region (Fc subunit). The Fc region used in the binding molecule and antibody of the present invention may be an Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0216] In some embodiments, the Fc region of the EGFR and cMet binding molecule of the present invention uses a "knobs-into-holes" technique (see, for example, John BBRidgway 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 domaininterface of a homodimer using a phage display library. J.Mol.Biol(1997, 270: pp. 26-35), this technique modifies the interface between the two chains of the EGFR and cMet binding molecule of the present invention to promote proper association between the two chains. Typically, this technique involves introducing a “protrusion” at the interface of one chain and a corresponding “cavity” at the interface of the other chain to be paired with, allowing the protrusion to be placed within the cavity. A 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 with. The protrusion can be constructed by replacing a small amino acid side chain from the CH3 domain interface of the heavy chain constant domain of one chain with a larger side chain (e.g., tyrosine or tryptophan). A compensating 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 with by replacing the large amino acid side chain with a smaller side chain (e.g., alanine or threonine).

[0217] In one embodiment, the Fc regions on both chains of the EGFR and cMet binding molecule of the present invention contain modifications that increase the binding affinity for Fc receptors. 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 molecule 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 molecule of the present invention, particularly within its CH2 region. In one embodiment, the EGFR and cMet binding molecule of the present invention includes an amino acid substitution at position 329 (EU number) of the heavy chain. In a specific embodiment, the amino acid substitution is P329G. In one embodiment, the EGFR and cMet binding molecule of the present invention includes amino acid substitutions at positions 234 and 235 (EU number) of the heavy chain. In one specific embodiment, the amino acid substitution is L234A and L235A (LALA mutation) (Armour KL et al., Recombinant human IgG molecules lacking Fcgamma receptor Ibinding and monocyte triggering activities). Eur J Immunol, 1999. 29(8): 2613-24). In one embodiment, the EGFR and cMet binding molecule of the present invention comprises amino acid substitutions at positions 234, 235, and 329 (EU number) of the heavy chain. In a specific embodiment, the EGFR and cMet binding molecule of the present invention comprises amino acid substitutions L234A, L235A, and P329G (EU number) in the heavy chain.

[0218] In one embodiment, the Fc region of the binding molecule and antibody of the present invention has a mutation YTE, namely a combination of mutations M252Y (Met252Tyr), S254T (Ser254Thr), and T256E (Thr256Glu) according to Kabat's EU index number, to provide an increased half-life.

[0219] In some embodiments, the immunoglobulin Fc region of the double-stranded multispecific antibody of the present invention comprises: An Fc strand with a Hole mutation (also called a clasp strand) comprising the sequence of SEQ ID NO: 137 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and An Fc strand (also called a knot strand) with a Knob mutation, which contains the sequence of SEQ ID NO: 138 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it.

[0220] Exemplary multispecific antibodies

[0221] In some embodiments, the present invention provides a multispecific antibody comprising 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 with it; and - 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 with it. Preferably, in some embodiments, (i) The first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 70 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 73, 75 or 74 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (ii) The first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 71 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 73, 75 or 74 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (iii) The first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 72 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 73, 75 or 74 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (iv) The first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 86 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 87 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it.

[0222] More preferably, in some embodiments, (i) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (ii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (iii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 87.

[0223] In other embodiments, this disclosure provides multispecific antibodies comprising a single polypeptide chain, wherein the polypeptide chain comprises a sequence selected from SEQ ID NO: 51-66 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or wherein the polypeptide chain comprises a sequence selected from SEQ ID NO: 67-68 and 76-83 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. Preferably, in some embodiments, the polypeptide chain comprises or is composed of sequences selected from SEQ ID NO: 51-56.

[0224] The properties of the EGFR and cMet binding molecules of this invention

[0225] EGFR affinity

[0226] The EGFR and cMet binding molecule of the present invention comprises an ISVD that binds EGFR with intermediate or low affinity. EGFR is expressed at low levels in normal tissues (e.g., skin). The EGFR and cMet binding molecule of the present invention, which binds EGFR with intermediate or low affinity, exhibits reduced on-target toxicity in normal tissues while still being able to target tumors expressing high levels of EGFR, thereby resulting in an improved safety profile.

[0227] In some embodiments, the ISVD contained in the binding molecule of the present invention that binds to human EGFR can also bind to cynomolgus monkey EGFR. For example, the ISVD that binds to human EGFR can have a similar K... D It binds to EGFR in cynomolgus monkeys. In this paper, the approximate K... D It refers to the two Ks being compared. D The difference between values ​​does not exceed 10 times, preferably not more than about 5 times, or more preferably not more than 2 times. The EGFR and cMet binding molecule of the present invention is able to bind to human EGFR and cynomolgus monkey EGFR. This cross-reactivity is advantageous because it allows for the administration and safety testing of the EGFR and cMet binding molecule of the present invention in cynomolgus monkeys during preclinical development.

[0228] cMet Affinity

[0229] The EGFR and cMet binding molecule of the present invention comprises an ISVD that specifically binds to cMet. In some embodiments, the EGFR and cMet binding molecule of the present invention comprises at least two ISVDs that specifically bind to different epitopes on cMet, which gives the EGFR and cMet binding molecule of the present invention better tumor targeting and internalization.

[0230] In some embodiments, the ISVD contained in the binding molecule of the present invention, which binds to human cMet, can be K D It binds to human cMet with an affinity lower 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. Alternatively, ISVDs binding to human cMet can bind at K... D It 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.

[0231] In some embodiments, the ISVD contained in the binding molecule of the present invention that binds to human cMet can also bind to cynomolgus monkey cMet. For example, the ISVD bound to human cMet can have a similar K D Binding to cynomolgus cMet. The EGFR and cMet binding molecule of the present invention is able to bind to both human cMet and cynomolgus cMet. This cross-reactivity is advantageous because it allows for the administration and safety testing of the EGFR and cMet binding molecule of the present invention in cynomolgus monkeys during preclinical development.

[0232] Simultaneously specifically binds to EGFR and cMet

[0233] The EGFR and cMet binding molecule of the present invention can bind to both EGFR and cMet targets simultaneously. Many tumors are known to co-express both EGFR and cMet; therefore, the EGFR and cMet binding molecule of the present invention, which has the ability to bind both EGFR and cMet simultaneously, is advantageous.

[0234] Internalization

[0235] The EGFR and cMet binding molecules of this invention can mediate efficient internalization. This is particularly useful for conjugates or conjugates because it ensures that the conjugates or conjugates are internalized into the cell and delivered to the lysosome, where the antibody molecules are subsequently degraded and the drug is released into the cell to exert its cellular effects, such as cytotoxicity.

[0236] Cellular internalization of the EGFR and cMet binding molecules of the present invention can be analyzed by contacting live cells with the EGFR and cMet binding molecules of the present invention and detecting the EGFR and cMet binding molecules of the present invention after a sufficient internalization time. Antibody molecules are determined not to be internalized when they remain on the cell surface (e.g., detected on the cell surface and / or not detected inside the cell). Detection of antibody molecules inside the cell (e.g., in the cytoplasm or organelles) determines that the antibody molecules have been internalized.

[0237] Compared to EGFR- or cMet-specific binding molecules, the EGFR and cMet-binding molecules of the present invention exhibit greater selectivity for tumor cells co-expressing both targets, thereby minimizing their adverse effects on normal tissues that do not show significant levels of EGFR and cMet co-expression.

[0238] In vitro activity

[0239] The EGFR and cMet binding molecule of this invention exhibits cytotoxic activity in vitro. Cytotoxic activity can be measured using an in vitro cell viability assay, such as the CellTiter-Glo® (Promega) assay. In some embodiments, the cells are cells expressing both EGFR and cMet.

[0240] In some embodiments, the EGFR and cMet binding molecules of the present invention can increase the killing effect on cells expressing significant amounts of both EGFR and cMet, such as tumor cells, compared to cells expressing low levels of EGFR and / or cMet. Cells expressing significant amounts of both EGFR and cMet can be identified by measuring the relative EGFR and cMet receptor density on the cell surface.

[0241] in vivo activity

[0242] The EGFR and cMet binding molecules of this invention can inhibit the development or progression of cancer in vivo. In some embodiments, the cancer may be a cancer expressing both or one of EGFR and cMet. Cancer cells may express one or both of EGFR and cMet on their cell surface. The cancer may be, for example, selected from lung cancer (e.g., squamous cell carcinoma of the lung, adenocarcinoma of the lung, non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), colon cancer, and pharyngeal squamous cell carcinoma.

[0243] IV. The fourth aspect of this disclosure: nucleic acids, vectors, hosts, and production methods

[0244] In a fourth aspect, this disclosure provides ISVDs, binding molecules and polypeptides encoding nucleic acids, vectors, host cells and production methods according to the first to third aspects of this disclosure.

[0245] In one embodiment, this disclosure provides a method for preparing the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody of the present invention, wherein the method includes culturing a host cell containing a nucleic acid encoding the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody, or an expression vector containing the nucleic acid, under conditions suitable for expressing a 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 one embodiment, the method further includes recovering the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody from the host cell (or host cell culture medium).

[0246] To recombinantly generate the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody of the present invention, the nucleic acid encoding the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody of the present invention is first isolated, and said nucleic acid is inserted into a vector for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes capable of specifically binding to the nucleic acid encoding the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody of the present invention.

[0247] The ISVD, EGFR-binding molecules, cMet-binding molecules, or multispecific antibodies of the present invention prepared as described herein can be purified using known prior art techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the ISVD, 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, etc.

[0248] V. Fifth aspect of this disclosure: Immunofusable fusion compounds, immunoconjugates, and antibody-drug conjugates (ADCs)

[0249] In a fifth aspect, this disclosure provides immunofusions, immunoconjugates, and antibody-drug conjugates comprising ISVDs, antibodies, and antigen-binding molecules according to the first to third aspects of this disclosure.

[0250] Immunofusions and Immunoconjugates

[0251] In one embodiment, this disclosure provides an immunofusion or immunoconjugate produced by fusing or conjugating an ISVD, antibody, and antigen-binding molecule according to the first to third aspects of this disclosure to a heterologous molecule.

[0252] In one embodiment, in the immunofusion, the antigen-binding molecule (such as an antibody) of the present invention is linked to a heterologous peptide or polypeptide molecule directly or via an amino acid linker. Heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that impart another functional activity to the fusion, or tagged peptides that facilitate the purification or detection of the immunofusion.

[0253] In one embodiment, in the immunoconjugate, the antigen-binding molecule (such as an antibody) of the present invention is conjugated to a therapeutic agent, diagnostic agent, or detectable agent. In the conjugate, chemical linkers can be used to covalently link different entities of the conjugate. In some cases, it is advantageous that the chemical linker is a "cleavable linker" that facilitates the release of the antigen-binding molecule polypeptide upon delivery to the target site. For example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used.

[0254] In embodiments where a therapeutic agent is conjugated, the therapeutic agent suitable for the conjugation includes, but is not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), drugs, or radioisotopes.

[0255] In embodiments conjugated with diagnostic or detectable agents, such conjugates can be used as part of clinical testing methods (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnostics and detections can be achieved by conjugating antibodies to detectable agents, including but not limited to a variety of enzymes such as horseradish peroxidase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0256] In some embodiments, therapeutic agents suitable for the conjugate include, but are not limited to, drugs (e.g., antitumor drugs); in other embodiments, diagnostic agents suitable for the conjugate include, but are not limited to, radiodiagnostic agents, fluorescent substances, or luminescent substances.

[0257] Antibody-drug conjugates (ADCs)

[0258] In some preferred embodiments, this disclosure provides antibody-drug conjugates (ADCs).

[0259] In some embodiments, this disclosure provides antibody-drug conjugates (ADCs) having formula (I0) or pharmaceutically acceptable salts or solvates thereof: Ab-(LD) p (I0) in: Ab is the binding molecule of the present invention, such as an antibody, such as the antibody or fragment thereof (e.g., antigen-binding fragment) that specifically binds to EGFR and / or cMet as described above. L is the connector; D represents a drug, such as an anti-tumor compound; 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. In some embodiments, Ab is an EGFR and cMet binding molecule according to a third aspect of this disclosure, particularly a multispecific antibody according to a third aspect of this disclosure. In some particularly preferred embodiments, the multispecific antibody comprises first and second polypeptide chains, wherein: (i) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (ii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (iii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 87.

[0260] It is understood that the -LD portion can be covalently linked to the Ab in any manner known in the art. In some embodiments, the -LD portion is covalently linked to the Ab via a sulfur (S) atom from the Ab, i.e., the -LD portion and the Ab are linked via -S-. In some embodiments, the sulfur atom originates from the opening of interchain disulfide bonds in the Ab. In some embodiments, the sulfur atom originates from (engineered or natural) cysteine ​​residues in the Ab.

[0261] 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) in: Ab is the binding molecule of the present invention, such as an antibody, such as the antibody or fragment thereof (e.g., antigen-binding fragment) that specifically binds to EGFR and / or cMet as described above. L is the connector; D is a drug, such as an anti-tumor compound; and p is an integer selected from 1 to 16, such as integers 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 derived from antibody Ab. In some embodiments, Ab is an EGFR and cMet binding molecule according to the third aspect of this disclosure, particularly a multispecific antibody according to the third aspect of this disclosure. In some particularly preferred embodiments, the multispecific antibody comprises first and second polypeptide chains, wherein: (i) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (ii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (iii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 87.

[0262] It can be understood that p refers to the number of -LDs linked to Ab in the antibody-drug conjugate molecule of formula (I0) or (I), which can also be called DAR.

[0263] In some embodiments, D in formula (I0) or (I) of the present invention can be any antitumor compound, as long as it has antitumor effects and has a structural portion that can be linked to the linker, without particular limitation. The antitumor compound can be a pharmaceutically active compound that acts on tumors. For antitumor compounds, preferably part or all of the linker can be cleaved within tumor cells, releasing the antitumor compound portion, thereby exhibiting an antitumor effect.

[0264] In some implementations, the antitumor compound may be, for example, a cytotoxic agent, such as camptothecin or aurestatin.

[0265] In some implementations, D has the structure shown in formula (D-1a) or formula (D-1b): Equation (D-1a) Where R 1a Selected from H and C1-C6 alkyl groups; R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ; R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and R 4a and R 5a Independently selected from H and C1-C4 alkyl groups; In some implementation schemes, R 1a For H; R 2a It is a C1-C6 alkyl group; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl; Equation (D-1b) Where R 1b R 2b R 3b R 4b R 5b and R 8b Each is independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; R 6b and R 7b Each is independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy; R 9b Selected from C 1-8 Alkyl groups and COOH; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and R 10b Selected from OH and H.

[0266] In some implementation schemes, R 1b R 4b and R 8b Each is independently selected from C 1-2 Alkyl; preferably methyl; R 2b R 3b and R 5b Each is independently selected from C 3-4 alkyl; R 6b and R 7b Each is independently selected from C 1-2 alkoxy groups; and R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H.

[0267] It can be understood that the wavy line in the structural formula indicates that the valence bond is connected to the rest of the molecule. For example, the wavy line in the D structural formula indicates that the valence bond is connected to L.

[0268] In some implementations, D has the structure shown in formula (D-2a) or formula (D-2b): Equation (D-2a), Where R 1a R 2a R 3a and R 4a As defined above; or Equation (D-2b) 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.

[0269] In some implementations, D has the structure shown in formula (D-3a) or (D-3b): (D-3a) or (D-3b).

[0270] In some implementations, D has the structure shown in formula (D-4a) or (D-4b): (D-4a) or (D-4b).

[0271] In some embodiments, in the ADC of this disclosure, the drug is Exatecan, Dxd, SN-38, monomethylauratestatin E (MMAE), or MMAF. The structural formula is shown below: , , , . In some implementations, -L- has the following structure: -Z-L1-L2-L3- in Z is selected from , , , , and , where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non-existent, , , and , where n1 and m1 are each independently an integer selected from 0 to 20, for example, an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8; L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and L3 is selected from: , , , and Where X is selected from -NH-, -O-, and -S-; R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; n3 and n4 are independently 1, 2, 3, 4, 5, or 6; and Z is connected to S on Ab, and L3 is connected to D.

[0272] In some implementations, -L- has the following structure: -Z-L1-L2-L3-

[0273] in

[0274] Z is selected from , , where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non-existent, and , where n1 is an integer independently selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8; L2 is a peptide residue composed of 2-8 amino acids; and L3 is selected from: , and , where R 1c 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.

[0275] 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.

[0276] In some implementation schemes, Z is selected from , , where m is 1, 2, 3, 4, 5, 6, 7 or 8.

[0277] In some implementation schemes, Z is selected from , or .

[0278] In some implementations, L1 is selected from non-existent, and , where n1 is an integer independently selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0279] In some implementations, L1 is selected from non-existent, and .

[0280] In some embodiments, L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6, or 7 amino acids.

[0281] In some embodiments, the amino acid residue or amino acid is preferably an L-amino acid. Moreover, in addition to α-amino acids, the amino acid residue or amino acid can be an amino acid residue or amino acid with structures such as β-alanine, ε-aminohexanoic acid, γ-aminobutyric acid, etc., and can also be a non-natural amino acid, such as an N-methylated amino acid.

[0282] 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).

[0283] In some implementations, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-.

[0284] In some implementations, L2 is selected from -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, and -Gly-Gly-Phe-Gly-. In some implementations, L2 is -Gly-. In some implementations, L2 is selected from -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, and -Glu-Val-Cit-.

[0285] It should be understood that L2 is connected to L1 or Z through the amino group of the amino acid on the left, and to L3 through the carbonyl group of the amino acid on the right, which is consistent with the explanation below.

[0286] In some implementations, L3 is selected from: , , , and , Where R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is selected independently from each of the following groups: , , and 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.

[0287] In some implementations, L3 is selected from: , , , and , The variables are as defined in this paper.

[0288] In some implementations, L3 is selected from: , , , and , The variables are as defined in this paper.

[0289] In some implementations, L3 is selected from: , and The variables are as defined in this paper.

[0290] In some embodiments, Su is selected from xylose, arabinose, xyuronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid, and mannuronic acid.

[0291] In some implementation schemes, Su is selected from .

[0292] In some implementations, Su is independently: .

[0293] In some implementation schemes, Su is independently .

[0294] In some implementation schemes, Su is independently .

[0295] In some implementations, L3 is selected from: , , , and .

[0296] In some implementations, L3 is selected from and .

[0297] In some implementations, L3 is selected from: , or .

[0298] It should be understood that L3 is connected to L2 via the amino group on the left and to D via the carbonyl group on the right, which is consistent with the explanation below.

[0299] In some implementations, -Z-L1-L2-L3- are each independently selected from the following structures: , , , , , , , , ,and .

[0300] Where each m is an integer selected from 1 to 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 group is connected to the S on the left side of Ab and to the D on the right side.

[0301] In some implementations, -Z-L1-L2-L3- are each independently selected from the following structures. , Where n1 is an integer independently selected from 0 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 8.

[0302] It should be understood that, unless otherwise specified and without contradiction in the context, for the ADCs of this invention, the left-hand bond of the divalent group shown herein is connected to an Ab group or a group near the Ab end, and the right-hand bond of the divalent group is connected to a D group or a group near the D end. For example, when L2 is In this case, the amino group on the left is connected to L1, and the carbonyl group on the right is connected to L3; In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, 2-4, or 2-6.

[0303] In some embodiments, the antibody-drug conjugate is selected from...

[0304] Wherein Ab is the 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, such as 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, 2-4, or 2-6. In some embodiments, the multispecific antibody is a multispecific antibody according to a third aspect of this disclosure. In some particularly preferred embodiments, the multispecific antibody comprises first and second polypeptide chains, wherein: (i) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (ii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (iii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 87.

[0305] It should be understood that the S atom linked to the Ab in the above ADC originates from the antibody Ab. The Ab breaks its disulfide bond (e.g., an interchain disulfide bond) under the action of a reducing agent such as TCEP, generating a thiol group (-SH), which then links to the terminal functional group of the linker, such as the maleimide moiety. In some embodiments, the S atom linked to the Ab originates from the cysteine ​​residue of the Ab.

[0306] It should be noted that the above and other technical solutions and one or more features of this disclosure can be arbitrarily combined to constitute technical solutions not directly described herein, and these undescribed technical solutions are also covered within the scope of this application.

[0307] Preparation of ADC molecules of the present invention

[0308] Another aspect of the present invention provides a method for preparing an ADC using the antibody of the present invention. In this invention, "ADC" is defined as an antibody coupled via a linker (L) to an active substance (D, also referred to as a payload) having biological and / or pharmaceutical activity. The method comprises coupling the antibody (Ab) of the present invention to one or more active substances D via one or more linkers (L) (e.g., as defined in the present invention). Preferably, the linker-active substance site is specifically coupled to the antibody.

[0309] In some embodiments, the method includes preparing an Ab for an ADC, comprising culturing a host cell containing a nucleic acid encoding the Ab (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector containing the nucleic acid, as provided above, under conditions suitable for expression of the Ab or its chain, as provided above, and optionally recovering the Ab from the host cell (or host cell culture medium).

[0310] In some implementations, the method includes the following steps: (a) Add antibody Ab to buffer solution, add reducing agent, and then incubate; (b) Adding a linker-loaded linker to the reaction solution in step (a) for coupling yields the crude product; and (c) Optionally, the crude product is purified to obtain the antibody-drug conjugate of the present invention; Ab is defined as above.

[0311] It should be understood that the linker-loaded compound reacts with Ab to provide the -LD portion in compound I, and the structure of the linker-loaded compound can be determined according to the prior art, provided that -LD is clearly defined.

[0312] In some implementations, the buffer solution in step a) is a PBS buffer, preferably with a pH of 5.0-9.0, for example 6.0-8.0.

[0313] In some implementations, the reducing agent in step a) is TCEP.

[0314] In some implementations, the connector-payload has the following structure: Z'-L1-L2-L3-D, where L1, L2, L3, and D are as defined above, and Z' is... or m is defined above, for example, as 1, 2, 3, 4, 5, 6, 7 or 8.

[0315] In some implementations, Z' is selected from , or .

[0316] In some implementations, for the synthesis where Z is , , or The ADC method also includes an additional hydrolysis step that opens the ring of maleimide.

[0317] In some implementations, the steps are performed under the specific reaction conditions disclosed in the embodiments.

[0318] It should be noted that implementation schemes obtained by varying the range or specific values ​​of the specific reaction conditions disclosed in the embodiments by 100%, 80%, 60%, 40%, 20%, or 10% are also under consideration in this invention.

[0319] VI. Sixth aspect of this disclosure: pharmaceutical compositions and pharmaceutical formulations, as well as combination products and kits

[0320] In some embodiments, this disclosure provides compositions comprising the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody or ADC described herein, preferably pharmaceutical compositions or pharmaceutical formulations. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition comprises the 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., chemotherapy agents, tumor vaccines, antibodies that bind to other specific antigens on tumor cells, other antibodies that deplete tumor cells).

[0321] In some embodiments, the compositions of the present invention are pharmaceutical compositions or pharmaceutical preparations comprising suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. 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.

[0322] The pharmaceutical compositions or formulations of the present invention may further comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. In the treatment of cancer, such active ingredients include, but are not limited to, anticancer agents and chemotherapeutic agents; in the treatment of 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.

[0323] In some embodiments, this disclosure also provides combination products comprising at least one of the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody or ADC of the present invention, or further comprising one or more other antitumor agents.

[0324] In some implementations, two or more components of the combined product may be administered to the subject sequentially, separately, or simultaneously.

[0325] In some embodiments, this disclosure also provides kits comprising the ISVD, EGFR-binding molecule, cMet-binding molecule, multispecific antibody, ADC, pharmaceutical composition, or combination product of the present invention, as well as optional package inserts providing instructions for administration.

[0326] In some embodiments, this disclosure also provides pharmaceutical articles comprising the ISVD, EGFR-binding molecule, cMet-binding molecule, multispecific antibody, ADC, pharmaceutical composition, or combination product of the present invention, optionally including a packaging insert for instructions of administration.

[0327] VII. Seventh aspect of this disclosure: Use and method

[0328] The ISVDs, EGFR-binding molecules, cMet-binding molecules, or multispecific antibodies containing them disclosed herein have in vitro and in vivo diagnostic and therapeutic uses. For example, these molecules can be administered in vitro or to cultured cells or to subjects, such as human subjects, to treat and / or diagnose EGFR antigen and / or cMet-related diseases, such as cancer.

[0329] In some embodiments, this disclosure provides a diagnostic method for detecting the presence of relevant EGFR and / or cMet antigens in biological samples, such as serum, semen, or urine, or tissue biopsy samples (e.g., from hyperplastic or cancerous lesions), either in vitro or in vivo. The diagnostic method comprises: (i) contacting a sample (and optionally, a control sample) with an ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody as described herein, under conditions that allow for interaction, or administering the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody to a subject; 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, the control sample). The formation of the complex indicates the presence of the relevant antigen and may indicate the suitability or need for the treatment described herein.

[0330] In some implementations, the relevant antigen is detected prior to treatment, for example, before initiating treatment or before a subsequent treatment after a treatment interval. Possible detection methods include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR techniques (e.g., RT-PCR), or in vivo imaging techniques. Generally, ISVD, EGFR-binding molecules, cMet-binding molecules, or multispecific antibodies used in in vivo and in vitro detection methods are directly or indirectly labeled with a detectable substance to facilitate the detection of bound or unbound conjugates. Suitable detectable substances include a variety of biologically active enzymes, cofactors, fluorescent substances, luminescent substances, paramagnetic (e.g., NMR-active) substances, and radioactive substances.

[0331] In some embodiments, the level and / or distribution of the relevant antigen in vivo are determined, for example, non-invasively (e.g., by using a suitable imaging technique (e.g., positron emission tomography (PET) scan) to detect the detectable marker ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody of the present invention). In one embodiment, for example, by detecting with PET reagents (e.g., 18 The present invention uses F-fluorodeoxyglucose (FDG) labeled in a detectable manner with ISVD, EGFR-binding molecules, cMet-binding molecules or multispecific antibodies to determine the level and / or distribution of related antigens in vivo.

[0332] In one embodiment, the present invention provides a diagnostic kit comprising the ISVD, EGFR-binding molecule, cMet-binding molecule or multispecific antibody described herein and instructions for use.

[0333] In some embodiments, this disclosure relates to the use of the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody or ADC of the present invention in vivo to treat cancer and inhibit the growth or metastasis of tumors expressing EGFR and / or cMet in subjects, thereby inhibiting or reducing cancer growth or metastasis. 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, such combination can be administered in any order or simultaneously.

[0334] Therefore, in one embodiment, the present invention provides a method for treating cancer, the method comprising administering to a subject a therapeutically effective amount of the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody or ADC described herein. In another embodiment, the present invention provides a method for preventing the development of cancer drug resistance in a subject, the method comprising administering to a subject a therapeutically effective amount of the ISVD, EGFR-binding molecule, cMet-binding molecule, or multispecific antibody or ADC described herein.

[0335] In some implementations, cancers treated with ISVD, EGFR-binding molecules, cMet-binding molecules, or multispecific antibodies include, but are not limited to, cancers expressing EGFR and / or cMet, such as lung cancer (e.g., squamous cell carcinoma of the lung, adenocarcinoma of the lung, non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), colon cancer, or pharyngeal squamous cell carcinoma.

[0336] The following embodiments are described to aid in understanding the invention. It is not intended, and should not be construed in any way, as limiting the scope of the invention.

[0337] Example

[0338] Example 1: Preparation and purification of reference antibody and reference antibody-drug conjugate.

[0339] The reference antibody JNJ-61186372 is a bispecific antibody targeting EGFR and cMet, composed of an anti-EGFR half-antibody and an anti-cMET half-antibody. JNJ-61186372 is generated based on the sequence and preparation method of SEQ ID NO:199-202 in patent WO2014081954A1.

[0340] The reference antibody ABT-700 is a monospecific IgG antibody targeting cMet, prepared based on the sequence SEQ ID NO: 86-87 and the method described in patent CN109562189B.

[0341] The reference 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 sequence SEQ ID NO: 59-62 in patent application number US2023 / 0183358A1 and the method described therein.

[0342] The reference antibody-drug conjugate ABBV399 is a monospecific antibody (ABT-700) targeting cMet conjugated with an MMAE molecule, which can be prepared based on the SEQ ID NO: 86-87 sequence and conjugation method in patent CN109562189B.

[0343] The reference antibody-drug conjugate AZD9592 (also referred to as AZD ADC in this article) is a bispecific antibody (RAA22 / B09-57) targeting EGFR and cMet conjugated with camptothecin molecules, which can be prepared based on the sequence of SEQ ID NO: 59-62 in patent application number US2023 / 0183358A1 and the conjugation method.

[0344] Example 2: Generation of ISVDs that specifically bind to EGFR and cMet, respectively

[0345] 2.1 Alpaca Immunization

[0346] Recombinant his-tagged human EGFR ECD protein (Aikon Biotech) and recombinant his-tagged human cMet ECD protein (Aikon Biotech) were used as target antigens for alpaca immunization. The immunization process was carried out as follows.

[0347] Two healthy adult alpacas (Aikon Biotechnology Co., Ltd.) were selected. 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) at a 1:1 ratio and immunized the alpacas via multiple subcutaneous injections into the cervical lymph nodes. The initial immunization was 500 μg, followed by 250 μg immunizations, for a total of 3-4 doses, with immunization intervals of 18-21 days. Seven days after the second and third immunizations, 5 ml of jugular venous blood was collected from the alpacas for ELISA serum titer determination. The results showed that the criteria for blood bank establishment were successfully met after three rounds of immunization, and blood bank establishment was arranged.

[0348] 2.2 Construction and selection of phage display libraries

[0349] As described in Example 2.1, after alpacas were immunized a third time with either EGFR or cMet, 100 ml of jugular venous 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 using the PrimeScript reverse transcription kit (Takara) as a template. Using the cDNA as a template, the first round of PCR amplification produced nucleic acid fragments of conventional IgG (VH) and pure heavy chain IgG (containing VHH as an ISVD) lacking the CH1 domain. These two types of nucleic acids were separated on an agarose gel, and the nucleic acid encoding VHH was extracted, purified, and then subjected to 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 E. coli cells, and the bacterial culture was frozen at -80°C.

[0350] Escherichia coli ER2738 bacterial culture was revived and inoculated into 100 ml of 2YT-A medium (Shanghai Sangon Biotech Co., Ltd.). Helper phages (New England Biolabs) were added for infection, and the cells were resuspended in 2×YT-AK medium (Shanghai Sangon Biotech Co., Ltd.) and incubated overnight at 37°C and 200 rpm. The culture supernatant was collected, and recombinant phages were prepared using the PEG / NaCl precipitation method. Anti-EGFR and anti-cMet phage display libraries were then prepared. Biotin-human EGFR and biotin-human cMet protein were used for enrichment and panning of the anti-EGFR and anti-cMet phage display libraries for subsequent ELISA positive clone selection.

[0351] 2.3 ELISA method for selecting positive clones and sequencing

[0352] The expression supernatant of the *Escherichia coli* ER2738 clones obtained through enrichment panning in Example 2.2 was detected using an ELISA assay. The detection procedure was performed as follows.

[0353] Dilute the antigen (human EGFR-his antigen or human cMet-his antigen) to 2 µg / mL with PBS buffer and coat a 96-well ELISA plate overnight at 4°C. Wash the antigen-coated plate 5 times with PBST, add PBST blocking buffer containing 5% skim milk, and block at room temperature for 1 hour. Wash 6 times with PBST, add the expression supernatant of *E. coli* ER2738 obtained in Example 2.2, and incubate at 37°C with shaking for 1 hour. Wash 6 times with PBST, add anti-M13-HRP secondary antibody (Aikon Biotechnology (Suzhou) Co., Ltd.) diluted in PBS, and shake at 37°C for 45 minutes. Wash 5 times with PBST, add TMB chromogenic buffer, and develop in the dark for 5-15 minutes. Add stop solution. Read the OD using a microplate reader. 450nm -OD 650nm Absorbance values. Bacterial clones with read values ​​greater than 1 were selected for Sanger sequencing. Single clones of ER2738 bacteria containing the corresponding positive VHH sequence were added to glycerol and frozen at -80°C.

[0354] 2.4 Production of VHH-his and VHH-Fc

[0355] Positive anti-EGFR VHH sequences and positive anti-cMET VHH sequences were obtained by PCR from anti-EGFR positive clones and anti-cMET positive clones, respectively. Tags were added to their C-termini (either a 6xHis tag or an Fc tag: hIgG1 isotype). These were then inserted into the expression vector pcDNA3.4 and transiently transfected into HEK-293F cells (hereinafter referred to as "293F cells"). The supernatant was collected. The 6xHis-tagged protein was initially purified using a Ni column and then 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 anti-EGFR tag VHH sequences: anti-EGFR VHH-his and anti-EGFR VHH-Fc; and two anti-cMet tag VHH sequences: anti-cMet VHH-his and anti-cMet VHH-Fc.

[0356] 2.5 FACS method for detecting antibody binding to target cells

[0357] The binding of the antibody expressed in the supernatant of transiently transfected HEK-293F to target cells was detected by FACS.

[0358] Specifically, 100 μL of anti-EGFR supernatant and 100 μL of anti-cMet supernatant obtained by transient transfection of HEK-293F in Example 2.4 were collected as test samples. The binding of the anti-EGFR supernatant to target cells CHO-S-EGFR cells or CHO-S cells was detected by FACS; and the binding of the anti-cMet supernatant to target cells CHO-S-cMet cells or CHO-S cells was detected by FACS. CHO-S-EGFR cells are an abbreviation for the CHO-S engineered cell line expressing human EGFR. CHO-S-cMet cells are an abbreviation for the CHO-S engineered cell line expressing human cMet.

[0359] FACS testing is performed as follows. Using 3 x 10 5 Cells were seeded at a density of 100 cells / well in 96-well plates and centrifuged at 300 g for 5 minutes at 4°C. 100 μl of the supernatant was added to each well and incubated for one hour. The secondary antibody PE-anti-human IgG (Invitrogen, Cat#:12-4998-82) was added and incubated at 4°C for half an hour. The mean fluorescence intensity (MFI) of the cells was measured using flow cytometry (Life Technologies) and analyzed using FlowJo. The results showed that all 23 initially screened anti-EGFR VHHs bound to target cells CHO-S-EGFR to varying degrees. All 18 initially screened anti-cMet VHHs bound to target cells CHO-S-cMet to varying degrees. The anti-EGFR and anti-cMet supernatants showed almost no binding to CHO-S cells.

[0360] Example 3: In vitro biological activity assay of each VHH as an ISVD

[0361] 3.1 FACS detection of VHH binding to cells

[0362] 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 (human lung adenocarcinoma cell line); in the anti-cMet VHH assay, the target cells used were cMet-expressing tumor cells EBC-1 (human lung squamous cell carcinoma cell line). FACS detection was performed as follows.

[0363] Target cells at 1.5 × 10 5Cells were seeded at a density of 100 cells / well in 96-well plates and centrifuged at 300g for 5 minutes at 4°C. The cells were resuspended in anti-EGFR VHH-Fc (375 nM, 3-fold serial dilutions) or anti-cMet VHH-Fc (50 nM, 4-fold serial dilutions) and incubated at 4°C for 1 hour. The cells were then incubated with the secondary antibody PE-anti-human IgG (eBioscience, cat#: 12-4998-82) at 4°C for 30 minutes, and the MFI was measured using flow cytometry (Life Technologies).

[0364] The detection results of anti-EGFR VHH-Fc are as follows: Figure 1 The results show that V-n5B10, V-n9B8, and V-n10A1, as candidates for anti-EGFR VHH, bind to target cells in a dose-dependent manner, with lower binding activity than the control antibody. In subsequent sections and other parts of this article, for ease of description, V-n5B10, V-n9B8, and V-n10A1 are sometimes simply referred to as 5B10, 9B8, and 10A1, respectively.

[0365] The detection results of anti-cMet VHH-Fc are as follows: Figure 2 As shown. V-n7A12, V-n9A2, and V-n9A10, as candidates for anti-cMet VHH, bind to target cells in a dose-dependent manner. In the following sections and other parts of this paper, for ease of description, the anti-cMET VHH effects of V-n7A12, V-n9A2, and V-n9A10 are sometimes simply referred to as 7A12, 9A2, and 9A10, respectively.

[0366] 3.2 Determination of the blocking effect of anti-cMet VHH-Fc on the binding of ligand HGF to target cell EBC-1.

[0367] EBC-1 cells were used as target cells at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 96-well plates and centrifuged at 300g for 5 minutes at 4°C. 50 μL of HGF-His ligand (1 μg / mL) (Beijing Lifetech Scientific 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, serially diluted 5-fold) was added, and the mixture was incubated for 1 hour. The secondary antibody iF647-anti-his (GenScript) was added and incubated at 4°C for half an hour. The MFI of the cells was measured using flow cytometry (LifeTechnologies).

[0368] The results are as follows Figure 3As shown, V-n7A12, V-n9A2, and V-n9A10 blocked the binding of EBC-1 cells to the ligand HGF, with MFI values ​​of 40640, 82172, and 77193 at their highest concentrations, respectively. The MFI ratios at the highest and lowest concentrations were 2.22, 1.12, and 1.14, respectively. These results indicate that candidate molecule V-n7A12 blocks the binding of EBC-1 cells to the ligand HGF in a dose-dependent manner. V-n9A2 and V-n9A10 weakly block the binding of EBC-1 cells to the ligand HGF.

[0369] 3.3 Epitope Identification

[0370] The surface plasmon resonance (SPR) technique was used to detect whether the epitopes binding to the anti-cMetVHH-Fc form of V-n7A12 and V-n9A2 antibodies were the same as those binding to the antigen cMet.

[0371] The V-n9A2 antibody was immobilized using a CM5 sensor chip (Cytiva). Human cMet antigen (300 nM) was injected onto the sensor chip at a flow rate of 50 μL / min for a binding phase of 200 seconds. Then, the anti-cMet antibody V-n7A12 was injected onto the sensor chip at a flow rate of 50 μL / min for a binding phase of 120 seconds.

[0372] The results are as follows Figure 4 As shown, V-n9A2 binds to the human cMet antigen without affecting 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.

[0373] 3.4 FACS detection of cellular endocytosis of each VHH-Fc

[0374] The internalization capacity of tumor target cells for the anti-EGFR VHH-Fc and anti-cMet VHH-Fc prepared in Example 2 was detected using the FACS method.

[0375] Prepare tumor target cells NCI-H1975 and EBC-1, with 1~1.5×10⁶ cells per well. 5Cells were seeded into 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, and the cells were incubated at 4°C for 30 min. The supernatant containing VHH-Fc was removed by centrifugation. The cells were divided into two groups and incubated at 4°C and 37°C for 4 hours, respectively. After incubation, PBS was immediately added to ice to terminate the endocytosis experiment. The secondary antibody PE-anti-human IgG (eBioscience, catalog number: 12-4998-82) was added, and the cells were incubated at 4°C for 30 min. The MFI of the cells was measured using flow cytometry (Life Technologies).

[0376] The internalization level of antibodies bound to the cell surface is calculated using the following formula: MFI of samples incubated at 4°C - MFI of samples incubated at 37°C.

[0377] Internalization rate % = 100% - (MFI of samples incubated at 37℃ / MFI of samples incubated at 4℃) × 100%.

[0378] The endocytosis results of tumor target cells NCI-H1975 for candidate molecules for anti-EGFR antibodies are as follows: Figure 5A As shown, the MFI of samples incubated at 4°C and 37°C at the tested antibody concentrations are illustrated. The endocytosis of tumor target cells EBC-1 with candidate anti-cMet antibodies is shown in the figure. Figure 5B As shown, the endocytosis MFI determined according to the above formula is displayed at the test antibody concentration.

[0379] 3.5 ELISA detection of cross-reactivity between each VHH-Fc and antigens from different species

[0380] Dilute human cMet-His antigen or cynomolgus monkey cMet-His antigen to 1 µg / mL with PBS buffer and coat a 96-well ELISA plate overnight at 4°C. Wash the antigen-coated plate three times with PBST (300 µL / well), add PBS containing 5% skim milk to 200 µL / well, and block at 37°C with shaking for 2 hours. Wash three times with PBST, add serially diluted anti-cMet VHH-Fc antibodies at 5-fold ratios, and incubate at 37°C with shaking for 1 hour. Wash three times with PBST, add PBS-diluted anti-human Fc-HRP secondary antibody (Abcam, CAT#: ab97225) at 100 µL / well, and incubate at 37°C with shaking for 45 minutes. Then wash three times with PBST (300 µL / well), add 100 µL of TMB chromogenic buffer to each well, and develop in the dark for 5-10 minutes. Add stop solution, with a volume of 50 μL / well. Read the OD value using a microplate reader.450nm -OD 650nm Absorbance value. ELISA test results are as follows: Figure 6 As shown in Table 1.

[0381] Table 1. ELISA detection of the binding of anti-cMet antibodies to human cMet antigen or monkey cMet antigen

[0382] Note: "-" indicates that EC cannot be fitted. 50 value.

[0383] The results showed that anti-cMet candidate molecules V-n7A12 and V-n9A2 exhibited cross-reactivity with human cMet antigen and cynomolgus monkey cMet antigen, and showed similar binding affinity for human and monkey cMet. However, the affinity of V-n9A10 for human cMet antigen differed significantly from its affinity for monkey cMet antigen.

[0384] Example 4. VHH Sequence Optimization and Characterization

[0385] 4.1 VHH Sequence Optimization

[0386] The original VHH sequence prepared in Example 2 was humanized using the "best-matching method". The amino acid sequence of the VHH framework region was compared and analyzed using a human germline V gene database to select the optimal 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. Several residues in the mutated framework region were restored, and post-translational modification (PTM) removal was performed as needed. The optimized sequence was reverse-translated and synthesized by Genewiz (Shanghai, China). It was then constructed into the pcDNA 3.4 expression vector to express monovalent humanized VHHs with a C-terminal fused human His tag. This yielded the humanized VHH clone protein.

[0387] Immunogenicity analysis was performed on humanized and PTM-removed VHH sequences. If a 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 synthesized by Genewiz (Shanghai, China) and then constructed into the pcDNA 3.4 expression vector to express VHHs with a C-terminal His tag fusion. This yielded VHH clones with low immunogenicity risk.

[0388] Sequence optimization for anti-EGFR VHH

[0389] Tables 2A to 2C below show the maternal anti-EGFR antibodies and their optimized sequences.

[0390] Table 2A. Anti-EGFR VHH-10A1 antibody and its optimized sequence

[0391] Table 2B. Anti-EGFR VHH 9B8 antibody and its optimized sequence

[0392] Table 2C. Anti-EGFR VHH 5B10 antibody and its optimized sequence

[0393] Sequence optimization against cMET VHH

[0394] Tables 2D to 2E below show the maternal anti-cMet antibodies and their optimized sequences.

[0395] Table 2D Anti-cMet VHH 9A2 antibody and its optimized sequence

[0396] Table 2E. Anti-cMet VHH 7A12 antibody and its optimized sequence

[0397] 4.2 Optimize antibody functional characterization

[0398] Using FACS binding assays, the binding of anti-EGFR VHH-His candidate antibody molecules V-n10A1, V-n9B8, and V-n5B10, and anti-cMet VHH-His candidate molecules V-n9A2 and V-n7A12, as well as their corresponding sequence-optimized molecules, to target cells MDA-MB-468 (triple-negative breast cancer cell line), EBC-1 cells (human non-small cell lung cancer cell line), MKN45 cell line (human gastric cancer cell line), or CHOK1 cells overexpressing EGFR or cMet was detected. The assays were performed as follows.

[0399] With 1.5×10 5Seed target cells at a density of 1 cell / well in 96-well plates and centrifuge at 300g for 5 minutes at 4°C. Resuspend the cells in the test antibody VHH-His or the reference antibody and incubate at 4°C for 1 hour. Add the secondary antibody iF647-anti-his (Genscript, 1:1000 dilution, catalog number: A01802-100) or APC-anti-his (BioLegend, 1:200, catalog number: 362605) and incubate at 4°C for 30 minutes or 45 minutes. Measure the MFI of the cells using flow cytometry (Life Technologies).

[0400] 1) Sequence optimization of anti-EGFR antibodies

[0401] 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, and the results are shown in Tables 3A and 3B.

[0402] Table 3A. Binding activity of various humanized anti-EGFR antibodies to target cells

[0403] Note: "-" indicates that EC cannot be fitted. 50 value.

[0404] Table 3B. Binding activities of each anti-EGFR antibody with target cells after PTM sequence removal optimization.

[0405] Note: "-" indicates that EC cannot be fitted. 50 value.

[0406] 2) Sequence optimization and modification of anti-cMet antibodies: 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, and the results are shown in Tables 3C to 3F below.

[0407] Table 3C. Binding activity of various humanized anti-cMet antibodies to target cells.

[0408] Note: "-" indicates that EC cannot be fitted. 50 value.

[0409] Table 3D. Binding activities of each anti-cMet antibody with target cells after PTM sequence removal optimization.

[0410] Table 3E. Binding activity of anti-cMet antibodies after first immunogenic removal to target cells.

[0411] Table 3F. Binding activity of anti-cMet antibodies after second immunogenic removal to target cells.

[0412] 4.4 SPR detection of anti-cMet VHH and anti-EGFR VHH

[0413] The binding affinity of anti-cMet VHH and anti-EGFR VHH to the target antigens was characterized using the SPR method.

[0414] For monovalent antibodies tagged with VHH_His, detection was performed according to Method 1 below. 10 μg / ml of antigen (EGFR antigen or cMet antigen from different species) was immobilized on a CM5 chip, and then each test antibody (serially diluted) was 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, regeneration was performed using 10 mM glycine (pH 2.0). The experimental data will be analyzed using a 1:1 binding model. The results are shown in Table 4A below.

[0415] For the bivalent antibody tagged with VHH_Fc, detection was performed according to Method 2 below. Each antibody (concentration 10 ug / ml) was captured using a Protein A chip, and then the antigen (EGFR antigen or cMet antigen from different species) was 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, regeneration was performed using 10 mM glycine (pH 2.0). The experimental data will be analyzed using a 1:1 binding model. The results are shown in Table 4B below.

[0416] Table 4A. SPR data of monovalent anti-cMet antibodies and anti-EGFR antibodies with VHH_His tag

[0417] Note: All Vn series antibodies tested in the table are in monovalent form of VHH with a His tag at the C-terminus, and were detected using Method 1; the control antibodies in the table were detected using Method 2.

[0418] Table 4B: SPR data of VHH_Fc-tagged bivalent anti-EGFR antibodies:

[0419] Note: All antibodies in the table are C-terminal Fc-tagged VHH dimers, detected using Method 2.

[0420] Example 5. Construction and characterization of anti-cMet dual epitope antibody

[0421] 5.1 Construction and preparation of anti-cMet biepisode antibodies

[0422] The M-1 molecule of the anti-cMet dual epitope of the present invention is in an asymmetric double-stranded form, having a first polypeptide chain (9A2-Fc chain) as shown in SEQ ID NO: 88 and a second polypeptide chain (hu7A12) as shown in SEQ ID NO: 89. b -Fc chain).

[0423] The M-2 molecule of the present invention, which is anti-cMet single epitope, is in a symmetrical double-stranded form, having a first polypeptide chain and a second polypeptide chain (9A2-Fc chain) as shown in SEQ ID NO: 90.

[0424] The M-3 molecule of this invention, which resists cMet single epitope, is in a symmetrical double-stranded form, having a first polypeptide chain and a second polypeptide chain (hu7A12) as shown in SEQ ID NO: 91. b -Fc chain).

[0425] The first / second polypeptide chains of M-1, M-2, and M-3 were constructed into pcDNA 3.4 expression vectors (one vector was generated if the antibody had a symmetrical structure; two vectors containing the encoding genes of the first and second polypeptide chains were generated if the antibody had an asymmetrical structure), and transfected into HEK293F cells. Cells were cultured for 3 days, and the culture supernatant was collected and loaded into a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody was eluted with acetate-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. Antibody concentration was measured using Nano Drop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC.

[0426] 5.2 FACS detection of the binding of biepisode antibodies to target cells

[0427] The binding of EBC-1 to anti-cMet molecules in tumor target cells was detected by FACS.

[0428] Prepare EBC-1 tumor target cells, with 1.5 × 10⁶ cells per well. 5Cells were seeded into 96-well plates. Anti-cMet M-1, M-2, and M-3 molecules (50 nM, 4-fold dilution) were added separately, and the cells were incubated at 4°C for 1 hour. The supernatant containing each VHH-Fc was removed by centrifugation. The secondary antibody PE-anti-human IgG (eBioscience, catalog number 12-4998-82) was added, and the cells were incubated at 4°C for 30 min. The MFI of the cells was then measured by flow cytometry (Life Technologies).

[0429] The results are as follows Figure 7 The results showed that, compared with the anti-cMet single epitope M-2 and M-3 molecules, tumor target cells EBC-1 had a stronger binding to the anti-cMet double epitope M-1 molecule, and the binding activity of the double epitope molecule to cells was also better than that of the control group ABT700.

[0430] 5.3 FACS detection of co-endocytosis of biepisode antibodies

[0431] The co-endocytosis of anti-cMet dual epitope molecule M-1 was detected using the FACS method.

[0432] Prepare EBC-1 tumor target cells, with 1.5 × 10⁶ cells per well. 5 Cells were seeded into 96-well plates. Anti-cMet M-1, M-2, and M-3 molecules (50 nM, 4-fold dilution) were added separately, and the cells were incubated at 4°C for 30 min. The supernatant containing each VHH-Fc was removed by centrifugation. The cells were divided into two groups and incubated at 4°C and 37°C for 4 hours, respectively. After incubation, PBS was immediately added to ice to stop the endocytosis experiment. Then, the secondary antibody PE-anti-human IgG (eBioscience, catalog number 12-4998-82) was added, and the cells were incubated at 4°C for 30 min. The MFI of the cells was measured by flow cytometry (Life Technologies).

[0433] The internalization level of antibodies bound to the cell surface is calculated using the following formula: MFI of samples incubated at 4°C - MFI of samples incubated at 37°C.

[0434] like Figure 8 The experimental results show that tumor target cells EBC-1 have a higher endocytosis rate for the anti-cMet dual epitope M-1 molecule (compared to 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.

[0435] Example 6: Generation and detection of multispecific anti-EGFR / cMet antibody molecules

[0436] 6.1 Generation of multispecific anti-EGFR / cMet antibody molecules

[0437] Based on the analysis and detection results of Examples 1-5 above, anti-EGFR VHH and anti-cMet VHH are combined to form single-chain or multi-chain multispecific antibodies.

[0438] Single-chain multispecific antibodies

[0439] The anti-EGFR VHH sequence and the anti-cMet VHH sequence are combined to form a single-chain antibody. Specifically, an anti-EGFR VHH sequence (V-n5B10, V-n9B8, V-n10A1) or its humanized VHH sequence is combined with two anti-cMet VHH sequences with the same or different epitopes (V-n7A12, V-n9A2) or their humanized VHH sequences to form a specific anti-EGFR / cMet antibody molecule. Specifically, a GGGGS peptide linker (SEQ ID NO: 43) is added to the C-terminus of the anti-EGFR VHH, followed by an anti-cMet VHH (first anti-cMet ISVD), then another GGGGSGGGGS peptide linker (SEQ ID NO: 44), and finally another anti-cMet VHH (second anti-cMet ISVD). Exemplary multispecific antibody molecules and their amino acid sequences formed by this method are shown in Table 5.

[0440] Table 5: Exemplary Single-Chain Multispecific Antibody Molecules

[0441] Note: In Table 5, V-n5B10, V-n9B8, and V-n10A1, which are used as anti-EGFR VHH, are abbreviated as 5B10, 9B8, and 10A1, respectively; and V-n7A12, V-n9A2, and V-n9A10, which are used as anti-cMet VHH, are abbreviated as 7A12, 9A2, and 9A10, respectively.

[0442] Optionally, the specific antibody molecule is linked to an ISVD that binds to human serum albumin, such as Alb8, to prolong 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 shown in SEQ ID NO: 43).

[0443] Specifically, taking the V-17 as an example, such as Figure 9As illustrated, a GGGGS peptide linker (SEQ ID NO: 43) is attached to the C-terminus of V-17, followed by an anti-HSA-Alb8 linker (SEQ ID NO: 45), thereby combining to form hu10A1-hu7A12-hu9A2-anti-HSA-Alb8 (also known 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 attached to the C-terminus, followed by an anti-HSA-Alb8 linker (SEQ ID NO: 45), forming 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), and V-21-anti-HSA-Alb8 (SEQ ID NO: 45), respectively. V-22-anti-HSA-Alb8 (SEQ ID NO: 79), 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).

[0444] The single-chain, multispecific anti-EGFR / cMet antibody described in this disclosure was constructed into the pcDNA 3.4 expression vector. It was transfected into HEK293F cells, cultured for 3 days, and the culture supernatant was collected and placed into Cytiva PrismA packing material (Cytiva) for purification. The antibody was eluted with acetate-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. The antibody concentration was measured using Nano Drop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[0445] Multi-chain multispecific antibodies

[0446] The VHH sequence of anti-EGFR and the VHH sequence of anti-cMet with the same or different epitopes are combined into polypeptide chains containing Fc subunits. The polypeptide chains can be further associated to form homologous or heterologous dimers to produce multispecific EGFR and cMet binding molecules in double-stranded form.

[0447] Figure 10 An exemplary multi-chain, multi-specific antibody structure is shown. 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 an LALA mutation and / or a Knob-into-hole structure. The prepared polypeptide chain includes sequences as shown in Table 6A. Table 6A: Exemplary sequences against EGFR-Fc

[0448] The VHH sequence of the anti-cMet is combined with the Fc subunit to form a polypeptide chain (anti-cMet1-anti-cMet2-Fc). Specifically, the C-terminus of the VHH sequence of the first anti-cMet is connected to the N-terminus of the same or different VHH sequence of the second anti-cMet via a peptide linker (such as GGGGSGGGGS shown in SEQ ID NO:42). The C-terminus of the VHH of the second anti-cMet is then combined with the Fc subunit via a hinge region to form a polypeptide chain. Optionally, the Fc subunit contains an LALA mutation and / or a Knob-into-hole structure. The prepared polypeptide chain includes the sequences shown in Table 6B. Table 6B: Exemplary sequences of anti-cMet1-anti-cMet2-Fc

[0449] Any of the above-mentioned anti-EGFR-Fc polypeptide chains can associate with any of the anti-cMet1-anti-cMet2-Fc polypeptide chains to form a dimer, for example, to synthesize the double-chain form of multispecific anti-EGFR / cMet antibodies shown in Table 7: Table 7: Examples of double-stranded multispecific anti-EGFR / cMet antibodies

[0450] Polypeptide chains 1 and 2 of the double-stranded multispecific anti-EGFR / cMet antibody described in this disclosure were constructed into the pcDNA 3.4 expression vector, transfected into HEK293F cells, cultured for 3 days, and the culture supernatant of the transfected cells was collected and loaded into a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody was eluted with acetate-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. The antibody concentration was measured using Nano Drop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[0451] 6.2 FACS Binding Detection

[0452] The binding of the above-mentioned trispecific anti-EGFR / cMet antibody molecules to target cells EBC-1 and NCI-H1975 cells (Chinese Academy of Sciences Cell Bank) was detected using the FACS method.

[0453] Prepare target cells separately, with 1.5 × 10⁶ cells per well. 5 Cells were seeded into 96-well plates. The trispecific antibodies prepared in Example 6.1 (250 nM, 3-fold dilution) were added, and the plates were incubated at 4°C for 1 h. The plates were then centrifuged at 300 g for 4 minutes to remove the supernatant. The plates were washed twice with FACS buffer (1% BSA). The secondary antibody PE-anti-human IgG (eBioscience, catalog number 12-4998-82) was added, and the plates were incubated at 4°C for 1 h. 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 flow cytometry (Life Technologies).

[0454] Depend on Figure 11 As shown, the V-17-Fc, V-20-Fc, V-23-Fc and V-26-Fc trispecific antibodies can bind to EBC-1 and NCI-H1975 cells.

[0455] 6.3 FACS Internalization Detection

[0456] The internalization capacity of the above-mentioned trispecific anti-EGFR / cMet antibody molecules in target cells EBC-1 and NCI-H1975 cells (Chinese Academy of Sciences Cell Bank) was detected by FACS method.

[0457] Prepare target cells separately, with 3 × 10 cells per well. 5 Cells were seeded into 96-well plates. The trispecific antibodies prepared in Example 6.1 (250 nM, 3-fold dilution) were added, and the cells were incubated at 4°C for 30 min. The supernatant containing the trispecific antibodies was removed by centrifugation. The cells were divided into two groups and incubated at 4°C and 37°C for 4 hours, respectively. After incubation, PBS was immediately added to ice to terminate the endocytosis experiment. After adding the secondary antibody and incubating at 4°C for 30 min, the MFI of the cells was measured using flow cytometry (Life Technologies). For molecules with the VHH-Fc pattern, the secondary antibody used was PE-anti-human IgG (eBioscience, catalog number: 12-4998-82); for molecules with the VHH and his tag, the secondary antibody used was APC-anti-his (BioLegend, catalog number: 362605).

[0458] The internalization level of antibodies bound to the cell surface is calculated using the following formula: MFI of samples incubated at 4°C - MFI of samples incubated at 37°C; The endocytosis results of 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 of the trispecific antibody in target cells MDA-MB-468, EBC-1, and NCI-H1975 are as follows: Figures 12-13 As shown.

[0459] like Figure 12 and Figure 13 As shown, target cells can endocytose three specific 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.

[0460] 6.4 SPR assay to detect the affinity of trispecific antibodies for human and cynomolgus monkey antigens

[0461] 6.4.1 SPR assay to detect the affinity of trispecific antibodies for human and cynomolgus monkey EGFR antigens

[0462] The affinity of trispecific anti-EGFR / cMet antibody molecules for human and cynomolgus monkey EGFR was detected using Cytiva via the SPR method (Beijing Yiqiao Shenzhou Technology Co., Ltd.).

[0463] The amino-conjugated antigens human EGFR-his (ECD, Met1-Ser645) and cynomolgus monkey EGFR-his (ECD, Met1-Ser645) (Beijing Yiqiao Shenzhou Technology Co., Ltd.) (20 μg / mL) were immobilized on a GLM sensor chip (Bio-Ray). The proportionally diluted trispecific anti-EGFR / cMet antibody molecules from Example 6.1 were injected into the sensor chip at a flow rate of 100 μL / min for a binding phase of 100 seconds, followed by a dissociation phase of 180 seconds. After each dissociation phase, the 10 mM glycine (pH 1.5) regeneration buffer was applied. The sensor maps of the blank surface and buffer channels were subtracted from the tested sensor maps. Langmuir analysis was performed using a 1:1 binding model. The affinity results of the candidate molecules for human and cynomolgus monkey EGFR are shown in Table 8.

[0464] The results showed that the candidate molecules exhibited similar binding and dissociation rates with human EGFR antigen and cynomolgus monkey EGFR antigen, and these trispecific anti-EGFR / cMet antibody molecules showed cross-reactivity with cynomolgus monkey EGFR.

[0465] Table 8. Affinity of SPR-detected trispecific antibodies to human and cynomolgus monkey EGFR antigens

[0466] 6.4.2 SPR assay to detect the affinity of trispecific antibodies for human and cynomolgus monkey cMet antigens

[0467] The amino-coupled antigens human cMet-his (ECD, Met1-Thr932) and cynomolgus monkey cMet-his (ECD, Met1-Thr932 (Beijing Yiqiao Shenzhou Technology Co., Ltd.)) (20 μg / mL) were immobilized on a GLM sensor chip (Bio-Rayet). The proportionally diluted trispecific anti-EGFR / cMet antibody molecule (500 nM) from Example 6.1 was injected onto the sensor chip at a flow rate of 30 μL / min, with a binding phase of 100 seconds followed by dissociation of 180 seconds. After each dissociation phase, 10 mM glycine (pH 1.5) was used to regenerate the buffer. The sensor maps of the blank surface and buffer channels were subtracted from the tested sensor maps. Experimental data were analyzed using a 1:1 binding model. The affinity results of the candidate molecules with human cMet and cynomolgus monkey cMet are shown in Table 9.

[0468] The results showed that the candidate molecules exhibited similar binding and dissociation rates with human cMet antigen and cynomolgus monkey cMet antigen, and these trispecific anti-EGFR / cMet antibody molecules cross-reacted with cynomolgus monkey cMet.

[0469] Table 9. Affinity of trispecific antibodies to human and cynomolgus monkey cMet antigens as determined by SPR

[0470] 6.5 Determination of the blocking effect of trispecific anti-EGFR / cMet antibody molecules on the binding of ligand HGF to target cell EBC-1.

[0471] EBC-1 cells were used as target cells at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 96-well plates and centrifuged at 300g for 5 minutes at 4°C. Trispecific anti-EGFR / cMet antibody (200 nM, 4-fold serial dilution) was added and incubated with the cells for 30 minutes. Then, HGF-His ligand (2 μg / mL) (Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added and incubated for 1 hour at 4°C. Finally, a secondary antibody, iF647-anti-his (GenScript), was added and incubated for 30 minutes at 4°C. The molecular weight fraction (MFI) of the cells was measured using flow cytometry (LifeTechnologies).

[0472] The results show that, Figure 14 As shown, the trispecific anti-EGFR / cMet antibody of the present invention can block the binding of EBC-1 cells to the ligand HGF, and its blocking activity is superior to that of ABT700 and BMK-AZD.

[0473] 6.6 FACS detection of the synergistic endocytosis of target cells by trispecific antibodies

[0474] The M-4 molecule serves as a control molecule. It is an asymmetric double-stranded Fc molecule, containing a first polypeptide chain (hu9A2-hu7A12-Fc) as shown in SEQ ID NO: 73 and a second polypeptide chain (anti-Covid-19 chain) as shown in SEQ ID NO: 92. The anti-Covid-19 chain does not bind to EGFR or cMET in target cells.

[0475] The M-5 molecule serves as a control molecule. It is an asymmetric double-stranded Fc molecule, containing a first polypeptide chain (anti-Covid-19 chain) as shown in SEQ ID NO: 93 and a second polypeptide chain (hu5B10-Fc) as shown in SEQ ID NO: 71. The anti-Covid-19 chain does not bind to EGFR or cMET in target cells.

[0476] The M-6 molecule serves as a control molecule. It is an asymmetric double-stranded Fc molecule, containing a first polypeptide chain (anti-Covid-19 chain) as shown in SEQ ID NO: 93 and a second polypeptide chain (hu9B8-Fc) as shown in SEQ ID NO: 72. The anti-Covid-19 chain does not bind to EGFR or cMET in target cells.

[0477] The M-7 molecule serves as a control molecule. It is an asymmetric double-stranded Fc molecule, containing a first polypeptide chain (anti-Covid-19 chain) as shown in SEQ ID NO: 93 and a second polypeptide chain (hu10A1-Fc) as shown in SEQ ID NO: 70. The anti-Covid-19 chain does not bind to EGFR or cMET in target cells.

[0478] The first / second polypeptide chains of the M-4, M-5, M-6, and M-7 molecules were constructed into pcDNA 3.4 expression vectors (one vector was generated if the antibody had a symmetrical structure; two vectors containing the encoding genes of the first and second polypeptide chains were generated if the antibody had an asymmetrical structure), and transfected into HEK293F cells. Cells were cultured for 3 days, and the culture supernatant was collected and loaded into a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody was eluted with acetate-sodium acetate solution (pH 3.5) and immediately neutralized with 2M Tris. Antibody concentration was measured using Nano Drop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC.

[0479] The enhanced internalization capacity of tumor target cells EBC-1 to trispecific anti-EGFR / cMet antibody was detected using the FACS method.

[0480] Prepare EBC-1 tumor target cells, with 3 × 10⁶ cells per well. 5 Cells were seeded into 96-well plates. Trispecific anti-EGFR / cMet antibodies V-20-Fc and V-23-Fc molecules, anti-cMet M-4, and anti-EGFR M-5, M-6, and M-7 molecules (40 nM, 5-fold dilution) were added separately. The plates were incubated at 4°C for 30 min, and the supernatant containing each VHH-Fc was removed by centrifugation. The cells were then divided into two groups and incubated at 4°C and 37°C for 4 hours, respectively. After incubation, PBS was immediately added to ice to terminate the endocytosis experiment. A secondary antibody, PE-anti-human IgG (eBioscience, catalog number 12-4998-82), was added, and the cells were incubated at 4°C for 30 min. The MFI of the cells was then measured using flow cytometry (Life Technologies).

[0481] The internalization level of antibodies bound to the cell surface is calculated using the following formula: MFI of samples incubated at 4°C - MFI of samples incubated at 37°C.

[0482] like Figure 15 The results showed that tumor target cells EBC-1 had a high endocytosis rate for the trispecific antibody molecules V-20-Fc and V-23-Fc (compared to anti-cMet M-4 and anti-EGFR M-5, M-6, and M-7 molecules).

[0483] 6.7 FACS detection of the synergistic binding of trispecific antibodies to target cells

[0484] NCI-H1975 cells were used at a density of 1.5 × 10⁶ cells per well. 5 Cells were seeded into 96-well plates. The trispecific antibody of this invention (200 nM, 5-fold dilution) was added, and the plates were incubated at 4°C for 1 hour. The plates were then centrifuged at 300 g for 4 minutes to remove the supernatant. The plates were washed twice with FACS buffer (1% BSA). The secondary antibody PE-anti-human IgG (eBioscience, catalog number 12-4998-82) was added, and the plates were 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).

[0485] Depend on Figure 16 As shown, V-23-Fc has a stronger binding ability compared to its corresponding control antibodies (M4 and M6), suggesting that the two targets have a synergistic binding effect.

[0486] Example 7. Preparation, characterization, and experiments of antibody-drug conjugates (ADCs)

[0487] The connectors-payloads used in some embodiments of this invention are known and / or commercially available in the prior art, or can be prepared according to the description herein. When the drawn structure differs from the actual situation, modifications or corrections to the structure should be permitted based on the actual circumstances.

[0488] General Synthesis Method A

[0489] 5.45 mg / ml of the antibody of the present invention was placed in an Eppendorf tube in 20 mM His-hac, 150 mM NaCl, pH 5.5. Six molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 5 mM) 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. Another 6 molar equivalents of TCEP (5 mM) were added to the mixture. The reaction mixture was then 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 replenished with 20 mM His-hac buffer. Six molar equivalents of the linker-loaded antibody (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 incubated at room temperature on a shaker for 2 hours (linker-load:antibody = 6:1). Conversion was assessed using HIC-HLPC, and purification was performed once conversion was complete. The reaction mixture was transferred to an ultrafiltration tube (MWCO 30 kDa), 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 by washing 10 times with 10 mM His-hac pH=5.0. Finally, the remaining solution was transferred and adjusted to the appropriate concentration.

[0490] When using Pro A for purification (Pro A product labeled 40 g / L capacity), wash with 10 CV 10 mM His-hac before use, then load the sample onto a gravity column filled with Pro A resin. Wash with at least 10 CV of 10 mM His-hac buffer containing 10% DMSO, and then wash with 50 CV of 10 mM His-hac buffer without DMSO to completely remove the free payload. Elute with acetic acid solution (50 mM) at pH 3 and immediately neutralize to pH 5.5–6.0 with 2 M Tris buffer (pH 12.0). 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 required concentration using centrifugal ultrafiltration (MWCO 30 kd ultrafiltration membrane).

[0491] Purity was determined by HIC and SEC-HPLC methods. Free linker-load was determined by RP-HPLC.

[0492] General methods and / or parameters for determining or detecting ADCs Size exclusion chromatography (SEC) method (for total ADC detection) SEC-HPLC method parameters Reversed-phase HPLC (RP HPLC) method (for the detection of free drugs) RP HPLC parameters

[0493] Perform elution according to the table below. HIC-HPLC method (for the detection of free antibodies and DAR distribution) HIC-HPLC conditions.

[0494]

[0495] Perform elution according to the table below.

[0496] 7.1 Preparation and Characterization of ADC Molecules

[0497] a) Preparation of ADC molecules coupled with Mal-PEG8-VA-PAB-Exatecan

[0498] Ab refers to the antibody V-26-Fc prepared in this 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, etc.

[0499] Following the above-described synthesis method A, V-26-Fc-VA-Exd was prepared using antibody V-26-Fc and linker-loaded Mal-PEG8-VA-PAB-Exatecan (CAS No.: 2679821-39-5; MedChemExpress, HY-147271). The MW was 96.37, the average Dar was 4.0, the yield was 63%, and the purity was 91.73%.

[0500] Following 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.

[0501] b) Preparation of ADC molecules coupled with MC-VC-PAB-MMAE

[0502] Ab refers to the antibody V-26-Fc prepared in this 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, etc.

[0503] Following the above-described synthesis method A, V-26-Fc-VC-MMAE was prepared using antibody V-26-Fc and linker-loaded MC-VC-PAB-MMAE (CAS No.: 646502-53-6; MedChemExpress, HY-15575).

[0504] Following the preparation method of V-26-Fc-VC-MMAE, 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.

[0505] c) Preparation of ADC molecules coupled with Mal-PEG8-VC-PAB-MMAE

[0506] Ab refers to the antibody V-26-Fc prepared in this 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, etc.

[0507] Following the above-described synthesis method A, V-26-Fc-PEG-VC-MMAE was prepared using antibody V-26-Fc and linker-loaded Mal-PEG8-VC-PAB-MMAE (CAS No.: 2353409-69-3; MedChemExpress, HY-141156).

[0508] Following the preparation method of V-26-Fc-PEG-VC-MMAE, 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.

[0509] d) Preparation of ADC molecules coupled with Mal-PEG8-EVC-PAB-MMAE

[0510] Ab refers to the antibody V-26-Fc prepared in this 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, etc.

[0511] Following the above-described synthesis method A, V-26-Fc-PEG-EVC-MMAE was prepared using antibody V-26-Fc and linker-loaded device Mal-PEG8-EVC-PAB-MMAE (prepared according to the preparation method of this application).

[0512] Following the preparation method of V-26-Fc-PEG-EVC-MMAE, 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.

[0513] e) Preparation of ADC molecules coupled with MC-EVC-PAB-MMAE

[0514] Ab refers to the antibody V-26-Fc prepared in this 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, etc.

[0515] Following the above-described synthesis method A, V-26-Fc-EVC-MMAE was prepared using antibody V-26-Fc and linker-loaded MC-EVC-PAB-MMAE (CAS No.: 2873452-49-2; MedChemExpress, HY-154915).

[0516] Following the preparation method of V-26-Fc-EVC-MMAE, 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.

[0517] f) Preparation of ADC molecules coupled with Mal-Gly-Exatecan-D-glucuronic acid:

[0518] Ab refers to the antibody V-23-Fc prepared in this 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, etc.

[0519] Following the above-described synthesis method A, V-23-Fc-Gluc-Exd (or V-23-Fc-Glu-Exd) was prepared using antibody V-23-Fc and linker-loaded Mal-Gly-Exatecan-D-glucuronic acid (CA No.: 2763252-25-9; MedChemExpress, HY-153179). The MW was 96, the average Dar was 4.0, the yield was 50%, and the purity was 84.31%.

[0520] Following 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.

[0521] The characterization data of the exemplary ADCs prepared in this application are as follows:

[0522] Preparation of the reference antibody-drug conjugate AZD9592

[0523] Add 15 equivalents of TCEP solution to the antibody (RAA22 / B09-57, also referred to as BMK-AZD or AZD Ab in this paper), mix well, and react at 37°C for 2 hours. Add DMA and 16 equivalents of the linker-loaded polymer (Mal-PEG8-amide-Val-Ala-(4-NH2)-Exatecan) dissolved in 10 mM DMA stock solution to the reaction solution, resulting in a final organic solvent content of 10%. Mix the reaction solution and continue reacting at 22°C in a shaker for 1 hour, then add N-acetylcysteine ​​to quench the reaction. Purify the coupled sample using an ultrafiltration concentrator, and transfer the buffer to the final ADC storage buffer (30 mM histidine, 30 mM arginine-HCl, containing 0.02% PS 80, pH 6.8). Filter the sample sterile using a 0.22 μm syringe filter.

[0524] The characterization data of the reference antibody-drug conjugates involved in this application are as follows:

[0525] Preparation of Mal-PEG8-EVC-PAB-MMAE in this application

[0526] (Mal-PEG8-EVC-PAB-MMAE)

[0527] Step 1:

[0528] (1) Compounds 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), along with HATU (188 mg) and triethylamine (66.7 mg), and the mixture was reacted under N2 protection for 4 h.

[0529] (2) After the reaction is complete, monitor the reaction solution with LCMS, add the reaction solution dropwise to water, and use EA (30 mL) to measure the reaction mixture. 2) Extract twice, combine the organic phases, and then rinse with saturated saline (30 mL). 3) Wash three times, dry with anhydrous sodium sulfate and concentrate to obtain 410 mg (88.29% yield) white solid.

[0530] Step Two:

[0531] (1) Compound HM-2039_2 (410 mg, 0.291 mmol, 1 eq) was added to DCM (4 mL), TFA (1 mL) was added, and the mixture was reacted for 1 h under nitrogen protection (0-10 °C).

[0532] (2) The reaction was monitored by LCMS until it ended. The crude product was obtained by low-temperature concentration to remove DCM. The crude product was passed through a reverse phase column to obtain the product: 200 mg, 54.86% yield, white solid.

[0533] Step 3:

[0534] (1) Dissolve HM-2039_3 (0.1g, 79.84 umol, 1 eq) and HM-2039_4 (CAS: 1818294-46-0) (51.86 mg, 83.83 umol, 1.05 eq) in DMF and stir the reaction solution for 2h.

[0535] (2) The reaction was monitored by LCMS until it ended. 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 MW 1756.08 and purity 99.18%.

[0536] 1H NMR (400 MHz, DMSO) δ 10.04 (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, 2H), 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).

[0537] 7.2 FACS binding detection of the ADCs of this invention

[0538] The binding of the above-mentioned trispecific anti-EGFR / cMet antibody molecules conjugated toxin molecules (i.e., payloads) to target cells MDA-MB-468, EBC-1, and NCI-H1975 cells (Chinese Academy of Sciences Cell Bank) after forming ADC molecules was detected by FACS method.

[0539] The experimental method was as described in Example 6.2. Target cells were prepared separately, with 1.5 × 10⁶ cells per well. 5Cells were seeded into 96-well plates. Test ADCs (200 nM or 40 nM, 5-fold dilution) were added, and the plates were incubated at 4°C for 1 hour. The plates were then centrifuged at 300g for 4 minutes to remove the supernatant. 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 the plates were 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 flow cytometry (Life Technologies).

[0540] Depend on Figure 17 As shown, V-26-Fc-PEG-EVC-MMAE, V-20-Fc-PEG-EVC-MMAE, and V-23-Fc-PEG-EVC-MMAE can bind to EBC-1 and NCI-H1975 cells. Similarly, V-23-Fc-VA-Exd and V-23-Fc-Gluc-Exd can bind to MDA-MB-468 and NCI-H1975 cells, with binding activity superior to the control groups AZD9592 and ABBV399.

[0541] 7.3 Cell killing detection of the ADCs of this invention

[0542] The killing effect of the above-mentioned trispecific anti-EGFR / cMet antibody molecules conjugated with toxins on target cells MDA-MB-468, EBC-1 and NCI-H1975 cells (Chinese Academy of Sciences Cell Bank) was detected by FACS method.

[0543] 100 μL of cells resuspended in cell culture medium (1 x 10⁻⁶ cells) 4 Cells were seeded into each well of a 96-well plate and incubated overnight at 37°C with 5% CO2. 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, along with positive controls ADCABBV399 and AZD9592, were added to the corresponding wells. Cells were cultured for 3–5 days. Then, 20 µL of CCK8 (ShanghaiLife iLab Biotech, catalog number: AC11L054) was added to each well and incubated at 37°C with 5% CO2 until color development. The absorbance at 450 nm was recorded using a Molecular Device (SpectraMax M5).

[0544] The formula for calculating cell viability is as follows: Cell viability percentage = [(As - Ab) / (Ac - Ab)] × 100. As = absorbance of experimental wells (cells, culture medium, CCK8, and ADC); Ab = absorbance of blank wells (culture medium and CCK8); Ac = absorbance of control wells (cells, culture medium, and CCK8).

[0545] The killing effects of V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE, and V-26-Fc-PEG-EVC-MMAE on EBC-1, NCI-H1975, and MDA-MB-468 are shown in Table 10A below. Figure 18A As shown, the kill effects of V-23-Fc-VA-Exd and V-23-Fc-Gluc-Exd on EBC-1 and NCI-H1975 are shown in Table 10B below. Figure 18B As shown in the figure. The results indicate that the killing effect of each ADC molecule on target cells is basically equal to or better than that of the positive control drugs (ABBV399 and AZD9592).

[0546] Table 10A. Cell killing effects of V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE, and V-26-Fc-PEG-EVC-MMAE on target cells

[0547] Note: "-" indicates that IC cannot be fitted. 50 value.

[0548] Table 10B. Cell killing effects of V-23-Fc-VA-Exd and V-23-Fc-Gluc-Exd on target cells

[0549] Note: "-" indicates that IC cannot be fitted. 50 value.

[0550] 7.4 Detection of cell binding and cell killing by PEG-modified and / or EVC-modified ADCs

[0551] Using V-26-Fc as the 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 previously (e.g., Examples 6.2 or 7.2), and the results are as follows. Figure 19 The cell killing assay procedure is as described in section 7.3, and the assay results are as follows. Figure 20 As shown.

[0552] Example 8. In vivo efficacy evaluation of multispecific anti-EGFR / cMet antibody-drug conjugates (ADCs)

[0553] The in vivo efficacy of trispecific anti-EGFR / cMet antibody-drug conjugates (ADCs) was evaluated using 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. Tumor cells were resuscitated with fresh culture medium. Cells in the logarithmic growth phase were collected. During cell collection, the culture medium was removed and the cells were washed twice, then resuspended in DPBS. The cells were then subcutaneously injected into BALB / c nu / nu mice. The seeding amounts for the EBC-1, NCI-H1975, NCI-H441, MDA-MB-468, FADU, and SW48 CDX models were 3 × 10⁻⁶. 6 / 100 μL, 3×10 6 / 100 μL, 1×10 7 / 100 μL, 1×10 7 / 100 μL, 5×10 6 / 100 μL and 5×10 6 / 100 μL. When the average tumor volume in mice reached 110-250 mm. 3 Mice were randomly assigned to groups based on tumor volume. The day of group assignment was defined as day 0. All CDX mouse models were administered the test drug via a single intravenous bolus injection on day 0. After administration began, mouse body weight was measured 1-2 times per week, tumor volume was measured 1-2 times per week, and animals were observed twice daily until the study endpoint. The formula for calculating tumor volume was: Tumor volume (millimeters in cubic meters) = 0.5 Tumor length Tumor width 2The antitumor efficacy of the test drug was evaluated by using changes in tumor size (based on baseline) to reflect tumor suppression. The formula for calculating changes in tumor size (based on baseline) is as follows: Tumor volume change = ( V t - V 0 ) / V 0 : The average tumor volume of mice in the test drug administration group on day t; : The average tumor volume of mice in the test drug administration group on day 0; 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 The test results are as follows Figure 21 As shown, V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE, and V-26-Fc-PEG-EVC-MMAE all exhibit strong tumor-suppressive effects. After intravenous injection, the tumor volume was significantly reduced, and the efficacy was better than that of the positive control drugs ABBV399 and AZD9592.

[0554] 8.2 In vivo efficacy of V-23-Fc-VA-Exd in the CDX model

[0555] The in vivo efficacy of V-23-Fc-VA-Exd was evaluated using EBC-1, NCI-H1975, NCI-H441, and MDA-MB-468 CDX mouse models. The results are as follows: Figure 22 As shown, V-23-Fc-VA-Exd exhibited superior in vivo tumor-inhibiting effects compared to the positive control drug AZD9592.

[0556] In the EBC-1 model, on day 54 after administration, V-23-Fc-VA-Exd and AZD9592 completely eliminated tumors at high doses; at low doses, V-23-Fc-VA-Exd was more effective than AZD9592.

[0557] In the NCI-H1975 model, on day 29 after administration, the tumor-suppressive 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.

[0558] In the NCI-H441 model, on day 45 after administration, V-23-Fc-VA-Exd was more effective than AZD9592 at the same dose; the tumor-suppressive effect of V-23-Fc-VA-Exd at 2 mg / kg was comparable to that of AZD9592 at 8 mg / kg.

[0559] In the MDA-MB-468 model, on day 42 post-dose, tumors were completely eliminated with V-23-Fc-VA-Exd and AZD9592 at 4 mg / kg and 8 mg / kg, respectively; the tumor volume change rate with V-23-Fc-VA-Exd at 2 mg / kg was -72%.

[0560] 8.3 In vivo efficacy of V-23-Fc-Gluc-Exd in the CDX model

[0561] Application of 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 using a mouse model. The results are as follows: Figure 23 As shown, V-23-Fc-Gluc-Exd exhibits superior in vivo tumor-inhibiting effects compared to the positive control drug.

[0562] In the SW48 model, FADU model, and NCI-H1975 model, V-23-Fc-Gluc-Exd showed better efficacy than the positive control drugs, including AZD9592 and ABBV399, at the same dosage.

[0563] In the EBC-1 CDX model, on day 36 after administration, V-23-Fc-Gluc-Exd was more effective than AZD9592 at a low dose (1 mg / kg), and tumors were completely eliminated in both groups of animals at a dose of 3 mg / kg.

[0564] This disclosure provides the following implementation scheme: 1. An immunoglobulin single variable domain (ISVD) that specifically binds to cMet, wherein the ISVD comprises or is composed of a VHH domain, wherein the VHH domain contains... (a) Three CDRs in the amino acid sequence shown in one of SEQ ID NO: 16, 39-40 and 121-130; (b) Three CDRs in one of the amino acid sequences shown in SEQ ID NO: 21, 42, and 134-136; or (c) Three CDRs in the amino acid sequence shown in SEQ ID NO: 26; Preferably, the VHH domain includes: (i) CDR1 comprising an amino acid sequence selected from one of SEQ ID NO: 18, 41 and 131-133 or composed thereof, CDR2 and CDR3 comprising an amino acid sequence selected from SEQ ID NO: 19 and SEQ ID NO: 20 or composed thereof; in particular, CDR1, CDR2 and CDR3 comprising an amino acid sequence selected from SEQ ID NO: 41, 19 and 20 or composed thereof; (ii) Containing the amino acid sequences of SEQ ID NO: 23-25 ​​or CDR1, CDR2, and CDR3 thereof, respectively; or (iii) Each of the amino acid sequences of SEQ ID NO: 28-30 or CDR1, CDR2 and CDR3 composed thereof; More preferably, the VHH domain includes: (a) A sequence of one of SEQ ID NO: 16, 39-40 and 121-130 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (b) A sequence of one of SEQ ID NO: 21, 42, and 134-136, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or (c) A sequence of SEQ ID NO: 26 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; More preferably, the VHH domain includes: (a) The amino acid sequence shown in one of SEQ ID NO: 16, 39-40 and 121-130; (b) The amino acid sequence shown in one of SEQ ID NO: 21, 42, and 134-136; or (c) The amino acid sequence shown in SEQ ID NO: 26, In particular, the VHH domain contains the amino acid sequence of SEQ ID NO:39 or 40, or contains the amino acid sequence of SEQ ID NO:42.

[0565] 2. An immunoglobulin single variable domain (ISVD) that specifically binds to EGFR, comprising or consisting of a VHH domain, wherein the VHH domain contains...

[0566] (a) Three CDRs in the amino acid sequence shown in one of SEQ ID NO: 1, 31 and 94-99; (b) Three CDRs in one of the amino acid sequences shown in SEQ ID NO: 6, 32, 100-102; or (c) Three CDRs in one of the amino acid sequences shown in SEQ ID NO: 11, 36, 84 and 105-114; Preferably, the VHH domain includes: (i) Each of the amino acid sequences of SEQ ID NO: 3-5 or CDR1, CDR2 and CDR3 composed thereof; (ii) CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 9, 34, or 103, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 10, 35, or 104; particularly, CDR1, CDR2, and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NO: 8, 34, and 35, respectively; or (iii) CDR1 comprising or composed of the amino acid sequence of SEQ ID NO: 13, CDR2 comprising or composed of the amino acid sequence of one of SEQ ID NO: 14, 38, 85, 115-120, and CDR3 comprising or composed of the amino acid sequence of SEQ ID NO: 15; in particular, CDR1, CDR2 and CDR3 comprising or composed of the amino acid sequences of SEQ ID NO: 13, 85 and 15, or SEQ ID NO: 13, 38 and 15, respectively; More preferably, the VHH domain includes: (a) A sequence of one of SEQ ID NO: 1, 31 and 94-99 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (b) A sequence of one of SEQ ID NO: 6, 32, 100-102, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or (c) A sequence of one of SEQ ID NO: 11, 36, 84 and 105-114 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; More preferably, the VHH domain includes: (a) The amino acid sequence shown in one of SEQ ID NO: 1, 31 and 94-99; (b) The amino acid sequence shown in one of SEQ ID NO: 6, 32, 100-102; or (c) The amino acid sequence shown in one of SEQ ID NO: 11, 36, 84 and 105-114; In particular, the VHH domain contains the amino acid sequence of SEQ ID NO:31, or contains the amino acid sequence of SEQ ID NO:32, or contains the amino acid sequence of SEQ ID NO:36, or contains the amino acid sequence of SEQ ID NO:84.

[0567] 3. A binding molecule comprising the ISVD according to embodiment 1 or 2.

[0568] 4. The binding molecule according to embodiment 3 comprises or consists of antibodies selected from: single-domain antibodies, nanobodies, heavy chain antibodies, monospecific antibodies, or multispecific antibodies.

[0569] 5. An antibody comprising at least one (e.g., 1, 2, 3, 4 or more) ISVD that specifically binds to cMet according to embodiment 1.

[0570] 6. The antibody according to embodiment 5, wherein the antibody further comprises at least one (e.g., 1, 2, 3, 4 or more) ISVD that specifically binds to EGFR, preferably the ISVD according to embodiment 2.

[0571] 7. An antibody according to any one of implementation schemes 5-6, wherein: (a) The antibody specifically binds to cMet and contains two ISVDs that specifically bind to the same epitope on cMet; or (b) The antibody specifically binds to cMet and includes two ISVDs that specifically bind to different epitopes on cMet.

[0572] 8. An antibody according to any one of implementation schemes 5-7, 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.

[0573] 9. An antibody according to any one of embodiments 5-8, wherein the antibody comprises first and second ISVDs that specifically bind to cMet, wherein the first and second ISVDs are respectively ISVDs according to embodiment 1 that specifically bind to the same epitope on cMet. Preferably, the first and second ISVDs comprise: (i) a CDR1 comprising an amino acid sequence selected from SEQ ID NO: 18 or 41 or composed thereof, and a CDR2 and CDR3 comprising amino acid sequences selected from SEQ ID NO: 19 and SEQ ID NO: 20 or composed thereof; or (ii) a CDR1, CDR2 and CDR3 comprising amino acid sequences selected from SEQ ID NO: 23-25 ​​or composed thereof; More preferably, the first and second ISVDs comprise, or are substantially composed of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in one of SEQ ID NO: 16, 39, 40 or SEQ ID NO: 21 or 42; More preferably, the first and second ISVDs comprise, or are substantially composed of, the amino acid sequences of SEQ ID NO: 16, 39, 40 or SEQ ID NO: 21 or 42.

[0574] 10. An antibody according to any one of embodiments 5-9, wherein the antibody comprises first and second ISVDs that specifically bind to cMet, wherein the first and second ISVDs are ISVDs according to embodiment 1 that specifically bind to different epitopes on cMet. Preferably, wherein: the first ISVD includes a first anti-cMet VHH domain, and the second ISVD includes a second anti-cMet VHH domain; or the first ISVD includes a second anti-cMet VHH domain, and the second ISVD includes a first anti-cMet VHH domain. The first anti-cMet VHH domain comprises: CDR1, CDR2 and CDR3 comprising amino acid sequences of SEQ ID NO: 23-25 ​​or composed thereof; and the second anti-cMet VHH domain comprises: CDR1 comprising amino acid sequences selected from SEQ ID NO: 18 or 41 or composed thereof, and CDR2 and CDR3 comprising amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20 or composed thereof. Preferably, the first anti-cMet VHH domain comprises, or is substantially composed of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, or is substantially composed of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16, 39, or 40; More preferably, the first anti-cMet VHH domain comprises, or is substantially composed of, the amino acid sequence of SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, or is substantially composed of, the amino acid sequence of SEQ ID NO: 16, 39 or 40.

[0575] Preferably, the first ISVD includes a first anti-cMet VHH domain, and the second ISVD includes a second anti-cMet VHH domain.

[0576] 11. An antibody according to any one of embodiments 5-10, wherein the antibody further comprises an anti-EGFR ISVD that specifically binds to EGFR, wherein the anti-EGFR ISVD is the ISVD according to embodiment 2. Preferably, the anti-EGFR ISVD comprises: (i) Containing amino acid sequences of SEQ ID NOs: 3, 4 and 5, or CDR1, CDR2 and CDR3 composed thereof; (ii) Containing amino acid sequences of SEQ ID NOs: 8, 9, and 10, or amino acid sequences of SEQ ID NOs: 8, 34, and 35, or amino acid sequences of SEQ ID NOs: 8, 103, and 104, or CDR1, CDR2, and CDR3 composed thereof; or (iii) Each of the amino acid sequences comprising SEQ ID NOs: 13, 14 and 15, or SEQ ID NOs: 13, 85 and 15, or SEQ ID NOs: 13, 38 and 15, or CDR1, CDR2 and CDR3 thereof. More preferably, the anti-EGFR ISVD: (a) Contains an amino acid sequence shown in one of SEQ ID NO: 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 with it, or is substantially composed of it, or is composed of it. (b) Contains, or is substantially composed of, an amino acid sequence shown in one of SEQ ID NO: 6, 32, 100-102, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such sequence; or (c) Contains an amino acid sequence shown in one of SEQ ID NO: 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 with it, or is substantially composed of it, or is composed of it. More preferably, the anti-EGFR ISVD: (a) Contains, is substantially composed of, or is composed of the amino acid sequence shown in SEQ ID NO: 31; (b) Contains, is substantially composed of, or is composed of the amino acid sequence shown in SEQ ID NO: 32; or (c) Contains, or is substantially composed of, or is composed of the amino acid sequence shown in SEQ ID NO: 84.

[0577] 12. An antibody according to any one of embodiments 5-11, wherein the antibody is in single-chain or double-chain form.

[0578] 13. An antibody according to any one of embodiments 5-12, wherein ISVDs located on the same polypeptide chain are linked 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).

[0579] 14. An antibody according to any one of embodiments 5-13, wherein the antibody further comprises a structural moiety that increases half-life, preferably an immunoglobulin Fc region or an ISVD that binds to human serum albumin, optionally wherein: - The immunoglobulin Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4 isotype; - The ISVD that binds to human serum albumin is an anti-HSA ISVD containing or composed of a VHH domain, wherein the VHH domain contains CDR1-3 of SEQ ID NO: 46-48.

[0580] 15. An antibody according to any one of embodiments 5-14, wherein the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus, The first polypeptide chain contains: a first ISVD that specifically binds to cMET and an immunoglobulin Fc region; The second polypeptide chain contains: a second ISVD that specifically binds to cMET and an immunoglobulin Fc region. Preferably, wherein: - The first polypeptide chain contains the sequence of SEQ ID NO: 88 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and - The second polypeptide chain contains the sequence of SEQ ID NO: 89 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. More preferably, the first polypeptide chain comprises or is composed of the sequence of SEQ ID NO: 88; and the second polypeptide chain comprises or is composed of the sequence of SEQ ID NO: 89.

[0581] 16. An antibody according to any one of embodiments 5-14, wherein the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus...

[0582] The first polypeptide chain contains: ISVD, which specifically binds to EGFR, and the Fc region of immunoglobulin. The second polypeptide chain includes: a first ISVD that specifically binds to cMET, a peptide linker, a second ISVD that specifically binds to cMET, and an immunoglobulin Fc region.

[0583] 17. An antibody according to any one of embodiments 5-14, wherein said antibody comprises a single polypeptide chain. The polypeptide chain comprises: a first ISVD that specifically binds to cMET, a second ISVD that specifically binds to cMET, an ISVD that specifically binds to EGFR, and optionally an ISVD that specifically binds to HSA. Preferably, from the N-terminus to the C-terminus, the polypeptide chain comprises: The ISVD specifically binds to EGFR, the first peptide linker, the first ISVD specifically binds to cMET, the second peptide linker, the second ISVD specifically binds to cMET, and optionally the third peptide linker and the ISVD specifically binds to HSA.

[0584] 18. The antibody according to embodiment 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 with it. - 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 with it; and Preferably, wherein: (i) The first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 70 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 73, 75 or 74 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (ii) The first polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 71 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and the second polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 73, 75, or 74 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. (iii) The first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 72 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 73, 75 or 74 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (iv) The first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 86 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 87 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it. More preferably, wherein: (i) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (ii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 73; (iii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO: 87.

[0585] 19. The antibody according to embodiment 17, wherein the antibody comprises a single polypeptide chain, wherein the polypeptide chain comprises a sequence selected from SEQ ID NO: 51-66 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; or wherein the polypeptide chain comprises a sequence selected from SEQ ID NO: 67-68 and 76-83 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; Preferably, the polypeptide chain comprises or consists of sequences selected from SEQ ID NO:51-56.

[0586] 20. The antibody according to any one of embodiments 5-19, which is a multispecific antibody that specifically binds to EGFR and cMet, and has one or more of the following characteristics:

[0587] (1) It binds to EGFR with moderate or low affinity, such as human EGFR; and specifically binds to cMet, such as human cMet; (2) It 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 ligand HGF, block the binding of ligand HGF to cMet on the cell surface; (4) Internalization of cMet-expressing cells; (5) Internalization of cells expressing EGFR; (6) It exhibits cross-reactivity with human EGFR and cynomolgus monkey EGFR; and also with human cMet and cynomolgus monkey cMet.

[0588] 21. An antibody comprising at least one EGFR-specific ISVD as described in embodiment 2, optionally further comprising at least one cMet-specific ISVD, preferably the cMet-specific ISVD as described in embodiment 1.

[0589] Preferably, the antibody has one or more of the following properties: (1) Binds to EGFR with low affinity, such as human EGFR; (2) It has cross-reactivity with human EGFR and cynomolgus monkey EGFR.

[0590] 22. An isolated nucleic acid encoding the ISVD described in embodiment 1 or 2, the binding molecule described in embodiment 3 or 4, or the antibody described in any one of embodiments 5-21.

[0591] 23. A vector comprising the nucleic acid of embodiment 22, preferably an expression vector.

[0592] 24. A host cell comprising the nucleic acid of embodiment 22 or the vector of embodiment 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 ISVD, and most preferably, the host cell is HEK 293 cell or CHO cell.

[0593] 25. A method of preparation, the method comprising culturing a host cell of embodiment 24, optionally recovering from the host cell or from a culture medium the ISVD of embodiment 1 or 2, the binding molecule of embodiment 3 or 4, or the antibody of any one of embodiments 5-21.

[0594] 26. An immunoconjugate or immunofusion comprising the ISVD of embodiment 1 or 2, the binding molecule of embodiment 3 or 4, or the antibody of any one of embodiments 5-21.

[0595] 27. Antibody-drug conjugates having formula (I0) or pharmaceutically acceptable salts or solvates thereof: Ab-(LD) p (I0) in: Ab is the ISVD described in embodiment 1 or 2, the binding molecule described in embodiment 3 or 4, or the antibody described in any one of embodiments 5-21; L is the connector; D represents a drug, such as an anti-tumor compound; p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0596] 28. The antibody-drug conjugate according to embodiment 27, or a pharmaceutically acceptable salt or solvation thereof, wherein the antibody-drug conjugate has formula (I): Ab-(SLD) p (I) in: Ab is the ISVD described in embodiment 1 or 2, the binding molecule described in embodiment 3 or 4, or the antibody described in any one of embodiments 5-21; L is the connector; D represents a drug, such as an anti-tumor compound; p is an integer selected from 1 to 16, for example, an integer selected from 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 In formula (I), S is sulfur derived from Ab.

[0597] 29. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 27 or 28, wherein the drug is a cytotoxic agent, such as a camptothecin or auratestatin.

[0598] 30. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 27-29, wherein D has the structure shown in formula (D-1a) or formula (D-1b): Equation (D-1a) Where R 1a Selected from H and C1-C6 alkyl groups; R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ; R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and R 4a and R 5a Independently selected from H and C1-C4 alkyl groups; or Equation (D-1b) Where R 1b R 2b R 3b R 4b R 5b and R 8b Each is independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; R 6b and R 7b Each is independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy; R 9b Selected from C 1-8 Alkyl groups and COOH; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and R 10b Selected from OH and H; In the D structure, the wavy line indicates that the valence bond is connected to L.

[0599] 31. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 30, wherein D has the structure of formula (D-1a), and wherein R 1a For H; R 2a It is a C1-C6 alkyl group; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl.

[0600] 32. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 30, wherein D has the structure of formula (D-1b), and wherein

[0601] R 1b R 4b and R 8b Each is independently selected from C 1-2 Alkyl; preferably methyl; R 2b R 3b and R 5b Each is independently selected from C 3-4 alkyl; R 6b and R 7b Each is independently selected from C 1-2 alkoxy groups; and R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H.

[0602] 33. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 27-32, wherein D has the structure shown in formula (D-2a) or formula (D-2b): Equation (D-2a), Where R 1a R 2a R 3a and R 4a As defined in equation (D-1a); or Equation (D-2b) Where R 1b R 2b R 3b R 4bR 5b R 6b R 7b R 8b R 9b and R 10b As defined in equation (D-1b).

[0603] 34. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 27-29, wherein D has the structure shown in formula (D-3a) or (D-3b): (D-3a) or (D-3b); Preferably, D has the structure shown in formula (D-4a) or (D-4b): (D-4a) or (D-4b).

[0604] 35. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 27-29, wherein the drug is Exatecan, Dxd, SN-38, monomethylaurestatin E (MMAE) or MMAF.

[0605] 36. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 27-35, wherein -L- has the following structure: -Z-L1-L2-L3- in Z is selected from , , , , and , where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non-existent, , , and , where n1 and m1 are each independently an integer selected from 0 to 20, for example, an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8; L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and L3 is selected from: , , , and Where X is selected from -NH-, -O-, and -S-; R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; n3 and n4 are independently 1, 2, 3, 4, 5, or 6; and Z is connected to S on Ab, and L3 is connected to D.

[0606] 37. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 36, wherein...

[0607] Z is selected from and , where m is 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, Z is selected from , and .

[0608] 38. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 36 or 37, wherein L1 is selected from those that do not exist. and , where n1 is an integer independently selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, L1 is selected from non-existent, and .

[0609] 39. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-38, wherein L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids; preferably, wherein each amino acid residue or amino acid is 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); 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).

[0610] 40. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-38, 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-.

[0611] 41. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-40, wherein L3 is selected from: , , , and , Where R 1c Each is independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is selected independently from each of the following groups: , , and 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.

[0612] 42. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-41, wherein L3 is selected from: , , , and ; Preferably, L3 is selected from: , , , and ; More preferably, L3 is selected from: , and .

[0613] 43. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-42, wherein each of Su is independently: ; Preferably, Su is independently , Alternatively, preferably, each Su is independently... .

[0614] 44. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 36-43, wherein L3 is selected from: , , , and ; Preferably, L3 is selected from and .

[0615] 45. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 36, wherein...

[0616] -Z-L1-L2-L3- are each independently selected from the following structures: , , , , , , , , ,and , Where each m is an integer selected from 1 to 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 group is connected to the S on the left side of Ab and to the D on the right side.

[0617] 46. ​​The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment 27 or 28, wherein the antibody-drug conjugate is an antibody-drug conjugate having a structure selected from:

[0618] Wherein Ab is the ISVD described in embodiment 1 or 2, the binding molecule described in embodiment 3 or 4, or the antibody described in any one of embodiments 5-21; and

[0619] p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0620] 47. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments 27-46, wherein the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, or 2-4 or 2-6.

[0621] 48. A pharmaceutical composition comprising the ISVD of embodiment 1 or 2, the binding molecule of embodiment 3 or 4, the antibody of any one of embodiments 5-21, the immunoconjugate or immunofusion of embodiment 26, or the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of any one of embodiments 27-47, and a pharmaceutically acceptable carrier, and optionally further comprising one or more additional pharmaceutically active peptides and / or compounds, for example, other therapeutic agents selected from inhibitors of oncolytic agents, cytotoxic agents, cytokines, and immune checkpoint molecules.

[0622] 49. Use of the ISVD of embodiment 1 or 2, the binding molecule of embodiment 3 or 4, the antibody of any one of embodiments 5-21, or the immunoconjugate or immunofusion of embodiment 26, or the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate of any one of embodiments 27-47, as a drug or for the preparation of a drug, wherein preferably the drug is used to treat cancer, for example selected from lung cancer (e.g., squamous cell carcinoma of the lung, adenocarcinoma of the lung, 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).

[0623] The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0624] Sequence List Overview:

Claims

1. 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 NO: 1, 31 and 94-99; (b) Three CDRs in one of the amino acid sequences shown in SEQ ID NO: 6, 32, 100-102; or (c) Three CDRs in one of the amino acid sequences shown in SEQ ID NO: 11, 36, 84 and 105-114; Preferably, the VHH domain includes: (i) Each of the amino acid sequences of SEQ ID NO: 3-5 or CDR1, CDR2 and CDR3 composed thereof; (ii) CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 9, 34, or 103, and CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 10, 35, or 104; particularly, CDR1, CDR2, and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NO: 8, 34, and 35, respectively; or (iii) CDR1 comprising or composed of the amino acid sequence of SEQ ID NO: 13, CDR2 comprising or composed of the amino acid sequence of one of SEQ ID NO: 14, 38, 85, 115-120, and CDR3 comprising or composed of the amino acid sequence of SEQ ID NO: 15; in particular, CDR1, CDR2 and CDR3 comprising or composed of the amino acid sequences of SEQ ID NO: 13, 85 and 15, or SEQ ID NO: 13, 38 and 15, respectively; More preferably, the VHH domain includes: (a) A sequence of one of SEQ ID NO: 1, 31 and 94-99 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (b) A sequence of one of SEQ ID NO: 6, 32, 100-102, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or (c) A sequence of one of SEQ ID NO: 11, 36, 84 and 105-114 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; More preferably, the VHH domain includes: (a) The amino acid sequence shown in one of SEQ ID NO: 1, 31 and 94-99; (b) The amino acid sequence shown in one of SEQ ID NO: 6, 32, 100-102; or (c) The amino acid sequence shown in one of SEQ ID NO: 11, 36, 84 and 105-114; In particular, the VHH domain contains the amino acid sequence of SEQ ID NO:31, or contains the amino acid sequence of SEQ ID NO:32, or contains the amino acid sequence of SEQ ID NO:36, or contains the amino acid sequence of SEQ ID NO:

84.

2. An immunoglobulin single variable domain (ISVD) that specifically binds to cMet, wherein the ISVD comprises or is composed of a VHH domain, wherein the VHH domain contains... (a) Three CDRs in the amino acid sequence shown in one of SEQ ID NO: 16, 39-40 and 121-130; (b) Three CDRs in one of the amino acid sequences shown in SEQ ID NO: 21, 42, and 134-136; or (c) Three CDRs in the amino acid sequence shown in SEQ ID NO: 26; Preferably, the VHH domain includes: (i) CDR1 comprising an amino acid sequence selected from one of SEQ ID NO: 18, 41 and 131-133 or composed thereof, CDR2 and CDR3 comprising an amino acid sequence selected from SEQ ID NO: 19 and SEQ ID NO: 20 or composed thereof; in particular, CDR1, CDR2 and CDR3 comprising an amino acid sequence selected from SEQ ID NO: 41, 19 and 20 or composed thereof; (ii) Containing the amino acid sequences of SEQ ID NO: 23-25 ​​or CDR1, CDR2, and CDR3 thereof, respectively; or (iii) Each of the amino acid sequences of SEQ ID NO: 28-30 or CDR1, CDR2 and CDR3 composed thereof; More preferably, the VHH domain includes: (a) A sequence of one of SEQ ID NO: 16, 39-40 and 121-130 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; (b) A sequence of one of SEQ ID NO: 21, 42, and 134-136, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; or (c) A sequence of SEQ ID NO: 26 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; More preferably, the VHH domain includes: (a) The amino acid sequence shown in one of SEQ ID NO: 16, 39-40 and 121-130; (b) The amino acid sequence shown in one of SEQ ID NO: 21, 42, and 134-136; or (c) The amino acid sequence shown in SEQ ID NO: 26, In particular, the VHH domain contains the amino acid sequence of SEQ ID NO:39 or 40, or contains the amino acid sequence of SEQ ID NO:

42.

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

4. An antibody comprising at least one (e.g., 1, 2, 3, 4 or more) ISVD that specifically binds to EGFR according to claim 1. Optionally, the antibody further comprises at least one (e.g., 1, 2, 3, 4 or more) ISVD that specifically binds to cMet, preferably the ISVD that specifically binds to cMet is the ISVD according to claim 2.

5. An immunoconjugate or immunofusion comprising the ISVD of claim 1 or 2, the binding molecule of claim 3, or the antibody of claim 4.

6. Antibody-drug conjugates having formula (I0) or pharmaceutically acceptable salts or solvates thereof: Ab-(LD) p (I0) in: Ab is the ISVD as described in claim 1 or 2, the binding molecule as described in claim 3, or the antibody as described in claim 4; L is the connector; D represents a drug, such as an anti-tumor compound; p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

7. A pharmaceutical composition comprising the ISVD of claim 1 or 2, the binding molecule of claim 3, the antibody of claim 4, the immunoconjugate or immunofusion of claim 5, or the antibody-drug conjugate of claim 6, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, and optionally further comprising one or more other pharmaceutically active peptides and / or compounds, for example, other therapeutic agents selected from inhibitors of oncolytic drugs, cytotoxic agents, cytokines, and immune checkpoint molecules.

8. Use of the ISVD of claim 1 or 2, the binding molecule of claim 3, the antibody of claim 4, or the immunoconjugate or immunofusion of claim 5, or the antibody-drug conjugate of claim 6, or a pharmaceutically acceptable salt or solvate thereof, as a medicine or for the preparation of a medicine, wherein preferably the medicine is used to treat cancer, for example selected from lung cancer (e.g., squamous cell carcinoma of the lung, adenocarcinoma of the lung, 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).

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