WO2019096294A1 - Thrombin antibody, antigen-binding fragment and pharmaceutical use thereof - Google Patents

Abstract

The present disclosure relates to a thrombin antibody, an antigen-binding fragment and the pharmaceutical use thereof. Furthermore, the present disclosure relates to a murine antibody, a chimeric antibody and a humanized antibody comprising the CDR region of the thrombin antibody, and a pharmaceutical composition comprising the thrombin antibody and/or the antigen-binding fragment thereof, as well as the use thereof as a drug. In particular, the present disclosure relates to thr use of the humanized thrombin antibody in preparing a drug for treating thrombin-related diseases or conditions.

Description

Thrombin antibody, antigen-binding fragment thereof, and medical use Technical field

The present disclosure relates to thrombin antibodies, antigen-binding fragments thereof, and further, the present disclosure also relates to chimeric antibodies, humanized antibodies comprising the CDR regions of the thrombin antibodies, and to the inclusion of the thrombin antibodies and antigens thereof. A pharmaceutical composition that binds to a fragment, and its use as a diagnostic agent and therapeutic agent for thrombin-related diseases.

Background technique

The statements herein are merely illustrative of the invention and are not intended to be

Blood coagulation is an important process to prevent bleeding from bleeding (hemostasis). However, blood clots that block blood flow through blood vessels (thrombus formation) or blood vessels (thromboembolism) deposited elsewhere in the body are a serious threat to health. Thrombosis (such as acute myocardial infarction (AMI), venous thrombosis, etc.) is a type of cardiovascular disease that seriously endangers human health and life. According to statistics from the World Health Organization in 2008, cardiovascular morbidity and mortality have now jumped to the top. The annual number of deaths from cardiovascular diseases in the world is about 17.33 million, accounting for 30% of the total number of deaths. The number of cardiovascular patients in China is 290 million, and the number of deaths per year is about 3.5 million, accounting for 41% of the total cause of death. According to the 2010 Global Disease Burden Study (GBD), stroke is the biggest cause of death for Chinese residents. Therefore, in recent years, research on effective drugs and methods for treating cardiovascular diseases has attracted more and more attention. Currently, some anticoagulant therapies can treat pathological blood coagulation, such as the use of traditional drug heparin, small molecule heparin or warfarin, or direct use of thrombin (Dhromiga) inhibitor dabigatran. A common drawback of these therapies is the increased risk of bleeding. The window between the effective dose of anticoagulant (to prevent thrombosis) and the safe dose (highest risk of no bleeding) is not large enough, and the window will be further reduced, taking into account differences in individual patient responses. Targeting thrombin and using thrombin antagonists to inhibit thrombus formation is one of the methods for clinical treatment of thrombosis.

The coagulation reaction is a complex signaling cascade in which thrombin is at the heart of it. Thrombin breaks down the fibrinogen of the circulatory system into fibrin monomers (polymerizable to form fibrin, the fibrous matrix of blood clots) and has many direct regulation of cells. As a serine protease, it triggers platelet deformation, releasing the platelet activators ADP, serotonin and thromboxane A2, as well as chemokines and growth factors. In addition, the adhesion molecules P-selectin and CD40 ligand are promoted to migrate to the surface of platelets, thereby activating integrin aIIb/b3. The latter binds to fibrinogen and von Willebrand factor (vWF), which in turn mediates platelet aggregation. Thrombin also stimulates procoagulant activity on the surface of platelets, which in turn promotes the expression of thrombin. In the culture of endothelial cells, thrombin promotes the release of vWF, the appearance of P-selectin in the plasma membrane and the production of chemokines. These reactions are thought to trigger the binding of platelets and leukocytes to the surface of endothelial cells in vivo. Endothelial cells then change shape and endothelial cell layer permeability increases. These reactions are expected to promote local exudation of plasma proteins and promote edema. In non-endothelial tissues, thrombin causes vasoconstriction by acting on smooth muscle cells. In in vitro culture of fibroblasts or vascular smooth muscle cells, thrombin regulates cytokine production and promotes mitosis, which triggers calcium signals and other responses in T lymphocytes. These cellular responses suggest that thrombin associates tissue damage with the hemostasis process, inflammatory response, and even the regulation of the body that enhances the immune response. These cellular responses also raise the possibility that in addition to tissue damage, thrombin from endothelial cells and other cell types may also play a role in leukocyte extravasation, vascular remodeling and/or angiogenesis. Therefore, thrombin has become a highly promising new anticoagulant and antithrombotic target.

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

Although there are currently a few patents for anti-thrombin antibodies, such as WO2013123591, WO2014153195, WO2014202992, WO2014202993, CN107043423A and WO2017133673. So far, no antithrombin antibody drugs have been marketed, and it is necessary to further develop and screen for more active antithrombin antibodies for clinical research and application.

Summary of the 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 disclosure provides a monoclonal antibody or antigen-binding fragment thereof, which binds to human thrombin, the monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light a chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 3, 4 and 9, respectively, said light chain variable region comprising SEQ ID NO: 11, respectively LCDR1, LCDR2 and LCDR3 shown in Figures 12 and 13, wherein SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 are amino acid sequences respectively having the following formula :

Wherein: X _{1 is} selected from H, L; X _{2 is} selected from Y, L; X _{3 is} selected from S, T; X _{4 is} selected from E, H; X _{5 is} selected from D, R; and X _{6 is} selected from I, M; X _{7 is} selected from A, T; X _{8 is} selected from S, Q, K; X _{9 is} selected from T, G; X _{10 is} selected from S, G; X _{11 is} selected from T, N, Y; and when X ₁ is H When X _{2 is} not Y, and X ₃ -X _{11 is} not selected from the following amino acids: X ₃ is S, X ₄ is E, X ₅ is D, X ₆ is I, X ₇ is A, and X ₈ is S. X ₉ is T, X ₁₀ is S, and X ₁₁ is T.

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

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

In some embodiments, in the monoclonal antibody or antigen-binding fragment thereof, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 3, 4, and 15, respectively. The chain variable region is a light chain variable region selected from any of the following ai:

a. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 17, 19 and 8, respectively,

b. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 6, 19 and 8, respectively,

c. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 17, 20 and 21, respectively,

d. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 6, 20 and 8, respectively,

e, a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 17, 7 and 8, respectively,

f. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 6, 7, and 21, respectively,

g, a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 6, 20 and 21, respectively,

h, a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 18, 7 and 8, respectively, and

i. A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 18, 7 and 22, respectively.

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

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

Wherein the heavy chain variable region sequence formula (SEQ ID NO: 10) is as follows:

The light chain variable region sequence formula (SEQ ID NO: 14) is as follows:

Wherein: X _{1 is} selected from H, L; X _{2 is} selected from Y, L; X _{3 is} selected from S, T; X _{4 is} selected from E, H; X _{5 is} selected from D, R; and X _{6 is} selected from I, M; X _{7 is} selected from A, T; X _{8 is} selected from S, Q, K; X _{9 is} selected from T, G; X _{10 is} selected from S, G; X _{11 is} selected from T, N, Y; and when X ₁ is H When X _{2 is} not Y, and X ₃ -X _{11 is} not selected from the following amino acids: X ₃ is S, X ₄ is E, X ₅ is D, X ₆ is I, X ₇ is A, and X ₈ is S. X ₉ is T, X ₁₀ is S, and X ₁₁ is T.

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

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

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

l a heavy chain variable region as set forth in SEQ ID NO: 16 and a light chain variable region as set forth in SEQ ID NO: 25;

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

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

o a heavy chain variable region as set forth in SEQ ID NO: 16 and a light chain variable region as set forth in SEQ ID NO: 28;

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

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

r, a heavy chain variable region as set forth in SEQ ID NO: 16 and a light chain variable region as set forth in SEQ ID NO: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 IgGl, IgG2, IgG3 or IgG4 or a variant thereof, and/or a light chain constant region comprising a human kappa, lambda chain or variant thereof; preferably The heavy chain constant region is a heavy chain constant region variant having at least 85% sequence identity to a human IgGl, IgG2, IgG3 or IgG4 sequence, for example, by amino acid mutations to enhance antibody function (eg, prolonging the half-life of the antibody in serum) a heavy chain constant region variant of IgG1, IgG2 or IgG4, preferably an antibody stability, etc., preferably a heavy chain constant region variant of IgG1, IgG2 or IgG4 of a YTE mutation, L234A and/or L235A mutation, or a S228P mutation; The light chain constant region is a light chain constant region variant having at least 85% sequence identity to a human κ, λ 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 at least 85% sequence identical to SEQ ID NO: 32, and/or as A light chain constant region of sequence SEQ ID NO: 33 or having at least 85% sequence identity to SEQ ID NO:33. Wherein the above heavy/light chain constant region having at least 85% sequence identity, preferably having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% sequence identity, more preferably an amino acid sequence having 90%, 95% or more sequence identity, the amino acid sequence having at least 85% sequence identity may It is obtained by one or more amino acid deletions, insertions or substitution mutations.

In some embodiments, in the aforementioned monoclonal antibody or antigen-binding fragment thereof, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (Dual antibody), a disulfide-stabilized V region (dsFv) and an antigen-binding fragment of a CDR-containing peptide.

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, the host cell being selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.

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

In another aspect, the present disclosure 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 as described above, or a recombinant vector as described above, or a host cell as described above, And one or more pharmaceutically acceptable carriers, diluents or excipients. In some embodiments, the pharmaceutical composition may contain from 0.01 to 99% by weight of the antibody or antigen-binding fragment thereof in a unit dose; in some embodiments, the pharmaceutical composition comprises a monoclonal antibody or antigen-binding fragment thereof in a unit dose. The amount is from 0.1 to 2000 mg; in other embodiments, the amount of the monoclonal antibody or antigen-binding fragment thereof in the unit dose of the pharmaceutical composition is from 1 to 1000 mg.

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

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

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 aforementioned monoclonal antibody or antigen-binding fragment thereof, or the foregoing A nucleic acid molecule, or a recombinant vector as described above, or a host cell as described above, or a pharmaceutical composition as described above, for treating or preventing 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 arterial disease or peripheral arterial disease; venous thrombosis, stroke and atherosclerosis caused by thrombosis are more preferred.

In another aspect, the present disclosure provides a 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, in preparation and coagulation Use of a therapeutic agent for an enzyme-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 arterial disease or peripheral arterial disease; venous thrombosis, stroke and atherosclerosis caused by thrombosis are most preferred.

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

In another aspect, the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof, or a combination thereof, which is useful for treating a disease, wherein the monoclonal antibody or antigen-binding fragment thereof is the monoclonal of any of the foregoing An antibody or antigen-binding fragment thereof, including but 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 arterial disease Or peripheral arterial disease; more preferably venous thrombosis, stroke and atherosclerosis caused by thrombosis.

In some embodiments, the binding of a thrombin monoclonal antibody or antigen-binding fragment thereof of the disclosure to thrombin does not inhibit or substantially inhibit a normal physiological response to vascular injury (ie, 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 disclosure have a well-selected activity that specifically recognizes thrombin.

In some embodiments, the present disclosure provides a novel thrombin antibody having high affinity, significant inhibitory activity against thrombus formation, and higher selective activity.

DRAWINGS

Figure 1: Detection of thrombin activity by thrombin antibody

Figure 2: Detection of normal human plasma activated partial thromboplastin time (APTT)

Detailed description of the invention

In order to more easily understand the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise defined explicitly herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The three-letter code and the one-letter code for amino acids used in the present disclosure are as described in J.biol.chem, 243, p3558 (1968).

"Monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, a variant variant antibody (eg, containing a naturally occurring mutation or a mutation produced during the manufacture of a monoclonal antibody preparation). In addition to the fact that these variants are typically present in minor amounts, the individual antibodies comprising the population are identical and/or bind to the same epitope. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (formulation) is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the identity of an antibody as obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies of the present disclosure can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods of using 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 "antibody (Ab)" as used in the present disclosure refers to an immunoglobulin (Ig), which is a tetrapeptide which is formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Chain structure. The immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, namely, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are a μ chain, a δ chain, a γ chain, an α chain, and an ε chain, respectively. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into a kappa chain or a lambda chain by the difference in the constant region, whereby the antibody can be classified into two types. Each class Ig of the five classes of Ig may have a kappa chain or a lambda chain. According to the difference of individual amino acids in the constant region of the λ chain, it can be further divided into four subtypes of λ1, λ2, λ3 and λ4.

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 IgGl, IgG2, IgG3, IgG4 or variants thereof.

The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions. The variable region comprises three hypervariable regions (HVRs) and four frameworks with relatively conserved framework regions (FR, also called framework regions, framework regions). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (LCVR or VL) and heavy chain variable region (HCVR or VH) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1 , FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. In some embodiments, the CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present disclosure conform to known Kabat numbering rules in number and position (Kabat et al. Sequences of Proteins of Immunological Interest, 5th edition, 1991, National Institutes of Health, Bethesda MD)).

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

The term "murine antibody" is a mouse-derived monoclonal antibody prepared according to the knowledge and skill in the art. The test subject is injected with an antigen, and then the hybridoma expressing the antibody having the desired sequence or functional property is isolated, and when the test subject is a mouse, the antibody produced is a mouse antibody. In one embodiment of the present disclosure, a "murine antibody" is a monoclonal antibody that binds to human thrombin prepared according to the knowledge and skill in the art. The test subject is injected with a thrombin antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional property is isolated. 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, a lambda chain or a variant thereof, or further comprising a murine IgGl, IgG2, The heavy chain constant region of IgG3 or a variant thereof.

The term "chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody. To establish a chimeric antibody, a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the murine variable region gene is The human constant region gene is ligated into a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic 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 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 comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or an amino acid An IgGl, IgG2 or IgG4 heavy chain constant region variant of a mutation (such as a YTE mutation or a back mutation, a L234A and/or L235A mutation, or a S228P mutation).

The term "humanized antibody", including CDR-grafted antibodies, refers to the transplantation of murine CDR sequences into human antibody variable region frameworks (ie, different types of human germline antibody framework sequences). The antibody produced in ). It is possible to overcome the heterologous reaction induced by the chimeric antibody by carrying a large amount of heterologous protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. The germline DNA sequences of the human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database (Internet http://www.vbase2.org), as well as in Kabat, EA et al., 1991 Sequences. Of Proteins of Immunological Interest, found in the 5th edition. To avoid a decrease in immunogenicity, the resulting human antibody variable region framework sequences can be subjected to minimal reverse or back mutations to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured by phage display. In one embodiment of the present disclosure, a human antibody variable region framework is selected by design, wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from a human germline heavy chain sequence, and a human germline light chain sequence. In order to avoid a decrease in the activity caused by a decrease in immunogenicity, the human antibody variable region can be subjected to minimal reverse mutation (reversion mutation, that is, the amino acid residue of the FR region derived from the human antibody is mutated to the original antibody. Corresponding position amino acid residues) to maintain activity.

The grafting of the CDRs may result in a decrease in the affinity of the antibody or antigen-binding fragment thereof to the antigen due to changes in the framework residues that are contacted with the antigen. Such interactions may be the result of high mutations in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids to the framework of humanized antibodies. Amino acid residues involved in antigen binding from a non-human antibody or antigen-binding fragment thereof can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be related. If the closest germline cannot be determined, the sequence can be compared to a subtype consensus sequence or a consensus sequence of a murine sequence with a high percent similarity. Rare framework residues are thought to be the result of high somatic mutations 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, a lambda chain or a variant thereof, and/or comprise a human IgGl, IgG2, Heavy chain constant region of IgG3, IgG4 or variants thereof. In one embodiment of the present disclosure, a human heavy chain constant region comprising IgGl, IgG2 or IgG4, or an IgGl, IgG2 or IgG4 comprising an amino acid mutation (such as a YTE mutation or a back mutation, a L234A and/or L235A mutation, or a S228P mutation) Heavy chain constant region variant.

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 (eg, thrombin). It has been shown that fragments of full length antibodies can be utilized to achieve antigen binding function of antibodies. Examples of the binding fragment contained in the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab') ₂ fragment, including a divalent fragment of two Fab fragments joined by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) an Fv fragment consisting of a single arm VH and VL domain of the antibody (v) a single domain or dAb fragment (Ward et al, (1989) Nature 341: 544-546), which consists of a VH domain, and also includes maxibody, minibody, intrabody (intrabody) , triple antibody, tetra antibody, v-NAR (v-new antigen receptor) and double scFv (see, eg, Hollinger and Hudson, 2005, Nature Biotechnology, 23.9: 1126-1136); and (vi) isolated complementarity determining regions ( CDR) or (vii) a combination of two or more isolated CDRs, optionally joined by a synthetic linker. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be used Recombination methods, which are connected by a synthetic linker, such that it can be produced as a unit in which the VL and VH regions are paired to form a unit price A single protein chain (referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879- 5883). Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art and in the same manner as for intact antibodies. Fragments of functional screening. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies. In some embodiments of the present disclosure, the antigen binding fragment comprises Fab, F(ab')2, Fab', single chain antibody (scFv), A polymerized V region (diabody), a disulfide-stabilized V region (dsFv), a CDR-containing peptide, and the like.

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

The Fab 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 a three-dimensional structure thereof with papain. Furthermore, the Fab can be produced by inserting a DNA encoding a Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express a Fab.

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

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 a three-dimensional structure thereof with pepsin. Further, the F(ab') 2 can be produced by linking the Fab' described below with a thioether bond or a disulfide bond.

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

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

The term "single-chain antibody", "single-chain Fv" or "scFv" is intended to encompass an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker. Molecule. Such scFv molecules can have the general structure: NH ₂ -VL- linker -VH-COOH or NH ₂ -VH- linker -VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variant thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) . Other linkers useful in the present disclosure are 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.

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

A diabody is an antibody fragment in which an scFv is dimerized, and is an antibody fragment having a bivalent antigen-binding activity. In the bivalent antigen binding activity, the two antigens may be the same or different.

The diabody of the present disclosure can be produced by obtaining the cDNA encoding the 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, and constructs the coding scFv. The DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector such that the amino acid sequence of the peptide linker is 8 residues or less, and then the expression vector is introduced into a prokaryote or eukaryotic expression vector. The organism is expressed as a diabody.

dsFv is obtained by linking a polypeptide in which one of amino acid residues 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 a three-dimensional structure prediction of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).

The dsFv of the present disclosure can be produced by obtaining the cDNA encoding the 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, and constructs a cDNA encoding dsFv. DNA, the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express dsFv.

A peptide comprising a CDR is constructed by one or more regions of a CDR comprising a VH or VL. Peptides comprising a plurality of CDRs can be joined directly or via a suitable peptide linker.

The CDR-containing peptide of the present disclosure can be produced by constructing a DNA encoding the CDRs of the 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. The DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express the peptide. The CDR-containing peptide 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 the variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain that does not have a CDR.

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 compared to the original protein or polypeptide, including one, two, three or on the basis of the original protein or polypeptide. Insertion, deletion or substitution of several amino acids.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a thrombin molecule). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

The terms "specifically binds", "selectively binds", "selectively binds" and "specifically binds" refers to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 ^-8 M, such as less than about 10 ^-9 M, 10 ^-10 M, 10 ^-11 M or less.

The term "KD" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, the antibodies of the present disclosure bind to thrombin with a dissociation equilibrium constant (KD) of less than about 10-7 M, such as less than about 10 ^-8 M, 10 ^-9 M, or ^10-10 M or less, for example, if a surface is used Plasma resonance (SPR) techniques were measured in a BIACORE instrument.

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

"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 affinity of the antibody for the antigen compared to the parent antibody without these alterations. improve.

When the term "competition" is used in the context of an antigen binding protein that competes for the same epitope (eg, neutralizing an antigen binding protein or a neutralizing antibody or an antibody that specifically binds), it means that competition between antigen binding proteins is passed through The following assays determine that an antigen binding protein (eg, an antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (eg, reduces) a reference antigen binding protein (eg, a ligand or reference antibody) from a common antigen (eg, a thrombin antigen or Specific binding of its fragment). Numerous types of competitive binding assays can be used to determine whether an antigen binding protein competes with another assay such as solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), Sandwich competition assay (see, eg, Stahli et al, 1983, Methods in Enzymology 9: 242-253); solid phase direct biotin-avidin EIA (see, eg, Kirkland et al, 1986, J. Immunol. 137: 3614-3619), solid Direct labeling assay, solid phase direct label sandwich assay (see, eg, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling with I-125 label RIA (see, eg, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, eg, 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 (which is on a solid surface or cell surface) that binds to a reference antigen binding protein with an unlabeled detection antigen binding protein and label. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein to be tested. Alternatively, competitive inhibition can be determined by immobilizing antigen-binding protein A, detecting a change in the labeled antigen signal that is pre-bound to the antigen-binding protein. Typically, this competitive inhibition assay confirms the location of antigen binding protein A and antigen binding protein B. An antigen binding protein identified by a competitive assay (competing antigen binding protein) includes: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and a table adjacent to an epitope that is sufficiently close to the binding of the reference antigen binding protein A binding antigen binding protein occurs where the two epitopes interfere with each other spatially. Typically, when a competing antigen binding protein is present in excess, the partially competitive antigen binding protein inhibits (eg, reduces) at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%). Reference antigen binding protein specifically binds to a common antigen. In the case of complete competition, 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. A suppression of less than 30% is considered to be non-competitive.

The term "nucleic acid molecule" as used herein refers to a DNA molecule and an RNA molecule. The nucleic acid molecule may be single stranded or double stranded, but is preferably a 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 the coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

The term "vector" refers to a construct that is 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, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA encapsulated in liposomes or RNA expression vectors 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 in which additional DNA segments can 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 (for example, a bacterial vector having an origin of replication of bacteria and an episomal mammalian vector) or can be integrated into the genome of the host cell after introduction into the host cell, thereby The host genome is replicated together (eg, a non-episomal mammalian vector).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as Cold Spring Harbor Antibody Technical Guide, Chapters 5-8 and 15. For example, a mouse can be immunized with human thrombin or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method. The antigen-binding fragment can also be prepared by a conventional method. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region. Human FR germline sequences can be obtained by aligning the IMGT human antibody variable region germline gene database and MOE software, available from the website http://www.imgt.org/, or from the Immunoglobulin Journal, 2001 ISBN 014441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include microorganisms (eg, bacteria), plant or animal cells. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

The antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more preferred 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 expressing antibodies that specifically bind to human thrombin. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium from which the antibody is secreted can be purified by a conventional technique. For example, purification is carried out using an A or G Sepharose FF column containing an adjusted buffer. The non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange. The resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.

"Administration," "administration," and "treatment" when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to an exogenous drug, therapeutic, diagnostic, or combination with an animal, Contact with a human, subject, cell, tissue, organ or biological fluid. "Administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells. "Administering," "administering," and "treating" also means treating the cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell. "Treatment", when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treatment" means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any 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, the patient having one or A variety of disease symptoms are known, and the therapeutic agents are known to have a therapeutic effect on these symptoms. Generally, a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any clinically measured extent. The amount of therapeutic agent (also referred to as "therapeutically effective amount") effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. Although embodiments of the present disclosure (eg, therapeutic methods or preparations) may be ineffective in ameliorating the symptoms of each target disease, any statistical test methods known in the art such as Student's t test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.

"Conservatively modified" or "conservative substitution or substitution" means that an amino acid in a protein or polypeptide is substituted with another amino acid having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), Such changes can often be made without altering the biological activity of the protein or other desired properties (eg, antigen affinity and/or specificity). It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. An exemplary conservative substitution is shown in the following table:

Primitive residue	Conservative substitution
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" includes 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 the diagnosis. An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. In one embodiment of the present disclosure, an "effective amount" is to obtain an elimination or reduction of risk, a reduction in severity, or a delay in the onset of a condition including, but not limited to, a condition, a complication thereof, and an intermediate pathology presented during the development of the condition. The amount of the drug, compound or pharmaceutical composition necessary for any one or more of the beneficial effects of the type of biochemical, histological and/or behavioral symptoms; in another embodiment, the "effective amount" is beneficial Or desired clinical outcome (including but not limited to, such as reducing the incidence of various thrombin-related disorders or ameliorating one or more symptoms of the disorder, reducing the dosage of other agents required to treat the disorder, enhancing the other The amount of the drug, compound or pharmaceutical composition necessary for the efficacy of the agent, and/or delaying the progression of the thrombin-related disorder in the patient.

"Exogenous" refers to a substance that is produced outside of a living being, cell or human, depending on the situation. "Endogenous" refers to a substance produced in a cell, organism or human body, depending on the circumstances.

"Homology" and "identity" are used interchangeably herein to refer to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position . The percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared × 100. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches in 100 positions in the two sequences Or homologous, then the two sequences are 95% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences. For example, the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are selected to give the largest match between the individual sequences over the entire length of each reference sequence. The following references relate to the BLAST algorithm often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, SF et al, (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al. (1997) Genome Res. 7: 649-656. Other conventional BLAST algorithms such as those provided by NCBI BLAST are also well known to those skilled in the art.

As used herein, the expression "cell", "cell line" and "cell culture" are used interchangeably and all such names include progeny. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a small portion of a particular portion of nucleic acid, RNA, and/or DNA is amplified as described, for example, in U.S. Patent No. 4,683,195. In general, it is desirable to obtain sequence information from the end or beyond of the target region such that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends 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 ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.

"Optional" or "optionally" means that the event or environment described subsequently may, but need not, occur, including where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a particular sequence may, but need not, be present.

"Pharmaceutical composition" means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients. The purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.

Further, the present disclosure includes an agent for treating a thrombin-related disease, the agent comprising the monoclonal antibody of the present disclosure or an antibody fragment thereof as an active ingredient.

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

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

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

The immunodetection or assay method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of immunoassay or assay methods include radioactive substance labeling immunological antibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods Wait.

The thrombin-related diseases described above can be diagnosed by detecting or measuring cells expressing thrombin using the monoclonal antibodies or antibody fragments of the present disclosure.

In order to detect cells expressing the polypeptide, a known immunodetection method can be used, and immunoprecipitation, fluorescent cell staining, immunohistochemical staining, or the like is preferably used. Further, a fluorescent antibody staining method or the like using the FMAT8100HTS system (Applied Biosystem) can be used.

In the present disclosure, a living sample for detecting or measuring thrombin is not particularly limited as long as it has a possibility of including a cell 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 of the present disclosure or an antibody fragment thereof may further contain an agent for performing an antigen-antibody reaction or an agent for detecting a reaction, depending on a desired diagnostic method. Agents for performing antigen-antibody reactions include buffers, salts, and the like. The reagents for detection include reagents commonly used in immunoassays or assay methods, such as labeled secondary antibodies that recognize the monoclonal antibodies, antibody fragments or conjugates thereof, substrates corresponding to the labels, and the like.

Detailed ways

The disclosure is further described below in conjunction with the examples, which are not intended to limit the scope of the disclosure. Experimental methods that do not specify specific conditions in the examples of the present disclosure are generally carried out according to conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual, Molecular Cloning Manual; or according to the conditions recommended by the raw material or commodity manufacturer. Reagents without specific source are routine reagents purchased from the market.

Example 1. h1601-008 increased thrombin selective activity

Further affinity maturation of h1601-008 (see h1601-008 described in WO2017133673A1), panning against thrombin and prothrombin, further ensuring that the binding activity of the antibody to thrombin is not reduced, further The selective activity of an antibody for binding of two molecules.

>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

Increased selection activity using M13 phage display technology. A codon-based primer (in the course of primer synthesis, a single codon consisting of wild-type codon and NNK) was introduced into each CDR, and a separate phage display library was constructed for each CDR. According to the length of the CDR, the NNK ratio that each CDR needs to be incorporated and the storage capacity of the library are adjusted. The specific scheme is shown in Table 2.

Table 2. Library storage capacity and NNK incorporation ratio design table

library	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 were ligated together by overlapping extension PCR to form a combinatorial library with a storage capacity greater than 1E9. The combinatorial library is packaged into phage for panning: after pre-incubation of the liquid phase with high concentration of prothrombin at room temperature, biotinylated thrombin binding, streptavidin magnetic bead capture, panning, elution Re-infect E. coli and perform the next cycle of panning. Each round of panning reduces the concentration of biotinylated thrombin by 2-5 fold. After 3 rounds of panning, each library picked a single clone for sequencing verification. It was found that HCDR3, LCDR1, LCDR2 and LCDR3 all have obvious amino acid enrichment. According to the degree of amino acid residue enrichment in CDR regions, some clones such as H4L9, H4L10, H4L11, H4L12, H4L13, H4L14, H4L15, H4L17, H4L18, H4L19 were selected. Construction of full-length immunoglobulin for mammalian cell expression.

The selected clones differed from h1601-008 on HCDR3, LCDR1, LCDR2 and LCDR3. The related HCDR3, LCDR1, LCDR2 and LCDR3 formulas and their corresponding light and heavy chain variable regions are described below.

The HCDR3 sequence of the obtained formula (SEQ ID NO: 9) was screened as follows:

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

In SEQ ID NOS: 9 and 10, X _{1 is} selected from H, L; X _{2 is} selected from Y, L; wherein X _{2 is} not Y when X ₁ is H.

The LCDR sequence obtained by the screening is as follows:

Further, the relevant light chain variable region sequence formula (SEQ ID NO: 14) was obtained as follows:

In SEQ ID NOS: 11 to 14, X _{3 is} selected from S, T; X _{4 is} selected from E, H; X _{5 is} selected from D, R; X _{6 is} selected from I, M; and X _{7 is} selected from A, T; _{8 is} selected from S, Q, K; X _{9 is} selected from T, G; X _{10 is} selected from S, G; X _{11 is} selected from T, N, Y, wherein LCDR1, LCDR2 and LCDR3 are not simultaneously KASEDIYNRLA, GATSLET and QQYWSTPWT .

The specific correlation sequences obtained include, but are not limited to, those described in Tables 3 and 4:

Table 3. Sequence of heavy chain variable regions as determined by affinity screening

Heavy chain variable region	X 1	X 2	VH sequence code	HCDR3 sequence included
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 screening

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 heavy chain variable region of the above antibody is selected from the heavy region of the human heavy chain IgG4/light chain kappa, and the heavy chain variable region and the light chain variable region, respectively, to form a complete antibody heavy and light chain, wherein the heavy chain The S228P mutation was made in the Fc fragment to increase the stability of the IgG4 antibody, and other mutations known in the art may be used to increase its performance.

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 purification of the cloned protein, the affinity of the antibody to thrombin and prothrombin was determined using biacore.

The performance and beneficial effects of the disclosed antibodies are verified by biochemical test methods below.

Test Example 1. ELISA experiment of thrombin antibody binding to human thrombin protein

The binding capacity of anti-thrombin antibodies was detected by an ELISA assay of antibodies with His-tagged human thrombin. The His-tagged thrombin labeled with the Biotin Labeling Kit (Tongren Chemical, Cat No. LK03) was immobilized into a 96-well microtiter plate by binding to streptavidin coated in an ELISA plate. The strength of the signal after the antibody is added is used to determine the binding activity of the antibody and thrombin. The specific experimental method is as follows.

Streptavidin (Sigma, Cat No. S4762-5MG) was diluted to a concentration of 5 μg/ml with PBS (Sigma, Cat No. P4417-100TAB) buffer at pH 7.4, and added to the 96-well volume in a volume of 50 μl/well. The plate was placed in an incubator at 37 ° C for 2 hours. After discarding the liquid, 200 μl/well of a 5% skim milk (bright skimmed milk powder) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 2.5 hours or at 4 ° C overnight (16-18 hours) for blocking. After the end of the blocking, the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), and then diluted to 0.5 by adding 50 μl/well of the sample diluent (pH 7.4 PBS containing 1% BSA). Gg/ml of biotin-labeled His-tagged human thrombin was incubated in a 37 °C incubator for 2 hours. After the incubation, discard the reaction solution in the plate, wash the plate with PBST 6 times, add 50 μl/well of different concentrations of the thrombin test antibody diluted with the sample dilution, and incubate in a 37 ° C incubator. 2 hours. After the end of the incubation, the plate was washed 5 times with PBST, and 100 μl/well of HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-035-003) diluted with the sample dilution was added, and incubated at 37 ° C for 1 hour. After washing the plate with PBST 5 times, add 50 μl/well TMB chromogenic substrate (KPL, Cat No. 52-00-03), incubate for 10-15 min at room temperature, and add 50 μl/well 1 M H ₂ SO _{4 to} terminate the reaction. The NOVOStar microplate reader reads the absorbance at a wavelength of 450 nm and calculates the binding EC50 value of the thrombin antibody to human thrombin. The results are shown in Table 6.

Table 6. Humanized antibody and thrombin binding activity

antibody	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 results showed that the humanized antibody obtained by the present disclosure had higher binding activity to human thrombin protein than that of h1601-008.

Test Example 2. ELISA experiment of thrombin antibody binding to human prothrombin protein

Prothrombin is a precursor to thrombin that is enzymatically activated. The binding ability of the anti-prothrombin antibody is detected by an ELISA 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 μg/ml with PBS (Sigma, Cat No. P4417-100TAB) buffer, pH 7.4, and added to 96-well in a volume of 100 μl/well. Place in the plate at 4 ° C overnight (16-18 hours). After discarding the liquid, 200 μl/well of a 5% skim milk (bright skimmed milk powder) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 2.5 hours for blocking. After the end of the blocking, the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), and 50 μl/well of the sample diluted with the sample dilution (pH 7.4 PBS containing 1% BSA) was added. The thrombin test antibody and the control antibody Ichorcumab (from WO2013088164) were exposed and incubated in a 37 ° C incubator for 1 hour. After the end of the incubation, the plate was washed 5 times with PBST, and 100 μl/well of HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-035-003) diluted with the sample dilution was added, and incubated at 37 ° C for 1 hour. After washing the plate with PBST 5 times, add 50 μl/well TMB chromogenic substrate (KPL, Cat No. 52-00-03), incubate for 10-15 min at room temperature, and add 50 μl/well 1 M H ₂ SO _{4 to} terminate the reaction. The NOVOStar microplate reader reads the absorbance at a wavelength of 450 nm and calculates the binding EC50 value of the thrombin antibody to human prothrombin.

Table 7. Binding activity of humanized antibodies to prothrombin

antibody

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 Detection of Thrombin Antibody Affinity Experiment

Human anti-capture antibody was covalently coupled to a CM5 biosensor chip according to the method described in the Human Anti-Capture Kit (Cat. #BR-1008-39, GE) specification (Cat. #BR-1000-12, GE The antibody is then affinity-captured, and then passed through the thrombin antigen His-tagged human thrombin on the surface of the chip, and the reaction signal is detected in real time using a Biacore instrument to obtain binding and dissociation curves, and the affinity values are obtained by fitting. After each cycle of dissociation was completed in the experiment, the biochip was washed and regenerated using the regeneration solution disposed in the human anti-capture kit. Affinity tests were performed on antibodies obtained by phage display technology screening. The results are shown in Table 8:

Table 8. Antibody biacore assay results

It can be seen from the above that the new antibody screened by the phage display technology has an obvious affinity for thrombin, and the affinity for prothrombin is also improved, but the above antibody selection activity is greatly increased. The selective activity of the antibodies was greater than 25, and the highest increase of H4L13 was 2.99 times (33.42/11.18) compared with h1601-008.

Test Example 4. Effect of thrombin antibody on thrombin activity

In this experiment, the effect of the disclosed antibodies on thrombinase activity was examined by testing the enzymatic activity of thrombin on its substrate S2228.

His-tagged human thrombin was diluted with PBS pH 7.4 to a concentration of 100 nM, 25 μl/well, and added to a black-walled clear bottom 96-well plate. The thrombin test antibody of the present disclosure was serially diluted with PBS to a concentration of 2000 nM to 62.5 nM (1:2 gradient dilution), 25 μl/well, and was also added to the plate. After incubation for 30-60 minutes at room temperature, S2228 (substrate for detecting thrombin activity, synthesized by Gil Biochemical (Shanghai) Co., Ltd.) was diluted with PBS to a concentration of 4 mM, 50 μl/well, and added to the plate of the previous step. The negative control was either the addition of thrombin alone or the addition of S2228 to the control wells. After incubating for 30 minutes at room temperature, the absorbance was read at a wavelength of 405 nm using a NOVOStar plate reader. The results are shown in Figure 1, where only thrombin and S2228 only represent negative OD values measured by the negative control wells, 0.625:1, 1.25:1, 2.5:1, 5:1, 10:1, 20:1 indicate the test The OD value measured at different molar ratios of antibody to thrombin.

The results in Figure 1 indicate that the binding of the antibody obtained by h1601-008 and phage display to thrombin does not affect its enzymatic activity on the substrate S2228.

Thrombin antibody pharmacodynamics experiment

Test Example 5. Normal human plasma APTT test

In this experiment, the effect of the disclosed antibodies on the activated partial thromboplastin time (activated partial thromboplastin time) was tested by co-incubating normal human plasma with IgG and different concentrations of thrombin antibodies.

IgG is a negative control, that is, a human immunoglobulin obtained by a conventional affinity chromatography method such as ProteinA purification; Ichorcumab (from WO2013088164) is a positive control, and h1601-008 and various concentrations of the test antibody of the present disclosure are detected.

The experimental results are shown in Figure 2. As the concentration of antibody obtained by antibody h1601-008 and phage display increased, the APTT value of normal human plasma was also prolonged. At the highest concentration of 3200 nM, the increase in APTT value reached a peak of 34 seconds, while the APTT value of the antibody obtained by phage display was significantly higher than that of h1601-008 at high concentration (Table 9).

Table 9. Increase in APTT value (seconds) for normal human plasma with different concentrations of thrombin antibody

The above described invention has been described in detail with the aid of the accompanying drawings and examples, and the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (20)

1. A monoclonal antibody or antigen-binding fragment thereof, which binds to human thrombin, the monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 3, 4 and 9, respectively, and the light chain variable region comprises LCDR1, LCDR2 and SEQ ID NOs: 11, 12 and 13, respectively. LCDR3, wherein the SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 are amino acid sequences respectively represented by the following formula:

   HCDR3 DHYX ₁ GNSYVFDX ₂ SEQ ID NO: 9;

   LCDR1 KAX ₃ X ₄ X ₅ X ₆ YNRLA SEQ ID NO: 11;

   LCDR2 GX ₇ TX ₈ LEX ₉ SEQ ID NO: 12;

   LCDR3 QQYWX ₁₀ X ₁₁ PWT SEQ ID NO: 13;

   Wherein: X _{1 is} selected from H, L; X _{2 is} selected from Y, L; X _{3 is} selected from S, T; X _{4 is} selected from E, H; X _{5 is} selected from D, R; and X _{6 is} selected from I, M; X _{7 is} selected from A, T; X _{8 is} selected from S, Q, K; X _{9 is} selected from T, G; X _{10 is} selected from S, G; X _{11 is} selected from T, N, Y; and when X ₁ is H When X _{2 is} not Y, and X ₃ -X _{11 is} not selected from the following amino acids: X ₃ is S, X ₄ is E, X ₅ is D, X ₆ is I, X ₇ is A, and X ₈ is S. X ₉ is T, X ₁₀ is S, and X ₁₁ is T.
2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 3, 4 and 15, respectively.
3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the light chain variable region LCDR1 sequence is selected from any of the sequences set forth in SEQ ID NOs: 6, 17, and 18, and the LCDR2 sequence is selected from, for example. SEQ ID NOs: 7, 19 and 20, the LCDR3 sequence is selected from any of the sequences set forth in SEQ ID NOs: 8, 21 and 22; wherein LCDR1, LCDR2 and LCDR3 are not simultaneously selected from the following sequence: LCDR1 It is selected from SEQ ID NO: 6, LCDR2 is selected from SEQ ID NO: 7 and LCDR3 is selected from SEQ ID NO: 8.
4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein said heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 3, 4 and 15, respectively, said light chain The variable region is a light chain variable region selected from any one of the following ai:

   a. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 17, 19 and 8, respectively;

   b. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 6, 19 and 8, respectively;

   c. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 17, 20 and 21, respectively;

   d. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 6, 20 and 8, respectively;

   e, a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 17, 7 and 8, respectively;

   f. a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 6, 7, and 21, respectively;

   g, a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOS: 6, 20 and 21, respectively;

   h, comprising a light chain variable region of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 18, 7 and 8, respectively;

   i. A light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 18, 7 and 22, respectively.
5. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: a heavy chain variable region as shown in SEQ ID NO: 10 and The light chain variable region shown by SEQ ID NO: 14.
6. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding fragment comprises: a heavy chain variable region as shown in SEQ ID NO: 16 and ID NO: The light chain variable region shown by 14.
7. 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 a heavy chain variable region and a light chain selected from any of the following jr Variable area:

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

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

   l a heavy chain variable region as set forth in SEQ ID NO: 16 and a light chain variable region as set forth in SEQ ID NO: 25;

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

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

   o a heavy chain variable region as set forth in SEQ ID NO: 16 and a light chain variable region as set forth in SEQ ID NO: 28;

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

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

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

   Preferably, the heavy chain constant region is a heavy chain constant region variant having at least 85% sequence identity to a human IgGl, IgG2, IgG3 or IgG4 sequence, preferably a YTE mutation, a L234A and/or L235A mutation, or a S228P a heavy chain constant region variant of a mutated 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 κ, λ 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 as set forth in SEQ ID NO: 33 or with SEQ ID NO :33 A light chain constant region having at least 85% sequence identity.
9. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antigen-binding fragment is a V region selected from the group consisting of Fab, Fab', F(ab')2, scFv, dimerization a disulfide-stabilized V region and an antigen-binding fragment of a CDR-containing peptide.
10. A nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.
11. A recombinant vector comprising the nucleic acid molecule of claim 10.
12. A host cell transformed with the recombinant vector of claim 11, the host cell being selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
13. A method for producing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which comprises culturing the host cell according to claim 12 in a growth suitable for the host cell The medium is cultured to form and accumulate the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and the accumulated monoclonal antibody or antigen-binding fragment thereof is recovered from the culture.
14. A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody of any one of claims 1 to 9 and/or an antigen-binding fragment thereof, or the nucleic acid molecule of claim 10, or as claimed 11. The recombinant vector of claim 11, or the host cell of claim 12, and one or more pharmaceutically acceptable carriers, diluents or excipients.
15. A method for immunodetection or assay of human thrombin, the method comprising the step of detecting or determining using a reagent comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.
16. 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 a thrombin-related disease.
17. 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 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; wherein said disease is preferred It is a thrombotic disease; more preferably venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral artery disease or peripheral arterial disease; most preferred is venous thrombosis, thrombosis Formation of stroke and atherosclerosis.
18. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the nucleic acid molecule according to claim 10, or the recombinant vector according to claim 11, or the method of claim 12. Use of a host cell, or a pharmaceutical composition according to claim 14, for the preparation of a medicament for a thrombin-related disease, wherein the disease is preferably a thrombotic disease; more preferably venous thrombosis and pulmonary embolism, arteries Stroke, thrombosis, stroke and peripheral arteries, atherosclerotic disease, cerebral arterial disease or peripheral arterial disease; venous thrombosis, stroke and atherosclerosis caused by thrombosis are most preferred.
19. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 as a medicament, and/or a nucleic acid molecule according to claim 10, and/or a recombination according to claim 11 a vector, and/or a host cell according to claim 12, and/or a composition according to claim 14; preferably, the medicament is for treating a thrombotic disease, including but not limited to : venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arterial disease or peripheral arterial disease; more preferably, the disease is venous thrombosis, thrombosis Caused by stroke and atherosclerosis.
20. A monoclonal antibody or antigen-binding fragment thereof, or a composition thereof, for use in the treatment of a disease, wherein the monoclonal antibody or antigen-binding fragment thereof is the monoclonal antibody or antigen thereof according to any one of claims 1 to 9. In combination with fragments, the diseases include, but are not limited to, thrombotic diseases; preferably venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arterial disease or peripheral arterial disease More preferred are venous thrombosis, stroke and atherosclerosis caused by thrombosis.

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