CN107043423B

CN107043423B - Thrombin antibody, antigen binding fragment thereof and medical application

Info

Publication number: CN107043423B
Application number: CN201710060629.9A
Authority: CN
Inventors: 应华; 刘佳建; 付雅媛; 张玲; 张昊颖; 孙嘉康; 张连山; 陶维康; 孙飘扬; 胡齐悦
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2016-02-05
Filing date: 2017-01-25
Publication date: 2021-02-26
Anticipated expiration: 2037-01-25
Also published as: CN107043423A

Abstract

The present invention relates to thrombin antibodies, antigen binding fragments thereof and medical uses thereof. Further, the present invention relates to chimeric antibodies, humanized antibodies comprising the CDR regions of the thrombin antibody, as well as pharmaceutical compositions comprising the thrombin antibody and antigen binding fragments thereof, and their use as anti-coagulant drugs. In particular, the invention relates to the use of a humanized thrombin antibody in the manufacture of a medicament for the treatment of a thrombin-mediated disease or condition.

Description

Thrombin antibody, antigen binding fragment thereof and medical application

Technical Field

The present invention relates to thrombin antibodies and antigen-binding fragments thereof, further, the present invention relates to chimeric antibodies, humanized antibodies comprising the CDR regions of said thrombin antibodies, pharmaceutical compositions comprising said thrombin antibodies and antigen-binding fragments thereof, and their use as anti-coagulant drugs.

Background

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

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

No antithrombin antibody drugs are currently on the market or enter the clinic, and relevant antibody patents are WO2013123591, WO2013088164, WO2014153195, WO2014202992 and WO 2014202993. The present invention provides a novel thrombin antibody having high affinity and a significant inhibitory activity against thrombus formation.

Disclosure of Invention

The invention provides a thrombin antibody or antigen-binding fragment thereof comprising 1 or more CDR region sequences selected from the group consisting of seq id nos: antibody heavy chain variable region HCDR region sequence: 7,8 and 9; and antibody light chain variable region LCDR region sequence: 10,11 and 12.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof according to the invention, wherein the thrombin antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9, respectively, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof according to the invention, wherein the thrombin antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, respectively, or an amino acid sequence having at least 95% sequence identity to SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof according to the invention, wherein the thrombin antibody or antigen-binding fragment thereof is a rabbit derived antibody or a functional fragment thereof.

In another preferred embodiment of the present invention, the thrombin antibody or antigen-binding fragment thereof according to the present invention, wherein the light chain variable region of the thrombin antibody or antigen-binding fragment thereof further comprises a light chain FR region of a rabbit-derived kappa chain or rabbit-derived kappa chain variant, or further comprises a light chain FR region of a rabbit-derived lambda chain or rabbit-derived lambda chain variant; and/or wherein the heavy chain variable region of the thrombin antibody or antigen-binding fragment thereof further comprises the heavy chain FR region of rabbit IgG or a variant thereof.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence of SEQ ID NO.5 and the light chain variable region sequence of SEQ ID NO. 6. The thrombin antibody described herein is an antibody of rabbit origin.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof of the invention, wherein the thrombin antibody or antigen-binding fragment thereof further comprises a light chain constant region of a kappa chain of rabbit origin or a variant thereof, or a light chain constant region of a lambda chain of rabbit origin or a variant thereof; and/or wherein said thrombin antibody further comprises a heavy chain constant region of rabbit IgG or a variant thereof.

In another preferred embodiment of the present invention, the thrombin antibody or antigen-binding fragment thereof according to the present invention is a chimeric antibody or a functional fragment thereof.

In another preferred embodiment of the present invention, the thrombin antibody or antigen-binding fragment thereof according to the present invention, wherein the thrombin antibody or antigen-binding fragment thereof is a humanized antibody or a functional fragment thereof.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof according to the invention, wherein the heavy chain FR region sequence of said humanized antibody or functional fragment thereof is derived from the combined sequences of human germline heavy chains IGHV3-66 x 01 and hjh 4.1.1 and mutated sequences thereof; wherein the humanized antibody or a functional fragment thereof comprises the FR1, FR2, FR3 region of human germline heavy chain IGHV3-66 x 01 and the FR4 region of hjh 4.1.1 and mutated sequences thereof.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof according to the invention, wherein the humanized antibody or functional fragment thereof comprises the heavy chain variable region as set forth in SEQ ID NO 13 or the heavy chain variable region as set forth in an amino acid sequence having at least 95% sequence identity to SEQ ID NO 13; preferably, the amino acid sequence has 0 to 10 amino acid changes from the amino acid sequence of SEQ ID NO 13.

In another preferred embodiment of the present invention, the thrombin antibody or antigen-binding fragment thereof according to the present invention, wherein the FR region of the heavy chain of said humanized antibody or functional fragment thereof has a back mutation of 0 to 10 amino acids in sequence, preferably said back mutation is selected from the group consisting of amino acid back mutations of V47I, S48G, R70K, L75V, A93V and Y91F.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof according to the invention, wherein the humanized antibody or functional fragment thereof comprises the heavy chain variable region as set forth in SEQ ID NO 16 or the heavy chain variable region as set forth in an amino acid sequence having at least 95% sequence identity to SEQ ID NO 16.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof of the invention, wherein the light chain FR region sequence of said humanized antibody or functional fragment thereof is derived from the combined sequence of human germline light chain templates IGLV4-60 x 01 and hjk4.1 and mutated sequences thereof; wherein the humanized antibody or a functional fragment thereof comprises FR1, FR2 and FR3 regions of human germline light chain IGLV4-60 x 01 and FR4 region of hjk4.1 and mutated sequences thereof.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof according to the invention, wherein the humanized antibody or functional fragment thereof comprises the light chain variable region sequence set forth in SEQ ID NO. 14 or the light chain variable region sequence set forth in an amino acid sequence having at least 95% sequence identity to SEQ ID NO. 14; the amino acid sequence has 0-10 amino acid changes with that of SEQ ID NO. 14.

In another preferred embodiment of the present invention, the thrombin antibody or antigen-binding fragment thereof according to the present invention, wherein the light chain FR region of said humanized antibody or functional fragment thereof has a back mutation of 0 to 10 amino acids in sequence, preferably said back mutation is an amino acid back mutation selected from the group consisting of K49E, P2L, H36Y, Y91H, Q1E and D89T.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof according to the invention, wherein the humanized antibody or functional fragment thereof comprises the light chain variable region of SEQ ID NO 14, 15 or 17 or the light chain variable region of an amino acid sequence having at least 95% sequence identity to SEQ ID NO 14, 15 or 17.

In another preferred embodiment of the invention, the thrombin antibody or antigen-binding fragment thereof according to the invention, wherein the humanized antibody or functional fragment thereof comprises (a) a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO 13, SEQ ID NO 16 and an amino acid sequence having at least 95% sequence identity to SEQ ID NO 13 or SEQ ID NO 16; or/and (b) a light chain variable region having a sequence selected from SEQ ID NO 14, 15 or 17 and an amino acid sequence having at least 95% sequence identity to SEQ ID NO 14, 15 or 17.

In another preferred embodiment of the present invention, the thrombin antibody or antigen-binding fragment thereof according to the present invention, wherein the thrombin antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

1) the heavy chain variable region of SEQ ID NO 13 and the light chain variable region of SEQ ID NO 14;

2) the heavy chain variable region of SEQ ID NO 13 and the light chain variable region of SEQ ID NO 15;

3) the heavy chain variable region of SEQ ID NO 16 and the light chain variable region of SEQ ID NO 15;

4) the heavy chain variable region of SEQ ID NO 16 and the light chain variable region of SEQ ID NO 14;

5) the heavy chain variable region of SEQ ID NO 13 and the light chain variable region of SEQ ID NO 17; and

6) the heavy chain variable region of SEQ ID NO 16 and the light chain variable region of SEQ ID NO 17.

In another preferred embodiment of the invention, the thrombin antibody or antigen binding fragment thereof according to the invention, wherein the thrombin antibody further comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4 and variants thereof, preferably comprises a heavy chain constant region of human IgG4 or a variant thereof, preferably a heavy chain constant region as depicted in, for example, SEQ ID NO:18 of the invention.

The chimeric or humanized antibody light chain further comprises a constant region of a human kappa, lambda chain or variant thereof, preferably, for example, the constant region depicted in SEQ ID NO 19 of the present invention.

In another preferred embodiment of the invention, the antigen binding fragment of the invention 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.

The invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a thrombin antibody or antigen-binding fragment thereof as described above, together with one or more pharmaceutically acceptable carriers, diluents or excipients.

The invention further provides a DNA molecule encoding the thrombin antibody or antigen-binding fragment thereof as described above.

The invention further provides an expression vector for the DNA molecule as described above.

The present invention further provides a host cell transformed with an expression vector as described above, said host cell being selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells.

The invention further provides the use of a thrombin antibody or antigen-binding fragment thereof as described above or a pharmaceutical composition as described above, in the manufacture of a medicament for the treatment of a thrombin-mediated disease or condition, preferably a thrombotic disease (which includes thrombosis and thromboembolism); 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.

Exemplary diseases that can be diagnosed using the antibodies of the invention include thrombotic diseases (which includes thrombosis and thromboembolism) that include any one or more of the following: venous thrombosis and pulmonary embolism, arterial thrombosis, stroke and peripheral arterial formation caused by thrombosis, atherosclerotic disease, cerebral arteriopathy or peripheral arteriopathy; .

In one aspect, the present invention provides methods of treating or preventing hypercholesterolemia and/or at least one of the following symptoms in a subject: venous thrombosis, thrombosis-induced stroke, and atherosclerosis, the method comprising administering to the individual an effective amount of an anti-thrombin antibody. The invention also provides the use of an effective amount of an anti-thrombin antibody that antagonizes extracellular or circulating thrombin in the manufacture of a medicament for treating or preventing thrombosis and/or at least one of the following conditions in a subject: pulmonary embolism, thrombosis induced stroke and atherosclerosis.

In one aspect, the present invention provides a reagent for detecting or measuring human thrombin, which comprises the above-described antibody or an antigen-binding fragment thereof.

Drawings

FIG. 1: the effect of the anti-thrombin antibody CH-1512 on human thrombin activity; the data result shows that the thrombin antibody CH-1512 does not influence the enzymolysis activity of the thrombin antibody on a substrate S2228 after being combined with thrombin;

FIG. 2: the effect of anti-thrombin antibody H1512-12 on human thrombin activity; the data result shows that the thrombin antibody H1512-12 does not influence the enzymolysis activity of the thrombin antibody on a substrate S2228 after being combined with thrombin;

FIG. 3: effect of different concentrations of thrombin antibody on normal human plasma APTT values; the data result shows that the APTT value of the normal human plasma is prolonged along with the increase of the concentration of the antibody H1512-12; the increase in APTT value of H1512-12 also peaked at 33.6 seconds at the highest concentration of 3200 nM;

FIG. 4: the test of the drug effect of the antibody in a rat body shows that the intravenous injection of the antibody H1512-12 has the influence on the formation of the thrombus of the abdominal main vein induced by ferric chloride; the data result shows that tail vein injection H1512-12 can obviously inhibit the generation of thrombus under the dosage of 6mg/kg and 12 mg/kg; the mean thrombus weights were reduced to 2.91 mg and 0.18 mg for the H1512-12-6mg/kg group and the H1512-12-12mg/kg group, respectively, with a statistically very significant difference (p <0.001) compared to the mean thrombus weight of 10.75 mg for the isotype antibody group (IgG-6 mg/kg);

FIG. 5: post-operative thrombus weight of cynomolgus monkey arteriovenous anastomosis model showed that at a dose of 6mg/kg, H1512-12 was able to significantly inhibit the formation of cynomolgus monkey thrombus, with a statistically very significant difference (. about.. p <0.001) compared to the average thrombus weight of the isotype antibody group (IgG-6 mg/kg).

Detailed Description

Term

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise 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 invention belongs.

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

The "antibody" of the present invention refers to an immunoglobulin, 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, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, 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 IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

In the present invention, the antibody light chain of the present invention may further comprise a light chain constant region comprising a human-or rabbit-derived kappa, lambda chain or a variant thereof.

In the present invention, the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising human or rabbit 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 regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues of the LCVR and HCVR regions of the antibody or antigen-binding fragment of the invention are in number and position in accordance with known Kabat numbering convention (LCDR1-3, HCDE2-3) or in accordance with Kabat and chothia numbering convention (HCDR 1).

The antibody of the present invention includes a rabbit-derived antibody, a chimeric antibody, a humanized antibody, and preferably a humanized antibody.

The term "Lagomorpha" refers to members of the taxonomic order Lagomorpha, including the lagomorphae (Leporidae) (e.g., hares and rabbits) and the lagomorphae (Ochotonidae) (rabbits). In a most preferred embodiment, the lagomorph is a rabbit. The term "rabbit" as used herein refers to an animal belonging to the family lagonidae (leporidae).

The term "rabbit derived antibody" is in the present invention a monoclonal antibody to 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 monoclonal antibodies that express antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the rabbit thrombin antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a rabbit kappa, lambda chain or a variant thereof, or further comprise a heavy chain constant region of a rabbit IgG or a variant thereof.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a rabbit-derived antibody with a constant region of a human antibody. The chimeric antibody is established, a rabbit anti-variable region gene can be obtained by using a phage display technology, the rabbit variable region gene and a human constant region gene are connected into a chimeric gene and then inserted into an expression vector, and finally, the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the invention, the antibody light chain of the thrombin chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the thrombin chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably a human IgG1, IgG2 or IgG4 heavy chain constant region, or an IgG1, IgG2 or IgG4 variant which uses amino acid mutations (such as YTE mutations) to prolong the half-life of the antibody in serum.

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting rabbit 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 rabbit 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 can be found in the "VBase" human germline sequence database (at the Internet)www.mrccpe.com.ac.uk/vbaseAvailable), and found in Kabat, e.a. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity. The humanized antibodies of the invention also include humanized antibodies after further affinity maturation of the CDRs by phage display. In a preferred embodiment of the invention, the rabbit CDR sequences of the humanized antibody to thrombin are selected from the group consisting of SEQ ID NO 7,8,9,10,11 or 12; human antibody variable region frameworks were selected by design, wherein the heavy chain FR region sequences on the antibody heavy chain variable region were derived from the combined sequences of human germline heavy chain IGHV3-66 x 01 and hjh 4.1.1; wherein the light chain FR region sequence of the variable region of the light chain of the antibody is derived from a human germlineHeavy chain IGLV4-60 x 01 and hjk4.1 combined sequence. To avoid reduced immunogenicity and resulting reduced activity, the human antibody variable regions may be subjected to minimal back mutations to maintain activity.

Grafting of CDRs can result in a decrease in affinity of the resulting thrombin antibody or antigen-binding fragment thereof for the antigen due to the framework residues that are contacted with the antigen. Such interactions may be the result of a high degree of somatic mutation. 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 thrombin 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 the rabbit 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 rabbit 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.

The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments that retain the ability of the antibody to specifically bind an antigen (e.g., thrombin). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')₂A fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) single domain or dAb fragments (Ward et al, (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) an isolated Complementarity Determining Region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by synthetic linkers using recombinant methods, allowing them to be generated as a pair in which the VL and VH regions are pairedA single protein chain which is a monovalent molecule (known as single chain fv (scFv); see, for example, Bird et al (1988) Science242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Such antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

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

The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat e.a. et al, (1991) Sequences of proteins of immunological interest, nih Publication 91-3242). As used herein, the Kabat definition of CDRs applies only to the

CDRs

1, 2, and 3 of the light chain variable domain (CDR L1, CDR L2, CDR L3 or L1, L2, L3), and the

CDRs

2 and 3 of the heavy chain variable domain (CDR H2, CDR H3 or H2, H3).

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 "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., epipope Mapping Protocols in Methods in Molecular B biology, volume 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^-7M, e.g. less than about 10^-8M、10^-9M or 10^-10M or less affinity (KD) binding.

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

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 nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of a host cell upon introduction into the host cell so as to be replicated along with the host genome (e.g., non-episomal mammalian vectors).

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

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, 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.

The engineered antibodies or antigen binding fragments of the invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains may be cloned and recombined into a GS expression vector. 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 an antigen binding 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" and "treatment," when applied to an animal, human, experimental 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" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by a reagent, 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 of the binding compounds of the invention, either internally or externally to a patient who has 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 useful 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 invention (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of each disease of interest, they should alleviate the symptoms of the disease of interest in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student's t-test, the chi-square test, the U-test by Mann and Whitney, the Kruskal-Wallis test (H-test), the Jonckhere-Terpstra test, and the Wilcoxon test.

"conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) so that changes can be made frequently without changing the biological activity 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.

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.

"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" 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, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned; two sequences are 95% homologous if there are 95 matches or homologies at 100 positions in the two sequences. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology.

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

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

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

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

Second, example and test

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

Example 1 preparation of Thrombin antigen and protein for detection

1. Related thrombin and prothrombin sequences

The sequence coding human prothrombin (h-prothrombin), mouse prothrombin (m-prothrombin) and cynomolgus monkey prothrombin (cyno-prothrombin) were synthesized by Asahi-crown biotech development Limited company of CRO (all the above prothrombin proteins were the template sequences designed by the present invention), His-tagged human prothrombin (h-prothrombin-His), Flag-tagged human prothrombin (h-prothrombin-Flag), His-tagged mouse prothrombin (m-prothrombin-His) and His-tagged cynomolgus monkey prothrombin (cyno-prothrombin-His) were cloned into pTT5 vector (Biovector, Cat #:102762), respectively, and transient expression was achieved in HEK293E cells. Prothrombin (prothrombin) obtained by recombinant expression is subjected to preliminary purification and in vitro activation to obtain thrombin (thrombomin), and is further purified, so that the obtained human thrombin (h-thrombomin-Flag) with Flag label is used for immunization, and the human thrombin (h-thrombomin-His) with His label, mouse thrombin (m-thrombomin-His) with His label and cynomolgus monkey thrombin (cyno-thrombomin-His) with His label are used for antibody test screened in vitro.

SEQ ID NO:1

Human prothrombin with Flag tag

MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRANTFLEEVRKGNLERECVEETCSYEEAFEALESSTATDVFWAKYTACETARTPRDKLAACLEGNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVCGQDQVTVAMTPRSEGSSVNLSPPLEQCVPDRGQQYQGRLAVTTHGLPCLAWASAQAKALSKHQDFNSAVQLVENFCRNPDGDEEGVWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDEDSDRAIEGRTATSEYQTFFNPR

SEQ ID NO:2

His-tagged human prothrombin

SEQ ID NO:3

Crab eating macaque prothrombin with his label

MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRASSGFLEEVFKGNLERECVEETCSYEEAFEALESSTATDAFWAKYTACETARTSRDTLAACLEGNCAEDLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTVRREECSIPVCGQDQVTVVMTPRSSVNLSLPSEECVPDRGRQYQGHLAVTTHGLPCLAWASAQAKALSKHQDFDSAVQLVENFCRNPDGDEEGVWCYVAGKPGDFEYCDLNYCEEAVDEETGDGLGEDPDRAIEGRTATSEYQTFFDPR

SEQ ID NO:4

Mouse prothrombin with his label

MSHVRGLGLPGCLALAALVSLVHSQHVFLAPQQALSLLQRVRRANSGFLEELRKGNLERECVEEQCSYEEAFEALESPQDTDVFWAKYTVCDSVRKPRETFMDCLEGRCAMDLGVNYLGTVNVTHTGIQCQLWRSRYPHKPEINSTTHPGADLKENFCRNPDSSTTGPWCYTTDPTVRREECSVPVCGQEGRTTVVMTPRSGGSKDNLSPPLGQCLTERGRLYQGNLAVTTLGSPCLPWNSLPAKTLSKYQDFDPEVKLVENFCRNPDWDEEGAWCYVAGQPGDFEYCNLNYCEEAVGEENYDVDESIAGRTTDAEFHTFFNEK

Note: the underlined part of the sequence is the thrombin sequence with the corresponding tag.

2. Purification and in vitro activation of prothrombin-related recombinant proteins, purification of thrombin-related recombinant proteins and recombinant antibodies

1) Purification steps of His-tagged human prothrombin, His-tagged mouse prothrombin and His-tagged cynomolgus monkey prothrombin:

and (4) centrifuging the cell expression supernatant sample at a high speed to remove impurities. The nickel column was equilibrated with PBS buffer (pH 7.4), washed 2-5 column volumes, and the supernatant sample was applied to a Ni Sepharose excel column at a flow rate. The column was washed with PBS buffer to A₂₈₀The reading is reduced to the baseline, the chromatographic column is washed by PBS +10mM imidazole to remove non-specifically bound impure proteins, the effluent is collected, finally the target protein is eluted by PBS containing 300mM imidazole, and the elution peak is collected. The collected eluate was concentrated and the sample buffer was exchanged for PBS solution using a desalting column for subsequent in vitro activation and further purification.

2) Purification step of the human prothrombin recombinant protein with Flag tag:

the sample was centrifuged at high speed to remove impurities and concentrated to the appropriate volume. The flag affinity column was equilibrated with 0.5 × PBS (pH 7.4) and washed 2-5 column volumes. And (4) loading the cell expression supernatant sample after impurity removal on a column. Wash column with 0.5 XPBS to A₂₈₀The reading dropped to baseline. The column was washed with PBS, and the contaminating proteins were washed and collected. The protein of interest was eluted with TBS buffer containing 100. mu.g/ml 3 XFlag polypeptide and collected for subsequent in vitro activation and further purification.

3) In vitro activation of a prothrombin-related recombinant protein and further purification of the thrombin-related protein after activation:

prothrombin obtained by the above-mentioned preliminary affinity chromatography (nickel column or Flag affinity column) was purified by the mass ratio of 100: 1 was mixed well with Ecarin enzyme and incubated overnight at room temperature. After the activated sample is centrifuged at high speed to remove the precipitate, it is further purified by ion exchange column and size exclusion chromatography SEC.

3.1) ion exchange

The his-labeled human thrombin, the his-labeled mouse thrombin and the his-labeled cynomolgus monkey thrombin were purified by using a cation exchange column SP HP column, and the flag-labeled human thrombin was further purified by using an anion exchange column QHP column. The cation exchange buffer solution A was 10mM PB, pH6.8 buffer solution, the buffer solution B was 10mM PB, pH6.8, 1M NaCl buffer solution, the anion exchange buffer solution A was 10mM Tris-HCl, pH8.5 buffer solution, the buffer solution B was 10mM Tris-HCl, pH8.5, 1M NaCl buffer solution.

The ion exchange column is equilibrated with 5 column volumes of buffer solution A, the activated thrombin sample exchanged to buffer solution A is loaded at a certain flow rate, and after loading is finished, the column is equilibrated with buffer solution A until the absorption value of A280 reaches the baseline. Eluting with buffer B from 0% to 80% concentration gradient within 20 column volume time, collecting each elution peak, identifying the component of the target protein by SDS-PAGE, and further verifying by mass spectrum and peptide map.

3.2) size exclusion chromatography

The SEC column (superdex75) was equilibrated with PBS (pH 7.4) in advance, the sample was loaded and eluted with PBS as a mobile phase, and each eluted peak was collected, and the fractions of the target protein were identified by SDS-PAGE and further confirmed by mass spectrometry and peptide mapping.

4) Purification of recombinant antibodies

And centrifuging the cell expression supernatant sample at a high speed to remove impurities, purifying the hybridoma expression supernatant by using a protein G column and purifying the recombinant antibody expression supernatant by using a protein A column. The column was washed with PBS (pH 7.4) until the a280 reading dropped to baseline. The target protein was eluted with 100mM acetic acid, pH3.0, and neutralized with 1M Tris-HCl, pH 8.0. After the eluted sample was appropriately concentrated, it was further purified by gel chromatography Superdex200(GE) equilibrated with PBS to remove the aggregates, collect the monomer peak, and split for future use.

Example 2 preparation of monoclonal antibodies against human Thrombin

1. Immunization

Anti-human thrombin antibodies were generated by immunizing rabbits. New Zealand white rabbits, female, 2-3kg are used for the experiment. (Shanghai Jia Dry Biotech Co., Ltd., animal production license number: SCXK (Shanghai) 2010-0028). A breeding environment: SPF grade. After the rabbit is purchased, breeding the rabbit in a laboratory environment for 1 week, and adjusting the light/dark period at 12/12 hours at the temperature of 20-25 ℃; the humidity is 40-60%. The rabbit which has been adapted to the environment is immunized. The immunizing antigen is Flag-tagged human thrombin (shown by double underlining of SEQ ID NO: 1).

Immunization protocol: the antigen was emulsified with Freund's adjuvant (Sigma Cat No. F5881/F5506): freund's complete adjuvant (CFA) was used for priming, and Freund's incomplete adjuvant (IFA) was used for boosting. The ratio of antigen to adjuvant was 1: 1500. mu.g/mouse (prime) and 250. mu.g/mouse (boost). On day 0, 500. mu.g/single emulsified antigen was injected subcutaneously at multiple points, and the immunization was boosted every two weeks for 6-8 weeks after the first immunization.

Blood was collected on days 22, 36, 50 and 64, and the antibody titer in rabbit serum was determined by ELISA. After 4-immunization, rabbits with high antibody titers and titers approaching the plateau were selected, and spleen and bone marrow cells were taken for RNA extraction and banking.

2. Construction of rabbit-derived phage single-chain antibody library

The spleen and bone marrow of rabbits were taken, and total RNA of tissues was extracted using Trizol (Invitrogen Cat No. 15596-018). Using PrimeScript^TMII 1st Strand cDNA Synthesis Kit (Takara Cat No.6210A) was used to obtain cDNA by reverse transcription. Primers for constructing libraries were designed and synthesized according to the report of Christoph Rader et al (J.biol.chem.2000,275:13668-13676) (Kingchi). Single-chain antigen-binding fragments were obtained by three rounds of PCR reactions. LA Tag (Takara Cat No. RR02MB) was used for all PCR reactions. Respectively amplifying cDNA as a template in the first round of PCR to obtain heavy chain variable region sequences and light chain variable region sequences; in the second round of PCR, the first round of products are used as templates, Sfi1 enzyme cutting site sequences are introduced at the 5 'end of the heavy chain variable region and the 3' end of the light chain variable region, and connecting sequences are introduced at the 3 'end of the heavy chain variable region and the 5' end of the light chain variable region; and the third round of PCR uses the heavy chain variable region and the light chain variable region of the second round of PCR product as a template together to carry out bridging PCR to obtain the single-chain antigen binding fragment with the heavy chain variable region in front and the light chain variable region in back.

The single-chain antigen binding fragment and the modified library construction vector pCantab5E (Amersham Biosciences/GE Cat No.27-9400-01) are subjected to enzyme digestion by Sfi1(NEB Cat No. # R0123L), and the obtained product is subjected to electrophoresis and then subjected to enzyme digestion by using a kit

The Gel Extraction Kit (Omega Cat No. D2500-02) was purified and recovered. Then, the DNA fragments were ligated with T4DNA ligase (NEB Cat No. # M0202L) at 16 degrees for 16 to 18 hours, purified and recovered by the above-mentioned kit, and finally eluted with deionized water. Mu.g of the ligation product was taken and 1 branch of electrotransformation-competent TG1 (L)ucigen Cat No.60502-2), setting the parameters of an electric converter (Bio Rad Micropulser) to 2.5kV, 200 omega and 25uF, and carrying out electric conversion. The transformation was repeated 10 times, plated and cultured in an inverted medium at 37 ℃ for 16 to 18 hours. All colonies were scraped off and mixed together, glycerol was added to a final concentration of 15%, and stored at-80 ℃ until use. The library has a storage capacity of more than 1 × 10⁸。

3. Screening phage single-chain antibody library to obtain positive monoclonal resisting human thrombin

The phage single-chain antibody library was packed and concentrated, and then subjected to three rounds of panning. Phage library (10)¹²～10¹³Pfu) was suspended in 1ml of 2% MPBS (PBS containing 2% skim milk powder) and 100. mu.l was added

M-280Streptavidin (Invitrogen Cat No.11206D), was repeatedly turned over on a turntable and blocked at room temperature for 1 hour. The tube was placed on a magnetic rack for 2 minutes, the Dynabeads were removed, and the phage library was transferred to a new tube. 2. mu.g/ml biotin-labeled his-tagged human thrombin (shown by double underlining of SEQ ID NO:2) was added to the phage pool after blocking, and the mixture was placed on a turntable and repeatedly turned over for 1 hour. At the same time, 100. mu.l of Dynabeads were suspended in 1ml of 2% MPBS, placed on a turntable and repeatedly turned over, and sealed at room temperature for 1 hour. Place the tube on a magnetic stand for 2 minutes and aspirate off the blocking solution. The blocked Dynabeads are added into a phage library and thrombin mixed solution with his labels, and the mixture is placed on a rotary table and repeatedly turned for 15 minutes. The tube was placed on a magnetic stand for 2 minutes and the mixture was aspirated. Dynabeads were washed 10 times with 1ml PBST (PBS containing 0.1% Tween-20), and 0.5ml 1MG/ml trypsin (Sigma Cat No. T1426-250MG) was added and placed on a turntable for repeated inversion incubation for 15 minutes for elution. The eluted phage were directly infected with E.coli TG1 in log phase, titered and amplified for concentration for the next round of panning. Second round panning phage library (10)¹¹～10¹²Pfu), biotin-labeled his-tagged human thrombin concentration was reduced to 0.5. mu.g/ml, binding time was shortened to 30 minutes, and PBST washing times were increased to 15 times. Phage library was elutriated in the third round (10)¹⁰～10¹¹Pfu), biotin-labeled his-tagged human thrombin concentration was reduced to 0.1. mu.g/ml, binding time was shortened to 30 minutes, and PBST washing times were increased to 20 times. After three rounds of panning, eluted phage-infected E.coli TG1 were plated and single clones were randomly picked for phage ELISA.

The clone is inoculated in a 96-hole deep-hole plate and cultured for 16-18 hours at 37 ℃. A small amount of the suspension was inoculated into another 96-well deep-well plate until the OD600 reached about 0.5, and M13K07 helper phage (NEB Cat No. N0315S) was added for packaging. The cells were centrifuged at 4000g for 10 minutes to remove the cells, and the culture broth was aspirated for human thrombin-binding ELISA (see test example 1). Screening to obtain a positive clone MAB-1512 as a key molecule for subsequent humanization. The positive clone strains are frozen in time and preserved and sent to a sequencing company for sequencing, and the amino acid sequence corresponding to the positive clone MAB-1512DNA sequence is determined as follows:

>MAB-1512VH

QSLEESGGRLITPGGSLTLTCTASGFSLSNYDMSWVRQAPGKGLEWIGMIETDGSIFHASWVNGRFTISKTATTVDLKMTSLTTEDTATYFCVRGSNDYDAYGYPYFTLWGQGTLVTVSS

SEQ ID NO：5

>MAB-1512VL

ELVLTQSPSASATLGASAKLTCTLSSAHETYTIDWYQQHQGEAPQYLMELKSDGSYNKGAGVPDRFSGSSSGADRYLMIPSVQADDEATYHCGADFSDGYVFGGGTQLTVTG

SEQ ID NO：6

note: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the FR sequences are in italics and the CDR sequences are underlined.

TABLE 1 CDR region sequences of the respective heavy and light chains

The positive clone MAB-1512 was subjected to molecular cloning, expression and purification in example 4 to obtain a chimeric antibody CH-1512, and binding activity was identified with respect to human thrombin and prothrombin (test example 2 and test example 3). Meanwhile, the chimeric antibody CH-1512 was examined for its cross-binding activity with thrombin of different species (see test example 4), affinity with thrombin of different species (see test example 5), and effect on human thrombin activity (see test example 6). The results of the measurements are shown in table 2 below and fig. 1.

TABLE 2 in vitro Activity of chimeric antibodies with different species of Thrombin and prothrombin

The results in Table 2 show that CH-1512 has strong binding to thrombin of different species, including human, murine, and simian thrombin, but weak binding to human prothrombin. The results in FIG. 1 show that CH-1512 does not affect the enzymatic activity of S2228 after binding to thrombin.

Example 3 humanization of Rabbit anti-human Thrombin monoclonal antibodies

1. Anti-human Thrombin antibody CH-1512 humanized framework selection

By comparing an IMGT human antibody heavy and light chain variable region germline gene database with MOE software, respectively selecting heavy and light chain variable region germline genes with high homology with the chimeric antibody CH-1512 as templates, respectively transplanting CDRs of the rabbit-derived antibody into corresponding human-derived templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Wherein the amino acid residues are determined and annotated by the Kabat numbering system.

The humanized heavy chain templates of the chimeric antibody CH-1512 are IGHV3-66 x 01 and hjh 4.1.1, the humanized light chain templates are IGLV4-60 x 01 and hjk4.1, and the humanized variable region sequences are as follows:

>H1512-1VH(CDR graft)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYDMSWVRQAPGKGLEWVSMIETDGSIFHASWVNGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSNDYDAYGYPYFTLWGQGTLVTVSS

SEQ ID NO：13

>H1512-1VL(CDR graft)

QPVLTQSSSASASLGSSVKLTCTLSSAHETYTIDWHQQQPGKAPRYLMKLKSDGSYNKGAGVPDRFSGSSSGADRYLTISNLQLEDEADYYCGADFSDGYVFGGGTKVEIK

SEQ ID NO：14

2. Design of back mutations

The back-mutation design of CH-1512 is shown in Table 3 below:

TABLE 3 CH-1512 Back-mutation design

Note: p2 is mutated back to L as denoted by P2L according to the Kabat numbering system.

Grafted, represents a rabbit antibody CDR-Grafted human germline FR region sequence.

Specific sequences after mutation are exemplified as follows:

H1512_VL.1D：

ELVLTQSSSASASLGSSVKLTCTLSSAHETYTIDWYQQQPGKAPRYLMELKSDGSYNKGAGVPDRFSGSSSGADRYLTISNLQLEDEATYHCGADFSDGYVFGGGTKVEIK

SEQ ID NO：15

H1512_VH.1F：

EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYDMSWVRQAPGKGLEWIGMIETDGSIFHASWVNGRFTISKDNSKNTVYLQMNSLRAEDTAVYFCVRGSNDYDAYGYPYFTLWGQGTLVTVSS

SEQ ID NO：16

H1512_VL.1E：

QLVLTQSSSASASLGSSVKLTCTLSSAHETYTIDWYQQQPGKAPRYLMELKSDGSYNKGAGVPDRFSGSSSGADRYLTISNLQLEDEATYHCGADFSDGYVFGGGTKVEIK

SEQ ID NO：17

the light and heavy chain variable regions after the back mutation were recombined, and the obtained antibody was cloned and expression-purified by the preparation method of example 4, and its binding activity to human thrombin protein was examined by the ELISA test of test example 3. The following antibodies (table 4) were finally selected according to the test results (table 5) for the next in vivo and in vitro activity test.

TABLE 4 humanized antibody light and heavy chain variable region sequences

Antibody numbering	Heavy chain variable region	Light chain variable region
			H1512-1	SEQ ID NO：13	SEQ ID NO：14
H1512-8	SEQ ID NO：13	SEQ ID NO：15
			H1512-12	SEQ ID NO：16	SEQ ID NO：17

The antibodies of the present invention were tested for their activity in binding to human thrombin and prothrombin (test examples 2 and 3), as well as for their cross-binding activity to thrombin of different species (see test example 4), their affinity to thrombin of different species (see test example 5), and their effect on human thrombin activity (see test example 6).

The test results show that the humanized antibodies H1512-12 and CH 1512 have strong binding force with thrombin of different species including human, murine and monkey thrombin. The results in FIG. 2 show that H1512-12, like CH 1512, does not affect the enzymatic activity of S2228, when bound to thrombin.

Example 4 preparation of recombinant and humanized antibodies

The antibody was selected from the combination of the constant region of human heavy chain IgG 4/light chain kappa with the variable regions, and the Fc region was mutated at S228P to increase the stability of the IgG4 antibody, or other mutations known in the art to increase its performance.

Heavy chain constant region:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：18

light chain constant region:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：19

1. molecular cloning of recombinant antibodies

And sequencing the candidate antibody molecules obtained by phage display screening to obtain a variable region coding gene sequence. Designing a primer head-tail primer by using a sequence obtained by sequencing, constructing each antibody VH/VK gene fragment by using a sequencing gene as a template through PCR, and carrying out homologous recombination with an expression vector pHr (signal peptide and hIgG4 constant region gene (CH1-FC/CL) fragment) to construct a recombinant antibody full-length expression plasmid VH-CH 1-FC-pHr/VK-CL-pHr.

2. Molecular cloning of humanized antibodies

The humanized antibody full-length expression plasmid VH-CH1-FC-pHr/VK-CL-pHr is constructed by performing homologous recombination on an antibody sequence after human source design and codon optimization to generate a coding gene sequence with human codon preference, designing primers and performing PCR to build each antibody VH/VK gene fragment, and then performing homologous recombination on the antibody VH/VK gene fragment and an expression vector pHr (with signal peptide and a constant region gene (CH1-FC/CL) fragment).

3. Expression and purification of recombinant and humanized antibodies

Plasmids expressing the light and heavy chains of the antibody respectively transfect HEK293E cells in a ratio of 1:1.5, and expression supernatant is collected after 6 days, and is subjected to high-speed centrifugation to remove impurities and purification by a protein A column. The column was washed with PBS until the a280 reading dropped to baseline. The target protein was eluted with 100mM acetic acid, pH3.0, and neutralized with 1M Tris-HCl, pH 8.0. After the eluted sample was appropriately concentrated, it was further purified by gel chromatography Superdex200(GE) equilibrated with PBS to remove the aggregates, collect the monomer peak, and split for future use.

The humanized antibody H1512-12 was obtained.

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

Test example 1 ELISA assay for binding of phage-displayed Single-chain antibody to human Thrombin protein

Streptavidin (Sigma, Cat No. S4762-5MG) was diluted to a concentration of 5. mu.g/ml with PBS buffer pH7.4 (Sigma, Cat No. P4417-100TAB), added to a 96-well plate at a volume of 50. mu.l/well, and placed in an incubator at 37 ℃ for 2 hours. After discarding the liquid, 200. mu.l/well of 5% skim milk (light skim milk powder) blocking solution diluted with PBS was added, and the block was incubated at 37 ℃ for 2.5 hours. After blocking was complete, the blocking solution was discarded, and the plate was washed 3 times with PBST buffer (pH7.4PBS containing 0.05% tween-20), then 50. mu.l/well of biotin-labeled his-tagged human thrombin protein (internal production, shown by double underlining of SEQ ID NO:2) diluted to 0.125. mu.g/ml with a sample diluent (pH7.4PBS containing 1% BSA) was added and placed at 4 ℃ for 16-18 hours. The reaction solution in the ELISA plate was discarded, the plate was washed with PBST 5 times, and then 100. mu.l/well of phage culture solution diluted 1:1 in sample diluent was added, followed by incubation for 1 hour in an incubator at 37 ℃. After incubation, the plates were washed 5 times with PBST and 100. mu.l/well diluted with sample diluent was addedMouse HRP/anti-M13conjugated secondary antibody (GE, Cat No.27-9421-01) was 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 5-10min, adding 50 μ l/well 1M H₂SO₄The reaction was stopped and absorbance was read at a wavelength of 450nm using a NOVOStar microplate reader.

Test example 2 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 human thrombin. His-tagged thrombin labeled with a biotin labeling kit (Dongchan chemical, Cat No. LK03) is fixed in a 96-well ELISA plate by binding with streptavidin coated in the ELISA plate, and the strength of a signal after the addition of an antibody is used for judging the binding activity of the antibody and the thrombin, and a specific experimental method is as follows.

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

TABLE 5 binding Activity of humanized antibodies and antigens

MAB-1512 humanized antibodies	EC50(nM)
		H1512-1	43.4
H1512-8	19.31
		H1512-12	0.16

The results show that the thrombin humanized antibody obtained by screening has higher binding activity with the human thrombin protein antigen.

Test example 3 ELISA assay for binding of Thrombin antibody to human Prothrombin protein

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

The his-tagged human prothrombin (SEQ ID NO:2) was diluted to a concentration of 10. mu.g/ml with PBS (Sigma, Cat No. P4417-100TAB) buffer, pH7.4, added to a 96-well plate at a volume of 100. mu.l/well, and left overnight (16-18 hours) at 4 ℃. After discarding the liquid, 200. mu.l/well of 5% skim milk (light skim milk powder) blocking solution diluted with PBS was added, and the block was incubated at 37 ℃ for 2.5 hours. Sealing ofAfter completion, the blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH7.4PBS containing 0.05% tween-20), 50. mu.l/well of the thrombin test antibody of the present invention diluted in a sample diluent (pH7.4PBS containing 1% BSA) was 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 an HRP-labeled secondary goat anti-human antibody (Jackson Immuno Research, Cat No. 109-. Washing the plate with PBST for 5 times, adding 50 μ l/well TMB chromogenic substrate (KPL, Cat No.52-00-03), incubating at room temperature for 10-15min, adding 50 μ l/well 1M H₂SO₄The reaction was stopped and the absorbance was read at a wavelength of 450nm using a NOVOStar microplate reader to calculate the EC50 value for the binding of thrombin antibody to human Pro thrombin.

Test example 4 Cross-binding experiments of anti-Thrombin antibodies with different classes of Thrombin

To test the in vitro binding capacity of the screened thrombin antibodies to thrombin from different species, mouse and cynomolgus monkey thrombin were used for in vitro binding assays.

His-tag antibody (GenScript, Cat No. A00174) was diluted to a concentration of 0.5. mu.g/ml with PBS buffer (Sigma, Cat No. P4417-100TAB) at pH7.4, added to a 96-well microplate at a volume of 100. mu.l/well, and placed in an incubator at 37 ℃ for 2 hours. After discarding the liquid, 200. mu.l/well of 5% skim milk (light skim milk powder) blocking solution diluted with PBS was added, and the cells were incubated at 37 ℃ for 2.5 hours or left overnight (16-18 hours) at 4 ℃ for blocking. After blocking was complete, the blocking solution was discarded, and after washing the plate 5 times with PBST buffer (pH7.4PBS containing 0.05% tween-20), 50. mu.l/well of his-tagged mouse thrombin protein (SEQ ID NO:3 double underlined) or his-tagged cynomolgus thrombin protein (SEQ ID NO:4 double underlined) diluted to 0.5. mu.g/ml with a sample diluent (pH7.4PBS containing 1% BSA) was added, and incubated at 37 ℃ for 2 hours in an incubator. After the incubation is finished, reaction liquid in the enzyme label plate is discarded, the plate is washed for 6 times by PBST, 50 mu l/hole of thrombin antibody to be detected with different concentrations diluted by sample diluent is added, and the mixture is incubated for 2 hours in an incubator at 37 ℃. After incubation, the plates were washed 5 times with PBST and HRP-labeled sheep diluted with sample diluent at 100. mu.l/well were addedAnti-human secondary antibody (Jackson Immuno Research, Cat No.109-035-003) was 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 H₂SO₄The reaction was stopped, absorbance was read at a wavelength of 450nm using a NOVOStar microplate reader, and the EC50 value for the binding of thrombin antibody to mouse and cynomolgus monkey thrombin was calculated. The results are shown in Table 6.

TABLE 6 in vitro Activity of humanized antibodies binding to different species of Thrombin

The results in Table 6 show that the humanized antibodies H1512-12, like CH 1512, bind strongly to thrombin of different species, including murine and simian thrombin.

Test example 5 BIAcore assay for thrombin antibody affinity

The human anti-capture antibody was covalently coupled to a CM5 biosensor chip (Cat. # BR-1000-12, GE) according to the method described in the specification of the human anti-capture kit (Cat. # BR-1008-39, GE) to affinity-capture the antibody to be detected, and then thrombin or prothrombin (his-labeled human thrombin or prothrombin) was passed over the surface of the chip, and a binding and dissociation curve was obtained by detecting the reaction signal in real time using a Biacore instrument, and affinity values were obtained by fitting, see table Y below. After each cycle of dissociation in the experiment was completed, the biochips were washed and regenerated with a regeneration solution prepared in a human anti-capture kit. The results are shown in Table 7.

TABLE 7 affinity of humanized antibodies to human thrombin and human prothrombin

Substrate	H1512-12 BIAcore KD(M)
		Human thrombin	2.79E-9
Human prothrombin	2.64E-8

The results show that the humanized antibody H1512-12 of the present invention has strong affinity with human thrombin, but weak affinity with human prothrombin.

Test example 6 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.

The his-tagged human thrombin was diluted with a PBS gradient at pH7.4 to a concentration of 100nM and 25. mu.l/well was added to a black-walled clear-bottomed 96-well plate. The thrombin test antibody of the invention was diluted with PBS in a gradient 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, sequence:

H-D-Phe-Pip-Arg-pNA.2HCl, synthesized by Gill Biochemical (Shanghai) Co., Ltd.) to a concentration of 4mM, 50. mu.l/well was added to the plate in 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. 2, in which thrombin and S2228 represent the OD values measured in the negative control wells, and 0.625:1, 1.25:1, 2.5:1, 5:1, 10:1, 20:1 represent the OD values measured at different molar ratios of the antibody to be tested to thrombin.

The results in FIG. 2 show that H1512-12 does not affect the enzymatic activity of S2228 after binding to thrombin.

Test example 7 APTT test on Normal human plasma

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

IgG is a negative control, i.e., human immunoglobulin obtained by purification using conventional affinity chromatography methods such as ProteinA; h1512-12 is the antibody to be detected with different concentrations in the invention.

The experimental results are shown in FIG. 3, and the APTT value of the normal human plasma is prolonged along with the increase of the concentration of the antibody H1512-12. The increase in APTT values also peaked at 33.6 seconds for H1512-12 at a maximum concentration of 3200nM (Table 8).

TABLE 8 increase in APTT values against normal human plasma for different concentrations of thrombin antibody

Test example 8 model of iron chloride-induced venous thrombosis

The thrombin antibody was injected into the tail vein to evaluate the effect of the antibody of the present invention on inhibition of thrombosis.

SD rats (purchased from shanghai sipel bikei laboratory animals llc) were acclimated in a laboratory environment for 7 days. After 20% urethane is injected into abdominal cavity for anesthesia, tail vein is injected for administration, and after 15 minutes of administration, 10% FeCl is infiltrated₃The 2 x 5 mm filter paper strip was attached to the exposed vein wall, the filter paper was removed after 3 minutes, the iron chloride-attached part was rinsed with an appropriate amount of physiological saline, the posterior vena cava blood vessel 10mm long above the iliac-lumbar vein below the renal vein was excised after 27 minutes, the thrombus in the lumen was peeled off, and the vessel was weighed on a ten-thousandth balance. Thrombus weight was recorded.

As shown in FIG. 4 and Table 9, the experimental results show that the tail vein injection of H1512-12 can significantly inhibit the generation of thrombus at the doses of 6mg/kg and 12 mg/kg. The average thrombus weights of the H1512-126 mg/kg group and the H1512-1212 mg/kg group were reduced to 2.91 mg and 0.18 mg, respectively, which were statistically significantly different from the average thrombus weight of 10.75 mg in the isotype antibody group (IgG-6 mg/kg).

TABLE 9 mean weights and standard errors of thrombi of each group

Test example 9 cynomolgus monkey arteriovenous anastomosis model

8 healthy cynomolgus monkeys (male and female halves, provided by Hainan Jingang biotechnology, Inc.) were acclimated in a laboratory environment for two weeks, weighed, and divided into 2 groups of 4 each based on weight balance. The patient is anesthetized with sutai, and then deep anesthesia is performed with pentobarbital. Before operation, the antibody medicine and the control medicine of the invention are injected into the lower limb vein of different groups slowly (the medicines and the dosage are shown in the table 10). The following steps were all performed on a 37 ℃ thermostatic operating table. The femoral artery and vein were isolated by surgery under the groin, with the left side then the right side. An arteriovenous cannula is operated. After the cannula was secured, the clamp was released (the left clamp was released 15 minutes after administration and the right clamp 25 minutes after administration) and timing was started simultaneously, and blood flow continued for 15 minutes. Clamping two ends of the intubation by using hemostatic forceps or a vascular clamp to prevent emboli from falling off, taking out the silicone tube, putting the silicone tube into a culture dish with normal saline, taking out the procoagulant line with thrombus by using surgical scissors, absorbing water for 3 seconds at two ends of the paper towel respectively, weighing by using a precision electronic scale, and calculating the net weight of the thrombus.

TABLE 10 cynomolgus monkey arteriovenous anastomosis model and postoperative thrombus weight

The experimental results are shown in FIG. 5, H1512-12 can significantly inhibit the generation of thrombi in macaques at the dose of 6mg/kg, and compared with the average thrombus weight of the negative antibody group (IgG-6mg/kg), the difference is very statistically significant.

The statistical methods are One-way ANOVA and student t test. P < 0.05, p < 0.01, p <0.001, compared to the isotype antibody group (IgG-6 mg/kg).

Example 10: pharmacokinetic testing of Thrombin antibodies of the invention

The blood concentration of the serum of the cynomolgus monkey after administration was measured by ELISA (see test example 4) using cynomolgus monkey as the test animal, and T1/2 and its major parameters of the test drug were calculated using Winnolin software and EXCEL. The pharmacokinetic behavior of the compounds of the invention in cynomolgus monkeys was studied and their pharmacokinetic profile was evaluated.

Experimental cynomolgus monkey, common grade, male, 3-5 years old, 3.5-4.5 kg, Chengdu Hua Xihai boundary medicine science and technology Limited company autotrophy. A breeding environment: a general area room. The laboratory environment adaptive feeding is carried out for not less than 3 days, the light/dark period is regulated for 12/12 hours, the temperature is 16-26 ℃, and the relative humidity is 40-70%. Cynomolgus monkeys were grouped into 3 groups. On the day of experiment, each cynomolgus monkey is injected with the tested medicine intravenously, and the administration dose is 3mg/kg and 10 mg/kg. The volume of intravenous injection is 10ml/kg, and the administration speed is 2-4 ml/min.

Blood sampling time points after administration were 0.25h,2h,4h,8h,1d,2d,3d,4d,7d,10d,14d,21d,28d,35 d. Taking 1mL of blood each time, anticoagulating heparin sodium, temporarily storing in an ice box, centrifuging at 1800g at 2-8 ℃ for 10 minutes, subpackaging, and storing at-70 ℃.

The blood concentration in serum was measured by ELISA and the T1/2 and its major parameters were calculated using Winnolin software and EXCEL as follows:

the pharmacokinetic parameters of the antibodies of the invention are shown in table 11 below:

TABLE 11 in vivo pharmacokinetic assay of the Thrombin antibody cynomolgus monkey

And (4) conclusion: the antibody of the invention has good drug absorption and obvious drug absorption effect.

Sequence listing

<110> Hengrui pharmaceuticals, Inc. of Jiangsu and Hengrui pharmaceuticals, Inc. of Shanghai

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

<130> 370019CG-360046

<160> 19

<170> PatentIn version 3.3

<210> 1

<211> 630

<212> PRT

<213> Artificial sequence

<220>

<223> Flag-tagged human prothrombin sequence

<400> 1

Met Ala His Val Arg Gly Leu Gln Leu Pro Gly Cys Leu Ala Leu Ala

1 5 10 15

Ala Leu Cys Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln

20 25 30

Gln Ala Arg Ser Leu Leu Gln Arg Val Arg Arg Ala Asn Thr Phe Leu

35 40 45

Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr

50 55 60

Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser Thr Ala Thr

65 70 75 80

Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr Pro

85 90 95

Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn Cys Ala Glu Gly Leu

100 105 110

Gly Thr Asn Tyr Arg Gly His Val Asn Ile Thr Arg Ser Gly Ile Glu

115 120 125

Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys Pro Glu Ile Asn Ser

130 135 140

Thr Thr His Pro Gly Ala Asp Leu Gln Glu Asn Phe Cys Arg Asn Pro

145 150 155 160

Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr Val

165 170 175

Arg Arg Gln Glu Cys Ser Ile Pro Val Cys Gly Gln Asp Gln Val Thr

180 185 190

Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser Val Asn Leu Ser Pro

195 200 205

Pro Leu Glu Gln Cys Val Pro Asp Arg Gly Gln Gln Tyr Gln Gly Arg

210 215 220

Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala

225 230 235 240

Gln Ala Lys Ala Leu Ser Lys His Gln Asp Phe Asn Ser Ala Val Gln

245 250 255

Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val

260 265 270

Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe Gly Tyr Cys Asp Leu

275 280 285

Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr Gly Asp Gly Leu Asp

290 295 300

Glu Asp Ser Asp Arg Ala Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr

305 310 315 320

Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly Glu Ala Asp Cys

325 330 335

Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu Asp Lys Thr Glu

340 345 350

Arg Glu Leu Leu Glu Ser Tyr Ile Asp Gly Arg Ile Val Glu Gly Ser

355 360 365

Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val Met Leu Phe Arg Lys

370 375 380

Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp

385 390 395 400

Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro Pro Trp Asp Lys Asn

405 410 415

Phe Thr Glu Asn Asp Leu Leu Val Arg Ile Gly Lys His Ser Arg Thr

420 425 430

Arg Tyr Glu Arg Asn Ile Glu Lys Ile Ser Met Leu Glu Lys Ile Tyr

435 440 445

Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp Arg Asp Ile Ala

450 455 460

Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser Asp Tyr Ile His Pro

465 470 475 480

Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser Leu Leu Gln Ala Gly

485 490 495

Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu Lys Glu Thr Trp Thr

500 505 510

Ala Asn Val Gly Lys Gly Gln Pro Ser Val Leu Gln Val Val Asn Leu

515 520 525

Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser Thr Arg Ile Arg Ile

530 535 540

Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Asp Glu Gly Lys Arg

545 550 555 560

Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys Ser

565 570 575

Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly Ile Val Ser Trp Gly Glu

580 585 590

Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr His Val Phe Arg

595 600 605

Leu Lys Lys Trp Ile Gln Lys Val Ile Asp Gln Phe Gly Glu Asp Tyr

610 615 620

Lys Asp Asp Asp Asp Lys

625 630

<210> 2

<211> 628

<212> PRT

<213> Artificial sequence

<220>

<223> His-tagged human prothrombin sequence

<400> 2

Met Ala His Val Arg Gly Leu Gln Leu Pro Gly Cys Leu Ala Leu Ala

1 5 10 15

Ala Leu Cys Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln

20 25 30

Gln Ala Arg Ser Leu Leu Gln Arg Val Arg Arg Ala Asn Thr Phe Leu

35 40 45

Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr

50 55 60

Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser Thr Ala Thr

65 70 75 80

Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr Pro

85 90 95

Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn Cys Ala Glu Gly Leu

100 105 110

Gly Thr Asn Tyr Arg Gly His Val Asn Ile Thr Arg Ser Gly Ile Glu

115 120 125

Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys Pro Glu Ile Asn Ser

130 135 140

Thr Thr His Pro Gly Ala Asp Leu Gln Glu Asn Phe Cys Arg Asn Pro

145 150 155 160

Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr Val

165 170 175

Arg Arg Gln Glu Cys Ser Ile Pro Val Cys Gly Gln Asp Gln Val Thr

180 185 190

Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser Val Asn Leu Ser Pro

195 200 205

Pro Leu Glu Gln Cys Val Pro Asp Arg Gly Gln Gln Tyr Gln Gly Arg

210 215 220

Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala

225 230 235 240

Gln Ala Lys Ala Leu Ser Lys His Gln Asp Phe Asn Ser Ala Val Gln

245 250 255

Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val

260 265 270

Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe Gly Tyr Cys Asp Leu

275 280 285

Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr Gly Asp Gly Leu Asp

290 295 300

Glu Asp Ser Asp Arg Ala Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr

305 310 315 320

Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly Glu Ala Asp Cys

325 330 335

Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu Asp Lys Thr Glu

340 345 350

Arg Glu Leu Leu Glu Ser Tyr Ile Asp Gly Arg Ile Val Glu Gly Ser

355 360 365

Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val Met Leu Phe Arg Lys

370 375 380

Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp

385 390 395 400

Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro Pro Trp Asp Lys Asn

405 410 415

Phe Thr Glu Asn Asp Leu Leu Val Arg Ile Gly Lys His Ser Arg Thr

420 425 430

Arg Tyr Glu Arg Asn Ile Glu Lys Ile Ser Met Leu Glu Lys Ile Tyr

435 440 445

Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp Arg Asp Ile Ala

450 455 460

Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser Asp Tyr Ile His Pro

465 470 475 480

Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser Leu Leu Gln Ala Gly

485 490 495

Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu Lys Glu Thr Trp Thr

500 505 510

Ala Asn Val Gly Lys Gly Gln Pro Ser Val Leu Gln Val Val Asn Leu

515 520 525

Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser Thr Arg Ile Arg Ile

530 535 540

Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Asp Glu Gly Lys Arg

545 550 555 560

Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys Ser

565 570 575

Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly Ile Val Ser Trp Gly Glu

580 585 590

Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr His Val Phe Arg

595 600 605

Leu Lys Lys Trp Ile Gln Lys Val Ile Asp Gln Phe Gly Glu His His

610 615 620

His His His His

625

<210> 3

<211> 626

<212> PRT

<213> Artificial sequence

<220>

<223> cynomolgus monkey prothrombin sequence with His label

<400> 3

Met Ala His Val Arg Gly Leu Gln Leu Pro Gly Cys Leu Ala Leu Ala

1 5 10 15

Ala Leu Cys Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln

20 25 30

Gln Ala Leu Ser Leu Leu Gln Arg Val Arg Arg Ala Ser Ser Gly Phe

35 40 45

Leu Glu Glu Val Phe Lys Gly Asn Leu Glu Arg Glu Cys Val Glu Glu

50 55 60

Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser Thr Ala

65 70 75 80

Thr Asp Ala Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr

85 90 95

Ser Arg Asp Thr Leu Ala Ala Cys Leu Glu Gly Asn Cys Ala Glu Asp

100 105 110

Leu Gly Thr Asn Tyr Arg Gly His Val Asn Ile Thr Arg Ser Gly Ile

115 120 125

Glu Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys Pro Glu Ile Asn

130 135 140

Ser Thr Thr His Pro Gly Ala Asp Leu Gln Glu Asn Phe Cys Arg Asn

145 150 155 160

Pro Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr

165 170 175

Val Arg Arg Glu Glu Cys Ser Ile Pro Val Cys Gly Gln Asp Gln Val

180 185 190

Thr Val Val Met Thr Pro Arg Ser Ser Val Asn Leu Ser Leu Pro Ser

195 200 205

Glu Glu Cys Val Pro Asp Arg Gly Arg Gln Tyr Gln Gly His Leu Ala

210 215 220

Val Thr Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala Gln Ala

225 230 235 240

Lys Ala Leu Ser Lys His Gln Asp Phe Asp Ser Ala Val Gln Leu Val

245 250 255

Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val Trp Cys

260 265 270

Tyr Val Ala Gly Lys Pro Gly Asp Phe Glu Tyr Cys Asp Leu Asn Tyr

275 280 285

Cys Glu Glu Ala Val Asp Glu Glu Thr Gly Asp Gly Leu Gly Glu Asp

290 295 300

Pro Asp Arg Ala Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr Gln Thr

305 310 315 320

Phe Phe Asp Pro Arg Thr Phe Gly Leu Gly Glu Ala Asp Cys Gly Leu

325 330 335

Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu Asp Lys Thr Glu Gly Glu

340 345 350

Leu Leu Glu Ser Tyr Ile Asp Gly Arg Ile Val Glu Gly Trp Asp Ala

355 360 365

Glu Ile Gly Met Ser Pro Trp Gln Val Met Leu Phe Arg Lys Ser Pro

370 375 380

Gln Glu Leu Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp Val Leu

385 390 395 400

Thr Ala Ala His Cys Leu Leu Tyr Pro Pro Trp Asp Lys Asn Phe Thr

405 410 415

Glu Asn Asp Leu Leu Val Arg Ile Gly Lys His Ser Arg Thr Arg Tyr

420 425 430

Glu Arg Asn Ile Glu Lys Ile Ser Met Leu Glu Lys Ile Tyr Ile His

435 440 445

Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp Arg Asp Ile Ala Leu Met

450 455 460

Lys Leu Lys Lys Pro Ile Thr Phe Ser Asp Tyr Ile His Pro Val Cys

465 470 475 480

Leu Pro Asp Arg Glu Thr Ala Ala Ser Leu Phe Gln Ala Gly Tyr Lys

485 490 495

Gly Arg Val Thr Gly Trp Gly Asn Leu Lys Glu Thr Trp Thr Thr Asn

500 505 510

Val Gly Lys Val Gln Pro Ser Val Leu Gln Val Val Asn Leu Pro Ile

515 520 525

Val Glu Arg Ser Val Cys Lys Asp Ser Thr Arg Ile Arg Ile Thr Asp

530 535 540

Asn Met Phe Cys Ala Gly Tyr Lys Pro Gly Glu Gly Lys Arg Gly Asp

545 550 555 560

Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys Asn Pro Leu

565 570 575

Asn Lys Arg Trp Tyr Gln Met Gly Ile Val Ser Trp Gly Glu Gly Cys

580 585 590

Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr His Val Phe Arg Leu Lys

595 600 605

Lys Trp Ile Gln Lys Val Ile Asp Gln Phe Gly Asp His His His His

610 615 620

His His

625

<210> 4

<211> 624

<212> PRT

<213> Artificial sequence

<220>

<223> His-tagged mouse prothrombin sequence

<400> 4

Met Ser His Val Arg Gly Leu Gly Leu Pro Gly Cys Leu Ala Leu Ala

1 5 10 15

Ala Leu Val Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln

20 25 30

Gln Ala Leu Ser Leu Leu Gln Arg Val Arg Arg Ala Asn Ser Gly Phe

35 40 45

Leu Glu Glu Leu Arg Lys Gly Asn Leu Glu Arg Glu Cys Val Glu Glu

50 55 60

Gln Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Pro Gln Asp

65 70 75 80

Thr Asp Val Phe Trp Ala Lys Tyr Thr Val Cys Asp Ser Val Arg Lys

85 90 95

Pro Arg Glu Thr Phe Met Asp Cys Leu Glu Gly Arg Cys Ala Met Asp

100 105 110

Leu Gly Val Asn Tyr Leu Gly Thr Val Asn Val Thr His Thr Gly Ile

115 120 125

Gln Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys Pro Glu Ile Asn

130 135 140

Ser Thr Thr His Pro Gly Ala Asp Leu Lys Glu Asn Phe Cys Arg Asn

145 150 155 160

Pro Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr

165 170 175

Val Arg Arg Glu Glu Cys Ser Val Pro Val Cys Gly Gln Glu Gly Arg

180 185 190

Thr Thr Val Val Met Thr Pro Arg Ser Gly Gly Ser Lys Asp Asn Leu

195 200 205

Ser Pro Pro Leu Gly Gln Cys Leu Thr Glu Arg Gly Arg Leu Tyr Gln

210 215 220

Gly Asn Leu Ala Val Thr Thr Leu Gly Ser Pro Cys Leu Pro Trp Asn

225 230 235 240

Ser Leu Pro Ala Lys Thr Leu Ser Lys Tyr Gln Asp Phe Asp Pro Glu

245 250 255

Val Lys Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Trp Asp Glu Glu

260 265 270

Gly Ala Trp Cys Tyr Val Ala Gly Gln Pro Gly Asp Phe Glu Tyr Cys

275 280 285

Asn Leu Asn Tyr Cys Glu Glu Ala Val Gly Glu Glu Asn Tyr Asp Val

290 295 300

Asp Glu Ser Ile Ala Gly Arg Thr Thr Asp Ala Glu Phe His Thr Phe

305 310 315 320

Phe Asn Glu Lys Thr Phe Gly Leu Gly Glu Ala Asp Cys Gly Leu Arg

325 330 335

Pro Leu Phe Glu Lys Lys Ser Leu Lys Asp Thr Thr Glu Lys Glu Leu

340 345 350

Leu Asp Ser Tyr Ile Asp Gly Arg Ile Val Glu Gly Trp Asp Ala Glu

355 360 365

Lys Gly Ile Ala Pro Trp Gln Val Met Leu Phe Arg Lys Ser Pro Gln

370 375 380

Glu Leu Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp Val Leu Thr

385 390 395 400

Ala Ala His Cys Ile Leu Tyr Pro Pro Trp Asp Lys Asn Phe Thr Glu

405 410 415

Asn Asp Leu Leu Val Arg Ile Gly Lys His Ser Arg Thr Arg Tyr Glu

420 425 430

Arg Asn Val Glu Lys Ile Ser Met Leu Glu Lys Ile Tyr Val His Pro

435 440 445

Arg Tyr Asn Trp Arg Glu Asn Leu Asp Arg Asp Ile Ala Leu Leu Lys

450 455 460

Leu Lys Lys Pro Val Pro Phe Ser Asp Tyr Ile His Pro Val Cys Leu

465 470 475 480

Pro Asp Lys Gln Thr Val Thr Ser Leu Leu Arg Ala Gly Tyr Lys Gly

485 490 495

Arg Val Thr Gly Trp Gly Asn Leu Arg Glu Thr Trp Thr Thr Asn Ile

500 505 510

Asn Glu Ile Gln Pro Ser Val Leu Gln Val Val Asn Leu Pro Ile Val

515 520 525

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

530 535 540

Met Phe Cys Ala Gly Phe Lys Val Asn Asp Thr Lys Arg Gly Asp Ala

545 550 555 560

Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys Ser Pro Phe Asn

565 570 575

Asn Arg Trp Tyr Gln Met Gly Ile Val Ser Trp Gly Glu Gly Cys Asp

580 585 590

Arg Lys Gly Lys Tyr Gly Phe Tyr Thr His Val Phe Arg Leu Lys Arg

595 600 605

Trp Ile Gln Lys Val Ile Asp Gln Phe Gly His His His His His His

610 615 620

<210> 5

<211> 120

<212> PRT

<213> mice

<400> 5

Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Ile Thr Pro Gly Gly Ser

1 5 10 15

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

20 25 30

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly

35 40 45

Met Ile Glu Thr Asp Gly Ser Ile Phe His Ala Ser Trp Val Asn Gly

50 55 60

Arg Phe Thr Ile Ser Lys Thr Ala Thr Thr Val Asp Leu Lys Met Thr

65 70 75 80

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

85 90 95

Asn Asp Tyr Asp Ala Tyr Gly Tyr Pro Tyr Phe Thr Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 6

<211> 112

<212> PRT

<213> mice

<400> 6

Glu Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Thr Leu Gly Ala

1 5 10 15

Ser Ala Lys Leu Thr Cys Thr Leu Ser Ser Ala His Glu Thr Tyr Thr

20 25 30

Ile Asp Trp Tyr Gln Gln His Gln Gly Glu Ala Pro Gln Tyr Leu Met

35 40 45

Glu Leu Lys Ser Asp Gly Ser Tyr Asn Lys Gly Ala Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg Tyr Leu Met Ile Pro

65 70 75 80

Ser Val Gln Ala Asp Asp Glu Ala Thr Tyr His Cys Gly Ala Asp Phe

85 90 95

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

100 105 110

<210> 7

<211> 5

<212> PRT

<213> mice

<400> 7

Asn Tyr Asp Met Ser

1 5

<210> 8

<211> 16

<212> PRT

<213> mice

<400> 8

Met Ile Glu Thr Asp Gly Ser Ile Phe His Ala Ser Trp Val Asn Gly

1 5 10 15

<210> 9

<211> 15

<212> PRT

<213> mice

<400> 9

Gly Ser Asn Asp Tyr Asp Ala Tyr Gly Tyr Pro Tyr Phe Thr Leu

1 5 10 15

<210> 10

<211> 12

<212> PRT

<213> mice

<400> 10

Thr Leu Ser Ser Ala His Glu Thr Tyr Thr Ile Asp

1 5 10

<210> 11

<211> 11

<212> PRT

<213> mice

<400> 11

Leu Lys Ser Asp Gly Ser Tyr Asn Lys Gly Ala

1 5 10

<210> 12

<211> 9

<212> PRT

<213> mice

<400> 12

Gly Ala Asp Phe Ser Asp Gly Tyr Val

1 5

<210> 13

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> H1512-1 VH (CDR grafted), humanized heavy chain variable region.

<400> 13

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 Val Ser Asn Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Met Ile Glu Thr Asp Gly Ser Ile Phe His Ala Ser Trp Val Asn

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gly Ser Asn Asp Tyr Asp Ala Tyr Gly Tyr Pro Tyr Phe Thr Leu

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> H1512-1 VL (CDR grafted), humanized light chain variable region.

<400> 14

Gln Pro Val Leu Thr Gln Ser Ser Ser Ala Ser Ala Ser Leu Gly Ser

1 5 10 15

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

20 25 30

Ile Asp Trp His Gln Gln Gln Pro Gly Lys Ala Pro Arg Tyr Leu Met

35 40 45

Lys Leu Lys Ser Asp Gly Ser Tyr Asn Lys Gly Ala Gly Val Pro Asp

50 55 60

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

65 70 75 80

Asn Leu Gln Leu Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Asp Phe

85 90 95

Ser Asp Gly Tyr Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 15

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> H1512-8/H1512-12, humanized light chain variable region sequence obtained after back mutation.

<400> 15

Glu Leu Val Leu Thr Gln Ser Ser Ser Ala Ser Ala Ser Leu Gly Ser

1 5 10 15

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

20 25 30

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

35 40 45

Glu Leu Lys Ser Asp Gly Ser Tyr Asn Lys Gly Ala Gly Val Pro Asp

50 55 60

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

65 70 75 80

Asn Leu Gln Leu Glu Asp Glu Ala Thr Tyr His Cys Gly Ala Asp Phe

85 90 95

Ser Asp Gly Tyr Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 16

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> H1512-8/H1512-12, humanized heavy chain variable region sequence obtained after back mutation.

<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 Val Ser Asn Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile Glu Thr Asp Gly Ser Ile Phe His Ala Ser Trp Val Asn

50 55 60

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

65 70 75 80

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

85 90 95

Arg Gly Ser Asn Asp Tyr Asp Ala Tyr Gly Tyr Pro Tyr Phe Thr Leu

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 17

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable region of H1512-12

<400> 17

Gln Leu Val Leu Thr Gln Ser Ser Ser Ala Ser Ala Ser Leu Gly Ser

1 5 10 15

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

20 25 30

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

35 40 45

Glu Leu Lys Ser Asp Gly Ser Tyr Asn Lys Gly Ala Gly Val Pro Asp

50 55 60

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

65 70 75 80

Asn Leu Gln Leu Glu Asp Glu Ala Thr Tyr His Cys Gly Ala Asp Phe

85 90 95

Ser Asp Gly Tyr Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 18

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain constant region, S228P mutation.

<400> 18

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> 19

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> kappa light chain constant region

<400> 19

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

1. A thrombin antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID No.10, SEQ ID No.11 and SEQ ID No. 12, respectively; wherein the antigen binding fragment is selected from the group consisting of Fab, Fab ', F (ab')₂Single chain antibodies, diabodies, dimerized V regions, and disulfide stabilized V regions.

2. The thrombin antibody or antigen-binding fragment thereof according to claim 1, wherein said thrombin antibody or antigen-binding fragment thereof is a rabbit derived antibody or antigen-binding fragment thereof.

3. The thrombin antibody or antigen-binding fragment thereof according to claim 2, wherein said thrombin antibody or antigen-binding fragment thereof further comprises a light chain FR region of a kappa chain of rabbit origin, or further comprises a light chain FR region of a lambda chain of rabbit origin; and/or wherein the thrombin antibody or antigen-binding fragment thereof further comprises the heavy chain FR region of a rabbit IgG.

4. The thrombin antibody or antigen-binding fragment thereof according to claim 1, wherein said thrombin antibody comprises the heavy chain variable region sequence of SEQ ID No.5 and the light chain variable region sequence of SEQ ID No. 6.

5. The thrombin antibody or antigen-binding fragment thereof according to claim 1, wherein said thrombin antibody further comprises a light chain constant region from a kappa chain of rabbit origin or a light chain constant region from a lambda chain of rabbit origin; and/or wherein the thrombin antibody further comprises a heavy chain constant region of a rabbit IgG.

6. The thrombin antibody or antigen-binding fragment thereof according to claim 1, wherein said thrombin antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof.

7. The thrombin antibody or antigen-binding fragment thereof according to claim 1, wherein said thrombin antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

8. The thrombin antibody or antigen-binding fragment thereof according to claim 7, wherein the heavy chain FR region sequence of said humanized antibody or antigen-binding fragment thereof is derived from the combined sequences of human germline heavy chains IGHV3-66 x 01 and hjh 4.1.1; wherein the humanized antibody comprises the FR1, FR2, FR3 region of human germline heavy chain IGHV3-66 x 01 and FR4 region of hjh 4.1.1.

9. The thrombin antibody or antigen-binding fragment thereof according to claim 8, wherein said humanized antibody or antigen-binding fragment thereof comprises the heavy chain variable region as set forth in SEQ ID NO 13.

10. The thrombin antibody or antigen-binding fragment thereof according to claim 8, wherein the heavy chain FR region sequence of said humanized antibody or antigen-binding fragment thereof has the amino acid back mutations of V47I, S48G, R70K, L75V, A93V and Y91F.

11. The thrombin antibody or antigen-binding fragment thereof according to claim 8, wherein said humanized antibody or antigen-binding fragment thereof comprises the heavy chain variable region as set forth in SEQ ID NO 16.

12. The thrombin antibody or antigen-binding fragment thereof according to claim 7, wherein the light chain FR region sequence of said humanized antibody or antigen-binding fragment thereof is derived from the combined sequences of human germline light chain templates IGLV4-60 x 01 and hjk 4.1; wherein the humanized antibody comprises the FR1, FR2, FR3 region of human germline light chain IGLV4-60 x 01 and the FR4 region of hjk 4.1.

13. The thrombin antibody or antigen-binding fragment thereof according to claim 12, wherein said humanized antibody or antigen-binding fragment thereof comprises the light chain variable region sequence set forth in SEQ ID No. 14.

14. The thrombin antibody or antigen-binding fragment thereof according to claim 12, wherein the light chain FR region sequence of said humanized antibody or antigen-binding fragment thereof has the amino acid back-mutations of K49E, P2L, H36Y, Y91H, Q1E and D89T or has the amino acid back-mutations of K49E, P2L, H36Y, Y91H and D89T.

15. The thrombin antibody or antigen-binding fragment thereof according to claim 12, wherein said humanized antibody or antigen-binding fragment thereof comprises the light chain variable region as set forth in SEQ ID No.15 or SEQ ID No. 17.

16. The thrombin antibody or antigen-binding fragment thereof according to claim 7, wherein said humanized antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region having the sequence of SEQ ID NO 13 or SEQ ID NO 16; and/or (b) a light chain variable region having the sequence SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 17.

17. The thrombin antibody or antigen-binding fragment thereof according to claim 7, wherein said thrombin antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

18. The thrombin antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein said thrombin antibody further comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3 or IgG4, and said thrombin antibody further comprises a light chain constant region selected from human kappa or lambda chains.

19. The thrombin antibody or antigen-binding fragment thereof according to claim 18, wherein said thrombin antibody comprises the heavy chain constant region of human IgG 4.

20. The thrombin antibody or antigen-binding fragment thereof according to claim 18, wherein said thrombin antibody comprises a light chain constant region as set forth in SEQ ID No. 19 and a heavy chain constant region as set forth in SEQ ID No. 18.

21. The thrombin antibody or antigen binding fragment thereof according to claim 1, wherein said antigen binding fragment is selected from the group consisting of Fab, Fab ', F (ab')₂scFv, and dsFv.

22. A pharmaceutical composition comprising a therapeutically effective amount of a thrombin antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, together with one or more pharmaceutically acceptable carriers, diluents or excipients.

23. A DNA molecule encoding the thrombin antibody or antigen-binding fragment thereof according to any one of claims 1 to 21.

24. An expression vector comprising the DNA molecule of claim 23.

25. A host cell transformed with the expression vector of claim 24, said host cell selected from the group consisting of prokaryotic cells and eukaryotic cells.

26. The host cell of claim 25, wherein the host cell is a eukaryotic cell.

27. The host cell of claim 25, wherein the host cell is a mammalian cell.

28. Use of the thrombin antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, or the pharmaceutical composition according to claim 22, or the DNA molecule according to claim 23, in the manufacture of a medicament for the treatment of a thrombin-mediated disease or disorder, wherein said thrombin-mediated disease or disorder is a thrombotic disease.

29. The use according to claim 28, wherein the thrombin mediated disease or condition is selected from the group consisting of venous thrombosis and pulmonary embolism, arterial thrombosis, thrombosis induced stroke and peripheral arterial formation, atherosclerotic diseases and cerebral arteriopathy.

30. The use according to claim 28, wherein the thrombin mediated disease or condition is peripheral arterial disease.

31. The use according to claim 28, wherein the thrombin mediated disease or condition is selected from the group consisting of venous thrombosis, stroke from thrombosis and atherosclerosis.

32. A reagent for detecting or determining human thrombin, the reagent comprising the thrombin antibody or antigen-binding fragment thereof according to any one of claims 1 to 21.