CN111094354A

CN111094354A - Thrombin antibody, antigen binding fragment thereof and medical application

Info

Publication number: CN111094354A
Application number: CN201880059071.5A
Authority: CN
Inventors: 应华; 毛浪勇; 孙嘉康; 胡齐悦; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2017-11-20
Filing date: 2018-11-19
Publication date: 2020-05-01
Anticipated expiration: 2038-11-19
Also published as: WO2019096294A1; CN111094354B; TW201922797A

Abstract

The present disclosure relates to thrombin antibodies, antigen binding fragments thereof, and medical uses. Further, the disclosure relates to murine, chimeric, humanized antibodies comprising the thrombin antibody CDR regions, as well as pharmaceutical compositions comprising the thrombin antibody and/or antigen binding fragments thereof, and their use as medicaments. In particular, the disclosure relates to the use of a humanized thrombin antibody in the manufacture of a medicament for the treatment of a thrombin-related disease or disorder.

Description

Thrombin antibody, antigen binding fragment thereof and medical application

Technical Field

The present disclosure relates to thrombin antibodies, antigen-binding fragments thereof, and further, chimeric antibodies, humanized antibodies comprising CDR regions of the thrombin antibodies, pharmaceutical compositions comprising the thrombin antibodies and antigen-binding fragments thereof, and uses thereof as a diagnostic agent and a therapeutic agent for thrombin-related diseases.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Blood coagulation is an important process for preventing bleeding (hemostasis) of damaged blood vessels. However, blood clots that block blood flow through a blood vessel (thrombosis) or that slough off and deposit on blood vessels elsewhere in the body (thromboembolism) represent a serious health threat. Thrombosis (e.g., Acute Myocardial Infarction (AMI), venous embolism, etc.) is a serious cardiovascular disease that endangers human health and life. The world health organization counted in 2008, by far, cardiovascular morbidity and mortality have leaped the first place. The number of cardiovascular disease deaths worldwide is about 1733 million people per year, accounting for 30% of the total deaths, 2.9 hundred million people in China, about 350 million deaths per year, accounting for 41% of the total deaths. In 2010, global disease burden research (GBD) statistics shows that stroke is the first leading cause of death of residents in China. Therefore, in recent years, people have attracted more and more attention to research into effective drugs and methods for treating cardiovascular diseases. Currently, some anticoagulant therapies can treat pathological blood coagulation, such as the use of traditional drugs heparin, small molecule heparin or warfarin, or the direct use of the Thrombin (Thrombin) inhibitor Dabigatran etexilate (Dabigatran), and the like. A common drawback of these therapies is the increased risk of bleeding. The window between the effective dose (to prevent thrombosis) and the safe dose (highest no bleeding risk) of many anticoagulant drugs is not large enough, and will be further narrowed to account for individual patient response differences. Thrombin is taken as a target, and the method for inhibiting the generation of thrombus by using a thrombin antagonist is one of the clinical methods for treating thrombus.

The coagulation reaction is a complex signaling cascade in which thrombin occupies a central place. Thrombin breaks down the fibrinogen of the circulatory system into fibrin monomers (which can polymerize to form fibrin, the fibrous matrix of blood clots) and has many direct controls on cells. As a serine protease, it triggers platelet deformation, releasing platelet activators ADP, serotonin and thromboxane a2, as well as chemokines and growth factors. In addition, adhesion molecules P-selectin and CD40 ligand are promoted to migrate to the surface of platelets, thereby activating integrin aIIb/b 3. The latter binds fibrinogen and von Willebrand factor (vWF), which in turn mediates platelet aggregation. Thrombin also stimulates procoagulant activity on the platelet surface, which in turn promotes thrombin expression. In endothelial cell cultures, thrombin facilitates vWF release, the appearance of P-selectin in the plasma membrane of the cell and chemokine production. These responses are thought to trigger the binding of platelets and leukocytes to the endothelial cell surface in vivo. The endothelial cells subsequently change shape and the permeability of the endothelial cell layer increases. These reactions are expected to promote local exudation of plasma proteins, promoting edema. In non-endothelial tissues, thrombin causes vasoconstriction by acting on smooth muscle cells. In vitro cultures of fibroblasts or vascular smooth muscle cells, thrombin regulates cytokine production and promotes mitosis, where it triggers calcium signaling and other responses. These cellular responses suggest that thrombin links tissue damage to the body's regulation of the hemostatic process, inflammatory responses, and even the enhancement of immune responses. These cellular responses also present a possibility: in addition to tissue damage, thrombin from endothelial cells and other cell types may play a role in leukocyte extravasation, vascular remodeling, and/or angiogenesis. Therefore, thrombin becomes a potential new anticoagulant and antithrombotic target.

An isolated anti-thrombin antibody molecule should inhibit thrombin in vivo without promoting or substantially promoting bleeding (bleeding) or hemorrhage (haemorrhage), i.e., the antibody molecule does not inhibit or substantially inhibit the normal physiological response to vascular injury (i.e., hemostasis). For example, hemostasis will not be inhibited or will be minimally inhibited (i.e., minimally inhibited, not affecting the health of the patient or requiring further intervention) by the antibody molecule. Bleeding is not increased or is minimally increased by antibody molecules.

Although there are currently a few patent publications of anti-thrombin antibodies, such as WO2013123591, WO2014153195, WO2014202992, WO2014202993, CN107043423A and WO 2017133673. To date, no antithrombin antibody drug is on the market, and further development and screening of antithrombin antibodies with better activity are necessary for relevant clinical research and application.

Disclosure of Invention

The present disclosure provides monoclonal antibodies or antigen-binding fragments thereof (also referred to as thrombin-binding proteins) that bind to the amino acid sequence or three-dimensional structure of the extracellular region of thrombin.

In one aspect, the present disclosure provides a monoclonal antibody, or antigen binding fragment thereof, that binds human thrombin, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain variable region comprising a heavy chain variable region amino acid sequence as set forth in SEQ ID NOs: 3.4 and 9, and HCDR1, HCDR2, and HCDR3, the light chain variable region comprising the amino acid sequence set forth in SEQ ID NOs: 11. 12 and 13, LCDR1, LCDR2 and LCDR3, wherein said LCDR is represented by SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13 are amino acid sequences respectively shown in the following general formula:

wherein: x₁Selected from H, L; x₂Selected from Y, L; x₃Selected from S, T; x₄Selected from E, H; x₅Selected from D, R; x₆Selected from I, M; x₇Selected from A, T; x₈Selected from S, Q, K; x₉Selected from T, G; x₁₀Selected from S, G; x₁₁Selected from T, N, Y; and when X₁When it is H, X₂Is not Y, and X₃-X₁₁Not simultaneously selected from the following amino acids: x₃Is S, X₄Is E, X₅Is D, X₆Is I, X₇Is A, X₈Is S, X₉Is T, X₁₀Is S and X₁₁Is T.

In some embodiments, the ratio of the affinity of the monoclonal antibody, or antigen-binding fragment thereof, and thrombin to the affinity of prothrombin (pro-thrombin) is greater than 25.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises a heavy chain variable region comprising a heavy chain variable region as set forth in SEQ ID NOs: 3. HCDR1, HCDR2 and HCDR3 shown in fig. 4 and 15; in some embodiments, the sequence of LCDR1 in the light chain variable region is selected from the group consisting of SEQ ID NOs: 6. 17 and 18, and the sequence of LCDR2 in the variable region of the light chain is selected from the group consisting of SEQ ID NOs: 7. 19 and 20, the sequence of LCDR3 in the light chain variable region is selected from the group consisting of SEQ ID NOs: 8. 21 and 22, wherein LCDR1, LCDR2 and LCDR3 are not simultaneously selected from the group consisting of: LCDR 1is selected from SEQ ID NO: 6. LCDR2 is selected from SEQ ID NO: 7 and LCDR3 are selected from SEQ ID NO: 8.

in some embodiments, in the monoclonal antibody or antigen-binding fragment thereof, the heavy chain variable region comprises a heavy chain variable region comprising a heavy chain variable region as set forth in SEQ ID NOs: 3.4 and 15, and HCDR1, HCDR2, and HCDR3, wherein the light chain variable region is selected from any one of the following a-i:

a. comprises the sequences respectively shown as SEQ ID NO: 17. LCDR1, LCDR2 and LCDR3 light chain variable regions shown at 19 and 8,

b. comprises the sequences respectively shown as SEQ ID NO: 6. LCDR1, LCDR2 and LCDR3 light chain variable regions shown at 19 and 8,

c. comprises the sequences respectively shown as SEQ ID NO: 17. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown at 20 and 21,

d. comprises the sequences respectively shown as SEQ ID NO: 6. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown at 20 and 8,

e. comprises the sequences respectively shown as SEQ ID NO: 17. LCDR1, LCDR2 and LCDR3 light chain variable regions shown in FIGS. 7 and 8,

f. comprises the sequences respectively shown as SEQ ID NO: 6. LCDR1, LCDR2 and LCDR3 light chain variable regions shown in FIGS. 7 and 21,

g. comprises the sequences respectively shown as SEQ ID NO: 6. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown at 20 and 21,

h. comprises the sequences respectively shown as SEQ ID NO: 18. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown in FIGS. 7 and 8, and

i. comprises the sequences respectively shown as SEQ ID NO: 18. LCDR1, LCDR2 and LCDR3 light chain variable regions shown at 7 and 22.

In some embodiments, the aforementioned monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO: 10 and the heavy chain variable region as set forth in SEQ ID NO: 14, or a light chain variable region as shown in fig. 14.

In some embodiments, the aforementioned monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 14, or a light chain variable region as shown in fig. 14.

Wherein the heavy chain variable region has the following general formula (SEQ ID NO: 10):

the light chain variable region has the following general sequence formula (SEQ ID NO: 14):

In some embodiments, the foregoing monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of j-r:

j. as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 23, a light chain variable region;

k. as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 24, a light chain variable region;

l, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 25;

m, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 26, a light chain variable region shown in seq id no;

n, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 27, a light chain variable region;

o, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 28;

p, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 29, a light chain variable region;

q, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 30; and

r, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 31, or a light chain variable region as shown in figure 31.

In some embodiments, the aforementioned monoclonal antibody or antigen-binding fragment thereof further comprises an antibody constant region. In some embodiments, wherein the antibody comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or variants thereof, and/or a light chain constant region comprising a human kappa, lambda chain, or variants thereof; preferably, the heavy chain constant region is a heavy chain constant region variant having at least 85% sequence identity to a human IgG1, IgG2, IgG3 or IgG4 sequence, such as a heavy chain constant region variant of IgG1, IgG2 or IgG4 that enhances antibody function (e.g., extends half-life of the antibody in serum, enhances antibody stability, etc.) by amino acid mutation, preferably a heavy chain constant region variant of ige, L234A and/or L235A, or S228P mutation of IgG1, IgG2 or IgG 4; the light chain constant region is a light chain constant region variant having at least 85% sequence identity to a human kappa and lambda chain sequence. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region as set forth in SEQ ID NO. 32 or having at least 85% sequence identity to SEQ ID NO. 32 and/or a light chain constant region as set forth in SEQ ID NO. 33 or having at least 85% sequence identity to SEQ ID NO. 33. Wherein the heavy/light chain constant region having at least 85% sequence identity is preferably an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, more preferably 90%, 95%, or 99% or more sequence identity, and the amino acid sequence having at least 85% sequence identity may be obtained by one or more amino acid deletion, insertion, or substitution mutation.

In some embodiments, the aforementioned monoclonal antibody, or antigen binding fragment thereof, is an antigen binding fragment selected from the group consisting of Fab, Fab ', F (ab')2, single chain antibody (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), and peptides comprising CDRs.

In another aspect, the present disclosure also provides a nucleic acid molecule encoding the aforementioned monoclonal antibody or antigen-binding fragment thereof.

In another aspect, the present disclosure also provides a recombinant vector comprising the aforementioned nucleic acid molecule.

In another aspect, the present disclosure also provides a host cell transformed with a recombinant vector according to the foregoing, said host cell being selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells.

In another aspect, the present disclosure also provides a method for producing the foregoing monoclonal antibody or antigen-binding fragment thereof, the method comprising culturing the foregoing host cell in a medium suitable for growth of the host cell to form and accumulate the foregoing monoclonal antibody or antigen-binding fragment thereof, and recovering the accumulated monoclonal antibody or antigen-binding fragment thereof from the culture.

In another aspect, the present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof according to the foregoing, or a nucleic acid molecule of the foregoing, or a recombinant vector of the foregoing, or a host cell of the foregoing, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the antibody or antigen-binding fragment thereof; in some embodiments, the unit dose of the pharmaceutical composition contains the monoclonal antibody or antigen-binding fragment thereof in an amount of 0.1 to 2000 mg; in still other embodiments, the unit dose of the pharmaceutical composition contains from 1 mg to 1000mg of the monoclonal antibody or antigen-binding fragment thereof.

In another aspect, the present disclosure also provides a method for the in vitro immunoassay or assay of human thrombin, the method comprising the step of detecting or assaying using a reagent comprising the aforementioned monoclonal antibody or antigen-binding fragment thereof.

In another aspect, the present disclosure also provides the use of the aforementioned monoclonal antibody or antigen-binding fragment thereof in the preparation of a diagnostic agent for thrombin-related diseases.

In some aspects, the present disclosure also provides a method of treating or preventing a thrombin-related disease, the method comprising administering to a subject a therapeutically or prophylactically effective amount of the foregoing monoclonal antibody or antigen-binding fragment thereof, or the foregoing nucleic acid molecule, or the foregoing recombinant vector, or the foregoing host cell, or the foregoing pharmaceutical composition, to treat or prevent a thrombin-related disease, wherein the disease includes, but is not limited to, a thrombotic disease; preferably venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; more preferred are venous thrombosis, stroke due to thrombosis and atherosclerosis.

In another aspect, the present disclosure also provides a use of the aforementioned monoclonal antibody or antigen-binding fragment thereof, or the aforementioned nucleic acid molecule, or the aforementioned recombinant vector, or the aforementioned host cell, or the aforementioned pharmaceutical composition for the preparation of a therapeutic agent for a thrombin-related disease, wherein the disease is preferably a thrombotic disease; more preferably venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; venous thrombosis, thrombosis induced stroke and atherosclerosis are most preferred.

In another aspect, the present disclosure also provides a monoclonal antibody or antigen binding fragment thereof of the foregoing, and/or a nucleic acid molecule of the foregoing, and/or a recombinant vector of the foregoing, and/or a host cell of the foregoing, and/or a composition of claim 14, as a medicament; preferably, the medicament is for the treatment of thrombotic disorders including, but not limited to: venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; more preferably, the disease is venous thrombosis, stroke due to thrombosis, and atherosclerosis.

In another aspect, the present disclosure also provides a monoclonal antibody or antigen-binding fragment thereof or a composition thereof useful for treating a disease, including but not limited to a thrombotic disease, wherein the monoclonal antibody or antigen-binding fragment thereof is the monoclonal antibody or antigen-binding fragment thereof of any one of the preceding claims; preferably venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; more preferred are venous thrombosis, stroke due to thrombosis and atherosclerosis.

In some embodiments, the binding of the thrombin monoclonal antibodies or antigen binding fragments thereof of the present disclosure to thrombin does not inhibit or substantially inhibit the normal physiological response to vascular injury (i.e., hemostasis).

In some embodiments, the thrombin monoclonal antibodies or antigen binding fragments thereof of the present disclosure have high specificity and high affinity for thrombin, greatly increasing APTT time.

In some embodiments, the thrombin monoclonal antibodies or antigen binding fragments thereof of the present disclosure have good selective activity for specifically recognizing thrombin.

In some embodiments, the present disclosure provides novel thrombin antibodies with high affinity, significant inhibitory activity against thrombus formation, and higher selectivity activity.

Drawings

FIG. 1: detection of thrombin Activity by Thrombin antibodies

FIG. 2: detection of Activated Partial Thromboplastin Time (APTT) in normal human plasma

Detailed Description

In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The three letter codes and the one letter codes for amino acids used in this disclosure are as described in j. diol. chem,243, p3558 (1968).

"monoclonal antibody" or "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g., antibodies containing naturally occurring mutations or mutations generated during the manufacture of monoclonal antibody preparations, which variants are typically present in minor amounts). Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (preparation) is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the identity of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies of the disclosure can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods, as well as other exemplary methods for preparing monoclonal antibodies, are described herein.

The present disclosure relates to an antibody (Ab) which is an immunoglobulin (immunoglobulin) having a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are connected by interchain disulfide bonds, and has different antigenicity, and thus, the immunoglobulin can be classified into five classes (classes), i.e., IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains thereof are respectively μ chain, δ chain, γ chain, α chain, and epsilon chain, and the same class Ig can be classified into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chains, such as IgG1, IgG2, IgG3, IgG4, and the light chain can be classified into either a κ chain or a λ chain according to the difference in the constant region, and thus, the antibody can be classified into two classes (types).

In the present disclosure, the antibody light chain of the present disclosure may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.

In the present disclosure, the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4, or variants thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region consists of a junction of 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs), which are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (LCVR or VL) and heavy chain variable region (HCVR or VH) consists of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3. In some embodiments, the amino acid residues of the CDRs of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the disclosure conform in number and position to known Kabat numbering conventions (Kabat et al Sequences of Proteins of Immunological Interest, (5 th edition, 1991, National Institutes of Health, Bethesda MD)).

Antibodies of the present disclosure include murine, chimeric, humanized, preferably humanized antibodies.

The term "murine antibody" is a monoclonal antibody of mouse origin prepared according to the knowledge and skill in the art. Prepared by injecting a test subject with an antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties, which antibodies are murine when the injected test subject is a mouse. In one embodiment of the present disclosure, a "murine antibody" is a monoclonal antibody that binds human thrombin prepared according to the knowledge and skill in the art. Preparation is accomplished by injecting a subject with a thrombin antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In one embodiment of the present disclosure, the murine thrombin antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or variant thereof.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing chimeric antibody, firstly establishing hybridoma secreting mouse-derived specific monoclonal antibody, then cloning variable region gene from mouse hybridoma cell, cloning constant region gene of human antibody according to the need, connecting mouse variable region gene and human constant region gene into chimeric gene, inserting into expression vector, and finally expressing chimeric antibody molecule in eukaryotic system or prokaryotic system. In a specific embodiment of the present disclosure, the antibody light chain of the thrombin chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain of the thrombin chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably comprises a heavy chain constant region of human IgG1, IgG2 or IgG4, or an IgG1, IgG2 or IgG4 heavy chain constant region variant using amino acid mutations (such as YTE mutation or back mutation, L234A and/or L235A mutation, or S228P mutation).

The term "humanized antibody", including CDR-grafted antibodies, refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework (i.e., a different type of human germline antibody framework sequence). Can overcome the heterogenous reaction induced by the chimeric antibody carrying a large amount of heterogenous protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA Sequences of, for example, human heavy and light chain variable region genes are available in the "VBase" human germline sequence database (Internet http:// www.vbase2.org), and are found in Kabat, E.A. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity. Humanized antibodies of the present disclosure also include humanized antibodies after further affinity maturation of the CDRs by phage display. In one embodiment of the disclosure, the human antibody variable region framework is designed and selected wherein the heavy chain FR region sequences on the antibody heavy chain variable region are derived from human germline heavy chain sequences, and human germline light chain sequences. In order to avoid the decrease in immunogenicity and the resulting decrease in activity, the variable region of the human antibody may be subjected to minimal reverse mutation (back mutation, i.e., mutation of amino acid residues in the FR region from which the human antibody is derived to amino acid residues at positions corresponding to those in the original antibody from which it was derived) to retain activity.

Grafting of CDRs can result in reduced affinity of the resulting antibody or antigen-binding fragment thereof for an antigen due to changes in framework residues that are contacted with the antigen. Such interactions may be the result of highly mutated somatic cells. Thus, there may still be a need to graft such donor framework amino acids to the framework of humanized antibodies. Amino acid residues from the non-human antibody or antigen-binding fragment thereof that are involved in antigen binding can be identified by examining the sequence and structure of the variable region of a murine monoclonal antibody. Residues in the CDR donor framework that differ from the germline can be considered related. If the closest germline cannot be determined, the sequence can be compared to a subtype consensus sequence or a consensus sequence of murine sequences with a high percentage of similarity. Rare framework residues are thought to be likely the result of somatic hypermutation and thus play an important role in binding.

In one embodiment of the present disclosure, the humanized antibody or antigen binding fragment thereof may further comprise a light chain constant region of a human kappa, lambda chain or variant thereof and/or a heavy chain constant region comprising a human IgG1, IgG2, IgG3, IgG4 or variant thereof. In one embodiment of the present disclosure, a human IgG1, IgG2, or IgG4 heavy chain constant region is included, or an IgG1, IgG2, or IgG4 heavy chain constant region variant using amino acid mutations (such as YTE mutations or back mutations, L234A and/or L235A mutations, or S228P mutations).

The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., thrombin). It has been shown that fragments of full-length antibodies can be used to achieve the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')₂A fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al, (1989) Nature 341: 544-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibody may beAre antibodies of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies. In some embodiments of the disclosure, the antigen binding fragments include Fab, F (ab ')2, Fab', single chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), peptides comprising CDRs, and the like.

Fab is an antibody fragment having a molecular weight of about 50,000Da and having an antigen binding activity among fragments obtained by treating an IgG antibody molecule with protease papain (which cleaves the amino acid residue at position 224 of the H chain), in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

The Fab of the present disclosure may be produced by treating a monoclonal antibody of the present disclosure, which specifically recognizes human thrombin and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, with papain. In addition, the Fab may be produced by inserting DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a eukaryote to express the Fab.

F (ab')2 is an antibody fragment having a molecular weight of about 100,000Da and having antigen binding activity and comprising two Fab regions linked at the hinge position, obtained by digestion of the lower part of the two disulfide bonds in the IgG hinge region with the enzyme pepsin.

The F (ab')2 of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure, which specifically recognizes human thrombin and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, with pepsin. Further, the F (ab ')2 can be produced by linking Fab' described below with a thioether bond or a disulfide bond.

Fab 'is an antibody fragment having a molecular weight of about 50,000Da and having an antigen-binding activity, which is obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F (ab') 2. The Fab 'of the present disclosure can be produced by treating the F (ab')2 of the present disclosure, which specifically recognizes thrombin and is bound to the amino acid sequence of the extracellular region or its three-dimensional structure, with a reducing agent such as dithiothreitol.

In addition, the Fab ' may be produced by inserting DNA encoding the Fab ' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a eukaryote to express the Fab '.

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker. Such scFv molecules can have the general structure: NH (NH)₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g.using 1-4 repeated variants (Holliger et al (1993), Proc. Natl. Acad. Sci. USA90: 6444-. Other linkers useful in the present disclosure are described by Alfthan et al (1995), Protein Eng.8: 725-.

The scFv of the present disclosure can be produced by the following steps: obtaining cDNA encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human thrombin and binds to the amino acid sequence of the extracellular region or the three-dimensional structure thereof, constructing DNA encoding scFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryote or a eukaryote to express scFv.

Diabodies are antibody fragments in which an scFv is dimerized, and are antibody fragments having bivalent antigen binding activity. In the divalent antigen binding activity, the two antigens may be the same or different.

Diabodies of the present disclosure can be produced by the following steps: obtaining cDNAs encoding VH and VL of the monoclonal antibody of the present disclosure, which specifically recognizes human thrombin and binds to the amino acid sequence of the extracellular region or the three-dimensional structure thereof, constructing DNA encoding scFv so that the amino acid sequence of the peptide linker is 8 residues or less in length, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryote or a eukaryote to express the diabody.

The dsFv is obtained by linking a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue via a disulfide bond between cysteine residues. The amino acid residue substituted with a cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to a known method (Protein Engineering,7,697 (1994)).

The dsFv of the present disclosure can be produced by the following steps: obtaining coding cDNA of VH and VL of the monoclonal antibody which specifically recognizes human thrombin and binds to the amino acid sequence of the extracellular region or the three-dimensional structure thereof, constructing DNA coding dsFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic organism or a eukaryotic organism to express dsFv.

A CDR-containing peptide is composed of one or more regions in a CDR that contains VH or VL. Peptides comprising multiple CDRs may be linked directly or via a suitable peptide linker.

The peptides of the present disclosure comprising CDRs may be produced by: the DNA encoding the CDRs of VH and VL of the monoclonal antibody of the present disclosure, which specifically recognizes human thrombin and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, is constructed, inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or a eukaryote to express the peptide. The CDR-containing peptides can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.

The term "antibody framework" as used herein refers to a portion of a variable domain, VL or VH, which serves as a scaffold for the antigen binding loops (CDRs) of that variable domain. It is essentially a variable domain without CDRs.

The term "amino acid mutation" or "amino acid difference" refers to the presence of an amino acid change or mutation in a variant protein or polypeptide as compared to the original protein or polypeptide, including the occurrence of 1, 2, 3 or several amino acid insertions, deletions or substitutions based on the original protein or polypeptide.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a thrombin molecule). Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, vol 66, g.e. morris, Ed. (1996).

The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is administered at a rate of about less than 10^-8M, e.g. less than about 10^-9M、10^-10M、10^-11M or less affinity (KD) binding.

The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the disclosure are administered at less than about 10 "7M, e.g., less than about 10^-8M、10^-9M or 10^-10M or less, binds thrombin, e.g., as determined in a BIACORE instrument using Surface Plasmon Resonance (SPR) techniques.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation equilibrium constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

An "affinity matured antibody" refers to an antibody that has one or more alterations in one or more hypervariable regions (CDRs) of the antibody, resulting in an increase in the affinity of the antibody for an antigen as compared to a parent antibody without the alterations.

The term "compete" when used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies or specifically binding antibodies) means competition between antigen binding proteins as determined by the following assay: the antigen binding protein to be detected (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., a thrombin antigen or fragment thereof). Numerous types of competitive binding assays are available for determining whether an antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, methods in Enzymology 9: 242-; solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J.Immunol.137: 3614-), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct labeling of RIA with a solid phase of I-125 label (see, e.g., Morel et al, 1988, mol. Immunol.25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al, 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol.32: 77-82). Typically, the assay involves the use of a purified antigen (either on a solid surface or on the surface of a cell) that binds to a test antigen binding protein with an unlabeled label and a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cells in the presence of the antigen binding protein to be detected. Alternatively, competitive inhibition can be measured by immobilizing antigen binding protein A and detecting a change in signal from the labeled antigen that is pre-bound to the antigen binding protein. Typically, such competitive inhibition assays will swap the positions of antigen binding protein a and antigen binding protein B for confirmation. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as a reference antigen binding protein; and an antigen binding protein that binds to an epitope sufficiently close to the epitope to which the reference antigen binding protein binds, said two epitopes sterically hindering the binding from occurring. Typically, a portion of a competing antigen binding protein inhibits (e.g., reduces) specific binding of at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%) of a reference antigen binding protein to a common antigen when the competing antigen binding protein is present in excess. In the complete competition, the binding of the reference antigen binding protein to the antigen is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or more. Inhibition of less than 30% is considered to be non-competitive.

The term "nucleic acid molecule" as used herein refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The term "vector" refers to a construct capable of delivering one or more genes or sequences of interest and preferably expressing it in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells. In one embodiment of the present disclosure, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of a host cell upon introduction into the host cell so as to be replicated along with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold spring harbor antibody protocols, chapters 5-8 and 15. For example, mice can be immunized with human thrombin or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibody or antigen binding fragment of the invention is genetically engineered to add one or more human FR regions to the CDR regions of non-human origin. Human FR germline sequences can be obtained from the website http:// www.imgt.org/or from the immunoglobulin journal, 2001ISBN012441351 by aligning the IMGT human antibody variable region germline gene database with the MOE software.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include microbial (e.g., bacterial), plant, or animal cells. Bacteria susceptible to transformation include members of the enterobacteriaceae family (enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (chinese hamster ovary cell line) and NS0 cells.

Engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains may be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expression of antibodies that specifically bind to human thrombin. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified by conventional techniques. For example, purification is carried out using an A or G Sepharose FF column containing a buffer adjusted. Non-specifically bound fractions are washed away. And eluting the bound antibody by using a pH gradient method, detecting the antibody fragment by using SDS-PAGE, and collecting. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

"administration," "administering," and "treating," when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating a cell in vitro and ex vivo by an agent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering a therapeutic agent, e.g., a composition comprising any one of the antibodies or antigen-binding fragments thereof of the present disclosure or a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, either internally or externally to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, to induce regression of such symptoms or to inhibit development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of each target disease, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined according to any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.

"conservative modification" or "conservative substitution or substitution" refers to the replacement of an amino acid in a protein or polypeptide with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that the alteration can be made without altering the biological activity or other desired characteristics (e.g., antigen affinity and/or specificity) of the protein. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., p. 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to abolish biological activity. Exemplary conservative substitutions are shown in the following table:

original residues	Conservative substitutions
Ala(A)	Gly；Ser
Arg(R)	Lys；His
Asn(N)	Gln；His；Asp

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

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. In one embodiment of the present disclosure, an "effective amount" is the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial results that eliminate or reduce the risk, lessen the severity, or delay the onset of a disorder (including, but not limited to, the disorder, its complications, and biochemical, histological, and/or behavioral symptoms of intermediate pathological phenotypes present during the development of the disorder); in another embodiment, an "effective amount" is the amount of a drug, compound, or pharmaceutical composition necessary to achieve a beneficial or desired clinical result (including, but not limited to, such as reducing the incidence of or ameliorating one or more symptoms of various thrombin-related disorders, reducing the dosage of other agents required to treat a disorder, enhancing the therapeutic efficacy of another agent, and/or delaying the progression of a thrombin-related disorder in a patient).

"exogenous" refers to a substance produced outside an organism, cell or human body as the case may be. "endogenous" refers to a substance produced in a cell, organism, or human body as the case may be.

"homology", "identity" are used interchangeably herein to refer to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned; two sequences are 95% homologous if there are 95 matches or homologies at 100 positions in the two sequences. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology. For example, the comparison may be performed by the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between the respective sequences over the entire length of the respective reference sequences. The following references refer to the BLAST algorithm often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS) Altschul, S.F. et al, (1990) J.mol.biol.215: 403-; gish, W. et al, (1993) Nature Genet.3: 266-; madden, T.L. et al, (1996) meth.Enzymol.266: 131-; altschul, S.F. et al, (1997) Nucleic Acids Res.25: 3389-3402; zhang, J. et al, (1997) Genome Res.7: 649-. Other conventional BLAST algorithms, such as provided by NCBI BLAST, are also well known to those skilled in the art.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of a particular portion of nucleic acid, RNA, and/or DNA are amplified as described, for example, in U.S. patent No. 4,683,195. In general, it is desirable to obtain sequence information from the ends of or beyond the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotide of the 2 primers may coincide with the end of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al (1987) Cold Spring Harbor Symp. Ouant. biol.51: 263; erlich editors, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used herein is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific portions of the nucleic acid.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but need not, be present.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

In addition, the present disclosure includes an agent for treating thrombin-related diseases, which comprises the monoclonal antibody or the antibody fragment thereof of the present disclosure as an active ingredient.

There is no limitation on the thrombin-related disease as long as it is a thrombin-related disease, e.g., a therapeutic response induced using the molecules of the present disclosure may inhibit coagulation by binding human thrombin and then inhibiting thrombin activity. Thus, the molecules of the present disclosure are very useful, when in preparations and formulations suitable for therapeutic applications, for those people who suffer from thrombotic disease; more preferably venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; venous thrombosis, stroke due to thrombosis, and atherosclerosis are most preferred.

Further, the present disclosure relates to a method for the immunodetection or assay of thrombin, a reagent for the immunodetection or assay of thrombin, a method for the immunodetection or assay of thrombin-expressing cells, and a diagnostic agent for the diagnosis of thrombin-related diseases, which comprise, as an active ingredient, the monoclonal antibody or antibody fragment of the present disclosure that specifically recognizes human thrombin and binds to the amino acid sequence of an extracellular region or a three-dimensional structure thereof.

In the present disclosure, the method for detecting or determining the amount of thrombin may be any known method. For example, it includes immunodetection or assay methods.

The immunoassay or measuring method is a method for detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a physicochemical method, and the like.

The above thrombin-related diseases can be diagnosed by detecting or measuring thrombin-expressing cells with the monoclonal antibody or antibody fragment of the present disclosure.

For detecting cells expressing the polypeptide, a known immunoassay method can be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, or the like is preferably used. In addition, a fluorescent antibody staining method using FMAT8100HTS system (Applied Biosystem) or the like can be used.

In the present disclosure, the living sample for detecting or measuring thrombin is not particularly limited as long as it has a possibility of containing cells expressing thrombin, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction, depending on the desired diagnostic method. Reagents for performing antigen-antibody reactions include buffers, salts, and the like. The reagent for detection includes reagents generally used in immunodetection or assay methods, such as a labeled secondary antibody recognizing the monoclonal antibody, an antibody fragment thereof or a binding substance thereof, a substrate corresponding to the label, and the like.

Detailed Description

The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure. The experimental methods of the present disclosure, in which specific conditions are not specified, are generally performed according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1 improvement of Thrombin-selective Activity of h1601-008

Further affinity maturation is carried out on h1601-008 (see h1601-008 described in WO2017133673A 1), panning is carried out on thrombin and prothrombin, and on the premise that the binding activity of the antibody on thrombin is not reduced, the selective activity of the antibody on the binding of two molecules is further improved.

>h1601-008 VH：(SEQ ID NO：1)

>h1601-008 VL：(SEQ ID NO：2)

TABLE 1.h1601-008 and its murine antibody mAb-1601 heavy and light chain CDR region sequences

The selection activity was increased by using M13 phage display technology. Mutations were introduced at each CDR using codon-based (codon-based) primers (during primer synthesis, individual codons were composed of wild-type codons and NNK), and a separate phage display library was constructed for each CDR. The proportion of NNK to be incorporated into each CDR and the size of library capacity of a required library are adjusted according to the length of the CDR, and the specific scheme is shown in Table 2.

TABLE 2 library capacity and NNK incorporation ratio design Table

Libraries	CDR Length	NNK ratio	Storage capacity
H1
	5	50％	>2E7
H2	17	20％	>1E8
H3	12	20％	>1E8
L1	11	30％	>1E8
L2	7	50％	>1E8
L3	9	50％	>1E8

The constructed 6 libraries are connected and combined together by a method of overlap extension PCR to form a combined library with the library capacity larger than 1E 9. Packaging the combinatorial library into phage, panning: pre-incubating the liquid phase and high-concentration prothrombin at room temperature, adding biotinylated thrombin for combination, capturing by streptavidin magnetic beads, elutriating, eluting, infecting escherichia coli again, and carrying out elutriation and screening in the next cycle. Each round of panning reduced the biotinylated thrombin concentration by a factor of 2-5. After 3 rounds of panning, a single clone was picked from each library for sequencing validation. HCDR3, LCDR1, LCDR2 and LCDR3 are all found to have obvious amino acid enrichment, partial clones such as H4L9, H4L10, H4L11, H4L12, H4L13, H4L14, H4L15, H4L17, H4L18 and H4L19 are selected and obtained according to the enrichment degree of amino acid residues in a CDR region, and full-length immunoglobulin is constructed to carry out mammalian cell expression.

The selected clones differed from h1601-008 in all of HCDR3, LCDR1, LCDR2, and LCDR 3. The relevant HCDR3, LCDR1, LCDR2 and LCDR3 formulas and their corresponding light and heavy chain variable regions are described below.

The general sequence formula (SEQ ID NO: 9) of the HCDR3 obtained by screening is as follows:

the related heavy chain variable region sequence formula (SEQ ID NO: 10) was obtained as follows:

in SEQ ID NO: x in 9 and 10₁Selected from H, L; x₂Selected from Y, L; wherein X₁Is H or X₂Is not Y.

The general formula of the LCDR sequence obtained by screening is as follows:

the related light chain variable region sequence was obtained as follows (SEQ ID NO: 14):

in SEQ ID NO: 11 to 14 in which X is₃Selected from S, T; x₄Selected from E, H; x₅Selected from D, R; x₆Selected from I, M; x₇Selected from A, T; x₈Selected from S, Q, K; x₉Selected from T, G; x₁₀Selected from S, G; x₁₁Is selected from T, N, Y, wherein LCDR1, LCDR2 and LCDR3 are not simultaneously respectivelyKASEDIYNRLA, GATSLET, and QQYWSTPWT.

Specific related sequences obtained include, but are not limited to, those described in tables 3 and 4:

TABLE 3 heavy chain variable region sequences determined by affinity screen

Heavy chain variable region	X ₁	X ₂	VH sequence code	Comprising the HCDR3 sequence
h1601-008	H	Y	SEQ ID NO：1	DHYHGNSYVFDY(SEQ ID NO：5)
H4	L	L	SEQ ID NO：16	DHYLGNSYVFDL(SEQ ID NO：15)

Antibody heavy chain variable region H4: (SEQ ID NO: 16)

TABLE 4 light chain variable region sequences determined by affinity screen

Antibody light chain variable region L9: (SEQ ID NO: 23)

Antibody light chain variable region L10: (SEQ ID NO: 24)

Antibody light chain variable region L11: (SEQ ID NO: 25)

Antibody light chain variable region L12: (SEQ ID NO: 26)

Antibody light chain variable region L13: (SEQ ID NO: 27)

Antibody light chain variable region L14: (SEQ ID NO: 28)

Antibody light chain variable region L15: (SEQ ID NO: 29)

Antibody light chain variable region L17: (SEQ ID NO: 30)

Antibody light chain variable region L18: (SEQ ID NO: 31)

The light chain variable region of the antibody is selected from the constant region of human heavy chain IgG 4/light chain kappa and is combined with each heavy chain variable region and light chain variable region respectively to form a complete antibody heavy chain and light chain, wherein the heavy chain is subjected to S228P mutation in the Fc segment to increase the stability of the IgG4 antibody, and other mutations known in the art can be selected to increase the performance of the IgG4 antibody.

Antibody heavy chain constant region: (SEQ ID NO: 32)

Light chain constant region: (SEQ ID NO: 33)

Illustratively, intact antibodies formed after affinity maturation and screening include:

TABLE 5 light and heavy chain variable region combinations of intact antibodies

After the cloned protein was purified, the affinity of the antibody for thrombin and prothrombin was determined using biacore.

The following biochemical test methods were used to verify the performance and beneficial effects of the antibodies of the present disclosure.

Test example 1 ELISA assay for binding of Thrombin antibody to human Thrombin protein

The binding of the anti-thrombin antibody was detected by ELISA assay of the antibody with His-tagged human thrombin. His-tagged thrombin labeled with a biotin labeling kit (Dongchan chemical, Cat No. LK03) is bound with streptavidin coated in an ELISA plate so as to be fixed in the 96-well ELISA plate, and the strength of a signal after the addition of an antibody is used for judging the binding activity of the antibody and the thrombin, and a specific experimental method is as follows.

Streptavidin (Sigma, Cat No. S4762-5MG) was diluted to a concentration of 5. mu.g/ml with PBS buffer pH7.4 (Sigma, Cat No. P4417-100TAB), added to a 96-well plate at a volume of 50. mu.l/well, and placed in an incubator at 37 ℃ for 2 hours. After discarding the liquid, 200. mu.l/well of 5% skim milk (light skim milk powder) blocking solution diluted with PBS was added, and the cells were incubated at 37 ℃ for 2.5 hours or left overnight (16-18 hours) at 4 ℃ for blocking. After blocking was complete, the blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH7.4PBS containing 0.05% tween-20), 50. mu.l/well of biotin-labeled His-tagged human thrombin diluted to 0.5. mu.g/ml with a sample diluent (pH7.4PBS containing 1% BSA) was added and incubated in an incubator at 37 ℃ for 2 hours. After the incubation is finished, reaction liquid in the enzyme label plate is discarded, the plate is washed for 6 times by PBST, 50 mu l/hole of thrombin antibody to be detected with different concentrations diluted by sample diluent is added, and the mixture is incubated for 2 hours in an incubator at 37 ℃. After incubation, the plates were washed 5 times with PBST, diluted with 100. mu.l/well of sample diluentA goat anti-human secondary antibody labeled with HRP (Jackson Immuno Research, Cat No. 109-. Washing the plate with PBST for 5 times, adding 50 μ l/well TMB chromogenic substrate (KPL, Cat No.52-00-03), incubating at room temperature for 10-15min, adding 50 μ l/well 1M H₂SO₄The reaction was stopped and the absorbance was read at a wavelength of 450nm using a NOVOStar microplate reader to calculate the EC50 value for the binding of thrombin antibody to human thrombin. The results are shown in Table 6.

TABLE 6 binding Activity of humanized antibodies with Thrombin

Antibodies	Binding ELISA EC50(ng/μl)
h1601-008	0.3747
H4L9	0.0197
H4L10	0.0212
H4L11	0.0182
H4L12	0.0186
H4L13	0.0187
H4L14	0.0268
H4L15	0.0468
H4L17	0.0342
H4L18	0.0319

The result shows that the humanized antibody obtained by screening in the disclosure has higher binding activity with human thrombin protein, and is greatly improved compared with h 1601-008.

Test example 2 ELISA assay for binding of Thrombin antibody to human Prothrombin protein

Prothrombin is a precursor to thrombin through enzymatic activation. The binding force of the antithrombin antibody is detected by ELISA (enzyme-Linked immunosorbent assay) of the antibody and human prothrombin, and the specific experimental method is as follows.

His-tagged human prothrombin (SEQ ID NO:2) was diluted to a concentration of 10. mu.g/ml with PBS (Sigma, Cat No. P4417-100TAB) buffer at pH7.4, added to a 96-well plate at a volume of 100. mu.l/well, and left overnight (16-18 hours) at 4 ℃. After discarding the liquid, 200. mu.l/well of 5% skim milk (light skim milk powder) blocking solution diluted with PBS was added, and the block was incubated at 37 ℃ for 2.5 hours. After blocking was complete the blocking solution was discarded and the plates were washed 5 times with PBST buffer (pH7.4PBS containing 0.05% tween-20), 50. mu.l/well of a test antibody against thrombin of the present disclosure and a control antibody Ichorcuumab (from WO2013088164) diluted in a sample diluent (pH7.4PBS containing 1% BSA) were added and incubated for 1 hour in an incubator at 37 ℃. After the incubation was completed, the plate was washed 5 times with PBST, and 100. mu.l/well of an HRP-labeled secondary goat anti-human antibody (Jackson Immuno Research, Cat No. 109-. Washing the plate with PBST for 5 times, adding 50 μ l/well TMB chromogenic substrate (KPL, Cat No.52-00-03), incubating at room temperature for 10-15min, adding 50 μ l/well 1M H₂SO₄Stopping reaction, reading absorption value at 450nm with NOVOStar microplate reader, calculating human blood coagulation of thrombin antibodyBinding EC50 values of the proenzyme.

TABLE 7 binding Activity of humanized antibodies with Prothrombin

Antibodies

Binding ELISA EC50(ng/μl)

Ichorcumab	0.282
h1601-008	0.1506
H4L9	0.0569
H4L10	0.0625
H4L11	0.0537
H4L12	0.0646
H4L13	0.0608
H4L14	0.0589
H4L15	0.0849
H4L17	0.0780
H4L18	0.0738

Test example 3 BIAcore assay for thrombin antibody affinity

The human anti-capture antibody is covalently coupled on a CM5 biosensing chip (Cat. # BR-1000-12, GE) according to the method described in the specification of a human anti-capture kit (Cat. # BR-1008-39, GE) so as to carry out affinity capture on the antibody to be detected, then human thrombin with a thrombin antigen His label flows on the surface of the chip, a Biacore instrument is used for detecting reaction signals in real time so as to obtain a binding and dissociation curve, and an affinity value is obtained through fitting. After each cycle of dissociation in the experiment was completed, the biochips were washed and regenerated with a regeneration solution prepared in a human anti-capture kit. The antibodies screened by the phage display technique were subjected to affinity testing, and the results are shown in table 8:

TABLE 8 results of antibody biacore assay

As is clear from the above, the novel antibody obtained by screening by the phage display technique has a remarkably improved affinity for thrombin and an improved affinity for prothrombin, but the selective activity of the antibody is greatly improved. The selective activity of the antibody is more than 25, wherein the H4L13 is improved by 2.99 times compared with H1601-008 (33.42/11.18).

Test example 4 Effect of Thrombin antibodies on Thrombin enzyme Activity

In the experiment, the influence of the antibody on the thrombin activity is detected by testing the enzymolysis activity of the thrombin on a substrate S2228.

His-tagged human thrombin was diluted in PBS gradient pH7.4 to a concentration of 100nM, 25. mu.l/well and added to a black-walled clear-bottom 96-well plate. Thrombin test antibodies of the disclosure were diluted in PBS to a concentration of 2000nM to 62.5nM (1:2 dilution gradient) 25. mu.l/well and also added to the plate. After incubation at room temperature for 30-60 minutes, S2228 (substrate for detecting thrombin activity, synthesized by Gill Biochemical (Shanghai) Co., Ltd.) was diluted with PBS to a concentration of 4mM, 50. mu.l/well, and added to the plate of the previous step. Negative controls were control wells with thrombin only, or S2228 only. After incubation at room temperature for 30 minutes, the absorbance was read at a wavelength of 405nm using a NOVOStar microplate reader. The results are shown in FIG. 1, in which only thrombin and only S2228 represent the OD values measured in the negative control wells, and 0.625:1, 1.25:1, 2.5:1, 5:1, 10:1 and 20:1 represent the OD values measured at different molar ratios of the test antibody to thrombin.

The results in FIG. 1 show that the antibody obtained from h1601-008 and phage display, after binding to thrombin, did not affect its enzymatic activity on substrate S2228.

Pharmacodynamic test of thrombin antibody

Test example 5 APTT test on Normal human plasma

The effect of the antibodies of the present disclosure on the APTT value (activated partial thrombin time) was tested in this experiment by incubating normal human plasma with IgG and thrombin antibodies at different concentrations.

IgG is a negative control, i.e., human immunoglobulin obtained by purification using conventional affinity chromatography methods such as ProteinA; ichorcumab (from WO2013088164) was a positive control, and h1601-008 and different concentrations of the antibodies to be tested according to the disclosure were detected.

As shown in FIG. 2, the APTT value of normal human plasma was also prolonged with the increase in the concentration of the antibody h1601-008 and the antibody obtained by phage display. The APTT value of h1601-008 at the highest concentration of 3200nM reaches a peak at 34 seconds, while the APTT value of the antibody obtained by phage display is significantly increased compared to that of h1601-008 at high concentration (Table 9).

TABLE 9 increase in APTT values (in seconds) against normal human plasma for different concentrations of thrombin antibody

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

A monoclonal antibody or antigen-binding fragment thereof that binds human thrombin, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 3.4 and 9, and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 11. 12 and 13, LCDR1, LCDR2 and LCDR3, wherein said LCDR is represented by SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13 are amino acid sequences respectively shown in the following general formula:

HCDR3 DHYX₁GNSYVFDX₂ SEQ ID NO：9；

LCDR1 KAX₃X₄X₅X₆YNRLA SEQ ID NO：11；

LCDR2 GX₇TX₈LEX₉ SEQ ID NO：12；

LCDR3 QQYWX₁₀X₁₁PWT SEQ ID NO：13；

wherein: x₁Selected from H, L; x₂Selected from Y, L; x₃Selected from S, T; x₄Selected from E, H; x₅Selected from D, R; x₆Selected from I, M; x₇Selected from A, T; x₈Selected from S, Q, K; x₉Selected from T, G; x₁₀Selected from S, G; x₁₁Selected from T, N, Y; and when X₁When it is H, X₂Is not Y, and X₃-X₁₁Not simultaneously selected from the following amino acids: x₃Is S, X₄Is E, X₅Is D, X₆Is I, X₇Is A, X₈Is S, X₉Is T, X₁₀Is S and X₁₁Is T.
The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3.4 and 15, HCDR1, HCDR2 and HCDR 3.
The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the light chain variable region LCDR 1sequence is selected from the group consisting of SEQ ID NOs: 6. 17 and 18, the LCDR2 sequence is selected from the group consisting of SEQ ID NOs: 7. 19 and 20, LCDR3 sequence selected from the group consisting of SEQ ID NO: 8. 21 and 22; wherein LCDR1, LCDR2 and LCDR3 are not simultaneously selected from the following sequences: LCDR 1is selected from SEQ ID NO: 6. LCDR2 is selected from SEQ ID NO: 7 and LCDR3 are selected from SEQ ID NO: 8.
the monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 3.4 and 15, and HCDR1, HCDR2, and HCDR3, wherein the light chain variable region is selected from any one of the following a-i:

a. comprises the sequences respectively shown as SEQ ID NO: 17. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown in fig. 19 and 8;

b. comprises the sequences respectively shown as SEQ ID NO: 6. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown in fig. 19 and 8;

c. comprises the sequences respectively shown as SEQ ID NO: 17. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown at 20 and 21;

d. comprises the sequences respectively shown as SEQ ID NO: 6. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown at 20 and 8;

e. comprises the sequences respectively shown as SEQ ID NO: 17. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown in fig. 7 and 8;

f. comprises the sequences respectively shown as SEQ ID NO: 6. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown in fig. 7 and 21;

g. comprises the sequences respectively shown as SEQ ID NO: 6. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown at 20 and 21;

h. comprises the sequences respectively shown as SEQ ID NO: 18. the light chain variable regions of LCDR1, LCDR2 and LCDR3 shown in fig. 7 and 8; and

i. comprises the sequences respectively shown as SEQ ID NO: 18. LCDR1, LCDR2 and LCDR3 light chain variable regions shown at 7 and 22.
The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: as shown in SEQ ID NO: 10 and the heavy chain variable region as set forth in SEQ ID NO: 14, or a light chain variable region as shown in fig. 14.
The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding fragment comprises: as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 14, or a light chain variable region as shown in fig. 14.
The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the monoclonal antibody or antigen-binding fragment thereof comprises any of the heavy chain variable region and the light chain variable region selected from the group consisting of j-r as follows:

j. as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 23, a light chain variable region;

k. as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 24, a light chain variable region;

l, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 25;

m, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 26, a light chain variable region shown in seq id no;

n, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 27, a light chain variable region;

o, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 28;

p, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 29, a light chain variable region;

q, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 30; and

r, as shown in SEQ ID NO: 16 and the heavy chain variable region as set forth in SEQ ID NO: 31, or a light chain variable region as shown in figure 31.
The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 7, comprising an antibody constant region, wherein the antibody heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or variants thereof, and/or the antibody light chain comprises a light chain constant region of human kappa, lambda chains, or variants thereof;

preferably, the heavy chain constant region is a heavy chain constant region variant having at least 85% sequence identity to a human IgG1, IgG2, IgG3 or IgG4 sequence, preferably a YTE mutant, L234A and/or L235A mutant, or S228P mutant of IgG1, IgG2 or IgG4, and/or

The light chain constant region is a light chain constant region variant having at least 85% sequence identity to a human kappa and lambda chain sequence;

more preferably, the antibody comprises a heavy chain constant region as set forth in SEQ ID NO. 32 or having at least 85% sequence identity to SEQ ID NO. 32, and a light chain constant region as set forth in SEQ ID NO. 33 or having at least 85% sequence identity to SEQ ID NO. 33.
The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antigen-binding fragment is an antigen-binding fragment selected from the group consisting of Fab, Fab ', F (ab')2, scFv, dimerized V regions, disulfide stabilized V regions, and peptides comprising CDRs.
A nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 9.
A recombinant vector comprising the nucleic acid molecule of claim 10.
A host cell selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells, transformed with the recombinant vector of claim 11.
A method for producing the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the method comprising culturing the host cell of claim 12 in a medium suitable for growth of the host cell to form and accumulate the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 9, and recovering the accumulated monoclonal antibody or antigen-binding fragment thereof from the culture.
A pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody and/or antigen-binding fragment thereof according to any one of claims 1 to 9, or a nucleic acid molecule according to claim 10, or a recombinant vector according to claim 11, or a host cell according to claim 12, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
A method for the immunoassay or determination of human thrombin, the method comprising the step of detecting or determining using a reagent comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 9.
Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for the preparation of a diagnostic agent for thrombin-related diseases.
A method of treating or preventing a thrombin-associated disease, the method comprising administering to a subject a therapeutically or prophylactically effective amount of the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-9, or the nucleic acid molecule of claim 10, or the recombinant vector of claim 11, or the host cell of claim 12, or the pharmaceutical composition of claim 14; wherein the disease is preferably a thrombotic disease; more preferably venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; venous thrombosis, thrombosis induced stroke and atherosclerosis are most preferred.
Use of a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or a nucleic acid molecule according to claim 10, or a recombinant vector according to claim 11, or a host cell according to claim 12, or a pharmaceutical composition according to claim 14, for the manufacture of a medicament for the treatment of a thrombin-related disease, preferably a thrombotic disease; more preferably venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; venous thrombosis, thrombosis induced stroke and atherosclerosis are most preferred.
A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and/or a nucleic acid molecule according to claim 10, and/or a recombinant vector according to claim 11, and/or a host cell according to claim 12, and/or a composition according to claim 14, as a medicament; preferably, the medicament is for the treatment of thrombotic disorders including, but not limited to: venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; more preferably, the disease is venous thrombosis, stroke due to thrombosis, and atherosclerosis.
A monoclonal antibody or antigen-binding fragment thereof or a composition thereof useful for treating a disease, including but not limited to a thrombotic disease, wherein the monoclonal antibody or antigen-binding fragment thereof is the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 9; preferably venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; more preferred are venous thrombosis, stroke due to thrombosis and atherosclerosis.