Anti-adam9 nanobody and use thereof

Abstract

The invention provides an anti-ADAM9 nano antibody and medical application thereof, and particularly provides the anti-ADAM9 nano antibody, a multispecific antigen binding molecule containing the nano antibody, a pharmaceutical composition containing the nano antibody and application of the pharmaceutical composition.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine and relates to an anti-ADAM9 nanobody and uses thereof, and specifically, to an anti-ADAM9 nanobody, a multispecific antigen-binding molecule comprising the nanobody, a pharmaceutical composition comprising the nanobody and uses thereof. Background Art

[0002] ADAM proteins (ADAM: disintegrin and metalloproteinase) are multifunctional proteins involved in the shedding of the extracellular domain of transmembrane proteins, cell adhesion and infiltration. The human genome contains 25 ADAMs (including four pseudogenes), and the 21 ADAMs include 13 proteolytic ADAMs that exhibit proteolytic activity and 8 non-proteolytic ADAMs. Proteolytic ADAMs have the metalloproteinase domain of matrix metalloproteinases (MMPs), and typical proteolytic ADAM proteins contain propeptides, metalloproteinases, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane and cytoplasmic domains. Among them, ADAM9 has proteolytic activity, is widely expressed in the human body, and regulates a variety of biological functions. It also plays an important role in various diseases, including neurodegenerative diseases, retinal diseases, inflammation and tumors. It is reported that ADAM9 participates in the development, invasion, metastasis and prognosis of various malignant tumors such as liver cancer, breast cancer, lung cancer, gastric cancer, kidney cancer, prostate cancer (ADAM9 Silencing Inhibits Breast Tumor Cell Invasion in Vitro. Biochimie, 2013, 95, 1371-1378.; Increased Expression of a Disintegrin and Metalloprotease-9 in Hepatocellular Carcinoma: Implications for Tumor Progression and Prognosis. Japanese Journal of Clinical Oncology, 2010, 40, 645-651.). In addition to tumor metastasis, ADAM9 plays an important role in tumor proliferation, angiogenesis, and even immune escape. Moreover, the overexpression of ADAM9 in solid tumors is related to invasive tumor phenotype and poor clinical prognosis. For example, ADAM9 mRNA expression is related to the tumor grade and histological type of glioma, and in patients with low-grade neoplasia, there is a significant correlation between the high expression of ADAM9 and poor clinical results. In addition, studies have shown that ADAM9 expression can be used as a prognostic indicator for patients with low-grade neoplasia and is also a potential therapeutic target. Kim et al. found that ADAM9 plays an important role in the proliferation and invasion of gastric cancer, and ADAM9 may be an effective therapeutic target for advanced gastric cancer (The Effect of Disintegrin-Metalloproteinase ADAM9 in Gastric Cancer Progression. Molecular Cancer Therapeutics, 2014, 13, 3074-3085.).Based on this, more and more researchers have initiated the drug development of ADAM9 as an anti-tumor therapeutic target (see patent documents US2016 / 0138113A1, US2015 / 0010575A1, US2010 / 0233079A1, WO 2016 / 077505A2 for details).

[0003] On the other hand, nanobody (Nb), namely heavy chain nanobody VHH (variable domain of heavy chain of heavy-chain antibody), is a nanobody consisting of only one heavy chain variable region obtained by cloning the variable region of heavy chain antibody (HCAb) that naturally lacks light chain in camels. Nanobody has the smallest functional antigen-binding fragment derived from HCAb in adult camels, has high stability and high affinity for antigen binding, and can interact with cavities or cracks on the surface of proteins and enzyme active sites, making it act like an inhibitor. Compared with the scFv of conventional four-chain antibodies, nanobody is equivalent to its corresponding scFv in terms of affinity, and surpasses scFv in terms of solubility, stability, resistance to aggregation, refoldability, expression yield, and ease of DNA manipulation, library construction and 3D structure determination. In addition, nanobody has a small molecular weight and strong tissue penetration, which makes it easy to enter the core of the lesion. Moreover, nanoantibodies can also provide new ideas for designing small molecule enzyme inhibitors from peptide mimetic drugs, and play an important role in the development and preparation of multivalent antibodies. Therefore, nanoantibodies have huge application potential in various fields such as immunoassays, diagnosis and treatment.

[0004] Based on the various potential advantages of the above-mentioned ADAM proteins and nanoantibodies, there is an urgent need to develop a new anti-ADAM9 nanoantibody with better binding efficiency for tumor target treatment. Summary of the invention

[0005] In view of the above needs, the present disclosure provides an anti-ADAM9 nanobody and uses thereof.

[0006] In the first aspect, the present disclosure provides an anti-ADAM9 nanobody, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises: a heavy chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and variants thereof, a heavy chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and variants thereof, and a heavy chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and variants thereof.

[0007] Preferably, the anti-ADAM9 Nanobody comprises: a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 10, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 11, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO: 12.

[0008] Preferably, the variant is a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 10-12 as a reference sequence; preferably, the substitutions are conservative amino acid substitutions.

[0009] Preferably, the anti-ADAM9 nanobody also comprises 4 FR regions, comprising: a heavy chain FR1 having the amino acid sequence shown in SEQ ID NO: 13, a heavy chain FR2 having the amino acid sequence shown in SEQ ID NO: 14, a FR3 having the amino acid sequence shown in SEQ ID NO: 15 or 16, and a FR4 having the amino acid sequence shown in SEQ ID NO: 17.

[0010] Preferably, the structure of the anti-ADAM9 nanobody is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0011] Preferably, the anti-ADAM9 Nanobody comprises a heavy chain variable region selected from the amino acid sequence shown in SEQ ID NO: 8 or 9 and variants thereof.

[0012] Preferably, the anti-ADAM9 Nanobody comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 8 or any variant thereof.

[0013] Preferably, the anti-ADAM9 Nanobody comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 9 or any variant thereof.

[0014] Preferably, the variant is an amino acid sequence that individually has at least 90% sequence identity compared to the amino acid sequence shown in SEQ ID NO: 8 or 9 as a reference sequence; more preferably, it is an amino acid sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to any one of SEQ ID NOs: 8-9.

[0015] Preferably, the nanobody is any one of an engineered antibody, a monospecific antibody, a multispecific antibody, a monovalent antibody, a multivalent antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0016] Preferably, the Nanobody comprises the heavy chain constant region sequence without the CH1 fragment.

[0017] Preferably, the Nanobody comprises a heavy chain constant region sequence having CH2 and CH3 fragments.

[0018] Preferably, the anti-ADAM9 nanobody is an anti-ADAM9 VHH-Fc fusion protein.

[0019] Preferably, the anti-ADAM9 VHH-Fc fusion protein comprises the human IgG1Fc fragment shown in SEQ ID NO.5.

[0020] Preferably, the anti-ADAM9 VHH-Fc fusion protein has a heavy chain sequence shown in SEQ ID NO.6 or 7.

[0021] Preferably, the dissociation constant (KD) of the nanobody binding to human ADAM9 is no greater than 10 -7 nM, the dissociation constant (KD) for binding to cynomolgus monkey ADAM9 is no greater than 10 -8 nM.

[0022] Optionally, the Nanobody binds or does not bind to monkey ADAM9 protein.

[0023] Optionally, the Nanobody binds or does not bind to mouse ADAM9 protein.

[0024] Preferably, the multispecific antibody is a "bispecific", "trispecific" or "tetraspecific".

[0025] Preferably, the Nanobody may further comprise a sequence of the heavy chain constant region of any antibody selected from human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it comprises a sequence of the heavy chain constant region of any antibody selected from human or murine antibodies IgG1, IgG2, IgG3 or IgG4.

[0026] Preferably, the Nanobody is a chimeric, or humanized, or fully humanized antibody.

[0027] Preferably, the nanobody is coupled to a therapeutic agent or a tracer; more preferably, the therapeutic agent is selected from a radioactive isotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from any one of a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent or a photosensitizer.

[0028] In a second aspect, the present disclosure further provides a multispecific antigen-binding molecule, which comprises the anti-ADAM9 nanobody and antigen-binding fragment thereof as described in any one of the first aspects.

[0029] Preferably, the multispecific antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module; wherein the first antigen-binding module comprises the anti-ADAM9 nanobody described in any one of the first aspects; and the second antigen-binding module specifically binds to antigens other than ADAM9 or binds to an ADAM9 antigen epitope different from the first antigen-binding module.

[0030] Preferably, the other antigens are selected from any one of CD3, PD-1, PD-L1, Her2, EpCAM, CD16, CD20, CD30, CD33, CD47, CD52, CD64, CD133, CEA, gpA33, Mucins, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL or FAP.

[0031] In a third aspect, the present disclosure provides an isolated nucleic acid molecule, which encodes the anti-ADAM9 nanobody described in any one of the first aspect, or the multispecific antigen-binding molecule described in any one of the second aspect.

[0032] In a fourth aspect, the present disclosure provides an expression vector comprising any isolated nucleic acid molecule described in the third aspect.

[0033] In a fifth aspect, the present disclosure provides a host cell comprising any nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect.

[0034] Preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from HEK293E or Chinese hamster ovary cell (CHO).

[0035] In a sixth aspect, the present disclosure provides a method for preparing the anti-ADAM9 nanobody as described in the first aspect, comprising culturing the host cell described in the fifth aspect under appropriate conditions and isolating the nanobody.

[0036] In the seventh aspect, the present disclosure provides a pharmaceutical composition comprising any one of the nanoantibodies described in any one of the first aspect, the multispecific antigen-binding molecules described in any one of the second aspect, the isolated nucleic acid molecules described in the third aspect, the expression vector described in the fourth aspect, and the host cell described in the fifth aspect.

[0037] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; more preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0038] In the eighth aspect, the present disclosure provides a method for preventing and / or treating tumor diseases or inflammatory diseases, comprising administering to an individual in need thereof an effective dose of the anti-ADAM9 nanoantibody described in any one of the first aspect, or the multispecific antigen-binding molecule described in any one of the second aspect, or the isolated nucleic acid molecule described in any one of the third aspect, or the expression vector described in any one of the fourth aspect, or the host cell described in any one of the fifth aspect, or the pharmaceutical composition described in any one of the seventh aspect.

[0039] Preferably, the tumor disease or inflammatory disease is a tumor disease or inflammatory disease in which ADAM9 is overexpressed.

[0040] Preferably, the tumor disease is bladder cancer, breast cancer (especially triple-negative breast cancer), cervical cancer, colorectal cancer (especially adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, melanoma or squamous cell carcinoma), brain cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, non-small cell lung cancer (especially squamous cell carcinoma, adenocarcinoma or large cell undifferentiated carcinoma), bone marrow cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, endometrial cancer, gallbladder cancer.

[0041] In the ninth aspect, the present disclosure provides the use of the anti-ADAM9 nanobody described in any one of the first aspect, or the multispecific antigen-binding molecule described in any one of the second aspect, or the isolated nucleic acid molecule described in any one of the third aspect, or the expression vector described in any one of the fourth aspect, or the host cell described in any one of the fifth aspect, or the pharmaceutical composition described in any one of the seventh aspect in the preparation of a medicament for preventing and / or treating tumor diseases or inflammatory diseases.

[0042] Preferably, the tumor disease or inflammatory disease is a tumor disease or inflammatory disease in which ADAM9 is overexpressed.

[0043] Preferably, the tumor disease is cancer.

[0044] Preferably, the cancer is selected from bladder cancer, breast cancer (especially triple-negative breast cancer), cervical cancer, colorectal cancer (especially adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, melanoma or squamous cell carcinoma), brain cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, non-small cell lung cancer (especially squamous cell carcinoma, adenocarcinoma or large cell undifferentiated carcinoma), bone marrow cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, endometrial cancer, gallbladder cancer.

[0045] More preferably, the cancer is selected from the group consisting of gastric cancer, non-small cell lung cancer, prostate cancer, brain cancer, pancreatic cancer, liver cancer, colorectal cancer, and triple-negative breast cancer.

[0046] In the tenth aspect, the present disclosure provides a pharmaceutical preparation comprising at least one component of the following substances as an active ingredient: the anti-ADAM9 nanobody described in any one of the first aspect, the multispecific antigen-binding molecule described in any one of the second aspect, the isolated nucleic acid molecule described in any one of the third aspect, the expression vector described in any one of the fourth aspect, the host cell described in any one of the fifth aspect, and the pharmaceutical composition described in any one of the seventh aspect.

[0047] Preferably, the pharmaceutical preparation can be used for screening, diagnosing or treating tumor diseases or inflammatory diseases.

[0048] In the eleventh aspect, the present disclosure provides a kit comprising at least one component of the following substances as an active ingredient: the anti-ADAM9 nanobody described in any one of the first aspect, the multispecific antigen-binding molecule described in any one of the second aspect, the isolated nucleic acid molecule described in any one of the third aspect, the expression vector described in any one of the fourth aspect, the host cell described in any one of the fifth aspect, and the pharmaceutical composition described in any one of the seventh aspect; and instructions for use.

[0049] In the twelfth aspect, the present disclosure provides a medicine kit comprising at least one component of the following substances as an active ingredient: the anti-ADAM9 nanobody described in any one of the first aspect, the multispecific antigen-binding molecule described in any one of the second aspect, the isolated nucleic acid molecule described in any one of the third aspect, the expression vector described in any one of the fourth aspect, the host cell described in any one of the fifth aspect, and the pharmaceutical composition described in any one of the seventh aspect; and instructions for use.

[0050] Beneficial Effects

[0051] As described in the first aspect to the twelfth aspect above, the present disclosure provides an anti-ADAM9 nanobody, which is specifically immunoreactive to the human ADAM9 receptor. In some embodiments, each VHH-Fc fusion protein prepared binds to the human ADAM9 protein, and from the EC50 point of view, each VHH-Fc fusion protein is better than the positive control antibody IMGC936. In some embodiments, the humanized antibody has a high affinity to the stable cell strain overexpressing the human ADAM9 antigen and the stable cell strain of the monkey ADAM9 antigen, among which the affinity and specificity of VHH3-Fc and VHH5-Fc are better. Moreover, each humanized antibody can bind to the human colon cancer cell HT-29. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1Shown is the total RNA electrophoresis of PMBC cell products of alpacas immunized with human ADAM9-ECD-His protein.

[0053] Figure 2 Shows the binding of VHH-Fc fusion protein samples to human ADAM9 protein DETAILED DESCRIPTION

[0054] Definitions and explanations of terms

[0055] Unless otherwise specified, the terms used herein have the meanings commonly understood by those of ordinary skill in the art. For terms that are explicitly defined herein, the meaning of the term is subject to the definitions.

[0056] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including, but not limited to, chimeric antibodies, humanized antibodies, fully human antibodies, heterologous conjugate antibodies (e.g., bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), antibody conjugates, and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments). In addition, unless otherwise indicated, the term "monoclonal antibody" (mAb) is meant to include intact antibody molecules that are capable of specifically binding to a target protein as well as incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack the Fc fragment of an intact antibody (which is cleared more quickly from animal circulation) and therefore lack Fc-mediated effector functions.

[0057] The "antibodies" described herein may be derived from any animal, including but not limited to humans and non-human animals, wherein the non-human animals may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0058] The term "natural antibody" herein refers to antibodies produced and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to non-natural antibodies obtained by techniques such as genetic engineering and antibody engineering, and illustratively, "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv, etc.), bispecific antibodies, and the like.

[0059] The term "monospecific" herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

[0060] The term "multispecific" herein refers to having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens. Thus, terms such as "bispecific", "trispecific", "tetraspecific" and the like refer to the number of different epitopes to which an antibody / antigen binding molecule can bind.

[0061] The term "valency" herein refers to the presence of a specified number of binding sites in an antibody / antigen binding molecule. Thus, the terms "monovalent", "divalent", "tetravalent" and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites and six binding sites in an antibody / antigen binding molecule, respectively.

[0062] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0063] As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. An antibody fragment can be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb comprising VH and VL domains; (vi) a dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) isolated complementarity determining regions (CDRs); and (ix) combinations of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, the two domains can be joined by a linker using recombinant methods, which enables them to be made into a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or by chemical peptide synthesis procedures known in the art.

[0064] As used herein, the term "ADAM9" includes any variant or isoform of ADAM9 naturally expressed by cells. The antibodies disclosed herein may cross-react with ADAM9 derived from non-human species. As another option, the antibody may also be human ADAM9-specific binding and may not show cross-reactivity with other species. ADAM9 or any variant or isoform thereof may be isolated from cells or tissues that naturally express them, or produced by recombinant techniques using techniques common in the art and those described herein. Preferably, anti-ADAM9 antibodies target human ADAM9 with a normal glycosylation pattern.

[0065] As used herein, the term "bispecific antibody" refers to an antibody that has monoclonal binding specificity to at least two different antigens, which is typically a human or humanized antibody. In the present disclosure, one of the binding specificities can be detected for an antigenic epitope of ADAM9, and the other can be detected for another antigenic epitope of ADAM9 or any other antigen other than ADAM9, such as cell surface proteins, receptors, receptor subunits, tissue-specific antigens, viral-derived proteins, virally encoded envelope proteins, bacterial-derived proteins, or bacterial surface proteins, etc.

[0066] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences of an immunoglobulin derived from one biological source (e.g., rat or mouse) and constant regions of an immunoglobulin derived from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al., 1986, Bio Techniques 4: 214-221; Gillies et al., 1985, J Immunol Methods 125: 191-202; the above are incorporated herein by reference.

[0067] As used herein, the term "heavy chain antibody" refers to an antibody lacking the light chain of a conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0068] As used herein, the term "nanobody" refers to a natural heavy chain antibody lacking a light chain present in camels. Cloning its variable region can obtain a single domain antibody consisting of only the variable region of the heavy chain, also known as VHH (Variable domain of heavy chain antibody), which is the smallest functional antigen-binding fragment. For further description of VHH and nanobodies, reference is made to the review article by Muyldermans (2001, Reviews in Molecular Biotechnology 74: 277-302). Nanobodies (particularly VHH sequences and partially humanized nanobodies) can be characterized in particular by the presence of one or more "characteristic residues" in one or more framework sequences. Further descriptions of nanobodies are found in, for example, WO 08 / 101985 and WO 08 / 142164, including humanization and / or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications that increase the half-life of nanobodies and their preparations. For a further general description of Nanobodies, reference is made to the prior art cited herein, for example as described in WO 08 / 020079 (page 16).

[0069] As used herein, the term "complementarity determining region" (CDR) refers to a hypervariable region found in both the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is called a framework region (FR). As understood in the art, the amino acid position representing the hypervariable region of an antibody can vary according to the context and various definitions known in the art, such as CDRs and sequences thereof confirmed according to the Kabat, IMGT, Chothia, Martin, and AHo numbering systems. Some positions within the variable domain can be considered as hybrid hypervariable positions, because these positions can be considered to be within a hypervariable region under a set of standards (such as IMGT or KABAT), and are considered to be outside the hypervariable region under different sets of standards (such as KABAT or IMGT). One or more of these positions can also be found in an extended hypervariable region. The present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each include four framework regions that are primarily in a sheet configuration, which are connected by three CDRs (CDR1, CDR2, and CDR3), which form a loop connecting the sheet structure, and in some cases form a part of the sheet structure. The CDRs in each chain are closely held together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen binding site of the antibody with the CDRs from other antibody chains (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference). For example, in this article, CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or its variable region); CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, the second CDR and the third CDR of the light chain variable region (VL), respectively, and these three CDRs constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).

[0070] As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation to the method by which the antibody is produced.

[0071] As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain (including the heavy chain of Fv, scFv or Fab) of an antibody. The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv or Fab).

[0072] The term "heavy chain constant region" herein refers to the carboxyl terminal portion of the heavy chain of an antibody, which is not directly involved in the binding of the antibody to the antigen, but exhibits effector functions, such as interactions with Fc receptors, which have a more conservative amino acid sequence relative to the variable domains of the antibody. The "heavy chain constant region" comprises at least one of the following: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment", the former having a structure substantially similar to that of a natural antibody constant region, while the latter only includes "a portion of a full-length heavy chain constant region". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from a CH1, Fc or CH3 domain.

[0073] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in the binding of the antibody to an antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0074] The term "Fc" herein refers to the antibody carboxyl terminal portion formed by papain hydrolysis of intact antibodies, typically comprising the CH3 and CH2 domains of antibodies. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of immunoglobulin heavy chains can vary slightly, the Fc region of human IgG heavy chains is generally defined as extending from the amino acid residue at Cys226 position or from Pro230 to its carboxyl terminal. The C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) can be, for example, removed during the production or purification of antibodies, or by recombinant engineering of nucleic acids encoding antibody heavy chains, and therefore, the Fc region may include or may not include Lys447.

[0075] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0076] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein are not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0077] The term "naked antibody" herein refers to an antibody that is not connected, fused or conjugated to another agent or molecule (e.g., a label or drug), a peptide or a polypeptide. In a specific embodiment, a naked antibody expressed by a mammalian host cell can be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell. In certain embodiments, when expressed by a host cell that does not have its own glycosylation machinery (e.g., glycosylase), the naked antibody is not glycosylated. In certain embodiments, the naked antibody is a complete antibody, while in other embodiments, the naked antibody is an antigen-binding fragment of a complete antibody, such as a Fab antibody.

[0078] The term "conjugated antibody" herein refers to an antibody that can be associated with a pharmaceutically acceptable carrier or diluent, which can be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody.

[0079] The term "diabody" herein refers to a bivalent, bispecific antibody that can bind to different epitopes on the same or different antigens.

[0080] As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced in one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored). For the purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires maximum alignment over the full length of the compared sequence. For example, a reference sequence for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence compared for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0081] The term "conservative amino acid" herein generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). Exemplarily, the amino acids within each of the following groups are conservative amino acid residues, and the replacement of amino acid residues within the group is a replacement of conservative amino acids:

[0082] (1) Acidic amino acids: Asp (D) and Glu (E);

[0083] (2) Basic amino acids: Lys (K), Arg (R) and His (H);

[0084] (3) hydrophilic uncharged amino acids: Ser (S), Thr (T), Asn (N), and Gln (Q);

[0085] (4) Aliphatic uncharged amino acids: Gly (G), Ala (A), Val (V), Leu (L) and Ile (I);

[0086] (5) non-polar uncharged amino acids: Cys (C), Met (M) and Pro (P);

[0087] (6) Aromatic amino acids: Phe (F), Tyr (Y) and Trp (W).

[0088] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0089] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0090] The term "IMGT numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature, 342:878-883).

[0091] As used herein, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by an antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not show specific binding to a particular antigen or epitope thereof will show a KD of greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope thereof. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein or carbohydrate.

[0092] As used herein, the term "antibody conjugate" refers to a coupling / conjugate formed by chemically bonding an antibody molecule directly or through a linker to another molecule, such as an antibody-drug conjugate (ADC), in which the drug molecule is the other molecule.

[0093] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules as described herein may contain naturally occurring or non-naturally occurring nucleotides.

[0094] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors disclosed herein contain polynucleotide sequences and additional sequence elements, such as for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express antibodies and antibody fragments of the present disclosure include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES) and polyadenylation signal sites to direct the efficient transcription of genes carried on the expression vector. The expression vectors disclosed herein may also contain the following polynucleotides, which encode markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0095] The term "host cell" herein refers to a cell into which an exogenous nucleic acid is introduced, including the offspring of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initially transformed cell are included herein.

[0096] The term "pharmaceutical composition" herein refers to a preparation which is in a form which permits the biological activity of the active ingredient contained therein to be effective, and which contains no additional ingredients which are unacceptably toxic to a subject to which the pharmaceutical composition is administered.

[0097] As used herein, the terms "subject," "object," and "patient" refer to an organism that is being treated for a particular disease or condition as described herein, such as cancer or an infectious disease. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as cattle, bison, buffalo, elk, and yaks, etc.), sheep, and horses, etc., that are being treated for a disease or condition (e.g., a cell proliferative disorder such as cancer or an infectious disease).

[0098] As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treatment subject, such as the progression of cell proliferative disorders (such as cancer or infectious diseases). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects who already have a disease or disease, as well as subjects who are susceptible to a disease or disease or subjects who intend to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0099] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that is effective in preventing or alleviating a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. "Effective amount" also refers to an amount of a compound sufficient to relieve symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevent, or alleviate these conditions. When an active ingredient is administered alone to an individual, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0100] The term "appropriate conditions" herein refers to conditions suitable for culturing various host cells, wherein the host cells include eukaryotic cells and prokaryotic cells.

[0101] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are both benign and malignant cancers.

[0102] The term "tumor" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

[0103] The term "anti-tumor agent" herein refers to anti-tumor drugs, which are a class of drugs for treating tumor diseases, including chemotherapy drugs, biological agents, etc.

[0104] The term "EC50" herein refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the antibody concentration at which 50% of its maximal effect is observed, and can be measured by methods known in the art.

[0105] Specific implementation plan

[0106] In the first aspect, the present disclosure provides an anti-ADAM9 nanobody, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises: a heavy chain CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and variants thereof, a heavy chain CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and variants thereof, and a heavy chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 12 and variants thereof.

[0107] In some embodiments, the anti-ADAM9 nanobody comprises: a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 10, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 11, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO: 12.

[0108] In some embodiments, the variant is a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 10-12 as a reference sequence; preferably, the substitutions are conservative amino acid substitutions.

[0109] In some embodiments, the anti-ADAM9 nanobody further comprises four FR regions, comprising: a heavy chain FR1 having the amino acid sequence shown in SEQ ID NO: 13, a heavy chain FR2 having the amino acid sequence shown in SEQ ID NO: 14, a FR3 having the amino acid sequence shown in SEQ ID NO: 15 or 16, and a FR4 having the amino acid sequence shown in SEQ ID NO: 17.

[0110] More specifically, the structure of the anti-ADAM9 nanobody is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0111] In some embodiments, the anti-ADAM9 Nanobody comprises a heavy chain variable region selected from the amino acid sequence shown in SEQ ID NO: 8 or 9 and variants thereof.

[0112] In one embodiment, the anti-ADAM9 Nanobody comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 8 or any variant thereof.

[0113] In one embodiment, the anti-ADAM9 Nanobody comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 9 or any variant thereof.

[0114] Specifically, the variant is an amino acid sequence that individually has at least 90% sequence identity compared to the amino acid sequence shown in SEQ ID NO: 8 or 9 as a reference sequence; more preferably, it is an amino acid sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to any one of SEQ ID NOs: 8-9.

[0115] In some embodiments, the nanobody is any one of an engineered antibody, a monospecific antibody, a multispecific antibody, a monovalent antibody, a multivalent antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0116] In some embodiments, the Nanobody comprises a heavy chain constant region sequence without the CH1 fragment.

[0117] In some embodiments, the Nanobody comprises a heavy chain constant region sequence having CH2 and CH3 fragments.

[0118] In some embodiments, the anti-ADAM9 nanobody is a chimeric antibody, more preferably, an anti-ADAM9 VHH-Fc fusion protein.

[0119] In some embodiments, the anti-ADAM9 VHH-Fc fusion protein comprises the human IgG1Fc fragment shown in SEQ ID NO.5.

[0120] In some embodiments, the anti-ADAM9 VHH-Fc fusion protein has a heavy chain sequence shown in SEQ ID NO.6 or 7.

[0121] In some embodiments, the dissociation constant (KD) of the Nanobody binding to human ADAM9 is no greater than 10 -7 nM, the dissociation constant (KD) for binding to cynomolgus monkey ADAM9 is no greater than 10 -8 nM.

[0122] Optionally, the Nanobody binds or does not bind to monkey ADAM9 protein.

[0123] Optionally, the Nanobody binds or does not bind to the mouse ADAM9 protein.

[0124] Optionally, the multispecific antibody is a "bispecific", "trispecific" or "tetraspecific".

[0125] In some embodiments, the Nanobody comprises a sequence of the heavy chain constant region of any antibody selected from human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it comprises a sequence of the heavy chain constant region of any antibody selected from human or murine antibodies IgG1, IgG2, IgG3 or IgG4.

[0126] In some embodiments, the Nanobody is a chimeric, or humanized, or fully humanized antibody.

[0127] In some embodiments, the Nanobody is coupled to a therapeutic agent or a tracer; more preferably, the therapeutic agent is selected from a radioactive isotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from any one of a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent or a photosensitizer.

[0128] In a second aspect, the present disclosure further provides a multispecific antigen-binding molecule, which comprises the anti-ADAM9 nanobody and antigen-binding fragment thereof as described in any one of the first aspects.

[0129] In some embodiments, the multispecific antigen-binding molecule comprises a first antigen-binding module and a second antigen-binding module; wherein the first antigen-binding module comprises the anti-ADAM9 nanobody described in any one of the first aspects; the second antigen-binding module specifically binds to antigens other than ADAM9 or binds to an ADAM9 antigen epitope different from the first antigen-binding module.

[0130] In some embodiments, the other antigen is any one selected from CD3, PD-1, PD-L1, Her2, EpCAM, CD16, CD20, CD30, CD33, CD47, CD52, CD64, CD133, CEA, gpA33, Mucins, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL or FAP.

[0131] In a third aspect, the present disclosure provides an isolated nucleic acid molecule, which encodes the anti-ADAM9 nanobody described in any one of the first aspect, or the multispecific antigen-binding molecule described in any one of the second aspect.

[0132] In a fourth aspect, the present disclosure provides an expression vector comprising any isolated nucleic acid molecule described in the third aspect.

[0133] In a fifth aspect, the present disclosure provides a host cell comprising any nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect.

[0134] Preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from HEK293E or Chinese hamster ovary cell (CHO).

[0135] In a sixth aspect, the present disclosure provides a method for preparing the anti-ADAM9 nanobody as described in the first aspect, comprising culturing the host cell described in the fifth aspect under appropriate conditions and isolating the nanobody.

[0136] In the seventh aspect, the present disclosure provides a pharmaceutical composition comprising any one of the nanoantibodies described in any one of the first aspect, the multispecific antigen-binding molecules described in any one of the second aspect, the isolated nucleic acid molecules described in the third aspect, the expression vector described in the fourth aspect, and the host cell described in the fifth aspect.

[0137] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; more preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0138] In the eighth aspect, the present disclosure provides a method for preventing and / or treating tumor diseases or inflammatory diseases, comprising administering to an individual in need thereof an effective dose of the anti-ADAM9 nanoantibody described in any one of the first aspect, or the multispecific antigen-binding molecule described in any one of the second aspect, or the isolated nucleic acid molecule described in any one of the third aspect, or the expression vector described in any one of the fourth aspect, or the host cell described in any one of the fifth aspect, or the pharmaceutical composition described in any one of the seventh aspect.

[0139] In some embodiments, the tumor disease or inflammatory disease is a tumor disease or inflammatory disease in which ADAM9 is overexpressed.

[0140] In some embodiments, the tumor disease is bladder cancer, breast cancer (especially triple-negative breast cancer), cervical cancer, colorectal cancer (especially adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, melanoma or squamous cell carcinoma), brain cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, non-small cell lung cancer (especially squamous cell carcinoma, adenocarcinoma or large cell undifferentiated carcinoma), bone marrow cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, endometrial cancer, gallbladder cancer.

[0141] In the ninth aspect, the present disclosure provides the use of the anti-ADAM9 nanobody described in any one of the first aspect, or the multispecific antigen-binding molecule described in any one of the second aspect, or the isolated nucleic acid molecule described in any one of the third aspect, or the expression vector described in any one of the fourth aspect, or the host cell described in any one of the fifth aspect, or the pharmaceutical composition described in any one of the seventh aspect in the preparation of a medicament for preventing and / or treating tumor diseases or inflammatory diseases.

[0142] In some embodiments, the tumor disease or inflammatory disease is a tumor disease or inflammatory disease in which ADAM9 is overexpressed.

[0143] In some embodiments, the neoplastic disease is cancer.

[0144] In some embodiments, the cancer is selected from bladder cancer, breast cancer (especially triple-negative breast cancer), cervical cancer, colorectal cancer (especially adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, melanoma or squamous cell carcinoma), brain cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, non-small cell lung cancer (especially squamous cell carcinoma, adenocarcinoma or large cell undifferentiated carcinoma), bone marrow cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, endometrial cancer, gallbladder cancer. More preferably, the cancer is selected from: gastric cancer, non-small cell lung cancer, prostate cancer, brain cancer, pancreatic cancer, liver cancer, colorectal cancer, triple-negative breast cancer.

[0145] In the tenth aspect, the present disclosure provides a pharmaceutical preparation comprising at least one component of the following substances as an active ingredient: the anti-ADAM9 nanobody described in any one of the first aspect, the multispecific antigen-binding molecule described in any one of the second aspect, the isolated nucleic acid molecule described in any one of the third aspect, the expression vector described in any one of the fourth aspect, the host cell described in any one of the fifth aspect, and the pharmaceutical composition described in any one of the seventh aspect.

[0146] In some embodiments, the pharmaceutical preparation can be used for screening, diagnosing or treating tumor diseases or inflammatory diseases.

[0147] In the eleventh aspect, the present disclosure provides a kit comprising at least one component of the following substances as an active ingredient: the anti-ADAM9 nanobody described in any one of the first aspect, the multispecific antigen-binding molecule described in any one of the second aspect, the isolated nucleic acid molecule described in any one of the third aspect, the expression vector described in any one of the fourth aspect, the host cell described in any one of the fifth aspect, and the pharmaceutical composition described in any one of the seventh aspect; and instructions for use.

[0148] In the twelfth aspect, the present disclosure provides a medicine kit comprising at least one component of the following substances as an active ingredient: the anti-ADAM9 nanobody described in any one of the first aspect, the multispecific antigen-binding molecule described in any one of the second aspect, the isolated nucleic acid molecule described in any one of the third aspect, the expression vector described in any one of the fourth aspect, the host cell described in any one of the fifth aspect, and the pharmaceutical composition described in any one of the seventh aspect; and instructions for use.

[0149] Example

[0150] By referring to some specific embodiments provided herein, a further understanding of the technical solution of the present disclosure can be obtained. These embodiments are only used to illustrate the present disclosure, and their purpose is not to limit the scope of the present disclosure in any way. Obviously, various modifications and changes can be made to the present disclosure without departing from the essence of the present disclosure, and therefore, these modifications and changes are also within the scope of the protection claimed in this application.

[0151] Example 1: Antigen, Immunization Scheme and Antibody Preparation

[0152] 1. Antigen

[0153] The protein antigen used for immunization is the 206-297 amino acids of the human ADAM9 recombinant protein (NCBI Reference Sequence: NP_003807.1) (SEQ ID NO: 1), i.e., the ADAM9 extracellular domain (ADAM9-ExtraCellular Domain, ADAM9-ECD, SEQ ID NO: 2), with 6 His tags at the C-terminus (SEQ ID NO: 3), i.e., human ADAM9-ECD-His protein (SEQ ID NO: 4), which is prepared and provided by Sanyou Biopharmaceuticals (Shanghai) Co., Ltd.

[0154] 2. Immunization regimen

[0155] The above human ADAM9-ECD-His protein was used for animal immunization. Specifically, two alpacas were selected for immunization, and each alpaca was immunized 4-5 times, and immunization was performed once every 2 weeks. The amount of antigen for each immunization was 0.5 mg ADAM9-ECD-His protein / mouse, and the immune adjuvant was Freund's complete adjuvant. The injection method was subcutaneous multi-point injection.

[0156] On the 14th day after the designated number of immunizations, blood was collected for serum titer testing.

[0157] Specifically, on the 14th day after the 4th or 5th immunization, 1 ml of peripheral blood was collected, serum samples were separated, and the titer and specificity of the nanobody against human ADAM9 in the serum were detected using an enzyme-linked immunosorbent assay (ELISA).

[0158] 3. Serum titer detection

[0159] Sample preparation

[0160] The serum of the immunized mice was taken and graded diluted with 5% PBST according to the dilution multiples listed in Table 1 to obtain samples of different concentrations.

[0161] 3.2. ELISA method to detect serum titer

[0162] (1) Coating: 2 μg / mL antigen, 30 μL per well, incubate at 4°C overnight, wash three times with PBST.

[0163] (2) Blocking: Block with 5% PBST at room temperature for 2 h, and wash the plate three times with PBST.

[0164] (3) Primary antibody: Add the above-mentioned serially diluted serum, and non-immune mouse serum as a negative control, 30 μL / well, incubate at room temperature for 1 h, and wash the plate 6 times with PBST.

[0165] (4) Secondary antibody: Serum samples were added with 1:5000 dilution of secondary antibody goat anti-mouse-IgG-HRP (Rockland, cat#609-103-123), diluted in 5% PBST; positive control group was added with 1:5000 dilution of secondary antibody goat anti-human IgG-HRP (Shanghai ELISA Biotechnology Co., Ltd., ml087062), diluted in 5% PBST. 30 μL / well, incubated at room temperature for 50 min, and washed 6 times with PBST.

[0166] (5) Termination: Add 30 μL / well of TMB (Suzhou Yaco Chemical Reagent Co., Ltd., cat# S0025) to develop color at room temperature for 5 min to 10 min, then add 30 μL / well of 2 M stop solution (Suzhou Yaco Chemical Reagent Co., Ltd.) to terminate the reaction. Use a microplate reader to read the OD450 data.

[0167] The test results are shown in Table 1 below. The titer of the 4th immunization against human ADAM9-ECD-His protein reached 64K, and the titer of the 5th immunization serum against human ADAM9-ECD-His protein reached 32K, and the serum titer test results were qualified. This shows that alpacas can be used for VHH antibody phage library construction.

[0168] Table 1. ELISA detection of alpaca serum antibody titers after immunization with human ADAM9 protein

[0169]

[0170] 4. Construction of phage antibody library

[0171] (1) After the immunization, 50 mL of peripheral blood was collected from alpacas with qualified serum antibody titers, and PBMCs were separated using lymphocyte separation solution. Total RNA was extracted using Trizol reagent, and the extracted product was identified by agarose gel electrophoresis. The results were as follows: Figure 1 As shown, there are three main RNA bands, namely 28S, 18S and 5S, which are used in subsequent experiments.

[0172] (2) According to the reverse transcription kit Prime Script TM cDNA was obtained using II 1st Strand cDNA Synthesis Kit (Takara, 6210A).

[0173] The nanobody (VHH) fragment was amplified by nested PCR. After the PCR amplification product was purified, it was ligated into the phage display vector pComb3Xss using restriction endonuclease SfiI (purchased from NEB, R0123L), and the ligation product was purified.

[0174] (3) Electroporate the cells at a ratio of 1-2 μg of purified product per 70 μL of TG1 competent cells. Electroporate the ligated product for a total of 7 electroporation transformations. Immediately after electroporate, add 1 mL of SOC medium to the electroporation cup for recovery and culture for 1 h, obtaining a total of 7.3 ml of TG1 cell recovery product.

[0175] (4) After electroporation, 10 μL was taken for dilution determination and reservoir capacity calculation. The TG1 bacterial solution was gradiently diluted 10 3 Times and 10 4 The number of transformants in the Nanobody library was determined by multiplying the number of transformants in the Nanobody library. 3 × and 10 4 × diluted TG1 bacterial solution was spread on a 150 mm plate, and the remaining bacterial solution was spread on 7 150 mm plates. The storage capacity was calculated to be 2.5×10 8 and 4×10 8 .

[0176] (5) 89 samples were sequenced, and the results showed that the insertion rate of the phage display library was higher than 90% and there were no repeated sequences, that is, the construction of the phage library was successful.

[0177] (6) The successfully sequenced sequences of the single-domain antibody library are converted into amino acid sequences to further analyze the diversity of its antibody genes; and by comparing the CDR region sequences of each clone one by one, it can be determined that the single-domain antibody library obtained by the above technical solution of the present invention exhibits high abundance.

[0178] Example 2: Screening of Alpaca Immune Antibody Library

[0179] Experimental Example 2.1: Screening of Alpaca Nanobody Library

[0180] The immunotube method was used for screening, as follows:

[0181] (1) Phage blocking: The alpaca nanobody library phage suspension prepared in Example 1 was blocked with 5% BSA;

[0182] (2) Coated immunotube: The human ADAM9-ECD-His used in the example was coated with 5% PBSM for blocking;

[0183] Add to 2 mL PBS, then add to the immunotube and incubate overnight at 4°C;

[0184] (3) Binding: incubating the coated and sealed immunotubes in the above steps, and binding and washing several times according to the commonly used methods of immunotube screening systems;

[0185] (4) Elution: elution of phages with 0.25% trypsin Trysin (Shanghai Biotechnology Co., Ltd.);

[0186] (5) Infection: The eluted phage solution was thoroughly mixed with logarithmic phase SS320 competent cells (Sanyou Biotechnology) and incubated at 37°C for 30 min. Spread on a plate containing 2x YT medium containing carbenicillin and tetracycline (2YT-Car+-Tet+, Sanyou Biotechnology) and cultured in a 37°C incubator overnight;

[0187] (6) Spot coating: The eluted phage solution was diluted 10-fold with logarithmic phase SS320 cells, incubated at 37°C for 30 min, mixed and then 2 μL was dropped on the plate and cultured in a 37°C incubator overnight.

[0188] The phage input and output were calculated, and the input phage was prepared by scraping and subjected to three rounds of screening. Each round of screening used a 3-fold gradient of antigen decreasing concentration to screen for antibody clones with higher affinity.

[0189] Experimental Example 2.2: Cell Screening Method

[0190] (1) Phage blocking: The phage selected in Experimental Example 2.1 was diluted 4 times with 5% FBS-PBS. 3 mL of 5% FBS was added to 1 mL of phage, and the mixture was placed on a four-dimensional rotator and mixed and blocked at room temperature for 1 h.

[0191] (2) Incubation: First, add the blocked phages to the negative screening cells; after binding, gently stand the cell culture bottle upright, use a disposable pipette to suck out the negatively screened phage library, and add it to the corresponding positive screening cell bottle, and incubate it in an orbital shaker (speed 80 rpm) at room temperature for 2 h;

[0192] Use a disposable pipette to suck up the blocked phage and slowly add it into the treated cell bottle along the side wall;

[0193] (3) Washing: discard the supernatant in the culture flask, add 5% FBS-PBS along the non-cell side to prevent the cells from being blown up; then place the cell plate upright so that the liquid covers the cells, shake on an orbital shaker (speed 80 rpm) for 1 min each time 5% FBS-PBS is added, then slowly stand the cell culture flask upright, carefully aspirate using a disposable pipette, and wash three times;

[0194] (4) Phage library elution: Add 2 mL of 100 mM glycine-HCl (pH 2.2), incubate on a shaker (80 rpm) at room temperature for 10 min, add 140 μL of 1 M Tris-base, and neutralize to a pH of 7-7.2.

[0195] Experimental Example 2.3: Detection of the positive rate of the selected set

[0196] The expression supernatant of the screening pool obtained from the above two rounds of screening was diluted 5-fold using 5% PBSM, and affinity test was performed by ELISA.

[0197] Specifically, the ELISA test steps are as follows:

[0198] (1) Coating: Add 2 μg / mL human ADAM9-ECD-His to a 96-well plate, 30 μL per well, incubate overnight at 4°C, and wash three times with PBST.

[0199] (2) Blocking: The multiwell plate was blocked with 5% PBSM at room temperature for 1 h and then washed three times with PBST.

[0200] (3) Primary antibody: Add the above-mentioned gradient diluted phage expression supernatant sample, 30 μL / well, incubate at room temperature for 1 h, and wash three times with PBST.

[0201] (4) Secondary antibody: Take the phage supernatant and add the secondary antibody Anti-M13-HRP diluted 1:20000, 30 μL / well in 5% PBSM, incubate at room temperature for 1 hour, and wash 6 times with PBST.

[0202] (5) Termination: Add 30 μL / well of TMB to develop color at room temperature for 5 min to 10 min, then add 2 M stop solution, 30 μL / well, to terminate the reaction.

[0203] Use a microplate reader to read the OD450 data.

[0204] According to the test results of the ELISA of the screening collection, monoclonal clones from the second and third rounds of the screening were selected for primary screening by VHHELISA. The human ADAM9-ECD-His antigen coating method was used for screening. A total of 872 clones were picked, and 207 positive clones with human-monkey cross-reactivity were obtained. After sequence analysis, 65 molecules with unique sequences were obtained, and 42 molecules with unique sequences were constructed.

[0205] Example 3: Preparation of anti-ADAM9 VHH-Fc fusion protein

[0206] The variable region sequence of VHH obtained in Example 2 and the human IgG1 Fc sequence were operably linked by genetic means (the human IgG1 Fc sequence is shown in SEQ ID NO: 5) and inserted into the expression vector BI3.4-huIgG1 (from Biointron). Plasmids were prepared according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).

[0207] The expression vector was transiently transfected into HEK293E cells (purchased from the cell bank of the Chinese Academy of Sciences Typical Culture Collection Committee) according to the instructions of PEI (purchased from Polysciences, catalog number: 24765-1), and cultured continuously for 5 days at 37°C using FreeStyle TM 293 (Thermofisher scientific, catalog number: 12338018), and the cell components were removed by centrifugation to obtain the culture supernatant containing VHH-Fc fusion protein. The culture supernatant was loaded onto a protein A chromatography column (protein A filler AT ProteinA Diamond and chromatography column BXK16 / 26 were purchased from Bogelon, catalog numbers: AA0273 and B-1620), washed with PBS phosphate buffer (pH 7.4) and then washed with 20mM PB, 1M NaCl (pH 7.2), and finally eluted with pH 3.4 citric acid buffer to collect the Fc-tagged antibodies eluted from the protein A chromatography column. The protein was neutralized with 1 / 10 volume of 1M Tris at pH 8.0 and dialyzed with PBS at 4°C overnight. The dialyzed protein was sterile filtered through a 0.22 μm filter membrane and packaged and stored at -80°C to obtain 42 full-length VHH1-42 Fc fusion proteins.

[0208] Example 4: Binding of VHH-Fc fusion protein to CHO-S cells expressing human ADAM9

[0209] Fluorescence activated cell sorting (FACS) was used to perform affinity testing and screening on the 42 full-length VHH-Fc obtained in Example 3 to further optimize the antibodies.

[0210] The specific steps of FACS testing are as follows:

[0211] (1) T75 cells were cultured in a culture flask until the logarithmic growth phase. For CHO-S cells expressing human ADAM9 that were grown on the wall, the culture medium was removed, the cells were washed twice with PBS buffer, and then the cells were digested with trypsin. After the digestion was terminated, the cells were washed twice with PBS buffer;

[0212] (2) Count the cells in step (1), and then resuspend the cell pellet to 4×10 in PBS (containing 2% (w / w) FBS) blocking solution. 6 cells / mL, added to a 96-well FACS reaction plate at 50 μL / well; then added the VHH-Fc fusion protein sample at 50 mL / well, incubated at 4°C for 1 hour, and then washed three times by centrifugation with PBS buffer;

[0213] (3) Add 100 μL / well of PE-labeled anti-human Fc secondary antibody (Abcam, 98596) at a dilution of 1:300 and incubate at 4°C for 30 min; wash five times with PBS buffer;

[0214] (4) FACS (FACS CantoTM, purchased from BD) was used to detect and analyze the results. Data were analyzed using software (CellQuest) to obtain the mean fluorescence intensity (MFI) of the cells;

[0215] (5) Samples with a MFI > 500 (3 times the background value) of the cell-only wells were selected to enter the candidate pool for the next round of humanization transformation.

[0216] After testing, the MFI results of each VHH-Fc are shown in Table 2 below. These results show that according to the MFI values, VHH3, VHH5, VHH24 and VHH36 can be used as candidate antibodies for the next step of humanization. Among them: the amino acid sequence of the VHH3-Fc fusion protein is shown in SEQ ID NO:5; the amino acid sequence of the VHH5-Fc fusion protein is shown in SEQ ID NO:6; the amino acid sequence of VHH3 is shown in SEQ ID NO:7; the amino acid sequence of VHH5 is shown in SEQ ID NO:8.

[0217] Table 2. VHH-Fc fusion protein samples and their corresponding MFI values

[0218]

[0219] Note: The MFI value of the well with only cells (cell only) is 166.2

[0220] Example 5: Binding of VHH-Fc fusion protein to human ADAM9 protein

[0221] The four antibodies screened in Example 4 were humanized. The binding of the VHH-Fc fusion protein to the human ADAM9 protein was detected by ELISA. The specific steps are as follows:

[0222] (1) The human ADAM9-ECD-His protein in Example 1 was diluted with PBS to a final concentration of 1 μg / mL, and then added to a 96-well ELISA plate at 50 μL / well. The plate was sealed with a plastic film and incubated at 4°C overnight. The plate was washed twice with PBS the next day, and blocked with blocking solution PBS (containing 2% (w / w) BSA) at room temperature for 2 h.

[0223] (2) Pour off the blocking solution and add 100 nM of VHH-Fc fusion protein to perform gradient dilutions, taking 50 μL / well, incubate at 37°C for 2 h, and then wash the plate three times with PBS.

[0224] (3) Add HRP (horseradish peroxidase) labeled secondary antibody (purchased from Sigma, catalog number: A0170), incubate at 37°C for 2 h, and wash the plate 5 times with PBS. Then add TMB substrate, 50 μL / well, incubate at room temperature for 30 min, and add stop solution 1.0 N HCl, 50 μL / well.

[0225] (4) OD450 nm value was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The binding results of VHH-Fc fusion protein and human ADAM9 protein are shown in Figure 2 As shown, antibody IMGC936 (IMMUNOMEDICS) was used as a positive control.

[0226] Figure 2 The results showed that all VHH-Fc fusion proteins bound to human ADAM9 protein as detected by ELISA, and from the EC50 point of view, all VHH-Fc fusion proteins were superior to the positive control antibody IMGC936.

[0227] Example 6: Binding of anti-ADAM9 nanobodies to cells expressing ADAM9 from different species

[0228] 1. Experimental Methods

[0229] (1) culturing cells that highly express ADAM9, specifically, including CHO-S cells that overexpress human ADAM-9, CHO-S cells that overexpress monkey ADAM-9, and human colon cancer cells HT-29 that express ADAM9, digesting with trypsin, and collecting the cells by centrifugation;

[0230] (2) After adjusting the cell density with FACS buffer (1× PBS containing 2% FBS), the cells were plated in 96-well U-bottom plates at 1×105 Up to 2×10 5 cells; centrifuge at 1200 g for 5 min, discard the supernatant; add 100 μL of antibody solution that has been gradient diluted with FACS buffer, and incubate at 4°C for 1 h; centrifuge at 1200 g for 5 min, discard the supernatant, wash the cells twice with PBS, add 100 μL of fluorescently labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Abcam, 98596, 1:300) prepared with FACS buffer to each well to resuspend the cells, and incubate at 4°C for 1 h.

[0231] (3) Centrifuge at 1200 g for 5 min and discard the supernatant; wash the cells twice with PBS and resuspend them in PBS.

[0232] (4) Detect the fluorescence signal using a flow cytometer (DxFlex) and analyze the EC binding of the nanoantibody to the cell 50 concentration.

[0233] 2. Experimental results

[0234] The affinity (EC50) of each humanized antibody to ADAM9 antigen-expressing cells was determined.

[0235] From the experimental results in Table 3, it can be seen that the humanized antibodies have high affinity to the stable cell lines overexpressing human ADAM9 antigen and the stable cell lines of monkey ADAM9 antigen, among which VHH3-Fc and VHH5-Fc have better affinity and specificity. Moreover, each humanized antibody can bind to human colon cancer cell HT-29.

[0236] Table 3. Affinity of humanized antibodies for human ADAM9 high-expressing cells (EC 50 value)

[0237]

[0238]

[0239] The above-mentioned embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be subject to the attached claims.

Claims

1. An anti-ADAM9 nanobody, comprising a heavy chain variable region, wherein the heavy chain variable region comprises: a heavy chain CDR1 having an amino acid sequence selected from SEQ ID NO: 10 and variants thereof, a heavy chain CDR2 having an amino acid sequence selected from SEQ ID NO: 11 and variants thereof, and a heavy chain CDR3 having an amino acid sequence selected from SEQ ID NO: 12 and variants thereof; Preferably, the heavy chain variable region of the anti-ADAM9 Nanobody comprises: a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 12; Preferably, the variant is a sequence having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 10-12 as a reference sequence; preferably, the substitutions are substitutions of conservative amino acids; Preferably, the anti-ADAM9 nanobody further comprises 4 FR regions, wherein the FR regions include: A heavy chain FR1 having the amino acid sequence shown in SEQ ID NO: 13, a heavy chain FR2 having the amino acid sequence shown in SEQ ID NO: 14, a FR3 having the amino acid sequence shown in SEQ ID NO: 15 or 16, and a FR4 having the amino acid sequence shown in SEQ ID NO: 17; Preferably, the structure of the anti-ADAM9 nanobody is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; Preferably, the anti-ADAM9 nanobody comprises a heavy chain variable region selected from the amino acid sequence shown in SEQ ID NO: 8 or 9 and variants thereof; Preferably, the variant is an amino acid sequence that has at least 90% sequence identity compared to the amino acid sequence shown in SEQ ID NO: 8 or 9 as a reference sequence; more preferably, it is an amino acid sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to any one of SEQ ID NOs: 8-9; Preferably, the nanobody is any one of an engineered antibody, a monospecific antibody, a multispecific antibody, a monovalent antibody, a multivalent antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

2. The anti-ADAM9 nanobody according to claim 1, wherein the anti-ADAM9 nanobody is an anti-ADAM9 VHH-Fc fusion protein; Preferably, the anti-ADAM9 VHH-Fc fusion protein comprises the human IgG1Fc fragment shown in SEQ ID NO.5; Preferably, the anti-ADAM9 VHH-Fc fusion protein has a heavy chain sequence shown in SEQ ID NO.6 or 7.

3. A multispecific antigen-binding molecule comprising the anti-ADAM9 nanobody or antigen-binding fragment thereof according to any one of the first aspects; Preferably, the multispecific antigen-binding molecule comprises a first antigen-binding moiety and a second antigen-binding moiety; wherein, The first antigen binding moiety comprises the anti-ADAM9 nanobody described in any one of the first aspects; the second antigen binding moiety specifically binds to antigens other than ADAM9 or binds to an ADAM9 antigen epitope different from the first antigen binding moiety; Preferably, the other antigens are selected from any one of CD3, PD-1, PD-L1, Her2, EpCAM, CD16, CD20, CD30, CD33, CD47, CD52, CD64, CD133, CEA, gpA33, Mucins, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL or FAP.

4. An isolated nucleic acid molecule encoding the anti-ADAM9 nanobody according to claim 1 or 2 or the multispecific antigen-binding molecule according to claim 3.

5. An expression vector comprising the isolated nucleic acid molecule of claim 4.

6. A host cell comprising any nucleic acid molecule according to claim 4 or the expression vector according to claim 5; Preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from HEK293E or Chinese hamster ovary cell CHO.

7. A method for preparing the anti-ADAM9 nanobody according to claim 1, comprising culturing the host cell according to the fifth aspect under appropriate conditions and isolating the nanobody.

8. A pharmaceutical composition comprising any one of the anti-ADAM9 nanobody according to claim 1 or 2, the multispecific antigen-binding molecule according to claim 3, the isolated nucleic acid molecule according to claim 4, the expression vector according to claim 5, and the host cell according to claim 6; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; More preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.

9. Use of the anti-ADAM9 nanobody according to claim 1 or 2, or the multispecific antigen-binding molecule according to claim 3, or the isolated nucleic acid molecule according to claim 4, or the expression vector according to claim 5, or the host cell according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing and / or treating a tumor disease or an inflammatory disease; Preferably, the tumor disease or inflammatory disease is a tumor disease or inflammatory disease in which ADAM9 is overexpressed; Preferably, the tumor disease is cancer; Preferably, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colorectal cancer, brain cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, non-small cell lung cancer, bone marrow cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, endometrial cancer, gallbladder cancer; More preferably, the cancer is selected from the group consisting of gastric cancer, non-small cell lung cancer, prostate cancer, brain cancer, pancreatic cancer, liver cancer, colorectal cancer, and triple-negative breast cancer.

10. A pharmaceutical preparation comprising at least one of the following substances as an active ingredient: the anti-ADAM9 nanobody according to claim 1 or 2, the multispecific antigen-binding molecule according to claim 3, the isolated nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, and the pharmaceutical composition according to claim 8; Preferably, the pharmaceutical preparation can be used for screening, diagnosing or treating tumor diseases or inflammatory diseases.

11. A kit comprising at least one of the following substances as an active ingredient: the anti-ADAM9 nanobody according to claim 1 or 2, the multispecific antigen-binding molecule according to claim 3, the isolated nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6 and the pharmaceutical composition according to claim 8; and instructions for use.

12. A medicine kit comprising at least one of the following substances as an active ingredient: the anti-ADAM9 nanobody according to claim 1 or 2, the multispecific antigen-binding molecule according to claim 3, the isolated nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6 and the pharmaceutical composition according to claim 8; and instructions for use; and instructions for use.

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