Anti-adamts-7 antibody and use thereof

Abstract

Provided are an anti-ADAMTS-7 antibody or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a pharmaceutical composition and a kit product, and a use of the antibody in preventing and / or treating ADAMTS-7-associated diseases. The antibody that specifically binds to ADAMTS-7 has an obvious effect of inhibiting atherosclerosis.

Description

Anti-adamts-7 antibodies and uses thereof

[0001] The present disclosure is based on and claims priority to Chinese Patent Application No. 202410772590.3, filed on June 14, 2024, the disclosure of which is hereby incorporated by reference in its entirety into the present disclosure. TECHNICAL FIELD

[0002] The present disclosure relates to the field of biomedicine, in particular to anti-ADAMTS-7 (A Disintegrin And Metalloproteinnase With Thrombospondin Type 1 Motif 7) antibodies and uses thereof. BACKGROUND

[0003] Cardiovascular diseases are 90% of primary lesions in blood vessels, in which atherosclerotic plaques cause lumen stenosis or plaque rupture, forming thrombus to block blood vessels, which is the main cause of acute myocardial infarction, stroke, and peripheral arterial disease, affecting a large number of people and causing high mortality and disability. For nearly a century, the understanding of the pathogenesis of atherosclerosis has mainly focused on the hyperlipidemia theory, so the main prevention and treatment means for the diagnosis and treatment of atherosclerosis-related coronary heart disease and stroke in clinical practice is lipid-lowering therapy.

[0004] However, in actual clinical application, statins, ezetimibe, PCSK9 inhibitors and other lipid-lowering drugs can only reduce about 1 / 3 of cardiovascular events, and a large number of people still have adverse cardiovascular events such as myocardial infarction and stroke even if their blood lipids are controlled to meet the standards. Therefore, for people with atherosclerosis-related diseases, there is an urgent need for theoretical innovation and breakthroughs in clinical practice to address the residual risk of cardiovascular events after lipid-lowering. SUMMARY

[0005] The inventors have found that ADAMTS-7 not only promotes the formation of neointima after vascular injury, leading to arterial lumen stenosis, but also plays a role in promoting atherosclerosis by degrading the protective factor matrix protein COMP of atherosclerosis. Importantly, ADAMTS-7 does not affect blood lipids in the above pathological processes. Therefore, ADAMTS-7 is considered a new target that is independent of lipid-lowering and is expected to reduce the high residual risk of atherosclerosis patients after using lipid-lowering drugs.

[0006] Anti-adamts-7 monoclonal antibodies

[0007] In one aspect, the present disclosure provides a monoclonal antibody against metalloproteinase ADAMTS-7, for which an inhibitory effect on atherosclerosis has been successfully observed in a mouse model of atherosclerosis.

[0008] Specifically, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to ADAMTS-7, wherein the antibody or the antigen-binding fragment thereof comprises the following complementarity-determining regions (CDRs):

[0009] (1) CDR-H1, CDR-H2 and CDR-H3 contained in a VH (heavy chain variable region) represented by SEQ ID NO: 7; and / or CDR-L1, CDR-L2 and CDR-L3 contained in a VL (light chain variable region) represented by SEQ ID NO: 9;

[0010] (2) CDR-H1, CDR-H2 and CDR-H3 contained in a VH represented by SEQ ID NO: 21; and / or CDR-L1, CDR-L2 and CDR-L3 contained in a VL represented by SEQ ID NO: 23;

[0011] (3) CDR-H1 represented by SEQ ID NO: 1, CDR-H2 represented by SEQ ID NO: 2 and CDR-H3 represented by SEQ ID NO: 3; and / or CDR-L1 represented by SEQ ID NO: 4, CDR-L2 represented by SEQ ID NO: 5 and CDR-L3 represented by SEQ ID NO: 6;

[0012] (4) CDR-H1 represented by SEQ ID NO: 1, CDR-H2 represented by SEQ ID NO: 16 and CDR-H3 represented by SEQ ID NO: 17; and / or CDR-L1 represented by SEQ ID NO: 18, CDR-L2 represented by SEQ ID NO: 5 and CDR-L3 represented by SEQ ID NO: 20;

[0013] (5) CDR-H1, CDR-H2 and CDR-H3 contained in a VH and / or CDR-L1, CDR-L2 and CDR-L3 contained in a VL, wherein the VH and / or VL contains a mutation in at least one CDR compared to the VH and / or VL described in (1) or (2), the mutation being substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids); or

[0014] (6) CDRs described below, which contain a mutation, which is a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids) in at least one CDR compared to the CDRs described in any one of (1) to (4).

[0015] In some embodiments, the substitution is a conservative substitution.

[0016] In some embodiments, the CDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering system.

[0017] In some embodiments, the CDRs described in item (6) are selected from the CDR-H1, CDR-H2, and CDR-H3 contained in SEQ ID NO: 21, SEQ ID NO: 32, and SEQ ID NO: 42.

[0018] In some embodiments, the CDRs described in item (6) are selected from the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, and SEQ ID NO: 41.

[0019] In some embodiments, the CDRs described in item (6) are selected from the CDR-H1, CDR-H2, and CDR-H3 contained in SEQ ID NO: 21, SEQ ID NO: 32, and SEQ ID NO: 42; and, the CDRs described in item (6) are selected from the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, and SEQ ID NO: 41.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0021] (1-1) a VH comprising the heavy chain complementarity determining regions contained in the VH set forth in SEQ ID NO: 7 or a variant thereof; and,

[0022] a VL comprising the light chain complementarity determining regions contained in the VL set forth in SEQ ID NO: 9 or a variant thereof;

[0023] (1-2) a VH comprising the heavy chain complementarity determining regions contained in the VH set forth in SEQ ID NO: 21 or a variant thereof; and,

[0024] a VL comprising the light chain complementarity determining regions contained in the VL set forth in SEQ ID NO: 23 or a variant thereof;

[0025] (1-3) a VH comprising three CDRs as follows: CDR-H1 is as shown in SEQ ID NO: 1 or a variant thereof, CDR-H2 is selected from the group consisting of a sequence as shown in SEQ ID NO: 2 or a variant thereof, and CDR-H3 is as shown in SEQ ID NO: 3 or a variant thereof; and,

[0026] a VL comprising three CDRs as follows: CDR-L1 is as shown in SEQ ID NO: 4 or a variant thereof, CDR-L2 is as shown in SEQ ID NO: 5 or a variant thereof, and CDR-L3 is as shown in SEQ ID NO: 6 or a variant thereof;

[0027] (1-4) a VH comprising three CDRs as follows: CDR-H1 is as shown in SEQ ID NO: 11 or a variant thereof, CDR-H2 is selected from the group consisting of a sequence as shown in SEQ ID NO: 16 or a variant thereof, and CDR-H3 is as shown in SEQ ID NO: 17 or a variant thereof; and,

[0028] a VL comprising three CDRs as follows: CDR-L1 is as shown in SEQ ID NO: 18 or a variant thereof, CDR-L2 is as shown in SEQ ID NO: 5 or a variant thereof, and CDR-L3 is as shown in SEQ ID NO: 20 or a variant thereof;

[0029] wherein the variant contains amino acid mutations, which are one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.

[0030] In some embodiments, the substitution is a conservative substitution.

[0031] In some embodiments, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence from which it is derived.

[0032] In some embodiments, the CDRs are defined by the Kabat, Chothia, or IMGT numbering system.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0034] VH comprising three CDRs as follows: CDR-H1 of the sequence set forth in SEQ ID NO: 1, CDR-H2 selected from the sequence set forth in SEQ ID NO: 2 and CDR-H3 of the sequence set forth in SEQ ID NO: 3, or, comprising CDR-H1, CDR-H2 and CDR-H3 contained in the VH set forth in SEQ ID NO: 32; and,

[0035] VL comprising three CDRs as follows: CDR-L1 of the sequence set forth in SEQ ID NO: 4, CDR-L2 of the sequence set forth in SEQ ID NO: 5, and CDR-L3 of the sequence set forth in SEQ ID NO: 6; or, comprising CDR-L1, CDR-L2 and CDR-L3 contained in the VL set forth in SEQ ID NO: 31.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0037] VH comprising three CDRs as follows: CDR-H1 of the sequence set forth in SEQ ID NO: 1, CDR-H2 selected from the sequence set forth in SEQ ID NO: 16 and CDR-H3 of the sequence set forth in SEQ ID NO: 17, or, comprising CDR-H1, CDR-H2 and CDR-H3 selected from the VH set forth in SEQ ID NO: 21 and SEQ ID NO: 42; and,

[0038] VL comprising three CDRs as follows: CDR-L1 of the sequence set forth in SEQ ID NO: 18, CDR-L2 of the sequence set forth in SEQ ID NO: 5 and CDR-L3 of the sequence set forth in SEQ ID NO: 20, or, comprising CDR-L1, CDR-L2 and CDR-L3 contained in the VL set forth in SEQ ID NO: 23, SEQ ID NO: 39 or SEQ ID NO: 41.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0040] (2-1) VH having the sequence set forth in SEQ ID NO: 7 or a variant thereof, and VL having the sequence set forth in SEQ ID NO: 9 or a variant thereof; or,

[0041] (2-2) VH having the sequence set forth in SEQ ID NO: 21 or a variant thereof, and VL having the sequence set forth in SEQ ID NO: 23 or a variant thereof;

[0042] wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or one or several substitutions, deletions, or additions of amino acids (e.g., 1, 2, 3, 4, or 5 substitutions, deletions, or additions of amino acids) compared to the sequence from which it is derived.

[0043] In some embodiments, the substitution is a conservative substitution.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0045] a VH having the sequence set forth in SEQ ID NO: 7, and a VL having the sequence set forth in SEQ ID NO: 9;

[0046] a VH having the sequence set forth in SEQ ID NO: 32, and a VL having the sequence set forth in SEQ ID NO: 29;

[0047] a VH having the sequence set forth in SEQ ID NO: 32, and a VL having the sequence set forth in SEQ ID NO: 30;

[0048] a VH having the sequence set forth in SEQ ID NO: 32, and a VL having the sequence set forth in SEQ ID NO: 31;

[0049] a VH having the sequence set forth in SEQ ID NO: 33, and a VL having the sequence set forth in SEQ ID NO: 29;

[0050] a VH having the sequence set forth in SEQ ID NO: 33, and a VL having the sequence set forth in SEQ ID NO: 30;

[0051] a VH having the sequence set forth in SEQ ID NO: 33, and a VL having the sequence set forth in SEQ ID NO: 31;

[0052] a VH having the sequence set forth in SEQ ID NO: 34, and a VL having the sequence set forth in SEQ ID NO: 29;

[0053] a VH having the sequence set forth in SEQ ID NO: 34, and a VL having the sequence set forth in SEQ ID NO: 30;

[0054] a VH having the sequence set forth in SEQ ID NO: 34, and a VL having the sequence set forth in SEQ ID NO: 31;

[0055] a VH having the sequence of SEQ ID NO: 35, and a VL having the sequence of SEQ ID NO: 29;

[0056] a VH having the sequence of SEQ ID NO: 35, and a VL having the sequence of SEQ ID NO: 30;

[0057] a VH having the sequence of SEQ ID NO: 35, and a VL having the sequence of SEQ ID NO: 31;

[0058] a VH having the sequence of SEQ ID NO: 36, and a VL having the sequence of SEQ ID NO: 29;

[0059] a VH having the sequence of SEQ ID NO: 36, and a VL having the sequence of SEQ ID NO: 30;

[0060] a VH having the sequence of SEQ ID NO: 36, and a VL having the sequence of SEQ ID NO: 31;

[0061] a VH having the sequence of SEQ ID NO: 21, and a VL having the sequence of SEQ ID NO: 23;

[0062] a VH having the sequence of SEQ ID NO: 42, and a VL having the sequence of SEQ ID NO: 37;

[0063] a VH having the sequence of SEQ ID NO: 42, and a VL having the sequence of SEQ ID NO: 38;

[0064] a VH having the sequence of SEQ ID NO: 42, and a VL having the sequence of SEQ ID NO: 39;

[0065] a VH having the sequence of SEQ ID NO: 42, and a VL having the sequence of SEQ ID NO: 40;

[0066] a VH having the sequence of SEQ ID NO: 42, and a VL having the sequence of SEQ ID NO: 41;

[0067] a VH having the sequence of SEQ ID NO: 43, and a VL having the sequence of SEQ ID NO: 37;

[0068] a VH having the sequence of SEQ ID NO: 43, and a VL having the sequence of SEQ ID NO: 38;

[0069] VH having the sequence of SEQ ID NO:43, and a VL having the sequence of SEQ ID NO:40;

[0070] VH having the sequence of SEQ ID NO:43, and a VL having the sequence of SEQ ID NO:40;

[0071] VH having the sequence of SEQ ID NO:43, and a VL having the sequence of SEQ ID NO:40;

[0072] VH having the sequence of SEQ ID NO:44, and a VL having the sequence of SEQ ID NO:37;

[0073] VH having the sequence of SEQ ID NO:44, and a VL having the sequence of SEQ ID NO:38;

[0074] VH having the sequence of SEQ ID NO:44, and a VL having the sequence of SEQ ID NO:39;

[0075] VH having the sequence of SEQ ID NO:44, and a VL having the sequence of SEQ ID NO:40;

[0076] VH having the sequence of SEQ ID NO:44, and a VL having the sequence of SEQ ID NO:41;

[0077] VH having the sequence of SEQ ID NO:45, and a VL having the sequence of SEQ ID NO:37;

[0078] VH having the sequence of SEQ ID NO:45, and a VL having the sequence of SEQ ID NO:38;

[0079] VH having the sequence of SEQ ID NO:45, and a VL having the sequence of SEQ ID NO:39;

[0080] VH having the sequence of SEQ ID NO:45, and a VL having the sequence of SEQ ID NO:40;

[0081] VH having the sequence of SEQ ID NO:45, and a VL having the sequence of SEQ ID NO:41;

[0082] VH having the sequence of SEQ ID NO:46, and a VL having the sequence of SEQ ID NO:37;

[0083] a VH having the sequence set forth in SEQ ID NO: 46, and a VL having the sequence set forth in SEQ ID NO: 37;

[0084] a VH having the sequence set forth in SEQ ID NO: 46, and a VL having the sequence set forth in SEQ ID NO: 38;

[0085] a VH having the sequence set forth in SEQ ID NO: 46, and a VL having the sequence set forth in SEQ ID NO: 39;

[0086] a VH having the sequence set forth in SEQ ID NO: 46, and a VL having the sequence set forth in SEQ ID NO: 40;

[0087] a VH having the sequence set forth in SEQ ID NO: 46, and a VL having the sequence set forth in SEQ ID NO: 41.

[0088] a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 37;

[0089] a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 38;

[0090] a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 39;

[0091] a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 40; or,

[0092] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0093] (2-1) a VH having the sequence set forth in SEQ ID NO: 7, and a VL having the sequence set forth in SEQ ID NO: 9;

[0094] (2-2) a VH having the sequence set forth in SEQ ID NO: 33, and a VL having the sequence set forth in SEQ ID NO: 29;

[0095] (2-3) a VH having the sequence set forth in SEQ ID NO: 33, and a VL having the sequence set forth in SEQ ID NO: 30;

[0096] (2-4) a VH having the sequence set forth in SEQ ID NO: 33, and a VL having the sequence set forth in SEQ ID NO: 31.

[0097] (2-5) a VH having the sequence set forth in SEQ ID NO: 34, and a VL having the sequence set forth in SEQ ID NO: 29;

[0098] (2-6) a VH having the sequence set forth in SEQ ID NO: 34, and a VL having the sequence set forth in SEQ ID NO: 30;

[0099] (2-7) a VH having the sequence set forth in SEQ ID NO: 34, and a VL having the sequence set forth in SEQ ID NO: 31;

[0100] (2-8) a VH having the sequence set forth in SEQ ID NO: 35, and a VL having the sequence set forth in SEQ ID NO: 29;

[0101] (2-9) a VH having the sequence set forth in SEQ ID NO: 35, and a VL having the sequence set forth in SEQ ID NO: 30;

[0102] (2-10) a VH having the sequence set forth in SEQ ID NO: 35, and a VL having the sequence set forth in SEQ ID NO: 31;

[0103] (2-11) a VH having the sequence set forth in SEQ ID NO: 36, and a VL having the sequence set forth in SEQ ID NO: 29;

[0104] (2-12) a VH having the sequence set forth in SEQ ID NO: 36, and a VL having the sequence set forth in SEQ ID NO: 30;

[0105] (2-13) a VH having the sequence set forth in SEQ ID NO: 36, and a VL having the sequence set forth in SEQ ID NO: 31;

[0106] (2-14) a VH having the sequence set forth in SEQ ID NO: 21, and a VL having the sequence set forth in SEQ ID NO: 23;

[0107] (2-15) a VH having the sequence set forth in SEQ ID NO: 43, and a VL having the sequence set forth in SEQ ID NO: 37;

[0108] (2-16) a VH having the sequence set forth in SEQ ID NO: 43, and a VL having the sequence set forth in SEQ ID NO: 38;

[0109] (2-17) a VH having the sequence set forth in SEQ ID NO: 44, and a VL having the sequence set forth in SEQ ID NO: 37;

[0110] (2-18) a VH having the sequence set forth in SEQ ID NO: 44, and a VL having the sequence set forth in SEQ ID NO: 38;

[0111] (2-19) a VH having the sequence set forth in SEQ ID NO: 45, and a VL having the sequence set forth in SEQ ID NO: 37;

[0112] (2-20) a VH having the sequence set forth in SEQ ID NO: 45, and a VL having the sequence set forth in SEQ ID NO: 38;

[0113] (2-21) a VH having the sequence set forth in SEQ ID NO: 46, and a VL having the sequence set forth in SEQ ID NO: 37;

[0114] (2-22) a VH having the sequence set forth in SEQ ID NO: 46, and a VL having the sequence set forth in SEQ ID NO: 38;

[0115] (2-23) a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 37;

[0116] (2-24) a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 38;

[0117] (2-25) a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 39;

[0118] (2-26) a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 40; or,

[0119] (2-27) a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 41.

[0120] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0121] a VH having the sequence set forth in SEQ ID NO: 7, and a VL having the sequence set forth in SEQ ID NO: 9;

[0122] a VH having the sequence set forth in SEQ ID NO: 35, and a VL having the sequence set forth in SEQ ID NO: 30;

[0123] a VH having the sequence set forth in SEQ ID NO: 21, and a VL having the sequence set forth in SEQ ID NO: 23;

[0124] a VH having the sequence set forth in SEQ ID NO: 43, and a VL having the sequence set forth in SEQ ID NO: 38;

[0125] a VH having the sequence set forth in SEQ ID NO: 46, and a VL having the sequence set forth in SEQ ID NO: 38;

[0126] a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 39; or,

[0127] a VH having the sequence set forth in SEQ ID NO: 47, and a VL having the sequence set forth in SEQ ID NO: 40.

[0128] In some embodiments, the antibody is a murine, chimeric, humanized, or fully human antibody.

[0129] In some embodiments, the antibody or antigen-binding fragment thereof comprises a constant region from or derived from a human immunoglobulin.

[0130] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a CH (heavy chain constant region) from or derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0131] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a CL (light chain constant region) from or derived from a human immunoglobulin (e.g., kappa or lambda).

[0132] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0133] (3-1) a HC (heavy chain) having the sequence set forth in SEQ ID NO: 11 or a variant thereof, and a LC (light chain) having the sequence set forth in SEQ ID NO: 13 or a variant thereof; or,

[0134] (3-2) a HC having the sequence set forth in SEQ ID NO: 25 or a variant thereof, and a LC having the sequence set forth in SEQ ID NO: 27 or a variant thereof.

[0135] The antibodies of the present disclosure have high binding activity to ADAMTS-7. In some embodiments, the affinity K D value of the antibodies of the present disclosure to ADAMTS-7 is 10 -6 M level, preferably 10 -7M level, preferably at 10 -8 M level, preferably at 10 -9 M level.

[0136] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a scFv, a Fab, a Fab', a Fab'-SH, a F(ab')2, an Fv fragment, a disulfide linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

[0137] The antibodies or antigen-binding fragments thereof of the present disclosure can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization of the antibodies or antigen-binding fragments thereof (e.g., labeling) will not adversely affect their binding to ADAMTS-7, in particular human ADAMTS-7. Thus, the antibodies or antigen-binding fragments thereof of the present disclosure are also intended to encompass such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present disclosure can be functionally linked (by chemical coupling, genetic fusion, noncovalent

[0138] In some embodiments, the antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof is labeled with a detectable marker, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.

[0139] The detectable label described in the present disclosure can be any substance that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dot, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), acridinium esters, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels. In some embodiments, such labels can be suitable for use in immunoassays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In some embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. In some embodiments, the detectable label can be attached to the antibody of the present disclosure or antigen-binding fragment thereof as described above by linkers of varying lengths to reduce potential steric hindrance.

[0140] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is an antitumor agent, such as a cytotoxic agent, a cytokine, a toxin, or a radionuclide.

[0141] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a substance that can improve the biological properties of the antibody, such as increasing the serum half-life, for example, can be a chemical group, such as polyethylene glycol (PEG), methyl or ethyl, or a sugar group, etc.

[0142] As one of the derivatives of the antibody, the present disclosure provides a multispecific antibody comprising the antibody of the present disclosure or antigen-binding fragment thereof. In some embodiments, the multispecific antibody comprises the antibody of the present disclosure or antigen-binding fragment thereof as a first antigen-binding domain, and further comprises at least one second antigen-binding domain against other targets. In some embodiments, each antigen-binding domain of the multispecific antibody retains the respective original binding specificity. In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

[0143] Preparation of antibodies

[0144] The antibody of the present disclosure can be prepared in various methods known in the art, for example, by obtaining a DNA molecule encoding the heavy chain and light chain genes of the antibody of the present disclosure through genetic engineering recombinant technology. The resulting DNA molecule is inserted into an expression vector, and then the host cell is transfected. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present disclosure is expressed.

[0145] Antigen-binding fragments of the present disclosure can be obtained via proteolytic hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). Alternatively, these antigen-binding fragments can be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab’ fragments can be directly obtained from host cells; Fab’ fragments can be chemically coupled to form F(ab’)2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). In addition, Fv, Fab or F(ab’)2 fragments can be isolated directly from recombinant host cell culture solutions. Those of ordinary skill in the art are fully aware of other techniques for making these antigen-binding fragments.

[0146] Accordingly, in another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present disclosure, or a heavy chain variable region and / or a light chain variable region thereof. In some embodiments, the nucleotide sequence is codon degenerate according to codon degeneracy in the art. In some embodiments, the nucleotide sequence is codon optimized.

[0147] In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein:

[0148] The nucleic acid molecule encoding an antibody variable region comprises:

[0149] (4-1) the nucleotide sequence set forth in SEQ ID NO: 8, and / or the nucleotide sequence set forth in SEQ ID NO: 10; or

[0150] (4-2) the nucleotide sequence set forth in SEQ ID NO: 22, and / or the nucleotide sequence set forth in SEQ ID NO: 24.

[0151] In some embodiments, the nucleic acid molecule encoding an antibody variable region comprises a sequence that is essentially identical to the nucleotide sequence set forth in (4-1) or (4-2) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or more sequence identity compared to the nucleotide sequence, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence), or a degenerate sequence of the nucleotide sequence set forth in (4-1) and (4-2).

[0152] In another aspect, the present disclosure provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present disclosure. In some embodiments, the vector of the present disclosure is, e.g., a plasmid, a virus, a bacteriophage, etc. Plasmids are the most commonly used vector form, but all other vector forms that serve similar functional roles and are known or knowable in the art are suitable for use in the present disclosure. See, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 and supplements thereto, Elsevier, N.Y.; and Rodriguez et al. (eds.), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth, Boston, MA. In some embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present disclosure in vivo in a subject (e.g., a mammal, e.g., a human).

[0153] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of the antibody or antigen-binding fragment thereof of the present disclosure and a second nucleotide sequence encoding a light chain or a light chain variable region thereof, wherein the first and second nucleotide sequences are present on the same or different vectors. When the first and second nucleotide sequences are present on different vectors, the vector of the present disclosure comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0154] In another aspect, the present disclosure provides a host cell comprising the isolated nucleic acid molecule or the vector of the present disclosure. The host cell can be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., E. coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell of the present disclosure is a mammalian cell, e.g., CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0155] In another aspect, the present disclosure provides a method of making the antibody or antigen binding fragment thereof, comprising culturing the host cell of the present disclosure under conditions that allow expression of the antibody or antigen binding fragment thereof, and recovering the antibody or antigen binding fragment thereof from the cultured host cell culture. In some embodiments, the method comprises the steps of culturing the host cell described in the present disclosure, collecting the culture supernatant, purifying, obtaining the antibody or antigen binding fragment thereof.

[0156] Therapeutic applications

[0157] The existing clinical prevention and treatment strategy for atherosclerosis is mainly lipid-lowering therapy with statins. Because statins have side effects such as inducing diabetes and rhabdomyolysis, they are usually used in combination with ezetimibe and PCSK9 inhibitors, on the one hand to reduce the dosage of statins and reduce the potential risk of side effects, and on the other hand to optimize the lipid-lowering effect. ADAMTS-7 is considered a new target that is independent of lipid-lowering and is expected to reduce the high residual risk of atherosclerotic patients after using lipid-lowering drugs.

[0158] In some embodiments, the anti-ADAMTS-7 antibody of the present disclosure is capable of:

[0159] (a) inhibiting ADAMTS-7 degradation of a substrate;

[0160] (b) inhibiting arterial lumen stenosis;

[0161] (c) inhibiting atherosclerotic plaques;

[0162] (d) inhibiting restenosis after stent implantation;

[0163] (e) inhibiting vascular calcification;

[0164] (f) inhibiting aortic aneurysm / dissection;

[0165] (g) inhibiting perioperative acute renal ischemia-reperfusion injury;

[0166] (h) inhibiting lupus nephritis;

[0167] (i) any combination of (a)-(h).

[0168] On the basis of the above, the present disclosure further provides pharmaceutical compositions comprising the anti-ADAMTS-7 antibodies of the present disclosure and therapies thereof.

[0169] In another aspect, the present disclosure provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells of the present disclosure, and pharmaceutically acceptable excipients.

[0170] The pharmaceutical compositions are used for preventing and / or treating ADAMTS-7 related diseases or disorders. In some embodiments, the ADAMTS-7 related diseases or disorders are autoimmune diseases (e.g., lupus nephritis), cardiovascular diseases or complications thereof (e.g., atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral arterial vascular disease, deep vein thrombosis, pulmonary embolism, restenosis after stent implantation, or vascular calcification, aortic aneurysm / dissection), perioperative acute renal ischemia-reperfusion injury.

[0171] In some embodiments, the pharmaceutical compositions can further comprise a second therapeutic agent, wherein the second therapeutic agent is a lipid lowering agent; preferably, the lipid lowering agent is selected from statins (e.g., simvastatin, atorvastatin, rosuvastatin, lovastatin, pravastatin, fluvastatin, pitavastatin), ezetimibe, PCSK9 inhibitors (e.g., evolocumab, alirocumab, inclisran). In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are provided as separate components or as a combined component with the second therapeutic agent. Thus, the antibodies or antigen-binding fragments thereof of the present disclosure and the second therapeutic agent can be administered simultaneously, separately or sequentially.

[0172] In another aspect, the present disclosure provides a kit product comprising the pharmaceutical compositions described herein, and optionally, instructions for use of the medicaments.

[0173] In some embodiments, the kit product further comprises a second therapeutic agent, wherein the second therapeutic agent is a lipid lowering agent. In some embodiments, the lipid lowering agent is selected from statins (e.g., simvastatin, atorvastatin, rosuvastatin, lovastatin, pravastatin, fluvastatin, pitavastatin), ezetimibe, PCSK9 inhibitors (e.g., evolocumab, alirocumab, inclisran).

[0174] In some embodiments, the kit is used for preventing and / or treating an ADAMTS-7 associated disease or disorder. In some embodiments, the ADAMTS-7 associated disease or disorder is an autoimmune disease (e.g., lupus nephritis), a cardiovascular disease or its complication (e.g., atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral arterial disease, deep vein thrombosis, pulmonary embolism, restenosis after stent implantation or vascular calcification, aortic aneurysm / dissection), or perioperative acute renal ischemia-reperfusion injury.

[0175] The antibody or antigen-binding fragment thereof of the present disclosure, the pharmaceutical composition of the present disclosure can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended administration mode and therapeutic use. The pharmaceutical composition of the present disclosure should be sterile and stable under the production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the necessary dosage of the antibody of the present disclosure in an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients, followed by sterilization by filtration. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) to facilitate storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0176] In addition, the antibody or antigen-binding fragment thereof of the present disclosure can be present in a pharmaceutical composition in unit dosage form to facilitate administration.

[0177] The antibody or antigen-binding fragment thereof, the pharmaceutical composition of the present disclosure can be administered by any suitable method known in the art. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled person will understand that the route and / or means of administration will vary depending on the intended purpose. In some embodiments, the antibody or antigen-binding fragment thereof, the pharmaceutical composition of the present disclosure is administered by intravenous infusion or injection.

[0178] In another aspect, the present disclosure provides the use of the antibody or antigen-binding fragment thereof, the pharmaceutical composition or the kit of the present disclosure in the preparation of a medicament for preventing and / or treating an ADAMTS-7 associated disease or disorder.

[0179] In some embodiments, the ADAMTS-7-associated disease or disorder is an autoimmune disease (e.g., lupus nephritis), a cardiovascular disease or its complication (e.g., atherosclerosis, coronary heart disease, heart infarction, stroke, peripheral arterial vascular disease, deep vein thrombosis, pulmonary embolism, restenosis after stent implantation, vascular calcification, aortic aneurysm / dissection, or acute ischemia-reperfusion injury of kidney during perioperative period).

[0180] In another aspect, the present disclosure provides the antibody or antigen-binding fragment thereof, the pharmaceutical composition or the kit product for use in preventing and / or treating an ADAMTS-7-associated disease or disorder.

[0181] In some embodiments, the ADAMTS-7-associated disease or disorder is an autoimmune disease (e.g., lupus nephritis), a cardiovascular disease or its complication (e.g., atherosclerosis, coronary heart disease, heart infarction, stroke, peripheral arterial vascular disease, deep vein thrombosis, pulmonary embolism, restenosis after stent implantation, vascular calcification, aortic aneurysm / dissection, or acute ischemia-reperfusion injury of kidney during perioperative period).

[0182] In another aspect, the present disclosure provides a method for preventing and / or treating an ADAMTS-7-associated disease or disorder, comprising the step of administering to an individual the antibody or antigen-binding fragment thereof, the pharmaceutical composition or the kit product of the present disclosure.

[0183] In some embodiments, the ADAMTS-7-associated disease or disorder is an autoimmune disease (e.g., lupus nephritis), a cardiovascular disease or its complication (e.g., atherosclerosis, coronary heart disease, heart infarction, stroke, peripheral arterial vascular disease, deep vein thrombosis, pulmonary embolism, restenosis after stent implantation, or vascular calcification, aortic aneurysm / dissection), or acute ischemia-reperfusion injury of kidney during perioperative period.

[0184] In some embodiments, the method further comprises the step of administering a second therapeutic agent, wherein the second therapeutic agent is a lipid-lowering drug; preferably, the antibody or antigen-binding fragment thereof and the second therapeutic agent are administered simultaneously or sequentially.

[0185] In some embodiments, the lipid-lowering drug is selected from statins (e.g., simvastatin, atorvastatin, rosuvastatin, lovastatin, pravastatin, fluvastatin, pitavastatin), ezetimibe, PCSK9 inhibitors (e.g., evolocumab, alirocumab, inclisran).

[0186] Detection applications

[0187] The antibodies or antigen-binding fragments thereof of the present disclosure are capable of specifically binding to ADAMTS-7 and can therefore be used to detect the presence or level of ADAMTS-7 in a sample.

[0188] Thus, in another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof of the present disclosure. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is detectably labeled. In a preferred embodiment, the kit further comprises a second antibody which specifically recognizes the antibody or antigen-binding fragment thereof of the present disclosure. Preferably, the second antibody also comprises a detectable label. In the present disclosure, the detectable label is as described previously.

[0189] In another aspect, the present disclosure provides a method of detecting the presence or level of ADAMTS-7 in a sample, comprising the step of using an antibody or antigen-binding fragment thereof of the present disclosure. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present disclosure is also detectably labeled. In another preferred embodiment, the method further comprises using a reagent detectably labeled to detect the antibody or antigen-binding fragment thereof of the present disclosure. The method can be used for diagnostic purposes, or non-diagnostic purposes (e.g. the sample is a cell sample, rather than a sample from a patient).

[0190] In certain embodiments, the method comprises contacting the sample with an antibody or antigen-binding fragment thereof of the present disclosure under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and ADAMTS-7, and detecting the formation of the complex.

[0191] In view of the strong correlation of ADAMTS-7 with atherosclerosis, as ADAMTS-7 is present in the vascular matrix of lesions, atherosclerosis can be diagnosed by detecting the presence or level of ADAMTS-7 in a sample. Thus, in certain embodiments, the method is used to diagnose atherosclerosis.

[0192] In some embodiments, the method comprises detecting the level of expression of ADAMTS-7 in a test sample from a subject, and comparing the level of expression to a reference value (e.g. a healthy control), wherein an increase in the level of expression compared to the reference value is indicative of atherosclerosis.

[0193] In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof of the present disclosure in the manufacture of a kit for detecting the presence or level of ADAMTS-7 in a sample and / or diagnosing atherosclerosis.

[0194] In another aspect, the present disclosure provides a diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate described herein, and instructions for use.

[0195] Definitions

[0196] Unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be understood by one of ordinary skill in the art. Also, the techniques used in the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory procedures, and the like are those known to those of ordinary skill in the corresponding art. Furthermore, the following definitions are provided for certain terms used herein.

[0197] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the above) through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The antibodies described herein encompass intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins incorporating an antibody, and any other modified configuration of the immunoglobulin molecule that exhibits the desired biological activity. An antibody can be composed of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). The antibody light chains can be assigned to a class, kappa (κ) and lambda (λ). The heavy chains can be assigned to a class based on the amino acid sequence of the constant regions, and the subclasses of these are defined by the amino acid sequences of the constant regions. Each of these chains is composed of structural domains. Within the light and heavy chains, the variable region is followed by a constant region, joined by a "J" region of about 12 or more amino acids. The heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains exhibit less inter-chain variability than the variable domains, but still exhibit some inter-chain variability. The variable domains of the heavy and light chains, VH and VL, can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains, VH and VL, form the antigen binding site of antibodies. The assignment of amino acids to each region or domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0198] The terms "Kabat numbering system" and like terms are known in the art to refer to a system of numbering the amino acid residues of the variable regions of the heavy and light chains of an antibody or antigen-binding fragment thereof. In some aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are generally found at amino acid positions 31 to 35, optionally including one or two additional amino acids after position 35 (termed 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are generally found at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In some embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0199] Chothia relates to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering system, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the location of insertions at H35A and H35B; if neither 35A nor 35B is present, the end of the loop is 32; if only 35A is present, the end of the loop is 33; if both 35A and 35B are present, the end of the loop is 34). The AbM hypervariable region represents a compromise between the Kabat CDRs and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.

[0200] or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill will readily be able to identify the CDRs defined by each numbering system. And, the correspondence between the different numbering systems is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0201] The term "antigen-binding fragment" of an antibody refers to a molecule other than the intact antibody that comprises a portion of the intact antibody that binds the antigen to which the intact antibody binds. For example, an antigen-binding fragment can be a polypeptide of a fragment of 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, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding capacity to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0202] The term "Fd fragment" means an antibody fragment consisting of VH and CHI domains; the term "dAb fragment" means an antibody fragment which consists of a VH domain (Ward et al., Nature 341 :544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CHI domains; the term "F(ab')2 fragment" means an antibody fragment which comprises two Fab fragments linked by disulfide bridges under the hinge region; the term "Fab' fragment" means the fragment obtained by reducing the disulfide bonds of a F(ab')2 fragment, consisting of an intact light chain and a Fd fragment (consisting of VH and CHI domains) of a heavy chain. The term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is commonly held to be the minimum fragment of an antibody that is capable of forming a complete antigen binding site. It is generally considered that the six CDRs confer antigen binding specificity to an antibody. However, even a single variable domain (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although it may do so at a lower affinity than the entire binding site. The term "Fc fragment" means an antibody fragment consisting of the second, third constant regions of the first heavy chain and the second, third constant regions of the second heavy chain, which are joined by disulfide bonds. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding. The term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Roseburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).Other linkers useful in the present application are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31 :94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VHand VLof the scFv.

[0203] Each of the above-mentioned antibody fragments retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen. In this context, one skilled in the art can use known and routine techniques to obtain an antigen-binding fragment of an antibody (e.g., the above-mentioned antibody fragments) from a given antibody (e.g., an antibody provided by the present disclosure), and screen the antigen-binding fragment of the antibody for specificity in the same manner as used for the intact antibody.

[0204] The terms "monoclonal antibody," "monoclonal," "mAb," have the same meaning and are used interchangeably, and refer to one antibody or one fragment of an antibody from a population of highly homogenous antibody molecules, i.e., a population of antibody molecules that are essentially identical except for naturally occurring mutations that can arise during production. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies are in contrast to monoclonal antibodies and generally comprise at least two or more different antibodies, which typically recognize different epitopes on an antigen. Moreover, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of highly homogenous antibodies and does not require production of the antibody by any particular method.

[0205] The term "multispecific antibody" refers to an antibody having multiple different antigen-binding specificities, including, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. A "bispecific antibody" refers to an antibody having two different antigen-binding specificities formed by conjugation of a first antibody (or fragment thereof) and a second antibody (or fragment thereof) or antibody analog via a conjugate arm, which includes but is not limited to chemical reaction, genetic fusion, and enzymatic. "Multispecific antibodies" include, for example, trispecific antibodies and tetraspecific antibodies, which are antibodies having three and four different antigen-binding specificities, respectively.

[0206] The term "chimeric antibody" refers to an antibody whose light chain or / and heavy chain is derived from one antibody (which can be derived from a certain species or belong to a certain antibody class or subclass) and the other part of the light chain or / and heavy chain is derived from another antibody (which can be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the binding activity to the target antigen. For example, the term "chimeric antibody" can include an antibody whose heavy and light chain variable regions are derived from a first antibody (e.g., human origin), while the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., murine origin). For example, an antibody produced by immunizing a fully human transgenic mouse can be referred to as a chimeric antibody, which is composed of a fully human variable region and a murine constant region.

[0207] The term "humanized antibody" refers to an antibody that can be prepared by replacing a part of a non-human antibody prepared by immunizing a mammal other than a human with a part of a human antibody. Specifically, it is known that it can be prepared by constructing a chimera having a gene encoding a human antibody constant region (Proc. Natl. Acad. Sci. (USA) (1987), vol. 84, p. 3439-3443, Journal of Immunology (1987), vol. 139, No. 1, p. 3521). The DNA sequence of the human constant region has been described in the prior art, and the constant region gene can be easily obtained from a known clone. Then, the DNA sequence encoding the variable region of the antibody can be fused to the human constant region. The isotype of the human constant region can be selected based on the desired effective function or antibody-dependent cellular cytotoxicity activity. Suitable isotypes are IgGl, IgG3, and IgG4. Both human light chain constant regions, kappa chain and lambda chain, can be used. Such humanized chimeric antibodies can be expressed by conventional methods.

[0208] The term "human antibody" refers to an antibody that can be produced by using mice in which constant region genes of human immunoglobulin are transferred (XenoMouse (Chemical Biology (2000), vol. 7, issue 8, p. R185-6), HuMAb-Mouse (Infection and Immunity (2002), vol. 70, issue 2, p. 612-9), TC mouse (Biotechnology and Genetics Enginnering Revew (2002), vol. 19, p. 73-82), and KM mouse (Cloning Stem Cells (2002), vol. 4, issue 1, p. 91-102)), in which the constant region genes of human immunoglobulin are transferred, and the target antibody can be mass-produced by isolating antibody-producing lymphocytes from the mice into hybridomas. It can be produced by a phage display method (FEBS Letter (1998), vol. 441, p. 20-24). In this method, by using phages in which human antibody genes are integrated into circular single-stranded DNA, a human antibody can be expressed on the surface of the phage in the form of fusion with the coat protein of the phage.

[0209] The term "variant", in the context of a polypeptide (including polypeptides), refers to a polypeptide or peptide that comprises an amino acid sequence that has been altered by the introduction of an amino acid residue substitution, deletion, or addition. In certain instances, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, and without limitation, a polypeptide can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, etc. A derivatized polypeptide or peptide can be produced by chemical modification using techniques known to those of ordinary skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Furthermore, a variant has similar, the same, or improved function as the polypeptide or peptide from which it is derived.

[0210] The term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue with an amino acid residue having a similar side chain, e.g., substitutions where the residue is replaced with an residues that is physically or functionally similar (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a preferred substitution is one in which the residue is replaced with another amino acid residue from the same side chain family. Methods of identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0211] The term "identity" is used in reference to the match between two polypeptide sequences or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is the number of matched positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Generally, the comparison is made over the full length of the sequences being compared. Such a comparison can be conveniently performed by means of the algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443-453, implemented as the Align program (DNAstar, Inc.), using, for example. The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as integrated into the ALIGN program (version 2.0), using a PAM120 weight residue table, 12 for the gap length penalty, and 4 for the gap penalty. In addition, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as implemented in the GAP program, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6.

[0212] The nomenclature used herein for the twenty conventional amino acids follows the conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present disclosure, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present disclosure, amino acids are generally represented by either the one-letter or three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0213] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its cognate antigen. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (K D ) or the half maximal effective concentration (EC 50 ) of the interaction.

[0214] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of antigen binding site / antigen complexes. Both the "association rate constant" (k a or k on ) and the "dissociation rate constant" (k dis or k off ) can be calculated from the concentrations and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 : 186-187). The ratio of k dis / k on is equal to the dissociation constant K D (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K D , k on and k dis values can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., ForteBio Octet method). In addition to this, the dissociation constant can be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0215] The term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection and direct the expression of elements of genetic material carried by the vector in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0216] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in cloning vectors, this sequence is the origin of replication, or autonomously replicating sequence, which permits the vector to replicate independent of the host chromosomal DNA. The term "expression vector" as used herein refers to a vector that comprises a recombinant polynucleotide operably linked to expression control sequences necessary for the expression of the nucleotide sequence to be expressed. Expression vectors comprise sufficient cis-acting elements for expression; additional elements can be provided by the host cell or an in vitro expression system.

[0217] The term "host cell" refers to a cell that can be used for the introduction of a vector.

[0218] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible, in pharmacological and / or physiological terms, with the subject and the active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure adjusting agents, agents that delay absorption, preservatives, stabilizers. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Osmotic pressure adjusting agents include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactose, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), and the like.

[0219] The term "prevent" refers to an approach taken to stop or delay the occurrence of a disease or disorder or symptoms (e.g., atherosclerosis) in a subject. As used herein, the term "treatment" refers to an approach taken to obtain a beneficial or desired clinical result. For the purposes of the present disclosure, a beneficial or desired clinical result includes, but is not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether or not detectable to the patient. Further, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0220] The term "subject" refers to a mammal, such as a primate, e.g., a human. In certain embodiments, the subject (e.g., human) has a disease addressed by the present disclosure, or is at risk of having a disease described above.

[0221] As used herein, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease refers to an amount that is sufficient to prevent, stop, or delay the occurrence of a disease; an effective amount for treating a disease refers to an amount that is sufficient to cure or at least partially arrest the disease and its complications in a patient already having a disease. Determining such an effective amount is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., the age, weight, and sex of the patient, the mode of administration of the drug, and other therapies that the patient may be undergoing, etc.

[0222] Combination therapy includes the use of an antibody or antigen-binding fragment thereof of the present disclosure in conjunction with one or more additional second therapeutic agents or other prophylactic or therapeutic modalities. In such combination therapies, the various active agents often have different mechanisms of action, and the combination therapy can result in a synergistic effect. Combination therapy can allow for lower dosages of one or more of the agents to be used, in order to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies can have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder, or condition. In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure can be administered sequentially with a second therapeutic agent. In other embodiments, an antibody or antigen-binding fragment thereof of the present disclosure can be administered simultaneously with a second therapeutic agent. An antibody or antigen-binding fragment thereof of the present disclosure can be used in combination with at least one other (active) agent in any manner.

[0223] In some embodiments, the combination therapy can reduce and / or slow the progression of atherosclerosis. In some embodiments, the combination therapy can reduce and / or slow atherosclerotic cardiovascular disease (ASCVD), CAD / CHD, cerebrovascular disease, and / or peripheral arterial disease (PAD). In some embodiments, any of the methods provided herein with respect to combination therapy can be used to reduce the risk of any one or more of these events. In some embodiments, any patient or subject at risk for one of these events is a subject identified to receive combination therapy.

[0224] The terms "administer," "administering," and the like, refer to methods used to deliver a drug, such as an antibody or antigen-binding fragment thereof, to the site of desired biological action (e.g., intravenous administration). Administration techniques useful for the agents and methods described herein can be found in, e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0225] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is administered at a frequency of at least once per week, at least once per two weeks, at least once per three weeks, at least once per four weeks, at least once per five weeks, at least once per six weeks, at least once per three months.

[0226] In some embodiments, the route of administration of the antibody or antigen-binding fragment thereof of the present disclosure is consistent with known methods, e.g., oral; injection via intravenous, intraperitoneal, intracerebral (into thin-walled tissues), intracerebroventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal, or intralesional routes; via sustained-release systems, or via implantation devices.

[0227] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is administered to a subject at least monthly for at least one year, e.g., at least 0.5, 12, 18, 24, 30, 36, 42, 48, 54, 60, or more months.

[0228] Advantages of the Invention

[0229] The present disclosure provides anti-ADAMTS-7 antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors, host cells, pharmaceutical compositions and kit products, which specifically bind to ADAMTS-7 and have significant inhibitory effect on atherosclerosis, providing a new solution for the residual risk of cardiovascular events after lipid-lowering therapy for atherosclerosis patients. BRIEF DESCRIPTION OF DRAWINGS

[0230] Figure 1 shows the western blot detection of the recognition ability of 313-mAb and 315-mAb to ADAMTS-7.

[0231] Figure 2 shows the process of establishing an atherosclerosis mouse model and administering the ADAMTS-7 monoclonal antibodies of the present disclosure.

[0232] Figure 3 shows the statistical results of body weight changes of atherosclerosis model mice at weeks 0, 4, 8 and 12 after the start of modeling.

[0233] Figure 4 shows the statistical results of cholesterol changes of atherosclerosis model mice at weeks 0, 4, 8 and 12 after the start of modeling.

[0234] Figure 5 shows the statistical results of triglyceride changes of atherosclerosis model mice at weeks 0, 4, 8 and 12 after the start of modeling.

[0235] Figure 6 shows the statistical results of aortic plaque area ratio of atherosclerosis model mice injected intraperitoneally with the ADAMTS-7 monoclonal antibodies of the present disclosure (10 mg / kg·week) for six weeks.

[0236] Figure 7 shows the experimental results of the effects of the ADAMTS-7 monoclonal antibodies of the present disclosure on vascular smooth muscle cell proliferation and migration. DETAILED DESCRIPTION

[0237] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, which are all conventional products that can be obtained by purchase.

[0238] Unless specifically indicated otherwise, the molecular biology experimental methods and immunoassay methods used in the present application are basically carried out according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes is in accordance with the conditions recommended by the product manufacturer.

[0239] The mouse is immunized by DP-9 vaccine peptide segment (patent: ZL201910914234.9), and the spleen cells are obtained to prepare hybridoma cells. The ADAMTS-7 protein is recognized by the supernatant of the hybridoma cells, and the vascular smooth muscle cell proliferation and migration experiment is carried out. The hybridoma cells with the function of inhibiting ADAMTS-7 are obtained, and the monoclonal antibody is further prepared from the hybridoma cells. The prevention and treatment effect on atherosclerosis is determined by a mouse atherosclerosis model, and mouse antibodies 313-mAb (also referred to as "Ab1") and 315-mAb (also referred to as "Ab2") are obtained.

[0240] The gene sequences encoding the heavy chain and light chain variable regions of 313-mAb and 315-mAb are respectively connected with the gene sequences encoding the heavy chain and light chain constant regions of human antibodies, and the chimeric antibodies 313-chimeric (also referred to as "cAb1") and 315-chimeric (also referred to as "cAb2") are obtained by recombination expression.

[0241] The above mouse antibody heavy chain and light chain CDR are respectively grafted to the FR framework region of a humanized template, and the corresponding variable region mutation is carried out. Among them, for 313-mAb, 3 light chains 313-mAb-L1 (non-mutation), 313-mAb-L2 and 313-mAb-L1a (the light chain variable region sequences are respectively shown as Seq ID No: 29-31), and 5 heavy chains 313-mAb-H1-313-mAb-H5 (H1 is non-mutation, and the heavy chain variable region sequences are respectively shown as Seq ID No: 32-36) are obtained. The light chains 313-mAb-L1, 313-mAb-L2 and 313-mAb-L1a are respectively cross combined with the heavy chains 313-mAb-H2-313-mAb-H5 to obtain 12 antibodies, and the naming rules of each antibody are Ab1-heavy chain number-light chain number; and for 315-mAb, 5 light chains 315-mAb-L1 (non-mutation), 315-mAb-L2, 315-mAb-L1a, 315-mAb-L1b and 315-mAb-L1c (the light chain variable region sequences are respectively shown as Seq ID No: 37-41), and 6 heavy chains 315-mAb-H1-315-mAb-H6 (H1 is non-mutation, and the heavy chain variable region sequences are respectively shown as Seq ID No: 42-47) are obtained. The light chains 315-mAb-L1 and 315-mAb-L2 are respectively cross combined with the heavy chains 315-mAb-H2-315-mAb-H6, and the three light chains 315-mAb-L1a-315-mAb-L1c are respectively combined with the heavy chain 315-mAb-H6, so as to obtain a total of 13 antibodies. The naming rules of each antibody are Ab2-heavy chain number-light chain number.

[0242] Validation of the antibodies produced according to the present disclosure in a mouse atherosclerosis model.

[0243] Sequence information table

[0244] Example 1 Antibody affinity experiment

[0245] HEK293 cells were transfected with control plasmid and overexpression plasmid with Flag tag, and proteins were extracted 48 hours later. Western blot was performed using Flag antibody (Flag Ab), 313-mAb and 315-mAb monoclonal antibodies, respectively. The results are shown in Figure 1. The results show that both 313-mAb and 315-mAb can recognize ADAMTS-7 protein, indicating that the antigen binds to the antibody.

[0246] Example 2 Antibody SPR analysis

[0247] HEK293F cells were transfected with ADAMTS-7 overexpression plasmid, and human ADAMTS-7 purified protein was obtained by purifying the cell supernatant. Surface plasmon resonance (SPR) analysis was performed using a Biacore 8K instrument. Specifically, ADAMTS-7 purified protein was used as the stationary phase to coat the chip at 1 μg per well; and candidate monoclonal antibodies were used as the mobile phase to determine the KD value of each monoclonal antibody with ADAMTS-7. The running program was as follows: binding for 120 s, dissociation for 360 s, and flow rate of 30 μl / min. The KD values of the candidate monoclonal antibodies are shown in Table 1. According to the SPR results, it can be seen that some of the modified candidate humanized monoclonal antibodies maintain a high affinity (E-09M).

[0248] Table 1: SPR results of humanized ADAMTS-7 monoclonal antibodies

[0249] Example 3

[0250] The experimental mice were male 8-week-old C57BL / 6 APOE - / Mice (purchased from the animal department of Peking University Health Science Center) weighing 21-23 g were used. As shown in Figure 2, a high-fat diet (D12108C) was used to induce atherosclerosis for 12 weeks starting at 8 weeks of age. After 6 weeks of high-fat induction, 10 mg / kg mouse IgG and 10 mg / kg ADAMTS-7 monoclonal antibodies 313-mAb or 315-mAb were intraperitoneally injected into the control and experimental groups of mice every week for 6 weeks, and the samples were taken at 12 weeks. The experimental groups were: control (mouse IgG), 313-mAb group, and 315-mAb group.

[0251] During the modeling process, the body weight of the mice was measured every week, and the results are shown in Figure 3. The blood was collected from the medial canthus of all mice at 0, 4, 8, and 12 weeks, and the plasma was obtained to determine the blood cholesterol (Zhongsheng Beikong Reagent Kit, Item No.: 4220) and triglycerides (Zhongsheng Beikong Reagent Kit, Item No.: 0220), and the results are shown in Figures 4 and 5. After the modeling was completed, the aortas of the mice were taken, cut along the midline, and stained with oil red O (Sigma), and the percentage of plaque area was calculated. Figure 6 shows the general view of the aortas and the percentage of plaque area in the two ADAMTS-7 monoclonal antibody treatment groups and the IgG control group.

[0252] Example 4 Effect of humanized anti-ADAMTS-7 antibodies on vascular smooth muscle cell proliferation and migration

[0253] The rat primary thoracic aortic smooth muscle complete culture medium (10% FBS, DMEM-LG, Gibco) was selected for the cell scratch experiment in a 12-well plate. After the cells grew on the plate bottom, the medium was changed to serum-free DMEM-LG and starved for 12 h. A scratch was made in the center of the well plate with a scratcher, and a photograph was taken under a microscope to record the scratch. The complete culture medium was replaced and different concentrations of antibodies (blank control vehicle group, human IgG control group, and candidate humanized ADAMTS-7 monoclonal antibodies were added to the culture medium at a concentration gradient of 0.5 / 1 / 2 μg / μl) were added. After incubation at 5% CO2 and 37°C for 12 h, the migration of smooth muscle cells at the scratch was recorded again. The migration area of each well after 12 h was calculated, and the IgG group was taken as 1 to calculate the relative migration inhibition rate of different candidate ADAMTS-7 humanized monoclonal antibodies.

[0254] The experimental results show (Figure 7) that Ab1-H4-L2, Ab2-H6-L1a, and Ab2-H6-L1b have the ability to significantly inhibit the proliferation and migration of rat primary thoracic aorta smooth muscle cells, and the degree of inhibition is in a dose-dependent manner. At a dose of 2 μg / μl, the three monoclonal antibodies can inhibit about 50% of cell migration. In Figure 7: vehicle is a blank control, IgG group is a negative control, Ab1 and hAb2 are positive controls, respectively. The last number of each group of data represents the dose (μg / μl), such as IgG-1 represents 1 μg / μl human IgG; Ab1-H4-L2-0.5 represents 0.5 μg / μl Ab1-H4-L2.

[0255] Although the specific embodiments of the present disclosure have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all the teachings disclosed, and such changes are within the scope of protection of the present disclosure. The entire scope of the present disclosure is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to ADAMTS-7, an disintegrin and metalloproteinase with thrombospondin type 1 motif 7, wherein, The antibody or antigen-binding fragment thereof comprises complementarity determining regions (CDRs) as follows: (1) CDR-H1, CDR-H2 and CDR-H3 contained in a VH (heavy chain variable region) represented by SEQ ID NO: 7; and / or CDR-L1, CDR-L2 and CDR-L3 contained in a VL (light chain variable region) represented by SEQ ID NO: 9; (2) CDR-H1, CDR-H2 and CDR-H3 contained in a VH represented by SEQ ID NO: 21; and / or CDR-L1, CDR-L2 and CDR-L3 contained in a VL represented by SEQ ID NO: 23; (3) CDR-H1 represented by SEQ ID NO: 1, CDR-H2 represented by SEQ ID NO: 2 and CDR-H3 represented by SEQ ID NO: 3; and / or CDR-L1 represented by SEQ ID NO: 4, CDR-L2 represented by SEQ ID NO: 5 and CDR-L3 represented by SEQ ID NO: 6; (4) CDR-H1 represented by SEQ ID NO: 1, CDR-H2 represented by SEQ ID NO: 16 and CDR-H3 represented by SEQ ID NO: 17; and / or CDR-L1 represented by SEQ ID NO: 18, CDR-L2 represented by SEQ ID NO: 5 and CDR-L3 represented by SEQ ID NO: 20; (5) CDR-H1, CDR-H2 and CDR-H3 contained in a VH and / or CDR-L1, CDR-L2 and CDR-L3 contained in a VL, wherein the VH and / or VL comprises at least one CDR having a mutation, which is a substitution, deletion or addition of one or several amino acids (e.g. a substitution, deletion or addition of 1, 2 or 3 amino acids) compared to the VH and / or VL of (1) or (2); preferably the substitution is a conservative substitution; or (6) CDRs having at least one CDR with a mutation, which is a substitution, deletion or addition of one or several amino acids (e.g. a substitution, deletion or addition of 1, 2 or 3 amino acids) compared to the CDRs of any one of (1) to (4); preferably the substitution is a conservative substitution; Preferably, the CDRs are selected from CDR-H1, CDR-H2 and CDR-H3 contained in SEQ ID NO: 21, SEQ ID NO: 32 and SEQ ID NO: 42; Preferably, the CDRs are selected from CDR-L1, CDR-L2 and CDR-L3 contained in SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39 and SEQ ID NO:

41. Preferably, the CDRs are selected from the group consisting of CDR-H1, CDR-H2 and CDR-H3 contained in SEQ ID NO: 21, SEQ ID NO: 32 and SEQ ID NO: 42; and, the CDRs in item (6) are selected from the group consisting of CDR-L1, CDR-L2 and CDR-L3 contained in SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39 and SEQ ID NO:

41. Preferably, the CDRs are defined according to IMGT, Kabat, Chothia or AbM numbering system.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising: (1-1) a VH comprising heavy chain complementarity determining regions contained in the VH set forth in SEQ ID NO: 7 or a variant thereof; and, a VL comprising light chain complementarity determining regions contained in the VL set forth in SEQ ID NO: 9 or a variant thereof; (1-2) a VH comprising heavy chain complementarity determining regions contained in the VH set forth in SEQ ID NO: 21 or a variant thereof; and, a VL comprising light chain complementarity determining regions contained in the VL set forth in SEQ ID NO: 23 or a variant thereof; (1-3) a VH comprising three CDRs as follows: CDR-H1 of the sequence set forth in SEQ ID NO: 1 or a variant thereof, CDR-H2 selected from the group consisting of the sequence set forth in SEQ ID NO: 2 or a variant thereof, and CDR-H3 of the sequence set forth in SEQ ID NO: 3 or a variant thereof, or, comprising CDR-H1, CDR-H2 and CDR-H3 contained in the VH set forth in SEQ ID NO: 32; and, a VL comprising three CDRs as follows: CDR-L1 of the sequence set forth in SEQ ID NO: 4 or a variant thereof, CDR-L2 of the sequence set forth in SEQ ID NO: 5 or a variant thereof, and CDR-L3 of the sequence set forth in SEQ ID NO: 6 or a variant thereof, or, comprising CDR-L1, CDR-L2 and CDR-L3 contained in the VL set forth in SEQ ID NO: 31; (1-4) a VH comprising three CDRs as follows: CDR-H1 of the sequence set forth in SEQ ID NO: 1 or a variant thereof, CDR-H2 selected from the group consisting of the sequence set forth in SEQ ID NO: 16 or a variant thereof, and CDR-H3 of the sequence set forth in SEQ ID NO: 17 or a variant thereof, or, comprising CDR-H1, CDR-H2 and CDR-H3 contained in the VH set forth in the group consisting of SEQ ID NO: 21 and SEQ ID NO: 42; and, (1-4) a VH comprising three CDRs as follows: CDR-H1 of the sequence set forth in SEQ ID NO: 1 or a variant thereof, CDR-H2 selected from the group consisting of the sequence set forth in SEQ ID NO: 16 or a variant thereof, and CDR-H3 of the sequence set forth in SEQ ID NO: 17 or a variant thereof, or, comprising CDR-H1, CDR-H2 and CDR-H3 contained in the VH set forth in the group consisting of SEQ ID NO: 21 and SEQ ID NO: 42; and, VL comprising the 3 CDRs as shown below: CDR-L1 of the sequence set forth in SEQ ID NO: 18 or a variant thereof, CDR-L2 of the sequence set forth in SEQ ID NO: 5 or a variant thereof, and CDR-L3 of the sequence set forth in SEQ ID NO: 20 or a variant thereof, or comprising the CDR-L1, CDR-L2 and CDR-L3 contained in a VL of SEQ ID NO: 23, SEQ ID NO: 39 or SEQ ID NO: 41 ; wherein said variant has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably said substitutions are conservative substitutions; said variant has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably said substitutions are conservative substitutions; said CDRs are defined by the Kabat, Chothia or IMGT numbering system.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising: (2-1 ) a VH having the sequence set forth in SEQ ID NO: 7 or a variant thereof, and a VL having the sequence set forth in SEQ ID NO: 9 or a variant thereof; or, (2-2) a VH having the sequence set forth in SEQ ID NO: 21 or a variant thereof, and a VL having the sequence set forth in SEQ ID NO: 23 or a variant thereof; wherein said variant has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably said substitutions are conservative substitutions; said antibody or antigen-binding fragment thereof comprises: a VH having the sequence set forth in SEQ ID NO: 7, and a VL having the sequence set forth in SEQ ID NO: 9; a VH having the sequence set forth in SEQ ID NO: 32, and a VL having the sequence set forth in SEQ ID NO: 29; a VH having the sequence set forth in SEQ ID NO: 32, and a VL having the sequence set forth in SEQ ID NO: 30; a VH having the sequence set forth in SEQ ID NO: 32, and a VL having the sequence set forth in SEQ ID NO: 31 ; a VH having the sequence set forth in SEQ ID NO: 33, and a VL having the sequence set forth in SEQ ID NO: 29; a VH having the sequence of SEQ ID NO:33, and a VL having the sequence of SEQ ID NO:30; a VH having the sequence of SEQ ID NO:33, and a VL having the sequence of SEQ ID NO:31 ; a VH having the sequence of SEQ ID NO:34, and a VL having the sequence of SEQ ID NO:29; a VH having the sequence of SEQ ID NO:34, and a VL having the sequence of SEQ ID NO:30; a VH having the sequence of SEQ ID NO:34, and a VL having the sequence of SEQ ID NO:31 ; a VH having the sequence of SEQ ID NO:35, and a VL having the sequence of SEQ ID NO:29; a VH having the sequence of SEQ ID NO:35, and a VL having the sequence of SEQ ID NO:30; a VH having the sequence of SEQ ID NO:35, and a VL having the sequence of SEQ ID NO:31 ; a VH having the sequence of SEQ ID NO:36, and a VL having the sequence of SEQ ID NO:29; a VH having the sequence of SEQ ID NO:36, and a VL having the sequence of SEQ ID NO:30; a VH having the sequence of SEQ ID NO:36, and a VL having the sequence of SEQ ID NO:31 ; a VH having the sequence of SEQ ID NO:21, and a VL having the sequence of SEQ ID NO:23; a VH having the sequence of SEQ ID NO:42, and a VL having the sequence of SEQ ID NO:37; a VH having the sequence of SEQ ID NO:42, and a VL having the sequence of SEQ ID NO:38; a VH having the sequence of SEQ ID NO:42, and a VL having the sequence of SEQ ID NO:39; a VH having the sequence of SEQ ID NO:42, and a VL having the sequence of SEQ ID NO:40; a VH having the sequence of SEQ ID NO:42, and a VL having the sequence of SEQ ID NO:41 ; a VH having the sequence of SEQ ID NO:43, and a VL having the sequence of SEQ ID NO:37; a VH having the sequence of SEQ ID NO:43, and a VL having the sequence of SEQ ID NO:38; a VH having the sequence of SEQ ID NO:43, and a VL having the sequence of SEQ ID NO:39; a VH having the sequence of SEQ ID NO:43, and a VL having the sequence of SEQ ID NO:40; a VH having the sequence of SEQ ID NO:43, and a VL having the sequence of SEQ ID NO:41 ; a VH having the sequence of SEQ ID NO: 44, and a VL having the sequence of SEQ ID NO: 37; a VH having the sequence of SEQ ID NO: 44, and a VL having the sequence of SEQ ID NO: 38; a VH having the sequence of SEQ ID NO: 44, and a VL having the sequence of SEQ ID NO: 39; a VH having the sequence of SEQ ID NO: 44, and a VL having the sequence of SEQ ID NO: 40; a VH having the sequence of SEQ ID NO: 44, and a VL having the sequence of SEQ ID NO: 41; a VH having the sequence of SEQ ID NO: 45, and a VL having the sequence of SEQ ID NO: 37; a VH having the sequence of SEQ ID NO: 45, and a VL having the sequence of SEQ ID NO: 38; a VH having the sequence of SEQ ID NO: 45, and a VL having the sequence of SEQ ID NO: 39; a VH having the sequence of SEQ ID NO: 45, and a VL having the sequence of SEQ ID NO: 40; a VH having the sequence of SEQ ID NO: 45, and a VL having the sequence of SEQ ID NO: 41; a VH having the sequence of SEQ ID NO: 46, and a VL having the sequence of SEQ ID NO: 37; a VH having the sequence of SEQ ID NO: 46, and a VL having the sequence of SEQ ID NO: 38; a VH having the sequence of SEQ ID NO: 46, and a VL having the sequence of SEQ ID NO: 39; a VH having the sequence of SEQ ID NO: 46, and a VL having the sequence of SEQ ID NO: 40; a VH having the sequence of SEQ ID NO: 46, and a VL having the sequence of SEQ ID NO: 41; a VH having the sequence of SEQ ID NO: 47, and a VL having the sequence of SEQ ID NO: 37; a VH having the sequence of SEQ ID NO: 47, and a VL having the sequence of SEQ ID NO: 38; a VH having the sequence of SEQ ID NO: 47, and a VL having the sequence of SEQ ID NO: 39; a VH having the sequence of SEQ ID NO: 47, and a VL having the sequence of SEQ ID NO: 40; or, a VH having the sequence of SEQ ID NO: 47, and a VL having the sequence of SEQ ID NO:

41.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, which is a murine, chimeric, humanized, or fully human antibody.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, which comprises a constant region from or derived from a human immunoglobulin. Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises or is derived from a CH (heavy chain constant region) of a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4); Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises or is derived from a CL (light chain constant region) of a human immunoglobulin (e.g., kappa or lambda).

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, which comprises: (3-1) a HC (heavy chain) having the sequence set forth in SEQ ID NO: 11 or a variant thereof, and a LC (light chain) having the sequence set forth in SEQ ID NO: 13 or a variant thereof; or, (3-2) a HC having the sequence set forth in SEQ ID NO: 25 or a variant thereof, and a LC having the sequence set forth in SEQ ID NO: 27 or a variant thereof.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein, The antibody or antigen-binding fragment thereof is selected from the group consisting of a scFv, a Fab, a Fab', a Fab'-SH, a F(ab')2, an Fv fragment, a disulfide linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein, The antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof is labeled with a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

9. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-8.

10. A vector comprising the isolated nucleic acid molecule of claim 9; preferably, the vector is a cloning vector or an expression vector.

11. A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

12. A method of preparing the antibody or antigen-binding fragment thereof of any one of claims 1-8, comprising the steps of: culturing the host cell of claim 11, collecting the culture supernatant, purifying, and obtaining the antibody or antigen-binding fragment thereof.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-8, the nucleic acid molecule of claim 9, the vector of claim 10, or the host cell of claim 11, and a pharmaceutically acceptable excipient; Preferably, the pharmaceutical composition is used for preventing and / or treating an ADAMTS-7-associated disease or disorder; preferably, the ADAMTS-7-associated disease or disorder is an autoimmune disease (e.g., lupus nephritis), a cardiovascular disease or its complication (e.g., atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral arterial vascular disease, deep vein thrombosis, pulmonary embolism, restenosis or vascular calcification after stent implantation, aortic aneurysm / dissection), perioperative acute ischemia-reperfusion injury of kidney.

14. A kit product comprising the pharmaceutical composition of claim 13, and optionally an instruction for use of the pharmaceutical composition. Preferably, the kit further comprises a second therapeutic agent, wherein the second therapeutic agent is a lipid lowering agent; preferably the lipid lowering agent is selected from statins (e.g., simvastatin, atorvastatin, rosuvastatin, lovastatin, pravastatin, fluvastatin, pitavastatin), ezetimibe, PCSK9 inhibitors (e.g., evolocumab, alirocumab, inclisran); Preferably, the kit is used for preventing and / or treating an ADAMTS-7 related disease or disorder; preferably the ADAMTS-7 related disease or disorder is an autoimmune disease (e.g., lupus nephritis), a cardiovascular disease or its complication (e.g., atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral arterial disease, deep vein thrombosis, pulmonary embolism, restenosis or vascular calcification after stent implantation, aortic aneurysm / dissection), acute ischemia-reperfusion injury of kidney during perioperative period.

15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-8, the pharmaceutical composition of claim 13, or the kit of claim 14 in the manufacture of a medicament for preventing and / or treating an ADAMTS-7 related disease or disorder; Preferably, the ADAMTS-7 related disease or disorder is an autoimmune disease (e.g., lupus nephritis), a cardiovascular disease or its complication (e.g., atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral arterial disease, deep vein thrombosis, pulmonary embolism, restenosis or vascular calcification after stent implantation, aortic aneurysm / dissection), acute ischemia-reperfusion injury of kidney during perioperative period.

16. A method for preventing and / or treating an ADAMTS-7 related disease or disorder, comprising the step of administering to a subject the antibody or antigen-binding fragment thereof of any one of claims 1-8, the pharmaceutical composition of claim 12, or the kit of claim 14; Preferably, the ADAMTS-7 related disease or disorder is an autoimmune disease (e.g., lupus nephritis), a cardiovascular disease or its complication (e.g., atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral arterial disease, deep vein thrombosis, pulmonary embolism, restenosis or vascular calcification after stent implantation, aortic aneurysm / dissection), acute ischemia-reperfusion injury of kidney during perioperative period. Preferably, the method further comprises the step of administering a second therapeutic agent, wherein the second therapeutic agent is a lipid lowering agent; preferably the antibody or antigen-binding fragment thereof and the second therapeutic agent are administered simultaneously or sequentially; Preferably, the lipid lowering agent is selected from statins (e.g., simvastatin, atorvastatin, rosuvastatin, lovastatin, pravastatin, fluvastatin, pitavastatin), ezetimibe, PCSK9 inhibitors (e.g., evolocumab, alirocumab, inclisran).

17. The antibody or antigen-binding fragment thereof of any one of claims 1-8, the pharmaceutical composition of claim 13, or the kit of claim 14, for use in the prevention and / or treatment of a disease or disorder associated with ADAMTS-7; Preferably, the disease or disorder associated with ADAMTS-7 is an autoimmune disease (e.g., lupus nephritis), a cardiovascular disease or its complication (e.g., atherosclerosis, coronary heart disease, myocardial infarction, stroke, peripheral arterial vascular disease, deep vein thrombosis, pulmonary embolism, restenosis or vascular calcification after stent implantation, aortic aneurysm / dissection), perioperative acute kidney ischemia-reperfusion injury.

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