CN111094354B - Thrombin antibody, antigen binding fragment thereof and medical application - Google Patents

Abstract

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

Description

Thrombin antibody, antigen binding fragment thereof and medical application

Technical Field

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

Background

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

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

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

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

Although there are currently a few patent publications of anti-thrombin antibodies, such as WO2013123591, WO2014153195, WO2014202992, WO2014202993, CN107043423A and WO2017133673. So far, no antithrombin antibody medicine is on the market, and further development and screening of antithrombin antibodies with better activity are needed for relevant clinical research and application.

Disclosure of Invention

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

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

wherein: x ₁ Selected from H, L; x ₂ Selected from Y, L; x ₃ Selected from S, T; x ₄ Selected from E, H; x ₅ Selected from D, R; x ₆ Selected from I, M; x ₇ Selected from A, T; x ₈ Is selected from the group consisting of S, Q,K；X ₉ selected from T, G; x ₁₀ Selected from S, G; x ₁₁ Selected from T, N, Y; and when X ₁ When it is H, X ₂ Is not Y and X ₃ -X ₁₁ Not simultaneously 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, 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 prothrombin (pro-thrombin) is greater than 25.

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

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

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

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

c. comprises the sequences respectively shown as SEQ ID NO: 17. 20 and 21, LCDR1, LCDR2 and LCDR3,

d. comprises the sequences respectively shown as SEQ ID NO: 6. 20 and 8, LCDR1, LCDR2 and LCDR3,

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

f. comprises the sequences respectively shown as SEQ ID NO: 6. 7 and 21, LCDR1, LCDR2 and LCDR3,

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

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

i. comprises the sequences respectively shown as SEQ ID NO: 18. 7 and 22, and LCDR1, LCDR2 and LCDR 3.

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

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

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

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

wherein: x ₁ Selected from H, L; x ₂ Is selected from Y, L; x ₃ Selected from S, T; x ₄ Selected from E, H; x ₅ Selected from D, R; x ₆ Selected from I, M; x ₇ Selected from A, T; x ₈ Selected from S, Q, K; x ₉ Selected from T, G; x ₁₀ Selected from S, G; x ₁₁ Selected from T, N, Y; and when X ₁ When is H, X ₂ Is not Y, and X ₃ -X ₁₁ Not simultaneously 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 represented by T.

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

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

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

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

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

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

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

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

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

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

In some embodiments, the aforementioned monoclonal antibody or antigen-binding fragment thereof further comprises an antibody constant region. In some embodiments, wherein the antibody comprises a heavy chain constant region of human IgG1, igG2, igG3, or IgG4, or a variant thereof, and/or comprises a light chain constant region of human kappa, lambda chains, or a variant thereof; preferably, the heavy chain constant region is a heavy chain constant region variant having at least 85% sequence identity to a human IgG1, igG2, igG3 or IgG4 sequence, e.g., a heavy chain constant region variant of IgG1, igG2 or IgG4 that enhances antibody function (e.g., extends half-life of the antibody in serum, enhances antibody stability, etc.) by amino acid mutation, preferably a YTE mutation, an L234A and/or L235A mutation, or an S228P mutation of IgG1, igG2 or IgG 4; the light chain constant region is a light chain constant region variant having at least 85% sequence identity to a human kappa and lambda chain sequence. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:32 or a sequence similar to that shown in SEQ ID NO:32, and/or a heavy chain constant region having at least 85% sequence identity to SEQ ID NO:33 or a sequence shown in SEQ ID NO:33 light chain constant region having at least 85% sequence identity. Wherein the heavy/light chain constant region having at least 85% sequence identity is preferably an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, more preferably 90%, 95%, or 99% or more sequence identity, and the amino acid sequence having at least 85% sequence identity may be obtained by one or more amino acid deletion, insertion, or substitution mutation.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Drawings

FIG. 1: detection of thrombin Activity by Thrombin antibodies

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

Detailed Description

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

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

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

The term "antibody (Ab)" as used in the present disclosure refers to immunoglobulin (Ig), which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes (classes), namely, igM, igD, igG, igA, and IgE, with their corresponding heavy chains being the μ chain, the δ chain, the γ chain, the α chain, and the ε chain, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified into kappa chains or lambda chains by the difference of constant regions, and thus antibodies can be classified into two types (types). Each of the five classes of Ig may have either a kappa chain or a lambda chain. Based on the difference of individual amino acids in the constant region of the lambda chain, the constant region can be divided into four subtypes (subtypes) of lambda 1, lambda 2, lambda 3 and lambda 4.

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

In the present disclosure, the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgG1, igG2, igG3, igG4 or variants thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region consists of a junction of 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs), which are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, and are also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (LCVR or VL) and heavy chain variable region (HCVR or VH) consists of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3. In some embodiments, the amino acid residues of the CDRs of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present disclosure are in numbers and positions conforming to known Kabat numbering conventions (Kabat et al Sequences of Proteins of Immunological Interest, (5 th edition, 1991, national Institutes of health, bethesda MD)).

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

The term "murine antibody" is a monoclonal antibody of mouse origin prepared according to the knowledge and skill in the art. Prepared by injecting a test subject with an antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties, which antibodies are murine when the injected test subject is a mouse. In one embodiment of the present disclosure, a "murine antibody" is a monoclonal antibody that binds human thrombin prepared according to the knowledge and skill in the art. Preparation is accomplished by injecting a subject with a thrombin antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In one embodiment of the present disclosure, the murine thrombin antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, igG2, igG3 or variant thereof.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing chimeric antibody, firstly establishing hybridoma secreting mouse-derived specific monoclonal antibody, then cloning variable region gene from mouse hybridoma cell, cloning constant region gene of human antibody according to the need, connecting mouse variable region gene and human constant region gene into chimeric gene, inserting into expression vector, and finally expressing chimeric antibody molecule in eukaryotic system or prokaryotic system. In a specific embodiment of the present disclosure, the antibody light chain of the thrombin chimeric antibody further comprises a light chain constant region of a human kappa or lambda chain or a variant thereof. The antibody heavy chain of the thrombin chimeric antibody further comprises a heavy chain constant region of human IgG1, igG2, igG3, igG4 or a variant thereof, preferably comprises a human IgG1, igG2 or IgG4 heavy chain constant region, or an IgG1, igG2 or IgG4 heavy chain constant region variant using amino acid mutations (e.g., YTE mutations or back mutations, L234A and/or L235A mutations, or S228P mutations).

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

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

In one embodiment of the present disclosure, the humanized antibody or antigen binding fragment thereof may further comprise a light chain constant region of a human kappa, lambda chain or variant thereof and/or a heavy chain constant region of a human IgG1, igG2, igG3, igG4 or variant thereof. In one embodiment of the disclosure, a human IgG1, igG2 or IgG4 heavy chain constant region is included, or an IgG1, igG2 or IgG4 heavy chain constant region variant using amino acid mutations (e.g., YTE mutations or back mutations, L234A and/or L235A mutations, or S228P mutations).

The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., thrombin). It has been shown that fragments of full-length antibodies can be used to achieve the antigen binding function of an antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) Single domain or dAb fragments (Ward et al, (1989) Nature341: 544-546), which consist of VH domains, also including maxibody (maxibody), minibody (minibody, intrabody (intrabody), triabody, tetrabody, v-NAR (v-new antigen receptor) and diabody (see e.g.Hollinger and Hudson,2005, nature Biotechnology,23.9, the antigen binding fragments include Fab, F (ab ') 2, fab', single chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), peptides comprising CDRs, and the like.

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

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

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

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

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

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

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker. Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, e.g. using 1-4 repeated variants (Holliger et al (1993), proc.natl.acad.sci.usa90: 6444-6448). Other linkers useful in the present disclosure are prepared by althan et al (1995), protein eng.8:725-731, choi et al (2001), eur.J. Immuno l.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Rovers et al (2001), cancer Immunol.

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

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

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

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

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

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

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

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

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

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

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

The term "KD" refers to the dissociation equilibrium constant for a particular antibody-antigen interaction. Typically, the antibodies of the disclosure are administered at less than about 10-7M, e.g., less than about 10 ^-8 M、10 ^-9 M or 10 ^-10 M or less dissociation equilibrium constant (KD) in combination with thrombin, e.g., as using Surface Plasmon Resonance (SPR) techniquesMeasured 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 (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation equilibrium constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

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

The term "compete" when used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies or specifically binding antibodies) means competition between antigen binding proteins as determined by the following assay: the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be detected prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., thrombin antigen or fragment thereof). Numerous types of competitive binding assays are available for determining whether an antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., stahli et al, 1983, methods in Enzymology 9; solid phase direct biotin-avidin EIA (see, e.g., kirkland et al, 1986, J.Immunol.137 3614-3619), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., harlow and Lane,1988, antibodies, A Laboratory Manual, cold Spring Harbor Press); direct labeling of RIA with a solid phase of I-125 label (see, e.g., morel et al, 1988, mol. Immunol.25; solid phase direct biotin-avidin EIA (see, e.g., cheung, et al, 1990, virogy176; and directly labeled RIA (Moldenhauer et al, 1990, scand. J. Immunol.32. Typically, the assay involves the use of a purified antigen (either on a solid surface or on the surface of a cell) that binds to a test antigen binding protein with an unlabeled label and a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cells in the presence of the antigen binding protein to be detected. Alternatively, competitive inhibition can be measured by immobilizing antigen binding protein A and detecting a change in signal from the labeled antigen that is pre-bound to the antigen binding protein. Typically, such competitive inhibition assays will swap the positions of antigen binding protein a and antigen binding protein B for confirmation. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as a reference antigen binding protein; and an antigen binding protein that binds to an epitope sufficiently close to the epitope to which the reference antigen binding protein binds, said two epitopes sterically hindering the binding from occurring. Typically, a portion of a competing antigen binding protein inhibits (e.g., reduces) specific binding of at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%) of a reference antigen binding protein to a common antigen when the competing antigen binding protein is present in excess. In the complete competition, the binding of the reference antigen binding protein to the antigen is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or more. Inhibition of less than 30% is considered to be non-competitive.

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

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

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold spring harbor antibody Experimental guidelines, chapters 5-8 and 15. For example, a mouse may be immunized with human thrombin or a fragment thereof, and the resulting antibody can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibody or antigen binding fragment of the invention is genetically engineered to add one or more human FR regions to the CDR regions of non-human origin. Human FR germline sequences can be identified from the website http: i/w w.imgt.org/or from J.Immunoglobulins, 2001ISBN 012441351.

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

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

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

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

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

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

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

"homology", "identity", which are used interchangeably herein, refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if there are 6 matches or homologies at 10 positions in two sequences when the sequences are optimally aligned, then the two sequences are 60% homologous; two sequences are 95% homologous if there are 95 matches or homologies at 100 positions in the two sequences. In general, the comparison is made when the two sequences are aligned to give the greatest percentage of homology. For example, the comparison may be performed by the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between the respective sequences over the entire length of the respective reference sequences. The following references relate to the BLAST algorithm often used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): altschul, s.f. et al, (1990) j.mol.biol.215:403-410; gish, w. et al, (1993) Nature genet.3:266 to 272; madden, t.l. et al, (1996) Meth Enzymol 266:131 to 141; altschul, s.f. 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 provided by NCBI BLAST, are also well known to those skilled in the art.

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

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

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

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

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

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

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

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

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

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

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

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

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

Detailed Description

The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure. The experimental methods without specifying specific conditions in the examples of the present disclosure are generally performed under conventional conditions such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1 improvement of thrombin Selective Activity of h1601-008

The method is characterized in that the method comprises the steps of performing further affinity maturation on h1601-008 (see h1601-008 described in WO2017133673A 1), performing panning on thrombin and prothrombin, and further improving the selective activity of the antibody on the combination of two molecules on the premise of ensuring that the combination activity of the antibody on the thrombin is not reduced.

＞h1601-008 VH：(SEQ ID NO：1)

＞h1601-008 VL：(SEQ ID NO：2)

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

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

TABLE 2 library capacity and NNK incorporation ratio design Table

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

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

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

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

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

in SEQ ID NO: x in 9 and 10 ₁ Selected from H, L; x ₂ Selected from Y, L; wherein X ₁ Is H or X ₂ Is not Y.

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

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

in SEQ ID NO:11 to 14 in which X is ₃ Selected from S, T; x ₄ Selected from E, H; x ₅ Selected from D, R; x ₆ Selected from I, M; x ₇ Selected from A, T; x ₈ Selected from S, Q, K; x ₉ Selected from T, G; x ₁₀ Selected from S, G; x ₁₁ Selected from T, N, Y, wherein LCDR1, LCDR2 and LCDR3 are not simultaneously eachKASEDIYNRLA, GATSLET, and QQYWSTPWT.

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

TABLE 3 heavy chain variable region sequences determined by affinity Screen

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

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

TABLE 4 light chain variable region sequences determined by affinity screen

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Streptavidin (Sigma, cat No. S4762-5 MG) was diluted to a concentration of 5. Mu.g/ml with PBS buffer pH7.4 (Sigma, cat No. P4417-100 TAB), added to a 96-well plate at a volume of 50. Mu.l/well, and placed in an incubator at 37 ℃ for 2 hours. After discarding the liquid, 200. Mu.l/well of 5% skim milk (light skim milk powder) blocking solution diluted with PBS was added, and the cells were incubated at 37 ℃ for 2.5 hours or left overnight (16-18 hours) at 4 ℃ for blocking. After blocking was complete, the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), and then 50. Mu.l/well of a sample diluent (pH 7.4 PBS containing 1% BSA) was added to dilute to 0.5. Mu.g/ml of biotin-labeled His-tagged human thrombin, followed by incubation in an incubator at 37 ℃ for 2 hours. After the incubation is finished, reaction liquid in the enzyme label plate is discarded, the plate is washed for 6 times by PBST, 50 mu l/hole of thrombin antibody to be detected with different concentrations diluted by sample diluent is added, and the mixture is incubated for 2 hours in an incubator at 37 ℃. After the incubation was completed, the plate was washed 5 times with PBST, and 100. Mu.l/well of HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, cat No. 109-035-003) diluted with the sample diluent was added and incubated at 37 ℃ for 1 hour. Washing the plate with PBST for 5 times, adding 50 μ l/well TMB chromogenic substrate (KPL, cat No. 52-00-03), incubating at room temperature for 10-15min, adding 50 μ l/well 1M H ₂ SO ₄ The reaction was stopped, and the absorbance was read at a wavelength of 450nm using a NOVOStar microplate reader to calculate the EC50 value for the binding of thrombin antibody to human thrombin. The results are shown in Table 6.

TABLE 6 binding Activity of humanized antibodies with Thrombin

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

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

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

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

With PBS (Sig) of pH7.4ma, cat No. p4417-100 TAB) buffer His-tagged human prothrombin (SEQ ID NO: 2) Diluted to 10. Mu.g/ml, added to a 96-well plate at a volume of 100. Mu.l/well and left overnight at 4 ℃ (16-18 hours). After discarding the liquid, 5% skim milk (light skim milk powder) diluted with PBS was added to the wells at 200. Mu.l/well, and the cells were incubated at 37 ℃ for 2.5 hours for blocking. After the blocking was completed, the blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), 50. Mu.l/well of a thrombin test antibody of the present disclosure and a control antibody Ichorcumab (from WO 2013088164) which were diluted in a gradient with a sample diluent (pH 7.4 PBS containing 1% BSA) were added, and incubated in an incubator at 37 ℃ for 1 hour. After the incubation was completed, the plate was washed 5 times with PBST, and 100. Mu.l/well of HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, cat No. 109-035-003) diluted with the sample diluent was added and incubated at 37 ℃ for 1 hour. Washing the plate with PBST for 5 times, adding 50 μ l/well TMB chromogenic substrate (KPL, cat No. 52-00-03), incubating at room temperature for 10-15min, adding 50 μ l/well 1M H ₂ SO ₄ The reaction was stopped, and the absorbance was read at a wavelength of 450nm using a NOVOStar microplate reader to calculate the EC50 value for the binding of thrombin antibody to human prothrombin.

TABLE 7 binding Activity of humanized antibodies with Prothrombin

Test example 3 BIAcore assay for thrombin antibody affinity assay

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

TABLE 8 results of antibody biacore assay

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

Test example 4 Effect of Thrombin antibodies on Thrombin enzyme Activity

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

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

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

Pharmacodynamic test of thrombin antibody

Test example 5 APTT test on Normal human plasma

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

IgG is a negative control, i.e., human immunoglobulin obtained by purification using conventional affinity chromatography methods such as ProteinA; ichorcumab (from WO 2013088164) was a positive control, and h1601-008 and different concentrations of the antibodies to be tested according to the disclosure were detected.

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

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

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

Sequence listing

<110> Hengrui pharmaceutical Co., ltd of Jiangsu

SHANGHAI HENGRUI PHARMACEUTICAL Co.,Ltd.

<120> thrombin antibodies, antigen-binding fragments thereof and medical uses

<130> 780088CPCT

<150> CN201711160510.5

<151> 2017-11-20

<160> 33

<170> SIPOSequenceListing 1.0

<210> 1

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1601-008 VH

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asn Ile Asn Ser Asp Gly Ser Ser Thr Tyr Tyr Leu Asp Ser Leu

50 55 60

Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Tyr His Gly Asn Ser Tyr Val Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 2

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> h1601-008 VL

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 3

<211> 5

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> mAb1601 HCDR1

<400> 3

Asp Tyr Tyr Met Ala

1 5

<210> 4

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> mAb1601 HCDR2

<400> 4

Asn Ile Asn Ser Asp Gly Ser Ser Thr Tyr Tyr Leu Asp Ser Leu Lys

1 5 10 15

Ser

<210> 5

<211> 12

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> mAb1601 HCDR3

<400> 5

Asp His Tyr His Gly Asn Ser Tyr Val Phe Asp Tyr

1 5 10

<210> 6

<211> 11

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> mAb1601 LCDR1

<400> 6

Lys Ala Ser Glu Asp Ile Tyr Asn Arg Leu Ala

1 5 10

<210> 7

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> mAb1601 LCDR2

<400> 7

Gly Ala Thr Ser Leu Glu Thr

1 5

<210> 8

<211> 9

<212> PRT

<213> mouse (Mus musculus)

<220>

<221> DOMAIN

<223> mAb1601 LCDR3

<400> 8

Gln Gln Tyr Trp Ser Thr Pro Trp Thr

1 5

<210> 9

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 HCDR3 formula

<220>

<221> UNSURE

<222> (4)..(4)

<223> The 'Xaa' at location 4 stands for His, or Leu.

<220>

<221> UNSURE

<222> (12)..(12)

<223> The 'Xaa' at location 12 stands for Tyr, or Leu.

<400> 9

Asp His Tyr Xaa Gly Asn Ser Tyr Val Phe Asp Xaa

1 5 10

<210> 10

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 heavy chain variable region general formula

<220>

<221> UNSURE

<222> (102)..(102)

<223> The 'Xaa' at location 102 stands for His, or Leu.

<220>

<221> UNSURE

<222> (110)..(110)

<223> The 'Xaa' at location 110 stands for Tyr, or Leu.

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asn Ile Asn Ser Asp Gly Ser Ser Thr Tyr Tyr Leu Asp Ser Leu

50 55 60

Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Tyr Xaa Gly Asn Ser Tyr Val Phe Asp Xaa Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 11

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 LCDR1 formula

<220>

<221> UNSURE

<222> (3)..(3)

<223> The 'Xaa' at location 3 stands for Ser, or Thr.

<220>

<221> UNSURE

<222> (4)..(4)

<223> The 'Xaa' at location 4 stands for Glu, or His.

<220>

<221> UNSURE

<222> (5)..(5)

<223> The 'Xaa' at location 5 stands for Asp, or Arg.

<220>

<221> UNSURE

<222> (6)..(6)

<223> The 'Xaa' at location 6 stands for Ile, or Met.

<400> 11

Lys Ala Xaa Xaa Xaa Xaa Tyr Asn Arg Leu Ala

1 5 10

<210> 12

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 LCDR2 formula

<220>

<221> UNSURE

<222> (2)..(2)

<223> The 'Xaa' at location 2 stands for Ala, or Thr.

<220>

<221> UNSURE

<222> (4)..(4)

<223> The 'Xaa' at location 4 stands for Ser, Gln, or Lys.

<220>

<221> UNSURE

<222> (7)..(7)

<223> The 'Xaa' at location 7 stands for Thr, or Gly.

<400> 12

Gly Xaa Thr Xaa Leu Glu Xaa

1 5

<210> 13

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 LCDR3 formula

<220>

<221> UNSURE

<222> (5)..(5)

<223> The 'Xaa' at location 5 stands for Ser, or Gly.

<220>

<221> UNSURE

<222> (6)..(6)

<223> The 'Xaa' at location 6 stands for Thr, Asn, or Tyr.

<400> 13

Gln Gln Tyr Trp Xaa Xaa Pro Trp Thr

1 5

<210> 14

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 variable region general formula

<220>

<221> UNSURE

<222> (26)..(26)

<223> The 'Xaa' at location 26 stands for Ser, or Thr.

<220>

<221> UNSURE

<222> (27)..(27)

<223> The 'Xaa' at location 27 stands for Glu, or His.

<220>

<221> UNSURE

<222> (28)..(28)

<223> The 'Xaa' at location 28 stands for Asp, or Arg.

<220>

<221> UNSURE

<222> (29)..(29)

<223> The 'Xaa' at location 29 stands for Ile, or Met.

<220>

<221> UNSURE

<222> (51)..(51)

<223> The 'Xaa' at location 51 stands for Ala, or Thr.

<220>

<221> UNSURE

<222> (53)..(53)

<223> The 'Xaa' at location 53 stands for Ser, Gln, or Lys.

<220>

<221> UNSURE

<222> (56)..(56)

<223> The 'Xaa' at location 56 stands for Thr, or Gly.

<220>

<221> UNSURE

<222> (93)..(93)

<223> The 'Xaa' at location 93 stands for Ser, or Gly.

<220>

<221> UNSURE

<222> (94)..(94)

<223> The 'Xaa' at location 94 stands for Thr, Asn, or Tyr.

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Xaa Xaa Xaa Xaa Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Xaa Thr Xaa Leu Glu Xaa Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Xaa Xaa Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 H4 HCDR3

<400> 15

Asp His Tyr Leu Gly Asn Ser Tyr Val Phe Asp Leu

1 5 10

<210> 16

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 H4 heavy chain variable region

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asn Ile Asn Ser Asp Gly Ser Ser Thr Tyr Tyr Leu Asp Ser Leu

50 55 60

Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp His Tyr Leu Gly Asn Ser Tyr Val Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Thr Val

115

<210> 17

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 LCDR1-1

<400> 17

Lys Ala Thr Glu Asp Met Tyr Asn Arg Leu Ala

1 5 10

<210> 18

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 LCDR1-2

<400> 18

Lys Ala Thr His Arg Met Tyr Asn Arg Leu Ala

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 LCDR2-1

<400> 19

Gly Thr Thr Gln Leu Glu Thr

1 5

<210> 20

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 LCDR2-2

<400> 20

Gly Ala Thr Lys Leu Glu Gly

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 LCDR3-1

<400> 21

Gln Gln Tyr Trp Gly Asn Pro Trp Thr

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 LCDR3-2

<400> 22

Gln Gln Tyr Trp Ser Tyr Pro Trp Thr

1 5

<210> 23

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 L9 light chain variable region

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Met Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Thr Thr Gln Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 24

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 L10 light chain variable region

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Thr Thr Gln Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 L11 light chain variable region

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Met Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Ala Thr Lys Leu Glu Gly Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Gly Asn Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 L12 light chain variable region

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr Glu Asp Met Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 27

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 L13 light chain variable region

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Ala Thr Lys Leu Glu Gly Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 28

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 L14 light chain variable region

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Gly Asn Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 29

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 L15 light chain variable region

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Ala Thr Lys Leu Glu Gly Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Gly Asn Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 30

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 L17 light chain variable region

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr His Arg Met Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 31

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 L18 light chain variable region

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Thr His Arg Met Tyr Asn Arg

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Ile Ile

35 40 45

Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 32

<211> 327

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 series antibody heavy chain constant region

<400> 32

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 33

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> DOMAIN

<223> 1601 series antibody light chain constant region

<400> 33

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims (16)

1. A monoclonal antibody or antigen-binding fragment thereof that binds human thrombin, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 3.4 and 15, and HCDR1, HCDR2 and HCDR3, and the light chain variable region

Comprises the sequences respectively shown as SEQ ID NO: 6. 20 and 8, LCDR1, LCDR2 and LCDR 3.

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein said monoclonal antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO:16 and the heavy chain variable region as set forth in SEQ ID NO:27, or a light chain variable region.

3. The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, comprising 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 human kappa, lambda chains, or a variant thereof.

4. The monoclonal antibody or antigen-binding fragment thereof of claim 3, wherein the heavy chain constant region is an L234A and/or L235A mutant, or an S228P mutant IgG1, igG2, or IgG4 heavy chain constant region variant.

5. The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody comprises a heavy chain constant region as set forth in SEQ ID NO. 32 and a light chain constant region as set forth in SEQ ID NO. 33.

6. The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antigen-binding fragment is an antigen-binding fragment selected from Fab, fab ', F (ab') 2, scFv, or a peptide comprising a CDR.

7. A nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

8. A recombinant vector comprising the nucleic acid molecule of claim 7.

9. A host cell transformed with the recombinant vector of claim 8, said host cell selected from the group consisting of prokaryotic cells and eukaryotic cells.

10. The host cell of claim 9, which is a eukaryotic cell.

11. The host cell of claim 9, which is a mammalian cell.

12. A method for producing the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the method comprising culturing the host cell of any one of claims 9 to 11 in a medium suitable for growth of the host cell to form and accumulate the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6, and recovering the accumulated monoclonal antibody or antigen-binding fragment thereof from the culture.

13. A pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody and/or antigen-binding fragment thereof according to any one of claims 1 to 6, or a nucleic acid molecule according to claim 7, or a recombinant vector according to claim 8, or a host cell according to any one of claims 9 to 11, and one or more pharmaceutically acceptable carriers, diluents or excipients.

14. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the nucleic acid molecule according to claim 7, or the recombinant vector according to claim 8, or the host cell according to any one of claims 9 to 11, or the pharmaceutical composition according to claim 13, for the manufacture of a medicament for the treatment of a thrombotic disorder.

15. Use according to claim 14, wherein the thrombotic disorder is selected from the group consisting of venous thrombosis and pulmonary embolism, arterial thrombosis, thrombosis induced stroke and peripheral arterial formation, atherosclerotic disorders, cerebral arteriopathy and peripheral arteriopathy.

16. Use according to claim 14, wherein the thrombotic disorder is selected from venous thrombosis, stroke due to thrombosis and atherosclerosis.

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