Antibody coupling medicine aiming at c-Met and application

Abstract

The invention relates to a c-Met-targeting antibody coupling medicine as well as a preparation method and application thereof, in particular to a nano antibody specifically bound with c-Met or an antigen binding fragment thereof, a polypeptide construct thereof, an antibody coupling medicine, a composition and medical application. The antibody coupling drug has excellent binding activity to c-Met protein, shows relatively good targeting and inhibitory activity to c-Met positive tumor cells, and is good in safety.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology drugs. Specifically, the present invention provides an antibody-drug conjugate targeting c-Met, a preparation method thereof, and uses thereof. Background Art

[0002] Cancer has become a major disease that seriously endangers human life and health and restricts social and economic development. Currently, conventional treatments for cancer mainly include a series of physical or chemical methods such as surgical resection, radiotherapy, and chemotherapy. However, conventional treatments have many limitations, such as low selectivity, easy recurrence, and large side effects. Antibody-drug conjugates (ADCs), as a new anti-cancer treatment strategy, have shown broad prospects in cancer treatment. It is a type of drug that combines the specificity of monoclonal antibodies with the potency of highly cytotoxic drugs. By preferentially targeting its payload to the tumor site, it can potentially reduce the severity of side effects. It is known as the "magic bullet" for cancer treatment, combining the powerful killing effect of traditional chemotherapy with the tumor targeting of antibody drugs. Currently, 15 ADC drugs have been approved worldwide, 7 for the treatment of hematological tumors and 8 for the treatment of solid tumors. Among them, 6 ADC drugs have been approved for marketing in China, providing new treatment options for cancer patients.

[0003] Although the efficacy of ADCs has been widely validated, obstacles such as the large size of monoclonal antibodies and low penetration into solid tumors still need to be overcome. Antibodies found in camelids (such as camels and alpacas) and cartilaginous fish that naturally lack light chains are called heavy-chain antibodies (HCAbs). Unlike traditional antibodies, which are composed of two heavy chains and two light chains, heavy-chain antibodies are composed only of heavy chains, including a variable region, a hinge region, and two constant regions (CH2 and CH3) that form their antigen-binding site, also known as the "VHH region" or nanobodies, which can independently bind to antigens. These characteristics of heavy-chain antibodies are expected to overcome the limitations of traditional antibodies due to their large size and complex structure. When used in ADCs, heavy-chain antibodies are expected to improve the penetration of ADCs into tissues, thereby enhancing the killing effect and reducing side effects.

[0004] c-MET, a member of the receptor tyrosine kinase (RTK) family, is closely associated with tumorigenesis, invasive growth, and metastasis. As a multifunctional transmembrane tyrosine kinase, c-MET is involved in regulating key cellular processes such as metabolism, differentiation, proliferation, migration, and the cell cycle, playing an important role in normal tissue development and repair. However, c-Met dysregulation has been observed in a variety of malignancies, including liver cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, and gastric cancer. Overexpression or mutation of c-Met leads to abnormally active signal transduction, thereby promoting tumor growth, angiogenesis, and cancer metastasis. Therefore, targeting c-Met has become an important target in the field of tumor treatment. Currently, treatment strategies mainly focus on the development of small molecule tyrosine kinase inhibitors and monoclonal antibodies. However, there are currently no ADCs specifically targeting c-Met on the market. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide an antibody-drug conjugate targeting c-Met. In some embodiments, the antibody-drug conjugate has Figure 1 The structure shown in FIG5 is a nano-antibody modified antibody, wherein the targeting portion is a nano-antibody Nano-A, which is derived from alpacas, and uses the anti-mitotic agent monomethyl oleate E (MMAE, a microtubule inhibitor) as a toxic payload, which is connected to the antibody's site-directed mutagenesis site through the lysosomal cleavable dipeptide valine-citrulline (Val-Cit), thereby obtaining an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate A4-VC-MMAE (DAR≈4) disclosed in the present application exhibits good binding activity to the c-Met protein. In vivo, A4-VC-MMAE has good targeting to the tumor site of a mouse model with high c-Met expression, can effectively inhibit the growth of mouse tumors, exhibits efficient anti-tumor activity, and has good safety. On the basis of the above, the technical solution of the present application involves the following aspects.

[0006] 1. Nanobodies

[0007] Based on phage display and screening technologies, alpaca antibodies against c-Met were isolated and obtained, which can specifically bind to c-Met-positive cells. In some embodiments, the isolated alpaca antibody "Nano-A" (SEQ ID NO: 1) has good binding activity to the c-Met antigen.

[0008] In one aspect, the present application provides a Nanobody or antigen-binding fragment thereof that specifically binds to c-Met, comprising the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

[0009] (a) CDR1 having: a sequence as shown in SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 2;

[0010] (b) CDR2 having: a sequence as shown in SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 3; and

[0011] (c) CDR3 having: a sequence as shown in SEQ ID NO: 4, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 4.

[0012] In some embodiments, the substitutions are conservative substitutions.

[0013] In some embodiments, the Nanobody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO: 2, a CDR2 as shown in SEQ ID NO: 3, and a CDR3 as shown in SEQ ID NO: 4.

[0014] In some embodiments, the CDR sequences are defined using the Kabat, IMGT, Chothia, or Abm numbering systems.

[0015] In some embodiments, the Nanobody comprises an amino acid sequence selected from the group consisting of:

[0016] (i) the sequence shown in SEQ ID NO: 1;

[0017] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 1; or

[0018] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1.

[0019] In some embodiments, the substitutions are conservative substitutions.

[0020] 2. Peptide Constructs

[0021] The present application further provides a polypeptide construct obtained by fusion of the nanobody with Fc.

[0022] Specifically, in another aspect, the present application provides a polypeptide construct that specifically binds to c-Met, which comprises the Nanobody or antigen-binding fragment thereof as described in any one of the foregoing.

[0023] In some embodiments, the polypeptide construct further comprises an immunoglobulin Fc domain.

[0024] In some embodiments, the immunoglobulin Fc domain is linked directly or via a peptide linker to the N-terminus or C-terminus of the Nanobody or antigen-binding fragment thereof.

[0025] In some embodiments, the immunoglobulin Fc domain is linked to the C-terminus of the Nanobody or antigen-binding fragment thereof directly or via a peptide linker.

[0026] In some embodiments, the immunoglobulin Fc domain is a human Fc domain or is derived from a human Fc domain. In some embodiments, the immunoglobulin Fc domain is selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 isotype.

[0027] In some embodiments, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) thereto.

[0028] In some embodiments, the immunoglobulin Fc domain is mutated. The mutation can provide a site for binding to a therapeutic agent (e.g., a cytotoxic drug). In some embodiments, the immunoglobulin Fc domain comprises a mutation at position S239 and / or K290, such as S239C and / or K290C.

[0029] In some embodiments, the immunoglobulin Fc domain is numbered according to the Kabat EU index.

[0030] In some embodiments, the polypeptide construct comprises or consists of the amino acid sequence shown in SEQ ID NO: 6 or 7.

[0031] In some embodiments, the polypeptide construct is a dimer, such as a homodimer or a heterodimer.

[0032] In some embodiments, the polypeptide construct comprises, but is not limited to, one or more of a single domain antibody, a single chain antibody, an antibody Fab, a full-length antibody protein, an antigen-binding fragment, a bispecific antibody, a multispecific antibody, a bi / multivalent single domain antibody, a bi / multivalent single chain antibody, and a bi / multivalent antibody Fab.

[0033] 3. Nucleic Acids, Vectors, Host Cells, and Expression Methods

[0034] In another aspect, the present application provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding the nanobody or its antigen-binding fragment or the polypeptide construct. The nucleic acid can be obtained using methods known in the art, for example, isolated from a phage display library, a yeast display library, an immune animal, an immortalized cell (e.g., a mouse B cell hybridoma cell, an EBV-mediated immortalized B cell) or chemically synthesized. The nucleic acid molecule can be codon-optimized for the host cell used for expression.

[0035] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0036] In some embodiments, the nucleic acid molecule is prepared as a recombinant nucleic acid. In some embodiments, the nucleic acid molecule is cloned into an expression vector. The expression vector may further include additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The recombinant nucleic acid comprising the nucleic acid can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination technology (such as polymerase chain reaction (PCR) technology) etc. (see Sambrook, J., EF Fritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nded. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The expression vector may also include a polynucleotide sequence encoding a polypeptide or protein that can promote detection and / or separation of the expressed antibody or antigen-binding fragment. Such polypeptides or proteins may include, but are not limited to, affinity tags (such as biotin, polyhistidine tags (His6) or glutathione S-transferase (GSH) tags), polypeptides comprising protease cleavage sites, and reporter proteins (such as fluorescent proteins). The nucleic acid molecule may be present in one or more vectors. In some embodiments, the expression vector is a DNA plasmid, such as a DNA plasmid for expression in bacteria, yeast, or mammalian cells. In other embodiments, the expression vector is a viral vector. In other embodiments, the expression vector is a phage vector or a phagemid vector.

[0037] In another aspect, the present application provides a host cell comprising at least one nucleic acid or vector as described above. In some embodiments, the host cell is used to express the Nanobody or its antigen-binding fragment or the polypeptide construct. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., Escherichia coli), eukaryotic cells (e.g., yeast, insect cells, mammalian cells).

[0038] Bacteria (e.g., E. coli BL21 (DE3)) are particularly advantageous for expressing smaller antigen-binding fragments. Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells (e.g., HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for expressing antibodies.

[0039] In another aspect, the present application provides a method for producing the Nanobody or its antigen-binding fragment or the polypeptide construct in a host cell, wherein the method comprises the following steps:

[0040] (1) transforming a host cell with at least one nucleic acid or expression vector described herein;

[0041] (2) culturing the transformed host cell under appropriate conditions to allow expression of the nucleic acid or expression vector, and

[0042] (3) Isolating and purifying the antibody or antigen-binding fragment thereof or the polypeptide construct from the host cell or culture medium.

[0043] In some embodiments, the host cell further comprises a chaperone plasmid that can help improve the solubility, stability and / or folding of the antibody or antibody fragment.Techniques for isolating and purifying antibodies from host cells are well known to those skilled in the art.

[0044] 4. Antibody-drug conjugates (ADCs)

[0045] The present application further provides an antibody-drug conjugate (ADC), comprising:

[0046] A targeting moiety selected from any of the polypeptide constructs described above;

[0047] Cytotoxic drug moiety; and

[0048] A linker is used to connect the targeting moiety and the cytotoxic drug moiety.

[0049] Cytotoxic drugs can be conjugated to disulfide bonds on antibodies, or site-specifically and stably conjugated to antibodies through engineered mutant cysteine ​​conjugation technology. Mutating amino acids at specific antibody sites to cysteine, which reacts with drug-linkers, can achieve site-specific conjugation, obtain highly uniform conjugates, and improve the therapeutic index of ADC drugs.

[0050] In some embodiments, the polypeptide construct is linked to the linker via a sulfhydryl group on a cysteine ​​residue.

[0051] In some embodiments, the polypeptide construct is linked to the linker via a sulfhydryl group on a cysteine ​​residue on the VHH.

[0052] In some embodiments, the polypeptide construct is linked to the linker via a sulfhydryl group on a cysteine ​​residue on a reduced disulfide bond in the hinge region.

[0053] In some embodiments, the polypeptide construct is linked to the linker via a sulfhydryl group on a cysteine ​​residue in the Fc domain.

[0054] In some embodiments, the polypeptide construct is linked to the linker via the sulfhydryl group on the cysteine ​​residues at positions 239 and / or 290 of the Fc domain.

[0055] In some embodiments, the cytotoxic drug is selected from the group consisting of a tubulin inhibitor and a DNA damaging drug.

[0056] In some embodiments, the tubulin inhibitor is selected from auristatins (e.g., MMAE, MMAF), maytansines (e.g., maytansine, maytansinol, DM1, DM4), taxanes (e.g., Taxol, Docetaxel, Cabazitaxel), vinca alkaloids (e.g., vinblastine, vincristine), eribulin, and colchicine.

[0057] In some embodiments, the DNA damaging agent is selected from DNA alkylating agents (calicheamicin γ11, N-acetyl-γ11 calicheamicin, anthramycin, PBD, dukamycin), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically, camptothecin, SN-38, Dxd, irinotecan, belotecan, topotecan, PNU-159682), doxorubicin, daunorubicin, etoposide, mitoxantrone) and amanitin.

[0058] In some embodiments, the cytotoxic drug is MMAE.

[0059] In some embodiments, the linker is a cleavable or non-cleavable linker.

[0060] In some embodiments, the cleavable linker is selected from the group consisting of a protease-sensitive, pH-sensitive, and glutathione-sensitive linker.

[0061] In some embodiments, the linker is selected from MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropionyl), Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Ala-Phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimidyl)-4-(pyridine-2- methyl) cyclohexane-1-carboxylate), 6-(2,5-dioxopyrrolidin-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidin-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), or SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate), and any combination thereof.

[0062] In some embodiments, the linker is MC-Val-Cit-PAB.

[0063] In some embodiments, each peptide chain of the polypeptide construct is linked to 0, 1, 2, 3, 4, or 5 of the following structures through a cysteine ​​residue in a VHH, a hinge region reduced disulfide bond, or a cysteine ​​residue in an Fc domain:

[0064]

[0065] In some embodiments, the antibody-drug conjugate is selected from:

[0066]

[0067] Wherein, x=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0068] Ab is the polypeptide construct described in any one of the above.

[0069] In some embodiments, the antibody-drug conjugate is:

[0070]

[0071] Wherein, x=1, 2, 3, 4, 5 or 6;

[0072] Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:6.

[0073] In some embodiments, the antibody-drug conjugate is:

[0074]

[0075] Wherein, x=1, 2, 3, 4, 5, 6, 7 or 8;

[0076] Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:7.

[0077] 5. Composition

[0078] In another aspect, the present application provides a composition comprising or consisting of one or more antibody-drug conjugates as described in any one of the foregoing.

[0079] In some embodiments, the composition has a DAR value of 1-10, e.g., 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1-8.5, 1-9, 1-9.5, 1-10, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1.5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 1.5-8.5, 1.5-9, 1.5-9.5, 1.5-10, 2-2.5, 2-3. 3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2-8.5, 2-9, 2-9.5, 2-10, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5-7, 2.5-7.5, 2.5-8, 2.5-8.5, 2.5-9, 2.5-9.5, 2.5-10, 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3-8.5, 3-9, 3-9.5, 3-10, 3.5-4 , 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 3.5-8.5, 3.5-9, 3.5-9.5, 3.5-10, 4-4.5, 4-5, 4-5.5, 4-6, 4.5-5, 4.5-5.5, 4.5- 6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 4.5-8.5, 4.5-9, 4.5-9.5, 4.5-10, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5-8.5, 5-9, 5-9.5, 5-10, 5.5-6, 5.5-6.5, 5 .5-7, 5.5-7.5, 5.5-8, 5.5-8.5, 5.5-9, 5.5-9.5, 5.5-10, 6-6.5, 6-7, 6-7.5, 6-8, 6-8.5, 6-9, 6-9.5, 6-10, 6.5-7, 6.5-7.5, 6.5-8, 6.5-8.5, 6.5-9, 6. 5-9.5, 6.5-10, 7-7.5, 7-8, 7-8.5, 7-9, 7-9.5, 7-10, 7.5-8, 7.5-8.5, 7.5-9, 7.5-9.5, 7.5-10, 8-8.5, 8-9, 8-9.5, 8-10, 8.5-9, 8.5-9.5, 8.5-10, 9-9.5, 9-10, 9.5-10. .

[0080] In another aspect, the present application provides a composition comprising or consisting of one or more of the following antibody-drug conjugates:

[0081]

[0082] Wherein, x=1, 2, 3, 4, 5, 6, 7 or 8;

[0083] Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:7.

[0084] In some embodiments, the composition has a DAR value of 1-8, e.g., 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1. 5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 2-2.5, 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5-7, 2.5-7.5, 2.5-8 , 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3.5-4, 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 4-4.5, 4-5, 4-5.5, 4-6, 4-6.5, 4-7, 4-7.5, 4-8, 4.5-5, 4 .5-5.5, 4.5-6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5.5-6, 5.5-6.5, 5.5-7, 5.5-7.5, 5.5-8, 6-6.5, 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-7.5, 7-8 or 7.5-8.

[0085] In some embodiments, the composition has a DAR value of 1-5, such as 3.5-4.5, such as 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5.

[0086] 6. Pharmaceutical Compositions

[0087] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate or composition described in any of the foregoing items, and an optional carrier or excipient.

[0088] The excipients may be those described in Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Non-limiting examples of suitable excipients include buffers, preservatives, binders, lubricants, disintegrants, chelating agents, surfactants, flavorings, sweeteners, coloring agents.

[0089] In some embodiments, suitable buffering agents include calcium bicarbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide and other calcium salts, etc. or combinations thereof.

[0090] In some embodiments, suitable preservatives include antioxidants such as α-tocopherol and ascorbate, and antimicrobial agents such as parabens, chlorobutanol, and phenol. Antioxidants may further include EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetyl cysteine.

[0091] In some embodiments, suitable binders include starches such as potato starch, corn starch, wheat starch; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatin; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinyl pyrrolidone (povidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol and water, or combinations thereof.

[0092] In some embodiments, suitable lubricants include metal stearates (e.g., magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (e.g., sodium stearyl fumarate), fatty acids (e.g., stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal lauryl sulfate (e.g., sodium lauryl sulfate, magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0093] In some embodiments, disintegrants can be non-effervescent disintegrants. Suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches, sweeteners, clays such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar fat, locust bean fat, karaya gum, pectin (pecitin) and tragacanth. In some embodiments, disintegrants can be effervescent disintegrants. Suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0094] In some embodiments, suitable flavoring agents may be selected from cinnamon oil; oil of wintergreen; peppermint oil; clover oil; hay oil; anise oil; eucalyptus oil; vanilla; citrus oils, such as lemon oil, orange oil, grape and grapefruit oil; and fruit flavors, including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple and apricot flavors.

[0095] In some embodiments, suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as the sodium salt; dipeptide sweeteners, such as aspartame; the dihydrochalcone compound, glycyrrhizin; Stevia Rebaudiana (stevioside); chlorinated derivatives of sucrose, such as sucralose; and sugar alcohols, such as sorbitol, mannitol, xylitol (sylitol), and the like.

[0096] In some embodiments, suitable colorants include food, drug, and cosmetic colors (FD&C), drug and cosmetic colors (D&C), and external drug and cosmetic colors (Ext. D&C).

[0097] In some embodiments, suitable chelating agents include ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium, and diammonium salts of EDTA; barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelates of EDTA; trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid Monohydrate; N,N-bis(2-hydroxyethyl)glycine; 1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid; 1,3-diaminopropane-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N′-diacetic acid; ethylenediamine-N,N′-dipropionic acid dihydrochloride; ethylenediamine-N,N′-bis(methylenephosphonic acid) hemihydrate; N-(2-hydroxyethyl)ethylenediamine-N , N′, N′-triacetic acid; ethylenediamine-N, N, N′, N′-tetrakis(methylenephosphonic acid); O, O′-bis(2-aminoethyl)ethylenediol-N, N, N′, N′-tetraacetic acid; N, N-bis(2-hydroxybenzyl)ethylenediamine-N, N-diacetic acid; 1,6-hexanediamine-N, N, N′, N′-tetraacetic acid; N-(2-hydroxyethyl)iminodiacetic acid; iminodiacetic acid; 1,2- Diaminopropane-N,N,N′,N′-tetraacetic acid; nitrilotriacetic acid; nitrilotripropionic acid; trisodium salt of nitrilotris(methylenephosphoric acid); 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]pentatriacontane hexahydrobromide; or triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid, etc.

[0098] In some embodiments, suitable diluents include water, glycerol, methanol, ethanol, and other biocompatible diluents.

[0099] In some embodiments, suitable surfactants include polysorbates, sodium lauryl sulfate, sodium stearyl fumarate, polyoxyethylene alkyl ethers, sorbitan fatty acid esters, polyethylene glycol (PEG), polyoxyethylene castor oil derivatives, sugar esters of fatty acids, glycerides of fatty acids, or combinations thereof.

[0100] Pharmaceutical compositions described herein can be formulated into various formulations and used in a variety of different ways, such as oral, rectal or parenteral administration. Term " parenteral " used herein can include intra-arterial, intracardiac, intraventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravenous, intravitreal, epidural, subcutaneous, inhalation, transdermal, through mucosa, sublingual, buccal and local (including epidermis, dermis, enema, eye drops, ear drops, intranasal, vaginal) administration. In some exemplary embodiments, route of administration can be by injection, such as intramuscular, intravenous, subcutaneous or intraperitoneal injection. Preparations for oral administration can include capsules, tablets, caplets, pills, lozenges, lozenges, powders and granules etc.

[0101] 7. Medical applications

[0102] In another aspect, the present application provides the use of the Nanobodies or antigen-binding fragments thereof, polypeptide constructs, antibody-drug conjugates, compositions or pharmaceutical compositions described herein in the preparation of drugs for treating cancer.

[0103] In another aspect, the present application provides a Nanobody or antigen-binding fragment thereof, polypeptide construct, antibody-drug conjugate, composition or pharmaceutical composition described herein for use in treating cancer.

[0104] In another aspect, the present application provides a method for treating cancer, comprising administering to a subject in need thereof an effective amount of the Nanobody or antigen-binding fragment thereof, polypeptide construct, antibody-drug conjugate, composition or pharmaceutical composition described herein.

[0105] In some embodiments, the cancer is c-Met positive.

[0106] In some embodiments, the cancer is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (eg, triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (eg, epithelial ovarian cancer), and cervical cancer.

[0107] Definition of terms

[0108] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures in molecular genetics, nucleic acid chemistry, cell culture, biochemistry, cell biology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0109] As used herein, the singular forms "a", "an" and "include" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, the terms "include", "comprise", "includes", "have", "contain" or their variations are open-ended and not exclusive or exhaustive.

[0110] As used herein, the terms "antibody" and "monoclonal antibody" refer to immunoglobulin molecules that are typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Each chain has variable regions, referred to as the heavy chain variable region (VH) and the light chain variable region (VL), respectively. Together, the VH and VL are responsible for binding to the antigen recognized by the antibody. Mammalian immunoglobulins have five major heavy chain classes (or isotypes), which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is a primary antibody produced by birds and reptiles that is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0111] The variable region of an antibody comprises a framework region (FR) and a hypervariable region (HVR), which are called "complementarity determining regions (CDRs)". The CDRs are primarily responsible for binding to the epitope of an antigen. VH and VL consist of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The allocation of amino acids to each region or domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0112] As used herein, the term "nanobody", "single-domain antibody", "VHH antibody" or "camelid antibody" refers to an antibody that has no additional antibody domains and has a single domain (variable region) that can specifically bind to an antigen or antigenic epitope. Nanobodies include, for example, VH Domain antibodies, V NAR Antibodies, Camelidae VHH antibodies and V L Domain antibodies. V NAR Antibodies are produced by cartilaginous fishes such as the nurse shark, Wobegon shark, spiny dogfish, and bamboo shark. Camelid VHH antibodies are produced by a variety of species, including camels, llamas, alpacas, dromedaries, and guanacos, and they produce heavy chain antibodies that naturally lack light chains.

[0113] As used herein, the term "Fc region" or "Fc domain" refers to the portion of the heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In some embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0114] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of lymphocytes or by cells transfected with the coding sequence of a single antibody. Monoclonal antibodies can be produced by methods known to those skilled in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0115] As used herein, the term "conservative variant" refers to a protein containing conservative amino acid substitutions that do not substantially affect or reduce the affinity of the protein. For example, a Nanobody or polypeptide construct that specifically binds to c-Met may comprise at most 1, at most 2, at most 5, at most 10, or at most 15 conservative substitutions and a polypeptide that specifically binds to c-Met. Conservative amino acid substitutions of functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0116] 1) Alanine (A), serine (S), threonine (T);

[0117] 2) Aspartic acid (D), glutamic acid (E);

[0118] 3) Asparagine (N), glutamine (Q);

[0119] 4) Arginine (R), Lysine (K);

[0120] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0121] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0122] As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0123] As used herein, the term "identity" refers to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0124] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and its antigen. The binding affinity between two molecules can be expressed as K. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as a molar concentration (M). K DThe smaller the value, the tighter the two molecules bind and the higher the affinity. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Bind to the antigen. K D The value can be determined by methods well known in the art, for example using surface plasmon resonance (SPR) in a BIACORE instrument.

[0125] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, and generally can include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0126] As used herein, the term "isolated" refers to a substance (eg, a nucleic acid molecule or a polypeptide) that is separated from its source or environment, ie, does not substantially contain any other components.

[0127] As used herein, the term "vector" is a medium for importing exogenous nucleic acid into a host cell, and when the vector is transformed into an appropriate host cell, the exogenous nucleic acid is amplified or expressed. The vector usually remains free, but can be designed to integrate a gene or part thereof into a chromosome of the genome. In this article, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc.

[0128] As used herein, the term "expression vector" refers to a vector capable of expressing DNA, and the DNA is operably connected to a regulatory sequence (such as a promoter, a ribosome binding site) that can affect expression of the DNA. Regulatory sequences can include promoter and terminator sequences, and optionally can include an origin of replication, a selective marker, an enhancer, a polyadenylation signal, etc. The expression vector can be a plasmid, a phage vector, a recombinant virus, or other vectors that, when introduced into a suitable host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, and are included in reproducible expression vectors in eukaryotic cells and / or prokaryotic cells and keep free expression vectors or are integrated into the expression vector of the host cell genome.

[0129] As used herein, the term "host cell" is a cell that is used to receive, maintain, replicate, or amplify a vector. A host cell can also be used to express a nucleic acid or a polypeptide encoded by the vector. The host cell can be a eukaryotic cell or a prokaryotic cell.

[0130] As used herein, the term "contacting" refers to direct physical association; including both solid and liquid forms.

[0131] As used herein, the term "cytotoxic drug" refers to any drug or compound that is capable of killing cells. "Cytotoxicity" refers to the toxicity of a molecule to the intended target cells, rather than to cells in the rest of the organism. In contrast, the term "toxicity" refers to the toxicity of a molecule to cells other than the intended target cells.

[0132] As used herein, the terms "subject," "patient," or "individual" include mammals and non-mammals. A mammal can be any member of the class Mammalia, including but not limited to humans; non-human primates such as chimpanzees, apes, or other monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs (or canines), and cats; laboratory animals including rodents such as rats, mice, and guinea pigs; and the like. Non-mammals can include birds, fish, and the like. In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. In some cases, the human can be an adult. In some cases, the human can be a child. In some cases, the human can be 0-17 years old. In some cases, the human can be 18-130 years old. In some cases, the subject can be male. In some cases, the subject can be female. In some cases, the subject is diagnosed with or suspected of having a disease. In some cases, the disease is cancer. The subject can be a patient or an individual. In some cases, subject, patient, or individual are used interchangeably.

[0133] As used herein, the terms "treat," "treat," "improve," or "alleviate" include alleviating or relieving the symptoms of a disease, inhibiting a disease, such as arresting the development of a disease, relieving a disease, causing regression of a disease, alleviating symptoms caused by a disease, or stopping the symptoms of a disease. The terms "treat," "treat," "improve," or "alleviate" may further include obtaining a therapeutic benefit. A therapeutic benefit may refer to the eradication of the disease being treated. Additionally, a therapeutic benefit may also be achieved by eradicating one or more physiological symptoms associated with the disease being treated, resulting in an observable improvement in the subject, although in some embodiments, the subject may still be suffering from the underlying disease.

[0134] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to a sufficient amount of the drug administered that will at least partially alleviate the symptoms of the disease being treated. The dosage regimen can be adjusted to provide the best desired response. For example, a single bolus injection can be administered, or several divided doses can be administered over time, or the dosage can be proportionally reduced or increased according to the treatment situation. It should be noted that the dosage value can vary with the type and severity of the disease to be alleviated, and can include single or multiple doses. It is further understood that for any particular individual, a specific dosage regimen should be adjusted over time according to individual needs and drug instructions or clinical physician professional judgment. In general, the effective dose is about 0.0001 to about 50 mg per kg body weight per day, for example, about 0.01 to about 10 mg / kg / day (single or divided administration). For a 70 kg person, this would add up to about 0.007 mg / day to about 3500 mg / day, for example, about 0.7 mg / day to about 700 mg / day. In some cases, dosage levels no higher than the lower limit of the aforementioned range may be sufficient, while in other cases, larger doses may still be employed without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses to be administered throughout the day.

[0135] Advantageous Effects of the Invention

[0136] The present application provides nanobodies, polypeptide constructs, and antibody-drug conjugates targeting c-Met, as well as compositions and uses thereof. The antibody-drug conjugates have excellent binding activity to the c-Met protein, exhibit good targeting and inhibitory activity against c-Met-positive tumor cells, and have good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1 The structure of A4-VC-MMAE is shown.

[0138] Figure 2 The band sizes of A4 identified by SDS-PAGE are shown (1: non-reducing, 2: reducing).

[0139] Figure 3 The results of ELISA assay for the binding activity of A4, A4-VC-MMAE, and the negative control 125s to the antigen c-Met are shown.

[0140] Figure 4 The results of the DAR value detection of A4-VC-MMAE using hydrophobic interaction chromatography (HIC) are shown.

[0141] Figure 5 The results of flow cytometry analysis of the internalization ability of A4 in BxPC3, HT-29, SCG7901, and SK-OV3 cells are shown.

[0142] Figure 6 The results of A4 endocytosis in HT-29 and BxPC3 cells were shown using a high-content imaging system.

[0143] Figure 7 Shown are the in vitro killing rate curves of A4-VC-MMAE against HT-29 and BxPC3 cells.

[0144] Figure 8 The in vitro killing rate curves of A4-VC-MMAE against BxPC3 cells (c-Met positive), BxPC3 (c-Met positive) + HCT116 (c-Met negative) = 1:1, and HCT116 (c-Met negative) are shown.

[0145] Figure 9 The results of in vivo targeting detection of A4 in BxPC3 and HT-29 tumor-bearing mice are shown.

[0146] Figure 10 The results show the inhibitory effect and body weight change of BxPC3 tumor-bearing mice after a single administration of A4-VC-MMAE. DETAILED DESCRIPTION

[0147] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0148] Sequence Listing

[0149] SEQ ID NO: describe SEQ ID NO: describe 1 Nano-A 5 Fc domain 2 Nano-A CDR1 6 Polypeptide construct A parent 3 Nano-A CDR2 7 Polypeptide construct A4 4 Nano-A CDR3

[0150] Example 1: Site-directed modification and binding activity of c-Met nanobodies

[0151] 1.1 In the early stage of the laboratory, a nanobody specific for c-Met was screened from the alpaca antibody library based on phage display technology and screening technology. The amino acid sequence is shown in Table 1. It was named "Nano-A".

[0152] Table 1. Amino acid sequence of Nanobody Nano-A

[0153]

[0154] The method described by KR Abhinandan et al. (Abhinandan KR, Martin AC. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol. 2008 Aug; 45(14): 3832-9. doi: 10.1016 / j.molimm.2008.05.022.) and IMGT (THE INTERNATIONAL IMMUNOGENETICS INFORMATION A dual strategy was used to determine the CDR sequences of Nano-A. The amino acid sequences of the variable region CDRs of Nano-A are shown in Table 2.

[0155] Table 2. Amino acid sequences of the variable region CDRs of Nano-A

[0156] name CDR sequences SEQ ID NO Nano-A CDR1 VCAMG 2 Nano-A CDR2 AINYSGERAFYAESVKG 3 Nano-A CDR3 DLGMTIDTMKIFEEYEY 4

[0157] 1.2 The screened Nanobody Nano-A VHH was expressed in tandem with the CH2 and CH3 segments of a conventional antibody Fc segment (SEQ ID NO: 5) to extend its half-life. The expression vector used was PTT5-H and was named "A Parent." Serine at position 239 was mutated to cysteine, and lysine at position 290 was mutated to cysteine, resulting in a designation of "A4." The amino acid sequences of A Parent and A4 (with Fc tag) are shown in Table 3.

[0158] Table 3. Amino acid sequences of parent A and A4 (with Fc tag)

[0159]

[0160]

[0161] Example 2: Transient expression and affinity chromatography purification of parent A and A4

[0162] 2.1 Obtaining Plasmids: Parent A and A4 plasmids were synthesized by Universal Biosystems (Anhui) Co., Ltd. The plasmids were transfected into DH-5α competent E. coli cells (Shenzhen Kangti) via rapid transfection. After a 90-second heat shock, the cells were cultured overnight at 37°C in an ampicillin-LB culture plate. Single colonies were selected and expanded in ampicillin-LB medium at 37°C, 220 rpm, for 15 hours. The plasmids were then extracted using an endotoxin-free plasmid extraction kit.

[0163] 2.2 Antibody Expression Use single plasmid to transiently transfect Expi-293F cells to express A parent and A4. Prepare at a density of 4×10 6 / mL, 95% viability, and a volume of 200mL of Expi-293F cells. 0.5mg of each antibody plasmid was filtered through a 0.22μm filter and added to 5mL of CD05 medium. Simultaneously, 2mg of PEI was added to 5mL of CD05 medium, immediately vortexed for 8 seconds, and allowed to stand for 2 minutes. 7mL of the PEI mixture was then added to the plasmid mixture, immediately vortexed for 8 seconds, and allowed to stand for 8 minutes. Finally, the PEI mixture was added dropwise to 200mL of cell suspension using a pipette, gently mixing. Incubate in a 5% CO2, 37°C incubator. After 4 hours, remove the cells and add 200mL of freestyle medium. Incubate in a 37°C incubator for expression for 7-8 days.

[0164] 2.3 Antibody Purification The cell expression supernatants of parent A and A4 were collected and centrifuged at 10,000 rpm for 25 min. The supernatants were filtered through 0.22 μm and set aside.

[0165] AKTA purification instrument operation: the software sets the flow rate to 8 mL / min and the maximum pressure to 0.3 MPa; first, flush the instrument pipeline thoroughly with B solution (100 mM citric acid monohydrate), reduce the flow rate to 2 mL / min, install the protein A medium-pressure chromatography column, and use 95% A solution (200 mM sodium hydrogen phosphate dodecahydrate) to equilibrate the protein A medium at a flow rate of 8 ml / min until the baseline level is stable. This process takes about 15 minutes; load the sample at a flow rate of 8 mL / min, and the UV value can be seen to rise and remain at a certain level. This peak is the penetration peak; after the loading is completed, it is equilibrated with 95% A solution again, and the peak value drops to the baseline level and stabilizes; elution is performed with 70% B solution. In this process, the peak value can be seen to first climb and then drop to the baseline. The process of elution peak formation is the process of eluting the target protein, and the eluate of this process is collected; 100% B solution is used to flush the pipeline to remove impurities, and then fill the pipeline and protein A column with 20% ethanol. Remove the column and store it at 4°C. The eluted protein was dialyzed against 20 mM PBS at 4°C for 24 h. Antibody concentration was determined using a microplate reader or BCA assay. If the antibody concentration was <0.5 mg / mL, the protein was concentrated using a 10Kd concentrator (Millipore) at 3000 rpm for 10 min. Aliquot the antibody and store at -20°C until use.

[0166] Example 3: Preparation of Antibody-Drug Conjugate A4-VC-MMAE

[0167] The antimitotic agent monomethyl aureusin E (MMAE, a microtubule inhibitor) was used as a toxic payload and linked to the site-directed mutagenesis site of the antibody via a lysosomal-cleavable MC-Val-Cit-PAB (maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl) linker to obtain an antibody-drug conjugate (e.g. Figure 1 The experimental details are as follows:

[0168] (1) Add 0.5 M EDTA to the antibody reaction system to make its working concentration reach 5 mM;

[0169] (2) Reduction: 10 eq of Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; BEYOTIME) was added to the reaction system and incubated at 37°C for 2 h. The disulfide bonds on the engineered antibody and the cysteine-activated sulfhydryl groups at the mutation site were exposed, and the buffer was replaced by ultrafiltration concentrator tubes (10Kd; Millipore);

[0170] (3) Oxidation: Add excess dehydroascorbic acid (DHAA) (50 eq) and incubate at room temperature for 3 h to reoxidize the reduced disulfide bonds and expose only the engineered cysteine ​​sites. Use ultrafiltration concentration tubes for buffer exchange to remove the oxidant DHAA.

[0171] (4) Conjugation: MC-vc-PAB-MMAE (MCE, CAS No.: 646502-53-6) was dissolved in DMSO to a stock concentration of 10 mM. The added DMSO accounted for 10% of the total reaction system. The mixture was vortexed and incubated at 4°C. 6 eq of MC-vc-PAB-MMAE was added to the reaction system of the A4 engineered antibody and incubated at 4°C for 4 h. Excess small molecules, cysteine, and impurities such as DMSO were removed using an ultrafiltration concentrator. This yielded the antibody-drug conjugate A4-VC-MMAE.

[0172] Example 4: SDS-PAGE identification of A4 band size

[0173] Load the precast gel (LabPAGE; 4-12%; 12 Wells; P41212) into the gel rack, place it in the electrophoresis tank, and add electrophoresis solution to cover the gel plate. After the sample is treated with the loading buffer, use a micropipette to add the marker, A4 non-reduced sample, and A4 reduced sample to the gel wells of the precast gel in sequence, with a sample volume of 10 μl. Among them, the protein marker (Thermo Scientific; 26616) is a pre-stained protein of known molecular weight. It does not need to be treated with the loading buffer and is directly added to the gel wells with a sample volume of 5 μl. Add 5 μl of loading buffer to the empty lane to prevent the adjacent lanes from diffusing into the empty lane. The voltage can be selected between 80 and 110 V, and stop until the bromophenol blue front indicator reaches the lower end of the electrophoresis tank. After the electrophoresis is completed, take out the gel plate and stain it with Coomassie Brilliant Blue for more than 10 minutes at room temperature. Then take out the colloid from the staining solution and put it into the decolorizing solution. Decolorize it multiple times until the background is clear and take pictures with a camera in a dark room. Figure 2 As shown: A4 antibody is about 80-90 kDa under non-reducing conditions and about 40-45 kDa under reducing conditions, which is consistent with the theoretical value. The band is clean and single, indicating that A4 antibody is highly pure.

[0174] Example 5: ELISA to detect the binding activity of A4, A4-VC-MMAE and antigen c-Met

[0175] The antigen c-Met (purchased from Sino Biological) was diluted to 1 μg / mL with PBS and plated on a 96-well plate, 100 μL per well, incubated at 4°C overnight, washed 5 times with PBST, blocked overnight with 2% BSA, removed the blocking solution, washed 5 times with PBST, and dried for 24 hours. A4, A4-VC-MMAE, and negative control 125s were diluted to a series of different concentrations and added to the 96-well plate coated with c-Met antigen, 100 μL per well, incubated at 37°C for 1 hour, and washed 5 times with PBST. Then, Goat Anti-Human IgG (HRP) secondary antibody (1:2000 dilution), incubated at 37 ° C for 30 minutes, washed 5 times with PBST, added substrate color development solution A / B solution 1:1 mixture, 100 μL / well, color development for about 10 minutes, showing a blue gradient change, then added 50 μL / well stop solution, the color turned to yellow, and the absorbance value was measured at 450nm / 630nm wavelength. The results showed that A4 and A4-VC-MMAE bound to the antigen c-Met in a concentration-dependent manner, and the conjugation of drugs did not affect the binding activity of the antibody. EC 50 All of them were at the ng / mL level (as shown in Table 4 ), and the negative control 125s did not show a specific binding trend (as shown in Table 4 ). Figure 3 shown).

[0176] Table 4. EC50 values ​​of A4 and A4-VC-MMAE (ELISA)

[0177] 125s A4 A4-VC-MMAE <![CDATA[EC 50 (μg / mL)]]> ～ 0.2166 0.3650 <![CDATA[LogEC 50 (μg / mL)]]> ～ -0.6643 -0.4377

[0178] Example 6: Identification of the Drug-Antibody Coupling Ratio (DAR) of A4-VC-MMAE

[0179] 6.1 UV spectrophotometer identification of the average DAR value of antibody-drug conjugates

[0180] (1) Dilute A4 and A4-VC-MMAE to 0.6 mg / mL, and dilute VC-PABC-MMAE to 80 μM;

[0181] (2) Measure the absorbance of VC-PABC-MMAE, A4, and A4-VC-MMAE at 280 nm and 248 nm three times and take the average value;

[0182] (3) According to the Beer-Lambert law formula: A = Ig(1 / T) = Kbc

[0183] A is the absorbance, T is the transmittance, which is the ratio of the transmitted light intensity to the incident light intensity, c is the concentration of the absorbing substance, and b is the absorption thickness. K is the molar extinction coefficient of the absorbing substance. Calculate the molar extinction coefficient of the absorbing substance at 280nm and 248nm.

[0184] (4) The concentrations of the antibody and small molecule drug (C) were calculated according to the following formula, and the average DAR value of A4-VC-MMAE was finally obtained to be 4.1;

[0185]

[0186]

[0187] The concentrations of the antibody and drug can be obtained respectively through the above two equations.

[0188]

[0189]

[0190] The DAR value calculation results of ADC are as follows:

[0191] DAR=c drug / c mAb .

[0192] 6.2 Average DAR values ​​of antibody-drug conjugates identified by hydrophobic interaction chromatography (HIC)

[0193] (1) A silica-based HPLC column (4.6 × 100 mm, 3.5 μm, Agilent) was used for HIC-UPLC (waters) analysis to determine the drug-antibody ratio (DAR);

[0194] (2) Antibodies and their conjugates were eluted by a 40-min linear gradient from buffer A (1.5 M ammonium sulfate, 50 mM sodium phosphate) to buffer B (80% sodium phosphate, 20% isopropanol), pH = 7.5, 0.5 mL / min, 25°C. Figure 4 As shown: A4-VC-MMAE has a DAR of 4 at pH = 7.5, which is consistent with the theoretical value and UV spectrophotometric value.

[0195] Example 7: Identification of the internalization performance of A4

[0196] 7.1 Flow cytometry detection of A4 internalization ability in BxPC3, HT-29, SCG7901, and SK-OV3 cells

[0197] BxPC3, HT-29, SCG7901, SK-OV3 (2×10 5 / well) and antibody (10 μg / mL) were resuspended in pre-cooled PBS, and three parallel samples were made. The samples were incubated at 4°C for 1 hour, and washed by centrifugation at 1200 rpm for 3 minutes. Two parallel samples were taken and resuspended in 2% FBS1640. The samples were incubated at 37°C for 30 minutes and 3 hours respectively, and washed by centrifugation at 1200 rpm for 3 minutes. Secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight TM 650; Invitrogen) was diluted 1:400, incubated at 4°C for 30 min, and analyzed by analytical flow cytometry (LSRFortessaX-20; BD). Figure 5 As shown: After 3 hours of incubation, the internalization rate of A4 in the four c-Met high-expressing cell lines was about 40% to 60%.

[0198] 7.2 Immunofluorescence detection of A4 internalization ability in HT-29 and BxPC3 cells

[0199] On the first day, colorectal cancer cell line HT-29 and pancreatic cancer cell line BxPC3 were plated at 10,000 cells / well and cultured overnight. On the second day, primary antibody (A4) was incubated for 3 and 1 hour in 2% FBS DMEM at a final concentration of 100 nM / L. Fixation: Wash three times with PBS, add 100 μL of 4% formaldehyde solution, and incubate for 20 minutes. Permeabilization: Wash three times with PBS, add 100 μL of immunofluorescence permeabilization buffer, and incubate for 5 minutes. Blocking: Wash twice with PBS, add 100 μL of 2% BSA, and incubate for 30 minutes. Secondary antibody: Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, diluted 1:2000, incubate at 4°C for 30 minutes. Nuclear staining: Wash three times with PBS, dilute DAPI 1:2000, mix by inversion, add 100 μL of DAPI, and incubate at room temperature for 10 minutes. The samples were loaded and photographed using a high-content cell screening imaging analysis system (Opera Phenix). Figure 6 As shown: In the HT-29 cell line, A4 has a good internalization effect. In the BxPC3 cell line, A4 has a good internalization effect.

[0200] Example 8: In vitro killing effect of A4-VC-MMAE

[0201] 8.1 Cytotoxicity of A4-VC-MMAE against BxPC3 and HT-29 cells (c-Met positive)

[0202] BxPC3 and HT-29 cells in the logarithmic growth phase were centrifuged, resuspended and counted, and 5000 cells / well were inoculated into a 96-well cell culture plate (Bioland) and cultured at 37°C overnight. Then, the ADC was diluted 2-fold (the diluent was 2% 1640 culture medium) with a total of 20 gradients starting at 500nM in the first well. Three parallel curves of ADC were made at 100μL / well. After incubation at 37°C for 72h, 10μL CCK8 reagent (MCE; cat.No:HY-K0301) was added to each well and cultured for 1-2h. The color reaction was observed and the absorbance at 450nm was measured with an enzyme-labeled instrument. During the experiment, a blank group (no cells) and a negative control group (no drug treatment) were set up, and the cell survival rate was calculated according to the following formula:

[0203] Cell killing rate = [(Ac-As) / (Ac-Ab)] × 100%

[0204] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution);

[0205] Ac: absorbance of control well (containing cells, culture medium, and CCK-8 solution, but no drug);

[0206] Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but not cells or drugs).

[0207] IC50 values ​​were calculated using GraphPad Prism software. Figure 7 As shown in Table 5, A4-VC-MMAE has IC50 values ​​of nanomolar level in BxPC3 and HT-29 cells.

[0208] Table 5. IC50 values ​​of A4-VC-MMAE (CCK8)

[0209] <![CDATA[IC 50 (nM / L)]]> <![CDATA[LogIC 50 ]]> BxPC3 0.07622 -1.118 HT-29 0.2444 -0.6119

[0210] 8.2A4-VC-MMAE's cytotoxicity against c-Met-positive cells, c-Met-positive:c-Met-negative cells (1:1), and c-Met-negative cells

[0211] The specific implementation method is the same as 7.1. Figure 8 As shown in Table 6, A4-VC-MMAE achieved a maximum tumor inhibition rate of 95% in c-Met-positive cells and 70% in a 1:1 ratio of c-Met-positive:c-Met-negative cells. There was virtually no significant tumor inhibition in c-Met-negative cells, suggesting a bystander effect. The IC50 values ​​of A4-VC-MMAE were similar in both the c-Met-positive and c-Met-positive:c-Met-negative ratios.

[0212] Table 6. IC50 values ​​of A4-VC-MMAE (CCK8)

[0213] <![CDATA[IC 50 (nM / L)]]> <![CDATA[LogIC 50 ]]> c-Met positive 0.07622 -1.118 c-Met positive: c-Met negative = 1:1 0.08493 -1.071 c-Met negative 336.8 2.527

[0214] Example 9: In vivo targeting of A4 in BxPC3 and HT-29 tumor-bearing mice

[0215] The antibody was coupled with Cyanine5.5 NHS ester (Luminex life science solutions) at a ratio of 1:10, and the mixture was rotated at 4°C overnight. TM Spin Desalting Columns,7K MWCO,0.5mL; Thermo Scientific TM ) was centrifuged to remove free fluorescein. Nude mice (5-6 weeks old, female) were selected, and BxPC3 and HT-29 were cultured at 2×10 6The cells were subcutaneously inoculated, and when the tumor volume reached about 80 mm3, the mice were evenly grouped according to the tumor size. 200 μg of antibody was injected into the tail vein of each mouse, and small animal fluorescence imaging (Caliper IVIS Lumina II) was performed on the 1st, 3rd, 5th and 7th days. Figure 9 As shown, A4 was enriched in the tumor sites of tumor-bearing mice on day 1. Subsequently, due to metabolism in the mouse body, the antibody in other nonspecific organ sites was metabolized, while the antibody in the tumor site remained significantly enriched on day 7. On day 7, the mice were dissected, and the heart, liver, spleen, lungs, kidneys, and tumors were removed. Fluorescence signals in each organ and tumor site were examined, revealing that it was primarily enriched in the liver and tumor sites, cautioning against liver toxicity.

[0216] Example 10: Single-injection tumor inhibition effect of A4-VC-MMAE

[0217] Nude mice (5-6 weeks old, female) were selected and BxPC3 was injected at a rate of 2×10 6 The number of cells was subcutaneously inoculated until the tumor volume reached 100 mm 3 Rats were divided into groups based on tumor size: A4-VC-MMAE 2.5μM, A4-VC-MMAE 5μM, and PBS. Tumor volume and body weight were monitored every 3-4 days after a single tail vein injection.

[0218] Tumor size was measured using calipers, and volume was calculated using the following formula: V = (W 2 × L) / 2, where V = tumor volume, W = smaller vertical diameter, and L = larger vertical diameter. When the tumor size reaches 1500 mm 3 , euthanize the mice. Figure 10 As shown: A4-VC-MMAE can inhibit 100mm at a dose of 2.5μM. 3 The growth of BxPC3 tumors was significantly different from that of the PBS group. At a dose of 5 μM, A4-VC-MMAE had a significant tumor inhibition effect and could completely eliminate 100 mm 3 BxPC3 tumors showed significant differences, without weight loss or other abnormalities.

[0219] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A nanobody or antigen-binding fragment thereof that specifically binds to c-Met, comprising the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences: (a) CDR1 having: a sequence as shown in SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 2; (b) CDR2 having: a sequence as shown in SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 3; and (c) CDR3 having: a sequence as shown in SEQ ID NO: 4, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 4; Preferably, the substitution is a conservative substitution; Preferably, the Nanobody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO: 2, a CDR2 as shown in SEQ ID NO: 3, and a CDR3 as shown in SEQ ID NO: 4; Preferably, the CDR sequences are defined using the Kabat, IMGT, Chothia or Abm numbering systems.

2. The Nanobody or antigen-binding fragment thereof according to claim 1, wherein the Nanobody comprises an amino acid sequence selected from the group consisting of: (i) the sequence shown in SEQ ID NO: 1; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 1; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 1; Preferably, the substitutions are conservative substitutions.

3. A polypeptide construct that specifically binds to c-Met, comprising the Nanobody or antigen-binding fragment thereof according to claim 1 or 2; Preferably, the polypeptide construct comprises an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is connected to the N-terminus or C-terminus of the Nanobody or antigen-binding fragment thereof directly or through a peptide linker; preferably, the immunoglobulin Fc domain is connected to the C-terminus of the Nanobody or antigen-binding fragment thereof directly or through a peptide linker; Preferably, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 5, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto; Preferably, the immunoglobulin Fc domain comprises or does not comprise a mutation at position S239 and / or K290, for example, the mutation comprises S239C and / or K290C; preferably, the immunoglobulin Fc domain is numbered according to the Kabat EU index; Preferably, the polypeptide construct comprises or consists of the amino acid sequence shown in SEQ ID NO: 6 or 7; Preferably, the polypeptide construct is a dimer; Preferably, the polypeptide construct comprises but is not limited to one or more of single domain antibodies, single chain antibodies, antibody Fab, full length antibody protein, antigen binding fragment, bispecific antibody, multispecific antibody, bi / multivalent single domain antibody, bi / multivalent single chain antibody and bi / multivalent antibody Fab.

4. An isolated nucleic acid molecule encoding the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, or the polypeptide construct according to claim 3. An expression vector comprising the nucleic acid molecule according to claim 4 . A host cell comprising the nucleic acid molecule according to claim 4 or the expression vector according to claim 5.

7. An antibody-drug conjugate (ADC), comprising: A targeting moiety selected from the polypeptide construct of claim 3; Cytotoxic drug moiety; and A linker is used to connect the targeting moiety and the cytotoxic drug moiety.

8. The antibody-drug conjugate according to claim 7, wherein the polypeptide construct is linked to the linker via a sulfhydryl group on a cysteine ​​residue; Preferably, the polypeptide construct is linked to the linker via the sulfhydryl group on the cysteine ​​residue in the VHH or Fc domain or the sulfhydryl group exposed by the cysteine ​​residue in the reduced disulfide bond in the hinge region; Preferably, the polypeptide construct is linked to the linker via the sulfhydryl groups of the cysteine ​​residues in the reduced disulfide bond of the VHH, hinge region or the cysteine ​​residues at positions 239 and / or 290 of the Fc domain.

9. The antibody-drug conjugate according to claim 7 or 8, wherein the cytotoxic drug is selected from the group consisting of a microtubule inhibitor and a DNA damaging drug; Preferably, the tubulin inhibitor is selected from auristatins (e.g., MMAE, MMAF), maytansines (e.g., maytansine, maytansinol, DM1, DM4), taxanes (e.g., Taxol, Docetaxel, Cabazitaxel), vinca alkaloids (e.g., vinblastine, vincristine), eribulin and colchicine; Preferably, the DNA damaging agent is selected from DNA alkylating agents (calicheamicin γ1l, N-acetyl-γ1I calicheamicin, anthramycin, PBD, dukamycin), DNA topoisomerase inhibitors (for example, camptothecin compounds (specifically, camptothecin, SN-38, Dxd, irinotecan, belotecan, topotecan, PNU-159682), doxorubicin, daunorubicin, etoposide, mitoxantrone) and amanitin; Preferably, the cytotoxic drug is MMAE.

10. The antibody-drug conjugate according to any one of claims 7 to 9, wherein the linker is a cleavable or non-cleavable linker; Preferably, the cleavable linker is selected from protease-sensitive, pH-sensitive and glutathione-sensitive linkers; Preferably, the linker is selected from MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropionyl), Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Ala-Phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimidyl)-4-(pyridin-2-ylthio) ) valerate), 6-(2,5-dioxopyrrolidin-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidin-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) or SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate), and any combination thereof; Preferably, the linker is MC-Val-Cit-PAB.

11. The antibody-drug conjugate according to any one of claims 7 to 10, wherein each peptide chain of the polypeptide construct is linked to 0, 1, 2, 3, 4 or 5 of the following structures via a cysteine ​​residue in a VHH, a hinge region reduced disulfide bond or a cysteine ​​residue in an Fc domain:

12. The antibody-drug conjugate according to any one of claims 7 to 11, which is selected from: in, x = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Ab is the polypeptide construct according to claim 3.

13. The antibody-drug conjugate according to any one of claims 7 to 12, which is: in, x = 1, 2, 3, 4, 5, or 6; Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:

6.

14. The antibody-drug conjugate according to any one of claims 7 to 12, which is: in, x = 1, 2, 3, 4, 5, 6, 7, or 8; Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:

7.

15. A composition comprising or consisting of one or more antibody-drug conjugates according to any one of claims 7 to 14; Preferably, the DAR value of the composition is 1-10, for example, 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1-8.5, 1-9, 1-9.5, 1-10, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1.5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 1.5-8.5, 1.5-9, 1.5-9.5, 1.5-10, 2-2.5, 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2-8.5, 2-9, 2-9.5, 2-10, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5 -7, 2.5-7.5, 2.5-8, 2.5-8.5, 2.5-9, 2.5-9.5, 2.5-10, 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3-8.5, 3-9, 3-9.5, 3-10, 3.5-4, 3 .5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 3.5-8.5, 3.5-9, 3.5-9.5, 3.5-10, 4-4.5, 4-5, 4-5.5, 4-6, 4.5-5, 4.5-5.5, 4.5-6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 4.5-8.5, 4.5-9, 4.5-9.5, 4.5-10, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5-8.5, 5-9, 5-9.5, 5-10, 5.5-6, 5.5-6.5, 5. 5-7, 5.5-7.5, 5.5-8, 5.5-8.5, 5.5-9, 5.5-9.5, 5.5-10, 6-6.5, 6-7, 6-7.5, 6-8, 6-8.5, 6-9, 6-9.5, 6-10, 6.5-7, 6.5-7.5, 6.5-8, 6.5-8.5, 6.5-9, 6. 5-9.5, 6.5-10, 7-7.5, 7-8, 7-8.5, 7-9, 7-9.5, 7-10, 7.5-8, 7.5-8.5, 7.5-9, 7.5-9.5, 7.5-10, 8-8.5, 8-9, 8-9.5, 8-10, 8.5-9, 8.5-9.5, 8.5-10, 9-9.5、9-10、9.5-10。.

16. A composition comprising or consisting of one or more antibody-drug conjugates according to claim 14; Preferably, the composition has a DAR value of 1-8, for example, 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1.5-6. 5, 1.5-7, 1.5-7.5, 1.5-8, 2-2.5, 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5-7, 2.5-7.5, 2.5-8, 3 -3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3.5-4, 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 4-4.5, 4-5, 4-5.5, 4-6, 4-6.5, 4-7, 4-7.5, 4-8, 4.5-5, 4. 5-5.5, 4.5-6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5.5-6, 5.5-6.5, 5.5-7, 5.5-7.5, 5.5-8, 6-6.5, 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-7.5, 7-8, 7.5-8; Preferably, the composition has a DAR value of 1-5, such as 3.5-4.5, such as 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.

5.

17. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 7 to 14 or the composition according to any one of claims 15 to 16, and an optional carrier or excipient.

18. Use of the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, the polypeptide construct according to claim 3, the antibody-drug conjugate according to any one of claims 7 to 14, the composition according to any one of claims 15 to 16, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for treating cancer; Preferably, the cancer is c-Met positive; Preferably, the cancer is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (eg, triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (eg, epithelial ovarian cancer), and cervical cancer.

19. The Nanobody or antigen-binding fragment thereof according to claim 1 or 2, the polypeptide construct according to claim 3, the antibody-drug conjugate according to any one of claims 7 to 14, the composition according to any one of claims 15 to 16, or the pharmaceutical composition according to claim 17, for use in treating cancer; Preferably, the cancer is c-Met positive; Preferably, the cancer is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (eg, triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (eg, epithelial ovarian cancer), and cervical cancer.

20. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, the polypeptide construct according to claim 3, the antibody-drug conjugate according to any one of claims 7 to 14, the composition according to any one of claims 15 to 16, or the pharmaceutical composition according to claim 17; Preferably, the cancer is c-Met positive; Preferably, the cancer is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (eg, triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (eg, epithelial ovarian cancer), and cervical cancer.

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