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) that include the immunoglobulin single variable domains. The disclosure also provides nucleic acids encoding the ISVD or binding molecules and vectors comprising the nucleic acids, as well as therapeutic applications of the ISVD or binding molecules and the ADCs.

Description

Technical Field

[0001] The present invention relates to an immunoglobulin single variable domain (ISVD) that specifically binds to EGFR and / or cMet, and an EGFR and / or cMet binding molecule and an antibody drug conjugate (ADC) comprising the immunoglobulin single variable domain. The present invention also relates to a nucleic acid encoding the ISVD or binding molecule and a vector comprising the nucleic acid, as well as the therapeutic application of the ISVD or binding molecule and the ADC. Background Art

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

[0003] cMet is a receptor with tyrosine kinase (RTK) activity expressed on epithelial cells, which plays a key role in cell proliferation, survival and migration. The binding ligand of cMet is hepatocyte growth factor (HGF). After binding to HGF, cMet dimerizes and activates the cMet pathway, promoting cell division, angiogenesis and immune regulation. Since cMet is highly expressed or mutated in many cancers, cMet has become an important drug target, and related anticancer drugs are being studied and developed.

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

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

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

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

[0008] Therefore, in a first aspect, the present disclosure provides an immunoglobulin single variable domain (ISVD) that specifically binds to cMet and a heavy chain antibody comprising the ISVD and a cMet binding molecule. In some embodiments, the cMet binding molecule is an anti-cMet bi-epitope antibody. In some embodiments, the cMet binding molecule further comprises a binding domain that binds to EGFR.

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

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

[0011] In a fourth aspect, the present disclosure provides nucleic acids encoding the ISVD, EGFR binding molecules, cMet binding molecules, EGFR and cMet binding molecules according to the first to third aspects of the present disclosure, vectors (preferably, expression vectors) comprising the nucleic acids, and host cells comprising the nucleic acids or the vectors. In some embodiments, the host 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 a HEK 293 cell or a CHO cell. The present disclosure also provides methods for preparing the ISVD, EGFR binding molecules, cMet binding molecules, EGFR and cMet binding molecules according to the first to third aspects of the present disclosure.

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

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

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

[0015] The ISVD specifically binding to EGFR and / or cMet and the EGFR and / or cMet binding molecules comprising the ISVD of the present invention have good tumor targeting, tumor tissue penetration ability and / or target cell endocytosis ability, etc., so the ISVD or binding molecule can be used as a targeting module of a conjugate or a coupling, and 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.

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

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

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

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

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

[0021] (4) It has bystander effects;

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

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

[0024] In some embodiments, the antibody drug conjugate according to the present invention also has one or more advantages selected from the following:

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

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

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

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

[0029] In some embodiments, the antibody drug conjugate according to the present invention also has one or more advantages selected from the following:

[0030] (11) Good product uniformity;

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

[0032] (13) It has good drugability.

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

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

[0035] Combined with the following Figure 1 When reading together, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the present invention, the drawings show the currently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0036] Figure 1The binding activity of anti-EGFR antibody to target cells was detected by FACS.

[0037] Figure 2 The binding activity of anti-cMet antibody to target cells was detected by FACS.

[0038] Figure 3 The blocking effect of anti-cMet VHH-Fc on the binding of the ligand HGF to the target cell EBC-1 is shown.

[0039] Figure 4 The anti-cMet antibodies V-n7A12 and V-n9A2 were shown to bind to different epitopes on the antigen cMet.

[0040] Figure 5A and 5B The endocytosis of each VHH-Fc by cells was detected by FACS.

[0041] Figure 6 The results of ELISA testing of whether the anti-cMet antibodies V-n7A12, V-n9A2, and V-n9A10 have cross-reactivity with human cMet antigen and cynomolgus monkey cMet antigen.

[0042] Figure 7 FACS detection of the binding of the bi-epitope antibody to target cells is shown.

[0043] Figure 8 Figure 3 shows the internalization of the bi-epitope antibody on target cells detected by FACS. Compared with the single epitope, the bi-epitope mediated synergistic endocytosis activity.

[0044] Fig. 9 Schematic diagram showing the molecular structure of a multispecific EGFR / cMet antibody in single-chain form.

[0045] Fig.10 Schematic diagram showing the molecular structure of a double-chain form of a multispecific EGFR / cMet antibody.

[0046] Fig.11 Binding of the trispecific antibody candidate molecules on target cells EBC-1 and NCI-H1975 cells is shown.

[0047] Fig.12 Internalization of single-chain trispecific antibody candidate molecules by target cells is shown.

[0048] Fig.13 Internalization of two-chain trispecific antibody candidate molecules by target cells is shown.

[0049] Fig.14 The trispecific anti-EGFR / cMet antibody was shown to block the binding of EBC-1 cells to the ligand HGF.

[0050] Fig.15 FACS detection of synergistic endocytosis of the trispecific antibodies mediated by anti-EGFR ISVD and anti-cMet ISVD on target cells is shown.

[0051] Fig.16 FACS detection of synergistic binding of trispecific antibodies mediated by anti-EGFR ISVD and anti-cMet ISVD on target cells is shown.

[0052] Fig.17 Shows the binding of ADC molecules to target cells.

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

[0054] Fig.19 Cell binding assays of ADC molecules with linkers of PEG and / or EVC are shown.

[0055] Fig. 20 Cell killing assays are shown for ADC molecules with linkers of PEG and / or EVC.

[0056] Fig.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.

[0057] Fig. 22 The in vivo efficacy of V-23-Fc-VA-Exd in the CDX model was shown.

[0058] Fig.23 The in vivo efficacy of V-23-Fc-Glu-Exd in the CDX model was demonstrated. DETAILED DESCRIPTION

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

[0060] definition

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

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

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

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

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

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

[0067] The domain that actually binds to the antigen in the antigen binding molecule is referred to herein as an "antigen binding site" or "antigen binding domain". "Domain" is a folded structure in a protein or polypeptide, generally speaking, responsible for a single function of a protein or polypeptide. For example, conventional antibodies and immunoglobulins form antigen binding domains on the surface of VH-VL dimers through 3 complementary determining regions (HCDR1-3) in their heavy chain variable region (VH) and 3 complementary determining regions (LCDR1-3) in the light chain variable region (VL), wherein 6 CDRs confer specific binding of antibodies to antigens. However, in some cases, a single immunoglobulin variable domain (e.g., heavy chain variable domain (VH) or light chain variable domain (VL), a heavy chain variable domain (VHH) derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) derived from fish IgNAR) can confer antigen binding. That is, the single variable domain does not need to interact with another variable domain, and can be independently used as an "antigen binding domain" for identifying and binding to a target antigen. Typically, through engineering modification, the "antigen-binding domain" of an antibody, including the above-mentioned single immunoglobulin variable domain and the variable domain pairs of conventional antibodies, can be added, removed or transferred to other proteins or polypeptides and still exert its antigen-binding function without losing the function of the rest of the protein or polypeptide and / or the rest of the domains.

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

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

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

[0071] In the present disclosure, the term "immunoglobulin single variable domain" (abbreviated as "ISVD") is used interchangeably with the term "single variable domain" to refer to an antibody polypeptide fragment that can specifically recognize and bind to an antigen of interest through a single variable domain, such as a single VHH domain or a single VH domain or a single VL domain, without pairing with additional immunoglobulin variable domains. For an ISVD composed of a VHH domain or a VH or VL domain, its structure can be considered to be composed of four framework regions ("FR") and three complementarity determining regions ("CDR"), the framework regions being referred to as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively; the four framework regions are interrupted by three complementarity determining regions or "CDRs", the complementarity determining regions being 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, four framework regions and three complementary 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 may be used alone in an isolated form, or as part of a larger protein to exert an antigen binding function. In the present 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; an ISVD that specifically binds to EGFR and cMet is also referred to as an anti-EGFR / cMet ISVD.

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

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

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

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

[0076] - Only a single domain is required to bind the antigen with high affinity and selectivity, so that there is no need for the presence of two separate domains, nor is there a need to ensure that the two domains are present in the appropriate spatial conformation and configuration (e.g. scFv generally requires the use of a specially designed linker);

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

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

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

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

[0081] - VHH domains are relatively small compared to conventional tetrapeptide antibodies and their antigen-binding fragments, and therefore exhibit higher tissue penetration and can be administered at higher doses;

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

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

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

[0085] The term "valency" refers to the number of antigen binding sites present in an antigen binding molecule (e.g., an antibody). Thus, "monovalent," "bivalent," "trivalent," and "tetravalent" antibodies refer to antibody molecules having 1, 2, 3, and 4 antigen binding sites, respectively.

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

[0087] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding of an antibody to an antigen. The variable domains of heavy and light chains generally have similar structures, including four conserved framework regions (FRs) and three complementary determining regions (CDRs). Since the CDR sequences are responsible for most antibody-antigen interactions, antibody variants that simulate the properties of known antibodies can be constructed by transforming the variable regions. In some cases, the CDR sequences from known antibodies can be transplanted to the framework regions of different antibodies with different properties, and 1 to several residue mutations can be performed as needed, such as back mutations to refine the desired properties of the antibody. In other cases, the variable domains of antibodies can be engineered to construct humanized, immunogenic and / or PTM (post-translational modification) removal 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 measured and screened in vitro or in vivo using methods known in the art and described herein. It will be appreciated that such functional variants of any variable region (eg, VH and / or VL region, VHH region) given herein are contemplated by the present invention.

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

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

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

[0091] As used herein, the term "epitope" refers to the part of an antigen to which an antibody specifically binds. An epitope can be composed of continuous and / or discontinuous amino acids that form a conformational space unit. For a discontinuous epitope, amino acids in different parts of the linear sequence of the antigen are closely adjacent in three-dimensional space by the folding of the protein molecule. Different antibodies that bind to the same antigen can be grouped with epitopes by competitive binding assays. Such competitive binding assays can be performed by methods known in the art, such as solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assays, or by the methods described in the embodiments herein.

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

[0093] In this article, the term "half-life extension domain" or the expression "half-life increasing binding part" are used interchangeably and refer to a chemical structure that can confer an increased circulating half-life to a molecule (e.g., antibody) bound thereto 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 connected to the binding molecule or antibody of the present invention by chemical conjugation or fusion according to its specific properties.

[0094] In some embodiments, the EGFR binding molecule described in the first aspect of the present disclosure, the cMet binding molecule described in the second aspect of the present disclosure, and the EGFR and cMet binding molecule described in the third aspect of the present disclosure comprise an ISVD that binds to human serum albumin as a half-life extension domain. In some embodiments, the ISVD that binds to human serum albumin is selected from the serum albumin binding portion of Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10, and Alb-23. In one embodiment, the serum albumin binding portion is Alb-8 or Alb-23 or a variant thereof, as shown on pages 7-9 of WO2012 / 175400. In some preferred embodiments, the ISVD that binds to human serum albumin comprises a CDR1 consisting of or consisting of the amino acid sequence shown in SEQ ID NO:46, a CDR2 consisting of or consisting of the amino acid sequence shown in SEQ ID NO:47, and a CDR3 consisting of or consisting of the amino acid sequence shown in SEQ ID NO:48. In some embodiments, the ISVD that binds human serum albumin comprises the sequence of SEQ ID NO: 45, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

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

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

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

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

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

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

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

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

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

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

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

[0106] Table A

[0107]

[0108]

[0109] Herein, "isolated" antibodies or antibody fragments refer to artificial antibodies or antibody fragments, recombinantly produced 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 are produced. 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 present invention are "isolated". In some embodiments, the isolated antibodies or antibody fragments are purified to more than 90%, 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC).

[0110] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0111] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which the recombinant polynucleotide is incorporated.

[0112] In this article, the terms "endocytosis" and "internalization" are used interchangeably and refer to the process in which the ligand / receptor complex is internalized and delivered to the cytosol or transferred to a suitable intracellular compartment, triggered by the binding of the ligand to the corresponding receptor on the cell surface. In some embodiments, the antibodies of the present invention trigger endocytosis mediated by EGFR and / or cMet receptors after binding to EGFR and / or cMet expressed on the cell surface. In this article, endocytosis and endocytosis rate can be measured by the methods described in the examples, for example, to characterize the endocytic activity of the antibody. In some embodiments, the antibodies of the present invention having endocytic activity can be used as a tool for carrying anti-tumor drugs into cancer cells in the ADC of the present invention.

[0113] The term "conjugate" or "conjugate" refers herein to a molecule formed by conjugating one or more immunoglobulin-related molecules or fragments thereof to one or more other molecules. A conjugate typically comprises at least one non-proteinaceous chemical structure portion, such as a chemical linker for achieving the conjugation. In some cases, the other molecules may be immunoglobulin-related molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-related molecules or fragments thereof. The one or more additional molecules may be the same or different from each other. For example, the other molecules may be target binding elements and / or effector elements, such as chemotherapeutic agents, toxins, drugs (such as immunotherapeutic agents), radioactive elements, probes or signaling molecules, etc.

[0114] "Antibody-drug conjugate (ADC)" refers to a compound obtained by connecting an antigen binding molecule and a (small molecule) drug through a linker. In this article, the term antibody drug conjugate or ADC includes its pharmaceutically acceptable salts and solvent compounds and other equivalent forms. The drug compound portion in the ADC may be referred to as "payload" or "toxin" in this article.

[0115] The term "connector" refers to a structural fragment that covalently links a drug (e.g., a small molecule drug) to an antigen binding molecule portion. It should be understood that the connection has a functional group that can form a key with the functional group of the antigen binding molecule before being connected to the antigen binding molecule. In some cases, the connection can also have a degradable portion and optionally a hydrophilicity adjustment module such as a PEG segment. In some embodiments of the ADC according to the present invention, the connection is preferably "degradable", whereby the ADC can be broken and released after being delivered to the target area (e.g., target tumor tissue site). Available such "degradable connection" includes, for example, an acid-labile connection, a peptidase-sensitive connection, a light-labile connection, a dimethyl connection, or a connection containing a disulfide.

[0116] The term "linker-payload" refers to a compound formed by the connection of a payload and a linker. In some cases, a linker-payload is used as an intermediate in the synthesis of an ADC.

[0117] The term "therapeutic agent" encompasses any substance effective in preventing or treating a disease, such as cancer, including chemotherapeutic agents, cytotoxic agents, immunomodulators (eg, immunosuppressants), other antibodies, small molecule drugs, angiogenesis inhibitors, or cytokines.

[0118] The term "drug" refers to a compound that is able to modulate biological processes, especially to alter or prevent pathological processes. In the present context, a drug preferably refers to an antitumor compound.

[0119] The term "small molecule drug" refers to a low molecular weight drug that can regulate biological processes, especially change or prevent pathological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10kD, usually less than 2kD and preferably less than 10kD, more preferably less than 500D. Small molecule drugs include, but are not limited to, organic molecules with a molecular weight as defined above, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less prone to eliciting immune responses than macromolecules.

[0120] "Anti-tumor compounds" are pharmaceutically active compounds that have effects on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773 and US5658920, camptothecin compounds such as Exatecan and Dxd (Exatecan derivatives), and auristatin compounds such as monomethyl auristatin E (MMAE) and MMAF.

[0121] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0122] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0123] The term "alkyl" as used herein refers to a fully saturated branched or unbranched hydrocarbon group. The alkyl group preferably contains 1 to 16 carbon atoms, such as 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0124] The term "alkenyl" refers to a straight or branched hydrocarbon group containing 2-16 carbon atoms and comprising at least one double bond and 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 include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, hexenyl, etc.

[0125] The term "alkynyl" refers to a straight or branched hydrocarbon group containing 2-16 carbon atoms and comprising at least one triple bond. Alkynyl groups preferably contain 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 include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc.

[0126] The term "halogen" or "halo" refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0127] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted by one or more halogen groups as defined herein. Halogenated alkyl may preferably be monohalogenated alkyl, dihalogenated alkyl or polyhalogenated alkyl (including perhalogenated alkyl). Monohalogenated alkyl groups may contain one iodine, bromine, chlorine or fluorine in the alkyl group. Dihalogenated alkyl and polyhalogenated alkyl groups may contain two or more identical halogen atoms or a combination of different halogenated groups in the alkyl group. Preferably, polyhalogenated alkyl groups contain up to 12, 10 or 8 or 6 or 4 or 3 or 2 halogen groups. Non-limiting examples of haloalkyl include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. Perhalogenated alkyl refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0128] The term "haloalkenyl" refers to an alkenyl as defined herein substituted with one or more halogen groups as defined herein. The term "haloalkynyl" refers to an alkynyl as defined herein substituted with one or more halogen groups as defined herein. The meaning of "halo" defined for "haloalkyl" is applicable to "haloalkenyl" and "haloalkynyl".

[0129] The terms "alkoxy" and "alkyl-O-" are used interchangeably to refer to an alkyl group as defined above attached via an oxygen atom. Preferably, the alkoxy group has 1 to 8 carbon atoms (C 1-8 Alkoxy), 1-6 carbon atoms (C 1-6 Alkoxy), 1-4 carbon atoms (C 1-4 Alkoxy) or 1-3 carbon atoms (C 1-3 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy, isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentyl, neopentyl, etc.), hexyloxy, heptyloxy, octyloxy, etc.

[0130] The term "amino acid" refers to naturally occurring and synthetic amino acids, amino acid analogs and artificially modified forms thereof. Amino acids may be L or D isomers. In the present disclosure, the 20 natural amino acids are represented by single-letter and three-letter abbreviations known in the art, for example: phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn; N), isoleucine (Ile; I), arginine (Arg; R), proline (Pro; P) and glutamine (Gln; Q). The remaining amino acids are expressed by 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. When not otherwise specified, the amino acids of the present invention refer to L-amino acids.

[0131] The term "penturonic acid" refers to a compound formed by oxidation of the primary hydroxyl group of a pentose as defined above to a carboxyl group. Examples of penturonic acids include, but are not limited to, xyluronic acid and arabinuronic acid.

[0132] The term "hexuronic acid" refers to a compound formed by oxidation of the primary hydroxyl group of a hexose as defined above to a carboxyl group. Examples of hexuronic acid include, but are not limited to, glucuronic acid, galacturonic acid, and mannuronic acid.

[0133] The term "optional" or "optionally": means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, when a group or structure is "optionally substituted", the group or structure may be substituted or may not be substituted.

[0134] "Pharmaceutically acceptable" herein refers to being able to be used for administration to an individual or subject without causing any biological or other effects that are not desirable, such as serious intolerable side effects. In the absence of contradictions in the context, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably herein.

[0135] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugate of the present invention, and the salt is not biologically or otherwise undesirable. The ADC conjugate of the present invention may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugate of the present invention and an organic or inorganic acid, and the organic or inorganic acid includes but is 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 and 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; organic base salts, such as ammonium salts formed by organic bases containing N groups.

[0136] The term "solvate" refers to an association formed by one or more solvent molecules and the ADC antibody-drug conjugate of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0137] The term "drug: antibody ratio" or "DAR" refers to the ratio of the drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety. In some embodiments described herein, the DAR can be determined by p in Formula I, for example, the DAR can be 1 to 16, 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 can also be calculated as the average DAR of the population of molecules in the product, i.e., the overall ratio of the drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in the product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), and this DAR is referred to as the average DAR herein. In some embodiments, the average DAR value of the conjugate of the invention is 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 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 , 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, and ranges with two of these values ​​as endpoints. It should be understood that when referring to an average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules, which comprises ADC molecules with the same and / or different DARs.

[0138] The term "median effective concentration (EC 50 )” refers to the concentration of a drug, antibody, ADC or toxicant that induces a response that is 50% between baseline and maximum after a specified exposure time. In the context of this application, EC 50 The unit is "nM".

[0139] The term "fluorescence activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). Instruments for performing FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACS 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).

[0140] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0141] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), carrier, stabilizer, etc., which is administered together with the active substance.

[0142] The term "drug combination", "combination product", "drug association" or "combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antigen binding molecules or ADC molecules of the present invention include pharmaceutically acceptable salts thereof, and (ii) other therapeutic agents) are applied to the patient in sequence as separate entities simultaneously, without specific time restrictions or at the same or different time intervals, wherein such administration provides two or more active agents at effective levels of prevention or treatment in the patient. In some embodiments, the antigen binding molecules or ADC molecules of the present invention and other therapeutic agents used in the drug combination are applied at levels not exceeding those when they are used alone. The term "fixed combination" means that two or more active agents are applied to the 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 each part can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in a separate formulation form, and its formulation form can be the same or different.

[0143] The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0144] The terms "tumor" and "cancer" are used interchangeably herein to refer to the physiological condition in mammals in which cell growth is unregulated and encompass both solid and liquid tumors, and encompass both malignant and benign tumors, and all pre-cancerous and cancerous cells and tissues.

[0145] As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual being treated. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, improving or alleviating the disease state, and alleviating or improving prognosis. In the case of tumor or cancer treatment, "treatment" covers anti-tumor biological effects that can be caused by human intervention (e.g., by the administration of a drug), including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0146] As used herein, "prevention" includes inhibition of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

[0147] The term "effective amount" refers to an amount or dosage of an antigen binding molecule or ADC molecule or composition or combination of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in a single or multiple doses. Depending on the desired effect, "therapeutically effective amount" and "preventive effective amount" may be included.

[0148] The term "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or ADC can vary according to a variety of factors such as disease state, age, sex and weight of the individual and the ability of the antibody or ADC to stimulate the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or ADC are less than the therapeutically beneficial effects. Relative to untreated subjects, a "therapeutically 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 a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.

[0149] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0150] The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

[0151] The present invention is described in detail below. It will be understood by those skilled in the art that, unless the context clearly indicates otherwise, any technical features described in any of the following sections, subsections or embodiments may be combined with any technical features described in any other sections, subsections or embodiments, and these combinations are all within the scope of the present invention.

[0152] I. First aspect of the present disclosure: ISVD that specifically binds to cMet and cMet-binding molecules comprising the ISVD

[0153] cMet (or c-Met) is the gene product of the proto-oncogene MET, which is encoded on chromosome 7 and recognizes only one known ligand, hepatocyte growth factor (HGF). The cMet protein is a receptor tyrosine kinase that is overexpressed or mutated in many tumor cell types and plays a key role in tumor cell proliferation, survival, invasion, metastasis and tumor angiogenesis. Inhibition of cMet can induce cell death in tumor cells that overexpress cMet protein or express constitutively activated cMet protein.

[0154] The first aspect of the present disclosure provides an ISVD that specifically binds to cMet (i.e., an anti-cMet ISVD) and a heavy chain antibody and a 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 to human cMet with medium 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.

[0155] The ISVD specifically binding to cMet of the present invention comprises three complementary determining regions from the N-terminus to the C-terminus, namely CDR1, CDR2 and CDR3.

[0156] In some embodiments, the immunoglobulin single variable domain (ISVD) that specifically binds to cMet of the present invention comprises

[0157] (a) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 16, 39-40 and 121-130;

[0158] (b) three CDRs in the amino acid sequence shown in SEQ ID NO:21 or one of SEQ ID NOs:42 and 134-136; or

[0159] (c) 3 CDRs in the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the ISVD that specifically binds to cMet of the present invention comprises a variant having a single or multiple CDR with no more than 1 to 3 amino acid changes in each CDR compared to the 3 CDRs in one of (a)-(c) above; wherein the amino acid changes are additions, deletions or conservative amino acid substitutions of amino acids. In some embodiments, the CDRs according to the present invention are defined according to AbM, Chothia, Kabat, IMGT or any combination thereof, preferably defined according to Kabat or AbM or a combination thereof.

[0160] In some embodiments, an ISVD of the invention that specifically binds cMet comprises a

[0161] (a) CDR1 shown in SEQ ID NO: 18 or a variant of CDR1 shown in SEQ ID NO: 18 with no more than 1 amino acid change (e.g., CDR1 shown in any one of SEQ ID NOs: 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 2 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 2 amino acid changes;

[0162] (b) CDR1 shown in SEQ ID NO:23 or a variant of CDR1 shown in SEQ ID NO:23 with no more than 1 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 2 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 2 amino acid changes; or

[0163] (c) CDR1 shown in SEQ ID NO:28 or a variant of CDR1 shown in SEQ ID NO:28 with no more than 1 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 2 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 2 amino acid changes;

[0164] The amino acid changes are amino acid additions, deletions or conservative amino acid substitutions.

[0165] In some embodiments, the present disclosure provides an immunoglobulin single variable domain (ISVD) that specifically binds cMet, wherein the ISVD comprises:

[0166] (a) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 18, 19 and 20, respectively;

[0167] (b) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 41, 19 and 20, respectively;

[0168] (c) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 131, 19 and 20, respectively;

[0169] (d) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 132, 19 and 20, respectively;

[0170] (e) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 133, 19 and 20, respectively; or

[0171] (f) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 23, 24 and 25, respectively. In some embodiments, the ISVD defined in (b) or (f) above is preferred.

[0172] In some embodiments, the ISVD of the present invention that specifically binds to cMet comprises or consists of a VHH. In some embodiments, the ISVD of the present invention that specifically binds to cMet comprises or consists of the following sequence:

[0173] (a) a sequence of one of SEQ ID NOs: 16, 39-40, and 121-130, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0174] (b) a sequence of one of SEQ ID NOs: 21, 42, and 134-136, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0175] (c) a sequence of SEQ ID NO:26, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0176] Preferably, the amino acid changes do not occur in the CDR regions. In some embodiments, the ISVD comprising the amino acid sequence of SEQ ID NO: 39, 40 or 42 is preferred.

[0177] In some embodiments, the present disclosure provides antibodies comprising an ISVD of the present invention that specifically binds to cMet, particularly an anti-cMet heavy chain antibody.

[0178] In some embodiments, the disclosure provides binding molecules comprising an ISVD of the invention that specifically binds to cMet. In some embodiments, the cMet binding molecule comprises or consists of an antibody selected from the group consisting of a single domain antibody, a nanobody, a VHH antibody, or a heavy chain antibody. In other embodiments, the cMet binding molecule is selected from a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0179] In some embodiments, the cMet-binding molecule of the present invention comprises at least one ISVD that specifically binds to cMet of the present invention, for example, it comprises two, three, four or more identical or different ISVDs that specifically bind to cMet of the present invention, preferably, it comprises two, three or four different ISVDs that specifically bind to cMet of the present invention. In some embodiments, the cMet-binding molecule provided by the present invention comprises two ISVDs that bind to different epitopes of cMet.

[0180] 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 VHH of camelid animals, the humanized VHH has a reduced human anti-camelid antibody response to the human body, thereby improving the safety of antibody application.

[0181] In some embodiments, the ISVD or cMet-binding molecule of the invention that specifically binds to cMet has one or more of the following properties:

[0182] (1) Binds to human cMet with medium or high affinity;

[0183] (2) specifically binds to cMet expressed on the cell surface;

[0184] (3) In the presence of HGF ligand, blocking the binding of HGF ligand to cMet on the cell surface;

[0185] (4) internalized by cells expressing cMet;

[0186] (5) Cross-reactivity with human cMet and cynomolgus monkey cMet;

[0187] (6) synergistic binding mediated by different cMet bi-epitopes;

[0188] (7) Cooperative endocytosis mediated by different cMet dual epitopes.

[0189] In some embodiments, the cMet binding molecules of the invention are in the form of monospecific, bispecific or multispecific antibody molecules. The multispecific antibody molecules can be, for example, trispecific antibody molecules comprising a first binding specificity for cMet and a second and third binding specificity for one or more other molecules.

[0190] In some embodiments, the cMet binding molecules of the invention comprise first and second ISVDs that specifically bind to the same epitope on cMet. In some embodiments, the first and second ISVDs are ISVDs according to the invention that specifically bind to the same epitope on cMet. In some further embodiments, the first and second ISVDs comprise: (i) a CDR1 comprising or consisting of an amino acid sequence selected from SEQ ID NO: 18 or 41, CDR2s comprising or consisting of the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and

[0191] CDR3; or (ii) CDR1s, CDR2s and CDR3s comprising or consisting of the amino acid sequences of SEQ ID NO: 23-25, respectively. In some further embodiments, the first and second ISVDs comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in one of SEQ ID NO: 16, 39, 40 or SEQ ID NO: 21 or 42, or consisting essentially of or consisting of the same. In some preferred embodiments, the first and second ISVDs comprise or consist essentially of or consist of the amino acid sequences of SEQ ID NO: 16, 39, 40 or SEQ ID NO: 21 or 42.

[0192] In some embodiments, the cMet binding molecules of the present invention comprise a first and a second ISVD that specifically bind to different epitopes on cMet. In some embodiments, the first and second ISVDs are ISVDs according to the present invention that specifically bind to different epitopes on cMet, respectively. In some embodiments, the first ISVD comprises a first anti-cMet VHH domain and the second ISVD comprises a second anti-cMet VHH domain, or vice versa, wherein the first and second cMet VHH domains are different from each other. In some embodiments, the first anti-cMet VHH domain comprises: CDR1, CDR2 and CDR3 comprising or consisting of an amino acid sequence of SEQ ID NOs: 23-25, respectively: and the second anti-cMet VHH domain comprises: CDR1 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 18 or 41, CDR2 and CDR3 comprising or consisting of an amino acid sequence of SEQ ID NOs: 19 and SEQ ID NOs: 20, respectively. In some further embodiments, the first anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:21 or 42; and the second anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:16, 39 or 40. In some further embodiments, the first anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, 39 or 40. In some preferred embodiments, the first ISVD comprises the first anti-cMet VHH domain, and the second ISVD comprises the second anti-cMet VHH domain. In some embodiments, the binding molecule is an anti-cMet bi-epitopic antibody.

[0193] In some embodiments, the cMet binding molecules of the present invention may be in single-chain or multi-chain form. In some embodiments, the binding molecules further comprise a peptide linker for connecting different domains (e.g., two or more ISVDs) located on the same polypeptide chain. In other embodiments, the binding molecules further comprise an immunoglobulin Fc region.

[0194] In some embodiments, the present disclosure provides an anti-cMet dual-epitope antibody, wherein the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus,

[0195] The first polypeptide chain comprises: a first ISVD that specifically binds to cMet and an immunoglobulin Fc region;

[0196] The second polypeptide chain comprises: a second ISVD that specifically binds to cMet and an immunoglobulin Fc region,

[0197] Preferably, wherein:

[0198] - the first polypeptide chain comprises the sequence of SEQ ID NO: 88, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0199] - the second polypeptide chain comprises the sequence of SEQ ID NO: 89, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto,

[0200] More preferably, the first polypeptide chain comprises or consists of the sequence of SEQ ID NO: 88; and the second polypeptide chain comprises

[0201] The sequence of SEQ ID NO:89 or consists of it.

[0202] In some embodiments, the cMet-binding molecules of the invention further comprise at least one ISVD that specifically binds to EGFR, preferably at least one (eg, 1) anti-EGFR ISVD according to the invention.

[0203] In some embodiments, the cMet-binding molecules of the present invention are linked to one or more other groups, residues or moieties at their N-terminus or C-terminus via one or more peptide linkers, wherein the one or more other groups, residues or moieties provide increased half-life, provide effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), compared to the corresponding cMet-binding molecules without the one or more other groups, residues or moieties linked.

[0204] In some embodiments, the one or more other groups, residues, moieties that provide increased half-life are selected from a polyethylene glycol molecule, a serum protein or a fragment thereof, a moiety that can bind to a serum protein (e.g., a serum albumin (such as human serum albumin)), a moiety that binds to a serum immunoglobulin (such as IgG), or an Fc domain.

[0205] In some embodiments, the cMet-binding molecule of the present invention is connected to an ISVD that binds to human serum albumin at its N-terminus or C-terminus via one or more peptide linkers. In some embodiments, the ISVD that binds to human serum albumin comprises a CDR1 consisting of or consisting of the amino acid sequence shown in SEQ ID NO: 46, a CDR2 consisting of or consisting of the amino acid sequence shown in SEQ ID NO: 47, and a CDR3 consisting of or consisting of the amino acid sequence shown in SEQ ID NO: 48. In some embodiments, the ISVD that binds to human serum albumin comprises a sequence of SEQ ID NO: 45, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0206] In certain embodiments, the cMet binding molecules provided herein are altered to increase or decrease the degree of their glycosylation. The addition or deletion of glycosylation sites of the cMet binding molecules can be conveniently achieved by changing the amino acid sequence to generate or remove one or more glycosylation sites. When the cMet binding molecule comprises an Fc region, the carbohydrates attached to the Fc region can be changed. In some applications, modifications to remove unwanted glycosylation sites can be useful, such as removing fucose modules to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, galactosylation modifications can be performed to regulate complement-dependent cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the cMet binding molecules provided herein to generate Fc region variants, so as to enhance the effectiveness of the cMet binding molecules of the present invention in treating cancer, for example.

[0207] II. Second aspect of the present disclosure: ISVD that specifically binds to EGFR and EGFR-binding molecules comprising the ISVD

[0208] Epidermal growth factor receptor (EGFR, ErbB1 or HER1) is a type I transmembrane glycoprotein encoded by the c-erbB1 proto-oncogene. EGFR is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases (RTKs), which includes HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Increased expression or kinase activity of EGFR is associated with a variety of human cancers, and therefore, EGFR is an attractive target for treating cancer.

[0209] In this specification, "EGFR" refers to EGFR from any species, and includes EGFR isoforms, fragments, variants or homologs from any species. Human EGFR is the protein shown in UniProt P00533. Alternative splicing of mRNA encoded by the human EGFR gene produces four isoforms: isoform 1-isoform 4. EGFR is a transmembrane protein that includes a large extracellular region, a single transmembrane domain, an intracellular juxtamembrane domain, a tyrosine kinase domain and a C-terminal regulatory region. The binding of EGFR to a ligand induces receptor dimerization and autophosphorylation of several tyrosine residues (Y992, Y1045, Y1068, Y1148 and Y1173) in the C-terminal regulatory region of EGFR. Abnormal EGFR expression / activity is associated with many diseases (e.g., cancer).

[0210] The second aspect of the present disclosure provides an ISVD (anti-EGFR ISVD) that specifically binds to EGFR and an EGFR binding molecule comprising the ISVD. In some embodiments, the anti-EGFR ISVD and EGFR binding molecules of the present invention have improved tissue permeability compared to conventional four-chain antibodies. In some embodiments, the anti-EGFR ISVD and EGFR binding molecules of the present invention bind to human EGFR with medium or low affinity. In this article, "medium affinity" refers to the binding affinity of the antibody for the target epitope, for example, as measured by surface plasmon resonance technology (SPR). D The value is equal to or higher than 1 nM, but less than 50 nM; "low affinity" means that the antibody has a binding affinity K of less than 1 nM for the target epitope. D Value greater than 50 nM. In some embodiments, D Compared with a "high-affinity" anti-EGFR ISVD or an EGFR-binding molecule comprising the ISVD having a value of less than 1 nM, especially less than 0.1 nM, the medium or low-affinity anti-EGFR ISVD or an EGFR-binding molecule comprising the ISVD of the present invention has at least one of the following advantages: (i) increased tumor tissue specificity; (ii) showing reduced on-target toxicity in normal tissues, such as skin toxicity; (iii) having an improved safety profile; and (iv) being more effective in treating cancer.

[0211] The immunoglobulin single variable domain (ISVD) that specifically binds to EGFR of the present invention comprises three complementary determining regions from the N-terminus to the C-terminus, namely CDR1, CDR2 and CDR3. In some embodiments, the immunoglobulin single variable domain (ISVD) that specifically binds to EGFR of the present invention comprises

[0212] (a) three CDRs in the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 31, and one of 94-99;

[0213] (b) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 6, 32, and 100-102; or

[0214] (c) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 11, 36, 84 and 105-114.

[0215] In some embodiments, the ISVD that specifically binds to EGFR of the present invention comprises, compared with the 3 CDRs described in one of (a)-(c) above, a variant having a single or multiple CDR with no more than 1 to 3 amino acid changes in each CDR, wherein the amino acid changes are additions, deletions or conservative amino acid substitutions of amino acids. In some embodiments, the CDRs according to the present invention are defined according to AbM, Chothia, Kabat, IMGT or any combination thereof, preferably defined according to Kabat or AbM or a combination thereof.

[0216] In some embodiments, an ISVD that specifically binds to EGFR of the invention comprises a

[0217] (a) CDR1 shown in SEQ ID NO:3 or a variant of CDR1 shown in SEQ ID NO:3 with no more than 1 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 2 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 2 amino acid changes;

[0218] (b) CDR1 shown in SEQ ID NO:8 or a variant of CDR1 shown in SEQ ID NO:8 with no more than 1 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 2 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 2 amino acid changes (e.g., CDR3 shown in SEQ ID NO:35 or 104); or

[0219] (c) CDR1 shown in SEQ ID NO: 13 or a variant of CDR1 shown in SEQ ID NO: 13 with no more than 1 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 2 amino acid changes (e.g., CDR2 shown in SEQ ID NO: 38 or one of SEQ ID NOs: 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 2 amino acid changes;

[0220] The amino acid changes are amino acid additions, deletions or conservative amino acid substitutions.

[0221] In some embodiments, the present invention provides an immunoglobulin single variable domain (ISVD) that specifically binds to EGFR, wherein the ISVD comprises:

[0222] (a) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 3, 4 and 5, respectively;

[0223] (b) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 8, 9 and 10, respectively;

[0224] (c) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 8, 34 and 35, respectively;

[0225] (d) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 8, 103 and 104, respectively;

[0226] (e) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 13, 14 and 15, respectively;

[0227] (f) a CDR1 comprising or consisting of SEQ ID No: 13, a CDR2 comprising or consisting of one of SEQ ID Nos: 115-120, and a CDR3 comprising or consisting of SEQ ID No: 15;

[0228] (g) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 13, 38 and 15, respectively; or

[0229] (h) CDR1, CDR2 and CDR3 comprising or consisting of SEQ ID Nos: 13, 85 and 15, respectively. In some embodiments, the ISVD defined in (a), (c) or (h) above is preferred. In some embodiments, the ISVD defined in (c) is more preferred.

[0230] In some embodiments, the ISVD that specifically binds to EGFR of the present invention comprises VHH or consists of VHH. In some embodiments, the ISVD that specifically binds to EGFR of the present invention comprises the following sequence or consists of the following sequence:

[0231] (a) a sequence of one of SEQ ID NOs: 1, 31, and 94-99, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0232] (b) a sequence of one of SEQ ID NOs: 6, 32, and 100-102, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0233] (c) a sequence of one of SEQ ID NOs: 11, 36, 84 and 105-114, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; preferably, the amino acid changes do not occur in the CDR regions. In some embodiments, the ISVD comprising the amino acid sequence of SEQ ID NOs: 31, 32, 84 or 36 is preferred. In other embodiments, the ISVD comprising the amino acid sequence of SEQ ID NO: 32 is more preferred.

[0234] In some embodiments, the present disclosure provides antibodies comprising an ISVD of the present invention that specifically binds to EGFR, particularly an anti-EGFR heavy chain antibody.

[0235] In some embodiments, the disclosure provides binding molecules that specifically bind to EGFR. In some embodiments, the EGFR binding molecules comprise an ISVD that specifically binds to EGFR of the present invention. In some embodiments, the EGFR binding molecules comprise or consist of an antibody selected from the following: a single domain antibody, a nanobody, a VHH antibody, or a heavy chain antibody. In other embodiments, the EGFR binding molecules are monospecific antibodies, bispecific antibodies, or multispecific antibodies.

[0236] In some embodiments, the EGFR binding molecules of the invention comprise at least one ISVD of the invention that specifically binds to EGFR, for example, they comprise two, three, four or more identical or different ISVDs of the invention that specifically bind to EGFR.

[0237] In some embodiments, the ISVD contained in the EGFR binding molecule of the present invention is preferably a humanized VHH. Compared with the VHH of camelid animals, the humanized VHH has a reduced human anti-camelid antibody response to the human body, thereby improving the safety of antibody application.

[0238] In some embodiments, the ISVD or EGFR binding molecule of the invention that specifically binds to EGFR has one or more of the following properties:

[0239] (1) Bind to human EGFR with moderate or low affinity;

[0240] (2) Cross-reactivity with human EGFR and cynomolgus monkey EGFR;

[0241] (3) specifically binds to EGFR expressed on the surface of tumor cells;

[0242] (4) Internalized by tumor cells expressing EGFR.

[0243] In some embodiments, the EGFR binding molecules of the present invention are in the form of bispecific or multispecific antibody molecules. The multispecific antibody molecules can be, for example, trispecific antibody molecules, which comprise a first binding specificity for EGFR and a second and third binding specificity for one or more other molecules. In some embodiments, the EGFR binding molecules of the present invention further comprise at least one ISVD that specifically binds to cMet, preferably at least one or two anti-cMet ISVDs according to the present invention.

[0244] In some embodiments, the EGFR binding molecules of the present invention are linked to one or more other groups, residues or moieties at their N-terminus or C-terminus via one or more peptide linkers, wherein the one or more other groups, residues or moieties provide increased half-life, provide effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), compared to the corresponding EGFR binding molecules without the one or more other groups, residues or moieties linked.

[0245] In some embodiments, the one or more other groups, residues, moieties that provide increased half-life are selected from a polyethylene glycol molecule, a serum protein or a fragment thereof, a moiety that can bind to a serum protein (e.g., a serum albumin (such as human serum albumin)), a moiety that binds to a serum immunoglobulin (such as IgG), or an Fc domain.

[0246] In some embodiments, the EGFR binding molecules of the present invention are connected to an ISVD that binds to human serum albumin at its N-terminus or C-terminus via one or more peptide linkers. In some embodiments, the ISVD that binds to human serum albumin, for example, comprises a CDR1 consisting of or consisting of the amino acid sequence shown in SEQ ID NO: 46, a CDR2 consisting of or consisting of the amino acid sequence shown in SEQ ID NO: 47, and a CDR3 consisting of or consisting of the amino acid sequence shown in SEQ ID NO: 48. In some embodiments, the ISVD that binds to human serum albumin comprises the sequence of SEQ ID NO: 45 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0247] In certain embodiments, the EGFR binding molecules provided herein are changed to increase or reduce the degree of its glycosylation. The addition or deletion of the glycosylation site of the EGFR binding molecule can be easily achieved by changing the amino acid sequence to produce or remove one or more glycosylation sites. When the EGFR binding molecule comprises an Fc region, the carbohydrate connected to the Fc region can be changed. In some applications, the modification of unwanted glycosylation sites can be useful, such as removing the fucose module to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, galactosylation modification can be performed to regulate complement-dependent cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the EGFR binding molecule provided herein to produce Fc region variants, so as to enhance the effectiveness of, for example, the EGFR binding molecules of the present invention for treating cancer.

[0248] III. The third aspect of the present disclosure: EGFR and cMet binding molecules

[0249] In a third aspect, the present disclosure provides EGFR and cMet binding molecules. Preferably, the binding molecule is a multispecific antibody capable of simultaneously binding EGFR and cMet. In some embodiments, the multispecific antibody according to the present invention has one or more of the following characteristics:

[0250] (1) Bind to EGFR, such as human EGFR, with moderate or low affinity;

[0251] (2) internalized by tumor cells expressing EGFR;

[0252] (2) specifically bind to cMet, such as human cMet;

[0253] (3) In the presence of HGF ligand, blocking the binding of HGF ligand to cMet on the cell surface;

[0254] (4) Internalized by tumor cells expressing cMet; in particular, synergistic internalization enhancement occurs when binding to different cMet epitopes;

[0255] (5) bind to tumor cells expressing both EGFR and cMet, and preferably exhibit synergistic binding activity;

[0256] (6) being internalized by tumor cells expressing both EGFR and cMet, and preferably exhibiting synergistic endocytic activity;

[0257] (7) Cross-reactivity with human EGFR and cynomolgus monkey EGFR;

[0258] (8) cross-reactivity with human cMet and cynomolgus monkey cMet; and

[0259] (9) Reduce tumor cell proliferation and metastasis.

[0260] In the past two decades, monoclonal antibodies have been established as anti-tumor therapeutic agents, and a variety of monoclonal antibodies targeting EGFR and cMet have been approved or are in clinical development, but acquired resistance generated by tumors limits their long-term efficacy. Since multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies) can specifically bind to different antigen epitopes, when designed to act on the signal transduction pathways of two or more different mediators at the same time, it is beneficial to avoid acquired resistance in tumors.

[0261] In some embodiments, the multispecific antibody of the present invention is an EGFR and cMet binding molecule, comprising at least one ISVD that specifically binds to EGFR as defined in the second aspect of the present disclosure (e.g., Section II herein), e.g., one or two ISVDs that bind to EGFR of the present invention; and further comprising at least one ISVD that binds to cMet as defined in the first aspect of the present disclosure (e.g., Section I herein), e.g., one or two ISVDs that bind to cMet of the present invention, optionally, wherein the ISVDs located on the same polypeptide chain are connected via one or more peptide linkers.

[0262] Generally, EGFR and cMet binding molecules comprising two or more ISVDs are also referred to herein as "multivalent" EGFR and cMet binding molecules. For example, a "bivalent" EGFR and cMet binding molecule may comprise one ISVD that binds EGFR and one ISVD that binds cMet, optionally connected by one peptide linker. A "trivalent" EGFR and cMet binding molecule may comprise one ISVD that binds EGFR and two ISVDs that bind cMet, optionally connected by two peptide linkers; or may comprise two ISVDs that bind EGFR and one ISVD that binds cMet, optionally connected by two peptide linkers. A "tetravalent" EGFR and cMet binding molecule may comprise two ISVDs that bind EGFR and two ISVDs that bind cMet, optionally connected by three peptide linkers; or may comprise one ISVD that binds EGFR and three ISVDs that bind cMet, optionally connected by three peptide linkers; or may comprise three ISVDs that bind EGFR and one ISVD that binds cMet, optionally connected by three peptide linkers, etc.

[0263] In a multivalent EGFR and cMet binding molecule, the two or more ISVDs may be the same or different, and may be directed against the same antigenic epitope of EGFR, the same antigenic epitope of cMet, or may be directed against different antigenic epitopes of EGFR, different antigenic epitopes of cMet; or any suitable combination thereof.

[0264] In some preferred embodiments, the multispecific EGFR and cMet binding molecules of the present invention comprise at least one (preferably one) ISVD against EGFR and two ISVDs against different epitopes on cMet. In other preferred embodiments, the multispecific EGFR and cMet binding molecules of the present invention comprise at least one (preferably one) ISVD against EGFR and two ISVDs against the same epitope of cMet.

[0265] Structural forms of the multispecific antibodies of the present invention

[0266] Multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies) can be divided into many categories according to different components and construction methods. For example, according to the basic symmetry of the left and right structure of the multispecific antibody, it can be divided into symmetrical structure and asymmetrical structure; according to whether the multispecific antibody has an IgG Fc region, it can be divided into antibody styles with Fc region and antibody styles without Fc region; according to the number of antigen binding sites in the multispecific antibody, it can be divided into bivalent, trivalent, tetravalent or more valent antibodies, etc.; according to the number of polypeptide chains constituting the multispecific antibody, it can be divided into single chain or multichain forms. See, for example, Brinkmann U. and Kontermann RE, The making of bispecific antibodies, Mabs, 2017, 9 (2): 182-212. These multispecific antibody structural forms known in the art are all considered in the present invention.

[0267] Single-chain multispecific antibodies

[0268] In some embodiments, the present disclosure provides EGFR and cMet binding molecules in single chain form (e.g., see Fig. 9 ), wherein at least one ISVD that specifically binds to EGFR and at least one ISVD that specifically binds to cMet are located on one polypeptide chain, wherein the ISVDs are connected via a peptide linker or directly connected.

[0269] In some embodiments, the present disclosure provides a multispecific antibody in single-chain form, comprising a single polypeptide chain, wherein the polypeptide chain comprises, from N-terminus to C-terminus:

[0270] (ISVD A ) n1 -(ISVD B ) n2 -(HLE) n3 -(ISVD A ) n4 -(ISVD B ) n5 -(HLE) n6， (I)

[0271] wherein n1, n2, n3, n4, n5 and n6 are independently selected from integers of 0, 1 or 2; wherein ISVD A and ISVD B Respectively represent ISVD domains that bind antigens A and B, wherein A and B are different from each other and are independently selected from EGFR and cMet; wherein HLE represents a serum albumin binding peptide as a half-life extension domain; wherein the symbol "-" represents connection through a peptide linker or direct connection, preferably a peptide linker of 5-15 amino acids in length. Each ISVD in formula (I)A The ISVDs may independently target the same epitope or different epitopes on the A antigen. B The same epitope or different epitopes on the B antigen may be targeted independently of each other. In some cases, it is preferred that the multispecific antibody has 2 to 6 valencies (i.e., n1+n2+n4+n5=2 to 6), more preferably, no more than 4 valencies (i.e., n1+n2+n4+n5=2 to 4), for example, 2 valencies, 3 valencies or 4 valencies.

[0272] The number of anti-EGFR ISVD domains and anti-cMet ISVD domains in the multispecific antibody may be equal or unequal. The multispecific antibody may contain or not contain an HLE domain as needed. In some embodiments, the number of anti-EGFR ISVD domains is no more than 4, preferably no more than 3, for example, 1. In some embodiments, the number of anti-cMet ISVD domains is no more than 4, preferably no more than 3, for example, 2. In some embodiments, the ratio of the number of anti-EGFR ISVD domains to anti-cMet ISVD domains is 1:1 or 1:2. In some embodiments, the antibody comprises 0 or 1 HLE.

[0273] In some embodiments, the present disclosure provides a multispecific antibody comprising a single polypeptide chain, wherein the polypeptide chain comprises, from N-terminus to C-terminus:

[0274] (i)ISVD A -ISVD B ;

[0275] (ii)ISVD A -ISVD B -ISVD B , where each ISVD B each targets the same epitope or preferably different epitopes of the B antigen;

[0276] (iii)ISVD B -ISVD A -ISVD B , where each ISVD B each targets the same epitope or preferably different epitopes of the B antigen;

[0277] Wherein preferably A represents EGFR and B represents cMet. In some embodiments, the polypeptide chain of (i)-(iii) comprises a HLE located at the N-terminus or preferably the C-terminus.

[0278] In some embodiments, the HLE is an anti-HSA ISVD that binds to human serum albumin. In some embodiments, the anti-HSA ISVD comprises a CDR1 consisting of or comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 consisting of or comprising the amino acid sequence of SEQ ID NO: 47, and a CDR3 consisting of or comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the ISVD that binds to human serum albumin comprises the sequence of SEQ ID NO: 45, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0279] In some embodiments, the present invention provides a multispecific antibody comprising a single polypeptide chain, wherein the polypeptide chain comprises:

[0280] (a) an ISVD that specifically binds to EGFR, preferably it is selected from the ISVD that specifically binds to EGFR according to the second aspect of the present disclosure; and (b) a first ISVD that specifically binds to cMet and a second ISVD that specifically binds to cMet, preferably respectively selected from the ISVD that specifically binds to cMet according to the first aspect of the present disclosure, wherein the first ISVD and the second ISVD are the same or different; preferably, the first ISVD and the second ISVD specifically bind to different epitopes of cMet. In some embodiments, the polypeptide chain further comprises an ISVD that specifically binds to HSA, preferably located at the N-terminus or C-terminus of the polypeptide chain. In some embodiments, preferably, from the N-terminus to the C-terminus, the polypeptide chain comprises: an ISVD that specifically binds to EGFR, a first peptide linker, a first ISVD that specifically binds to cMet, a second peptide linker, a second ISVD that specifically binds to cMet, and optionally a third peptide linker and an ISVD that specifically binds to HSA.

[0281] Two-chain multispecific antibodies

[0282] In some embodiments, the present disclosure provides EGFR and cMet binding molecules in double-chain form (e.g., see Fig.10 ), which 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, optionally wherein the ISVDs located on the same polypeptide chain are connected via a peptide linker or directly connected.

[0283] In some embodiments, the present disclosure provides a multispecific antibody comprising a first polypeptide chain and a second polypeptide chain, wherein

[0284] The first polypeptide chain comprises from N-terminus to C-terminus: (ISVD A ) n1 -(ISVD B ) n2 -HLE-(ISVD A ) n3 -(ISVD B ) n4， (II)

[0285] The second polypeptide chain comprises from N-terminus to C-terminus: (ISVD B ) m1 -(ISVD A ) m2 -HLE-(ISVD B ) m3 -(ISVD A ) m4， (III)

[0286] wherein n1, n2, n3 and n4 and m1, m2, m3 and m4 are independently selected from integers of 0, 1 or 2;

[0287] ISVD A and ISVD B Respectively represent ISVD domains that bind antigens A and B, wherein A and B are different from each other and are independently selected from EGFR and cMet; wherein HLE represents an immunoglobulin Fc region as a half-life extension domain, especially a human IgG1 or IgG4 Fc region; wherein the symbol "-" represents connection through a peptide linker or direct connection, preferably a peptide linker with a length of 5-15 amino acids. Each ISVD in formula (II) and formula (III) A The ISVDs may independently target the same epitope or different epitopes on the A antigen. B The same epitope or different epitopes on the B antigen may be targeted independently of each other. In some cases, it is preferred that the multispecific antibody has 2 to 6 valencies (i.e., the sum of n1, n2, n3 and n4 and m1, m2, m3 and m4 is 2-6), more preferably, no more than 4 valencies, for example, 2 valencies, 3 valencies or 4 valencies.

[0288] The number of anti-EGFR ISVD domains and anti-cMet ISVD domains in the multispecific antibody may be equal or unequal. In some embodiments, the number of anti-EGFR ISVD domains is no more than 4, preferably no more than 3, for example, 1. In some embodiments, the number of anti-cMet ISVD domains is no more than 4, preferably no more than 3, for example, 2. In some embodiments, the ratio of the number of anti-EGFR ISVD domains to anti-cMet ISVD domains is 1:1 or 1:2.

[0289] In some preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0290] The first polypeptide chain comprises from N-terminus to C-terminus: (ISVD A )-HLE ，

[0291] The second polypeptide chain comprises from N-terminus to C-terminus: (ISVD B )-HLE ；

[0292] or,

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

[0294] The second polypeptide chain comprises from N-terminus to C-terminus: (ISVD B )-HLE ；

[0295] Each ISVD B each targets the same epitope or preferably different epitopes of the B antigen;

[0296] or,

[0297] The first polypeptide chain comprises from N-terminus to C-terminus: (ISVD A )-HLE ；

[0298] The second polypeptide chain comprises from N-terminus to C-terminus: (ISVD B )-(ISVD B )-HLE ，

[0299] Each ISVD B each targets the same epitope or preferably different epitopes of the B antigen;

[0300] Preferably, A represents EGFR, and B represents cMet.

[0301] Due to the dimerization of the Fc region of immunoglobulins, the first and second polypeptide chains of the above-mentioned multispecific antibodies can associate to form a heterodimer, thereby generating a multispecific binding molecule in a double-stranded form. Preferably, in order to promote the heterodimerization of the first and second polypeptide chains, Knob-into-hole mutations can be introduced into the Fc regions of the first and second polypeptide chains, such as (T366W / T366S, L368A, Y407V) mutations or (T366Y / Y407T) mutations. Preferably, the Fc region contains an amino acid sequence derived from human IgG1 or IgG4, and more preferably, the Fc region further contains mutations that reduce or eliminate Fcγ receptor binding, such as LALA mutations.

[0302] In some specific embodiments, the EGFR and cMet binding molecule of the present invention is in a double-stranded form and comprises:

[0303] (a) an ISVD that specifically binds to EGFR is located on one polypeptide chain, preferably the ISVD is selected from the ISVDs that specifically bind to EGFR according to the second aspect of the present disclosure, wherein an Fc region (Fc subunit) is linked to the C-terminus of the ISVD; and

[0304] (b) a first ISVD that specifically binds to cMet and a second ISVD that specifically binds to cMet are located on the other chain from the N-terminus to the C-terminus, preferably selected from the ISVDs that specifically bind to cMet according to the first aspect of the present disclosure, wherein the first ISVD and the second ISVD are the same or different; preferably, the first ISVD and the second ISVD specifically bind to different epitopes of cMet; optionally, the first ISVD and the second ISVD are linked via one or more peptide linkers,

[0305] wherein an Fc region (Fc subunit) is linked to the C-terminus of the second ISVD.

[0306] In some specific embodiments, the EGFR and cMet binding molecule of the present invention is a double-stranded multispecific antibody, which comprises a first polypeptide chain and a second polypeptide chain. Among them, 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.

[0307] Example Antigenic Domain Combinations

[0308] In some embodiments of the above-mentioned multispecific antibodies according to the present invention, preferably, the multispecific antibodies according to the present invention comprise at least one anti-cMet ISVD according to the first aspect of the present disclosure (e.g., as defined in Part I) and / or at least one anti-EGFR ISVD according to the second aspect of the present disclosure (e.g., as defined in Part II).

[0309] In some further preferred embodiments, the multispecific antibody according to the present invention comprises a first and a second ISVD that specifically bind to the same epitope on cMet. In some embodiments, the first and the second ISVD are respectively anti-cMet ISVDs according to the first aspect of the disclosure that specifically bind to the same epitope on cMet. In some further embodiments, the first and the second ISVD comprise: (i) a protein selected from

[0310] In some further embodiments, the first and second ISVDs comprise a CDR1 consisting of or consisting of an amino acid sequence of SEQ ID NO: 18 or 41, a CDR2 and a CDR3 consisting of or consisting of an amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or (ii) a CDR1, a CDR2 and a CDR3 consisting of or consisting of an amino acid sequence of SEQ ID NO: 23-25, respectively. In some further embodiments, the first and second ISVDs comprise a CDR1 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 1

[0311] In some preferred embodiments, the first and second ISVDs comprise, consist essentially of, or consist of an amino acid sequence with 96%, 97%, 98% or 99% identity. In some preferred embodiments, the first and second ISVDs comprise, consist essentially of, or consist of an amino acid sequence of SEQ ID NO: 16, 39, 40 or SEQ ID NO: 21 or 42.

[0312] In other further preferred embodiments, the multispecific antibody according to the present invention comprises a first and a second ISVD that specifically bind to different epitopes on cMet. In some embodiments, the first and second ISVDs are anti-cMet ISVDs according to the first aspect of the present disclosure that specifically bind to different epitopes on cMet, respectively. In some embodiments, the first ISVD comprises a first anti-cMet VHH domain and the second ISVD comprises a second anti-cMet VHH domain, or vice versa, wherein the first and second cMet VHH domains are different from each other. In some embodiments, the first anti-cMet VHH domain comprises: CDR1, CDR2 and CDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 23-25, respectively: and the second anti-cMet VHH domain comprises: CDR1 comprising or consisting of an amino acid sequence selected from SEQ ID NO: 18 or 41, CDR2 and CDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 20, respectively. In some further embodiments, the first anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:21 or 42; and the second anti-cMet VHH domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:16, 39 or 40. In some further embodiments, the first anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 21 or 42; and the second anti-cMet VHH domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, 39 or 40. In some preferred embodiments, the first ISVD comprises the first anti-cMet VHH domain, and the second ISVD comprises the second anti-cMet VHH domain.

[0313] In some preferred embodiments of the above-mentioned multispecific antibodies of the present invention comprising a first and a second ISVD that bind to the same cMet epitope or different cMet epitopes, the antibody further comprises at least one (preferably 1) anti-EGFR ISVD according to the second aspect of the present disclosure. In some embodiments, the anti-EGFR ISVD comprises:

[0314] (i) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 3, 4 and 5, respectively;

[0315] (ii) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 8, 9 and 10, or the amino acid sequences of SEQ ID NOs: 8, 34 and 35, or the amino acid sequences of SEQ ID NOs: 8, 103 and 104, respectively; or

[0316] (iii) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 13, 14 and 15, or the amino acid sequences of SEQ ID NOs: 13, 85 and 15, or the amino acid sequences of SEQ ID NOs: 13, 38 and 15, respectively;

[0317] More preferably, the anti-EGFR ISVD:

[0318] (a) comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 1, 31, and 94-99, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0319] (b) comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 6, 32, 100-102, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; or

[0320] (c) comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in one of SEQ ID NOs: 11, 36, 84, and 105-114, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0321] More preferably, the anti-EGFR ISVD:

[0322] (a) comprising, or consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO: 31;

[0323] (b) comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 32; or

[0324] (c) comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 84.

[0325] In some embodiments, the disclosure provides a single-chain multispecific antibody comprising an anti-EGFR ISVD in combination with a first and a second anti-cMet ISVD selected from:

[0326] (a) the anti-EGFR ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 1, 6 or 13 and the first anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 21 and the second anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 16; or

[0327] (b) the anti-EGFR ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 1, 6 or 13 and the first anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 16 and the second anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 21; wherein the CDRs are preferably according to the Kabat definition,

[0328] Preferably, the antibody comprises an anti-EGFR ISVD in combination with a first and a second anti-cMet ISVD selected from:

[0329] (a) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 1, 6 or 13 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 21 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 16; or

[0330] (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 N-terminus to C-terminus, the anti-EGFR ISVD, a peptide linker, the first anti-cMet ISVD, a peptide linker and the second anti-cMet ISVD.

[0331] In some embodiments, the present disclosure provides a two-chain multispecific antibody comprising an anti-EGFR ISVD in combination with a first and a second anti-cMet ISVD selected from:

[0332] (a) the anti-EGFR ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 31, 32 or 84 and the first anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 39;

[0333] (b) the anti-EGFR ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 31, 32 or 84 and the first anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 39 and the second anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 39;

[0334] (c) the anti-EGFR ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 31, 32 or 84 and the first anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 42; or

[0335] (d) the anti-EGFR ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 31, 32 or 84 and the first anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 21 and the second anti-cMet ISVD comprises 3 CDRs of the amino acid sequence of SEQ ID NO: 40; wherein the CDRs are preferably according to the Kabat definition,

[0336] Preferably, the antibody comprises an anti-EGFR ISVD in combination with a first and a second anti-cMet ISVD selected from:

[0337] (a) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO:31, 32 or 84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:42 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:39; or

[0338] (b) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 31, 32 or 84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 39 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 39;

[0339] (c) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO: 31, 32 or 84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 42 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO: 42; or

[0340] (d) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO:31, 32 or 84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:21 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:40;

[0341] Still more preferably, the antibody comprises a combination of an anti-EGFR ISVD and a first and a second anti-cMet ISVD selected from the group consisting of:

[0342] (a) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO:31 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:42 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:39;

[0343] (b) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO:32 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:42 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:39;

[0344] (c) the anti-EGFR ISVD comprises the amino acid sequence of SEQ ID NO:84 and the first anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:21 and the second anti-cMet ISVD comprises the amino acid sequence of SEQ ID NO:40;

[0345] More preferably, the antibody comprises: an anti-EGFR ISVD comprising the amino acid sequence of SEQ ID NO:31, a first anti-cMet ISVD comprising the amino acid sequence of SEQ ID NO:42, and a second anti-cMet ISVD comprising the amino acid sequence of SEQ ID NO:39.

[0346] In some further embodiments of this two-chain multispecific antibody, the antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from N-terminus to C-terminus, the first polypeptide chain comprises: an ISVD that specifically binds to EGFR and an immunoglobulin Fc region, and the second polypeptide chain comprises: a first ISVD that specifically binds to cMet, a peptide linker, a second ISVD that specifically binds to cMet, and an immunoglobulin Fc region.

[0347] Exemplary Peptide Linkers

[0348] In some embodiments of the multispecific antibodies of the present invention, the antibodies comprise a peptide linker. In this article, a "peptide linker" used in the binding molecules of the present invention and the antibodies of the present invention refers to a short amino acid sequence composed of natural amino acids. There is no particular restriction on the length or flexibility of the peptide linker used in the binding molecules of the present invention and the antibodies of the present invention. The peptide linker used in the binding molecules of the present invention and the antibodies of the present invention can be any suitable amino acid sequence, in particular an amino acid sequence of 1 to 50, preferably 1 to 30, for example 1 to 10 amino acid residues. In some embodiments, the peptide linker is essentially composed of glycine (G) and serine (S) residues, preferably, comprising one or more repeats of a peptide motif such as a GGGGS (SEQ ID NO: 43) motif (e.g., comprising the formula (Gly-Gly-Gly-Gly-Ser) n, wherein n can be 1, 2, 3, 4, 5, 6, 7 or greater), for example, GGGGSGGGGS (SEQ ID NO: 44). In some embodiments, the peptide linker used to connect 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, one skilled in the art will be able to determine the optimal peptide linker for use in a binding molecule of the invention or an antibody of the invention after limited routine experimentation.

[0349] Exemplary Immunoglobulin Fc Regions

[0350] In some embodiments of the multispecific antibodies of the invention, the antibodies comprise an Fc region (Fc subunit). The Fc region useful in the binding molecules and antibodies of the invention may be an Fc region derived from IgG1, IgG2, IgG3 or IgG4.

[0351] In some embodiments, the Fc region of the EGFR and cMet binding molecules of the present invention uses the "knobs-into-holes" technology (see, for example, John BB Ridgway et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, 1996. 9 (7): p. 617-21; Shane Atwell et al., Stable heterodimers form remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol, 1997. 270: p. 26-35), which can remodel the interface between the two chains of the EGFR and cMet binding molecules of the present invention to promote the correct association of the two chains of the EGFR and cMet binding molecules of the present invention. Generally, the technology involves introducing a "protrusion" at the interface of one chain and introducing a corresponding "cavity" at the interface of the other chain to be paired therewith, so that the protrusion can be placed in the cavity. The preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to be paired. The protrusion can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size as the protrusion are constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired by replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine).

[0352] In one embodiment, the Fc region on both chains of the EGFR and cMet binding molecules of the present invention comprises a modification of the binding affinity for the Fc receptor. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In one embodiment, the modification reduces the effector function of the EGFR and cMet binding molecules of the present invention. In a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the modification is within the Fc region of the EGFR and cMet binding molecules of the present invention, particularly within its CH2 region. In one embodiment, the EGFR and cMet binding molecules of the present invention comprise an amino acid replacement at position 329 (EU numbering) of the heavy chain. In a specific embodiment, the amino acid replacement is P329G. In one embodiment, the EGFR and cMet binding molecules of the present invention comprise amino acid replacements at positions 234 and 235 (EU numbering) of the heavy chain. In a specific embodiment, the amino acid substitutions are L234A and L235A (LALA mutations) (Armour KL et al., Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol, 1999. 29 (8): 2613-24). In one embodiment, the EGFR and cMet binding molecules of the present invention comprise amino acid substitutions at positions 234, 235 and 329 of the heavy chain (EU numbering). In a specific embodiment, the EGFR and cMet binding molecules of the present invention comprise amino acid substitutions L234A, L235A and P329G (EU numbering) in the heavy chain.

[0353] In one embodiment, the Fc region used in the binding molecules and antibodies of the invention has the mutation YTE, i.e., a combination of the mutations M252Y (Met252Tyr), S254T (Ser254Thr) and T256E (Thr256Glu) numbered according to the EU index of Kabat, to provide an increased half-life.

[0354] In some embodiments, the immunoglobulin Fc region used in the two-chain multispecific antibody of the invention comprises:

[0355] An Fc chain with a Hole mutation (also referred to as a clasp chain) comprising the sequence of SEQ ID NO: 137, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and

[0356] An Fc chain with a Knob mutation (also referred to as a knob chain) comprising the sequence of SEQ ID NO: 138, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0357] Exemplary multispecific antibodies

[0358] In some embodiments, the present invention provides a multispecific antibody comprising a first polypeptide chain and a second polypeptide chain, wherein:

[0359] - the first polypeptide chain comprises a sequence selected from SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:86, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0360] - the second polypeptide chain comprises a sequence selected from SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 87, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. Preferably, in some embodiments,

[0361] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:70, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:73, 75 or 74, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0362] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:71, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:73, 75 or 74, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0363] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:72, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:73, 75 or 74, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0364] (iv) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:86, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO:87, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0365] More preferably, in some embodiments,

[0366] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0367] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0368] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain comprises, or consists of the amino acid sequence of SEQ ID NO: 87.

[0369] In other embodiments, the disclosure provides a multispecific antibody 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 thereto; 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 thereto. Preferably, in some embodiments, the polypeptide chain comprises or consists of a sequence selected from SEQ ID NO: 51-56.

[0370] Properties of the EGFR and cMet Binding Molecules of the Invention

[0371] EGFR affinity

[0372] The EGFR and cMet binding molecules of the present invention comprise ISVDs that bind to EGFR with moderate or low affinity. EGFR is expressed at low levels in normal tissues (e.g., skin). The EGFR and cMet binding molecules of the present invention that bind to EGFR with moderate or low affinity show reduced on-target toxicity in normal tissues while still being able to target tumors that express high levels of EGFR, resulting in an improved safety profile.

[0373] In some embodiments, the ISVD that binds to human EGFR contained in the binding molecule of the present invention can also bind to cynomolgus monkey EGFR. For example, the ISVD that binds to human EGFR can be used with a similar K D Binds to cynomolgus monkey EGFR. In this article, the similar K D refers to the two K being compared D The difference between the values ​​is no more than 10-fold, preferably no more than about 5-fold, or more preferably no more than 2-fold. The EGFR and cMet binding molecules of the present invention are capable of binding to both human EGFR and cynomolgus monkey EGFR, and this cross-reactivity is advantageous because it allows dosing and safety testing of the EGFR and cMet binding molecules of the present invention in cynomolgus monkeys during preclinical development.

[0374] cMet affinity

[0375] The EGFR and cMet binding molecules of the present invention comprise an ISVD that specifically binds to cMet. In some embodiments, the EGFR and cMet binding molecules of the present invention comprise at least two ISVDs that specifically bind to different epitopes on cMet, which enables the EGFR and cMet binding molecules of the present invention to have better tumor targeting and internalization.

[0376] In some embodiments, the ISVD that binds to human cMet contained in the binding molecules of the invention can be expressed as D The ISVD may bind to human cMet with an affinity of less than 80 nM, 50 nM, 20 nM, 15 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM or 2.5 nM. Alternatively, the ISVD may bind to human cMet with an affinity of less than 80 nM, 50 nM, 20 nM, 15 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM or 2.5 nM. D Binds to human cMet with an affinity of 1 to 20 nM, 1 to 15 nM, 1 to 10 nM, 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, 1 to 3 nM, 1 to 2.5 nM, or 2 to 2.5 nM.

[0377] In some embodiments, an ISVD that binds to human cMet contained in a binding molecule of the invention may also bind to cynomolgus monkey cMet. For example, an ISVD that binds to human cMet may be expressed as a protein having a similar K D Binding to cynomolgus monkey cMet. The EGFR and cMet binding molecules of the present invention are able to bind to both human cMet and cynomolgus monkey cMet, and this cross-reactivity is advantageous because it allows dosing and safety testing of the EGFR and cMet binding molecules of the present invention in cynomolgus monkeys during preclinical development.

[0378] Simultaneously and specifically binds to EGFR and cMet

[0379] The EGFR and cMet binding molecules of the present invention can bind to both EGFR and cMet targets simultaneously. It is known that many tumors co-express both EGFR and cMet, and therefore, the EGFR and cMet binding molecules of the present invention having the ability to bind to both EGFR and cMet simultaneously are expected to be advantageous.

[0380] Internalization

[0381] The EGFR and cMet binding molecules of the present invention can mediate efficient internalization. This is particularly useful for conjugates or couplings because it ensures that the conjugate or coupling is internalized into the cell and delivered to the lysosomes, where the antibody molecule is subsequently degraded and the drug is released into the cell to exert the cellular effect of the drug, such as cytotoxicity.

[0382] Internalization of the EGFR and cMet binding molecules of the invention by cells can be analyzed by contacting living cells with the EGFR and cMet binding molecules of the invention and detecting the EGFR and cMet binding molecules of the invention after a sufficient internalization time. When the antibody molecule is retained on the cell surface (e.g., detected on the cell surface, and / or not detected in the cell), it is determined that the antibody molecule has not been internalized by the cell. When the antibody molecule is detected in the cell (e.g., located in the cytoplasm or organelles), it is determined that the antibody molecule has been internalized.

[0383] When compared to EGFR monospecific binding molecules or cMet monospecific binding molecules, the internalization mediated by the EGFR and cMet binding molecules of the present invention exhibits greater selectivity for tumor cells that co-express both targets, thereby minimizing its adverse effects in normal tissues that do not show significant levels of EGFR and cMet co-expression.

[0384] In vitro activity

[0385] The EGFR and cMet binding molecules of the present invention have cytotoxic activity in vitro. Cytotoxic activity can be measured using in vitro cell viability assays, such as (Promega) assay. In some embodiments, the cell is a cell that expresses both EGFR and cMet.

[0386] In some embodiments, the EGFR and cMet binding molecules of the invention are capable of increasing the killing of cells, such as tumor cells, that express significant amounts of both EGFR and cMet, compared to cells that express low levels of EGFR and / or cMet. Cells that express significant amounts of both EGFR and cMet can be determined by measuring the relative EGFR and cMet receptor density on the cell surface.

[0387] In vivo activity

[0388] The EGFR and cMet binding molecules of the present invention can inhibit the development or progression of cancer in vivo. In some embodiments, the cancer can be a cancer that expresses both or one of EGFR and cMet. Cancer cells can express one or both of EGFR and cMet on the cell surface. The cancer can be, for example, selected from lung cancer (e.g., squamous cell 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.

[0389] IV. Fourth Aspect of the Disclosure: Nucleic Acids, Vectors, Hosts, and Production Methods

[0390] In a fourth aspect, the present disclosure provides nucleic acids encoding, vectors, host cells and production methods of the ISVDs, binding molecules and polypeptides according to the first to third aspects of the present disclosure.

[0391] In one embodiment, the disclosure provides a method for preparing an ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody of the invention, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody, and optionally isolating the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody. In a certain embodiment, the method further comprises recovering the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody from the host cell (or host cell culture medium).

[0392] To recombinantly produce an ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody of the invention, nucleic acid encoding the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody of the invention is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to nucleic acids encoding the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody of the invention.

[0393] The ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody of the invention prepared as described herein can be purified by 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 particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art. The purity of the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody of the 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.

[0394] V. Fifth Aspect of the Disclosure: Immunofusions, Immunoconjugates, and Antibody Drug Conjugates (ADCs)

[0395] In a fifth aspect, the present disclosure provides immunofusions, immunoconjugates and antibody drug conjugates comprising the ISVD, antibodies and antigen binding molecules according to the first to third aspects of the present disclosure.

[0396] Immunofusions and immunoconjugates

[0397] In one embodiment, the present disclosure provides an immunofusion or immunoconjugate produced by fusing or conjugating the ISVD, antibody and antigen binding molecule according to the first to third aspects of the present disclosure to a heterologous molecule.

[0398] In one embodiment, in an immune fusion, an antigen binding molecule (such as an antibody) of the invention is connected to a heterologous peptide or polypeptide molecule directly or through an amino acid linker. Heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that confer another functional activity to the fusion, or tag peptides that facilitate purification or detection of the immune fusion.

[0399] In one embodiment, in immunoconjugates, the antigen binding molecules of the present invention (such as antibodies) are conjugated with therapeutic agents or diagnostic agents or detectable agents. In conjugates, chemical linkers can be used to covalently link different entities of conjugates. In some cases, advantageously, chemical linkers are "cleavable linkers" that are beneficial to antigen binding molecule polypeptides and released after being delivered to the target site. For example, acid labile linkers, peptidase sensitive linkers, light labile linkers, dimethyl linkers or disulfide-containing linkers can be used.

[0400] In embodiments conjugated to a therapeutic agent, therapeutic agents suitable for use in the conjugate include, but are not limited to, a cytotoxin (eg, a cytostatic or cytocidal agent), a drug, or a radioisotope.

[0401] In embodiments conjugated to diagnostic or detectable agents, such conjugates can be used as part of a clinical test method (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnosis and detection can be achieved by coupling the antibody to a detectable agent, including but not limited to a variety of enzymes, such as horseradish peroxidase; prosthetic groups, such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron emission tomography techniques.

[0402] In some embodiments, therapeutic agents suitable for use in the conjugate include, but are not limited to, drugs (eg, anti-tumor drugs); in other embodiments, diagnostic agents suitable for use in the conjugate include, but are not limited to, radioactive diagnostic agents, fluorescent substances, or luminescent substances.

[0403] Antibody Drug Conjugates (ADC)

[0404] In some preferred embodiments, the present disclosure provides antibody-drug conjugates (ADCs).

[0405] In some embodiments, the present disclosure provides a compound having formula (I 0) or a pharmaceutically acceptable salt or solvate thereof:

[0406] Ab-(LD) p (I 0 )

[0407] in:

[0408] Ab is a binding molecule of the present invention, such as an antibody, such as the above-mentioned antibody or a fragment thereof (such as an antigen binding fragment) that specifically binds to EGFR and / or cMet;

[0409] L is a linker;

[0410] D is a drug, such as an anti-tumor compound; and

[0411] p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, Ab is an EGFR and cMet binding molecule according to the third aspect of the present disclosure, especially a multispecific antibody according to the third aspect of the present disclosure. In some particularly preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0412] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0413] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0414] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain comprises, or consists of the amino acid sequence of SEQ ID NO: 87.

[0415] It is understood that the -LD portion can be covalently linked to Ab by any means known in the art. In some embodiments, the -LD portion is covalently linked to Ab by a sulfur (S) atom from Ab, i.e., the -LD portion and Ab are connected by -S-. In some embodiments, the sulfur atom is derived from the opening of the interchain disulfide bond of Ab. In some embodiments, the sulfur atom is from (engineered or natural) cysteine ​​in Ab.

[0416] In some embodiments, the present invention provides an antibody-drug conjugate (ADC) having formula (I) or a pharmaceutically acceptable salt or solvate thereof:

[0417] Ab-(SLD) p (I)

[0418] in:

[0419] Ab is a binding molecule of the present invention, such as an antibody, such as the above-mentioned antibody or a fragment thereof (such as an antigen binding fragment) that specifically binds to EGFR and / or cMet;

[0420] L is a linker;

[0421] D is a drug, such as an anti-tumor compound; and

[0422] p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12. It should be understood that S in formula (I) is sulfur from antibody Ab. In some embodiments, Ab is an EGFR and cMet binding molecule according to the third aspect of the present disclosure, especially a multispecific antibody according to the third aspect of the present disclosure. In some particularly preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0423] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0424] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0425] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain comprises, or consists of the amino acid sequence of SEQ ID NO: 87.

[0426] It is understood that p refers to the formula (I 0 ) or (I) the number of -LDs to which Ab is linked, which can also be referred to as DAR.

[0427] In some embodiments, the present invention is 0) or D in (I) can be any antitumor compound, as long as it has an antitumor effect and has a structural part that can be connected to the linker, without any particular limitation. The antitumor compound can be a pharmaceutically active compound that has an effect on tumors. For the antitumor compound, part or all of the linker can preferably be cut off in tumor cells to release the antitumor compound part, thereby showing an antitumor effect.

[0428] In some embodiments, the anti-tumor compound can be, for example, a cytotoxic agent, such as a camptothecin or an auristatin.

[0429] In some embodiments, D has a structure shown in formula (D-1a) or formula (D-1b):

[0430]

[0431] Where R 1a Selected from H and C 1 -C 6 alkyl;

[0432] R 2a Selected from H, halogen, C 1 -C 6 Alkyl, C 1 -C 6 Haloalkyl, -OR 5a and-SR 5a ;

[0433] R 3a Selected from H, halogen, CN, C 1 -C 6 Alkyl, C 1 -C 6 Haloalkyl and -OR 5a ;and

[0434] R 4a and R 5a Independently selected from H and C 1 -C 4 alkyl;

[0435] In some embodiments, R 1a H; R 2a C 1 -C 6 Alkyl; R 3a is halogen, preferably -F; R 4a C 1 -C 4 Alkyl, preferably ethyl;

[0436]

[0437] Where R 1b , R 2b , R 3b , R 4b , R 5b and R 8b Each independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0438] R 6b and R 7b Each independently selected from C 1-8 Alkoxy, such as methoxy, ethoxy or propoxy;

[0439] R 9b Selected from C 1-8 Alkyl and COOH; preferably C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and

[0440] R 10b Selected from OH and H.

[0441] In some embodiments, R 1b , R 4b and R 8b Each independently selected from C 1-2 Alkyl; preferably methyl;

[0442] R 2b , R 3b and R 5b Each independently selected from C 3-4 alkyl;

[0443] R 6b and R 7b Each independently selected from C 1-2 alkoxy; and

[0444] R 9b Selected from C 1-4 Alkyl and R 10b OH; or R 9b is COOH and R 10b For H.

[0445] It is understood that the wavy line in the structural formula indicates that the valence bond is connected to the rest of the molecule. For example, the wavy line in the structural formula of D indicates that the valence bond is connected to L.

[0446] In some embodiments, D has a structure shown in formula (D-2a) or formula (D-2b):

[0447]

[0448] Where R 1a , R 2a , R 3a and R 4a as defined above; or

[0449]

[0450] 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.

[0451] In some embodiments, D has a structure shown in formula (D-3a) or (D-3b):

[0452]

[0453] In some embodiments, D has a structure shown in formula (D-4a) or (D-4b):

[0454]

[0455] In some embodiments, in the ADC of the present disclosure, the drug is Exatecan, Dxd, SN-38, monomethyl auristatin E (MMAE) or MMAF. The structural formula is shown below:

[0456]

[0457] In some embodiments, -L- has the structure: -ZL 1 -L 2 -L 3 -

[0458] in

[0459] Z is selected from wherein m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0460] L 1 Select from non-existence, wherein n1 and m1 are each independently an integer selected from 0-20, such as an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0461] L 2is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and

[0462] L 3 Selected from: wherein X is selected from -NH-, -O- and -S-; R 1c Each independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 alkoxy, halogen, nitro and cyano; Su is each independently selected from pentose, pentose uronic acid, hexose and hexose uronic acid; n2 is 0, 1, 2, 3 or 4; n5 is 0, 1, 2 or 3; n3 and n4 are independently 1, 2, 3, 4, 5 or 6; and

[0463] Among them, Z is connected to S on Ab, L 3 Connect with D.

[0464] In some embodiments, -L- has the structure: -ZL 1 -L 2 -L 3 -

[0465] in

[0466] Z is selected from wherein m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0467] L 1 Select from non-existence, wherein n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0468] L 2 is a peptide residue consisting of 2-8 amino acids; and

[0469] L 3 Selected from: Where R 1c Selected from: H and C 1 -C 6 alkyl; n2 is 1, 2, 3 or 4; and n3 and n4 are independently 1, 2, 3, 4, 5 or 6.

[0470] It should be understood that the above-ZL 1 -L 2 -L 3 -, Z is connected to Ab, for example, to S on Ab, L 3 Connect with D.

[0471] In some embodiments, Z is selected from wherein m is 1, 2, 3, 4, 5, 6, 7 or 8.

[0472] In some embodiments, Z is selected from

[0473] In some embodiments, L 1 Select from non-existence, wherein n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0474] In some embodiments, L 1 Select from non-existence,

[0475] In some embodiments, L 2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids. 2 is an amino acid residue. In some embodiments, L 2 It is a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids.

[0476] In some embodiments, the amino acid residue or amino acid is preferably an L-amino acid. In addition, in addition to α-amino acids, the amino acid residue or amino acid can be an amino acid residue or amino acid of a structure such as β-alanine, ε-aminocaproic acid, γ-aminobutyric acid, etc., and can also be a non-natural amino acid, such as an N-methylated amino acid.

[0477] 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).

[0478] In some embodiments, L 2Selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-.

[0479] In some embodiments, L 2 is selected from the group consisting of -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, and -Gly-Gly-Phe-Gly-. In some embodiments, L 2 In some embodiments, L 2 Selected from -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit- and -Glu-Val-Cit-.

[0480] It should be understood that L 2 Through the amino group of the left amino acid and L 1 or Z, through the carbonyl group of the right amino acid and L 3 connection, which is consistent with the explanation below.

[0481] In some embodiments, L 3 Selected from:

[0482]

[0483] Where R 1c Each independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkoxy, halogen, nitro and cyano; Su are each independently selected from n2 is 0, 1, 2, 3 or 4; n5 is 0, 1, 2 or 3; and n3 and n4 are independently 1, 2, 3, 4, 5 or 6.

[0484] In some embodiments, L 3 Selected from:

[0485]

[0486] wherein the variables are as defined herein.

[0487] In some embodiments, L 3 Selected from:

[0488]

[0489] wherein the variables are as defined herein.

[0490] In some embodiments, L 3 Selected from: wherein the variables are as defined herein.

[0491] In some embodiments, Su is selected from the group consisting of xylose, arabinose, xyluronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid, and mannuronic acid.

[0492] In some embodiments, Su is selected from

[0493] In some embodiments, Su are each independently:

[0494]

[0495] In some embodiments, Su are each independently In some embodiments, Su are each independently In some embodiments, L 3 Selected from:

[0496] In some embodiments, L 3 Selected from In some embodiments, L 3 Selected from: It should be understood that L 3 Through the left amino group and L 2 connected, connected to D via the carbonyl group on the right, which is consistent with the following explanation.

[0497] In some embodiments, -ZL 1 -L 2 -L 3 - are independently selected from the following structures:

[0498]

[0499]

[0500]

[0501] wherein m is each independently an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0502] 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 wherein the left side of the group is connected to S on Ab and the right side is connected to D.

[0503] In some embodiments, -ZL 1 -L 2 -L 3- Each independently selected from the following structures

[0504]

[0505] wherein n1 is independently an integer selected from 0-8, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 8.

[0506] It should be understood that, unless otherwise specified and not contradictory according to the context, for the ADCs of the present invention, the left-hand bond of the divalent group shown herein is connected to Ab or a group near the end of Ab, and the right-hand bond of the divalent group is connected to D or a group near the end of D. For example, when L 2 for When the left amino group is 1 Connect, the carbonyl group on the right side is connected to L 3 connect;

[0507] In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, or 2-4, or 2-6.

[0508] In some embodiments, the antibody-drug conjugate is selected from

[0509]

[0510]

[0511] Wherein Ab is a binding molecule of the present invention, such as a multispecific antibody, preferably V-17-Fc, V-20-Fc, V-23-Fc and V-26-Fc; p is as defined above, for example, p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Preferably, the antibody-drug conjugate has an average DAR of, for example, 2-10, 6-10, 4-8, 7-9 or 2-4 or 2-6. In some embodiments, the multispecific antibody is a multispecific antibody according to the third aspect of the present disclosure. In some particularly preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0512] (i) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 71; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0513] (ii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72; and the second polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 73;

[0514] (iii) the first polypeptide chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 86; and the second polypeptide chain comprises, or consists of the amino acid sequence of SEQ ID NO: 87.

[0515] It should be understood that the S atom connected to Ab in the above ADC comes from the antibody Ab. Ab opens disulfide bonds (e.g., interchain disulfide bonds) under the action of a reducing agent such as TCEP to generate a thiol group -SH, which is then connected to the terminal functional group of the linker, such as a maleimide portion. In some embodiments, the S atom connected to Ab comes from the cysteine ​​of Ab.

[0516] It should be noted that the above-mentioned and other technical solutions of the present disclosure and one or more features thereof can be arbitrarily combined to constitute technical solutions not directly described in this document, and these technical solutions not directly described are also included in the scope of disclosure of this application.

[0517] Preparation of ADC molecules of the present invention

[0518] Another aspect of the present invention provides a method for preparing ADC using the antibody of the present invention. "ADC" in the present invention is defined as an antibody coupled to an active substance (D, also referred to as a payload) having biological and / or pharmaceutical activity via a linker (L). 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 herein). Preferably, the linker-active substance is site-specifically coupled to the antibody.

[0519] In some embodiments, the method includes preparing an Ab for ADC, which comprises culturing a host cell comprising a nucleic acid encoding the Ab (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid under conditions suitable for expression of the Ab or its chains, as provided above, and optionally recovering the Ab from the host cell (or host cell culture medium).

[0520] In some embodiments, the method comprises the steps of:

[0521] (a) adding antibody Ab to a buffer solution, adding a reducing agent, and then incubating;

[0522] (b) adding a linker-payload to the reaction solution in step (a) for coupling to obtain a crude product; and

[0523] (c) optionally purifying the crude product to obtain the antibody drug conjugate of the present invention;

[0524] wherein Ab is as defined above.

[0525] It should be understood that the linker-payload reacts with Ab to provide the -LD portion in the compound of Formula I, and in the case where -LD is clearly defined, the structure of the linker-payload can be determined based on the prior art.

[0526] In some embodiments, the buffer solution of step a) is a PBS buffer, preferably, the pH thereof is 5.0-9.0, such as 6.0-8.0.

[0527] In some embodiments, the reducing agent of step a) is TCEP.

[0528] In some embodiments, the linker-payload has the following structure: Z'-L 1 -L 2 -L 3 -D, where L 1 , L 2 , L 3 , D is as defined above, Z' is m is as defined above, for example 1, 2, 3, 4, 5, 6, 7 or 8.

[0529] In some embodiments, Z' is selected from

[0530] In some embodiments, for the synthesis of wherein Z is The method for producing an ADC further comprises an additional hydrolysis step to open the maleimide ring.

[0531] In some embodiments, the steps are performed under the specific reaction conditions disclosed in the Examples.

[0532] It should be noted that embodiments in which the ranges or specific values ​​of the specific reaction conditions disclosed in the examples are varied by 100%, 80%, 60%, 40%, 20% or 10% are also contemplated by the present invention.

[0533] VI. Sixth Aspect of the Disclosure: Pharmaceutical Compositions and Pharmaceutical Preparations, Combination Products and Kits

[0534] In some embodiments, the present disclosure provides a composition comprising an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC as described herein, preferably the composition is a pharmaceutical composition or pharmaceutical preparation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition comprises an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC of the present invention, and a combination of one or more other therapeutic agents (e.g., chemotherapeutic drugs, tumor vaccines, antibodies that bind to other specific antigens on tumor cells, other antibodies that consume tumor cells).

[0535] In some embodiments, the composition of the present invention is a pharmaceutical composition or pharmaceutical preparation, which contains suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art. As used herein, "pharmaceutical carriers" include any and all solvents, dispersion media, isotonic agents, and absorption delay agents that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.

[0536] The pharmaceutical composition or formulation of the present invention may also contain more than one active ingredient, which is required for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. When used for the treatment of cancer, such active ingredients include but are not limited to anticancer agents and chemotherapeutic agents; when used for 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 an amount effective for the intended use.

[0537] In some embodiments, the present disclosure also provides combination products comprising at least one ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody or ADC of the present invention, or further comprising one or more other anti-tumor agents.

[0538] In some embodiments, two or more components of the combination product may be co-administered to a subject sequentially, separately or simultaneously.

[0539] In some embodiments, the disclosure also provides a kit comprising an ISVD, EGFR binding molecule, cMet binding molecule, multispecific antibody, ADC, pharmaceutical composition or combination product of the invention, and optionally a package insert directing administration.

[0540] In some embodiments, the present disclosure also provides a pharmaceutical product comprising the ISVD, EGFR binding molecule, cMet binding molecule, multispecific antibody, ADC, pharmaceutical composition, combination product of the present invention, optionally further comprising a package insert directing administration.

[0541] VII. Seventh Aspect of the Disclosure: Uses and Methods

[0542] The ISVDs, EGFR binding molecules, cMet binding molecules, or multispecific antibodies comprising the same disclosed herein have in vitro and in vivo diagnostic uses as well as therapeutic uses. For example, these molecules can be administered to cultured cells in vitro or ex vivo or to subjects, e.g., human subjects, to treat and / or diagnose EGFR antigen and / or cMet-related diseases, such as cancer.

[0543] In some embodiments, the present disclosure provides a diagnostic method for detecting the presence of relevant EGFR and / or cMet antigens in a biological sample, such as serum, semen or urine or a tissue biopsy sample (e.g., from a hyperproliferative or cancerous lesion) in vitro or in vivo. The diagnostic method comprises: (i) contacting the sample (and optionally, a control sample) with an ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody as described herein or administering the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody to a subject under conditions that allow interaction to occur and (ii) detecting the formation of a complex between the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody and the sample (and optionally, a control sample). The formation of a complex indicates the presence of the relevant antigen and can indicate the suitability or need for a treatment described herein.

[0544] In some embodiments, the relevant antigen is detected before treatment, for example, before initiation of treatment or before a treatment after a treatment interval. Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR techniques (e.g., RT-PCR) or in vivo imaging techniques. Generally, the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody used in the in vivo and in vitro detection methods is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound conjugates. Suitable detectable substances include a variety of biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances, paramagnetic (e.g., nuclear magnetic resonance active) substances and radioactive substances.

[0545] In some embodiments, the level and / or distribution of an antigen of interest is determined in vivo, e.g., non-invasively (e.g., by detecting an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody of the invention that is detectably labeled using a suitable imaging technique (e.g., positron emission tomography (PET) scanning). In one embodiment, for example, by detecting a PET agent (e.g., 18 The level and / or distribution of the relevant antigen can be determined in vivo using an ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody of the invention detectably labeled with F-fluorodeoxyglucose (FDG).

[0546] In one embodiment, the invention provides a diagnostic kit comprising an ISVD, an EGFR binding molecule, a cMet binding molecule, or a multispecific antibody described herein and instructions for use.

[0547] In some embodiments, the present disclosure relates to using the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody or ADC of the present invention to treat cancer in vivo and inhibit the growth or metastasis of tumors expressing EGFR and / or cMet in a subject, thereby inhibiting or reducing the growth or metastasis of cancer. The EGFR binding molecule, cMet binding molecule or multispecific antibody or ADC of the present invention can be used alone. Alternatively, the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody or ADC of the present invention can be administered in combination with other cancer therapeutics / preventives. When the ISVD, EGFR binding molecule, cMet binding molecule or multispecific antibody or ADC of the present invention is administered in combination with one or more other drugs, the combination can be administered in any order or simultaneously.

[0548] Thus, in one embodiment, the invention provides a method of treating cancer, the method comprising administering to a subject a therapeutically effective amount of an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC as described herein. In another embodiment, the invention provides a method of preventing the emergence of cancer drug resistance in a subject, the method comprising administering to a subject a therapeutically effective amount of an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody or ADC as described herein.

[0549] In some embodiments, cancers treated with an ISVD, EGFR binding molecule, cMet binding molecule, or multispecific antibody include, but are not limited to, cancers expressing EGFR and / or cMet, e.g., lung cancer (e.g., lung squamous cell carcinoma, lung adenocarcinoma, non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), colon cancer, or pharyngeal squamous cell carcinoma.

[0550] The following examples are described to assist understanding of the present invention. The examples are not intended to and should not be interpreted in any way as limiting the scope of protection of the present invention.

[0551] Example

[0552] Example 1 Preparation and purification of reference benchmark antibodies and reference benchmark antibody-drug conjugates

[0553] The reference antibody JNJ-61186372 is a bispecific antibody targeting EGFR and cMet, consisting of an anti-EGFR half antibody and an anti-cMET half antibody. JNJ-61186372 is generated based on the SEQ ID NO:199-202 sequence and preparation method in patent WO2014081954A1.

[0554] The reference benchmark antibody ABT-700 is a monospecific IgG antibody targeting cMet, which is prepared based on the SEQ ID NO:86-87 sequence and the method described in patent CN109562189B.

[0555] The reference benchmark antibody RAA22 / B09-57 (also referred to herein as BMK-AZD, or AZD Ab) is a bispecific antibody targeting EGFR and cMet, composed of an anti-EGFR half antibody and an anti-cMET half antibody, and is prepared based on the SEQ ID NO:59-62 sequence in patent application No. US2023 / 0183358A1 and the method described therein.

[0556] The reference benchmark antibody-drug conjugate ABBV399 is a monospecific antibody (ABT-700) targeting cMet coupled to an MMAE molecule, which can be prepared based on the SEQ ID NO:86-87 sequence and coupling method in patent CN109562189B.

[0557] The reference benchmark antibody-drug conjugate AZD9592 (also referred to herein as AZD ADC) is a bispecific antibody (RAA22 / B09-57) targeting EGFR and cMet coupled to a camptothecin molecule, which can be prepared based on the SEQ ID NO:59-62 sequence and coupling method in patent application No. US2023 / 0183358A1.

[0558] Example 2 Generation of ISVDs that specifically bind to EGFR and cMet, respectively

[0559] 2.1 Alpaca Immunity

[0560] Recombinant his-tagged human EGFR ECD protein (Aikangde Biotechnology) and recombinant his-tagged human cMet ECD protein (Aikangde Biotechnology) were used as target antigens for alpaca immunization. The immunization process was performed as follows.

[0561] Two healthy adult alpacas (Aikangde Biotechnology Co., Ltd.) were selected, and 0.5 mg of the target antigen (recombinant his-tagged human EGFR ECD protein or recombinant his-tagged human cMet ECD protein) was mixed with Gerbu adjuvant (GERBU biochemicals GmbH) in a 1:1 ratio, and the alpacas were immunized by subcutaneous multiple injections in the cervical lymph nodes. The first immunization was 500 μg, and the subsequent immunization was 250 μg, for a total of 3-4 doses, with an immunization interval of 18-21 days. 7 days after the second and third immunizations, 5 ml of jugular vein blood was taken from the alpaca for ELISA serum titer detection. The results showed that the standards for blood collection and library construction were successfully met after three rounds of immunization, and blood collection and library construction were arranged.

[0562] 2.2 Construction and selection of phage display libraries

[0563] As described in Example 2.1, after the alpaca was immunized with EGFR or cMet for the third time, 100 ml of alpaca jugular vein blood was collected and peripheral blood mononuclear cells (PBMC) were extracted by centrifugation. Total RNA was extracted from PBMC, and cDNA was generated by reverse transcription using the PrimeScript reverse transcription kit (Takara) using RNA as a template. Using cDNA as a template, the first round of PCR amplification produced nucleic acid fragments of conventional IgG (VH) and pure heavy chain IgG lacking the CH1 domain (including VHH as ISVD). These two types of nucleic acids were separated on an agarose gel, and the encoding nucleic acid containing VHH was extracted and purified, and then a second round of PCR amplification was performed 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 electrotransformed into competent Escherichia coli cells, and the bacterial solution was frozen at -80°C.

[0564] E. coli ER2738 bacterial suspension was revived and inoculated into 100 ml 2YT-A medium (Shanghai Bioengineering Company), helper phage (New England Biolabs) was added for infection, and the bacteria were resuspended in 2×YT-AK medium (Shanghai Bioengineering Company) and cultured overnight at 37°C 200rpm. The culture supernatant was collected, and recombinant phages were prepared by PEG / NaCl precipitation method, and anti-EGFR phage display library and anti-cMet phage display library were prepared respectively. Biotin-human EGFR protein and biotin-human cMet protein were used to enrich and pan the anti-EGFR phage display library and the anti-cMet phage display library for subsequent ELISA positive clone selection.

[0565] 2.3 ELISA method to select positive clones and sequencing

[0566] The expression supernatant of the E. coli ER2738 clone obtained by enrichment and panning in Example 2.2 was detected by ELISA binding test. The detection process was carried out as follows.

[0567] Take the antigen (human EGFR-his antigen or human cMet-his antigen) and dilute it to 2 μg / mL with PBS buffer, coat the 96-well ELISA plate, and incubate at 4°C overnight. Wash the antigen-coated plate 5 times with PBST, add PBST blocking solution containing 5% skim milk, and block at room temperature for 1 hour. Wash 6 times with PBST, add the expression supernatant of Escherichia coli ER2738 obtained in Example 2.2, and incubate at 37°C with shaking for 1 hour. Wash 6 times with PBST, add anti-M13-HRP secondary antibody diluted with PBS (Aikand Biotechnology (Suzhou) Co., Ltd.), and shake at 37°C for 45 minutes. Wash 5 times with PBST, add TMB colorimetric solution, and color for 5-15 minutes in the dark. Then add the stop solution. Read the plate with an enzyme reader and measure the OD 450nm -OD 650nm Absorbance value. Select bacterial clones with a reading value greater than 1 and send them to Sanger sequencing. Select ER2738 bacterial monoclonal clones containing the corresponding positive VHH sequence, add glycerol, and freeze in a -80℃ refrigerator.

[0568] 2.4 Production of VHH-HIS and VHH-FC

[0569] 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, and tags were added to their C-termini (6xHis tags or Fc tags were added to the C-termini: hIgG1 isotype), and then inserted into the expression vector pcDNA3.4, transiently transfected HEK-293F cells (hereinafter also referred to as "293F cells"), and the supernatant was collected. After the 6xHis-tagged protein was initially purified using a Ni column, the protein was further purified using an ion exchange column; the hFc fusion protein was purified using a Protein A column. The purity of the VHH antibody was determined by SDS-PAGE and SEC-HPLC. Thus, two tagged VHHs for anti-EGFR were obtained: anti-EGFR VHH-his and anti-EGFR VHH-Fc; and two tagged VHHs for anti-cMet were obtained: anti-cMet VHH-his and anti-cMet VHH-Fc.

[0570] 2.5 FACS detection of antibody binding to target cells

[0571] The binding of antibodies expressed in the supernatant of transiently transfected HEK-293F to target cells was detected by FACS.

[0572] Specifically, 100 μL of the anti-EGFR supernatant obtained by transient transfection of HEK-293F in Example 2.4 and 100 μL of the anti-cMet supernatant were collected as samples to be tested. The binding of the anti-EGFR supernatant to the target cells CHO-S-EGFR cells or CHO-S cells was detected by FACS; and the binding of the anti-cMet supernatant to the target cells CHO-S-cMet cells or CHO-S cells was detected by FACS. CHO-S-EGFR cells are the abbreviation of CHO-S engineered cell lines expressing human EGFR. CHO-S-cMet cells are the abbreviation of CHO-S engineered cell lines expressing human cMet.

[0573] FACS assay was performed as follows. 5The cells were plated in 96-well plates at a density of 10 cells / well and centrifuged at 300 g for 5 minutes at 4°C. The supernatant to be tested (100 μl / well) was added and incubated for one hour. After adding the secondary antibody PE-anti-human IgG (invitrogen, Cat#: 12-4998-82) and incubating at 4°C for half an hour, the mean fluorescence intensity (MFI) of the cells was measured by flow cytometry (Life Technologies) and analyzed by FlowJo. The results showed that the 23 anti-EGFR VHHs screened in the initial screening all bound to the target cell CHO-S-EGFR to varying degrees. The 18 anti-cMet VHHs screened in the initial screening all bound to the target cell CHO-S-cMet to varying degrees. The anti-EGFR supernatant and anti-cMet supernatant had almost no binding to CHO-S cells.

[0574] Example 3 In vitro biological activity detection of each VHH as ISVD

[0575] 3.1 FACS detection of VHH binding to cells

[0576] 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.

[0577] The target cells were 1.5×10 5 Cells were plated in 96-well plates at a density of 10 cells / well and centrifuged at 300 g for 5 minutes at 4°C. Anti-EGFR VHH-Fc (375 nM, 3-fold serial dilution) or anti-cMet VHH-Fc (50 nM, 4-fold serial dilution) was added and resuspended, and incubated at 4°C for 1 hour. The secondary antibody PE-anti-human IgG (eBioscience, cat#: 12-4998-82) was added and incubated at 4°C for half an hour, and the MFI of the cells was measured by flow cytometry (Life Technologies).

[0578] The results of anti-EGFR VHH-Fc assay are as follows Figure 1 As anti-EGFR VHH candidates, V-n5B10, V-n9B8, and V-n10A1 bind to target cells in a dose-dependent manner, and their binding activity is lower than that of the control antibody. In the following and other parts of this article, sometimes for the convenience of description, the anti-EGFR VHHs of V-n5B10, V-n9B8, and V-n10A1 are also referred to as 5B10, 9B8, and 10A1, respectively.

[0579] The results of anti-cMet VHH-Fc assay are as follows Figure 2 As shown. V-n7A12, V-n9A2, and V-n9A10 as anti-cMet VHH candidates bind to target cells in a dose-dependent manner. In the following and other parts of this article, sometimes for the convenience of description, the anti-cMET VHHs of V-n7A12, V-n9A2, and V-n9A10 are also referred to as 7A12, 9A2, and 9A10, respectively.

[0580] 3.2 Determination of the blocking effect of anti-cMet VHH-Fc on the binding of ligand HGF to target cell EBC-1

[0581] The target cells EBC-1 cells were cultured at 1.5×10 5 The cells were plated in a 96-well plate at a density of 10 cells / well and centrifuged at 300 g for 5 minutes at 4°C. The ligand 50 μL HGF-His (1 μg / mL) (Beijing Biopsies Technology Co., Ltd.) was added and incubated for 1 hour, and then 50 μL anti-cMet VHH-Fc V-n7A12, V-n9A2 or V-n9A10 (100 nM, 5-fold gradient dilution) was added, mixed and incubated for 1 hour. The secondary antibody iF647-anti-his (GenScript) was added and incubated at 4°C for half an hour, and the MFI of the cells was measured by flow cytometry (Life Technologies).

[0582] The results are as follows Figure 3 As shown, V-n7A12, V-n9A2 and V-n9A10 blocked the binding of EBC-1 cells to the ligand HGF, and the MFI values ​​at the highest concentration were 40640, 82172 and 77193, respectively. The MFI ratios at the highest concentration and the lowest concentration were 2.22, 1.12 and 1.14, respectively. The above results show that the candidate molecule V-n7A12 blocked the binding of EBC-1 cells to the ligand HGF in a dose-dependent manner. V-n9A2 and V-n9A10 weakly blocked the binding of EBC-1 cells to the ligand HGF.

[0583] 3.3 Epitope identification

[0584] Surface Plasmon Resonance (SPR) technology was used to detect whether the epitopes bound by the anti-cMet VHH-Fc forms of V-n7A12 and V-n9A2 antibodies were the same as those of the antigen cMet.

[0585] 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, with 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, with a binding phase of 120 seconds.

[0586] The results are as follows Figure 4 As shown, V-n9A2 binds to the human cMet antigen and does not affect the binding of V-n7A12 to the human cMet antigen. This indicates that the anti-cMet antibodies V-n7A12 and V-n9A2 bind to different epitopes on the antigen cMet.

[0587] 3.4 FACS detection of cell internalization of each VHH-Fc

[0588] The internalization ability of tumor target cells to the anti-EGFR VHH-Fc and anti-cMet VHH-Fc prepared in Example 2 was detected by FACS method.

[0589] Prepare tumor target cells NCI-H1975 and EBC-1, with the number of cells per well being 1 to 1.5 × 10 5 cells were plated on a 96-well plate. Anti-EGFR VHH-Fc (125 nM, 3-fold dilution) or anti-cMet VHH-Fc (50 nM, 4-fold dilution) prepared in Example 2 was added respectively, incubated at 4°C for 30 min, and the supernatant containing each VHH-Fc was removed by centrifugation. The cells were divided into 2 groups and incubated at 4°C and 37°C for 4 hours respectively. After the incubation, PBS in an ice bath was immediately added to terminate the endocytosis experiment. After adding the second antibody PE-anti-human IgG (eBioscience, catalog number: 12-4998-82) and incubating at 4°C for 30 min, the MFI of the cells was measured using a flow cytometer (Life Technologies).

[0590] The internalization level of cell surface-bound antibody was calculated using the following formula:

[0591] Endocytosis = MFI of sample incubated at 4°C - MFI of sample incubated at 37°C.

[0592] Internalization rate % = 100% - (MFI of the sample incubated at 37°C / MFI of the sample incubated at 4°C) × 100%.

[0593] The results of the internalization of the candidate molecules for anti-EGFR antibodies in the tumor target cell NCI-H1975 are as follows Figure 5A , which shows the MFI of samples incubated at 4°C and the MFI of samples incubated at 37°C at the tested antibody concentrations. Figure 5B, which shows the endocytic MFI determined according to the above formula at the tested antibody concentrations.

[0594] 3.5 ELISA to detect cross-reactivity of each VHH-Fc with antigens from different species

[0595] Human cMet-His antigen or cynomolgus monkey cMet-His antigen was diluted to 1 μg / mL with PBS buffer and coated on a 96-well ELISA plate at 4°C overnight. The antigen-coated plate was washed 3 times with PBST (300 μL / well), PBS containing 5% skim milk was added to 200 μL / well, and the plate was blocked at 37°C for 2 hours. Washed 3 times with PBST, anti-cMet VHH-Fc diluted in a 5-fold gradient was added, and the plate was incubated at 37°C for 1 hour with shaking. Washed 3 times with PBST, anti-human Fc-HRP secondary antibody (Abcam, CAT#: ab97225) diluted in PBS was added to each well at a volume of 100 uL / well, and then incubated at 37°C for 45 minutes with shaking. Then washed 3 times with PBST (300 μL / well), TMB color development solution was added to the well at 100 uL / well, and color was developed for 5-10 minutes in the dark. Then add the stop solution, the volume of the stop solution is 50uL / well. Read the microplate reader and measure the OD 450nm -OD 650nm Absorbance value. ELISA test results are as follows Figure 6 And as shown in Table 1.

[0596] Table 1. ELISA detection of the binding of anti-cMet antibodies to human cMet antigen or monkey cMet antigen

[0597]

[0598] Note: “-” indicates that EC cannot be fitted 50 value.

[0599] The results showed that the anti-cMet candidate molecules V-n7A12 and V-n9A2 showed cross-reactivity with human cMet antigen and cynomolgus monkey cMet antigen, and exhibited similar binding affinities to human and monkey cMet. However, the affinity of V-n9A10 for human cMet antigen was significantly different from that for monkey cMet antigen.

[0600] Example 4. VHH sequence optimization and characterization

[0601] 4.1 VHH sequence optimization

[0602] The original VHH sequence prepared in Example 2 was humanized using the "best match method". The amino acid sequence of the VHH framework region was compared and analyzed using the human germline V gene database to select the best germline sequence. Using the Kabat CDR definition, the VHH CDR sequence was used to replace the best matching human CDR sequence to generate a humanized VHH sequence. Multiple residues in the framework region were backmutated, and post-translational modification (PTM) was removed as appropriate. The optimized sequence was reverse translated and sent to GeneWeichi (Shanghai, China) for gene synthesis. It was then constructed into a pcDNA 3.4 expression vector to express monovalent humanized VHHs with a C-terminal fusion human His tag. Humanized VHH cloned protein was thus obtained.

[0603] The immunogenicity of the humanized and PTM-removed VHH sequences was analyzed. If the sequence had a high risk, the TCE (T cell epitope) epitope of the sequence was mutated to eliminate or reduce the immunogenicity risk. The mutated sequence was sent to GeneWeizhi (Shanghai, China) for gene synthesis and then constructed into the pcDNA 3.4 expression vector to express VHHs with a C-terminal His tag. Thus, a VHH cloned protein with a low immunogenicity risk was obtained.

[0604] Sequence optimization of anti-EGFR VHH

[0605] Each parent anti-EGFR antibody and its optimized sequence are shown in Tables 2A to 2C below.

[0606] Table 2A. Anti-EGFR VHH-10A1 antibody and its optimized sequence

[0607]

[0608] Table 2B. Anti-EGFR VHH 9B8 antibody and its optimized sequence

[0609]

[0610] Table 2C. Anti-EGFR VHH 5B10 antibody and its optimized sequence

[0611]

[0612]

[0613] Sequence optimization of anti-cMET VHH

[0614] Each parent anti-cMet antibody and its optimized sequence are shown in Tables 2D to 2E below.

[0615] Table 2D Anti-cMet VHH 9A2 antibody and its optimized sequence

[0616]

[0617] Table 2E. Anti-cMet VHH 7A12 antibody and its optimized sequence

[0618]

[0619] 4.2 Functional characterization of optimized antibodies

[0620] Using FACS binding assay, the binding of each anti-EGFR VHH-His candidate antibody molecule V-n10A1, V-n9B8 and V-n5B10 and the anti-cMet VHH-His candidate molecule V-n9A2 and V-n7A12 and 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 assay was performed as follows.

[0621] 1.5×10 5 The target cells were plated in a 96-well plate at a density of 10 cells / well and centrifuged at 300g for 5 minutes at 4°C. The test antibody VHH-His or the reference antibody was added and resuspended, and incubated at 4°C for 1 hour. The secondary antibody iF647-anti-his (Genscript, 1:1000 dilution, catalog number: A01802-100) or APC-anti-his (BioLegend, 1:200, catalog number: 362605) was added, incubated at 4°C for half an hour or 45 minutes, and the MFI of the cells was measured by flow cytometry (Life Technologies).

[0622] 1) Sequence optimization and modification of anti-EGFR antibodies

[0623] After sequence optimization and modification of the parent 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.

[0624] Table 3A. Binding activity of each humanized anti-EGFR antibody to target cells

[0625]

[0626] Note: “-” indicates that EC cannot be fitted 50 value.

[0627] Table 3B. Binding activity of each anti-EGFR antibody to target cells after PTM removal sequence optimization

[0628]

[0629] Note: “-” indicates that EC cannot be fitted 50 value.

[0630] 2) Sequence optimization and modification of anti-cMet antibodies:

[0631] After sequence optimization and modification of the parent 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.

[0632] Table 3C. Binding activity of each humanized anti-cMet antibody to target cells

[0633]

[0634] Note: “-” indicates that EC cannot be fitted 50 value.

[0635] Table 3D. Binding activity of each anti-cMet antibody to target cells after PTM removal sequence optimization

[0636]

[0637] Table 3E. Binding activity of anti-cMet antibodies after first immunogenicity removal to target cells

[0638]

[0639] Table 3F. Binding activity of anti-cMet antibodies after second immunogenicity removal to target cells

[0640]

[0641] 4.4 SPR detection of anti-cMet VHH and anti-EGFR VHH

[0642] Using the SPR method, the binding affinity of anti-cMet VHH and anti-EGFR VHH to the target antigen was characterized.

[0643] For monovalent antibodies with VHH_His tags, the detection was performed according to the following method 1. 10ug / ml of antigen (EGFR antigen or cMet antigen of different species) was fixed on the CM5 chip, and then each antibody to be tested was injected at a flow rate of 30μL / min (antibodies were diluted in a concentration gradient), and the binding time was set to 120s and the dissociation time was set to 200s. After the dissociation, 10mM glycine (pH 2.0) was used for regeneration. The experimental data will be analyzed using a 1:1 binding model. The results are shown in Table 4A below.

[0644] For bivalent antibodies with VHH_Fc tags, the detection was performed according to the following method 2. Each antibody to be tested was captured with a Protein A chip (concentration of 10ug / ml), and then the antigen (EGFR antigen of different species or cMet antigen) was injected at a flow rate of 30μL / min, and the binding time was set to 120s and the dissociation time was set to 200s. After the dissociation, it was regenerated with 10mM glycine (pH 2.0). The experimental data will be analyzed using a 1:1 binding model. The results are shown in Table 4B below.

[0645] Table 4A. SPR data of monovalent anti-cMet and anti-EGFR antibodies with VHH His tags

[0646] Antibody Name antigen ka(1 / Ms) kd(1 / s) KD(M) V-n5B10 Human EGFR 2.32E+05 5.36E-03 2.32E-08 V-n5B10 Cyno EGFR 5.22E+05 3.84E-03 7.36E-09 V-n10A1 Human EGFR 6.91E+05 2.58E-02 3.74E-08 V-n10A1 Cyno EGFR 5.81E+05 1.00E-02 1.72E-08 V-n7A12 Human cMet 5.09E+05 1.53E-04 3.01E-10 V-n7A12 Cyno cMet 4.58E+04 4.39E-03 9.60E-08 V-n9A2 Human cMet 1.81E+05 2.90E-03 1.60E-08 V-n9A2 Cyno cMet 1.02E+05 7.20E-03 7.03E-08 ABT700 Human cMet 1.08E+05 7.38E-05 6.81E-10 ABT700 Cyno cMet 1.22E+05 7.14E-05 5.84E-10 BMK-AZD Human EGFR 1.90E+05 3.10E-03 1.63E-08 BMK-AZD Cyno EGFR 5.91E+05 2.63E-02 4.45E-08 BMK-AZD Human cMet 2.88E+05 6.23E-04 2.16E-09 BMK-AZD Cyno cMet 3.58E+05 1.15E-03 3.22E-09

[0647] Note: All Vn series antibodies tested in the table are monovalent forms of VHH with a His tag at the C-terminus, and are detected using method 1; the control antibodies in the table are detected using method 2.

[0648] Table 4B: SPR data of VHH_Fc-tagged bivalent anti-EGFR antibodies:

[0649]

[0650] Note: All antibodies in the table are in the dimer form of VHH with an Fc tag at the C-terminus and are detected using method 2.

[0651] Example 5. Construction and characterization of anti-cMet dual-epitope antibodies

[0652] 5.1 Construction and preparation of anti-cMet bi-epitope antibodies

[0653] The anti-cMet dual-epitope M-1 molecule of the present invention is an asymmetric double-chain form, which has a first polypeptide chain (9A2-Fc chain) shown in SEQ ID NO: 88 and a second polypeptide chain (hu7A12 b -Fc chain).

[0654] The anti-cMet single-epitope M-2 molecule of the present invention is in a symmetric double-chain form, which has a first polypeptide chain and a second polypeptide chain (9A2-Fc chain) shown in SEQ ID NO: 90.

[0655] The anti-cMet single-epitope M-3 molecule of the present invention is in a symmetric double-chain form, which has a first polypeptide chain and a second polypeptide chain (hu7A12 b -Fc chain) shown in SEQ ID NO: 91.

[0656] The first / second polypeptide chains of the above M-1, M-2, and M-3 are constructed into the pcDNA 3.4 expression vector (if the antibody is symmetric, one vector is generated; if the antibody is asymmetric, two vectors containing the coding genes of the first and second polypeptide chains are generated), and transfected into HEK293F cells. The cells are cultured for 3 days, and the culture supernatant of the transfected cells is collected and loaded onto a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody is eluted with an acetic acid-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. The antibody concentration is measured using a Nano Drop. The protein purity is determined by SDS-PAGE and analytical HPLC-SEC.

[0657] 5.2 FACS detection of the binding of the bispecific antibody to target cells

[0658] The binding of the tumor target cell EBC-1 to the anti-cMet molecule is detected by FACS.

[0659] Prepare the tumor target cell EBC-1, with 1.5×10 5 cells per well, and seed them on a 96-well plate. Add the anti-cMet M-1, M-2, and M-3 molecules (50 nM, 4-fold dilution) respectively, incubate at 4°C for 1 hour, and centrifuge to remove the supernatant containing each VHH-Fc. Add the secondary antibody PE-anti-human IgG (eBioscience, catalog number 12-4998-82), incubate at 4°C for 30 min, and then measure the MFI of the cells using a flow cytometer (Life Technologies).

[0660] The results are as Figure 7 shown. The results indicate that: compared with the anti-cMet single-epitope M-2 and M-3 molecules, the tumor target cell EBC-1 has stronger binding to the anti-cMet bispecific M-1 molecule, and the binding activity of the bispecific molecule to the cells is also superior to that of the control group ABT700.

[0661] 5.3 FACS detection of the co-endocytosis of the bispecific antibody

[0662] The FACS method was used to detect the synergistic endocytosis of the anti-cMet dual-epitope molecule M-1.

[0663] Prepare tumor target cells EBC-1, with the number of cells per well being 1.5×10 5 cells were plated on a 96-well plate. Anti-cMet M-1, M-2, and M-3 molecules (50 nM, 4-fold dilution) were added respectively, incubated at 4°C for 30 min, and the supernatant containing each VHH-Fc was removed by centrifugation. The cells were evenly divided into 2 groups and incubated at 4°C and 37°C for 4 hours respectively. After the incubation, PBS in an ice bath was immediately added to terminate the endocytosis experiment. Subsequently, the secondary antibody PE-anti-human IgG (eBioscience, catalog number 12-4998-82) was added and incubated at 4°C for 30 min, and the MFI of the cells was measured by flow cytometry (Life Technologies).

[0664] The internalization level of cell surface-bound antibody was calculated using the following formula:

[0665] Endocytosis = MFI of sample incubated at 4°C - MFI of sample incubated at 37°C.

[0666] like Figure 8 The experimental results show that the tumor target cell EBC-1 has a higher endocytosis of the anti-cMet dual-epitope M-1 molecule (compared with the anti-cMet single-epitope M-2 and M-3 molecules), and the anti-cMet dual-epitope M-1 molecule has a better endocytosis effect than ABT700.

[0667] Example 6 Generation and detection of multispecific anti-EGFR / CMet antibody molecules

[0668] 6.1 Generation of multispecific anti-EGFR / CMet antibody molecules

[0669] According to the analysis and detection results of the above-mentioned Examples 1 to 5, the anti-EGFR VHH and the anti-cMet VHH are combined together to form a single-chain or multi-chain multispecific antibody.

[0670] SCMA

[0671] The anti-EGFR VHH sequence and the anti-cMet VHH sequence are combined into a single-chain antibody form. Specifically, an anti-EGFR VHH sequence (V-n5B10, V-n9B8, V-n10A1) or a humanized VHH sequence thereof and two anti-cMet VHH sequences of the same or different epitopes (V-n7A12, V-n9A2) or a humanized VHH sequence thereof are combined to form a specific anti-EGFR / cMet antibody molecule. Among them, 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), and then a GGGGSGGGGS peptide linker (SEQ ID NO: 44) is added, and then another anti-cMet VHH (second anti-cMet ISVD) is added. The exemplary multispecific antibody molecules and their amino acid sequences formed by the combination of this method are shown in Table 5.

[0672] Table 5: Exemplary single-chain multispecific antibody molecules

[0673]

[0674] Note: In Table 5, V-n5B10, V-n9B8, and V-n10A1 as anti-EGFR VHHs are abbreviated as 5B10, 9B8, and 10A1, respectively; V-n7A12, V-n9A2, and V-n9A10 as anti-cMet VHHs are abbreviated as 7A12, 9A2, and 9A10, respectively.

[0675] Optionally, the specific antibody molecule is linked to an ISVD that binds to human serum albumin, such as Alb8, to extend the half-life of the specific anti-EGFR / cMet antibody molecule. For example, the C-terminus of the specific antibody molecule is linked to an ISVD that binds to human serum albumin, such as Alb8 (SEQ ID NO: 45), via a peptide linker (such as GGGGS as shown in SEQ ID NO: 43).

[0676] Specifically, taking V-17 as an example, Fig. 9As shown in the example, a GGGGS peptide linker (SEQ ID NO: 43) was connected to the C-terminus of V-17, followed by an anti-HSA-Alb8 (SEQ ID NO: 45), thereby forming hu10A1-hu7A12-hu9A2-anti-HSA-Alb8 (also referred to as "V-17-anti-HSA-Alb8"). Similarly, for the other specific antibody molecules described in Table 5, a GGGGS peptide linker (SEQ ID NO: 43) was connected to the C-terminus, followed by an anti-HSA-Alb8 (SEQ ID NO: 45), to form V-1-anti-HSA-Alb8, V-3-anti-HSA-Alb8, V-7-anti-HSA-Alb8, V-9-anti-HSA-Alb8, V-13-anti-HSA-Alb8, V-15-anti-HSA-Alb8, V-16-anti-HSA-Alb8 (SEQ ID NO: 67), V-17-anti-HSA-Alb8 (SEQ ID NO: 68), V-18-anti-HSA-Alb8 (SEQ ID NO: 76), V-19-anti-HSA-Alb8 (SEQ ID NO: 77), V-20-anti-HSA-Alb8 (SEQ ID NO: 78), V-21-anti-HSA-Alb8 (SEQ ID NO: 79), V-22-anti-HSA-Alb8 (SEQ ID NO: 80), V-23-anti-HSA-Alb8 (SEQ ID NO: 81), V-24-anti-HSA-Alb8 (SEQ ID NO: 82), V-25-anti-HSA-Alb8 (SEQ ID NO: 83), V-26-anti-HSA-Alb8 (SEQ ID NO: 84), V-27-anti-HSA-Alb8 (SEQ ID NO: 85), V-28-anti-HSA-Alb8 (SEQ ID NO: 86), V-29-anti-HSA-Alb8 (SEQ ID NO: 87), V-30-anti-HSA-Alb8 (SEQ ID NO: NO:78), V-21-anti-HSA-Alb8 (SEQ ID NO:79), V-22-anti-HSA-Alb8 (SEQ ID NO:80), V-23-anti-HSA-Alb8 (SEQ ID NO:81), V-24-anti-HSA-Alb8 (SEQ ID NO:82), V-25-anti-HSA-Alb8 (SEQ ID NO:83).

[0677] The single-chain multi-specific anti-EGFR / cMet antibody described in the present disclosure was constructed into the pcDNA 3.4 expression vector. It was transfected into HEK293F cells, the cells were cultured for 3 days, and the culture supernatant of the transfected cells was collected and loaded into Cytiva PrismA filler (Cytiva) for purification. The antibody was eluted with acetic acid-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. The antibody concentration was measured with Nano Drop. The protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[0678] Multi-chain multispecific antibodies

[0679] The anti-EGFR VHH sequence and the anti-cMet VHH sequence of the same or different epitopes are respectively combined into polypeptide chains containing Fc subunits, and the polypeptide chains can further associate to form homologous or heterologous dimers to generate multispecific EGFR and cMet binding molecules in the form of two chains.

[0680] Fig.10 An exemplary multi-chain multi-specific antibody structure is shown. Specifically, the anti-EGFR VHH sequence and the Fc subunit form a polypeptide chain (anti-EGFR-Fc), and optionally the Fc subunit contains a LALA mutation and / or a Knob-into-hole structure, and the prepared polypeptide chain includes the sequence shown in Table 6A:

[0681] Table 6A: Exemplary sequences of anti-EGFR-Fc

[0682] Domain splicing Serial Number hu10A1-Fc SEQ ID NO:70 hu5B10-Fc SEQ ID NO:71 hu9B8-Fc SEQ ID NO:72 <![CDATA[hu10A1 b -Fc]]> SEQ ID NO:86

[0683] The anti-cMet VHH sequence and the Fc subunit form a polypeptide chain (anti-cMet1-anti-cMet2-Fc), specifically, the C-terminus of the first anti-cMet VHH sequence is connected to the N-terminus of the same or different second anti-cMet VHH sequence through a peptide linker (GGGGSGGGGS as shown in SEQ ID NO:42), and the C-terminus of the second anti-cMet VHH is then connected to the Fc subunit through the hinge region to form a polypeptide chain, and optionally the Fc subunit contains a LALA mutation and / or a Knob-into-hole structure. The prepared polypeptide chain includes the sequence shown in Table 6B:

[0684] Table 6B: Exemplary sequences of anti-cMet1-anti-cMet2-Fc

[0685] Domain splicing Serial Number hu9A2-hu7A12-Fc SEQ ID NO:73 hu9A2-hu9A2-Fc SEQ ID NO:74 hu7A12-hu7A12-Fc SEQ ID NO:75 9A2-hu7A12-Fc SEQ ID NO:69 <![CDATA[9A2-hu7A12 b -Fc]]> SEQ ID NO:87

[0686] Any of the above anti-EGFR-Fc polypeptide chains can be associated with any of the anti-cMet1-anti-cMet2-Fc polypeptide chains to form a dimer, for example, to synthesize a double-chain multispecific anti-EGFR / cMet antibody as shown in Table 7:

[0687] Table 7: Examples of multispecific anti-EGFR / cMet antibodies in two-chain format

[0688]

[0689]

[0690] The polypeptide chain 1 and polypeptide chain 2 of the double-chain multispecific anti-EGFR / cMet antibody described in the present 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 acetic acid-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. The antibody concentration was measured with Nano Drop. The protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[0691] 6.2 FACS BINDING Detection

[0692] The binding of the above trispecific anti-EGFR / cMet antibody molecules to the target cells EBC-1 and NCI-H1975 cells (Cell Bank of the Chinese Academy of Sciences) was detected by FACS method.

[0693] Prepare target cells separately, with 1.5×10 cells per well. 5 cells were plated on a 96-well plate. Each trispecific antibody prepared in Example 6.1 (250 nM, 3-fold dilution) was added, incubated at 4°C for 1 h, and then centrifuged at 300 g for 4 minutes to remove the supernatant. The plate was washed twice by centrifugation with FACS buffer (1% BSA). After adding the secondary antibody PE-anti-human IgG (eBioscience, catalog number 12-4998-82) and incubating at 4°C for 1 h, the plate was washed twice by centrifugation, and finally the cells were resuspended with FACS buffer (1% BSA), and the MFI of the cells was measured by flow cytometry (Life Technologies).

[0694] Depend on Fig.11 As shown, the V-17-Fc, V-20-Fc, V-23-Fc and V-26-Fc trispecific antibodies were able to bind to EBC-1 and NCI-H1975 cells.

[0695] 6.3 FACS endocytosis detection

[0696] The internalization ability of the target cells EBC-1 and NCI-H1975 cells (Cell Bank of Chinese Academy of Sciences) to the above-mentioned trispecific anti-EGFR / cMet antibody molecules was detected by FACS method.

[0697] Prepare target cells separately, with 3×10 cells per well. 5cells, spread on a 96-well plate. Add each trispecific antibody prepared in Example 6.1 (250nM, 3-fold dilution), incubate at 4°C for 30min, and remove the supernatant containing the trispecific antibodies by centrifugation. Divide the cells into 2 groups and incubate at 4°C and 37°C for 4 hours, respectively. After the incubation, immediately add PBS in an ice bath to terminate the endocytosis experiment. After adding the second antibody and incubating at 4°C for 30min, the MFI of the cells was measured by flow cytometry (Life Technologies). For molecules in the VHH-Fc mode, PE-anti-human IgG (eBioscience, Catalog No.: 12-4998-82) was used as the second antibody; for VHH molecules with his tag, APC-anti-his (BioLegend, Catalog No.: 362605) was used as the second antibody.

[0698] The internalization level of cell surface-bound antibody was calculated using the following formula:

[0699] Endocytosis = MFI of samples incubated at 4°C − MFI of samples incubated at 37°C;

[0700] The results of endocytosis of the candidate molecules of the trispecific antibody V-1, V-3, V-7, V-9, V-13, V-15, V-17-Fc, V-20-Fc, V-23-Fc and V-26-Fc by the target cells MDA-MB-468, EBC-1 and NCI-H1975 are shown in Figure 12-13 shown.

[0701] like Fig.12 and Fig.13 As shown, target cells were able to internalize trispecific anti-EGFR / cMet antibody molecules V-1, V-3, V-7, V-9, V-13, V-15, V-17-Fc, V-20-Fc, V-23-Fc and V-26-Fc.

[0702] 6.4SPR detection of the affinity of trispecific antibodies to human and cynomolgus monkey antigens

[0703] 6.4.1 SPR detection of the affinity of trispecific antibodies for human and cynomolgus monkey EGFR antigens

[0704] The affinity of trispecific anti-EGFR / cMet antibody molecules to human and cynomolgus monkey EGFR (Beijing Sino Biological Technology Co., Ltd.) was detected by SPR method using Cytiva.

[0705] The amino-coupled 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 fixed on the GLM sensor chip (Bio-Rad). The trispecific anti-EGFR / cMet antibody molecules of Example 6.1 diluted in proportion were injected into the sensor chip at a flow rate of 100 μL / min, with a binding phase of 100 seconds and then a dissociation of 180 seconds. After each dissociation phase, 10 mM glycine (pH 1.5) was used to regenerate the buffer. The sensorgrams of the blank surface and the buffer channel were subtracted from the tested sensorgrams. The experimental data were subjected to Langmuir analysis using a 1:1 binding model. The affinity results of the candidate molecules to human and cynomolgus monkey EGFR are shown in Table 8.

[0706] The results showed that the candidate molecules showed similar binding and dissociation rates with human EGFR antigen and cynomolgus monkey EGFR antigen, and these trispecific anti-EGFR / cMet antibody molecules had cross-reactivity with cynomolgus monkey EGFR.

[0707] Table 8. Affinity of trispecific antibodies to human and cynomolgus monkey EGFR antigens detected by SPR

[0708]

[0709] 6.4.2 SPR detection of the affinity of trispecific antibodies for human and cynomolgus monkey cMet antigens

[0710] The amino-coupled antigens human cMet-his (ECD, Met1-Thr932) and cynomolgus monkey cMet-his (ECD, Met1-Thr932 (Beijing Sino Biological Technology Co., Ltd.) (20 μg / mL) were immobilized on the GLM sensor chip (Bio-Rad). The trispecific anti-EGFR / cMet antibody molecules (500 nM) of Example 6.1 diluted in proportion were injected onto the sensor chip at a flow rate of 30 μL / min, with a binding phase of 100 seconds and then a dissociation phase of 180 seconds. After each dissociation phase, 10 mM glycine (pH 1.5) was used to regenerate the buffer. The sensorgrams of the blank surface and the buffer channel were subtracted from the sensorgrams tested. The experimental data were analyzed using a 1:1 binding model. The affinity results of the candidate molecules to human cMet and cynomolgus monkey cMet are shown in Table 9.

[0711] The results showed that the candidate molecules showed similar binding and dissociation rates with human cMet antigen and cynomolgus monkey cMet antigen, and these trispecific anti-EGFR / cMet antibody molecules had cross-reactivity with cynomolgus monkey cMet.

[0712] Table 9. Affinity of trispecific antibodies for human and cynomolgus monkey cMet antigens detected by SPR

[0713]

[0714]

[0715] 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

[0716] The target cells EBC-1 cells were cultured at 1.5×10 5 Cells were plated in a 96-well plate at a density of 10 cells / well and centrifuged at 300g for 5 minutes at 4°C. Trispecific anti-EGFR / cMet antibody (200nM, 4-fold gradient dilution) was added and incubated with the cells for 30 minutes, and then the ligand HGF-His (2μg / mL) (Beijing Sino Biological Technology Co., Ltd.) was added and incubated at 4°C for 1 hour. The secondary antibody iF647-anti-his (GenScript) was added and incubated at 4°C for half an hour, and the MFI of the cells was measured by flow cytometry (Life Technologies).

[0717] The results show that if Fig.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 the blocking activity is superior to that of ABT700 and BMK-AZD.

[0718] 6.6 FACS detection of the synergistic endocytosis of trispecific antibodies on target cells

[0719] The M-4 molecule serves as a control molecule, which is in the form of an asymmetric two-chain Fc, having a first polypeptide chain (hu9A2-hu7A12-Fc) shown in SEQ ID NO:73 and a second polypeptide chain (anti-Covid-19 chain) shown in SEQ ID NO:92, wherein the anti-Covid-19 chain does not bind to the target cell EGFR or cMET.

[0720] The M-5 molecule serves as a control molecule, which is in the form of an asymmetric two-chain Fc, having a first polypeptide chain (anti-Covid-19 chain) shown in SEQ ID NO:93 and a second polypeptide chain (hu5B10-Fc) shown in SEQ ID NO:71, wherein the anti-Covid-19 chain does not bind to the target cell EGFR or cMET.

[0721] The M-6 molecule serves as a control molecule, which is in the form of an asymmetric two-chain Fc, having a first polypeptide chain (anti-Covid-19 chain) shown in SEQ ID NO:93 and a second polypeptide chain (hu9B8-Fc) shown in SEQ ID NO:72, wherein the anti-Covid-19 chain does not bind to the target cell EGFR or cMET.

[0722] The M-7 molecule serves as a control molecule, which is in the form of an asymmetric two-chain Fc, having a first polypeptide chain (anti-Covid-19 chain) shown in SEQ ID NO:93 and a second polypeptide chain (hu10A1-Fc) shown in SEQ ID NO:70, wherein the anti-Covid-19 chain does not bind to the target cell EGFR or cMET.

[0723] The first / second polypeptide chains of the above-mentioned M-4, M-5, M-6, and M-7 molecules were constructed into the pcDNA3.4 expression vector (if the antibody is a symmetrical structure, one vector is generated; if the antibody is an asymmetric structure, two vectors containing the first and second polypeptide chain encoding genes are generated, respectively), and transfected into HEK293F cells. The cells were cultured for 3 days, and the culture supernatant of the transfected cells was collected and loaded into a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody was eluted with acetic acid-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. The antibody concentration was measured with Nano Drop. The protein purity was determined by SDS-PAGE and analytical HPLC-SEC.

[0724] The enhanced internalization ability of the trispecific anti-EGFR / cMet antibody by the tumor target cell EBC-1 was detected by FACS.

[0725] Prepare tumor target cells EBC-1, with 3×10 cells per well. 5 cells were plated on a 96-well plate. Trispecific anti-EGFR / cMet antibodies V-20-Fc, 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 respectively, incubated at 4°C for 30 min, and the supernatant containing each VHH-Fc was removed by centrifugation. The cells were evenly divided into 2 groups and incubated at 4°C and 37°C for 4 hours, respectively. After the incubation, PBS in an ice bath was immediately added to terminate the endocytosis experiment. After adding the secondary antibody PE-anti-human IgG (eBioscience, catalog number 12-4998-82) and incubating at 4°C for 30 min, the MFI of the cells was measured by flow cytometry (Life Technologies).

[0726] The internalization level of cell surface-bound antibody was calculated using the following formula:

[0727] Endocytosis = MFI of sample incubated at 4°C - MFI of sample incubated at 37°C.

[0728] like Fig.15 The results showed that the tumor target cell EBC-1 had a higher internalization of the trispecific antibody molecules V-20-Fc and V-23-Fc (compared with the anti-cMet M-4, and the anti-EGFR M-5, M-6, and M-7 molecules).

[0729] 6.7 FACS detection of synergistic binding of trispecific antibodies to target cells

[0730] NCI-H1975 cells were cultured at 1.5 × 10 cells per well. 5 cells were plated on a 96-well plate. The trispecific antibody of the present invention (200 nM, 5-fold dilution) was added, incubated at 4°C for 1 hour, and then centrifuged at 300 g for 4 minutes to remove the supernatant. The plate was washed twice by centrifugation with FACS buffer (1% BSA). The secondary antibody PE-anti-human IgG (eBioscience, catalog number 12-4998-82) was added and incubated at 4°C for 1 hour, and then the plate was washed twice by centrifugation. Finally, the cells were resuspended with FACS buffer (1% BSA), and the MFI of the cells was measured by flow cytometry (Life Technologies).

[0731] Depend on Fig.16 As shown, V-23-Fc has stronger binding ability than its corresponding control antibodies (M4 and M6), indicating that the two targets have synergistic binding effects.

[0732] Example 7. Preparation, characterization and experiments of antibody-drug conjugates (ADCs)

[0733] The linker-payloads used in the examples of the present invention are known in the prior art and / or commercially available, or can be prepared as described herein. When the drawn structure is inconsistent with the actual situation, it should be allowed to modify or correct the structure according to the actual situation.

[0734] General Synthesis Method A

[0735] 5.45mg / ml of the antibody of the present invention in 20mM His-hacn, 150mM NaCl, pH5.5 was placed in an Eppendorf tube. 6 molar equivalents of TCEP (Tris (2-carboxyethyl) phosphine hydrochloride, 5mM concentration) were added to the antibody buffer (TCEP: antibody = 6: 1). The Eppendorf tube containing the reaction mixture was placed on an oscillator (x500 rpm) and reacted at 37°C for 2 hours. Another 6 molar equivalents of TCEP (5mM) were added to the mixture. The reaction mixture was placed on an oscillator (500rpm) and shaken for another 2 hours at 37°C. Then, ultrafiltration (MWCO 30kd) was performed to remove TCEP, and the buffer was supplemented with 20mM His-hac buffer. 6 molar equivalents of linker-payload (5mg / ml, in DMA) were added dropwise to the fully reduced antibody, while the DMA concentration was kept below 20% (v / v). The reaction was kept on a shaker at room temperature for 2 hours (linker-payload: antibody = 6:1). The conversion rate was detected by HIC-HLPC, and purification was performed when the conversion was complete. The reaction mixture was transferred to an ultrafiltration tube (MWCO 30kd), and the sample was centrifuged at 10,000 rpm for 5 minutes to half the solution volume, and supplemented to the original volume with His-hac buffer (containing 10% DMA). Discard the flow-through. Repeat the washing step 10 times. Then repeat the washing step 10 times with 10mM His-hac pH = 5.0 for solution replacement, and finally transfer the remaining solution and adjust it to the appropriate concentration.

[0736] When another method Pro A is used for purification (ProA product marked 40g / L capacity), wash with 10CV 10mM His-hac before use, and then load the sample onto a gravity column filled with Pro A resin. Wash with at least 10CV of 10mM His-hac buffer containing 10% DMSO, and 50CV of 10mM His-hac buffer without DMSO to completely remove the free payload. Then elute with pH 3 acetic acid solution (concentration 50mM) and immediately neutralize with 2M Tris buffer (pH 12.0) to pH 5.5-pH 6.0. Combine the conjugate solution in an Eppendorf tube. Measure the concentration (Nanodrop, A280). Use 10mM His-Hac (pH 6) as a blank. If the final concentration is less than expected, concentrate to the required concentration using a centrifugal ultrafiltration method (MWCO 30kd ultrafiltration membrane).

[0737] Purity was determined by HIC and SEC-HPLC methods. Free linker-payload was determined by RP-HPLC method. General methods and / or parameters for determining or testing ADCs Size Exclusion Chromatography (SEC) Method (for total ADC detection) SEC-HPLC Method Parameters

[0738]

[0739] ◆Reversed Phase HPLC (RP HPLC) method (for free drug detection) RP HPLC method parameters

[0740]

[0741] Elute according to the following table

[0742] Time(Min) Mobile phase A (%) Mobile phase B (%) 0.0 75.0 25.0 2.0 75.0 25.0 10.0 5.0 95.0 12.0 5.0 95.0 14.0 5.0 95.0 16.0 80.0 20.0 18.0 80.0 20.0

[0743] ◆HIC-HPLC method (for free antibody and DAR distribution detection) HIC-HPLC conditions.

[0744]

[0745] Elute according to the following table

[0746] Time(Min) Mobile phase A (%) Mobile phase B (%) 0.0 100.0 0.0 7.0 100.0 0.0 23.0 0.0 100.0 26.0 0.0 100.0 26.5 100.0 0.0 35.0 100.0 0.0

[0747] 7.1 Preparation and Characterization of ADC Molecules

[0748] a) Preparation of ADC molecules coupled with Mal-PEG8-VA-PAB-Exatecan

[0749]

[0750] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It can be understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10, etc.

[0751] According to the above-mentioned synthesis method A, antibody V-26-Fc and linker-payload Mal-PEG8-VA-PAB-Exatecan (CAS No.: 2679821-39-5; MedChemExpress, HY-147271) were used to prepare V-26-Fc-VA-Exd with MW 96.37, average Dar 4.0, yield 63%, and purity 91.73%.

[0752] According to the preparation method of V-26-Fc-VA-Exd, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-VA-Exd, V-20-Fc-VA-Exd, and V-23-Fc-VA-Exd.

[0753] b) Preparation of ADC molecules coupled with MC-VC-PAB-MMAE

[0754]

[0755] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It can be understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10, etc.

[0756] According to the above-mentioned synthesis method A, V-26-Fc-VC-MMAE was prepared using antibody V-26-Fc and linker-payload MC-VC-PAB-MMAE (CAS No.: 646502-53-6; MedChemExpress, HY-15575).

[0757] According to the V-26-Fc-VC-MMAE preparation method, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-VC-MMAE, V-20-Fc-VC-MMAE, and V-23-Fc-VC-MMAE.

[0758] c) Preparation of ADC molecules coupled with Mal-PEG8-VC-PAB-MMAE

[0759]

[0760] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It can be understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10, etc.

[0761] According to the above-mentioned synthesis method A, V-26-Fc-PEG-VC-MMAE was prepared using antibody V-26-Fc and linker-payload Mal-PEG8-VC-PAB-MMAE (CAS No.: 2353409-69-3; MedChemExpress, HY-141156).

[0762] According to the V-26-Fc-PEG-VC-MMAE preparation method, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-PEG-VC-MMAE, V-20-Fc-PEG-VC-MMAE, and V-23-Fc-PEG-VC-MMAE.

[0763] d) Preparation of ADC molecules coupled with Mal-PEG8-EVC-PAB-MMAE

[0764]

[0765] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It can be understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10, etc.

[0766] According to the above-mentioned synthesis method A, V-26-Fc-PEG-EVC-MMAE was prepared using antibody V-26-Fc and linker-payload Mal-PEG8-EVC-PAB-MMAE (prepared according to the preparation method of the present application).

[0767] According to the V-26-Fc-PEG-EVC-MMAE preparation method, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-PEG-EVC-MMAE, V-20-Fc-PEG-EVC-MMAE, and V-23-Fc-PEG-EVC-MMAE.

[0768] e) Preparation of ADC molecules coupled with MC-EVC-PAB-MMAE

[0769]

[0770] Ab is the antibody V-26-Fc prepared in the present application, and p is mainly 4. It can be understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10, etc.

[0771] According to the above-mentioned synthesis method A, V-26-Fc-EVC-MMAE was prepared using antibody V-26-Fc and linker-payload MC-EVC-PAB-MMAE (CAS No.: 2873452-49-2; MedChemExpress, HY-154915).

[0772] According to the V-26-Fc-EVC-MMAE preparation method, the antibody V-26-Fc was replaced with V-17-Fc, V-20-Fc, and V-23-Fc, respectively, to prepare V-17-Fc-EVC-MMAE, V-20-Fc-EVC-MMAE, and V-23-Fc-EVC-MMAE.

[0773] f) Preparation of ADC molecules conjugated with Mal-Gly-Exatecan-D-glucuronic acid:

[0774]

[0775] Ab is the antibody V-23-Fc prepared in the present application, and p is mainly 4. It can be understood that p can also be other positive integers, such as 1, 2, 3, 5, 6, 7, 8, 9 or 10, etc.

[0776] According to the above-mentioned synthesis method A, V-23-Fc-Gluc-Exd (or V-23-Fc-Glu-Exd) was prepared using antibody V-23-Fc and linker-payload Mal-Gly-Exatecan-D-glucuronic acid (CA No.: 2763252-25-9; MedChemExpress, HY-153179), MW 96, average Dar 4.0, yield 50%, purity 84.31%.

[0777] According to the preparation method of V-23-Fc-Gluc-Exd, the antibody V-23-Fc was replaced with V-26-Fc, V-17-Fc, and V-20-Fc, respectively, to prepare V-26-Fc-Gluc-Exd, V-17-Fc-Gluc-Exd, and V-23-Fc-Gluc-Exd.

[0778] The characterization data of the exemplary ADCs prepared in this application are as follows:

[0779]

[0780] Preparation of reference benchmark antibody drug conjugate AZD9592

[0781] 15 equivalents of TCEP solution were added to the antibody (RAA22 / B09-57, also referred to herein as BMK-AZD, or AZD Ab) solution, mixed well and reacted at 37°C for 2 hours. DMA and 16 equivalents of linker-payload (Mal-PEG8-amide-Val-Ala-(4-NH2)-Exatecan) dissolved from DMA into a 10mM mother solution were added to the reaction solution, and the organic solvent accounted for 10% in the final reaction system. The reaction solution was mixed and placed in a 22°C constant temperature shaker for further reaction for 1 hour, and then N-acetylcysteine ​​was added to quench the reaction. The coupled sample was purified using an ultrafiltration concentration tube and exchanged into the final ADC storage buffer (30mM histidine, 30mM arginine-HCl, containing 0.02% PS 80, pH6.8). The sample was sterilized by filtering with a 0.22μm syringe filter.

[0782] The characterization data of the reference antibody drug conjugate involved in this application are as follows:

[0783] Molecular ID MW Free Payload Average Dar value Yield (%) purity(%) AZD9592 96.37 1.2％ Average Dar 5.9 95.82％ 98.57％ ABBV399 96.36 <1％ Average Dar 3.0 27.6％ 100％

[0784] Preparation of Mal-PEG8-EVC-PAB-MMAE in this application

[0785]

[0786] Step 1:

[0787]

[0788] (1) Compound HM-2039_1 (CAS: 13726-84-6) (100 mg, 0.33 mmol, 1 eq) and HN-078A_7 (Catalog: HY-100374, CAS: 644981-35-1) (388.86 mg, 0.346, 1.05 eq) were added to DMF (6 mL), HATU (188 mg) and triethylamine (66.7 mg) were added, and the mixture was stirred at room temperature for 2 h. 2 The reaction was allowed to proceed for 4 h under protection.

[0789] (2) After the reaction was completed as monitored by LCMS, the reaction solution was dropped into water and extracted twice with EA◆ (30 mL*2). The organic phases were combined and washed three times with saturated brine (30 mL*3), dried over anhydrous sodium sulfate and concentrated to obtain 410 mg (88.29% yield) of a white solid.

[0790] Step 2:

[0791]

[0792] (1) Compound HM-2039_2 (410 mg, 0.291 mmol, 1 eq) was added to DCM (4 mL), and TFA (1 mL) was added. The mixture was reacted under nitrogen protection (0-10° C.) for 1 h.

[0793] (2) After the reaction was completed as monitored by LCMS, the crude product was concentrated at low temperature to remove DCM. The crude product was passed through a reverse phase column to obtain the product: 200 mg, 54.86% yield, as a white solid.

[0794] Step 3:

[0795]

[0796] (1) HM-2039_3 (0.1 g, 79.84 umol, 1 eq) and HM-2039_4 (CAS: 1818294-46-0) (51.86 mg, 83.83 umol, 1.05 eq) were dissolved in DMF, and the reaction solution was stirred for 2 h.

[0797] (2) After the reaction was completed as monitored by LCMS, the reaction solution was directly prepared (TFA in water, ACN) to obtain 30 mg (23.54% yield) of the target product as a white solid with a MW of 1756.08 and a purity of 99.18%.

[0798] 1 H NMR (400MHz, DMSO) δ10.04(s,1H),8.35-8.25(m,0.28H),8.25-8.15(m,1H),8.13-8.03(m,1.5H),7.94-7.87(m,0.4H),7.76-7.70(m,1H), 7.67-7.63(m,0.4H),7.62-7.57(m,1.7H),7.38-7.25(m,7H),7.22-7 .14(m,1H),7.04(s,2H),5.99(s,1H),5.66–5.20(m,2H),5.15-4.95( s,2H),4.80–4.15(m,8H),4.10-3.90(m,2H),3.63-3.56(m,5H),3.52 -3.48(m,28H),3.28-3.24(m,4H),3.22-3.18(m,5H),3.16-3.10(m,2 H),3.10–2.84(m,9H),2.50-2.40(m,2H),2.30-2.20(m,3H),2.20-1. 69(m,10H),1.67-1.30(m,7H),1.08–0.98(m,7H),0.89–0.76(m,26H).

[0799] 7.2 FACS binding assay of ADCs of the present invention

[0800] The FACS method was used to detect the binding of the above-mentioned trispecific anti-EGFR / cMet antibody molecules to the target cells MDA-MB-468, EBC-1 and NCI-H1975 cells (Cell Bank of the Chinese Academy of Sciences) after they were coupled to the toxin molecules (i.e., the payload) to form ADC molecules.

[0801] The test method is as described in Example 6.2. Prepare target cells separately, with 1.5×10 cells per well. 5 cells were plated on a 96-well plate. Test ADCs (200 nM or 40 nM, 5-fold dilution) were added, incubated at 4°C for 1 h, and then centrifuged at 300 g for 4 minutes to remove the supernatant. The plate was washed twice by centrifugation with FACS buffer (1% BSA). The secondary antibody (PE-anti-human IgG (eBioscience, Cat. No.: 12-4998-82)) was added and incubated at 4°C for 1 h, then the plate was washed twice by centrifugation, and finally the cells were resuspended with FACS buffer (1% BSA), and the MFI of the cells was measured by flow cytometry (Life Technologies).

[0802] Depend on Fig.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 cells and NCI-H1975 cells, and the binding activity is better than that of the control group AZD9592 and ABBV399.

[0803] 7.3 Cell Killing Assay of ADCs of the Invention

[0804] The FACS method was used to detect the killing effect of the above-mentioned trispecific anti-EGFR / cMet antibody molecules coupled with toxins to form ADC molecules on target cells MDA-MB-468, EBC-1 and NCI-H1975 cells (cell bank of Chinese Academy of Sciences).

[0805] 100ul of cells (1x10 4 ) were inoculated into each well of a 96-well plate and incubated at 37°C, 5% CO 2Incubate overnight in an incubator. The next day, add 100ul of serially diluted V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE and V-26-Fc-PEG-EVC-MMAE and positive control ADC ABBV399 and AZD9592 to the corresponding wells. Continue to culture the cells for 3-5 days. Then, add 20μL CCK8 (Shanghai Life iLabBiotech, Cat. No.: AC11L054) to each well and incubate at 37°C, 5% CO 2 The cells were cultured in an incubator until color was developed and the absorbance at 450 nm was recorded using an enzyme reader (Molecular Device, SpectraMax M5).

[0806] The calculation formula of 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).

[0807] The killing results 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 Tables 10A and Fig.18A As shown in Table 10B and Table 10C, the killing results of V-23-Fc-VA-Exd and V-23-Fc-Gluc-Exd on EBC-1 and NCI-H1975 are shown in Table 10B and Table 10D. Fig.18B The results show 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).

[0808] Table 10A. Killing of target cells by V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE and V-26-Fc-PEG-EVC-MMAE

[0809]

[0810] Note: “-” means IC cannot be fitted 50 value.

[0811] Table 10B. Killing of target cells by V-23-Fc-VA-Exd and V-23-Fc-Gluc-Exd

[0812]

[0813] Note: “-” means IC cannot be fitted 50 value.

[0814] 7.4 Cell Binding and Cell Killing Assays of PEGylated and / or EVC-modified ADCs

[0815] Taking V-26-Fc as the representative of the antibody part, 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 coupled respectively. The FACS binding test method was as described above (such as Example 6.2 or 7.2), and the test results were as follows. Fig.19 The cell killing test steps are as described in Section 7.3 and the test results are as follows. Fig. 20 shown.

[0816] Example 8. In vivo efficacy evaluation of multispecific anti-EGFR / cMet antibody drug conjugates (ADCs)

[0817] EBC-1, NCI-H1975, NCI-H441 (lung adenocarcinoma cells), MDA-MB-468, FADU (head and neck cancer cells), and SW48 (colon adenocarcinoma cells) CDX mouse models were used to evaluate the in vivo efficacy of trispecific anti-EGFR / cMet antibody molecules coupled to toxin molecules to form ADC molecules. Fresh medium was used to revive each tumor cell. Cells in the logarithmic growth phase were collected. During the cell collection process, the medium was removed and washed twice, and then resuspended in DPBS. The cells were then injected subcutaneously into BALB / c nu / nu mice. The inoculum size for EBC-1, NCI-H1975, NCI-H441, MDA-MB-468, FADU, and SW48 CDX models was 3×10 6 / 100μL, 3×10 6 / 100μL, 1×10 7 / 100μL, 1×10 7 / 100μL, 5×10 6 / 100μL and 5×10 6 / 100μL. When the average tumor volume of mice reaches 110-250mm 3At the time of the study, the mice were randomly divided into groups according to the tumor volume. The day of grouping was defined as day 0. Each CDX mouse model was administered with a single intravenous push, and the test article was administered intravenously on D0. After the start of administration, the mice were weighed 1-2 times a week, the tumor volume was measured 1-2 times, and the animals were observed twice a day until the end of the study. The formula for calculating tumor volume is: Tumor volume (cubic millimeters) = 0.5*tumor length*tumor width 2 The change in tumor size (based on the baseline) is used to reflect the tumor inhibition and to evaluate the anti-tumor efficacy of the test article. The calculation formula for the change in tumor size (based on the baseline) is as follows:

[0818] Tumor volume change = (V t -V 0 ) / V 0 ×100%

[0819] V t : The average tumor volume of mice in the test article administration group on day t;

[0820] V 0 : Average tumor volume of mice in the test article administration group on day 0;

[0821] 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

[0822] The test results are as follows Fig.21 As shown, V-20-Fc-PEG-EVC-MMAE, V-23-Fc-PEG-EVC-MMAE and V-26-Fc-PEG-EVC-MMAE all have strong tumor inhibitory effects. After intravenous injection of the drug, the tumor volume was significantly reduced, and the efficacy was better than that of the positive control drugs ABBV399 and AZD9592.

[0823] In vivo efficacy of 8.2V-23-Fc-VA-Exd in the CDX model

[0824] The in vivo efficacy of V-23-Fc-VA-Exd was evaluated using EBC-1, NCI-H1975, NCI-H441, and MDA-MB-468CDX mouse models. Fig. 22 As shown, V-23-Fc-VA-Exd exhibited an in vivo tumor inhibition effect that was superior to the positive control drug AZD9592.

[0825] In the EBC-1 model, on day 54 after administration, tumors were completely eliminated by V-23-Fc-VA-Exd and AZD9592 at high doses; at low doses, V-23-Fc-VA-Exd was more effective than AZD9592.

[0826] In the NCI-H1975 model, on the 29th day after administration, the tumor inhibitory effect of V-23-Fc-VA-Exd at 2 mg / kg was comparable to that of AZD9592 at 4 mg / kg; at the same dose, the efficacy of V-23-Fc-VA-Exd was superior to that of AZD9592.

[0827] In the NCI-H441 model, at the same dose, on day 45 after administration, the efficacy of V-23-Fc-VA-Exd was superior to that of AZD9592; the tumor inhibitory effect of V-23-Fc-VA-Exd at 2 mg / kg was comparable to that of AZD9592 at 8 mg / kg.

[0828] In the MDA-MB-468 model, on day 42 after administration, V-23-Fc-VA-Exd and AZD9592 at 4 mg / kg and 8 mg / kg completely eliminated tumors; the tumor volume change rate of V-23-Fc-VA-Exd at 2 mg / kg was -72%.

[0829] In vivo efficacy of 8.3V-23-Fc-Gluc-Exd in the CDX model

[0830] SW48 CDX (colorectal cancer model, EGFR ++ &cMet + ), FADU CDX (head and neck cancer model, EGFR +++ &cMet + / - )、EBC-1CDX (non-small cell lung cancer model, EGFR ++ &cMet +++ ) and NCI-H1975 CDX (non-small cell lung cancer model, EGFR + &cMet + ) mouse model to evaluate the in vivo efficacy of V-23-Fc-Gluc-Exd. Fig.23 As shown, V-23-Fc-Gluc-Exd exhibited an in vivo tumor inhibition effect superior to that of the positive control drug.

[0831] In the SW48 model, FADU model, and NCI-H1975 model, V-23-Fc-Gluc-Exd was more effective than positive control drugs, including AZD9592 and ABBV399, at the same dose.

[0832] In the EBC-1CDX model, at day 36 after administration, V-23-Fc-Gluc-Exd was more effective than AZD9592 at a low dose (1 mg / kg), and tumors in both groups of animals were completely eliminated at a dose of 3 mg / kg.

[0833] The above describes exemplary embodiments of the present invention. It should be understood by those skilled in the art that these disclosures are merely exemplary and that various other substitutions, adaptations and modifications may be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0834]

[0835]

[0836]

[0837]

[0838]

[0839]

[0840]

[0841]

[0842]

[0843]

[0844]

[0845]

[0846]

[0847]

[0848]

Claims

1. An immunoglobulin single variable domain (ISVD) that specifically binds to cMet, wherein the ISVD comprises or consists of a VHH domain, wherein the VHH domain comprises (a) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 16, 39-40 and 121-130; (b) three CDRs in the amino acid sequence shown in one of SEQ ID NOs: 21, 42 and 134-136; or (c) 3 CDRs in the amino acid sequence shown in SEQ ID NO:26; Preferably, the VHH domain comprises: (i) a CDR1 comprising or consisting of an amino acid sequence selected from one of SEQ ID NOs: 18, 41, and 131-133, and a CDR2 and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NOs: 19 and 20, respectively; in particular, a CDR1, a CDR2, and a CDR3 comprising or consisting of an amino acid sequence of SEQ ID NOs: 41, 19, and 20, respectively; (ii) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 23-25, respectively; or (iii) CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences of SEQ ID NOs: 28-30, respectively; More preferably, the VHH domain comprises: (a) a sequence of one of SEQ ID NOs: 16, 39-40, and 121-130, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (b) a sequence of one of SEQ ID NOs: 21, 42, and 134-136, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (c) a sequence of SEQ ID NO:26, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; More preferably, the VHH domain comprises: (a) the amino acid sequence shown in one of SEQ ID NOs: 16, 39-40 and 121-130; (b) the amino acid sequence shown in one of SEQ ID NOs: 21, 42 and 134-136; or (c) the amino acid sequence shown in SEQ ID NO: 26, In particular, the VHH domain comprises the amino acid sequence of SEQ ID NO: 39 or 40, or comprises the amino acid sequence of SEQ ID NO:

42.

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

84.

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

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

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

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

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

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

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

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

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

84.

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

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

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

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

89.

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

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

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

87.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

33. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 27 to 32, wherein D has a structure represented by formula (D-2a) or formula (D-2b): Where R 1a , R 2a , R 3a and R 4a As defined in formula (D-1a); or Where R 1b , R 2b , R 3b , R 4b , R 5b , R 6b , R 7b , R 8b , R 9b and R 10b As defined in formula (D-1b).

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

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

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

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

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

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

41. The antibody drug conjugate according to any one of claims 36 to 40, or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: Where R 1c Each independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkoxy, halogen, nitro and cyano; Su are each independently selected from n2 is 0, 1, 2, 3 or 4; n5 is 0, 1, 2 or 3; and n3 and n4 are independently 1, 2, 3, 4, 5 or 6.

42. The antibody drug conjugate according to any one of claims 36 to 41, or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: Preferably, L3 is selected from: More preferably, L3 is selected from:

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

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

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

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

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

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