WO2023078391A1 - 结合c-Met的抗体及其应用 - Google Patents

结合c-Met的抗体及其应用 Download PDF

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WO2023078391A1
WO2023078391A1 PCT/CN2022/129875 CN2022129875W WO2023078391A1 WO 2023078391 A1 WO2023078391 A1 WO 2023078391A1 CN 2022129875 W CN2022129875 W CN 2022129875W WO 2023078391 A1 WO2023078391 A1 WO 2023078391A1
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cancer
seq
amino acid
acid sequence
antigen
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PCT/CN2022/129875
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English (en)
French (fr)
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甄子朋
杜秀贞
张正平
赵伟
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正大天晴药业集团股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the disclosure relates to the field of molecular biology technology, in particular to an antibody binding to c-Met or an antigen-binding fragment thereof and applications thereof.
  • c-Mesenchymal-epithelial transition factor is a kind of receptor tyrosine kinase, which is composed of a 50kDa extracellular chain ( ⁇ chain) and a 145kDa transmembrane chain ( ⁇ chain). ) is a heterodimer of about 190 kDa.
  • the transmembrane chain includes SEMA domain (sema homology region, SEMA), PSI domain (plexin semaphorin-integrin, PSI), four immunoglobulin-like repeat domains (immunoglobulin-like regions in plexins and transcription factors , IPT), a transmembrane domain, a juxtamembrane domain (JM), a tyrosine kinase domain (TK) and a carboxy-terminal tail region (Carboxyl terminal, CT).
  • SEMA domain semaphorin-integrin
  • PSI plexin semaphorin-integrin
  • IPT immunoglobulin-like repeat domains
  • JM juxtamembrane domain
  • TK tyrosine kinase domain
  • CT Carboxyl terminal
  • c-Met is a receptor expressed on the cell surface, in which the SEMA domain is one of the important elements for ligand binding, and is considered to be the binding site for its ligand hepatocyte growth factor (HGF), which is composed of Mesenchymal cells, fibroblasts and smooth muscle cells synthesize and activate HGF/c-Met signaling through a paracrine mechanism to exert its biological functions.
  • HGF hepatocyte growth factor
  • HGF/c-Met signaling pathway is abnormally activated, which promotes the growth of tumor cells , invasion, migration and angiogenesis.
  • Abnormal expression of c-Met gene exists in many cancer types, such as brain cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, lung cancer, liver cancer and so on.
  • Immunotherapeutics such as antibodies that bind to c-Met can block the binding between HGF and c-Met. As a potential therapeutic target, some antibodies targeting c-Met have been developed, but they are still limited and more available options are needed.
  • IgG antibodies in camelids in addition to IgG1 with a traditional four-chain structure, also naturally exist heavy chain-only antibodies (HcAb) IgG2 and IgG3 that do not contain light chains. Only a single variable region domain ( VHH ) in the heavy chain antibody can specifically bind to the antigen, and has a high affinity for the antigen.
  • HcAb heavy chain-only antibodies
  • VHH variable region domain
  • VHH domains as part of antibodies or antigen-binding fragments has more significant advantages than conventional antibody fragments (such as scFv, Fab, etc.), such as only requiring a single domain to specifically bind antigens with high affinity, Easy to transform into multivalent and multispecific forms, VHH domains are highly soluble and have no tendency to aggregate, VHH molecules are small and have high tissue permeability, VHH does not need to be paired with light chains, in the composition There is no light and heavy chain mismatch problem with bispecific antibodies or multispecific antibodies, and so on.
  • conventional antibody fragments such as scFv, Fab, etc.
  • the present disclosure provides single variable domains, antibodies or antigen-binding fragments thereof, or fusion proteins that bind c-Met.
  • the present disclosure also provides nucleic acids, vectors, cells, preparation methods, compositions and uses encoding the provided single variable domains, antibodies or antigen-binding fragments thereof, or fusion proteins.
  • the disclosure provides an isolated single variable domain that binds c-Met, said single variable domain comprising CDR1, CDR1, CDR2 and CDR3.
  • the disclosure provides an isolated single variable domain that binds c-Met, said single variable domain comprising the following complementarity determining regions: (a) CDR1 comprising SEQ ID NO:5, 8, 11, The amino acid sequence shown in 14, 17 or 20; (b) CDR2, it comprises the amino acid sequence shown in SEQ ID NO:6, 9, 12, 15, 18 or 21; And (c) CDR3, it comprises SEQ ID NO : the amino acid sequence shown in 7, 10, 13, 16, 19 or 22.
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds c-Met, said antibody or antigen-binding fragment comprising a single variable domain, wherein said single variable domain comprises SEQ ID NO: 24 CDR1, CDR2 and CDR3 of the amino acid sequence shown in , 26, 28, 30, 32 or 34.
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds c-Met, said antibody or antigen-binding fragment comprising a single variable domain, wherein said single variable domain comprises the following complementarity determining regions: (a) CDR1, it comprises the aminoacid sequence shown in SEQ ID NO:5,8,11,14,17 or 20; (b) CDR2, it comprises SEQ ID NO:6,9,12,15,18 or 21 the amino acid sequence shown; and (c) CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 7, 10, 13, 16, 19 or 22.
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds c-Met, said antibody or antigen-binding fragment comprising a single variable domain, wherein said single variable domain is identical to a single variable domain of the disclosure. Either of the variable domains binds the same epitope.
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds c-Met, said antibody or antigen-binding fragment comprising a single variable domain, wherein said single variable domain is identical to a single variable domain of the disclosure. Either of the variable domains competes for c-Met binding.
  • the single variable domain is a VHH , preferably a camelid VHH or a humanized VHH .
  • the antibody or antigen-binding fragment thereof is chimeric or humanized.
  • the single variable domain is camelid or humanized.
  • the antibody or antigen-binding fragment thereof comprises an IgG Fc. In some embodiments, the antibody or antigen-binding fragment thereof comprises the Fc of a human IgG, preferably the Fc of a human IgGl, IgG2, IgG3 or IgG4.
  • the disclosure provides fusion proteins comprising an antibody of the disclosure, or an antigen-binding fragment thereof.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody of the present disclosure, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.
  • the disclosure provides an isolated nucleic acid encoding an antibody of the disclosure or an antigen-binding fragment thereof.
  • the disclosure provides a vector comprising an isolated nucleic acid of the disclosure.
  • the disclosure provides a host cell comprising an isolated nucleic acid or vector of the disclosure.
  • the present disclosure provides a method for preparing the disclosed antibody or antigen-binding fragment thereof, comprising: culturing the host cell of the present disclosure, and isolating the expressed antibody or antigen-binding fragment thereof, wherein the vector is an expression vector.
  • the present disclosure provides a use of an antibody of the present disclosure, or an antigen-binding fragment thereof, in the manufacture of a medicament for treating a subject suffering from a tumor.
  • the present disclosure provides the use of the fusion protein of the present disclosure in the manufacture of a medicament for treating a subject suffering from a tumor.
  • the present disclosure provides a use of a pharmaceutical composition of the present disclosure for the manufacture of a medicament for treating a subject suffering from a tumor.
  • the disclosure provides methods of detecting or measuring c-Met in a sample comprising contacting the sample with an antibody of the disclosure or an antigen-binding fragment thereof and detecting or measuring the bound complex.
  • the present disclosure provides a method of inhibiting, reducing or blocking c-Met signaling in a cell, comprising administering to the cell an effective amount of an antibody or antigen-binding fragment thereof, fusion protein, or drug of the present disclosure combination.
  • the present disclosure provides a method for inhibiting the growth of tumor cells or killing tumor cells, comprising administering to the tumor cells an effective amount of the disclosed antibody or its antigen-binding fragment, fusion protein, or pharmaceutical composition.
  • the disclosure provides a method of treating a subject with a tumor comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, fusion protein, or pharmaceutical composition of the disclosure.
  • Fig. 1A is the binding curve of anti-human c-Met V H H-Fc chimeric antibody and H1993 cells detected by flow cytometry;
  • Fig. 1B is the binding curve of anti-human c-Met V H H-Fc chimeric antibody and MKN45 cells detected by flow cytometry;
  • Fig. 1C is the binding curve of anti-human c-Met V H H-Fc chimeric antibody and KP4 cells detected by flow cytometry;
  • Fig. 1D is the binding curve of anti-human c-Met V H H-Fc chimeric antibody and 4MBr-5 cells detected by flow cytometry;
  • Figure 2A is the result of detecting the blocking effect of anti-human c-Met V H H H-Fc chimeric antibody on the combination of HGF and c-Met by competition ELISA;
  • Fig. 2B is the result of detecting the blocking effect of anti-human c-Met V H H H-Fc chimeric antibody on the combination of HGF ⁇ and c-Met by competition ELISA;
  • Figure 3 is the detection result of the inhibitory effect of anti-human c-Met V H H H-Fc chimeric antibody on HGF-stimulated c-Met phosphorylation in H441 cells;
  • Fig. 4 is the result of the inhibitory effect of anti-human c-Met V H H H-Fc chimeric antibody on KP4 cell proliferation detected by CCK8 method;
  • antibody is used in the broadest sense encompassing a variety of antibody structures including, but not limited to, monoclonal, polyclonal, and multispecific antibodies (e.g., bispecific, trispecific) so long as they demonstrate the desired antigen-binding activity.
  • Antigen-binding fragment refers to a fragment that retains the antigen-binding function of a full-length antibody, including Fab, Fab', F(ab') 2 , scFv, Fv, Fd, Fd', single variable domain (e.g., VHH ) and other antibody fragments known in the art, or fragments of any modification known in the art to the above fragments.
  • variable domain refers to the domain of an antibody that is involved in the binding of the antibody to an antigen.
  • Each variable domain of a native antibody consists essentially of four framework regions and three complementarity determining regions.
  • the four "framework regions” are referred to in the art and herein as “framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3” or “FR3”, and “framework region Region 4" or “FR4"; said framework regions are referred to in the art and herein as “complementarity determining region 1" or “CDR1”, “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3” or The three complementarity determining region CDRs of "CDR3" are spaced apart.
  • variable domains confer specificity to an antibody for an antigen by having an antigen-binding site.
  • single variable domain refers to a variable domain having the ability to specifically bind an antigenic epitope without pairing with another variable domain.
  • the antigen binding site of a single variable domain is usually formed by three CDRs (CDR1, CDR2 and CDR3), present on a single domain.
  • the single variable domain may be a heavy chain variable domain (e.g., VH ) or a suitable fragment thereof; so long as it is capable of forming a single antigen-binding unit (i.e., a functional domain consisting essentially of the single variable domain).
  • antigen-binding unit such that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).
  • Another example of a single variable domain is the " VHH domain” of Camelidae, also known as a VHH domain, VHH or VHH.
  • VH domain is used to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (which are referred to herein as “ VH domains” or “ VH” ) and The light chain variable domains (which are referred to herein as “ VL domains” or “ VL ”) present in conventional four-chain antibodies are distinguished.
  • the VHH domains specifically bind epitopes without the need for other antigens binding domain (this is different from the VH or VL domain in conventional four-chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain).
  • the VHH domain is composed of a single structure A small, stable and efficient antigen recognition unit formed by domains.
  • framework region (FR) or "framework region” residues are those amino acid residues of a variable domain other than the CDR residues defined herein.
  • CDRs Complementarity Determining Regions
  • HVRs Hypervariable Regions
  • Native four-chain antibodies typically contain six CDRs, three in the heavy chain variable region (heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3) and three in the light chain variable region (light chain CDR1, light chain CDR2 and light chain CDR3).
  • Heavy chain-only antibodies or single variable domains typically have three CDRs (CDR1, CDR2 and CDR3).
  • CDR3 is the most diverse of the three CDRs and is believed to play a unique role in conferring fine specificity to antibodies.
  • the basis for the "contact" definition of the CDRs is the analysis of available complex crystal structures.
  • the boundaries of the CDRs of the same antibody variable region obtained based on different methods may be different, that is, the CDR sequences of the same antibody variable region defined by different methods may be different. Therefore, when it comes to defining an antibody with a specific CDR sequence defined by certain divisions in the present disclosure, the scope of the antibody also covers conversion to any other definition (for example, a combination of one or more of the definitions of IMGT, Chothia, AbM, etc. ) CDR sequence defined antibody.
  • human consensus framework region or "acceptor human framework” is a framework representing the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework region sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroups of sequences are those in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include: for V L , the subgroups may be subgroups ⁇ I, ⁇ II, ⁇ III or ⁇ IV as described by Kabat et al., supra.
  • the subgroup may be subgroup I, subgroup II or subgroup III as described by Kabat et al.
  • the human consensus framework can be derived from the specific residues above, for example, by aligning the donor framework sequence to a collection of various human framework sequences, when the human framework residues are compared to the donor framework
  • An acceptor human framework "derived from" a human consensus framework region may comprise its identical amino acid sequence when selecting human framework residues based on their homology, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less.
  • Fc domain or “Fc” is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least part of the constant region.
  • the term includes native sequence Fc and variant Fc.
  • the C-terminal lysine of Fc (Lys447 according to the EU numbering system) may or may not be present.
  • EC50 refers to the effective concentration, 50% of the maximal response of a certain compound molecule (eg, antibody or antigen-binding fragment thereof).
  • IC50 refers to the inhibitory concentration, the 50% maximal response of a certain compound molecule (eg, antibody or antigen-binding fragment thereof). Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.
  • K a or "association rate constant” refers to the association rate constant for a molecule of a compound (eg, an antibody or antigen-binding fragment thereof) that binds to an antigen to form a complex.
  • Kd or “dissociation rate constant” refers to the dissociation rate constant for a molecule of a compound (eg, an antibody or antigen-binding fragment thereof) to dissociate from a complex.
  • KD or “equilibrium dissociation constant” refers to a titration measurement at equilibrium, or the value obtained by dividing the dissociation rate constant ( Kd ) by the association rate constant ( Ka ). The binding affinities between a compound molecule and an antigen are expressed using Ka , Kd and KD . Methods for determining the values of Ka , Kd and KD are well known in the art.
  • pharmaceutically acceptable refers to substances, such as carriers or diluents, which do not abrogate the biological activity or properties of the compounds described herein. Such a substance is administered to an individual without causing an undesired biological effect or interacting in a deleterious manner with any component of the composition in which it is contained.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, Salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc. and combinations thereof are well known to those skilled in the art (Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329). Except for carriers incompatible with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.
  • treatment refers to an attempt to alter the natural course of disease in an individual, and may be clinical intervention for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, amelioration or palliation of the disease state, and regression or improved prognosis.
  • terapéuticaally effective amount refers to the amount of a compound, pharmaceutical composition or combination of drugs necessary to provide a therapeutic benefit to a subject.
  • subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, ovines, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.
  • the subject according to the present disclosure is a human.
  • the terms “patient” or “subject” are used interchangeably.
  • telomere binding means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • a compound molecule e.g., an antibody or antigen-binding fragment thereof
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • isolated refers to a compound of interest, such as an antibody or antigen-binding fragment thereof, VHH , or nucleic acid, that has been separated from its natural environment.
  • sequence identity also called identity.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as illustrated in the following non-limiting examples.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl.
  • “about” means within a range of acceptable error for a particular value, as judged by one of ordinary skill in the art, which depends in part on how the value is measured or determined, ie, the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, about can mean up to 20% (ie, ⁇ 20%), such as within ⁇ 10%, within ⁇ 5%, within ⁇ 2%, within ⁇ 1%, or within ⁇ 1% of the particular numerical range given. fluctuate within 0.5%. Also, particularly for biological systems or methods, the term can mean up to an order of magnitude or up to 5 times a value. When a specific value is given in the disclosure or claims, unless otherwise stated, the meaning of "about” should be considered to be within an acceptable error range for the specific value.
  • phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine Amide is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic acid is Asp or D; Glutamic acid is Glu or E; Cysteine is Cys or C; Tryptophan Acid is Trp or W; Arginine is Arg or R; Glycine is Gly or G.
  • the present disclosure provides isolated single variable domains that bind c-Met, such as human c-Met.
  • Single variable domains provide more available options for the development of drugs targeting c-Met or drug construction.
  • the single variable domain has many desired therapeutic properties, such as good affinity for human c-Met, ability to block the binding of ligand HGF or its ⁇ chain to c-Met, and inhibition of c-Met induced by HGF stimulation.
  • the single variable domain binds c-Met comprising one, two or all three CDRs of the variable region sequence set forth in SEQ ID NO:24. In a specific embodiment, said single variable domain binds c-Met comprising CDR1, CDR2 and CDR3 of the variable region sequence shown in SEQ ID NO:24. In some embodiments, the single variable domain binds c-Met comprising one, two or all three CDRs of the variable region sequence set forth in SEQ ID NO:26. In a specific embodiment, said single variable domain binds c-Met comprising CDR1, CDR2 and CDR3 of the variable region sequence shown in SEQ ID NO:26.
  • the single variable domain binds c-Met comprising one, two or all three CDRs of the variable region sequence set forth in SEQ ID NO:28. In a specific embodiment, said single variable domain binds c-Met comprising CDR1, CDR2 and CDR3 of the variable region sequence shown in SEQ ID NO:28. In some embodiments, the single variable domain binds c-Met comprising one, two or all three CDRs of the variable region sequence set forth in SEQ ID NO:30. In a specific embodiment, said single variable domain binds c-Met comprising CDR1, CDR2 and CDR3 of the variable region sequence shown in SEQ ID NO:30.
  • the single variable domain binds c-Met comprising one, two or all three CDRs of the variable region sequence set forth in SEQ ID NO:32. In a specific embodiment, said single variable domain binds c-Met, which comprises CDR1, CDR2 and CDR3 of the variable region sequence shown in SEQ ID NO:32. In some embodiments, the single variable domain binds c-Met comprising one, two or all three CDRs of the variable region sequence set forth in SEQ ID NO:34. In a specific embodiment, said single variable domain binds c-Met comprising CDR1, CDR2 and CDR3 of the variable region sequence shown in SEQ ID NO:34. In some embodiments, the single variable domain is of a camelid. In some embodiments, the single variable domain is humanized. In some embodiments, the single variable domain comprises an acceptor human framework.
  • the single variable domain binds c-Met comprising at least one, at least two or all three CDRs selected from: (a) comprising SEQ ID NO:5, 8, 11, CDR1 of the amino acid sequence shown in 14, 17 or 20; (b) CDR2 comprising the amino acid sequence shown in SEQ ID NO: 6, 9, 12, 15, 18 or 21; and (c) comprising SEQ ID NO: 7, 10 , 13, 16, 19 or 22 of the amino acid sequence shown in the CDR3.
  • the single variable domain binds c-Met comprising the following complementarity determining regions: (a) CDR1 comprising SEQ ID NO: 5, 8, 11, 14, 17 or 20 Amino acid sequence; (b) CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 6, 9, 12, 15, 18 or 21; and (c) CDR3, which comprises SEQ ID NO: 7, 10, 13, 16 , the amino acid sequence shown in 19 or 22.
  • the single variable domain is of a camelid.
  • the single variable domain is humanized.
  • the single variable domain comprises an acceptor human framework.
  • the single variable domain binds c-Met, comprising: CDR1 comprising the amino acid sequence shown in SEQ ID NO:5, CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and comprising SEQ ID NO: CDR3 of the amino acid sequence shown in ID NO:7.
  • the single variable domain binds c-Met, comprising: CDR1 comprising the amino acid sequence shown in SEQ ID NO:8, CDR2 comprising the amino acid sequence shown in SEQ ID NO:9, and comprising SEQ ID NO: CDR3 of the amino acid sequence shown in ID NO:10.
  • the single variable domain binds c-Met, comprising: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 11, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12, and comprising SEQ ID NO: 12 CDR3 of the amino acid sequence shown in ID NO:13.
  • the single variable domain binds c-Met, comprising: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 14, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 15, and comprising SEQ ID NO: 15 CDR3 of the amino acid sequence shown in ID NO:16.
  • the single variable domain binds c-Met, comprising: CDR1 comprising the amino acid sequence shown in SEQ ID NO:17, CDR2 comprising the amino acid sequence shown in SEQ ID NO:18, and comprising SEQ ID NO:18 CDR3 of the amino acid sequence shown in ID NO:19. In some embodiments, the single variable domain binds c-Met, comprising: CDR1 comprising the amino acid sequence shown in SEQ ID NO:20, CDR2 comprising the amino acid sequence shown in SEQ ID NO:21, and comprising SEQ ID NO:21 CDR3 of the amino acid sequence shown in ID NO:22. In some embodiments, the single variable domain is of a camelid. In some embodiments, the single variable domain is humanized. In some embodiments, the single variable domain comprises an acceptor human framework.
  • the single variable domain binds c-Met comprising: CDR1 of the amino acid sequence shown in SEQ ID NO:5, CDR2 of the amino acid sequence shown in SEQ ID NO:6, and SEQ ID NO: CDR3 of the amino acid sequence shown in 7.
  • the single variable domain binds c-Met comprising: CDR1 of the amino acid sequence shown in SEQ ID NO:8, CDR2 of the amino acid sequence shown in SEQ ID NO:9, and SEQ ID NO: CDR3 of the amino acid sequence shown in 10.
  • the single variable domain binds c-Met comprising: CDR1 of the amino acid sequence shown in SEQ ID NO: 11, CDR2 of the amino acid sequence shown in SEQ ID NO: 12, and SEQ ID NO: CDR3 of the amino acid sequence shown in 13.
  • the single variable domain binds c-Met comprising: CDR1 of the amino acid sequence shown in SEQ ID NO:14, CDR2 of the amino acid sequence shown in SEQ ID NO:15, and SEQ ID NO: CDR3 of the amino acid sequence shown in 16.
  • the single variable domain binds c-Met comprising: CDR1 of the amino acid sequence shown in SEQ ID NO:17, CDR2 of the amino acid sequence shown in SEQ ID NO:18, and SEQ ID NO: CDR3 of the amino acid sequence shown in 19.
  • the single variable domain binds c-Met comprising: CDR1 of the amino acid sequence shown in SEQ ID NO:20, CDR2 of the amino acid sequence shown in SEQ ID NO:21, and SEQ ID NO: CDR3 of the amino acid sequence shown in 22.
  • the single variable domain is of a camelid.
  • the single variable domain is humanized.
  • the single variable domain comprises an acceptor human framework.
  • the single variable domain binds c-Met comprising at least 85%, 86%, 87% of the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32, or , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences; optionally, all Said single variable domain comprises the specific CDR1, CDR2 and/or CDR3 sequences described above.
  • the single variable domain binds c-Met comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • said single variable domain comprises: comprising SEQ ID NO: CDR1 of the amino acid sequence shown in 5, CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:7.
  • the single variable domain binds c-Met comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • said single variable domain comprises: comprising SEQ ID NO: CDR1 of the amino acid sequence shown in 8, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10.
  • the single variable domain binds c-Met comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • said single variable domain comprises: comprising SEQ ID NO: CDR1 of the amino acid sequence shown in 11, CDR2 comprising the amino acid sequence shown in SEQ ID NO:12, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:13.
  • the single variable domain binds c-Met comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • said single variable domain comprises: comprising SEQ ID NO: CDR1 of the amino acid sequence shown in 14, CDR2 comprising the amino acid sequence shown in SEQ ID NO:15, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:16.
  • the single variable domain binds c-Met comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • said single variable domain comprises: comprising SEQ ID NO: CDR1 of the amino acid sequence shown in 17, CDR2 comprising the amino acid sequence shown in SEQ ID NO:18, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:19.
  • the single variable domain binds c-Met comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • said single variable domain comprises: comprising SEQ ID NO: CDR1 of the amino acid sequence shown in 20, CDR2 comprising the amino acid sequence shown in SEQ ID NO:21, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:22.
  • a single variable domain amino acid sequence that is 99% or 100% identical contains a substitution (e.g., a conservative substitution), insertion, or deletion relative to the reference sequence, but the single variable domain comprising the sequence retains the ability to bind c-Met .
  • a total of 1-18, 1-16, 1-14, 1-12, 1-10 in the amino acid sequence selected from SEQ ID NO: 24, 26, 28, 30, 32 and 34 , 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acids are substituted, inserted and/or deleted.
  • substitutions, insertions or deletions occur in regions outside of the CDRs (ie, in FRs). In some embodiments, substitutions, insertions or deletions occur in CDR regions, eg, one, two or three of CDR1, CDR2, CDR3. In some embodiments, substitutions, insertions or deletions occur in CDR regions and non-CDR regions.
  • the single variable domain comprises an amino acid sequence selected from SEQ ID NO: 24, 26, 28, 30, 32 and 34, including post-translational modifications of the sequence.
  • the single variable domain binds c-Met comprising the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34.
  • the single variable domain (including any of the embodiments described above, eg, a single variable domain comprising a particular CDR1, CDR2 and/or CDR3 described above) is a VHH .
  • the VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the single variable domain (including any of the embodiments described above, eg, a single variable domain comprising a particular CDR1, CDR2 and/or CDR3 described above) is a humanized VHH .
  • a VHH derived from a non-human can be generated by substituting one or more amino acid residues in the original VHH amino acid sequence with one or more amino acid residues present at the corresponding position in a human conventional four-chain antibody VH domain.
  • Humanized Humanization can desirably reduce immunogenicity.
  • the single variable domain binds c-Met, the amino acid sequence of which is shown in SEQ ID NO: 24, 26, 28, 30, 32 or 34.
  • the disclosure also provides a single variable domain that binds the same epitope as any of the single variable domains of the disclosure.
  • the single variable domain binds to the same epitope as the single variable domain comprising the amino acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32 or 34.
  • the single variable domains that bind the same epitope are camelid or humanized.
  • Single variable domains can be competitively screened for binding to the same epitope using routine techniques known to those skilled in the art. For example, competition studies can be performed to obtain single variable domains that compete with each other for antigen binding. Accordingly, the present disclosure also provides a single variable domain that competes for c-Met binding with any of the single variable domains described in the present disclosure. In some specific embodiments, the single variable domain competes for c-Met binding with a single variable domain comprising the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34. Binding to c-Met can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assays, or any other method known in the art. In some embodiments, the single variable domain that competes for binding to c-Met is camelid or humanized.
  • a single variable domain according to any one of the above embodiments may comprise, alone or in combination, one or more of the following properties:
  • the single variable domain binds c-Met. In some embodiments, the single variable domain blocks the binding of HGF or its beta chain to c-Met. In some embodiments, the single variable domain inhibits c-Met phosphorylation induced by HGF stimulation.
  • the present disclosure provides some exemplary single variable domains that bind c-Met.
  • the amino acid sequences of the CDRs (CDR1, CDR2, and CDR3) of exemplary single variable domains provided by the present disclosure are provided in Table S1 below, and the full-length amino acid sequences of exemplary single variable domains are provided in Table S2 below .
  • the present disclosure provides isolated antibodies or antigen-binding fragments thereof that bind c-Met, such as human c-Met, said antibodies or antigen-binding fragments thereof comprising at least one (one or more) single variable domain(s) of the present disclosure .
  • the c-Met-binding antibody or antigen-binding fragment thereof provided by the present disclosure has many desired therapeutic properties, such as having good affinity for human c-Met, being able to block the binding of the ligand HGF or its ⁇ chain to c-Met, One or more of inhibiting c-Met phosphorylation caused by HGF stimulation, having good binding properties to cells with different c-Met expression levels, killing tumor cells, etc.
  • the antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 24 One, two or all three CDRs of a variable region sequence.
  • said antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 24 CDR1, CDR2 and CDR3 of the variable region sequences shown.
  • the antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 26 One, two or all three CDRs of a variable region sequence.
  • said antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 26 CDR1, CDR2 and CDR3 of the variable region sequences shown. In some embodiments, the antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 28 One, two or all three CDRs of a variable region sequence. In a specific embodiment, said antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 28 CDR1, CDR2 and CDR3 of the variable region sequences shown.
  • the antibody or antigen-binding fragment thereof binds to c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 30 One, two or all three CDRs of a variable region sequence.
  • said antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 30 CDR1, CDR2 and CDR3 of the variable region sequences shown.
  • the antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 32 One, two or all three CDRs of a variable region sequence.
  • said antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO:32 CDR1, CDR2 and CDR3 of the variable region sequences shown. In some embodiments, the antibody or antigen-binding fragment thereof binds to c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO: 34 One, two or all three CDRs of a variable region sequence. In a specific embodiment, said antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising SEQ ID NO:34 CDR1, CDR2 and CDR3 of the variable region sequences shown. In some embodiments, the single variable domain is of a camelid. In some embodiments, the single variable domain is humanized. In some embodiments, the single variable domain comprises an acceptor human framework.
  • the antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising at least one selected from the group consisting of, At least two or all three CDRs: (a) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, 8, 11, 14, 17 or 20; (b) comprising SEQ ID NO: 6, 9, 12, 15 , the CDR2 of the amino acid sequence shown in 18 or 21; and (c) the CDR3 comprising the amino acid sequence shown in SEQ ID NO: 7, 10, 13, 16, 19 or 22.
  • the antibody or antigen-binding fragment thereof binds c-Met and comprises at least one (one or more) single variable domain comprising the following complementarity determining regions: (a ) CDR1, which comprises the amino acid sequence shown in SEQ ID NO:5, 8, 11, 14, 17 or 20; (b) CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 6, 9, 12, 15, 18 or 21 and (c) CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 7, 10, 13, 16, 19 or 22.
  • the single variable domain is of a camelid.
  • the single variable domain is humanized.
  • the single variable domain comprises an acceptor human framework.
  • the single variable domain comprises: (a) CDR1 comprising the amino acid sequence shown in SEQ ID NO:5, CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and comprising SEQ ID NO: CDR3 of the amino acid sequence shown in 7; (b) CDR1 comprising the amino acid sequence shown in SEQ ID NO:8, CDR2 comprising the amino acid sequence shown in SEQ ID NO:9, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:10 (c) CDR1 comprising the amino acid sequence shown in SEQ ID NO:11, CDR2 comprising the amino acid sequence shown in SEQ ID NO:12, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:13; (d) comprising SEQ ID CDR1 of the amino acid sequence shown in NO:14, CDR2 comprising the amino acid sequence shown in SEQ ID NO:15, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:16; (e) comprising the amino acid sequence shown in SEQ ID NO:5
  • the single variable domain comprises: (a) CDR1 of the amino acid sequence shown in SEQ ID NO:5, CDR2 of the amino acid sequence shown in SEQ ID NO:6, and the CDR2 of the amino acid sequence shown in SEQ ID NO:7 CDR3 of the amino acid sequence; (b) CDR1 of the amino acid sequence shown in SEQ ID NO:8, CDR2 of the amino acid sequence shown in SEQ ID NO:9, and CDR3 of the amino acid sequence shown in SEQ ID NO:10; (c) SEQ ID CDR1 of the amino acid sequence shown in NO:11, CDR2 of the amino acid sequence shown in SEQ ID NO:12, and CDR3 of the amino acid sequence shown in SEQ ID NO:13; (d) CDR1 of the amino acid sequence shown in SEQ ID NO:14, CDR2 of the amino acid sequence shown in SEQ ID NO:15, and CDR3 of the amino acid sequence shown in SEQ ID NO:16; (e) CDR1 of the amino acid sequence shown in SEQ ID NO:
  • the single variable domain comprises at least 85%, 86%, 87%, 88%, 89% of the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences.
  • the single variable domain comprises at least 85%, 86%, 87%, 88%, 89% of the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, and the single variable domain comprises the above-mentioned specific CDR1, CDR2 and/or CDR3 sequences.
  • the single variable domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the single variable domain comprises: comprising the amino acid sequence shown in SEQ ID NO:5 CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:7.
  • the single variable domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the single variable domain comprises: comprising the amino acid sequence shown in SEQ ID NO:8 CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO:9, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:10.
  • the single variable domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the single variable domain comprises: comprising the amino acid sequence shown in SEQ ID NO:11 CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO:12, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:13.
  • the single variable domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the single variable domain comprises: comprising the amino acid sequence shown in SEQ ID NO:14 CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO:15, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:16.
  • the single variable domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the single variable domain comprises: comprising the amino acid sequence shown in SEQ ID NO:17 CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO:18, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:19.
  • the single variable domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the single variable domain comprises: comprising the amino acid sequence shown in SEQ ID NO:20 CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO:21, and CDR3 comprising the amino acid sequence shown in SEQ ID NO:22.
  • a total of 1-18, 1-16, 1-14, 1-12, 1-10 in the amino acid sequence selected from SEQ ID NO: 24, 26, 28, 30, 32 and 34 , 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or 1-2 amino acids are substituted, inserted and/or deleted.
  • substitutions, insertions or deletions occur in regions outside of the CDRs (ie, in FRs).
  • substitutions, insertions or deletions occur in CDR regions, eg, one, two or three of CDR1, CDR2, CDR3.
  • substitutions, insertions or deletions occur in CDR regions and non-CDR regions.
  • the single variable domain comprises an amino acid sequence selected from SEQ ID NO: 24, 26, 28, 30, 32 and 34, including post-translational modifications of the sequence.
  • the single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34.
  • the antibodies may be monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies, trispecific antibodies), or antibodies of other configurations known in the art.
  • the antigen-binding fragment may be a single variable domain.
  • said antibody is a monospecific antibody.
  • said antibody is a bispecific antibody.
  • the single variable domain (including any of the embodiments described above, eg, a single variable domain comprising a particular CDR1, CDR2 and/or CDR3 described above) is a VHH .
  • the VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the single variable domain (including any of the embodiments described above, eg, a single variable domain comprising a particular CDR1, CDR2 and/or CDR3 described above) is a humanized VHH .
  • a VHH derived from a non-human can be generated by substituting one or more amino acid residues in the original VHH amino acid sequence with one or more amino acid residues present at the corresponding position in a human conventional four-chain antibody VH domain.
  • Humanized Humanization can desirably reduce immunogenicity.
  • the antibody or antigen-binding fragment thereof is of a camelid. In some embodiments, the antibody or antigen-binding fragment thereof is chimeric. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG constant region, preferably a human IgG constant region, more preferably a human IgG1 constant region, a human IgG2 constant region, a human IgG3 constant region or a human IgG4 constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG Fc.
  • the antibody or antigen-binding fragment thereof comprises the Fc of human IgG; preferably, the Fc is the Fc of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the antibody or antigen-binding fragment thereof is humanized. In some embodiments, the antibody or antigen-binding fragment thereof comprises a receptor human framework.
  • the antibody or antigen-binding fragment thereof comprises at least 85%, 86%, 87%, 88%, 89% Amino acid sequences that are %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the antibody or antigen-binding fragment thereof comprises at least 85%, 86%, 87%, 88%, 89% %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence, and the antibody or antigen-binding fragment thereof comprises Specific CDR1, CDR2 and/or CDR3 sequences in the single variable domains of the present disclosure.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 37, 39, 41, 43, 45, or 47. In some embodiments, the antibody or antigen-binding fragment thereof comprises at least one polypeptide chain, wherein the amino acid sequence of at least one polypeptide chain is shown in SEQ ID NO: 37, 39, 41, 43, 45 or 47. In some embodiments, the antibody or antigen-binding fragment thereof comprises two identical polypeptide chains, wherein the amino acid sequence of each polypeptide chain is set forth in SEQ ID NO: 37, 39, 41, 43, 45 or 47.
  • the disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope as any of the antibodies or antigen-binding fragments thereof of the disclosure.
  • the antibody or antigen-binding fragment thereof binds to the same epitope as the antibody or antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 37, 39, 41, 43, 45 or 47.
  • the antibodies or antigen-binding fragments thereof that bind the same epitope are camelid, chimeric or humanized.
  • Antibodies, or antigen-binding fragments thereof can be competitively screened for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition studies can be performed to obtain antibodies or antigen-binding fragments thereof that compete with each other for antigen binding. Accordingly, the present disclosure also provides antibodies or antigen-binding fragments thereof that compete with any of the antibodies or antigen-binding fragments thereof of the present disclosure for binding to c-Met. In some specific embodiments, the antibody or antigen-binding fragment thereof competes for binding to c-Met with an antibody or antigen-binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO: 37, 39, 41, 43, 45 or 47.
  • Binding to c-Met can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assays, or any other method known in the art.
  • the antibody or antigen-binding fragment thereof that competes for binding to c-Met is camelid, chimeric, or humanized.
  • the antibody or antigen-binding fragment thereof according to any of the above embodiments may include, alone or in combination, one or more of the following properties:
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the antibody or antigen-binding fragment thereof binds c-Met, eg, monkey c-Met and/or human c-Met. In some embodiments, the antibody or antigen-binding fragment thereof binds cynomolgus c-Met. In some embodiments, the antibody or antigen-binding fragment thereof binds human c-Met. In some embodiments, the antibody or antigen-binding fragment thereof has the following binding affinity (K D ) for human c-Met: between about 1E-11M to about 1E-07M, about 1E-10M to about 1E-08M , or in the range of about 2.00E-10M to about 8.25E-09M.
  • K D binding affinity
  • the antibody or antigen-binding fragment thereof has a binding affinity ( KD ) for human c-Met of about 1E-07M or less, about 1E-08M or less, about 8.25E-09M or less, about 1E-09M or less, about 2.00E-10M or less, about 1E-10M or less, or about 1E-11M or less.
  • the binding affinity KD of an antibody or antigen-binding fragment thereof provided herein is measured by Biacore.
  • the antibody or antigen-binding fragment thereof blocks the binding of HGF or its beta chain to c-Met.
  • the antibody or antigen-binding fragment thereof inhibits c-Met phosphorylation induced by HGF stimulation.
  • the antibody or antigen-binding fragment thereof exerts ADCC effects on tumor cells.
  • the tumor cells are tumor cells expressing c-Met.
  • the present disclosure provides exemplary antibodies or antigen-binding fragments thereof, such as monospecific antibodies (including chimeric antibodies 1B-3B11-Fc, 1B-1B6-Fc, 1B-1C7-Fc, 1B-1A8-Fc, 1B- 1B2-Fc and 3B-1C7-Fc), a single variable domain is fused to the Fc of human IgG1 to form a homodimer through Fc.
  • monospecific antibodies including chimeric antibodies 1B-3B11-Fc, 1B-1B6-Fc, 1B-1C7-Fc, 1B-1A8-Fc, 1B- 1B2-Fc and 3B-1C7-Fc
  • a single variable domain is fused to the Fc of human IgG1 to form a homodimer through Fc.
  • the amino acid sequences of exemplary antibodies are provided in Table S3 below.
  • the single variable domains of the present disclosure can be used to construct any desired fusion protein, thereby endowing the fusion protein with the property of targeting c-Met or other single variable domains, antibodies or antigen binding thereof The properties that the fragment has. Accordingly, the present disclosure provides isolated fusion proteins comprising at least one (one or more) single variable domain(s) of the present disclosure, or an antibody or antigen-binding fragment thereof.
  • the fusion protein includes the exemplary CDR sequence, single variable domain sequence or antibody sequence of the present disclosure, such as one, two or three of any single variable domain in Table S1 CDR sequence, any single variable domain sequence in Table S2, or any antibody sequence in Table S3; optionally, the fusion protein can undergo any modification known in the art (such as glycosylation modification, chemical modification, etc. ).
  • the same or different single variable domains can be selected.
  • the present disclosure provides a pharmaceutical composition comprising an antibody of the present disclosure or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.
  • the present disclosure also provides a pharmaceutical composition, which comprises the fusion protein of the present disclosure, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition which comprises the fusion protein of the present disclosure, and a pharmaceutically acceptable carrier.
  • the present disclosure provides isolated nucleic acids encoding the single variable domains described in the present disclosure, or antibodies or antigen-binding fragments thereof.
  • the present disclosure also provides isolated nucleic acids encoding the fusion proteins described in the present disclosure.
  • the nucleic acid encodes a single variable domain, such as 1B-3B11-Fc, 1B-1B6-Fc, 1B-1C7-Fc, 1B-1A8-Fc, 1B-1B2-Fc, or 3B-1C7 - Single variable domain of Fc.
  • the nucleic acid encodes an antibody or antigen-binding fragment thereof, such as 1B-3B11-Fc, 1B-1B6-Fc, 1B-1C7-Fc, 1B-1A8-Fc, 1B-1B2-Fc, or 3B- 1C7-Fc.
  • nucleic acid sequences of some single variable domains, antibodies or antigen-binding fragments thereof are exemplarily listed in the sequence listing.
  • the present disclosure provides vectors comprising the isolated nucleic acids.
  • the vector is a cloning vector; in other embodiments, the vector is an expression vector.
  • the expression vector may optionally be any expression vector capable of expressing the single variable domain of the present disclosure, or an antibody or an antigen-binding fragment thereof.
  • the expression vector may optionally be any expression vector capable of expressing the fusion protein described in the present disclosure.
  • the expression vector is pcDNA3.1.
  • the present disclosure provides host cells comprising the nucleic acids of the present disclosure or the vectors, which are suitable host cells for cloning or expressing single variable domains, or antibodies or antigen-binding fragments thereof.
  • the present disclosure also provides a host cell comprising the nucleic acid of the present disclosure or the vector, which is a suitable host cell for cloning or expressing the fusion protein.
  • the host cell is a prokaryotic cell.
  • the host cell is a eukaryotic cell.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for making single variable domains, antibodies or antigen-binding fragments thereof, or fusion proteins.
  • Mammalian cells are, for example, Chinese Hamster Ovary (CHO) cells, CHO-S cells.
  • the present disclosure provides a method for preparing a single variable domain, the method comprising: cultivating a host cell comprising a nucleic acid encoding a single variable domain of the disclosure, recovering the single variable domain from the host cell or host cell culture medium variable domain.
  • the present disclosure also provides a method of producing an antibody or antigen-binding fragment thereof, the method comprising: culturing a host cell comprising a nucleic acid encoding an antibody of the disclosure or an antigen-binding fragment thereof, recovering the host cell or host cell culture medium from the host cell or host cell culture medium. The antibody or antigen-binding fragment thereof.
  • the present disclosure also provides a method for preparing a fusion protein, the method comprising: culturing a host cell comprising a nucleic acid encoding the fusion protein of the present disclosure, and recovering the fusion protein from the host cell or host cell culture medium.
  • the nucleic acid encoding the single variable domain, or an antibody or an antigen-binding fragment thereof is inserted into a vector for further cloning or / and expression.
  • the nucleic acid encoding the fusion protein is inserted into a vector for further cloning or/and expression in host cells.
  • the nucleic acid can be obtained by various methods well known in the art, such as gene splicing and chemical synthesis.
  • the present disclosure provides uses of the single variable domains, antibodies or antigen-binding fragments thereof, or fusion proteins of the present disclosure.
  • the present disclosure also provides uses of the pharmaceutical compositions of the present disclosure.
  • the present disclosure provides the use of a single variable domain of the present disclosure in the manufacture of a medicament for treating a subject suffering from a tumor.
  • the present disclosure also provides a use of an antibody of the present disclosure or an antigen-binding fragment thereof in the manufacture of a medicament for treating a subject suffering from a tumor.
  • the present disclosure also provides the use of the fusion protein of the present disclosure in the preparation of a medicament for treating a subject suffering from a tumor.
  • the present disclosure also provides the use of the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a subject suffering from a tumor.
  • the present disclosure provides methods of detecting or measuring c-Met in a sample comprising contacting the sample with a single variable domain of the present disclosure, or an antibody or antigen-binding fragment thereof, and detecting or measuring the bound complex.
  • the disclosure also provides methods of detecting or measuring c-Met in a sample comprising contacting the sample with a fusion protein of the disclosure and detecting or measuring the bound complex.
  • the present disclosure provides methods of inhibiting, reducing or blocking c-Met signaling in a cell comprising administering to the cell an effective amount of a single variable domain described in the present disclosure, or an antibody or antigen-binding fragment thereof.
  • the present disclosure also provides the use of the single variable domain of the present disclosure, or an antibody or an antigen-binding fragment thereof, in the preparation of a medicament for inhibiting, reducing or blocking c-Met signal transduction in cells.
  • the present disclosure provides methods of inhibiting, reducing or blocking c-Met signaling in a cell comprising administering to the cell an effective amount of a fusion protein described in the present disclosure.
  • the present disclosure also provides the use of the fusion protein of the present disclosure in preparing a medicament for inhibiting, reducing or blocking c-Met signal transduction in cells.
  • the present disclosure provides methods of inhibiting, reducing or blocking c-Met signaling in a cell comprising administering to the cell an effective amount of a pharmaceutical composition described in the present disclosure.
  • the present disclosure also provides the use of the pharmaceutical composition of the present disclosure in the preparation of a medicament for inhibiting, reducing or blocking c-Met signal transduction in cells.
  • the cells are tumor cells.
  • the present disclosure provides a method for inhibiting the growth of tumor cells or killing tumor cells, which comprises administering to the tumor cells an effective amount of the single variable domain of the present disclosure, or an antibody or an antigen-binding fragment thereof.
  • the present disclosure also provides the use of the single variable domain, or the antibody or antigen-binding fragment thereof in the preparation of a drug for inhibiting tumor cell growth or killing tumor cells.
  • the present disclosure provides a method for inhibiting the growth of tumor cells or killing tumor cells, which comprises administering an effective amount of the fusion protein of the present disclosure to the tumor cells.
  • the present disclosure also provides the use of the fusion protein in the preparation of drugs for inhibiting tumor cell growth or killing tumor cells.
  • the present disclosure provides a method for inhibiting the growth of tumor cells or killing tumor cells, which comprises administering an effective amount of the pharmaceutical composition of the present disclosure to the tumor cells.
  • the present disclosure also provides the use of the pharmaceutical composition in the preparation of drugs for inhibiting tumor cell growth or killing tumor cells.
  • the present disclosure provides methods of treating a subject having a tumor comprising administering to the subject a therapeutically effective amount of a single variable domain, or an antibody or antigen-binding fragment thereof, described in the present disclosure.
  • the present disclosure also provides a method of treating a subject having a tumor comprising administering to the subject a therapeutically effective amount of a fusion protein described in the present disclosure.
  • the present disclosure also provides a method of treating a subject having a tumor comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described in the present disclosure.
  • Those in need of treatment include those already with the disease or condition as well as those at risk of developing the disease or condition and for the purpose of preventing, delaying or attenuating the disease or condition.
  • the aforementioned tumor is a c-Met expressing tumor. In some embodiments, the aforementioned tumor is a c-Met overexpressing tumor. In some embodiments, the aforementioned tumor is a solid tumor. In some embodiments, the aforementioned tumor is a non-solid tumor.
  • the aforementioned tumor is brain cancer, head and neck cancer, lung cancer, esophageal cancer, pharyngeal cancer, nasal cancer, tongue cancer, oral cancer, thyroid cancer, skin cancer, bone cancer, soft tissue sarcoma, myeloma, gastric cancer, gastric cancer Esophageal junction adenocarcinoma, breast cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lymphoma cancer, thymus cancer, bladder cancer, vaginal cancer, testicular cancer, uterine cancer, cervical cancer, colorectal cancer, anal cancer, papillary cancer, prostate cancer, ovarian cancer, epithelial cell carcinoma, or blood tumors.
  • the lung cancer is non-small cell lung cancer.
  • the brain cancer is glioblastoma.
  • Embodiment 1 An isolated antibody or antigen-binding fragment thereof that binds c-Met and comprises at least one single variable domain, wherein said single variable domain comprises a complementarity determination district:
  • CDR1 which comprises the amino acid sequence shown in SEQ ID NO: 5, 8, 11, 14, 17 or 20;
  • CDR2 comprising the amino acid sequence shown in SEQ ID NO: 6, 9, 12, 15, 18 or 21;
  • CDR3 which comprises the amino acid sequence shown in SEQ ID NO: 7, 10, 13, 16, 19 or 22.
  • Embodiment 2 The isolated antibody or antigen-binding fragment thereof of embodiment 1, wherein said single variable domain comprises:
  • CDR1 comprising the amino acid sequence shown in SEQ ID NO:5
  • CDR2 comprising the amino acid sequence shown in SEQ ID NO:6
  • CDR3 comprising the amino acid sequence shown in SEQ ID NO:7;
  • CDR1 comprising the amino acid sequence shown in SEQ ID NO:8
  • CDR2 comprising the amino acid sequence shown in SEQ ID NO:9
  • CDR3 comprising the amino acid sequence shown in SEQ ID NO:10
  • CDR1 comprising the amino acid sequence shown in SEQ ID NO:11
  • CDR2 comprising the amino acid sequence shown in SEQ ID NO:12
  • CDR3 comprising the amino acid sequence shown in SEQ ID NO:13;
  • CDR1 comprising the amino acid sequence shown in SEQ ID NO:14
  • CDR2 comprising the amino acid sequence shown in SEQ ID NO:15
  • CDR3 comprising the amino acid sequence shown in SEQ ID NO:16;
  • CDR1 comprising the amino acid sequence shown in SEQ ID NO:17
  • CDR2 comprising the amino acid sequence shown in SEQ ID NO:18
  • CDR3 comprising the amino acid sequence shown in SEQ ID NO:19
  • CDR1 comprising the amino acid sequence shown in SEQ ID NO:20
  • CDR2 comprising the amino acid sequence shown in SEQ ID NO:21
  • CDR3 comprising the amino acid sequence shown in SEQ ID NO:22.
  • Embodiment 3 The isolated antibody or antigen-binding fragment thereof according to embodiment 2, wherein said single variable domain comprises Amino acid sequences that are at least 85% identical.
  • Embodiment 4 The isolated antibody or antigen-binding fragment thereof of embodiment 3, wherein said single variable domain comprises the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34.
  • Embodiment 5 The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-4, wherein said single variable domain is a VHH .
  • Embodiment 6 The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-5, wherein said antibody or antigen-binding fragment thereof is chimeric or humanized.
  • Embodiment 7 The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-6, wherein said antibody or antigen-binding fragment thereof comprises the Fc of a human IgG, preferably human IgGl, IgG2, IgG3 or IgG4 Fc.
  • Embodiment 8 The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-7, wherein said antibody or antigen-binding fragment thereof comprises SEQ ID NO: 37, 39, 41, 43, 45 or The amino acid sequence shown in 47.
  • Embodiment 9 The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-8, wherein said antibody or antigen-binding fragment thereof may comprise, alone or in combination, one of the following property characteristics or several:
  • Embodiment 10 Binding to human c-Met or/and monkey c-Met;
  • Embodiment 11 Blocking the binding of HGF or its beta chain to c-Met;
  • Embodiment 12 Inhibition of c-Met phosphorylation induced by HGF stimulation
  • Embodiment 13 Exercising ADCC effects on tumor cells.
  • Embodiment 14 A fusion protein comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-9.
  • Embodiment 15 A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-9 or the fusion protein of embodiment 10, and a pharmaceutically acceptable carrier.
  • Embodiment 16 An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of embodiments 1-9 or the fusion protein of embodiment 10.
  • Embodiment 17 A vector comprising the isolated nucleic acid of embodiment 12.
  • Embodiment 18 A host cell comprising the nucleic acid of embodiment 12 or the vector of embodiment 13.
  • Embodiment 19 A method of preparing the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-9, comprising culturing the host cell of embodiment 14, isolating the expressed antibody or Its antigen-binding fragment, wherein the vector is an expression vector.
  • Embodiment 20 Use of the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-9 in the manufacture of a medicament for treating a subject suffering from a tumor.
  • Embodiment 21 Use of the fusion protein of embodiment 10 for the manufacture of a medicament for the treatment of a subject suffering from a tumor.
  • Embodiment 22 Use of the pharmaceutical composition of embodiment 11 for the manufacture of a medicament for the treatment of a subject suffering from a tumor.
  • Embodiment 23 The use according to any one of embodiments 16-18, wherein the tumor is brain cancer, head and neck cancer, lung cancer, esophageal cancer, pharyngeal cancer, nasal cancer, tongue cancer, oral cavity cancer, thyroid cancer, Skin cancer, bone cancer, soft tissue sarcoma, myeloma, gastric cancer, gastroesophageal junction adenocarcinoma, breast cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lymph cancer, thymus cancer, bladder cancer, vaginal cancer, testicular cancer, Cancer of the uterus, cervix, colorectum, anus, papillary, prostate, ovary, epithelial cell, or blood tumors.
  • the tumor is brain cancer, head and neck cancer, lung cancer, esophageal cancer, pharyngeal cancer, nasal cancer, tongue cancer, oral cavity cancer, thyroid cancer, Skin cancer, bone cancer, soft tissue sarcoma, myeloma, gastric
  • Embodiment 24 A method of detecting or measuring c-Met in a sample comprising contacting the sample with the antibody or antigen-binding fragment thereof of any one of embodiments 1-9 and detecting or measuring the binding complex thing.
  • Embodiment 25 A method of inhibiting, reducing or blocking c-Met signaling in a cell comprising administering to the cell an effective amount of the antibody or antigen binding thereof of any one of embodiments 1-9 fragment, the fusion protein of embodiment 10, or the pharmaceutical composition of embodiment 11.
  • Embodiment 26 A method of inhibiting the growth of tumor cells or killing tumor cells, comprising administering to the tumor cells an effective amount of the antibody or antigen-binding fragment thereof according to any one of embodiments 1-9, embodiment 10
  • Embodiment 27 A method of treating a subject with a tumor comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 1-9, performing The fusion protein described in Scheme 10, or the pharmaceutical composition described in Embodiment 11.
  • Embodiment 28 The method according to embodiment 22 or 23, wherein the tumor is brain cancer, head and neck cancer, lung cancer, esophageal cancer, pharyngeal cancer, nasal cancer, tongue cancer, oral cancer, thyroid cancer, skin cancer, bone cancer Carcinoma, soft tissue sarcoma, myeloma, gastric cancer, gastroesophageal junction adenocarcinoma, breast cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lymphatic cancer, thymus cancer, bladder cancer, vaginal cancer, testicular cancer, uterine cancer, cervical cancer cancer, colorectal cancer, anal cancer, papillary cancer, prostate cancer, ovarian cancer, epithelial cell cancer, or blood tumors.
  • the tumor is brain cancer, head and neck cancer, lung cancer, esophageal cancer, pharyngeal cancer, nasal cancer, tongue cancer, oral cancer, thyroid cancer, skin cancer, bone cancer Carcinoma, soft tissue sarcoma, myel
  • the recombinant human c-Met-Fc fusion protein (SinoBiological, product catalog 10692-H02H) was mixed and emulsified with complete Freund's adjuvant at a volume ratio of 1:1, and the Bactrian camel was immunized by subcutaneous multi-point injection for the first time; Zhou, the recombinant human c-Met-Fc fusion protein and incomplete Freund's adjuvant were mixed and emulsified according to the volume ratio of 1:1, and booster immunization was carried out. Serum was collected after the 4th or 5th immunization to detect the titer of anti-human c-Met antibody. After multiple rounds of immunization, the peripheral blood of Bactrian camels was collected, and peripheral blood mononuclear cells (PBMC) were isolated.
  • PBMC peripheral blood mononuclear cells
  • Total RNA was extracted from PBMCs (from 1.1) using TRIzol TM reagent. The quality of the extracted total RNA was evaluated by 1% agarose gel electrophoresis and quantified by measuring the absorbance at 260nm and 280nm, the ratio of OD 260nm /OD 280nm should be between 1.8-2.0.
  • the total RNA was reverse-transcribed into cDNA using the cDNA synthesis kit PrimeScript TM II 1st Strand cDNA Synthesis Kit (TAKARA, catalog No. 6210A) according to the instructions.
  • TAKARA TM II 1st Strand cDNA Synthesis Kit
  • the specific method is as follows: use cDNA as a template, use primers Call001 (SEQ ID NO: 1) and Call002 (SEQ ID NO: 2) for the first One round of PCR amplification, and the amplified DNA product short fragments were purified with a gel recovery kit (QIAGEN, catalog No. 28706).
  • V-Back (SEQ ID NO: 3): GATGTGCAGCTGCAGGAGTCTGGRGGAGG
  • V-Fwd (SEQ ID NO: 4): CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT
  • the amplified VHH coding fragment was digested with PstI/NotI endonuclease, it was inserted into the phagemid vector pMECS (NTCC Plasmid Vector Strain Cell Gene Collection Center, Product Catalog No.pMECS) to construct a recombinant vector , and electroporated into Escherichia coli TG1 (Lucigen, catalog No. 60502-1). Take a small portion of the transformed bacterial solution and spread it on a selective plate containing 100 ⁇ g/mL ampicillin after dilution. The library capacity is calculated by colony counting method, and 100 clones are randomly selected for sequencing to evaluate the quality of the library.
  • pMECS NTCC Plasmid Vector Strain Cell Gene Collection Center, Product Catalog No.pMECS
  • the rest of the bacterial solution was coated on a selective plate containing 100 ⁇ g/mL ampicillin, and the bacterial lawn of the colony was scraped off the plate, supplemented with glycerol, and frozen at -80°C as the original seed of the library.
  • the stock library was amplified to the logarithmic growth phase, and M13KO7 helper phage (New England Biolabs, catalog No. N0315S) was added for library amplification, shaken at 200 rpm overnight at 28°C.
  • the VHH phage display library was panned by solid phase panning. Immobilize the recombinant c-Met-Avitage TM (ACRO, catalog No.MET-H82E1) on a high-adsorption microtiter plate, and after blocking, add the phage obtained in 1.4 into the well plate and incubate at 37°C for 1-2h. Use phosphate Tween buffered saline (PBST) to wash 10 times to remove non-specifically bound phages. After washing with PBS, the bound phages are eluted with trypsin and washed with 4-(2-aminoethyl)benzene.
  • ACRO c-Met-Avitage TM
  • PBST phosphate Tween buffered saline
  • AEBSF Sulfonyl fluoride hydrochloride
  • the positive clones 1B-1B2, 1B-3B11, 1B-1C7, 1B-1B6, 1B-1A8 and 3B-1C7 were screened.
  • the nucleotide sequence of the VHH of 1B-3B11 is shown in SEQ ID NO:23, the amino acid sequence is shown in SEQ ID NO:24; the nucleotide sequence of the VHH of 1B-1B6 is shown in SEQ ID NO:24 As shown in ID NO:25, the amino acid sequence is shown in SEQ ID NO:26; the nucleotide sequence of the VHH of 1B-1C7 is shown in SEQ ID NO:27, and the amino acid sequence is shown in SEQ ID NO:28;
  • the nucleotide sequence of the VHH of 1B-1A8 is shown in SEQ ID NO:29, and the amino acid sequence is shown in SEQ ID NO:30; the nucleotide sequence of the VHH of 1B-1B2 is shown in SEQ ID NO:31 As shown, the amino acid sequence is shown
  • VHH sequences of the screened positive clones were connected to the human Fc region to construct a VHH -Fc chimeric antibody. Specifically, insert the V H H sequence obtained by sequencing in 2.2 into the pCDNA3.1 eukaryotic expression vector containing the human IgG1 Fc region, and use the Expifectamine TM CHO Transfection Kit transient expression system (Thermo Fisher Scientific Inc., catalog No.A29129 ) to express these VHH -Fc chimeric antibodies.
  • the full-length amino acid sequence of 1B-3B11-Fc is shown in SEQ ID NO:37, and the nucleotide sequence is shown in SEQ ID NO:36; the full-length amino acid sequence of 1B-1B6-Fc is shown in SEQ ID NO :39, the nucleotide sequence is as shown in SEQ ID NO:38; the full-length amino acid sequence of 1B-1C7-Fc is as shown in SEQ ID NO:41, and the nucleotide sequence is as shown in SEQ ID NO:40;
  • the full-length amino acid sequence of 1B-1A8-Fc is shown in SEQ ID NO:43, and the nucleotide sequence is shown in SEQ ID NO:42; the full-length amino acid sequence of 1B-1B2-Fc is shown in SEQ ID NO:45
  • the nucleotide sequence is shown in SEQ ID NO:44; the full-length amino acid sequence of 3B-1C7-Fc is shown in SEQ ID NO:47, and the nucle
  • Affinity detection of anti-human c-Met V H H-Fc chimeric antibody was performed using Biomolecular Interaction Analysis System (GE, Biacore T200).
  • Amino-conjugated Anti-hIgG (Fc) antibody (GE, catalog No. BR-1008-39) to CM5 sensor chip, using running buffer (137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 12H 2 O , 1.8mM KH 2 PO 4 , 0.05% surfactant P-20 (w/v), pH 7.4) diluted anti-human c-Met V H H-Fc chimeric antibody to 1 ⁇ g/mL, 30 ⁇ l/min flow rate through the experimental channel capture.
  • the software BiaControl Software 2.0 collects data signals in real time, the software BiaEvaluation Software 2.0 data analysis, and uses Langmuir 1:1 model fitting to calculate the association rate constant K a (1/Ms), dissociation rate constant K d (1/s), equilibrium Dissociation constant K D (M) value.
  • the detection results are shown in Table 1, 1B-1B2-Fc, 1B-3B11-Fc, 1B-1C7-Fc, 1B-1B6-Fc, 1B-1A8-Fc, 3B-1C7-Fc and human c-Met protein were all They have high affinity and all have cross-reaction with cynomolgus monkey c-Met protein.
  • Example 4 Binding of anti-human c-Met V H H-Fc chimeric antibody to natural cell lines expressing c-Met
  • Flow cytometry was used to detect the binding of anti-human c-Met V H H-Fc chimeric antibody to target cells with different expression levels of c-Met, among which H1993 cells (Beina Biotechnology, product catalog BNCC342186) had high expression of c-Met Level of human lung adenocarcinoma cell line, MKN45 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog CBP60541) is a human gastric cancer cell line with medium expression level of c-Met, KP4 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog CBP60541) is a human pancreatic cancer cell line with low expression level of c-Met.
  • Example 5 Blocking of receptor/ligand binding by anti-human c-Met V H H-Fc chimeric antibody and antibody epitope competition analysis
  • HGF ⁇ HGF ⁇ chain
  • NP_000592.3 Val495-Ser7228
  • IC 50 values were calculated (Table 4).
  • 1B-3B11-Fc, 1B-1B2-Fc, 1B-1C7-Fc, 1B-1B6-Fc, 1B-1A8-Fc, 3B-1C7-Fc can all block HGF and c-Met.
  • 1B-3B11-Fc, 1B-1C7-Fc, 1B-1B6-Fc, 1B-1A8-Fc can block the binding of HGF ⁇ to c-Met receptor
  • 1B-1B2-Fc, 3B-1C7- Fc cannot block the binding of HGF ⁇ to c-Met receptor.
  • Epitope competition analysis was performed using Biomolecular Interaction System (Fortebio, catalog Octet RED96).
  • Use Anti-Penta-HIS (HIS1K) sensor (Fortebio, product catalog No.18-5120), use running buffer to dilute c-Met-His protein (SinoBiological, product catalog 10692-H08H) to about 5 ⁇ g/mL, the sensor Immerse in the diluted antigen sample, and control the solidification height at about 1nm by adjusting the binding time. Then interact with the first antibody A and the second antibody B in sequence, and detect the binding signal of the B antibody to determine whether the two antibodies recognize the same epitope. The results are shown in Table 5-1 and Table 5-2.
  • the judgment criteria are: a value >60% indicates that the two antibodies do not compete at all; a value between 20% and 60% indicates that the two antibodies partially compete (possibly the epitope has Crossover); values ⁇ 20% indicate perfect competition between the 2 antibodies. If the self-reaction signal (gray part) ⁇ 20%, the data is judged to be valid.
  • antibodies 1B-3B11-Fc and 1B-1B2-Fc, 1B-3B11-Fc and 1B-1C7-Fc, 1B-3B11-Fc and 1B-1B6-Fc, 1B-1A8-Fc and 1B-1B2-Fc , 1B-1A8-Fc and 1B-1C7-Fc, 1B-1A8-Fc and 1B-1B6-Fc, 1B-1B2-Fc and 1B-1C7-Fc, 1B-1B2-Fc and 1B-1B6-Fc, 1B - 1C7-Fc and 1B-1B6-Fc can simultaneously bind to different epitopes of the c-Met antigen.
  • Example 6 Inhibition of c-Met phosphorylation in H441 cells by anti-human c-Met V H H H-Fc chimeric antibody
  • H441 cells 100 ⁇ l/well, 10000cells/well inoculated into 96-well plate, after incubation for 6-16h, starve overnight in serum-free medium, discard the supernatant, add 45 ⁇ l/well anti-human c-Met V H H-Fc chimeric antibody (initial concentration 1000nM, 10-fold serial dilution, 7 concentrations), incubated for 30min, then added 5 ⁇ l HGF to stimulate for 15min. Discard the supernatant, add 100 ⁇ l of freshly prepared 1 ⁇ lysis buffer, incubate for 10 min, and use the AlphaLISA module to read the plate.
  • Example 7 Proliferation inhibition of KP4 cells by anti-human c-Met V H H-Fc chimeric antibody
  • KP4 cells were added to a 96-well cell culture plate at 1000 cells/well, and cultured in a 37°C, 5% CO 2 carbon dioxide cell incubator. After the cells adhered to the wall, anti-human c-Met V H H-Fc Chimeric antibody (initial concentration 900nM, 2-fold serial dilution, 7 concentrations) was added to the cells, and after 120 h of incubation, 10 ⁇ l of CCK-8 solution was added to each well, and after incubation for 1 h, the absorbance at 450 nm was measured with a microplate reader.
  • anti-human c-Met V H H-Fc Chimeric antibody initial concentration 900nM, 2-fold serial dilution, 7 concentrations
  • Proliferation inhibition rate 100% - cell survival rate.

Abstract

涉及结合c-Met的抗体及其应用；具体地，提供了结合c-Met的单可变结构域、抗体或其抗原结合片段、或者融合蛋白；以及编码它们的核酸、包含该核酸的载体、包含该载体的细胞、包含它们的药物组合物，还提供了其检测、制药和治疗用途。

Description

结合c-Met的抗体及其应用 技术领域
本公开涉及分子生物技术领域，具体涉及结合c-Met的抗体或其抗原结合片段及其应用。
背景技术
间质表皮转化因子(c-Mesenchymal-epithelial transition factor，c-Met)是受体酪氨酸激酶的一种，其是由50kDa的胞外链(α链)与145kDa的跨膜链(β链)连结而成的约190kDa的异二聚体。跨膜链(β链)包括SEMA结构域(sema homology region，SEMA)、PSI结构域(plexin semaphorin-integrin，PSI)、4个免疫球蛋白样重复结构域(immunoglobulin-like regions in plexins and transcription factors，IPT)、一个跨膜域、一个近膜域(juxtamembrane domain，JM)、酪氨酸激酶结构域(tyrosine kinase，TK)和一个羧基末端的尾部区域(Carboxyl terminal，CT)。c-Met是表达在细胞表面的受体，其中SEMA结构域是配体结合的重要元素之一，被认为是其配体肝细胞生长因子(hepatocyte growth factor，HGF)的结合位点，HGF由间充质细胞、成纤维细胞和平滑肌细胞合成，通过旁分泌机制激活HGF/c-Met信号，发挥其生物学功能。
在多种肿瘤中，由于c-Met和HGF的过表达，且HGF和c-Met形成的旁分泌和自分泌正反馈回路，造成HGF/c-Met信号通路异常活化，促进了肿瘤细胞的生长、侵袭、迁移和血管新生。许多癌种类型中都存在c-Met基因的异常表达，诸如脑癌、乳腺癌、结直肠癌、胃癌、头颈癌、肺癌、肝癌等。免疫治疗剂如与c-Met结合的抗体可阻断HGF和c-Met之间的结合。作为潜在的治疗靶点，已有一些靶向c-Met的抗体被开发，但仍然是有限的，需要更多可用的选择。
骆驼科动物体内的IgG抗体，除传统四链结构的IgG1外，还天然存在不含有轻链的仅重链抗体(HcAb)IgG2和IgG3。仅重链抗体中的单个可变区结构域(V HH)能特异性结合抗原，且对抗原具有较高的亲和力。基于其独特性，使用V HH结构域作为抗体或抗原结合片段的一部分，具有比常规抗体片段(如scFv、Fab等)更加显著的优势，例如仅需要单一结构域以高亲和力特异结合抗原、易于改造成多价及多特异性形式、V HH结构域高度可溶且无聚集趋势、V HH分子小从而具有较高的组织渗透性、V HH不需要与轻链配对、在组成双特异性抗体或多特异性抗体时不存在轻重链错配问题，等等。
发明内容
本公开提供了结合c-Met的单可变结构域、抗体或其抗原结合片段、或者融合蛋白。本公开还提供了编码所提供的单可变结构域、抗体或其抗原结合片段、或者融合蛋白的核酸、载体、细胞、制备方法、组合物及用途。
一方面，本公开提供了结合c-Met的分离的单可变结构域，所述单可变结构域包含SEQ ID NO:24、26、28、30、32或34所示氨基酸序列的CDR1、CDR2和CDR3。
一方面，本公开提供了结合c-Met的分离的单可变结构域，所述单可变结构域包含以下互补决定区：(a)CDR1，其包含SEQ ID NO:5、8、11、14、17或20所示的氨基酸序列；(b)CDR2，其包含SEQ ID NO:6、9、12、15、18或21所示的氨基酸序列；和(c)CDR3，其包含SEQ ID NO:7、10、13、16、19或22所示的氨基酸序列。
一方面，本公开提供了结合c-Met的分离的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含单可变结构域，其中所述单可变结构域包含SEQ ID NO:24、26、28、30、32或34所示氨基酸序列的CDR1、CDR2和CDR3。
一方面，本公开提供了结合c-Met的分离的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含单可变结构域，其中所述单可变结构域包含以下互补决定区：(a)CDR1，其包含SEQ ID NO:5、8、11、14、17或20所示的氨基酸序列；(b)CDR2，其包含SEQ ID NO:6、9、12、15、18或21所示的氨基酸序列；和(c)CDR3，其包含SEQ ID NO:7、10、13、16、19或22所示的氨基酸序列。
一方面，本公开提供了结合c-Met的分离的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含单可变结构域，其中所述单可变结构域与本公开的单可变结构域中任一者结合相同表位。
一方面，本公开提供了结合c-Met的分离的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含单可变结构域，其中所述单可变结构域与本公开的单可变结构域中任一者竞争结合c-Met。
在一些实施方案中，所述单可变结构域是V HH，优选是骆驼科动物的V HH或人源化的V HH。
在一些实施方案中，所述抗体或其抗原结合片段是嵌合的或人源化的。在一些实施方案中，所 述单可变结构域是骆驼科动物的或人源化的。
在一些实施方案中，所述抗体或其抗原结合片段包含IgG的Fc。在一些实施方案中，所述抗体或其抗原结合片段包含人IgG的Fc，优选为人IgG1、IgG2、IgG3或IgG4的Fc。
一方面，本公开提供了融合蛋白，其包含本公开的抗体或其抗原结合片段。
一方面，本公开提供了药物组合物，其包含本公开的抗体或其抗原结合片段，以及药学上可接受的载体。
一方面，本公开提供了分离的核酸，其编码本公开的抗体或其抗原结合片段。
一方面，本公开提供了载体，其包含本公开的分离的核酸。
一方面，本公开提供了宿主细胞，其包含本公开的分离的核酸或载体。
一方面，本公开提供了制备本公开的抗体或其抗原结合片段的方法，包括：培养本公开的宿主细胞，分离所表达的所述抗体或其抗原结合片段，其中载体为表达载体。
另一方面，本公开提供了本公开的抗体或其抗原结合片段在制备治疗患有肿瘤的受试者的药物中的用途。
另一方面，本公开提供了本公开的融合蛋白在制备治疗患有肿瘤的受试者的药物中的用途。
另一方面，本公开提供了本公开的药物组合物在制备治疗患有肿瘤的受试者的药物中的用途。
另一方面，本公开提供了检测或测量样品中的c-Met的方法，其包括使所述样品与本公开的抗体或其抗原结合片段接触并且检测或测量结合复合物。
另一方面，本公开提供了抑制、减少或阻断细胞中的c-Met信号传导的方法，其包括向所述细胞施用有效量的本公开的抗体或其抗原结合片段、融合蛋白、或者药物组合物。
另一方面，本公开提供了抑制肿瘤细胞生长或杀伤肿瘤细胞的方法，其包括向所述肿瘤细胞施用有效量的本公开的抗体或其抗原结合片段、融合蛋白、或者药物组合物。
另一方面，本公开提供了治疗患有肿瘤的受试者的方法，其包括向所述受试者施用治疗有效量的本公开的抗体或其抗原结合片段、融合蛋白、或者药物组合物。
附图说明
图1A为流式细胞术检测抗人c-Met V HH-Fc嵌合抗体与H1993细胞的结合曲线；
图1B为流式细胞术检测抗人c-Met V HH-Fc嵌合抗体与MKN45细胞的结合曲线；
图1C为流式细胞术检测抗人c-Met V HH-Fc嵌合抗体与KP4细胞的结合曲线；
图1D为流式细胞术检测抗人c-Met V HH-Fc嵌合抗体与4MBr-5细胞的结合曲线；
图2A为竞争ELISA法检测抗人c-Met V HH-Fc嵌合抗体对HGF与c-Met结合的阻断作用的结果；
图2B为竞争ELISA法检测抗人c-Met V HH-Fc嵌合抗体对HGFβ与c-Met结合的阻断作用的结果；
图3为抗人c-Met V HH-Fc嵌合抗体对H441细胞中HGF刺激c-Met磷酸化的抑制作用的检测结果；
图4为CCK8法检测抗人c-Met V HH-Fc嵌合抗体对KP4细胞增殖的抑制作用的结果；
具体实施方式
定义
除非另有说明，本公开中所用的下列术语具有下列含义。一个特定的术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通的含义去理解。
术语“抗体”以最广泛的含义使用，涵盖包括但不限于单克隆抗体、多克隆抗体和多特异性抗体(例如双特异性抗体、三特异性抗体)的多种抗体结构，只要它们显示希望的抗原结合活性。
“抗原结合片段”是指保留全长抗体的抗原结合功能的片段，包括Fab、Fab’、F(ab’) 2、scFv、Fv、Fd、Fd’、单可变结构域(例如V HH)和本领域己知的其他抗体片段，或将上述片段进行任何本领域己知修饰的片段。
术语“可变结构域”或“可变区”是指抗体的涉及该抗体与抗原结合的结构域。天然抗体的每个可变结构域基本上由四个骨架区和三个互补决定区组成。四个“骨架区”在本领域中和在本文中分别称为“骨架区1”或“FR1”、“骨架区2“或“FR2”、“骨架区3”或“FR3”、及“骨架区4”或“FR4”；所述骨架区由本领域及本文中分别称为“互补决定区1“或“CDR1”、“互补决定区2“或“CDR2”、及“互补决定区3”或“CDR3”的三个互补决定区CDR间隔开。因此，可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。可变结构域因具有抗原结合 位点而赋予抗体对抗原的特异性。
术语“单可变结构域”是指具有能够在不与其他可变结构域配对的情况下特异性结合抗原表位的可变结构域。单可变结构域的抗原结合位点通常由三个CDR(CDR1、CDR2和CDR3)形成，存在于单个结构域上。在一些情况下，单可变结构域可以是重链可变结构域(例如V H)或其合适的片段；只要它能够形成单个抗原结合单元(即基本上由单可变结构域组成的功能性抗原结合单元，这样单抗原结合结构域就不需要与另一个可变结构域相互作用即可形成功能性抗原结合单元)。单可变结构域的另一个实例为骆驼科的“V HH结构域”，亦称为VHH结构域、V HH或VHH。
使用术语“V HH结构域”以将这些可变结构域与存在于常规四链抗体中的重链可变结构域(其在本文中称为“V H结构域”或“V H)以及存在于常规四链抗体中的轻链可变结构域(其在本文中称为“V L结构域”或“V L”)进行区分。V HH结构域特异性结合表位而无需其他抗原结合结构域(此与常规四链抗体中的V H或V L结构域不同，在该情况下表位由V L结构域与V H结构域一起识别)。V HH结构域为由单一结构域形成的小型稳定及高效的抗原识别单元。
术语“骨架区”(FR)或“框架区”残基是除了本文定义的CDR残基之外的那些可变结构域的氨基酸残基。
“互补决定区”(CDR)，也称为“高变区”(HVR)。天然四链抗体通常包含六个CDR，三个在重链可变区中(重链CDR1、重链CDR2和重链CDR3)，三个在轻链可变区中(轻链CDR1、轻链CDR2和轻链CDR3)。仅重链抗体或单可变结构域通常具有三个CDR(CDR1、CDR2和CDR3)。CDR3在三种CDR中最具多样性，并且据信在赋予抗体精细特异性方面发挥了独特的作用。
当前有许多方法来划分定义CDR。其中，Kabat定义基于序列可变性划分CDR，并且是最常用的(Elvin A.Kabat,et al,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,Md.(1991))；而Chothia定义则基于结构环的位置(Cyrus Chothia,et al,Canonical Structures for the Hypervariable Regions of Immunoglobulins,J.Mol.Biol.196:901-917(1987))。AbM定义是Kabat定义和Chothia定义之间的折衷方案，并且被Oxford Molecular的AbM抗体建模软件使用。“接触(contact)”定义CDR的基础是对可用的复合物晶体结构的分析。然而，应当注意的是，基于不同的方法划分定义获得的同一抗体可变区的CDR的边界可能有所差异，即不同方法划分定义的同一抗体可变区的CDR序列可能有所不同。因此，在涉及用本公开某些划分定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了转换为其他任意定义(例如IMGT、Chothia、AbM等定义中的一种或几种的结合)的CDR序列限定的抗体。
术语“人共有框架区”或“受体人框架”是代表人免疫球蛋白V L或V H框架区序列的选择中最常出现的氨基酸残基的框架。一般而言，人免疫球蛋白V L或V H序列的选择来自可变结构域序列的亚组。一般而言，序列的亚组是Kabat等人,Sequences of Proteins of ImmunologicalInterest,第5版,Public Health Service,National Institutes of Health,Bethesda,Md.(1991)中的亚组。实例包括：对于V L，亚组可以是Kabat等人(出处同上)所述的亚组κI、κII、κIII或κIV。另外，对于V H，亚组可以是Kabat等人所述的亚组I、亚组II或亚组III。或者，人共有区框架可衍生自上文特定的残基，比如，通过将供体框架区序列与各种人框架区序列的集合进行比对，当根据人框架区残基与供体框架区的同源性来选择人框架区残基时“衍生自”人共有框架区的受体人框架可包含其相同的氨基酸序列，或它可含有预先存在的氨基酸序列变化。在一些实施方案中，预先存在的氨基酸变化的数目为10或更小、9或更小、8或更小、7或更小、6或更小、5或更小、4或更小、3或更小或2或更小。
术语“Fc结构域”或“Fc”用来定义免疫球蛋白重链的C端区域，其包含至少部分恒定区。该术语包括天然序列Fc和变体Fc。Fc的C末端赖氨酸(根据EU编号系统的Lys447)可存在或不存在。
术语EC 50是指有效浓度，某化合物分子(例如，抗体或其抗原结合片段)的50％最大应答。术语“IC 50”是指抑制浓度，某化合物分子(例如，抗体或其抗原结合片段)的50％最大应答。EC 50和IC 50均可以通过ELISA或FACS分析或本领域已知的任何其他方法进行测量。
术语“K a”或“结合速率常数”是指某化合物分子(例如，抗体或其抗原结合片段)与抗原结合以形成复合物的结合速率常数。术语“K d”或“解离速率常数”是指某化合物分子(例如，抗体或其抗原结合片段)从复合物中解离的解离速率常数。术语“K D”或“平衡解离常数”是指在滴定测量中在平衡时、或者通过将解离速率常数(K d)除以结合速率常数(K a)所获得的值。使用K a、K d和K D表示某化合物分子和抗原之间的结合亲和力。确定K a、K d和KD的值的方法为本领域熟知。
术语“药学上可接受的”指不消除本文所述的化合物的生物学活性或性质的物质，如载体或稀释剂。这类物质被施用于个体不导致不希望的生物学作用或者不以有害方式与包含它的组合物中的任何组分相互作用。
术语“药学上可接受的载体”包括任何和所有的溶剂、分散介质、包衣材料、表面活性剂、抗氧化剂、防腐剂(例如抗菌剂、抗真菌剂)、等渗剂、吸收延迟剂、盐、防腐剂、药物稳定剂、粘合剂、赋形剂、崩解剂、润滑剂、甜味剂、矫味剂、染料等和其组合，这是本领域技术人员所熟知的(Remington's Pharmaceutical Sciences,18th Ed.Mack Printing Company,1990,pp.1289-1329)。除了与活性成分不相容的载体外，在治疗或药物组合物中考虑使用任何常规载体。
术语“治疗”指试图改变治疗个体中疾病的自然进程，并且可以是为了预防或在临床病理学的过程期间实施的临床干预。治疗的期望效果包括但不限于预防疾病的发生或复发，缓解症状，降低疾病的任何直接或间接病理学后果，预防转移，减缓疾病进展率，改善或减轻疾病状态，及消退或改善的预后。
如本文所用，术语“治疗有效量”是指向受试者提供治疗性益处所必需的化合物、药物组合物或药物组合的量。
术语“受试者”包括任何人类或非人动物。术语“非人动物”包括所有的脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类、绵羊、犬、猫、马、牛、鸡、两栖动物、爬行动物等。优选地，根据本公开的受试者是人。除非标明，术语“患者”或“受试者”可以互换使用。
术语“特异性结合”或“特异性的结合”意为结合对于抗原而言是选择性的并且可以与不需要或非特异性的相互作用区别开。某化合物分子(例如，抗体或其抗原结合片段)与特定抗原决定簇的结合能力可通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其它技术测量，例如表面等离子体共振(SPR)技术(在Biacore仪器上分析)。
术语“分离的”是指已经从其天然环境中分离的目标化合物，例如抗体或其抗原结合片段、V HH或核酸。
术语序列“同一性”，也称一致性。两序列间的百分比同一性为序列共有的相同位置数的函数(即％同一性＝相同位置数/位置总数×100)，其中需考虑产生两序列的最优比对需要引入的缺口数和每个缺口的长度。如下述非限制性实施例所示，可以使用数学算法完成序列的比较和两序列间百分比同一性的测定。可以使用E.Meyers和W.Miller的算法(Comput.Appl.Biosci.,4:11-17(1988))测定两氨基酸序列间的百分比同一性，该算法已收入到ALIGN程序(版本2.0)中，其使用PAM120残基权重表，缺口长度罚分为12，缺口罚分为4。此外，可以使用Needleman和Wunsch的算法(J.Mol.Biol.484 453(1970))测定两氨基酸序列的百分比同一性，该算法已掺入到GCG软件包(可在www.gcg.com获得)中的GAP程序中，其使用Blossum 62矩阵或PAM250矩阵，缺口权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6。
如本文所用，“约”表示在本领域普通技术人员判定的对特定值可以接受的误差范围内，其部分取决于如何测量或测定该值，即测量系统的限制。例如，“约”按照本领域实践可表示1倍或超过1倍标准偏差以内。或者，约可以表示多至20％(即±20％)的范围，例如在所给定的具体数值范围±10％范围、±5％范围、±2％范围内、±1％范围内或±0.5％范围内波动。此外，特别对于生物学系统或方法，该术语可以表示多至一个数量级或多至某值的5倍。当本公开或权利要求中给出特定值时，除非另有说明，“约”的含义应认为是在该特定值的可接受的误差范围内。
蛋白中的氨基酸残基缩写如下：苯丙氨酸是Phe或F；亮氨酸是Leu或L；异亮氨酸是Ile或I；甲硫氨酸是Met或M；缬氨酸是Val或V；丝氨酸是Ser或S；脯氨酸是Pro或P；苏氨酸是Thr或T；丙氨酸是Ala或A；酪氨酸是Tyr或Y；组氨酸是His或H；谷氨酰胺是Gln或Q；天冬酰胺是Asn或N；赖氨酸是Lys或K；天冬氨酸是Asp或D；谷氨酸是Glu或E；半胱氨酸是Cys或C；色氨酸是Trp或W；精氨酸是Arg或R；甘氨酸是Gly或G。
本公开的各个方面将在下述部分中进一步详细描述。
单可变结构域
本公开提供了结合c-Met(诸如人c-Met)的分离的单可变结构域。单可变结构域为靶向c-Met药物的开发或药物构建提供了更多可用的选择。所述单可变结构域具有诸多期望的治疗特性，例如对人c-Met具有良好的亲和力、能够阻断配体HGF或其β链与c-Met的结合、抑制HGF刺激引起的c-Met磷酸化等中的一种或几种。
在一些实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:24所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:24所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:26所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:26所示可变区序列的CDR1、CDR2和CDR3。 在一些实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:28所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:28所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:30所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:30所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:32所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:32所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:34所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:34所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述单可变结构域是骆驼科动物的。在一些实施方案中，所述单可变结构域是人源化的。在一些实施方案中，所述单可变结构域包含受体人框架。
在一些实施方案中，所述单可变结构域结合c-Met，其包含选自以下的至少一个、至少两个或全部三个CDR：(a)包含SEQ ID NO:5、8、11、14、17或20所示氨基酸序列的CDR1；(b)包含SEQ ID NO:6、9、12、15、18或21所示氨基酸序列的CDR2；和(c)包含SEQ ID NO:7、10、13、16、19或22所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含以下互补决定区：(a)CDR1，其包含SEQ ID NO:5、8、11、14、17或20所示的氨基酸序列；(b)CDR2，其包含SEQ ID NO:6、9、12、15、18或21所示的氨基酸序列；和(c)CDR3，其包含SEQ ID NO:7、10、13、16、19或22所示的氨基酸序列。在一些实施方案中，所述单可变结构域是骆驼科动物的。在一些实施方案中，所述单可变结构域是人源化的。在一些实施方案中，所述单可变结构域包含受体人框架。
在一些实施方案中，所述单可变结构域结合c-Met，其包含：包含SEQ ID NO:5所示氨基酸序列的CDR1，包含SEQ ID NO:6所示氨基酸序列的CDR2，和包含SEQ ID NO:7所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：包含SEQ ID NO:8所示氨基酸序列的CDR1，包含SEQ ID NO:9所示氨基酸序列的CDR2，和包含SEQ ID NO:10所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：包含SEQ ID NO:11所示氨基酸序列的CDR1，包含SEQ ID NO:12所示氨基酸序列的CDR2，和包含SEQ ID NO:13所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：包含SEQ ID NO:14所示氨基酸序列的CDR1，包含SEQ ID NO:15所示氨基酸序列的CDR2，和包含SEQ ID NO:16所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：包含SEQ ID NO:17所示氨基酸序列的CDR1，包含SEQ ID NO:18所示氨基酸序列的CDR2，和包含SEQ ID NO:19所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：包含SEQ ID NO:20所示氨基酸序列的CDR1，包含SEQ ID NO:21所示氨基酸序列的CDR2，和包含SEQ ID NO:22所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域是骆驼科动物的。在一些实施方案中，所述单可变结构域是人源化的。在一些实施方案中，所述单可变结构域包含受体人框架。
在一些实施方案中，所述单可变结构域结合c-Met，其包含：SEQ ID NO:5所示氨基酸序列的CDR1，SEQ ID NO:6所示氨基酸序列的CDR2，和SEQ ID NO:7所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：SEQ ID NO:8所示氨基酸序列的CDR1，SEQ ID NO:9所示氨基酸序列的CDR2，和SEQ ID NO:10所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：SEQ ID NO:11所示氨基酸序列的CDR1，SEQ ID NO:12所示氨基酸序列的CDR2，和SEQ ID NO:13所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：SEQ ID NO:14所示氨基酸序列的CDR1，SEQ ID NO:15所示氨基酸序列的CDR2，和SEQ ID NO:16所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：SEQ ID NO:17所示氨基酸序列的CDR1，SEQ ID NO:18所示氨基酸序列的CDR2，和SEQ ID NO:19所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域结合c-Met，其包含：SEQ ID NO:20所示氨基酸序列的CDR1，SEQ ID NO:21所示氨基酸序列的CDR2，和SEQ ID NO:22所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域是骆驼科动物的。在一些实施方案中，所述单可变结构域是人源化的。在一些实施方案中，所述单可变结构域包含受体人框架。
在一些实施方案中，所述单可变结构域结合c-Met，其包含与SEQ ID NO:24、26、28、30、32或34所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；任选地，所述单可变结构域包含上述特定CDR1、 CDR2和/或CDR3序列。
在一些实施方案中，所述单可变结构域结合c-Met，其包含与SEQ ID NO:24所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:5所示氨基酸序列的CDR1，包含SEQ ID NO:6所示氨基酸序列的CDR2，和包含SEQ ID NO:7所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域结合c-Met，其包含与SEQ ID NO:26所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:8所示氨基酸序列的CDR1，包含SEQ ID NO:9所示氨基酸序列的CDR2，和包含SEQ ID NO:10所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域结合c-Met，其包含与SEQ ID NO:28所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:11所示氨基酸序列的CDR1，包含SEQ ID NO:12所示氨基酸序列的CDR2，和包含SEQ ID NO:13所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域结合c-Met，其包含与SEQ ID NO:30所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:14所示氨基酸序列的CDR1，包含SEQ ID NO:15所示氨基酸序列的CDR2，和包含SEQ ID NO:16所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域结合c-Met，其包含与SEQ ID NO:32所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:17所示氨基酸序列的CDR1，包含SEQ ID NO:18所示氨基酸序列的CDR2，和包含SEQ ID NO:19所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域结合c-Met，其包含与SEQ ID NO:34所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:20所示氨基酸序列的CDR1，包含SEQ ID NO:21所示氨基酸序列的CDR2，和包含SEQ ID NO:22所示氨基酸序列的CDR3。
在一些实施方案中，具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的单可变结构域氨基酸序列相对于参考序列含有取代(例如，保守取代)、插入或缺失，但包含该序列的单可变结构域保留了结合c-Met的能力。在一些实施方案中，在选自SEQ ID NO:24、26、28、30、32和34所示的氨基酸序列中总共1-18、1-16、1-14、1-12、1-10、1-9、1-8、1-7、1-6、1-5、1-4、1-3或1-2个氨基酸被取代、插入和/或缺失。在一些实施方案中，取代、插入或缺失发生在CDR之外的区域中(即，FR中)。在一些实施方案中，取代、插入或缺失发生在CDR区域，例如发生CDR1、CDR2、CDR3中的一个、两个或三个。在一些实施方案中，取代、插入或缺失发生在CDR区域和非CDR区域。任选地，所述单可变结构域包含选自SEQ ID NO:24、26、28、30、32和34所示的氨基酸序列，包括该序列的翻译后修饰。
在一些实施方案中，所述单可变结构域结合c-Met，其包含SEQ ID NO:24、26、28、30、32或34所示的氨基酸序列。
在一些实施方案中，所述单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)是V HH。在一些具体的实施方案中，V HH从N末端至C末端具有以下结构：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
在一些实施方案中，所述单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)是人源化的V HH。源自非人的V HH可通过以人常规四链抗体V H结构域中相应位置处存在的一个或多个氨基酸残基置换原始V HH氨基酸序列中的一个或多个氨基酸残基而经“人源化”。人源化可以期望的降低免疫原性。
在一些实施方案中，所述单可变结构域结合c-Met，其氨基酸序列如SEQ ID NO:24、26、28、30、32或34所示。
本公开还提供了与本公开的单可变结构域中任一者结合相同表位的单可变结构域。在一些具体 的实施方案中，所述单可变结构域与包含SEQ ID NO:24、26、28、30、32或34所示氨基酸序列的单可变结构域结合相同的表位。在一些实施方案中，所述结合相同表位的单可变结构域是骆驼科动物的或人源化的。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选单可变结构域。例如，可进行竞争研究，以获得彼此竞争与抗原结合的单可变结构域。因此，本公开还提供了与本公开所述的单可变结构域中任一者竞争结合c-Met的单可变结构域。在一些具体的实施方案中，所述单可变结构域与包含SEQ ID NO:24、26、28、30、32或34所示氨基酸序列的单可变结构域竞争结合c-Met。可以通过ELISA、流式细胞术、表面等离子体共振(SPR)测定或本领域已知的任何其他方法测量与c-Met的结合。在一些实施方案中，所述竞争结合c-Met的单可变结构域是骆驼科动物的或人源化的。
在一些实施方案中，根据上述实施方案中的任一种的单可变结构域可单独地或组合地包括下述性质特征中的一种或几种：
(i)结合人c-Met或/和猴c-Met；
(ii)阻断HGF或其β链与c-Met的结合；或
(iii)抑制HGF刺激引起的c-Met磷酸化。
在一些实施方案中，所述单可变结构域结合c-Met。在一些实施方案中，所述单可变结构域阻断HGF或其β链与c-Met的结合。在一些实施方案中，所述单可变结构域抑制HGF刺激引起的c-Met磷酸化。
本公开提供了一些示例性的结合c-Met的单可变结构域。本公开提供的示例性的单可变结构域的CDR(CDR1、CDR2和CDR3)的氨基酸序列提供于下表S1中，示例性的单可变结构域的全长氨基酸序列提供于下表S2中。
表S1：示例性的单可变结构域CDR序列
Figure PCTCN2022129875-appb-000001
表S2：示例性的单可变结构域全长序列
Figure PCTCN2022129875-appb-000002
Figure PCTCN2022129875-appb-000003
抗体或其抗原结合片段
本公开提供了结合c-Met(诸如人c-Met)的分离的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含至少一个(一个或多个)本公开的单可变结构域。
本公开提供的结合c-Met的抗体或其抗原结合片段具有诸多期望的治疗特性，例如对人c-Met具有良好的亲和力、能够阻断配体HGF或其β链与c-Met的结合、抑制HGF刺激引起的c-Met磷酸化、对不同c-Met表达水平的细胞具有良好的结合性能、杀伤肿瘤细胞等中的一种或几种。
在一些实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:24所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:24所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:26所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:26所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:28所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:28所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:30所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:30所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:32所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:32所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:34所示可变区序列的一个、两个或全部三个CDR。在一个具体的实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含SEQ ID NO:34所示可变区序列的CDR1、CDR2和CDR3。在一些实施方案中，所述单可变结构域是骆驼科动物的。在一些实施方案中，所述单可变结构域是人源化的。在一些实施方案中，所述单可变结构域包含受体人框架。
在一些实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含选自以下的至少一个、至少两个或全部三个CDR：(a)包含SEQ ID NO:5、8、11、14、17或20所示氨基酸序列的CDR1；(b)包含SEQ ID NO:6、9、12、15、18或21所示氨基酸序列的CDR2；和(c)包含SEQ ID NO:7、10、13、16、19或22所示氨基酸序列的CDR3。在一些实施方案中，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个(一个或多个)单可变结构域，所述单可变结构域包含以下互补决定区：(a)CDR1，其包含SEQ ID NO:5、8、11、14、17或20所示的氨基酸序列；(b)CDR2，其包含SEQ ID NO:6、9、12、15、18或21所示的氨基酸序列；和(c)CDR3，其包含SEQ ID NO:7、10、13、16、19或22所示的氨基酸序列。在一些实施方案中，所述单可变结构域是骆驼科动物的。在一些实施方案中，所述单可变结构域是人源化的。在一些实施方案中，所述单可变结构域包含受体人框架。
在一些实施方案中，所述单可变结构域包含：(a)包含SEQ ID NO:5所示氨基酸序列的CDR1，包含SEQ ID NO:6所示氨基酸序列的CDR2，和包含SEQ ID NO:7所示氨基酸序列的CDR3；(b)包 含SEQ ID NO:8所示氨基酸序列的CDR1，包含SEQ ID NO:9所示氨基酸序列的CDR2，和包含SEQ ID NO:10所示氨基酸序列的CDR3；(c)包含SEQ ID NO:11所示氨基酸序列的CDR1，包含SEQ ID NO:12所示氨基酸序列的CDR2，和包含SEQ ID NO:13所示氨基酸序列的CDR3；(d)包含SEQ ID NO:14所示氨基酸序列的CDR1，包含SEQ ID NO:15所示氨基酸序列的CDR2，和包含SEQ ID NO:16所示氨基酸序列的CDR3；(e)包含SEQ ID NO:17所示氨基酸序列的CDR1，包含SEQ ID NO:18所示氨基酸序列的CDR2，和包含SEQ ID NO:19所示氨基酸序列的CDR3；或(f)包含SEQ ID NO:20所示氨基酸序列的CDR1，包含SEQ ID NO:21所示氨基酸序列的CDR2，和包含SEQ ID NO:22所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域是骆驼科动物的。在一些实施方案中，所述单可变结构域是人源化的。在一些实施方案中，所述单可变结构域包含受体人框架。
在一些实施方案中，所述单可变结构域包含：(a)SEQ ID NO:5所示氨基酸序列的CDR1，SEQ ID NO:6所示氨基酸序列的CDR2，和SEQ ID NO:7所示氨基酸序列的CDR3；(b)SEQ ID NO:8所示氨基酸序列的CDR1，SEQ ID NO:9所示氨基酸序列的CDR2，和SEQ ID NO:10所示氨基酸序列的CDR3；(c)SEQ ID NO:11所示氨基酸序列的CDR1，SEQ ID NO:12所示氨基酸序列的CDR2，和SEQ ID NO:13所示氨基酸序列的CDR3；(d)SEQ ID NO:14所示氨基酸序列的CDR1，SEQ ID NO:15所示氨基酸序列的CDR2，和SEQ ID NO:16所示氨基酸序列的CDR3；(e)SEQ ID NO:17所示氨基酸序列的CDR1，SEQ ID NO:18所示氨基酸序列的CDR2，和SEQ ID NO:19所示氨基酸序列的CDR3；或(f)SEQ ID NO:20所示氨基酸序列的CDR1，SEQ ID NO:21所示氨基酸序列的CDR2，和SEQ ID NO:22所示氨基酸序列的CDR3。在一些实施方案中，所述单可变结构域是骆驼科动物的。在一些实施方案中，所述单可变结构域是人源化的。在一些实施方案中，所述单可变结构域包含受体人框架。
在一些实施方案中，所述单可变结构域包含与SEQ ID NO:24、26、28、30、32或34所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。在一些实施方案中，所述单可变结构域包含与SEQ ID NO:24、26、28、30、32或34所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包含上述特定CDR1、CDR2和/或CDR3序列。
在一些实施方案中，所述单可变结构域包含与SEQ ID NO:24所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:5所示氨基酸序列的CDR1，包含SEQ ID NO:6所示氨基酸序列的CDR2，和包含SEQ ID NO:7所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域包含与SEQ ID NO:26所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:8所示氨基酸序列的CDR1，包含SEQ ID NO:9所示氨基酸序列的CDR2，和包含SEQ ID NO:10所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域包含与SEQ ID NO:28所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:11所示氨基酸序列的CDR1，包含SEQ ID NO:12所示氨基酸序列的CDR2，和包含SEQ ID NO:13所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域包含与SEQ ID NO:30所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:14所示氨基酸序列的CDR1，包含SEQ ID NO:15所示氨基酸序列的CDR2，和包含SEQ ID NO:16所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域包含与SEQ ID NO:32所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:17所示氨基酸序列的CDR1，包含SEQ ID NO:18所示氨基酸序列的CDR2，和包含SEQ ID NO:19所示氨基酸序列的CDR3。
在一些实施方案中，所述单可变结构域包含与SEQ ID NO:34所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述单可变结构域包括：包含SEQ ID NO:20所示氨基酸序列的CDR1，包含SEQ ID NO:21所示氨基酸序列的CDR2，和包含SEQ ID NO:22所示氨基酸序列的CDR3。
在一些实施方案中，具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、 95％、96％、97％、98％、99％或100％同一性的单可变结构域氨基酸序列相对于参考序列含有取代(例如，保守取代)、插入或缺失，但包含含有该序列的单可变结构域的抗体或其抗原结合片段保留了结合c-Met的能力。在一些实施方案中，在选自SEQ ID NO:24、26、28、30、32和34所示的氨基酸序列中总共1-18、1-16、1-14、1-12、1-10、1-9、1-8、1-7、1-6、1-5、1-4、1-3或1-2个氨基酸被取代、插入和/或缺失。在一些实施方案中，取代、插入或缺失发生在CDR之外的区域中(即，FR中)。在一些实施方案中，取代、插入或缺失发生在CDR区域，例如发生CDR1、CDR2、CDR3中的一个、两个或三个。在一些实施方案中，取代、插入或缺失发生在CDR区域和非CDR区域。任选地，所述单可变结构域包含选自SEQ ID NO:24、26、28、30、32和34所示的氨基酸序列，包括该序列的翻译后修饰。
在一些实施方案中，所述单可变结构域包含SEQ ID NO:24、26、28、30、32或34所示的氨基酸序列。
当抗体或其抗原结合片段包含的单可变结构域是两个或两个以上时，可以选择相同或不同的单可变结构域。所述抗体可以是单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体、三特异性抗体)或本领域已知的其他构型的抗体。所述抗原结合片段可以是单可变结构域。在一个具体的实施方案中，所述抗体是单特异性抗体。在一个具体的实施方案中，所述抗体是双特异性抗体。
在一些实施方案中，所述单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)是V HH。在一些具体的实施方案中，V HH从N末端至C末端具有以下结构：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
在一些实施方案中，所述单可变结构域(包括上述任何实施方案，例如上述包含特定CDR1、CDR2和/或CDR3的单可变结构域)是人源化的V HH。源自非人的V HH可通过以人常规四链抗体V H结构域中相应位置处存在的一个或多个氨基酸残基置换原始V HH氨基酸序列中的一个或多个氨基酸残基而经“人源化”。人源化可以期望的降低免疫原性。
在一些实施方案中，所述抗体或其抗原结合片段是骆驼科动物的。在一些实施方案中，所述抗体或其抗原结合片段是嵌合的。在一些实施方案中，所述抗体或其抗原结合片段包含IgG恒定区，优选包含人IgG恒定区，更优选包含人IgG1恒定区、人IgG2恒定区、人IgG3恒定区或人IgG4恒定区。在一些实施方案中，所述抗体或其抗原结合片段包含IgG的Fc。在一些实施方案中，所述抗体或其抗原结合片段包含的人IgG的Fc；优选地，所述Fc为人IgG1、IgG2、IgG3或IgG4的Fc。在一些实施方案中，所述抗体或其抗原结合片段是人源化的。在一些实施方案中，所述抗体或其抗原结合片段包含受体人框架。
在一些实施方案中，所述抗体或其抗原结合片段包含与SEQ ID NO:37、39、41、43、45或47所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。在一些实施方案中，所述抗体或其抗原结合片段包含与SEQ ID NO:37、39、41、43、45或47所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，并且所述抗体或其抗原结合片段包含本公开所述单可变结构域中特定CDR1、CDR2和/或CDR3序列。在一些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:37、39、41、43、45或47所示的氨基酸序列。在一些实施方案中，所述抗体或其抗原结合片段包含至少一条多肽链，其中至少一条多肽链的氨基酸序列如SEQ ID NO:37、39、41、43、45或47所示。在一些实施方案中，所述抗体或其抗原结合片段包含两条相同的多肽链，其中每条多肽链的氨基酸序列如SEQ ID NO:37、39、41、43、45或47所示。
本公开还提供了与本公开的抗体或其抗原结合片段中任一者结合相同表位的抗体或其抗原结合片段。在一些具体的实施方案中，所述抗体或其抗原结合片段与包含SEQ ID NO:37、39、41、43、45或47所示氨基酸序列的抗体或其抗原结合片段结合相同的表位。在一些实施方案中，所述结合相同表位的抗体或其抗原结合片段是骆驼科动物的、嵌合的或人源化的。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体或其抗原结合片段。例如，可进行竞争研究，以获得彼此竞争与抗原结合的抗体或其抗原结合片段。因此，本公开还提供了与本公开所述的抗体或其抗原结合片段中任一者竞争结合c-Met的抗体或其抗原结合片段。在一些具体的实施方案中，所述抗体或其抗原结合片段与包含SEQ ID NO:37、39、41、43、45或47所示氨基酸序列的抗体或其抗原结合片段竞争结合c-Met。可以通过ELISA、流式细胞术、表面等离子体共振(SPR)测定或本领域已知的任何其他方法测量与c-Met的结合。在一些实施方案中，所述竞争结合c-Met的抗体或其抗原结合片段是骆驼科动物的、嵌合的或人源化的。
在一些实施方案中，根据上述实施方案中的任一种的抗体或其抗原结合片段可单独地或组合地 包括下述性质特征中的一种或几种：
(i)结合人c-Met或/和猴c-Met；
(ii)阻断HGF或其β链与c-Met的结合；
(iii)抑制HGF刺激引起的c-Met磷酸化；
(iv)对肿瘤细胞发挥抗体依赖的细胞介导的细胞毒性(ADCC)作用。
在一些实施方案中，所述抗体或其抗原结合片段结合c-Met，例如猴c-Met和/或人c-Met。在一些实施方案中，所述抗体或其抗原结合片段结合食蟹猴c-Met。在一些实施方案中，所述抗体或其抗原结合片段结合人c-Met。在一些实施方案中，所述的抗体或其抗原结合片段对人c-Met具有下述结合亲和力(K D)：在约1E-11M至约1E-07M、约1E-10M至约1E-08M、或约2.00E-10M至约8.25E-09M的范围内。在一些实施方案中，所述抗体或其抗原结合片段对人c-Met具有下述结合亲和力(K D)：约1E-07M或更小、约1E-08M或更小、约8.25E-09M或更小、约1E-09M或更小、约2.00E-10M或更小、约1E-10M或更小、或者约1E-11M或更小。在一些实施方案中，通过Biacore来测量本公开提供的抗体或其抗原结合片段的结合亲和力K D。
在一些实施方案中，所述抗体或其抗原结合片段阻断HGF或其β链与c-Met的结合。
在一些实施方案中，所述抗体或其抗原结合片段抑制HGF刺激引起的c-Met磷酸化。
在一些实施方案中，所述抗体或其抗原结合片段对肿瘤细胞发挥ADCC作用。在一些实施方案中，所述肿瘤细胞为表达c-Met的肿瘤细胞。
本公开提供了示例性的抗体或其抗原结合片段，如单特异性抗体(包括嵌合抗体1B-3B11-Fc、1B-1B6-Fc、1B-1C7-Fc、1B-1A8-Fc、1B-1B2-Fc和3B-1C7-Fc)，将单可变结构域与人IgG1的Fc融合，通过Fc形成同二聚体。示例性的抗体的氨基酸序列提供于下表S3中。
表S3：示例性的抗体全长序列
Figure PCTCN2022129875-appb-000004
Figure PCTCN2022129875-appb-000005
融合蛋白
本公开的单可变结构域、和抗体或其抗原结合片段可用于构建任何需要的融合蛋白，从而赋予融合蛋白靶向结合c-Met的特性或其他单可变结构域、抗体或其抗原结合片段具有的特性。因此，本公开提供了包含至少一个(一个或多个)本公开的单可变结构域、或者抗体或其抗原结合片段的分离的融合蛋白。
一些具体的实施方案中，所述融合蛋白包括本公开示例性的CDR序列、单可变结构域序列或抗体序列，例如表S1中任一单可变结构域中的一个、两个或三个CDR序列，表S2中任一单可变结构域序列，或者表S3中任一抗体序列；任选地，该融合蛋白可经过任何本领域已知的修饰(例如糖基化修饰、化学修饰等)。
当融合蛋白包含的单可变结构域是两个或两个以上时，可以选择相同或不同的单可变结构域。
药物组合物
本公开提供了药物组合物，其包含本公开的抗体或其抗原结合片段，以及药学上可接受的载体。本公开还提供了药物组合物，其包含本公开的融合蛋白，以及药学上可接受的载体。在一些实施方案中，将1B-3B11-Fc、1B-1B6-Fc、1B-1C7-Fc、1B-1A8-Fc、1B-1B2-Fc和3B-1C7-Fc抗体的任意一个或几个，以及药学上可接受的载体制成药物组合物。
分离的核酸
本公开提供了分离的核酸，其编码本公开所述的单可变结构域、或者抗体或其抗原结合片段。本公开还提供了分离的核酸，其编码本公开所述的融合蛋白。在一些实施方案中，所述核酸编码单可变结构域，如1B-3B11-Fc、1B-1B6-Fc、1B-1C7-Fc、1B-1A8-Fc、1B-1B2-Fc或3B-1C7-Fc的单可变结构域。在一些实施方案中，所述核酸编码抗体或其抗原结合片段，如1B-3B11-Fc、1B-1B6-Fc、1B-1C7-Fc、1B-1A8-Fc、1B-1B2-Fc或3B-1C7-Fc。
序列表中示例性的列举了一些单可变结构域、抗体或其抗原结合片段的核酸序列。
载体
本公开提供了包含所述的分离的核酸的载体。在一些实施方案中，所述的载体为克隆载体；在 另一些实施方案中，所述的载体为表达载体。所述表达载体可选的能够表达本公开所述单可变结构域、或者抗体或其抗原结合片段的任意表达载体。所述表达载体可选的能够表达本公开所述融合蛋白的任意表达载体。一个具体的例子，表达载体为pcDNA3.1。
宿主细胞
本公开提供包含本公开所述核酸或所述载体的宿主细胞，宿主细胞为用于克隆或表达单可变结构域、或者抗体或其抗原结合片段的适当宿主细胞。本公开还提供包含本公开所述核酸或所述载体的宿主细胞，宿主细胞为用于克隆或表达融合蛋白的适当宿主细胞。在一些实施方案中，宿主细胞为原核细胞。在另一些实施方案中，宿主细胞为真核细胞。在一些实施方案中，宿主细胞选自酵母细胞、哺乳细胞或适用于制备单可变结构域、抗体或其抗原结合片段、或者融合蛋白的其他细胞。哺乳细胞例如为中国仓鼠卵巢(CHO)细胞、CHO-S细胞。
制备单可变结构域、抗体或其抗原结合片段、或融合蛋白的方法
本公开提供了制备单可变结构域的方法，所述方法包括：培养包含编码本公开单可变结构域的核酸的宿主细胞，从所述宿主细胞或宿主细胞培养基中回收所述单可变结构域。本公开还提供了制备抗体或其抗原结合片段的方法，所述方法包括：培养包含编码本公开抗体或其抗原结合片段的核酸的宿主细胞，从所述宿主细胞或宿主细胞培养基中回收所述抗体或其抗原结合片段。本公开还提供了制备融合蛋白的方法，所述方法包括：培养包含编码本公开融合蛋白的核酸的宿主细胞，从所述宿主细胞或宿主细胞培养基中回收所述融合蛋白。
为了产生所述的单可变结构域、或者抗体或其抗原结合片段，将编码所述单可变结构域、或者抗体或其抗原结合片段的核酸插入载体，用于在宿主细胞中进一步克隆或/和表达。为了产生所述的融合蛋白，将编码所述融合蛋白的核酸插入载体，用于在宿主细胞中进一步克隆或/和表达。所述核酸可以采用基因拼接、化学合成等多种本领域所熟知的方法获取。
用途
本公开提供了本公开的单可变结构域、抗体或其抗原结合片段、或者融合蛋白的用途。本公开还提供了本公开的药物组合物的用途。
本公开提供了本公开的单可变结构域在制备治疗患有肿瘤的受试者的药物中的用途。本公开还提供了本公开的抗体或其抗原结合片段在制备治疗患有肿瘤的受试者的药物中的用途。本公开还提供了本公开的融合蛋白在制备治疗患有肿瘤的受试者的药物中的用途。本公开还提供了本公开的药物组合物在制备治疗患有肿瘤的受试者的药物中的用途。
本公开提供了检测或测量样品中的c-Met的方法，其包括使所述样品与本公开的单可变结构域、或者抗体或其抗原结合片段接触并且检测或测量结合复合物。本公开还提供了检测或测量样品中的c-Met的方法，其包括使所述样品与本公开的融合蛋白接触并且检测或测量结合复合物。
本公开提供了抑制、减少或阻断细胞中的c-Met信号传导的方法，其包括向所述细胞施用有效量的本公开所述的单可变结构域、或者抗体或其抗原结合片段。本公开还提供了本公开的单可变结构域、或者抗体或其抗原结合片段在制备抑制、减少或阻断细胞中的c-Met信号传导的药物中的用途。本公开提供了抑制、减少或阻断细胞中的c-Met信号传导的方法，其包括向所述细胞施用有效量的本公开所述的融合蛋白。本公开还提供了本公开的融合蛋白在制备抑制、减少或阻断细胞中的c-Met信号传导的药物中的用途。本公开提供了抑制、减少或阻断细胞中的c-Met信号传导的方法，其包括向所述细胞施用有效量的本公开所述的药物组合物。本公开还提供了本公开的药物组合物在制备抑制、减少或阻断细胞中的c-Met信号传导的药物中的用途。一些实施方案中，所述细胞为肿瘤细胞。
本公开提供了抑制肿瘤细胞生长或杀伤肿瘤细胞的方法，其包括向所述肿瘤细胞施用有效量的本公开的单可变结构域、或者抗体或其抗原结合片段。本公开还提供了所述单可变结构域、或者抗体或其抗原结合片段在制备抑制肿瘤细胞生长或杀伤肿瘤细胞的药物中的用途。本公开提供了抑制肿瘤细胞生长或杀伤肿瘤细胞的方法，其包括向所述肿瘤细胞施用有效量的本公开的融合蛋白。本公开还提供了所述融合蛋白在制备抑制肿瘤细胞生长或杀伤肿瘤细胞的药物中的用途。本公开提供了抑制肿瘤细胞生长或杀伤肿瘤细胞的方法，其包括向所述肿瘤细胞施用有效量的本公开的药物组合物。本公开还提供了所述药物组合物在制备抑制肿瘤细胞生长或杀伤肿瘤细胞的药物中的用途。
本公开提供了治疗患有肿瘤的受试者的方法，其包括向所述受试者施用治疗有效量的本公开所述的单可变结构域、或者抗体或其抗原结合片段。本公开还提供了治疗患有肿瘤的受试者的方法， 其包括向所述受试者施用治疗有效量的本公开所述的融合蛋白。本公开还提供了治疗患有肿瘤的受试者的方法，其包括向所述受试者施用治疗有效量的本公开所述的药物组合物。需要治疗的受试者包括那些已经患有疾病或病状的受试者，以及可能患疾病或病状并且其目的是预防、延迟或减弱疾病或病状的受试者。
在一些实施方案中，上述肿瘤为表达c-Met的肿瘤。在一些实施方案中，上述肿瘤为c-Met过表达的肿瘤。在一些实施方案中，上述肿瘤为实体瘤。在一些实施方案中，上述肿瘤为非实体瘤。在一些实施方案中，上述肿瘤为脑癌、头颈癌、肺癌、食管癌、咽癌、鼻癌、舌癌、口腔癌、甲状腺癌、皮肤癌、骨癌、软组织肉瘤、骨髓瘤、胃癌、胃食管结合部腺癌、乳腺癌、肝癌、肾癌、胰腺癌、脾癌、淋巴癌、胸腺癌、膀胱癌、阴道癌、睾丸癌、子宫癌、宫颈癌、结直肠癌、肛门癌、乳头状癌、前列腺癌、卵巢癌、上皮细胞癌或血液瘤。在一些实施方案中，所述肺癌为非小细胞肺癌。在一些实施方案中，所述脑癌为胶质母细胞瘤。
本公开还提供了以下一些具体的实施方案，但本公开的保护范围不限于此：
实施方案1.一种分离的抗体或其抗原结合片段，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个单可变结构域，其中所述单可变结构域包含以下互补决定区：
a)CDR1，其包含SEQ ID NO:5、8、11、14、17或20所示的氨基酸序列；
b)CDR2，其包含SEQ ID NO:6、9、12、15、18或21所示的氨基酸序列；和
c)CDR3，其包含SEQ ID NO:7、10、13、16、19或22所示的氨基酸序列。
实施方案2.根据实施方案1所述的分离的抗体或其抗原结合片段，其中所述单可变结构域包含：
a)包含SEQ ID NO:5所示氨基酸序列的CDR1，包含SEQ ID NO:6所示氨基酸序列的CDR2，和包含SEQ ID NO:7所示氨基酸序列的CDR3；
b)包含SEQ ID NO:8所示氨基酸序列的CDR1，包含SEQ ID NO:9所示氨基酸序列的CDR2，和包含SEQ ID NO:10所示氨基酸序列的CDR3；
c)包含SEQ ID NO:11所示氨基酸序列的CDR1，包含SEQ ID NO:12所示氨基酸序列的CDR2，和包含SEQ ID NO:13所示氨基酸序列的CDR3；
d)包含SEQ ID NO:14所示氨基酸序列的CDR1，包含SEQ ID NO:15所示氨基酸序列的CDR2，和包含SEQ ID NO:16所示氨基酸序列的CDR3；
e)包含SEQ ID NO:17所示氨基酸序列的CDR1，包含SEQ ID NO:18所示氨基酸序列的CDR2，和包含SEQ ID NO:19所示氨基酸序列的CDR3；或
f)包含SEQ ID NO:20所示氨基酸序列的CDR1，包含SEQ ID NO:21所示氨基酸序列的CDR2，和包含SEQ ID NO:22所示氨基酸序列的CDR3。
实施方案3.根据实施方案2所述的分离的抗体或其抗原结合片段，其中所述单可变结构域包含与SEQ ID NO:24、26、28、30、32或34所示氨基酸序列具有至少85％的同一性的氨基酸序列。
实施方案4.根据实施方案3所述的分离的抗体或其抗原结合片段，其中所述单可变结构域包含SEQ ID NO:24、26、28、30、32或34所示的氨基酸序列。
实施方案5.根据实施方案1-4中任一项所述的分离的抗体或其抗原结合片段，其中所述单可变结构域是V HH。
实施方案6.根据实施方案1-5中任一项所述的分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段是嵌合的或人源化的。
实施方案7.根据实施方案1-6中任一项所述的分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含人IgG的Fc，优选为人IgG1、IgG2、IgG3或IgG4的Fc。
实施方案8.根据实施方案1-7中任一项所述的分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含SEQ ID NO:37、39、41、43、45或47所示的氨基酸序列。
实施方案9.根据实施方案1-8中任一项所述的分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段可单独地或组合地包括下述性质特征中的一种或几种：
实施方案10.结合人c-Met或/和猴c-Met；
实施方案11.阻断HGF或其β链与c-Met的结合；
实施方案12.抑制HGF刺激引起的c-Met磷酸化；
实施方案13.对肿瘤细胞发挥ADCC作用。
实施方案14.一种融合蛋白，其包含实施方案1-9中任一项所述的抗体或其抗原结合片段。
实施方案15.一种药物组合物，其包含实施方案1-9中任一项所述的抗体或其抗原结合片段或 者实施方案10所述的融合蛋白，以及药学上可接受的载体。
实施方案16.一种分离的核酸，其编码实施方案1-9中任一项所述的抗体或其抗原结合片段或者实施方案10所述的融合蛋白。
实施方案17.一种载体，其包含实施方案12所述的分离的核酸。
实施方案18.一种宿主细胞，其包含实施方案12所述的核酸或实施方案13所述的载体。
实施方案19.一种制备实施方案1-9中任一项所述的分离的抗体或其抗原结合片段的方法，包括：培养实施方案14所述的宿主细胞，分离所表达的所述抗体或其抗原结合片段，其中载体为表达载体。
实施方案20.实施方案1-9中任一项所述的分离的抗体或其抗原结合片段在制备治疗患有肿瘤的受试者的药物中的用途。
实施方案21.实施方案10所述的融合蛋白在制备治疗患有肿瘤的受试者的药物中的用途。
实施方案22.实施方案11所述的药物组合物在制备治疗患有肿瘤的受试者的药物中的用途。
实施方案23.根据实施方案16-18中任一项所述的用途，其中所述肿瘤为脑癌、头颈癌、肺癌、食管癌、咽癌、鼻癌、舌癌、口腔癌、甲状腺癌、皮肤癌、骨癌、软组织肉瘤、骨髓瘤、胃癌、胃食管结合部腺癌、乳腺癌、肝癌、肾癌、胰腺癌、脾癌、淋巴癌、胸腺癌、膀胱癌、阴道癌、睾丸癌、子宫癌、宫颈癌、结直肠癌、肛门癌、乳头状癌、前列腺癌、卵巢癌、上皮细胞癌或血液瘤。
实施方案24.一种检测或测量样品中的c-Met的方法，其包括使所述样品与实施方案1-9中任一项所述的抗体或其抗原结合片段接触并且检测或测量结合复合物。
实施方案25.一种抑制、减少或阻断细胞中的c-Met信号传导的方法，其包括向所述细胞施用有效量的实施方案1-9中任一项所述的抗体或其抗原结合片段、实施方案10所述的融合蛋白、或者实施方案11所述的药物组合物。
实施方案26.一种抑制肿瘤细胞生长或杀伤肿瘤细胞的方法，其包括向所述肿瘤细胞施用有效量的实施方案1-9中任一项所述的抗体或其抗原结合片段、实施方案10所述的融合蛋白、或者实施方案11所述的药物组合物。
实施方案27.一种治疗患有肿瘤的受试者的方法，其包括向所述受试者施用治疗有效量的实施方案1-9中任一项所述的抗体或其抗原结合片段、实施方案10所述的融合蛋白、或者实施方案11所述的药物组合物。
实施方案28.根据实施方案22或23所述的方法，其中所述肿瘤为脑癌、头颈癌、肺癌、食管癌、咽癌、鼻癌、舌癌、口腔癌、甲状腺癌、皮肤癌、骨癌、软组织肉瘤、骨髓瘤、胃癌、胃食管结合部腺癌、乳腺癌、肝癌、肾癌、胰腺癌、脾癌、淋巴癌、胸腺癌、膀胱癌、阴道癌、睾丸癌、子宫癌、宫颈癌、结直肠癌、肛门癌、乳头状癌、前列腺癌、卵巢癌、上皮细胞癌或血液瘤。
尽管为了清楚理解的目的，已经通过举例说明和实施例相当详细地描述了前述发明，但是根据本公开的教义，本领域的普通技术人员将显而易见的是，可另外对本公开进行某些改变和修改而不背离所附权利要求的精神和范围。以下实施例仅以说明方式提供，而并不起限制作用。本领域的技术人员将容易地识别多种非关键性参数，所述参数可发生改变或修改以产生基本上类似的结果。
实施例1：抗c-Met V HH噬菌体展示文库构建
1.1动物免疫
将重组人c-Met-Fc融合蛋白(SinoBiological，产品目录10692-H02H)与完全弗氏佐剂按照体积比1:1混合乳化，对双峰驼进行首次皮下多点注射免疫；之后每间隔2周，将重组人c-Met-Fc融合蛋白与不完全弗氏佐剂按照体积比1:1混合乳化后进行加强免疫。在第4或第5次免疫之后取血清检测抗人c-Met抗体的滴度。在多轮免疫后取双峰驼外周血，分离外周血单个核细胞(PBMC)。
1.2 RNA提取
使用TRIzol TM试剂从PBMC(来自1.1)中提取总RNA。提取的总RNA用1％琼脂糖凝胶电泳来评估质量，并通过测量260nm和280nm下的吸光度来定量，OD 260nm/OD 280nm的比值应在1.8-2.0之间。
1.3 V HH扩增
用cDNA合成试剂盒PrimeScript TM II 1st Strand cDNA Synthesis Kit(TAKARA，产品目录No.6210A)按照说明，将总RNA逆转录成cDNA。使用巢式PCR法扩增骆驼抗体的可变区(V HH)序列，具体方法如下：以cDNA为模板，使用引物Call001(SEQ ID NO:1)和Call002(SEQ ID NO:2)进行第一轮PCR扩增，扩增得到的DNA产物短片段用胶回收试剂盒(QIAGEN，产品目录No.28706) 纯化。以第一轮PCR产物为模板，使用引物V-Back(SEQ ID NO:3)和V-Fwd(SEQ ID NO:4)进行第二轮PCR扩增，扩增得到的DNA产物即为V HH编码片段，用胶回收试剂盒(QIAGEN，产品目录No.28706)进行纯化。
Call001(SEQ ID NO:1)：GTCCTGGCTGCTCTTCTACAAGG
Call002(SEQ ID NO:2)：GGTACGTGCTGTTGAACTGTTCC
V-Back(SEQ ID NO:3)：GATGTGCAGCTGCAGGAGTCTGGRGGAGG
V-Fwd(SEQ ID NO:4)：CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT
1.4 V HH噬菌体文库构建
将扩增得到的V HH编码片段经PstI/NotI内切酶酶切后，插入噬菌粒载体pMECS(NTCC质粒载体菌种细胞基因保藏中心，产品目录No.pMECS)中，构建成重组载体，并电转入大肠杆菌TG1(Lucigen，产品目录No.60502-1)中。取一小部分转化后的菌液稀释后涂布含有100μg/mL氨苄青霉素的选择性平板，通过菌落计数法来计算文库容量，并随机挑取100个克隆测序来评估文库质量。其余菌液涂布含有100μg/mL氨苄青霉素的选择性平板，从平板上刮下菌落的菌苔，补充甘油后冻存于-80℃作为文库原种。将文库原种扩增到对数生长期，加入M13KO7辅助噬菌体(New England Biolabs，产品目录No.N0315S)进行文库扩增，28℃，200rpm振摇过夜。将菌液离心取上清，加入上清1/4体积的PEG6000/NaCl溶液(20％PEG6000(w/v)，2.5M NaCl)，冰上孵育1-2小时沉淀噬菌体，离心收取噬菌体沉淀，用PBS重悬后加20％甘油保存于-80℃作为V HH噬菌体展示文库。
实施例2：抗人c-Met V HH筛选
2.1淘选
采用固相淘选的方式对V HH噬菌体展示文库进行淘选。将重组c-Met-Avitage TM(ACRO，产品目录No.MET-H82E1)固定在高吸附酶标板上，封闭后将1.4中获得的噬菌体加入孔板中37℃孵育1-2h。采用磷酸盐吐温缓冲液(PBST)清洗10次以去除非特异性结合的噬菌体，PBS清洗后，用胰蛋白酶(trypsin)将结合的噬菌体洗脱下来，用4-(2-氨乙基)苯磺酰氟盐酸盐(AEBSF)将酶中和，感染大肠杆菌TG1扩增后进行下一轮文库淘选。通过2-3轮淘选富集后，将收集的噬菌体感染对数生长期大肠杆菌TG1，涂布含20％(w/v)葡萄糖和100μg/mL氨苄青霉素的选择性平板。挑取单克隆进行培养，异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达制备上清。
2.2阳性克隆鉴定
通过针对人c-Met-His(SinoBiological，产品目录10692-H08H)的间接ELISA方法对挑选的克隆进行阳性鉴定。将人c-Met-His或对照蛋白人Fc蛋白(ACRO，产品目录No.FCC-H5214)包被在高吸附酶标板上，然后用封闭液封闭，加2.1中制备的上清37℃孵育1小时，清洗后加HRP缀合的抗HA标签二抗(GenScript，产品目录No.A01296)，37℃孵育0.5小时，清洗5次，加显色底物进行显色，检测450nm波长及650nm参比波长处光吸收信号值。挑选仅结合人c-Met-His且信号值比较高的阳性克隆进行保种并测序。
筛选获得阳性克隆1B-1B2、1B-3B11、1B-1C7、1B-1B6、1B-1A8和3B-1C7。经序列分析，1B-3B11的V HH的核苷酸序列如SEQ ID NO:23所示，氨基酸序列如SEQ ID NO:24所示；1B-1B6的V HH的核苷酸序列如SEQ ID NO:25所示，氨基酸序列如SEQ ID NO:26所示；1B-1C7的V HH的核苷酸序列如SEQ ID NO:27所示，氨基酸序列如SEQ ID NO:28所示；1B-1A8的V HH的核苷酸序列如SEQ ID NO:29所示，氨基酸序列如SEQ ID NO:30所示；1B-1B2的V HH的核苷酸序列如SEQ ID NO:31所示，氨基酸序列如SEQ ID NO:32所示；3B-1C7的V HH的核苷酸序列如SEQ ID NO:33所示，氨基酸序列如SEQ ID NO:34所示。
实施例3：抗人c-Met V HH-Fc嵌合抗体的制备
3.1 V HH-Fc嵌合抗体的制备
将筛选的阳性克隆的V HH序列与人Fc区连接，构建V HH-Fc嵌合抗体。具体地，将2.2中测序获得的V HH序列插入含有人IgG1Fc区的pCDNA3.1真核表达载体中，使用Expifectamine TM CHO Transfection Kit瞬转表达系统(Thermo Fisher Scientific Inc.，产品目录No.A29129)表达这些V HH-Fc嵌合抗体。
经序列分析，1B-3B11-Fc的全长氨基酸序列如SEQ ID NO:37所示，核苷酸序列如SEQ ID NO:36所示；1B-1B6-Fc的全长氨基酸序列如SEQ ID NO:39所示，核苷酸序列如SEQ ID NO:38所示；1B-1C7-Fc的全长氨基酸序列如SEQ ID NO:41所示，核苷酸序列如SEQ ID NO:40所示；1B-1A8-Fc的全长氨基酸序列如SEQ ID NO:43所示，核苷酸序列如SEQ ID NO:42所示；1B-1B2-Fc的全长氨 基酸序列如SEQ ID NO:45所示，核苷酸序列如SEQ ID NO:44所示；3B-1C7-Fc的全长氨基酸序列如SEQ ID NO:47所示，核苷酸序列如SEQ ID NO:46所示。
3.2表面等离子共振技术测定V HH-Fc嵌合抗体与人、食蟹猴c-Met的亲和力
使用生物分子相互作用分析系统(GE，Biacore T200)进行抗人c-Met V HH-Fc嵌合抗体的亲和力检测。氨基偶联Anti-hIgG(Fc)抗体(GE，产品目录No.BR-1008-39)至CM5传感芯片，使用运行缓冲液(137mM NaCl，2.7mM KCl，10mM Na 2HPO 4·12H 2O，1.8mM KH 2PO 4，0.05％surfactant P-20(w/v)，pH 7.4)稀释抗人c-Met V HH-Fc嵌合抗体至1μg/mL，30μl/min流速通过实验通道进行捕获。使用运行缓冲液稀释人c-Met-His(SinoBiological，产品目录10692-H08H)或食蟹猴c-Met-His(SinoBiological，产品目录90304-C08H)至100nM、50nM、25nM、12.5nM、6.25nM和3.125nM，50μl/min流速结合，结合时间设置为200s，之后停止进样进行解离，解离时间设置为800s。软件BiaControl Software 2.0实时采集数据信号，软件BiaEvaluation Software 2.0数据分析，用Langmuir 1:1模型拟合，计算结合速率常数K a(1/Ms)、解离速率常数K d(1/s)、平衡解离常数K D(M)值。检测结果如表1所示，1B-1B2-Fc、1B-3B11-Fc、1B-1C7-Fc、1B-1B6-Fc、1B-1A8-Fc、3B-1C7-Fc与人c-Met蛋白均有较高的亲和力，且均与食蟹猴c-Met蛋白有交叉反应。
表1 V HH-Fc嵌合抗体与人、食蟹猴c-Met的亲和力
Figure PCTCN2022129875-appb-000006
实施例4：抗人c-Met V HH-Fc嵌合抗体与表达c-Met的天然细胞系的结合
4.1流式细胞术测定抗人c-Met V HH-Fc嵌合抗体与细胞的结合
采用流式细胞术检测抗人c-Met V HH-Fc嵌合抗体与c-Met不同表达水平的靶细胞的结合，其中H1993细胞(北纳生物，产品目录BNCC342186)为c-Met高表达水平的人肺腺癌细胞系，MKN45细胞(南京科佰生物科技有限公司，产品目录CBP60541)为c-Met中等表达水平的人胃癌细胞系，KP4细胞(南京科佰生物科技有限公司，产品目录CBP60541)为c-Met低表达水平的人胰腺癌细胞系。用梯度稀释(初始浓度100nM，5倍梯度稀释，7个浓度)的抗人c-Met V HH-Fc嵌合抗体孵育2×10 5个靶细胞，冰上孵育1小时后清洗细胞，加PE标记的抗人IgG Fc抗体(Jackson Immuno Research，产品目录No.109-116-170)，冰上孵育0.5小时，清洗细胞后使用流式细胞仪(Thermo Fisher Scientific Inc.，Attune NXT)检测。结果如图1A-1C、表2所示，1B-1B2-Fc、1B-3B11-Fc、1B-1C7-Fc、1B-1B6-Fc、1B-1A8-Fc、3B-1C7-Fc与c-Met不同表达水平的靶细胞均有较高的结合能力。
表2抗人c-Met V HH-Fc嵌合抗体与靶细胞的结合
Figure PCTCN2022129875-appb-000007
4.2流式细胞术测定抗人c-Met V HH-Fc嵌合抗体与表达猕猴c-Met的细胞的结合
采用流式细胞术检测抗人c-Met V HH-Fc嵌合抗体与猕猴上皮细胞4MBr-5(上海青旗生物技术发展公司，产品目录BFN60807362)的结合，方法参考4.1。结果如图1D、表3所示，1B-1B2-Fc、1B-3B11-Fc、1B-1C7-Fc、1B-1B6-Fc、1B-1A8-Fc和3B-1C7-Fc均可有效靶向结合表达c-Met的猕猴上皮细胞4MBr-5。
表3抗人c-Met V HH-Fc嵌合抗体与猕猴上皮细胞的结合
Figure PCTCN2022129875-appb-000008
实施例5：抗人c-Met V HH-Fc嵌合抗体对受体/配体结合的阻断以及抗体表位竞争分析
5.1抗人c-Met V HH-Fc嵌合抗体对HGF、HGFβ与c-Met受体结合的阻断
用竞争ELISA的方法分析抗人c-Met V HH-Fc嵌合抗体是否可以阻断肝细胞生长因子(HGF)与c-Met受体的结合。具体地，将2μg/mL人c-Met-His蛋白(SinoBiological，产品目录10692-H08H)包被在高吸附酶标板上，然后用3％(w/v)BSA封闭液封闭，清洗后加入80ng/mL生物素缀合的HGF(SinoBiological，产品目录10463-HNAS)和抗人c-Met V HH-Fc嵌合抗体(初始浓度100nM，5倍梯度稀释，7个浓度)，25℃孵育2小时，清洗后加HRP缀合的链霉亲和素(eBioscience，产品目录No.18-4100-51)，25℃孵育1小时，清洗5次，加100μl/孔TMB底物溶液(TIANGEN，产品目录No.PA107)进行显色，检测450nm波长及650nm参比波长处光吸收信号值，拟合曲线(图2A)并计算IC 50值(表4)。
用同样的方法分析抗人c-Met V HH-Fc嵌合抗体是否可以阻断HGF的β链(HGFβ，NP_000592.3，Val495-Ser728)与c-Met受体的结合。HGFβ的氨基酸序列如SEQ ID NO:35所示。检测结果拟合曲线(图2B)并计算IC 50值(表4)。
从检测结果看，1B-3B11-Fc、1B-1B2-Fc、1B-1C7-Fc、1B-1B6-Fc、1B-1A8-Fc、3B-1C7-Fc均可以阻断HGF与c-Met受体的结合；1B-3B11-Fc、1B-1C7-Fc、1B-1B6-Fc、1B-1A8-Fc可以阻断HGFβ与c-Met受体的结合，1B-1B2-Fc、3B-1C7-Fc不能阻断HGFβ与c-Met受体的结合。
表4抗人c-Met V HH-Fc嵌合抗体对HGF、HGFβ与c-Met受体结合的阻断
Figure PCTCN2022129875-appb-000009
5.2表面等离子共振技术检测抗人c-Met V HH-Fc不同克隆间的竞争关系
使用生物分子相互作用系统(Fortebio，产品目录Octet RED96)进行表位竞争关系分析。使用Anti-Penta-HIS(HIS1K)传感器(Fortebio，产品目录No.18-5120)，使用运行缓冲液稀释c-Met-His蛋白(SinoBiological，产品目录10692-H08H)至5μg/mL左右，将传感器浸入稀释后的抗原样品中，通过调整结合时间将固化高度控制在1nm左右。然后依次与第一个抗体A和第二个抗体B相互作用，检测B抗体结合信号以判定两个抗体是否识别同一表位。结果如表5-1和表5-2所示，判定标准为：数值>60％表示2个抗体完全不竞争；数值在20％-60％之间表示2个抗体部分竞争(可能表位有交叉)；数值<20％表示2个抗体完全竞争。自反应信号(灰色部分)<20％，判定数据有效。
从表5-1中数据可得出，1B-3B11-Fc与1B-1A8-Fc两个抗体完全竞争；1B-3B11-Fc和1B-1A8-Fc与其他三个候选抗体之间没有明显的竞争关系，分别属于不同的表位。因此，抗体1B-3B11-Fc和1B-1B2-Fc、1B-3B11-Fc和1B-1C7-Fc、1B-3B11-Fc和1B-1B6-Fc、1B-1A8-Fc和1B-1B2-Fc、1B-1A8-Fc和1B-1C7-Fc、1B-1A8-Fc和1B-1B6-Fc、1B-1B2-Fc和1B-1C7-Fc、1B-1B2-Fc和1B-1B6-Fc、1B-1C7-Fc和1B-1B6-Fc可同时结合c-Met抗原的不同表位。
从表5-2中数据可得出，3B-1C7-Fc与1B-1B2-Fc两个抗体完全竞争。因此，结合表5-1数据结果，可得出：抗体1B-1A8-Fc和3B-1C7-Fc、3B-1C7-Fc和1B-1C7-Fc、3B-1C7-Fc和1B-1B6-Fc可 同时结合c-Met抗原的不同表位。
表5-1表面等离子共振技术表位差异分析
Figure PCTCN2022129875-appb-000010
表5-2表面等离子共振技术表位差异分析
Figure PCTCN2022129875-appb-000011
实施例6：抗人c-Met V HH-Fc嵌合抗体对H441细胞中c-Met磷酸化作用的抑制
用Phospho-c-Met(Tyr1234/1235)AlphaLISA SureFire Ultra Assay Kit(PerkinElmer，产品目录ALSU-PC-MET-A500)检测抗体在H441细胞(北纳生物，产品目录BNCC292357)中对HGF刺激产生的c-Met磷酸化的抑制作用。根据供应商说明，取H441细胞，100μl/孔，10000cells/孔接种至96孔板，孵育6-16h后，无血清培养基饥饿过夜，弃上清，加入45μl/孔抗人c-Met V HH-Fc嵌合抗体(初始浓度1000nM，10倍梯度稀释，7个浓度)，孵育30min，再加入5μl HGF刺激15min。弃上清，加入新鲜配制的1×裂解缓冲液100μl，孵育10min后，使用AlphaLISA模块读板。结果如图3、表6所示，1B-1B2-Fc、1B-3B11-Fc、1B-1C7-Fc、1B-1B6-Fc、1B-1A8-Fc、3B-1C7-Fc均可以有效抑制H441细胞中由HGF刺激引起的c-Met下游磷酸化。
表6抗人c-Met V HH-Fc嵌合抗体对H441细胞c-Met磷酸化抑制
Figure PCTCN2022129875-appb-000012
实施例7：抗人c-Met V HH-Fc嵌合抗体对KP4细胞的增殖抑制
用Cell Counting Kit 8(DOJINDO Laboratories，Lot.SH797)法检测抗体对KP4细胞增殖的抑制作用。具体地，将KP4细胞以1000cells/孔加入96孔细胞培养板，置于37℃，5％CO 2二氧化碳细胞培养箱中培养，待细胞贴壁后，将抗人c-Met V HH-Fc嵌合抗体(初始浓度900nM，2倍梯度稀释，7个浓度)加入细胞中，培养120h后，每孔加入10μl CCK-8溶液，孵育1h后使用酶标仪测量450nm处的吸光度。结果如图4、表7所示，1B-3B11-Fc、1B-1C7-Fc、1B-1B6-Fc、1B-1A8-Fc、3B-1C7-Fc均可以有效抑制KP4细胞的增殖。
细胞存活率计算公式：
细胞存活率＝[(As-Ab)/(Ac-Ab)]×100％，
As＝实验孔吸光度(含有细胞、培养基、CCK-8和待测抗体的孔的吸光度)，
Ab＝空白孔吸光度(含有培养基和CCK-8的孔的吸光度)，
Ac＝对照孔吸光度(含有细胞、培养基和CCK-8的孔的吸光度)；
增殖抑制率＝100％-细胞存活率。
表7抗人c-Met V HH-Fc嵌合抗体对KP4细胞的增殖抑制
Figure PCTCN2022129875-appb-000013
Figure PCTCN2022129875-appb-000014

Claims (18)

  1. 一种分离的抗体或其抗原结合片段，所述抗体或其抗原结合片段结合c-Met，并且包含至少一个单可变结构域，其中所述单可变结构域包含以下互补决定区：
    (a)CDR1，其包含SEQ ID NO:11、8、5、14、17或20所示的氨基酸序列；
    (b)CDR2，其包含SEQ ID NO:12、9、6、15、18或21所示的氨基酸序列；和
    (c)CDR3，其包含SEQ ID NO:13、10、7、16、19或22所示的氨基酸序列。
  2. 根据权利要求1所述的分离的抗体或其抗原结合片段，其中所述单可变结构域包含：
    (a)包含SEQ ID NO:11所示氨基酸序列的CDR1，包含SEQ ID NO:12所示氨基酸序列的CDR2，和包含SEQ ID NO:13所示氨基酸序列的CDR3；
    (b)包含SEQ ID NO:8所示氨基酸序列的CDR1，包含SEQ ID NO:9所示氨基酸序列的CDR2，和包含SEQ ID NO:10所示氨基酸序列的CDR3；
    (c)包含SEQ ID NO:5所示氨基酸序列的CDR1，包含SEQ ID NO:6所示氨基酸序列的CDR2，和包含SEQ ID NO:7所示氨基酸序列的CDR3；
    (d)包含SEQ ID NO:14所示氨基酸序列的CDR1，包含SEQ ID NO:15所示氨基酸序列的CDR2，和包含SEQ ID NO:16所示氨基酸序列的CDR3；
    (e)包含SEQ ID NO:17所示氨基酸序列的CDR1，包含SEQ ID NO:18所示氨基酸序列的CDR2，和包含SEQ ID NO:19所示氨基酸序列的CDR3；或
    (f)包含SEQ ID NO:20所示氨基酸序列的CDR1，包含SEQ ID NO:21所示氨基酸序列的CDR2，和包含SEQ ID NO:22所示氨基酸序列的CDR3。
  3. 根据权利要求2所述的分离的抗体或其抗原结合片段，其中所述单可变结构域包含与SEQ ID NO:24、26、28、30、32或34所示氨基酸序列具有至少85％的同一性的氨基酸序列，或者
    所述单可变结构域包含SEQ ID NO:24、26、28、30、32或34所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的分离的抗体或其抗原结合片段，其中所述单可变结构域是V HH；可选地，所述抗体或其抗原结合片段是嵌合的或人源化的。
  5. 根据权利要求1-4中任一项所述的分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含人IgG的Fc，优选为人IgG1、IgG2、IgG3或IgG4的Fc；可选地，所述抗体或其抗原结合片段包含SEQ ID NO:37、39、41、43、45或47所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的分离的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段可单独地或组合地包括下述性质特征中的一种或几种：
    (a)结合人c-Met或/和猴c-Met；
    (b)阻断HGF或其β链与c-Met的结合；
    (c)抑制HGF刺激引起的c-Met磷酸化；
    (d)对肿瘤细胞发挥ADCC作用。
  7. 一种融合蛋白，其包含权利要求1-6中任一项所述的抗体或其抗原结合片段。
  8. 一种药物组合物，其包含权利要求1-6中任一项所述的抗体或其抗原结合片段或者权利要求7所述的融合蛋白，以及药学上可接受的载体。
  9. 一种分离的核酸，其编码权利要求1-6中任一项所述的抗体或其抗原结合片段或者权利要求7所述的融合蛋白。
  10. 一种载体，其包含权利要求9所述的分离的核酸。
  11. 一种宿主细胞，其包含权利要求9所述的核酸或权利要求10所述的载体。
  12. 一种制备权利要求1-6中任一项所述的分离的抗体或其抗原结合片段的方法，包括：培养权利要求11所述的宿主细胞，分离所表达的所述抗体或其抗原结合片段，其中载体为表达载体。
  13. 权利要求1-6中任一项所述的分离的抗体或其抗原结合片段、权利要求7所述的融合蛋白或者权利要求8所述的药物组合物在制备治疗患有肿瘤的受试者的药物中的用途；优选地，所述肿瘤为脑癌、头颈癌、肺癌、食管癌、咽癌、鼻癌、舌癌、口腔癌、甲状腺癌、皮肤癌、骨癌、软组织肉瘤、骨髓瘤、胃癌、胃食管结合部腺癌、乳腺癌、肝癌、肾癌、胰腺癌、脾癌、淋巴癌、胸腺癌、膀胱癌、阴道癌、睾丸癌、子宫癌、宫颈癌、结直肠癌、肛门癌、乳头状癌、前列腺癌、卵巢癌、上皮细胞癌或血液瘤。
  14. 一种检测或测量样品中的c-Met的方法，其包括使所述样品与权利要求1-6中任一项所述的抗体或其抗原结合片段接触并且检测或测量结合复合物。
  15. 一种抑制、减少或阻断细胞中的c-Met信号传导的方法，其包括向所述细胞施用有效量的 权利要求1-6中任一项所述的抗体或其抗原结合片段、权利要求7所述的融合蛋白、或者权利要求8所述的药物组合物。
  16. 一种抑制肿瘤细胞生长或杀伤肿瘤细胞的方法，其包括向所述肿瘤细胞施用有效量的权利要求1-6中任一项所述的抗体或其抗原结合片段、权利要求7所述的融合蛋白、或者权利要求8所述的药物组合物。
  17. 一种治疗患有肿瘤的受试者的方法，其包括向所述受试者施用治疗有效量的权利要求1-6中任一项所述的抗体或其抗原结合片段、权利要求7所述的融合蛋白、或者权利要求8所述的药物组合物。
  18. 根据权利要求16或17所述的方法，其中所述肿瘤为脑癌、头颈癌、肺癌、食管癌、咽癌、鼻癌、舌癌、口腔癌、甲状腺癌、皮肤癌、骨癌、软组织肉瘤、骨髓瘤、胃癌、胃食管结合部腺癌、乳腺癌、肝癌、肾癌、胰腺癌、脾癌、淋巴癌、胸腺癌、膀胱癌、阴道癌、睾丸癌、子宫癌、宫颈癌、结直肠癌、肛门癌、乳头状癌、前列腺癌、卵巢癌、上皮细胞癌或血液瘤。
PCT/CN2022/129875 2021-11-05 2022-11-04 结合c-Met的抗体及其应用 WO2023078391A1 (zh)

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