Nanobodies targeting c-met, drug conjugates and uses thereof

Abstract

The application provides a nanobody targeting c-MET, a drug conjugate and use thereof. Specifically, the application provides a specific nanobody against c-MET. The application also provides a drug conjugate constructed based on the nanobody. The nanobody of the application has a small molecular weight, can quickly penetrate tumor tissues, has high endocytosis activity, has better toxin delivery capacity, and can be used for treating c-MET related diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. Specifically, it relates to nanobodies targeting c-MET, drug conjugates, and their uses. Background Technology

[0002] Nanobodies (Nb), also known as variable domain of heavy chain antibody (VHH), contain only one heavy chain variable region (VHH) and CH2 and CH3 regions. Compared to other antibodies, the light chain is naturally absent. Nanobody crystals are 2.5 nm in diameter and 4 nm in length, representing the smallest naturally occurring fragments capable of binding to antigens.

[0003] Antibody-drug conjugates (ADCs) are targeted biological agents that conjugate target-specific monoclonal antibodies to highly cytotoxic small-molecule drugs via specific linkers. Using the monoclonal antibody as a carrier, these cytotoxic drugs are efficiently delivered to target tumor cells in a targeted manner. Compared to chemotherapy drugs, ADCs can more precisely identify diseased cells, reduce damage to normal cells, and broaden the therapeutic window. Compared to traditional antibodies or antibody fragments, ADCs enhance therapeutic efficacy due to carrying highly active cytotoxic drugs.

[0004] Currently, treatment options for c-MET-mediated tumors include: 1) small molecule kinase inhibitors, such as crizotinib (Pfizer) and cabozantinib (Exelixis), which are approved for the treatment of non-small cell lung cancer (NSCLC) and medullary thyroid carcinoma, respectively; 2) c-MET antibodies; and 3) c-MET antibody-drug conjugates (ADCs). Traditional small molecule inhibitors suffer from drug resistance issues, significantly shortening patient response times. Currently, no c-MET monoclonal antibodies have been approved for marketing. Continued development of c-MET antibodies and corresponding ADCs for treating tumors is needed to overcome the limitations of existing technologies, such as increasing tumor penetration while improving binding and endocytic abilities, overcoming drug resistance, and reducing adverse reactions.

[0005] Therefore, there is a need in this field to develop an anti-c-MET antibody with high binding and endocytosis capabilities and low adverse reactions. Summary of the Invention

[0006] The purpose of this invention is to provide nanobodies, drug conjugates, and their uses that target c-MET.

[0007] In a first aspect of the invention, an anti-c-MET nanobody is provided, wherein the complementarity-determining region (CDR) of the VHH chain of the nanobody is selected from the group consisting of:

[0008] (1) CDR1 shown in SEQ ID NO:3,

[0009] CDR2 shown in SEQ ID NO:4, and

[0010] CDR3 as shown in SEQ ID NO:5;

[0011] and,

[0012] (2) CDR1 shown in SEQ ID NO:6

[0013] CDR2 shown in SEQ ID NO:7, and

[0014] CDR3 as shown in SEQ ID NO:8.

[0015] In another preferred embodiment, the CDR region of the nanobody VHH chain contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above sequences.

[0016] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence that has optionally been added, deleted, modified, and / or substituted at least one amino acid and is capable of retaining c-MET binding affinity.

[0017] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, more preferably 1-2, and even more preferably 1.

[0018] In another preferred embodiment, the VHH chain of the nanobody further includes a framework region (FR).

[0019] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0020] In another preferred embodiment, the frame region FR is of human, mouse, rabbit, or camel origin.

[0021] In another preferred embodiment, the nanobody is bound to human, mouse, or monkey-derived c-MET.

[0022] In another preferred embodiment, the nanobody is capable of endocytosis in cells expressing the c-MET antigen.

[0023] In another preferred embodiment, the VHH chain of the nanobody has an amino acid sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology with the amino acid sequence shown in SEQ ID NO:1 or 2.

[0024] In another preferred embodiment, the VHH chain of the nanobody has one or more amino acid sequences as shown in SEQ ID NO:1 or 2.

[0025] In another preferred embodiment, the nanobody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0026] In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, or a combination thereof.

[0027] In another preferred embodiment, the nanobody blocks the binding of MET and HGF.

[0028] In another preferred embodiment, the nanobody comprises the Fc segment of an antibody, preferably the Fc segment of IgG, and more preferably the Fc segment of human IgG.

[0029] In another preferred embodiment, the nanobody has an amino acid sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology to the amino acid sequence shown in SEQ ID NO:9 or 10.

[0030] In another preferred embodiment, the nanobody sequence is as shown in SEQ ID NO:9 or 10.

[0031] In a second aspect of the invention, a nanoantibody fusion protein is provided, the nanoantibody fusion protein having a structure from the N-terminus to the C-terminus as shown in Formula I:

[0032] Z1-Z2-L-Z3 (Formula I)

[0033] In the formula,

[0034] Z1 is the VHH chain of the anti-c-MET nanobody as described in the first aspect of the present invention;

[0035] Z2 is the Fc segment of an immunoglobulin;

[0036] L represents the connector sequence;

[0037] Z3 is the immunomodulatory molecular component.

[0038] In another preferred embodiment, the immunomodulatory molecule is an immune-activating molecule, such as a cytokine.

[0039] In a third aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a protein selected from the group consisting of: the anti-c-MET nanobody described in the first aspect of the invention, the nanobody fusion protein described in the second aspect of the invention, or a combination thereof.

[0040] In another preferred embodiment, the polynucleotide includes DNA, RNA, or cDNA.

[0041] In a fourth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide described in the third aspect of the invention.

[0042] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0043] In another preferred embodiment, the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, or retrovirus.

[0044] In a fifth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the fourth aspect of the invention, or having the polynucleotide described in the third aspect of the invention integrated into its genome.

[0045] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0046] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0047] In a sixth aspect of the invention, a method for generating anti-c-MET nanobodies is provided, comprising the steps of:

[0048] (a) Culturing the host cells described in the fifth aspect of the invention under conditions suitable for the production of nanobodies, thereby obtaining a culture containing the said anti-c-MET nanobodies; and

[0049] (b) Isolating or recovering the anti-c-MET nanobody from the culture; and

[0050] (c) Optionally, purify and / or modify the anti-c-MET nanobody obtained in step (b).

[0051] In a seventh aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0052] (a) the anti-c-MET nanobody as described in the first aspect of the present invention; and

[0053] (b) The conjugation portion of the nanobody, wherein the conjugation portion is selected from the group consisting of: detectable markers, drugs, or combinations thereof.

[0054] In another preferred embodiment, the immunoconjugate is an antibody-drug conjugate.

[0055] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0056] In another preferred embodiment, the detectable marker is a chemical marker, a biological marker, or a combination thereof.

[0057] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0058] In another preferred embodiment, the biomarker is biotin, avidin, or an enzyme label.

[0059] In another preferred embodiment, the drug is a small molecule drug, a biological factor, or a combination thereof.

[0060] In another preferred embodiment, the drug is a cytotoxic drug (toxin).

[0061] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0062] In another preferred embodiment, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and microtubule inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0063] In another preferred embodiment, the toxin is selected from the group consisting of: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, methamphetamine, pyrine, pyrine A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxychloroquine. Anthraxone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachomycin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0064] In another preferred embodiment, the toxin is MMAE.

[0065] In another preferred embodiment, the coupling portion is a detectable marker.

[0066] In another preferred embodiment, the detectable marker includes a radionuclide, wherein the radionuclide comprises:

[0067] (i) a diagnostic isotope selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and / or

[0068] (ii) Therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or combinations thereof.

[0069] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, viral capsid proteins (VLPs), liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinoflavin (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0070] In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) anti-c-MET nanobody as described in the first aspect of the invention. The multivalent meaning is that the amino acid sequence of the immunoconjugate contains a plurality of repeating anti-c-MET nanobodies as described in the first aspect of the invention.

[0071] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0072]

[0073] in:

[0074] nAb is an anti-c-MET nanobody as described in the first aspect of the present invention;

[0075] LU represents a chemical bond or linker;

[0076] D is a drug;

[0077] p is the average number of drugs conjugated in the antibody-drug conjugate, and p is a value selected from 1 to 10.

[0078] In another preferred embodiment, p is 2 to 6, preferably 3 to 4, and more preferably 3.8 to 4.0.

[0079] In another preferred embodiment, the LU is a maleimide hexanoyl-valine-citrulline (MC-Val-Cit) linker.

[0080] In an eighth aspect of the invention, a multispecific antibody is provided, the multispecific antibody comprising the anti-c-MET nanobody described in the first aspect of the invention.

[0081] In another preferred embodiment, the multispecific antibody comprises a heavy chain constant region.

[0082] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0083] In a ninth aspect of the present invention, a recombinant protein is provided, said recombinant protein having:

[0084] (i) sequences of nanobodies as described in the first aspect of the present invention, nanobodies fusion proteins as described in the second aspect of the present invention, and multispecific antibodies as described in the eighth aspect of the present invention; and

[0085] (ii) Tag sequences that assist in expression and / or purification.

[0086] In another preferred embodiment, the tag sequence includes a 6His tag and an HA tag.

[0087] In another preferred embodiment, the recombinant protein specifically binds to the c-MET protein.

[0088] In a tenth aspect of the present invention, a pharmaceutical composition comprising:

[0089] (i) the anti-c-MET nanobody as described in the first aspect of the present invention, or the fusion protein as described in the second aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention, or the multispecific antibody as described in the eighth aspect of the present invention, or the recombinant protein as described in the ninth aspect of the present invention, or a combination thereof; and

[0090] (ii) Pharmaceutically acceptable carriers.

[0091] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0092] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating tumors selected from the group consisting of: colon cancer, renal chromophobe carcinoma, renal papillary cell carcinoma, mesothelioma, pancreatic cancer, prostate cancer, ovarian germ cell cancer, thyroid cancer, gastric cancer, esophageal cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), malignant glioma, liver cancer, bladder cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, osteosarcoma, angiosarcoma, or combinations thereof.

[0093] In another preferred embodiment, the cancer is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, stomach cancer, etc.

[0094] In another preferred embodiment, the conjugation portion of the immunoconjugate is a drug, a toxin, and / or a therapeutic isotope.

[0095] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating immune system diseases or tumor diseases.

[0096] In another preferred embodiment, the other drugs for treating immune system diseases or tumor diseases are selected from the group consisting of: budesonide, fluticasone, beclomethasone, mometasone furoate, salbutamol, theophylline, formoterol, tiotropium bromide, sulfasalazine, methotrexate, cyclophosphamide, fluorouracil, bleomycin, anastrozole, or combinations thereof.

[0097] In an eleventh aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: anti-c-MET nanobodies as described in the first aspect of the invention, fusion proteins as described in the second aspect of the invention, immunoconjugates as described in the seventh aspect of the invention, multispecific antibodies as described in the eighth aspect of the invention, recombinant proteins as described in the ninth aspect of the invention, pharmaceutical compositions as described in the tenth aspect of the invention, or combinations thereof, wherein the active ingredient is used for (a) preparing diagnostic reagents, diagnostic plates, or kits; and / or (b) preparing medicaments for the prevention and / or treatment of diseases.

[0098] In another preferred embodiment, the detection reagent, detection plate, or kit is used for:

[0099] (1) Detect c-MET protein in the sample; and / or

[0100] (2) Detect tumor cells expressing c-MET protein.

[0101] In another preferred embodiment, the detection reagent, detection plate, or kit is used to diagnose c-MET-related diseases.

[0102] In another preferred embodiment, the detection includes flow cytometry and cell immunofluorescence detection.

[0103] In another preferred embodiment, the disease is a c-MET-related disease.

[0104] In another preferred embodiment, the disease includes cancer.

[0105] In another preferred embodiment, the cancers include solid tumors and hematologic malignancies.

[0106] In another preferred embodiment, the cancer is selected from the group consisting of: colon cancer, renal chromophobe carcinoma, renal papillary cell carcinoma, mesothelioma, pancreatic cancer, prostate cancer, ovarian germ cell cancer, thyroid cancer, gastric cancer, esophageal cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), malignant glioma, liver cancer, bladder cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, osteosarcoma, angiosarcoma, or combinations thereof.

[0107] In another preferred embodiment, the cancer is selected from the group consisting of: lung cancer, colorectal cancer, pancreatic cancer, breast cancer, kidney cancer, stomach cancer, etc.

[0108] In a twelfth aspect of the present invention, a method for detecting c-MET protein in a sample is provided, the method comprising the steps of:

[0109] (1) Contact the sample with the anti-c-MET nanobody as described in the first aspect of the present invention;

[0110] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of c-MET protein in the sample.

[0111] In a thirteenth aspect of the present invention, a c-MET protein detection reagent is provided, the detection reagent comprising:

[0112] (i) the anti-c-MET nanobody described in the first aspect of the present invention, or the immunoconjugate described in the seventh aspect of the present invention, or the recombinant protein described in the ninth aspect of the present invention; and

[0113] (ii) A detectable carrier.

[0114] In another preferred embodiment, the coupling portion of the immunoconjugate is a diagnostic isotope.

[0115] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0116] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0117] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0118] In another preferred embodiment, the test reagent is in liquid or powder form (such as aqueous solution, injection, lyophilized powder, tablet, lozenge, or inhaler).

[0119] In a fourteenth aspect of the present invention, a detection kit for c-MET protein is provided, the kit containing the immunoconjugate described in a seventh aspect of the present invention or the detection reagent described in a thirteenth aspect of the present invention, and an instruction manual.

[0120] In another preferred embodiment, the specification states that the kit is used for non-invasive detection of c-MET expression in a test subject.

[0121] In a fifteenth aspect of the invention, a method for treating a disease associated with c-MET is provided, the method comprising administering to a desired subject an anti-c-MET nanobody as described in a first aspect of the invention, an immunoconjugate as described in a seventh aspect of the invention, a recombinant protein as described in a ninth aspect of the invention, or a pharmaceutical composition as described in a tenth aspect of the invention.

[0122] In another preferred embodiment, the object includes a human or a non-human mammal.

[0123] In another preferred embodiment, the non-human mammals include rodents (such as mice and rabbits) and non-human primates (such as monkeys).

[0124] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0125] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0126] Figure 1A-1D The antibody binding activity detected by ELISA is shown.

[0127] Figure 2A-2D The binding activity of the antibody to the CHO-K1-MET cell surface antigen was demonstrated by flow cytometry.

[0128] Figure 3 The binding activity of c-MET nanobody to NCI-H1975 cells was demonstrated.

[0129] Figures 4A-4B The results of antibody endocytosis activity assays in CHO-K1-MET and NCI-H1975 cells are shown respectively.

[0130] Figure 5 The results showed that the KY301-09 and KY301-39 antibodies blocked the binding of MET to HGF.

[0131] Figures 6A-6C The binding ability of KY301-09 and KY301-39 antibodies to human, mouse, and monkey c-MET antigens was demonstrated, respectively.

[0132] Figures 7A-7D The results show that the c-MET ADC is primarily composed of DAR4 products.

[0133] Figures 8A-8H The in vitro killing activity of c-MET ADC against a variety of tumor cells was demonstrated.

[0134] Figures 9A-9B The study showed the in vivo antitumor effect of c-MET ADC in a mouse model of xenografted HCT116 colon cancer and the changes in mouse body weight.

[0135] Figure 9C-9D The study demonstrated the in vivo antitumor effect of c-MET ADC in a mouse model of xenografted NCI-H1975 lung adenocarcinoma and the changes in mouse body weight. Detailed Implementation

[0136] Through extensive and in-depth research, the inventors have developed, for the first time, an anti-c-MET nanobody and its drug conjugate. The nanobody of this invention has a small molecular weight, enabling rapid penetration into tumor tissue; it also possesses high endocytic activity, resulting in better toxin delivery capabilities. The drug conjugate of this invention exhibits uniform DAR values ​​and high purity of the product DAR4 molecule. Based on these findings, this invention was completed.

[0137] the term

[0138] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0139] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0140] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0141] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0142] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0143] c-MET

[0144] The c-MET gene is located on human chromosome 7 (7Q21-Q31), comprising 21 exons and 20 introns, encoding a protein of approximately 120 kDa. The translated product is processed into a heterodimer, composed of α and β chains linked by disulfide bonds, and consists of an extracellular domain, a transmembrane helical domain, and an intracellular domain. The extracellular domain contains three distinct functional regions: a SEMA domain, a disulfide-bonded cysteine-rich domain (PSI), and four immunoglobulin regions (IPTs). The main function of the intracellular domain is to initiate autophosphorylation to activate downstream signaling, positively regulating the catalytic activity of tyrosine kinases.

[0145] c-MET is a ligand for hepatocyte growth factor (HGF). Under normal circumstances, the binding of HGF and c-MET mediates embryogenesis, tissue regeneration, wound healing, and the formation of nerves and muscles. However, in cancer cells, abnormal activation of the HGF / c-MET pathway (closely related to c-MET gene mutations, overexpression, and amplification) stimulates numerous downstream signaling pathways such as PI3K / AKT, Ras / MAPK, JAK / STAT, SRC, and Wnt / β-catenin, thereby promoting tumor formation, invasive growth, and metastasis.

[0146] Aberrant activation of c-MET protein can occur through multiple mechanisms, mainly including MET exon 14 skipping mutations, MET amplification, and MET protein overexpression. These pathways are involved in tumor proliferation, migration, and invasion, and are also important factors in the metastasis of tumor cells to distant sites. Aberrant regulation of c-MET protein occurs in various solid tumors (lung cancer, gastric cancer, liver cancer, breast cancer, skin cancer, colorectal cancer, etc.).

[0147] Nanobody

[0148] As used herein, the terms "anti-c-MET nanobody of the present invention" and "anti-c-MET nanobody" are used interchangeably and both refer to the nanobody that specifically recognizes and binds to c-MET (including human MET) according to the first aspect of the present invention, and are particularly preferred to be nanobodies with amino acid sequences of the VHH chain as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0149] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0150] As used herein, the terms "nanobody" and "VHH" have the same meaning, referring to the variable region of the heavy chain of a monoclonal antibody. A nanobody (VHH) is the smallest antigen-binding fragment with complete function. Typically, an antibody lacking both the light chain and the constant region 1 (CH1) of the heavy chain is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0151] As used herein, the term "heavy chain antibody" refers to an antibody containing only the heavy chain. Some antibodies found in the blood of camels are "heavy chain antibodies" lacking the light chain. The heavy chain antibody of this invention comprises a heavy chain variable region (VHH) and heavy chain constant regions CH2 and CH3. The heavy chain antibody of this invention can be an antibody derived from an animal (e.g., camel-derived) that is naturally lacking both the light chain and heavy chain constant region 1 (CH1); or it can be a recombinant antibody obtained by recombination of the nanobody (VHH) of this invention with the heavy chain constant region. The heavy chain antibody of this invention may contain a constant region derived from, for example, IgG1, IgG2, IgG3, or IgG4, preferably derived from the constant region of IgG1.

[0152] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are closely packed together through the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0153] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions (CDR1, CDR2, and CDR3). Various methods exist for CDR partitioning, including the IMGT method, Kabat method, Chothia method, and VBASE2 method. In one embodiment, the IMGT method is used for all CDR partitioning methods mentioned in this invention.

[0154] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0155] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0156] In this invention, the terms "recombinant protein of the present invention," "fusion protein of the present invention," or "peptide of the present invention" are used interchangeably and all refer to peptides that specifically bind to c-MET proteins, such as proteins or peptides having the VHH chain of the nanobody of the present invention. They may or may not contain initiating methionine.

[0157] The heavy chain variable regions of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology to the CDRs identified herein.

[0158] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0159] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0160] The term "antibody of the present invention" refers to a polypeptide having c-MET protein-binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0161] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0162] The present invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the nanobodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0163] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0164] Table A

[0165] Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0166] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0167] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0168] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0169] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0170] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0171] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0172] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0173] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0174] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0175] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0176] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0177] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0178] Immunoconjugates

[0179] The present invention also provides immunoconjugates (ADCs) based on the antibodies of the present invention, such as nanobody-drug conjugates (NDCs).

[0180] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0181] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0182] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0183] Nanobodies can be conjugated with drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0184] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0185] In one embodiment, the ADC of the present invention uses a maleimide hexanoyl-valine-citrulline (MC-Val-Cit) linker.

[0186] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0187] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0188] In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0189] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0190] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0191] In some embodiments, the method of the present invention includes binding an antibody to a adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0192] The ADC prepared from nanobodies provided by this invention has a uniform drug-to-antibody ratio (DAR). In some embodiments, the drug-to-antibody ratio is in the range of 2 to 4, for example 2 to 2.5, 2.5 to 3, 3 to 3.2, 3.2 to 3.4, 3.4 to 3.6, 3.6 to 3.8, or 3.8 to 4.

[0193] Pharmaceutical Composition

[0194] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0195] The pharmaceutical compositions of the present invention can be directly used to bind c-MET protein molecules, and therefore can be used to treat tumors. Furthermore, other therapeutic agents can be used simultaneously.

[0196] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described nanobody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0197] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0198] For antibody-drug conjugates (ADCs), because the nanobody-drug conjugates provided by this invention can target specific cell populations and bind to cell surface-specific proteins (antigens), thereby releasing the drug into the cells in its active form through conjugate endocytosis or drug infiltration, the nanobody-drug conjugates of this invention can be used to treat target diseases. The aforementioned antibody-drug conjugates can be administered to subjects (e.g., humans) in therapeutically effective amounts via appropriate routes. Subjects requiring treatment may be patients at risk or suspected of having a condition related to the activity or expression level of a specific antigen. Such patients can be identified through routine physical examinations.

[0199] When treated with the nanobody-drug conjugate of the present invention, delivery can be performed using methods conventional in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally by direct injection or by using an infusion pump.

[0200] Labeled nanobodies

[0201] In a preferred embodiment of the invention, the nanobody carries a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0202] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, anti-c-MET nanobodies are labeled with colloidal gold to obtain colloidal gold-labeled nanobodies.

[0203] The anti-c-MET nanobody of the present invention has excellent specificity and high potency.

[0204] Detection methods

[0205] The present invention also relates to a method for detecting c-MET protein. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of c-MET protein in the dissolved samples.

[0206] Book

[0207] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0208] Reagent test kit

[0209] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention or a detection plate. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0210] This invention also provides a detection kit for detecting c-MET levels, comprising an antibody that recognizes the c-MET protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0211] application

[0212] As described above, the nanobody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of c-MET-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of c-MET.

[0213] The main advantages of this invention include:

[0214] 1) The nanobody structure of this invention is smaller, with only one heavy chain variable region, and a molecular weight of only 15KD. The molecular weight of the nanobody after fusion with FC is approximately 80KD, which is about half the molecular weight of traditional antibodies (150KD). Therefore, nanobodies can recognize hidden epitopes of antigens and have better binding specificity, tissue penetration, and low immunogenicity. At the same time, nanobodies have higher stability, are easier to modify and optimize, and are suitable for large-scale production.

[0215] 2) More uniform DAR distribution: Traditional antibody molecules have four pairs of disulfide bonds, and the DAR distribution range after toxin conjugation is 0-8. This invention modifies the nanobody by fusing the FC end, resulting in two pairs of disulfide bonds between the two heavy chains. After toxin conjugation by reducing these disulfide bonds, the DAR distribution range is 0-4, which is more uniform. Furthermore, the nanobody of this invention has a higher proportion of DAR4 product, with a DAR4 product purity >90%.

[0216] 3) The high specificity and high endocytic activity of the c-MET nanobody of the present invention are key factors for the efficacy of ADCs. Hengrui Medicine's c-MET antibody has a maximum endocytosis rate of 53% in tumor cells after 2 hours. In the same time period, the endocytosis rate of the c-MET nanobody of the present invention in tumor cells is above 70%.

[0217] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. The MET-BMK2 sequence is derived from Regeneron patent US11,142,578B, and MET-BMK4 is the MET portion monoclonal antibody in the marketed drug Amivantamab-vmjw. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0218] Example 1. Preparation method of anti-c-MET nanobodies

[0219] Alpacas were immunized with c-MET antigen, and total RNA was extracted from their peripheral lymphoid blood. cDNA was obtained by RT-PCR, and the target fragment was amplified using the cDNA as a template. The target fragment was ligated to a phage vector after enzyme digestion, electroporated into TG1 competent cells, and then packaged by helper phage infection to obtain a phage library. Anti-c-MET nanobodies were panned from the phage display library using c-MET protein, and positive monoclonal antibodies were screened by ELISA. After sequencing, they could be expressed, purified, and validated.

[0220] Antibodies used for activity detection are typically expressed by fusion with the human IgG FC region; here, human IgG1 FC is selected.

[0221] Example 2. Antigen-antibody binding assay (ELISA)

[0222] This invention utilizes enzyme-linked immunosorbent assay (ELISA) to detect the binding ability of anti-c-MET nanobodies to c-MET antigens.

[0223] Specific experimental steps:

[0224] 1. Coating antigen (hMET-His): Dilute the antigen to 1 μg / mL with PBS, add 100 μL of diluted antigen per well to the ELISA plate, and incubate overnight at 4°C.

[0225] 2. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times with a plate washer.

[0226] 3. Blocking: Add 300 μL of PBS containing 3% BSA to each well and block at 37°C for 2 h.

[0227] 4. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times with a plate washer.

[0228] 5. Primary antibody incubation: Dilute the antibody with PBS containing 1% BSA to a maximum concentration of 2 μg / mL, then serially dilute it 4-fold to 8 concentrations. Add 100 μL to each well and incubate at room temperature for 1 h.

[0229] 6. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times with a plate washer.

[0230] 7. Incubate the secondary antibody: Add 100 μL of secondary antibody (1:10000 dilution) to each well and incubate at 37℃ for 30 min.

[0231] 8. Washing: Add 300 μL of 0.05% PBST to each well and wash 4 times with a plate washer.

[0232] 9. Add 100 μL of TMB to each well and develop color at 25°C in the dark for 5-10 minutes.

[0233] 10. Termination: Add 50 μL of TMB colorimetric stop solution to each well to terminate the reaction.

[0234] 11. Detection: Read the plate at 450 nm using a TECAN microplate reader.

[0235] 12. Data Processing: GraphPad Prism software was used to fit antibody concentration and OD450 to obtain a four-parameter fitting curve, and EC was calculated. 50 .

[0236] Experimental results are as follows Figure 1A-1D As shown in Table 1, MET-BMK2 is a positive control antibody targeting c-MET and binds strongly to the human c-MET antigen. By comparing the strength of antigen-antibody binding activity, EC was excluded. 50 Antibodies with a concentration ≥0.10 μg / mL, such as KY301-06, KY301-08, KY301-10, KY301-11, KY301-15, KY301-20, KY301-27, KY301-28, KY301-29, KY301-30, KY301-33, and KY301-34. The screened c-MET antibodies all exhibited strong binding activity, such as KY301-02, KY301-04, KY301-05, KY301-07, KY301-09, KY301-12, KY301-13, KY301-14, KY301-16, KY301-17, KY301-18, KY301-19, KY301-21, KY301-22, KY301-23, KY301-24, KY301-25, KY301-26, KY301-31, KY301-32, KY301-36, KY301-37, KY301-39, and KY301-40.

[0237] Table 1. ECG binding between anti-c-MET antibody and MET antigen 50 value

[0238] KY301-02 0.0046 KY301-22 0.0066 KY301-04 0.0035 KY301-23 0.0022 KY301-05 0.0061 KY301-24 0.0073 KY301-06 0.64 KY301-25 0.02 KY301-07 0.023 KY301-26 0.029 KY301-08 0.72 KY301-27 0.19 KY301-09 0.0058 KY301-28 0.68 KY301-10 0.75 KY301-29 0.10 KY301-11 0.35 KY301-30 5.53 KY301-12 0.0041 KY301-31 0.01 KY301-13 0.0065 KY301-32 0.015 KY301-14 0.012 KY301-33 0.10 KY301-15 0.11 KY301-34 5.59 KY301-16 0.0032 KY301-36 0.013 KY301-17 0.0041 KY301-37 0.012 KY301-18 0.0071 KY301-39 0.0085 KY301-19 0.0063 KY301-40 0.0057 KY301-20 0.11 MET-BMK2 0.0067 KY301-21 0.011

[0239] Example 3. Detection of the binding activity of anti-c-MET nanobodies to antigen-overexpressing cells

[0240] The binding activity of c-MET nanobodies, positive controls, and isotype controls to c-MET-overexpressing cells (CHO-K1-MET) was tested.

[0241] Experimental procedures: Prepare test cells in the logarithmic growth phase in advance. Digest the cells with trypsin, centrifuge, collect the cells, resuspend the cells in PBS, and adjust the cell count to 2*102. 6The concentration was set at 100 μL / well of cell suspension and 100 μL / well of c-MET antibody diluent. The maximum antibody concentration was 20 μg / mL. The cells were serially diluted 4-fold with PBS containing 1% BSA to obtain eight concentrations. The cell suspension and antibody diluent were thoroughly mixed and incubated at 4°C in the dark for 1 h. After incubation, the cells were centrifuged at 4°C and 400g for 5 min and the supernatant was discarded. 200 μL of PBS containing 1% BSA was added to each well, and the cells were centrifuged at 4°C and 400g for 5 min, and the supernatant was discarded. This process was repeated twice. PE anti-human IgG Fc Antibody antibody diluent (1:100 dilution) was added to each well, and the cells were mixed by pipetting. The cells were incubated at 4°C in the dark for 30 min. After incubation, the cells were centrifuged at 4°C and 400g for 5 min and the supernatant was discarded. Add 200 μL of PBS containing 1% BSA to each well, centrifuge at 4°C and 400g for 5 minutes, discard the supernatant, and repeat twice. Finally, resuspend the cells in 100 μL of PBS containing 1% BSA to each well, and detect the median fluorescence intensity (Median-PE) of the expressed cells using flow cytometry. Use GraphPadPrism software to fit antibody concentration and fluorescence value to obtain a four-parameter fitting curve.

[0242] Experimental results are as follows Figure 2A-2D As shown: c-MET nanobodies and c-MET overexpressing cells CHO-K1-MET have different degrees of binding activity. Antibodies with relatively weak binding activity were excluded, such as KY301-12, KY301-14, KY301-16, KY301-17, KY301-23, KY301-24, KY301-25, KY301-26, and KY301-32. The screened c-MET antibodies showed strong binding activity to CHO-K1-MET, such as KY301-02, KY301-04, KY301-05, KY301-07, KY301-09, KY301-13, KY301-18, KY301-19, KY301-21, KY301-22, KY301-31, KY301-36, KY301-37, KY301-39, and KY301-40.

[0243] Example 4. Detection of the binding activity of anti-c-MET nanobodies to tumor cells

[0244] The binding activity of c-MET nanobody, positive control, and isotype control to NCI-H1975 tumor cells (human lung adenocarcinoma cells) was tested.

[0245] Experimental procedures: Prepare test cells in the logarithmic growth phase in advance. Digest the cells with trypsin, centrifuge, collect the cells, resuspend the cells in PBS, and adjust the cell count to 2*102. 6 The concentration was 10 μg / mL. 100 μL of cell suspension was added to each well of a 96-well cell culture plate, along with 100 μL of c-MET antibody dilution buffer. The antibody concentration was 10 μg / mL. The cell suspension and antibody dilution buffer were thoroughly mixed and incubated at 4°C in the dark for 1 h. After incubation, the cells were centrifuged at 4°C and 400g for 5 min and the supernatant was discarded. 200 μL of PBS containing 1% BSA was added to each well, and the cells were centrifuged at 4°C and 400g for 5 min, and the supernatant was discarded. This process was repeated twice. PE anti-human IgG Fc Antibody antibody dilution buffer (1:100 dilution) was added to each well, and the cells were mixed by pipetting. The cells were incubated at 4°C in the dark for 30 min. After incubation, the cells were centrifuged at 4°C and 400g for 5 min and the supernatant was discarded. 200 μL of PBS containing 1% BSA was added to each well, and the cells were centrifuged at 4°C and 400g for 5 min, and the supernatant was discarded. This process was repeated twice. Finally, 100 μL of 1% BSA in PBS was added to each well to resuspend the cells, and the median fluorescence intensity (Median-PE) of the expression in the cells was detected by flow cytometry.

[0246] Experimental results are as follows Figure 3 As shown, all c-MET nanobodies exhibited binding activity in NCI-H1975 cells, with stronger binding ability than the positive control antibody. Antibodies KY301-07, KY301-36, and KY301-37 were excluded based on the strength of their binding activity. Antibodies KY301-02, KY301-04, KY301-05, KY301-09, KY301-13, KY301-18, KY301-19, KY301-21, KY301-22, KY301-31, KY301-39, and KY301-40 were retained for further screening and evaluation.

[0247] Example 5. Detection of endocytic activity of anti-c-MET nanobodies in cells

[0248] The endocytic activity of c-MET nanobodies, positive controls, and isotype controls in antigen-overexpressing cells CHO-K1-MET and tumor cells NCI-H1975 was tested.

[0249] Experimental procedures: Prepare test cells in the logarithmic growth phase in advance. Digest the cells with trypsin, centrifuge, collect the cells, resuspend the cells in PBS, and adjust the cell count to 2*102. 6The concentration was set at 100 μL / well. 100 μL of cell suspension and 100 μL / well of c-MET antibody dilution were added to each well of a 96-well cell culture plate. The maximum antibody concentration was 10 μg / mL, diluted with complete culture medium (two identical plates were prepared for each cell type). After thoroughly mixing the cell suspension and antibody dilution, both identical cell plates were treated in the same way and incubated at 4°C in the dark for 1 h. After incubation, the cells were centrifuged at 4°C and 400g for 5 min and the supernatant was discarded. 200 μL of complete culture medium was added to each well, and the cells were centrifuged at 4°C and 400g for 5 min, and the supernatant was discarded. This process was repeated twice. 100 μL / well of complete culture medium was added to all cell plates and the cells were mixed by pipetting. Cells for internalization efficiency were incubated at 37°C for 2 h, and control cells were incubated at 4°C for 2 h. After incubation, all cell plates were centrifuged at 4°C and 400g for 5 min and the supernatant was discarded. Add 200 μL of PBS containing 1% BSA to each well, centrifuge at 4°C and 400g for 5 minutes, discard the supernatant, and repeat twice. Add 100 μL of PE anti-human IgG Fc Antibody antibody dilution buffer (1:100 dilution) to each well, and incubate at 4°C in the dark for 30 minutes. After incubation, centrifuge at 4°C and 400g for 5 minutes and discard the supernatant. Finally, resuspend the cells in 100 μL / well of PBS containing 1% BSA to each cell plate, and detect the median fluorescence intensity (Medium-PE) expressed in the cells using flow cytometry. Calculate: Internalization rate (%) = [1 - (Internalized cells Medium-PE) / (Control cells Medium-PE)] x 100%.

[0250] Experimental results are as follows Figure 4A As shown in the figure. Analysis of the endocytosis results indicates that c-MET nanobodies exhibited varying degrees of endocytosis in CHO-K1-MET cells. Antibodies with endocytosis rates greater than 55%, such as KY301-02, KY301-09, KY301-31, KY301-39, and KY301-40, underwent further endocytosis evaluation in NCI-H1975 cells. The results are as follows... Figure 4B The c-MET nanobodies all exhibited strong endocytosis activity in NCI-H1975 cells, with endocytosis rates exceeding 70%. Based on the endocytosis behavior of the antibodies in these two cell types, KY301-09 and KY301-39, which had relatively high endocytosis rates, were selected for further evaluation. The antibody sequences are shown in SEQ ID NO:9 and SEQ ID NO:10.

[0251] Example 6. In vitro affinity detection of anti-c-MET nanobodies

[0252] In this embodiment, surface plasmon resonance (SPR) technology was used to detect the affinity of the antibody for the c-MET antigen.

[0253] Specific experimental steps:

[0254] 1. Antibody capture: The experiment used 1×PBS (containing 0.05% Tween 20, pH 7.4) buffer as the test buffer. Each antibody was diluted with PBS buffer to 1 μg / mL, and the flow rate was set to 10 μL / min. The antibody was directly captured onto the test surface of the Protein A chip and captured for 60 seconds. The capture amount could reach about 400 RM.

[0255] 2. Sample testing conditions: A total of 8 analytical concentrations were set for the samples, with concentration gradients of 0 nM, 7.81 nM, 15.62 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM. The flow rate for sample analysis was set to 30 μL / min, the binding time to 120 s, and the dissociation time to 1200 s.

[0256] 3. Regeneration conditions: Gly-HCl buffer (pH 1.5) was selected as the regeneration buffer. The flow rate was set to 30 μL / min, and the regeneration time was 30 s. After regeneration, the chip was stabilized for 60 s before analyzing the next sample.

[0257] 4. Determination of Kinetic Parameters: The experiment employed multi-cycle operation, with the analysis time plotted on the x-axis and the response value on the y-axis. The obtained data were fitted using BIAcore T200 analysis software, employing a 1:1 Langmuir binding model to determine the binding rate constant, dissociation rate constant, and binding-dissociation constant, among other kinetic constants. The results are shown in Table 2.

[0258] Table 2. Binding affinity and kinetic constants of anti-c-MET nanobodies to antigens.

[0259]

[0260] Example 7. Ligand (HGF) blocking assay (ELISA) for anti-c-MET nanobodies

[0261] The ability of c-MET nanobodies, positive controls, and isotype controls to competitively bind to hMET antigen with ligand HGF was tested.

[0262] Specific experimental steps:

[0263] 1. Coating antigen: Dilute the antigen to 1 μg / mL with PBS, add 100 μL to each well, and incubate overnight at 4°C.

[0264] 2. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times with a plate washer.

[0265] 3. Blocking: Add 300 μL of PBS containing 3% BSA to each well and block at 37°C for 2 h.

[0266] 4. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times with a plate washer.

[0267] 5. Primary antibody and HGF co-incubation: The antibody and HA-tagged HGF were diluted with PBS containing 1% BSA to a maximum antibody concentration of 2 μg / mL. The antibody was then serially diluted 4-fold to obtain 8 concentrations, with 50 μL added to each well. 100 μg / mL of HGF-HA was then added to each well, and the mixture was incubated at room temperature for 1 hour.

[0268] 6. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times with a plate washer.

[0269] 7. Incubate the secondary antibody: Add 100 μL of anti-HA secondary antibody dilution solution (1:10000) to each well and incubate at 37°C for 30 min.

[0270] 8. Washing: Add 300 μL of 0.05% PBST to each well and wash 4 times with a plate washer.

[0271] 9. Add 100 μL of TMB to each well and develop color at 25°C in the dark for 5-10 minutes.

[0272] 10. Termination: Add 50 μL of TMB colorimetric stop solution to each well to terminate the reaction.

[0273] 11. Detection: Read the plate at 450 nm using a TECAN microplate reader.

[0274] 12. Data processing: The antibody concentration and OD450 were fitted using GraphPad Prism software to obtain a four-parameter fitting curve.

[0275] Experimental results are as follows Figure 5 As shown in the figure. Compared with the isotype control group, KY301-09 and KY301-39 both had strong HGF blocking effects, and their blocking ability was stronger than that of the positive control MET-BMK2.

[0276] Example 8. Cross-species detection assay (ELISA) of anti-c-MET nanobodies with human, mouse, and monkey antigens.

[0277] The binding ability of c-MET nanobodies, positive controls, and isotype controls to human, mouse, and monkey c-MET antigens was tested to determine the cross-species situation of antigens.

[0278] Specific experimental steps:

[0279] 1. Coating antigen: Dilute human, mouse and monkey c-MET antigens to 1 μg / mL with PBS, and add 100 μL of each antigen to the corresponding 96-well plate. Incubate overnight at 4°C.

[0280] 2. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times with a plate washer.

[0281] 3. Blocking: Add 300 μL of PBS containing 3% BSA to each well and block at 37°C for 2 h.

[0282] 4. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times with a plate washer.

[0283] 5. Primary antibody incubation: Dilute the antibody with PBS containing 1% BSA to a maximum concentration of 2 μg / mL, then serially dilute it 4-fold to obtain 8 concentrations, adding 50 μL to each well. Add 100 μg / mL of HGF-HA to each well and incubate at room temperature for 1 hour.

[0284] 6. Washing: Add 300 μL of 0.05% PBST to each well and wash 3 times with a plate washer.

[0285] 7. Incubate the secondary antibody: Add 100 μL of secondary antibody (1:10000 dilution) to each well and incubate at 37℃ for 30 min.

[0286] 8. Washing: Add 300 μL of 0.05% PBST to each well and wash 4 times with a plate washer.

[0287] 9. Add 100 μL of TMB to each well and develop color at 25°C in the dark for 5-10 minutes.

[0288] 10. Termination: Add 50 μL of TMB colorimetric stop solution to each well to terminate the reaction.

[0289] 11. Detection: Read the plate at 450 nm using a TECAN microplate reader.

[0290] 12. Data processing: The antibody concentration and OD450 were fitted using GraphPad Prism software to obtain a four-parameter fitting curve.

[0291] Experimental results are as follows Figures 6A-6C As shown in Table 3, KY301-09, KY301-39, and the positive control antibody MET-BMK2 have strong binding ability to human and monkey c-MET antigens. At the same time, KY301-09 also has a strong binding ability to mouse c-MET antigen.

[0292] Table 3 shows the ECG binding of c-MET antibodies to human, mouse, and monkey-derived c-MET antigens. 50 value

[0293]

[0294] Example 9. Anti-c-MET nanobody-conjugated toxin Vc-MMAE

[0295] The process of anti-c-MET nanobody-conjugated toxin Vc-MMAE involves three main steps: reduction, conjugation, and purification.

[0296] (1) Reagent preparation

[0297]

[0298] (2) Reaction process

[0299] A. Restoration

[0300] With the reduction system and antibody mass fixed, the dosage of TCEP reducing agent was 8 eq, i.e., 8 times the amount of antibody. The final concentration of EDTA-2Na in the reduction system was 5 mM. The dosage volumes of each component were calculated based on the reaction system, and the final volume was made up with His-HAc buffer. The mixed solution was then reduced in a 37°C metal bath for 4 hours at a rotation speed of 200 rpm.

[0301] B. Coupling

[0302] The feed equivalent of VcMMAE was fixed at 9 eq. The feed volume was calculated based on the concentration of VcMMAE stock solution. DMSO solvent was added to make the final concentration in the system 10%. After thorough mixing, the reduced solution was slowly added and reacted in a metal bath at 4℃ for 2 hours at a speed of 200 rpm.

[0303] C. Termination

[0304] The reaction was terminated with L-Cysteine ​​solution. The L-Cysteine ​​feed equivalent was 7 eq. The feed volume was calculated based on the stock solution concentration.

[0305] D. Purification

[0306] The resulting coupled solution was centrifuged at 10000g for 5 min, the precipitate was discarded, and this process was repeated 2-3 times. The product was then transferred to 10kDa 14mL ultrafiltration tubes (for example, if the total volume of the above product is 40mL, it should be divided into four 10kDa 14mL ultrafiltration tubes). The sample was concentrated by centrifugation at 3500g / min for 15 min. His-HAc (30mM, pH 5.5) containing 10% DMSO was added to 14mL, and the solution was centrifuged at 3500g / min for 15 min, for a total of 4 centrifugations. Then, His-HAc (30mM, pH 5.5) was added to 14mL, and the solution was centrifuged at 3500g / min for 15 min, for a total of 16 centrifugations. The liquid in the ultrafiltration tube was collected and rinsed with 1mL of His-HAc (30mM, pH 5.5). This rinsed liquid was then combined with the previously collected liquid, mixed thoroughly, and used for subsequent analysis.

[0307] E. ADC concentration and DAR value were detected, and the recovery rate was calculated.

[0308] The test results are shown in Table 4.

[0309] Table 4. DAR values ​​and product content of anti-c-MET nanobody-conjugated MMAE products.

[0310] KY301-09 100％ ND ND N / A KY301-09-MMAE 1.779％ 6.371％ 91.850％ 3.80 KY301-39 100％ ND ND N / A KY301-39-MMAE 1.454％ 4.607％ 93.939％ 3.85

[0311] DAR distribution map (see...) Figures 7A-7D .

[0312] Example 10. In vitro cell activity evaluation of anti-c-MET nanobody-drug conjugate (c-MET ADC)

[0313] The in vitro killing ability of c-MET ADC was tested in various tumor cell types, including NCI-H1993 (human non-small cell lung cancer cells), HT29 (human colon cancer cells), MDA-MB-231 (human breast cancer cells), 786-O, NCI-H1975, HCT116, MDA-MB-468 (human breast cancer cells), and BXPC-3 (human orthotopic pancreatic adenocarcinoma cells). Cell viability was assessed using the CellTiter-Glo2.0 Cell Viability Assay.

[0314] Specific experimental steps:

[0315] 1. Preparation before the experiment: Prepare the test cells in the logarithmic growth phase in advance.

[0316] 2. Cell Plating (one day in advance): Digest and collect the cells to be tested, and prepare a cell suspension (20,000 / mL) using complete culture medium (RPMI 1640 + 10% FBS + 1% P / S). Add 100 μL of the cell suspension to each well of a 96-well black transparent plate. Discard the wells at the edge of the plate and add 100 μL of PBS to each well. Place the plated cell plate in an incubator overnight to allow the cells to adhere.

[0317] 3. ADC preparation: Dilute the ADC with complete culture medium. Use 120 μg / mL for the first well, and then perform serial dilutions of 4-fold to obtain a total of 6 concentrations.

[0318] 4. Incubation of ADC: Add 20 μL of ADC dilution buffer to each well of a 96-well cell culture plate and incubate at 37°C and 5% CO2 for 6 days.

[0319] 5. Detection: After incubation, add 100 μL / well of CellTiter-Glo2.0Reagent detection solution to the 96-well cell culture plate, let stand for 15 min, and wait for the cells to completely lyse.

[0320] 6. Plate reading: The TECAN microplate reader detects the luminescence value of the test sample, constructs a curve based on the luminescence value, and calculates the IC50. 50 .

[0321] Experimental results are as follows Figures 8A-8H As shown in Table 5, c-MET ADC exhibits strong in vitro killing activity in various tumor cell types, and this activity is dose-dependent. These results indicate that c-MET ADC possesses broad-spectrum tumor-killing capabilities and remains effective even at low concentrations.

[0322] Table 5. IC50 of c-MET ADC in different tumor cells 50 value

[0323]

[0324] Example 11. Pharmacodynamic evaluation of anti-c-MET nanobody-conjugated drug in NCI-H1975 and HCT116 immunodeficient mouse xenograft models

[0325] 1. Detection of the antitumor efficacy of c-MET ADC in NCG mouse xenograft HCT116 colon cancer tumor model

[0326] Human colon cancer cells HCT116 expressing c-MET were inoculated into female NCG mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., 5-6 weeks old, 18-21g, divided into three groups: a PBS group, a low-dose group (2mg / kg) of KY301-09-MMAE, and a high-dose group (6mg / kg) of KY301-39-MMAE, with 5 mice in each group). The tumors were inoculated subcutaneously in the right anterior flank, and the tumors had grown to 100-150mm. 3 Mice were divided into left and right groups and administered either ADC or PBS via tail vein injection once a week for two weeks. Tumor volume and body weight were measured twice a week, and the relationship between changes in body weight and tumor volume and administration time was recorded.

[0327] Tumor volume (V) is calculated as (length × width) 2 ) / 2.

[0328] The tumor growth inhibition rate (TGI%) is calculated using the following formula:

[0329] Tumor growth inhibition rate = (1 - change in tumor volume in the drug-treated group / change in tumor volume in the control group) × 100%

[0330] The test results are as follows Figure 9A As shown, on Day 16, at a dose of 2 mg / kg, tumor growth was significantly inhibited, with KY301-09-MMAE and KY301-39-MMAE showing tumor inhibition rates of approximately 40%. At higher doses (6 mg / kg), the inhibitory effect of ADC on tumor volume was further significantly enhanced, with inhibition rates exceeding 95%. The overall tumor inhibition rate is shown in Table 6. These results indicate that c-MET ADC can effectively inhibit tumor growth in a dose-dependent manner. Mouse body weight changes are shown below. Figure 9B As shown, compared with the PBS control group, the mice in the high-dose experimental group did not show a decreasing trend in body weight; their body weight remained stable and slightly increased.

[0331] Table 6. Tumor inhibition rate of c-MET ADC in HCT116 xenografted NCG mice

[0332]

[0333] 2. Detection of the antitumor efficacy of c-MET ADC in NCG mouse xenografted NCI-H1975 lung adenocarcinoma tumor model

[0334] Fifteen female NCG mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., 5-6 weeks old, 18-21g) were subcutaneously inoculated in the right anterior flank with human lung adenocarcinoma cells NCI-H1975 expressing c-MET. The tumors were allowed to grow to 100-150 mm. 3At approximately 24:00 AM, mice were randomly divided into three groups of five mice each. The low-dose (2 mg / kg) and high-dose (6 mg / kg) groups of PBS, KY301-09-MMAE, and KY301-39-MMAE were administered via tail vein injection once weekly for two weeks. Tumor volume and body weight were measured twice weekly, and the relationship between changes in body weight and tumor volume and the administration time was recorded. The experimental results are as follows: Figure 9C As shown, at a low dose of 2 mg / kg, both c-MET ADCs exhibited tumor growth inhibition rates exceeding 96%, and the overall tumor inhibition rates are shown in Table 7. These results demonstrate that low-dose c-MET ADCs can significantly inhibit tumor growth. Mouse body weight changes are shown in... Figure 9D As shown, compared with the PBS control group, the body weight of mice in the high-dose experimental group tended to be stable.

[0335] Table 7. Tumor inhibition rate of c-MET ADC in NCI-H1975 xenografted NCG mice.

[0336]

[0337] Table 8. Amino acid sequences of the variable region of nanobodies

[0338]

[0339] Table 9. CDR sequences of nanobodies

[0340]

[0341] Table 10. Nanobody-FC Fusion Sequence

[0342]

[0343] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-c-MET nanobody, characterized in that, The complementarity-determining region (CDR) of the VHH chain of the nanobody is selected from the following group: (1) CDR1 shown in SEQ ID NO: 3, CDR2 shown in SEQ ID NO: 4, and CDR3 as shown in SEQ ID NO: 5; and, (2) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO:

8.

2. The nanobody as described in claim 1, characterized in that, The amino acid sequence of the VHH chain of the anti-c-MET nanobody is shown in SEQ ID NO: 1 or 2.

3. The nanobody as described in claim 1, characterized in that, The nanobody further comprises an Fc segment of an antibody, and the sequence of the nanobody is shown in SEQ ID NO: 9 or 10.

4. A nanobody fusion protein, characterized in that, The nanoantibody fusion protein has the structure shown in Formula I from the N-terminus to the C-terminus: Z1-Z2-L-Z3 (Formula I) In the formula, Z1 is the VHH chain of the anti-c-MET nanobody as described in claim 1; Z2 is the Fc segment of an immunoglobulin; L represents the connector sequence; Z3 is the immunomodulatory molecular component.

5. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of the anti-c-MET nanobody of claim 1.

6. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell contains the expression vector of claim 6, or the polynucleotide of claim 5 is integrated into its genome.

8. A method for generating anti-c-MET nanobodies, characterized in that, Including the following steps: (a) Culturing the host cells of claim 7 under conditions suitable for the production of nanobodies, thereby obtaining a culture containing the anti-c-MET nanobodies; and (b) Isolating or recovering the anti-c-MET nanobody from the culture; and (c) Optionally, purify and / or modify the anti-c-MET nanobody obtained in step (b).

9. An immunoconjugate, characterized in that, This immunoconjugate contains: (a) The anti-c-MET nanobody as described in claim 1; and (b) The conjugation portion of the nanobody, wherein the conjugation portion is selected from the group consisting of: detectable markers, drugs, or combinations thereof.

10. The immunoconjugate as described in claim 9, characterized in that, The drug in question is MMAE.

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