Anti-ADAM9 antibodies and their uses

Abstract

This invention relates to an anti-ADAM9 antibody and its uses. The anti-ADAM9 antibody or its antigen-binding fragment provided by this invention has good affinity and endocytic activity, and shows promising potential for drug development.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to antibodies or antigen-binding fragments thereof that specifically bind to ADAM9. This invention also relates to methods for preparing anti-ADAM9 antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the anti-ADAM9 antibody or antigen-binding fragments thereof, vectors containing the nucleic acid molecules, host cells, and compositions containing the anti-ADAM9 antibody or antigen-binding fragments thereof. This invention further relates to the use of anti-ADAM9 antibodies or antigen-binding fragments thereof and compositions containing them. Background Technology

[0002] ADAM9 is a cell membrane surface protein with protease activity that regulates tumor cell proliferation and invasion. Its integrin dissociation domain can bind to integrins, regulating tumor cell adhesion. Its metalloprotein domain possesses protease activity, capable of cleaving and releasing extracellular segments of membrane proteins, regulating tumor migration, invasion and metastasis, immune evasion, and extracellular matrix degradation. ADAM9 enzymatic activity is higher in tumor tissues than in normal tissues, and it is highly expressed in non-small cell lung cancer and gastric cancer, and at high levels in ovarian cancer, breast cancer, and pancreatic cancer. Compared to HER2 and Trop2, ADAM9 shows a clear preference for expression in the same tumor tissue, suggesting that targeting ADAM9 kills cells within a specific cellular spectrum. This could differentiate the target population for HER2 and Trop2 ADC drugs, allowing for the utilization of ADAM9's tumor-specific high expression to effectively inhibit ADAM9 enzymatic activity and precisely target and kill tumor cells through conjugated toxins. Summary of the Invention

[0003] The purpose of this invention is to provide an antibody or antigen-binding fragment thereof that has a strong binding affinity to ADAM9, and to provide its uses based on the antibody or fragment thereof.

[0004] On the one hand, this invention provides an antibody or antigen-binding fragment thereof that has binding specificity to detegrin-like metalloproteinase 9 (ADAM9). The anti-ADAM9 antibody or antigen-binding fragment of this invention exhibits many excellent properties, including high affinity and specificity in binding to human ADAM9 protein, as well as good endocytic activity and inhibition of ADAM9 protease activity, showing promising drug development potential.

[0005] The present invention provides an anti-ADAM9 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 respectively, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 respectively.

[0006] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises at least a heavy chain variable region and a light chain variable region, both of which include the aforementioned CDRs and spaced framework regions (FRs), with the domains arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0007] In some embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is of animal origin, such as mouse origin, or is a chimeric antibody; preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 6, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 6. In some specific embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6.

[0008] In some embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the heavy chain variable region comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising a heavy chain FR region of human IgG1, IgG2 or IgG4; and the light chain variable region comprises a light chain FR region derived from human κ chain, λ chain or its variants, preferably comprising a light chain FR region derived from human κ chain.

[0009] In some embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 11-16, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences shown in SEQ ID NO: 11-16; the light chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 17-23, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences shown in SEQ ID NO: 17-23.

[0010] In some specific embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein: (1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; (2) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18. (3) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 19, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19. (4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17. (5) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 20, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; (6) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 14; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 21, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21; (7) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 22; or (8) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 16, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 23, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23.

[0011] In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 17. In other preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 18. In other preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19. In other preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 17. In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20. In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21. In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22. In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23.

[0012] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region.

[0013] In some embodiments, the antibody or its antigen-binding fragment comprises a human or mouse constant region, preferably a human or mouse heavy chain constant region and / or a light chain constant region; preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, and / or a light chain constant region of human κ, λ chains or variants thereof.

[0014] In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 5. In one embodiment, the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 10.

[0015] It should be understood that sequence variants of these constant region structural domains may also be used, for example, to contain one or more amino acid modifications, where the amino acid sites are identified by the EU indexing system of Kabat et al. (1991).

[0016] In some embodiments, the antibody is any form of monoclonal antibody (including full-length monoclonal antibody), polyclonal antibody, multispecific antibody (e.g., bispecific antibody), bifunctional antibody, murine antibody, fully or partially humanized antibody, or chimeric antibody. In one specific embodiment, the antibody is a monoclonal antibody. In a preferred embodiment, the antibody is a chimeric antibody. In another preferred embodiment, the antibody is a humanized antibody.

[0017] In some implementations, the antibody is a full-length antibody.

[0018] In some embodiments, the anti-ADAM9 antibody of the present invention is a complete antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. In other embodiments, the anti-ADAM9 antibody of the present invention covers only its antigen-binding portion, such as: Fab, Fab', Fd, Fd', Fv, dAb, F(ab')2, scFv, diabodies, linear antibodies, or dsFv.

[0019] In some embodiments, the antigen-binding fragment is an antigen-binding fragment of an antibody, such as Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, BsFv, dsFv, (dsFv)2, or dAb.

[0020] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain.

[0021] On the other hand, the present invention provides a biomaterial comprising: (i) A nucleic acid molecule encoding the anti-ADAM9 antibody or its antigen-binding fragment as described in this invention; (ii) a vector comprising the nucleic acid molecule described in (i); and / or (iii) A host cell comprising the nucleic acid molecule described in (i) and / or the vector described in (ii), or the host cell being transformed or transfected by the nucleic acid molecule described in (i) and / or the vector described in (ii).

[0022] In some embodiments, the nucleic acid molecule may be an isolated nucleic acid molecule. In some embodiments, the nucleic acid molecule is DNA, such as cDNA, genomic DNA, or recombinant DNA. In other embodiments, the nucleic acid molecule is RNA, such as mRNA or hnRNA. Those skilled in the art, based on the anti-ADAM9 antibody or its antigen-binding fragment disclosed in this application, can deduce the DNA or RNA sequence encoding the amino acid sequence and configure suitable expression elements thereon, enabling the DNA or RNA molecule to express the antibody or its antigen-binding fragment of the present invention.

[0023] In some implementations, the vector is an expression vector, such as a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector.

[0024] In some embodiments, the host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells. In some embodiments, the host cell is prokaryotic, such as *Escherichia coli*. In other embodiments, the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells. In some embodiments, the host cell is another cell suitable for preparing antibodies or their antigen-binding fragments.

[0025] On the other hand, the present invention provides a method for preparing the anti-ADAM9 antibody or its antigen-binding fragment as described herein, comprising: (1) Chemical synthesis method: prepared by synthesizing the amino acid sequence of the anti-ADAM9 antibody or its antigen-binding fragment according to the present invention; (2) Bio-preparation method: culturing the host cells as described above, and optionally isolating and / or purifying the antibodies from the obtained culture.

[0026] On the other hand, the present invention provides a composition comprising the anti-ADAM9 antibody or its antigen-binding fragment described herein and / or the biological material described herein, and optionally a pharmaceutically acceptable carrier or excipient.

[0027] In some embodiments, the composition is a pharmaceutical composition.

[0028] On the other hand, the present invention provides the use of the anti-ADAM9 antibody or its antigen-binding fragment, the biomaterials described herein, and / or the compositions described herein in the preparation of medicaments for treating tumor diseases.

[0029] In some implementations, the tumor disease is selected from at least one of the group consisting of gastric cancer, lung cancer, colorectal cancer, prostate cancer, breast cancer, and pancreatic cancer.

[0030] In some embodiments, the anti-ADAM9 antibody of the present invention or its antigen-binding fragment, biological materials (including nucleic acid molecules, vectors, host cells) and / or compositions of the present invention may also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the purposes described herein, such as for the treatment of the related oncological diseases mentioned herein.

[0031] On the other hand, the present invention provides a method for treating tumor diseases, comprising administering to a subject in need a preventatively effective amount or a therapeutically effective amount of the anti-ADAM9 antibody or its antigen-binding fragment thereof, the biological material (including nucleic acid molecules, carriers, host cells), or the composition thereof. Preferably, the subject is a mammal; more preferably, the subject is a human.

[0032] On the other hand, the present invention provides the anti-ADAM9 antibody or its antigen-binding fragment thereof, the biological material (including nucleic acid molecules, carriers, host cells) or the composition thereof, which can be used as a drug or for treatment.

[0033] In some implementations, the drug is a drug for treating tumor diseases.

[0034] On the other hand, the present invention provides a kit comprising the anti-ADAM9 antibody or its antigen-binding fragment described herein, the biological material (including nucleic acid molecules, vectors, host cells) or the composition described herein.

[0035] The invention is further illustrated in the following figures and specific embodiments. However, these figures and specific embodiments should not be considered as limiting the scope of the invention, and modifications readily apparent to those skilled in the art will be included within the spirit of the invention and the scope of protection of the appended claims. Attached Figure Description

[0036] Figure 1 Results of immune huADAM9-his antigen activity verification.

[0037] Figure 2 Results of serum titer testing in multiple immunized mice.

[0038] Figure 3A , Figure 3Band Figure 3C Results of chimeric antibody binding to cells.

[0039] Figure 4 Results of chimeric antibody endocytosis activity assay.

[0040] Figure 5 Affinity detection results of humanized antibody and hADAM9-his protein.

[0041] Figure 6A and Figure 6B Results of humanized antibody binding to cells.

[0042] Figure 7 Results of endocytic activity of humanized antibodies on NCI-H1703 cells.

[0043] Figure 8 Results of the binding activity assay of humanized antibody with ADAM9 family proteins ADAM8, ADAM10, ADAM15, and ADAM17.

[0044] Figure 9 Results of detection of the inhibitory effect of humanized antibody on the enzyme activity of ADAM9 protein.

[0045] Figure 10 Results of assay for the activity of humanized antibodies in inhibiting cell protein shedding. Detailed Implementation

[0046] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, the invention will be practiced using conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of this art. For the purposes of this invention, the following terms are defined below.

[0047] Abbreviations and Definitions Unless otherwise stated, the following terms shall have the meanings described below. Other terms or abbreviations shall have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields.

[0048] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.”

[0049] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0050] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y” and should be used to provide clear support for both meanings or either meaning.

[0051] As used herein, the term "antibody" or "immunoglobulin" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (knO) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, and ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain consists of a variable region (VH) and a constant region (CH). The constant region consists of three domains (CH1, CH2, and CH3). The light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of the heavy / light chain pairs form the antigen-binding sites.

[0052] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the antibody variable region that are primarily responsible for binding to the antigen. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as those defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering rule (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16;9:2278), etc. For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system.

[0053] Unless otherwise stated, the variable region and CDR sequence in the embodiments of the present invention are governed by the "Kabat" numbering rule. Although a numbering system (such as Kabat) is used to define amino acid residues in a specific implementation, the corresponding technical solutions of other numbering systems are considered equivalent.

[0054] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody, other than the CDR residues as defined above. The term "antibody" is not limited to any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0055] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab' F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragment, scFv, comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Exemplary examples include: (i) a Fab fragment having VL, CL, VH, and CH1 domains and a disulfide bond between the heavy and light chains; (ii) a Fab' fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) an Fd fragment having VH and CH1 domains; (iv) an Fd' fragment having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) an Fv fragment having VL and VH domains of one arm of the antibody; (vi) a dAb fragment consisting of a VH domain; and (vii) a hingeless antibody comprising at least VL, VH, and C1 domains. (viii) F(ab)2 fragment, which is a bivalent fragment containing two Fab' fragments linked by a disulfide bridge in the hinge region; (ix) Single-chain antibody molecule (scFv) refers to a molecule containing an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH; (x) "Diabodies" have two antigen-binding sites, including a heavy chain variable domain (VH) and a light chain variable domain (VL) linked together in the same polypeptide chain; (xi) "Linear antibody" contains a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptide form a pair of antigen-binding regions; (xii) dsFv refers to a fragment formed by replacing one amino acid residue in VH and VL with a cysteine ​​residue and then linking the polypeptides together via SS bonds between those cysteine ​​residues.

[0056] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0057] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions.

[0058] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0059] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0060] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from one source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0061] The monoclonal antibodies of this invention can be prepared using a variety of techniques, such as hybridoma technology (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al., Nature 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0062] Antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and purified by immunoaffinity chromatography to achieve immunospecific antibody purification. For purification of immunoglobulins, see, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0063] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Typically, at least one or two, but usually all three (heavy and / or light immunoglobulin chains) of the receptor CDR of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDR is called the "donor," and the immunoglobulin providing the framework is called the "receptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., mouse) antibody, and the receptor framework can be a naturally occurring human framework or a sequence having approximately 85%, 90%, 95%, 99%, or higher identity compared to it. Humanized antibodies typically retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, and reactivity. Donor antibodies can be mouse, rat, rabbit, or non-human primate antibodies with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0064] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids, phage particles, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site. Based on the given nucleic acid sequence, those skilled in the art can easily construct a suitable vector that enables the nucleic acid to be replicated or expressed.

[0065] As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0066] The term "antigen" refers to a molecule or molecular moiety that can be selectively recognized by antigen-binding protein molecules (such as antibodies) or bound by a binding agent. An antigen may have one or more epitopes that can interact with different antigen-binding protein molecules (such as antibodies).

[0067] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 The equilibrium dissociation constant (K) of M or less D ( ) binds to antigens or epitopes. In some embodiments, the antibody binds to the K antigen. D This antibody binds to the K+ of non-specific antigens (such as BSA, casein). D 10% or less (e.g., 1%). K can be measured using known methods. DFor example, measurements can be taken using FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or its epitope may be cross-reactive to other related antigens, such as to corresponding antigens from other species (homologous) (e.g., humans or monkeys, such as the cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno) and the chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or the common marmoset (Callithrix jacchus) (commonmarmoset, marmoset).

[0068] The term "non-binding" means that the antibody cannot bind to an antigen or its epitope within that antigen in the manner described above for specific binding. For example, when the antibody binds at approximately 1 × 10⁻⁶... -6 M or a larger equilibrium dissociation constant (K) D () Binds to antigens or their epitopes within antigens.

[0069] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0070] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions when optimally aligned. During alignment, gaps may be introduced where necessary to achieve the maximum percentage of sequence identity, but any conserved substitutions are not considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0071] The antibodies or antigen-binding fragments thereof of the present invention may contain amino acid mutations and / or conserved modifications. The antibodies or antigen-binding fragments thereof of the present invention also include sequences having more than 95% identity with the specifically provided amino acid sequences. Furthermore, the antibodies or antigen-binding fragments thereof of the present invention also include variants thereof.

[0072] The sequence “variant” used in this article refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0073] Amino acids are organic compounds that contain both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in their precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). The fact that the same amino acid can be encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0074] Amino acid mutations can be amino acid substitutions, deletions, insertions, and / or additions. In some embodiments, an amino acid mutation is the substitution of one or more amino acids, such as a single amino acid substitution or a combination of multiple amino acid substitutions. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. The amino acid substitutions of the present invention optionally include conservative substitutions of amino acids.

[0075] "Variations of conserved substitutions" or "conserved amino acid substitutions" refer to amino acid substitutions known to those skilled in the art that such substitutions generally do not alter the biological activity of the resulting molecule. Generally, it is generally accepted by those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity. Conserved substitutions can be made by amino acid substitutions with chemically similar side chains, such as: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0076] "Nucleic acid" or "polynucleotide" refers to a polymer molecule composed of a single nucleotide: adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA, or modifications thereof. Nucleic acid molecules can be natural or synthetic nucleic acid molecules, or a combination of one or more natural nucleic acid molecules with one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0077] "Isolated nucleic acid molecules" are nucleic acid molecules that have been identified and separated from at least one contaminating nucleic acid molecule. Isolated nucleic acid molecules differ from their naturally occurring form or environment. Therefore, isolated nucleic acid molecules are distinct from nucleic acid molecules present in their natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express antibodies, for example, where the chromosomal location of the nucleic acid molecule differs from its chromosomal location in natural cells.

[0078] The term "pharmaceutical composition" means a mixture containing one or more antigen-binding molecules or antibodies described herein, along with other chemical components, such as physiological / pharmaceutical carriers and excipients.

[0079] The term "pharmaceuticalally acceptable carrier" refers to a component in a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the test subject. Pharmaceutically acceptable carriers include, but are not limited to, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, and suspending agents.

[0080] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless explicitly stated otherwise, the terms “patient” or “subject” are used interchangeably herein. As used herein, the term “cynomolgus monkey” refers to the cynomolgus monkey (Macacafascicularis). In some embodiments, the individual or subject is a human.

[0081] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0082] "Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention intended to be applied to the individual being treated, and may be implemented for preventative purposes or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, the molecules disclosed herein are used to delay the onset of disease or slow its progression.

[0083] As used herein, the term "therapeutic effective dose" or "effective dose" refers to the amount of an anti-ADAM9 antibody or its antigen-binding fragment, administered alone or in combination with another therapeutic agent, that effectively prevents or alleviates the disease or condition to be treated. A therapeutic effective dose further refers to the amount of the antibody or its antigen-binding fragment sufficient to cause symptom relief, such relief being, for example, treatment, cure, prevention, or alleviation of an associated medical condition, or an increase in the rate of treatment, cure, prevention, or alleviation of the symptom. The effective dose for a specific patient can vary depending on various factors, such as the disease to be treated, the patient's overall health condition, the route and dosage of administration, and the severity of side effects. An effective dose may be the maximum dose or administration regimen that avoids significant side effects or toxicity. When administered to an individual as a single active ingredient, the therapeutic effective dose refers to that single ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, continuously, or simultaneously.

[0084] Example The present invention will be further illustrated by specific embodiments below, but the scope of the present invention is not limited thereto.

[0085] Unless otherwise specified, the reagents and instruments used in this invention are all conventional reagents and instruments that can be obtained commercially; the methods used are all conventional techniques, and those skilled in the art can undoubtedly complete the experiments and obtain the corresponding results based on the contents of the specification.

[0086] Example 1: Validation of Immunogen Activity The protein antigen used for immunization was recombinant ADAM9 protein, namely human ADAM9-his protein (catalog number: AD9-H52H7), purchased from Beijing Biosys Biotechnology Co., Ltd., containing the antigenic active fragment Ala 206-Asp 697. The experimental steps for verifying its activity were as follows: First, antigen coating was performed. Serially diluted huADAM9-his antigen solutions were prepared using PBS solution and coated overnight at 4°C in 96-well plates. After coating the 96-well plates overnight, the plates were washed three times with PBST solution and then blocked with antigen. The antigen was blocked with PBS (containing 3% BSA) at room temperature for 1 hour. Then, the ADAM9 antibody (Biointron, catalog number: 467DDD001) was quantitatively diluted with DPBS (containing 1% BSA) solution and coated at 37°C for 1 hour. Discard the reaction solution, wash three times with PBST solution, add 100 μl of secondary antibody (HRP-Goat Anti-Human IgG, F(ab')2 Secondary Antibody source: Jackson, catalog number: 109-035-006) to each well, and incubate at 37°C for 1 hour. Finally, discard the reaction solution, wash three times with PBST solution; add TMB (Abcam, catalog number: ab171523) solution and stop solution successively, gently shake until a yellow color appears, and then measure the OD value at 450 nm.

[0087] The results of the activity verification of the human ADAM9-his antigen used in the immunization were as follows: Figure 1 As shown, the huADAM9-his antigen has good antigen-antibody binding activity and can be used as a mouse immunogen in later experiments.

[0088] Example 2: Preparation and screening of anti-ADAM9 antibodies Five female Balb / C mice, aged 6-8 weeks, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and used for animal immunization with the huADAM9-his protein described in Example 1. The initial immunization used Freund's complete adjuvant (Sigma, catalog number: F5881-6x10ML) at a dose of 50 μg / mouse. Subsequent immunizations used Freund's incomplete adjuvant (Sigma, catalog number: F5506-6x10ML) at a dose of 25 μg / mouse. The injection method was subcutaneous injection at multiple sites (sc). Serum titers were monitored, and mice with high antibody titers were selected for a pulse immunization with 50 μg huADAM9-his protein via intraperitoneal injection (ip). Fusion was performed 3 days later. The experimental animals and immunization information are shown in Table 1 below.

[0089] Table 1. Experimental animals and immunization information

[0090] Mice immunized using the above method were subjected to serum titer testing using huADAM9-his antigen. The experimental steps were as follows: A 1 μg / ml solution of huADAM9-his antigen (AcroBiosystem, catalog number: AD9-H52H7) was prepared using PBS and coated in 96-well plates overnight at 4°C. The overnight coated 96-well plates were washed three times with PBST solution. The antigen was blocked with PBS (containing 3% BSA) and kept at room temperature for 1 hour. The serum solution was diluted with DPBS (containing 1% BSA). The negative control was serum from unimmunized mice. The positive control wells contained ADAM9 antibody (Biointron, catalog number: 467DDD001). The reaction was carried out at room temperature for 1 hour. Discard the reaction solution, wash three times with PBST solution, add secondary antibody (Goat anti-Ms IgG F(ab')2 Secondary Antibody, source: Invitrogen, catalog number: 31436), add secondary antibody (HRP-Goat Anti-Human IgG, F(ab')2 Secondary Antibody, source: Jackson, catalog number: 109-035-006) to the positive control well, and incubate at room temperature for 1 hour. Finally, discard the reaction solution, wash three times with PBST solution, add TMB (Abcam, catalog number: ab171523) solution and stop solution successively, gently shake until yellow, and then detect the OD value at 450nm. Serum titer results from multiple immunized mice are shown below. Figure 2 As shown in the figure, the results indicate that the immune response of mouse No. 5 was good.

[0091] As described above, after titer testing of mice immunized with huADAM9-his protein, high-titer Balb / c mice were selected for pulse immunization to initiate the fusion experiment. Splenic cells and SP2 / 0 mouse myeloma cells were mixed at a 3:1 ratio, centrifuged, and co-precipitated. After removing the supernatant, the cells were resuspended in an appropriate amount of fusion buffer, and the mixed cell density was adjusted to 1-2 × 10⁻⁶ cells / mL. 7 Cells were electrofused at 100 cells / ml (BTX, ECM2001). After fusion, the cells were incubated in the electrode holder for 5 minutes, then added to 1×HAT selective DMEM medium (Gibco, catalog number: 11995-065) containing 20% ​​FBS at a concentration of 2.0×10⁶ cells / ml. 4Cells / well were seeded into 96-well flat-bottom cell culture plates and cultured at 37°C in a 5% CO2 incubator for 7 days. The medium containing HAT (Sigma, catalog number: H0262-10VL) in the 96-well cell culture plates was replaced with DMEM selective medium containing 1×HT (Sigma, catalog number: H0137-10VL). After 10 days, the supernatant was collected, and the binding ability to CHO-K1 human ADAM9 overexpressing cell lines was detected by FACS. Positive clones were transferred to 24-well plates. The supernatant was screened and identified by FACS (CHO-K1 human ADAM9 overexpressing cell lines) and ELISA (huADAM9-his protein). Positive clones were selected for 1-2 rounds of subcloning, and hybridoma monoclonal cells stably expressing specific anti-ADAM9 antibodies were screened again. Based on this, the cell lines with the highest expression levels were selected, and cell samples and Trizol samples were cryopreserved. Total RNA was extracted from ADAM9-positive hybridoma monoclonal cells obtained through screening, and a transcriptome sequencing library was constructed for high-throughput sequencing. The sequencing data were analyzed using bioinformatics and compared with the IMGT database to obtain the full-length, functional antibody heavy chain and light chain variable region gene sequences. The VH and VL genes were synthesized and cloned into mammalian expression vectors containing the constant regions of human antibodies. The recombinant plasmids were transfected into CHO cells for antibody expression. The cell culture supernatant was collected and purified by Protein A affinity chromatography to obtain the anti-ADAM9 chimeric antibody.

[0092] Example 3: Detection of chimeric antibody affinity The interaction between the antibody and ADAM9 protein was determined using a ForteBio molecular interaction analyzer. The specific method is as follows: Samples were prepared using 0.05% Tween 20 PBS as PBST buffer. The antibody was diluted to 5 μg / ml in the antibody sample wells. Simultaneously, Human ADAM9 Protein, His Tag (Acro, catalog number: AD9-H52H7-50 μg) was diluted to 100 nM using PBST, followed by six 2-fold dilutions. These concentration gradients were then added to the antigen sample wells. Anti-human IgG Fc Capture (AHC) Dip and Read Biosensors were used as detection probes. The regeneration solution was pH 1.5 and 100 mM glycine.

[0093] The affinity assay results for the chimeric antibodies are shown in Table 2. The results show that the three chimeric antibodies can bind to human ADAM9 protein, with an affinity K0. D The (M) values ​​are 2.39E-09, 2.19E-09, and 1.73E-09, respectively.

[0094] Table 2. Affinity detection of chimeric antibodies with human ADAM9

[0095] Example 4: Detection of chimeric antibody binding to cells The binding of the antibody to the ADAM9-overexpressing cell line or the naturally ADAM9-expressing NCI-H1703 cells was detected by flow cytometry. The ADAM9 human / cyno-overexpressing cell line or the naturally ADAM9-expressing NCI-H1703 cells were seeded in 96-well plates, and the test antibody prepared with 2% FBS (Gibco) PBS buffer was added. The initial concentration was 30 μg / mL, and the cells were serially diluted 5-fold to a total of 8 concentration points. The blank wells were filled with 2% FBS PBS as a negative control. The cells were incubated at 4°C for 1 h, and then the secondary antibody was added. After incubation, the cells were analyzed by flow cytometry.

[0096] Results of chimeric antibody binding to cells: Figure 3A , Figure 3B and Figure 3C As shown, the results indicate that chimeric antibodies 12F5B12, 19G5G9, and 18C7F9 can bind to human / cyno ADAM9 overexpressing cell lines or NCI-H1703 cells that naturally express ADAM9.

[0097] Example 5: Chimeric antibody endocytosis activity Flow cytometry was used to detect the endocytic activity of the antibodies. NCI-H1703 cells were seeded in 96-well plates, and 100 μL of diluted antibody (final concentration 15 μg / mL) was added. After thorough mixing, the cells were incubated for 30 min, centrifuged, and washed twice with 200 μL of PBS containing 0.5% FBS. The cells were resuspended and cultured at 4℃ and 37℃ for 1, 2, and 4 h, respectively. Samples were taken at each time point, centrifuged at 4℃, and incubated with secondary antibody (goat anti-human AF647, Thermo Fisher Scientific) for 30 min at 4℃ before flow cytometry analysis. The results of the chimeric antibody endocytic activity assay are shown below. Figure 4 As shown, the results indicate that all three chimeric antibodies can mediate endocytosis.

[0098] Based on the experimental results, the 12F5B12 antibody, which has good affinity and endocytic activity, was selected for subsequent humanization.

[0099] The sequence of antibody 12F5B12, after sequencing, is shown below: Heavy chain variable region (VH): EVQLVETGGGLVQPKGSLKVSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRDLDYYGSSYAFAYWGQGTLVTVSA (SEQ ID NO: 1) VH CDR1: NNVMN (SEQ ID NO: 2) VH CDR2: RIRSKSNNYEIYYADSVKD (SEQ ID NO: 3) VH CDR3: DLDYYGSSYAFAY (SEQ ID NO: 4) Heavy chain constant region (CH): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5) Light chain variable region (VL): DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVFNAKTLAEGVPSRFSGGGSGTQFSLKINSLQPEDFGTYYCQHHYVIPLTFGAGTKLELK (SEQ ID NO: 6) VL CDR1: RASENIYSYLA (SEQ ID NO: 7) VL CDR2: NAKTLAE (SEQ ID NO: 8) VL CDR3: QHHYVIPLT (SEQ ID NO: 9) Light chain constant region (CL): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10) Example 6: Preparation of Humanized Antibodies The heavy chain variable region sequence of the human-mouse chimeric antibody ADAM9 antibody 12F5B12 was compared with the human antibody gene sequence library (NCBI Ig BLAST) to identify corresponding sequences of human germline antibodies with similar heavy chain variable region sequences. Among these, 13 amino acids differed in the FR region of the heavy chain.

[0100] Based on the framework sequence optimization, the humanized antibody heavy chain variable region sequence was designed as follows: VH1: EVQLVESGGGLVQPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVGRIRSKSNNYEIYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS (SEQ ID NO: 11) VH2: EVQLVESGGGLVQPGGSLRVSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS (SEQ ID NO: 12) VH3: EVQLVESGGGLVKPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS(SEQ ID NO: 13) VH4: EVQLVESGGGLVKPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS(SEQ ID NO: 14) VH5: EVQLVESGGGLVKPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS(SEQ ID NO: 15) VH6: EVQLVESGGGLVKPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVGRIRSKSNNYEIYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS(SEQ ID NO: 16) By comparing the light chain variable region sequence of the human-mouse chimeric antibody ADAM9 antibody 12F5B12 with the human antibody gene sequence library (NCBI Ig BLAST), sequences corresponding to the variable regions of human germline antibodies with similar light chain variable region sequences to those of human-mouse chimeric antibody 12F5B12 were identified. Among these, 16 amino acids differed in the light chain FR region.

[0101] Based on the framework sequence optimization, the humanized antibody light chain variable region sequence was designed as follows: VL1: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVFNAKTLAEGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQHHYVIPLTFGQGTKLEIK (SEQ ID NO: 17) VL2: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPQLLVFNAKTLAEGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQHHYVIPLTFGQGTKLEIK (SEQ ID NO: 18) VL3: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPQLLVFNAKTLAEGVPSRFSGSGSGTQFSLTINSLQPEDFATYYCQHHYVIPLTFGQGTKLEIK (SEQ ID NO: 19) VL4: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPKLLVFNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVIPLTFGGGTKVEIK(SEQ ID NO: 20) VL5: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVFNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVIPLTFGGGTKVEIK(SEQ ID NO: 21) VL6: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIFNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVIPLTFGGGTKVEIK(SEQ ID NO: 22) VL7: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPKLLVFNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVIPLTFGGGTKVEIK(SEQ ID NO: 23) The combinations of the heavy chain variable region and light chain variable region of the humanized antibody of the present invention are shown in Table 3.

[0102] Table 3. Humanized anti-ADAM9 antibody combinations

[0103] Example 7: Affinity detection of humanized antibody with hADAM9-his protein The binding of humanized antibodies to ADAM9 protein was detected by ELISA. A PBS-prepared hADAM9-his protein (Acro, catalog number: AD9-H52H7) solution was added to a 96-well plate and incubated overnight at 4°C. After washing with PBST and blocking with 3% BSA, 15 μg / mL antibody working solution was added and the plates were set up in duplicate and incubated at 37°C. The plates were then washed with PBST, and HRP-labeled secondary antibody (HRP-GoatAnti-Human IgG, F(ab')2 Secondary Antibody, source: Jackson, catalog number: 109-035-006) was added and incubated at 37°C. After washing with PBST again, TMB (Abcam, catalog number: ab171523) was added for color development, and the reaction was terminated with stop solution. Finally, the absorbance at 450 nm was measured using a microplate reader.

[0104] Affinity assay results of humanized antibody and hADAM9-his protein are as follows: Figure 5 As shown, the results indicate that all humanized antibodies can bind to the hADAM9-His antigen, and the affinity of 12F5B12-4 for the hADAM9-His antigen is similar to that of the parent antibody 12F5B12.

[0105] Example 8: Detection of humanized antibody binding to cells The binding of humanized antibodies to ADAM9-overexpressing cell lines or naturally ADAM9-expressing NCI-H1703 cells was detected by flow cytometry. ADAM9-overexpressing cell lines and naturally ADAM9-expressing NCI-H1703 cells were seeded in 96-well plates. The test antibody, prepared with 2% FBS in PBS, was added at an initial concentration of 30 μg / mL, with 8 concentration spots obtained through a 5-fold serial dilution. Blank wells contained 2% FBS (Gibco) in PBS buffer as a negative control. The plates were incubated at 4°C for 1 h. Secondary antibodies were then added, and the plates were incubated before flow cytometry analysis.

[0106] Humanized antibody binding detection, such as Figure 6A and Figure 6B As shown, the humanized antibodies 12F5B12-1, 12F5B12-1, 12F5B12-2, 12F5B12-3, and 12F5B12-4 can bind to human ADAM9 overexpressing cell lines or NCI-H1703 cells that naturally express ADAM9, and their binding ability is comparable to that of the parent 12F5B12. Example 9: Endocytotic activity of humanized antibody in NCI-H1703 cells at 37°C The endocytic activity of the antibody in NCI-H1703 cells was detected by flow cytometry. NCI-H1703 cells were seeded in 96-well plates, and 100 μL of diluted antibody (final concentration 15 μg / mL) was added. After thorough mixing, the cells were incubated for 30 min, centrifuged, and washed twice with 200 μL of PBS containing 0.5% FBS. The cells were then resuspended and cultured at 4℃ and 37℃ for 1, 2, and 4 h, respectively. At each time point, samples were centrifuged at 4℃, added with secondary antibody, incubated at 4℃ for 30 min, and then analyzed by flow cytometry.

[0107] The endocytic activity of humanized antibodies on NCI-H1703 cells at 37°C is as follows Figure 7 As shown, the ADAM9 humanized antibody exhibits significant endocytic activity at 37°C, which is comparable to that of the parent antibody 12F5B12.

[0108] Example 10: Detection of binding activity of humanized antibody with ADAM9 family proteins ADAM8, ADAM10, ADAM15, and ADAM17 The ELISA method was used to coat 96-well plates with PBS-prepared solutions of ADAM8 (Acro, catalog number: AD8-H5223), ADAM10 (R&D Systems, catalog number: 936-AD-020), ADAM15 (Sino Biological, catalog number: 10517-H08H), and ADAM17 (R&D Systems, catalog number: 930-ADB-010) family proteins overnight at 4°C. After washing with PBST and blocking with 3% BSA, 15 μg / mL antibody working solution was added and double-duplicates were set up. The plates were then incubated at 37°C. The sample was then washed with PBST, incubated with HRP-labeled secondary antibody (HRP-Goat Anti-Human IgG, F(ab')2 Secondary Antibody, source: Jackson, catalog number: 109-035-006) at 37°C, washed again with PBST, and then incubated with TMB (Abcam, catalog number: ab171523) for color development. The reaction was terminated with stop solution, and the absorbance at 450 nm was measured using an ELISA reader.

[0109] Test results as follows Figure 8 As shown, both the maternal antibody and the humanized antibody bind to the human ADAM9 protein, but do not bind to the human ADAM8, ADAM10, ADAM15, or ADAM17 proteins.

[0110] Example 11: Detection of the inhibitory effect of humanized antibody on the enzymatic activity of ADAM9 protein The inhibitory effect of the antibody on the enzymatic activity of ADAM9 protein was evaluated using fluorescence resonance energy transfer (FRET). A pH 9.0 buffer containing 25 mM Tris, 2.5 μM ZnCl2, and 0.005% (w / v) Brij-35 was prepared. hADAM9 was then diluted to 80 μg / mL and the antibody to 120 μg / mL using this buffer. The antigen and antibody solutions were mixed in equal volumes and incubated at room temperature for 15 min. Simultaneously, Mca-PLAQAV-Dpa-RSSSR-NH2 Fluorogenic Peptide Substrate (R&D Systems, catalog number: ES003) was diluted to 20 μM. An equal volume of the hADAM9-antibody mixture was then mixed with the substrate solution to achieve a final concentration of 20 μg / mL hADAM9, 30 μg / mL antibody, and 10 μM substrate. A control group was also included, consisting only of substrate and no hADAM9. After incubation at 37℃ for 30 min, 100 μL of reactants and controls were added to a black ELISA plate. The fluorescence intensity was detected using a fluorescence plate reader with an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Finally, a relevant curve was plotted based on the fluorescence values.

[0111] Test results as follows Figure 9 As shown, compared with hIgG1, both the humanized antibody 12F5B12 and the maternal antibody 12F5B1 showed a decrease in fluorescence value, indicating that the antibody has an enzyme activity inhibitory effect, and there was no significant difference in the value before and after humanization.

[0112] Example 12: Detection of the activity of humanized antibodies in inhibiting cell protein shedding CHOK1-hADAM9 cells were placed in 6-well plates. The experimental wells were transfected with hEphB4-AP-Puro plasmid using PEI transfection reagent (Polysciences, catalog number: 23966-1), and the control wells were transfected with Luc-GFP plasmid. Cells were co-cultured for 6 hours. After 6 hours, the supernatant was discarded, and 2 ml of complete culture medium was added. 1 ml of Opti-MEM serum-free culture medium (Gibco) and 5 μg / ml of either the test antibody protein or the IgG1 control antibody protein (Novoprotein, catalog number: NC002) were added. 48 hours after transfection, the supernatant was collected and transferred to centrifuge tubes, then placed on ice. The supernatant was mixed with an equal volume of 2 mg / mL 4-nitrophenyl phosphate disodium hexahydrate (Merck, catalog number: N2765-50TAB) and incubated at 37°C for 1.5 hours. The absorbance at 405 nm was measured using a microplate reader.

[0113] Test results as follows Figure 10As shown, compared to the transiently transfected Luc-GFP control plasmid, CHOK1-hADAM9 cells transfected with the hEPHB4-AP plasmid exhibited increased alkaline phosphatase activity in the supernatant. The maternal antibody 12F5B12 and the humanized antibody reduced the alkaline phosphatase activity in the supernatant, suggesting that they inhibited the cleavage of hEPHB4-AP by hADAM9 cells.

Claims

1. An anti-ADAM9 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

2. The anti-ADAM9 antibody or its antigen-binding fragment according to claim 1, wherein: (1) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 1; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 6; (2) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 17; (3) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 18; (4) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 19; (5) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 17; (6) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 20; (7) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 14; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 21; (8) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 22; or (9) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 16; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:

23.

3. The anti-ADAM9 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody is any form of monoclonal antibody, polyclonal antibody, multispecific antibody, bifunctional antibody, murine antibody, fully or partially humanized antibody, or chimeric antibody; and / or The antigen-binding fragment is an antigen-binding fragment of an antibody.

4. The anti-ADAM9 antibody or antigen-binding fragment thereof of claim 3, wherein, The antigen-binding fragment is Fab, Fab', Fd, Fd', Fv, dAb, F(ab')2, scFv, a biantibody, a linear antibody, or dsFv.

5. The anti-ADAM9 antibody or its antigen-binding fragment according to claim 3, wherein, The antibody or its antigen-binding fragment contains a human or mouse-derived heavy chain constant region and / or light chain constant region; The antibody or its antigen-binding fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a light chain constant region of the κ, λ chain or its variants; or, The antibody is a monoclonal antibody, wherein the heavy chain constant region of the monoclonal antibody is the heavy chain constant region of IgG1, IgG2, IgG3, IgG4 or their variants and / or the light chain constant region of the monoclonal antibody is the light chain constant region of κ, λ chains or their variants.

6. The anti-ADAM9 antibody or its antigen-binding fragment according to claim 5, wherein, The heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; and / or the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:

10.

7. A biomaterial comprising: (i) A nucleic acid molecule encoding an anti-ADAM9 antibody or an antigen-binding fragment thereof as described in any one of claims 1-6; (ii) a vector comprising the nucleic acid molecule described in (i); and / or (iii) A host cell comprising the nucleic acid molecule described in (i) and / or the vector described in (ii), or the host cell being transformed or transfected by the nucleic acid molecule described in (i) and / or the vector described in (ii).

8. A method for preparing the anti-ADAM9 antibody or its antigen-binding fragment according to any one of claims 1-6, comprising: (1) Chemical synthesis method: prepared by synthesizing the amino acid sequence of the anti-ADAM9 antibody or its antigen-binding fragment according to any one of claims 1 to 6; (2) Bio-preparation method: culturing the host cells of claim 7, and optionally isolating antibodies from the obtained culture and / or purifying the antibodies.

9. A composition comprising the anti-ADAM9 antibody or its antigen-binding fragment as described in any one of claims 1-6 and / or the biological material as described in claim 7, and optionally a pharmaceutically acceptable carrier or excipient.

10. Use of the anti-ADAM9 antibody or its antigen-binding fragment as described in any one of claims 1-6, the biomaterial of claim 7, and / or the composition of claim 9 in the preparation of a medicament for treating tumor diseases; wherein the tumor disease is selected from at least one of the group consisting of gastric cancer, lung cancer, colorectal cancer, prostate cancer, breast cancer, and pancreatic cancer.

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