CN107043423A - Anti-thrombin antibody, its antigen-binding fragment and medical usage - Google Patents

Abstract

The present invention relates to anti-thrombin antibody, its antigen-binding fragment and medical usage.Further, the present invention relates to chimeric antibody, the humanized antibody for including the anti-thrombin antibody CDR region, and the pharmaceutical composition comprising anti-thrombin antibody and its antigen-binding fragment, and it is used as the purposes of anticoagulant.Especially, the purposes in being used to treat the disease of thrombin-mediated or the medicine of illness is being prepared the present invention relates to a kind of anti-thrombin antibody of humanization.

Description

Anti-thrombin antibody, its antigen-binding fragment and medical usage

Technical field

The present invention relates to anti-thrombin antibody and its antigen-binding fragment, further, the present invention relates to comprising described solidifying Chimeric antibody, the humanized antibody of hemase antibody CDR region, the invention further relates to include the anti-thrombin antibody and its antigen binding The pharmaceutical composition of fragment, and it is used as the purposes of anticoagulant.

Background technology

Blood clotting is to prevent the significant process of damaged blood vessels bleeding (hemostasis).However, obstruction blood stream intravascular (thrombus Formed) or the blood clot of the blood vessel (thromboembolism) that is deposited on other locations in the body after coming off be serious threat to health.Blood Bolt disease (such as acute myocardial infarction (AMI), venous blood embolism) is that a class seriously jeopardizes human health and the cardiovascular disease of life Disease.World Health Organization 2008 counts, to current, and cardiovascular morbidity and the death rate have leapt to the first position.The annual heart in the world The people of angiosis death toll about 17,330,000, accounts for dead sum 30%, the Chinese people of cardiovascular patient 2.9 hundred million, annual death about 3,500,000 People, accounts for total cause of death 41%.Global disease burden (GBD) statistics in 2010, apoplexy is the big cause of the death of China resident first. So in recent years, the medicine and method of the effective treatment angiocardiopathy of research cause people and more and more paid close attention to.At present, Some anticoagulant therapies can treat pathology blood clotting, such as using conventional medicament heparin, low molecular weight or warfarin, Or use direct thrombin inhibitor dabigatran etcxilate (Dabigatran) etc..The common defects of these therapies are increase bleedings Risk.Window between many anticoagulant effective doses (prevention thrombosis) and safe dose (highest is without bleeding risk) is not It is enough big, it is contemplated that the response difference of individual patient, the window will further reduce.Using fibrin ferment as target spot, fibrin ferment antagonism is used Generation of the agent to suppress thrombus is one of method of clinical treatment thrombus.

Coagulation test is a complicated signal cascade process, and fibrin ferment (Thrombin) occupies core status wherein. Fibrin ferment by the fibrinogenolysis of the circulatory system be fibrin monomer (polymerizable formation fibrin, blood clotting Fibre substrate), also there are many direct regulations and controls to cell.As a kind of serine protease, it triggers blood platelet and deformed upon, releases Put inducer of platelet activation ADP, thrombocytin and thromboxane A2, and chemotactic factor (CF) and growth factor.In addition, adhesion is also promoted Molecule palatelet-selectin and CD40L move to platelet surface, and then activate integrin aIIb/b3.The latter's binding fiber albumen Former and vWF ELISA (von Willebrand factor, vWF), and then the aggregation of mediating platelet.Fibrin ferment is also The procoagulant activity on stimulating platelet surface, promotes the expression of fibrin ferment in turn.In the culture of endothelial cell, blood coagulation enzymatic Enter vWF release, the appearance of the palatelet-selectin of cytoplasma membrane and the generation of chemotactic factor (CF).These reactions are considered as blood in trigger body The combination of platelet and leucocyte and endothelial cell surface.Endothelial cell changes shape, the increase of endothelial layer permeability immediately.This A little reactions are expected to ooze out the part for promoting plasma protein, promote oedema.In non-endothelial tissue, fibrin ferment is by acting on Smooth muscle cell causes vessel retraction.In the in vitro culture of fibroblast or vascular smooth muscle cells, blood coagulation enzyme adjustment is thin The generation of intracellular cytokine simultaneously promotes mitosis, and it triggers Ca2+ oscillations and other reactions in T lymphocytes.These cell effects Show that the Adjust System of tissue damage and hemostasis, inflammatory reaction, even booster immunization response has been associated in one by fibrin ferment Rise.These cell effects it is also proposed a kind of possibility：In addition to tissue damaged, the blood coagulation of endothelial cell and other types cell Enzyme may also play certain role in Leukocyte extravasation, vascular remodeling and/or angiogenesis.Therefore, fibrin ferment turns into One great potential, new anticoagulation anti-bolt target.

There is presently no the listing of antithrombase antibody drug or into clinic, related antibody patent has WO2013123591, WO2013088164, WO2014153195, WO2014202992 and WO2014202993.The present invention is provided A kind of affinity is high, and the new anti-thrombin antibody with remarkable inhibiting activity is generated to thrombus.

The content of the invention

The present invention provides a kind of anti-thrombin antibody or its antigen-binding fragment, and it is selected from following CDR comprising one or more Region sequence or the amino acid sequence with it with least 95% sequence identity：Heavy chain of antibody variable region HCDR region sequences：SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9；With antibody light chain variable region LCDR region sequences：SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the anti-thrombin antibody is comprising respectively such as SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:Shown in 9 HCDR1, HCDR2 and HCDR3, or with SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 have at least 95% sequence same The amino acid sequence of one property.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein described anti-thrombin antibody or its antigen-binding fragment are comprising respectively such as SEQ ID NO:10, SEQ ID NO:11 Hes SEQ ID NO:LCDR1, LCDR2 and LCDR3 shown in 12, or with SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 have the amino acid sequence of at least 95% sequence identity.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein described anti-thrombin antibody or its antigen-binding fragment are rabbit source antibody or its function fragment.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the light chain variable district of described anti-thrombin antibody or its antigen-binding fragment further includes rabbit source κ chains or rabbit source κ chains The light chain FR areas of variant or further the light chain FR areas comprising rabbit source λ chains or rabbit source λ chain variants；And/or wherein described blood coagulation The weight chain variable district of enzyme antibody or its antigen-binding fragment further includes rabbit IgG or the heavy chain FR areas of its variant.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein described anti-thrombin antibody or its antigen-binding fragment include SEQ ID NO:5 weight chain variabl area sequence and SEQ ID NO:6 light-chain variable sequence.Described anti-thrombin antibody is rabbit source antibody herein.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein described anti-thrombin antibody or its antigen-binding fragment further the constant region of light chain comprising rabbit source κ chains or its variant, Or the constant region of light chain of rabbit source λ chains or its variant；And/or wherein described anti-thrombin antibody further comprising rabbit IgG or The heavy chain constant region of its variant.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein described anti-thrombin antibody or its antigen-binding fragment are chimeric antibody or its function fragment.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein described anti-thrombin antibody or its antigen-binding fragment are humanized antibody or its function fragment.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the heavy chain FR region sequences of described humanized antibody or its function fragment derive from human germline heavy IGHV3-66*01 With hjh4.1 composite sequence and its mutant nucleotide sequence；Wherein described humanized antibody or its function fragment include human germline heavy IGHV3-66*01 FR1, FR2, FR3 area and hjh4.1 FR4 areas and its mutant nucleotide sequence.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the humanized antibody or its function fragment include SEQ ID NO:Weight chain variable district shown in 13, or with SEQ ID NO:13 have the weight chain variable district at least shown in the amino acid sequence of 95% sequence identity；It is preferred that with SEQ ID NO:13 The amino acid that amino acid sequence has 0-10 changes.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the heavy chain FR region sequences of described humanized antibody or its function fragment have the back mutation of 0-10 amino acid, it is excellent The back mutation is selected to be selected from V47I, S48G, R70K, L75V, A93V and Y91F amino acid back mutation.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the humanized antibody or its function fragment include SEQ ID NO:Weight chain variable district shown in 16, or with SEQ ID NO:16 have the weight chain variable district at least shown in the amino acid sequence of 95% sequence identity.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the light chain FR region sequences of described humanized antibody or its function fragment derive from people's germline light chain template IGLV4- 60*01 and hjk4.1 composite sequence and its mutant nucleotide sequence；Wherein described humanized antibody or its function fragment include ethnic group It is light chain IGLV4-60*01 FR1, FR2, FR3 area and hjk4.1 FR4 areas and its mutant nucleotide sequence.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the humanized antibody or its function fragment include SEQ ID NO:Light-chain variable sequence shown in 14, or and SEQ ID NO:14 have the light-chain variable sequence at least shown in the amino acid sequence of 95% sequence identity；It is described with SEQ ID NO:14 amino acid sequence has 0-10 amino acid change.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the light chain FR region sequences of described humanized antibody or its function fragment have the back mutation of 0-10 amino acid, it is excellent The back mutation is selected to be selected from K49E, P2L, H36Y, Y91H, Q1E and D89T amino acid back mutation.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the humanized antibody or its function fragment include SEQ ID NO:14、SEQ ID NO:15 or SEQ ID NO:17 Shown light chain variable district, or with SEQ ID NO:14、SEQ ID NO:15 or SEQ ID NO:17 have at least 95% sequence Light chain variable district shown in the amino acid sequence of homogeneity.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the humanized antibody or its function fragment include (a) weight chain variable district, the sequence of the weight chain variable district is selected from SEQ ID NO:13、SEQ ID NO:16 and with SEQ ID NO:13 or SEQ ID NO:16 have at least 95% sequence same The amino acid sequence of property；Or/and (b) light chain variable district, the sequence of the light chain variable district is selected from SEQ ID NO:14、SEQ ID NO:15 or SEQ ID NO:17 and with SEQ ID NO:14、SEQ ID NO:15 or SEQ ID NO:17 have at least 95% The amino acid sequence of sequence identity.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein described anti-thrombin antibody or its antigen-binding fragment are included and be selected from the weight chain variable district and light chain variable of the following group Area：

1)SEQ ID NO:13 weight chain variable district and SEQ ID NO:14 light chain variable district；

2)SEQ ID NO:13 weight chain variable district and SEQ ID NO:15 light chain variable district；

3)SEQ ID NO:16 weight chain variable district and SEQ ID NO:15 light chain variable district；

4)SEQ ID NO:16 weight chain variable district and SEQ ID NO:14 light chain variable district；

5)SEQ ID NO:13 weight chain variable district and SEQ ID NO:17 light chain variable district；With

6)SEQ ID NO:16 weight chain variable district and SEQ ID NO:17 light chain variable district.

In presently preferred embodiment, anti-thrombin antibody of the present invention or its antigen binding fragment Section, wherein the anti-thrombin antibody further includes the light chain constant selected from humanized IgG 1, IgG2, IgG3, IgG4 and its variant Area, preferably comprises humanized IgG 4 or the heavy chain constant region of its variant, SEQ ID NO preferably for example of the present invention:Heavy chain shown in 18 Constant region.

The chimeric antibody or humanized antibody light chain further include people source κ, λ chain or the constant region of its variant, preferably Such as SEQ ID NO of the present invention:Constant region shown in 19.

In presently preferred embodiment, antigen-binding fragment of the present invention is to be selected from Fab, Fab', F (ab') 2, single-chain antibody (scFv), the V areas (double antibody) of dimerization, disulfide-stabilized V areas (dsFv) and include CDR's The antigen-binding fragment of peptide.

The present invention further provides a kind of pharmaceutical composition, it contains the anti-thrombin antibody as described above of therapeutically effective amount Or its antigen-binding fragment, and one or more pharmaceutically acceptable carriers, diluent or excipient.

Anti-thrombin antibody as described above or DNA points of its antigen-binding fragment are encoded the present invention further provides a kind of Son.

The present invention further provides a kind of expression vector of DNA molecular as described above.

The present invention further provides a kind of host cell of expression vector conversion as described above, the host cell is selected from Prokaryotic and eukaryotic, preferably eukaryotic, more preferably mammalian cell.

The present invention further provides a kind of anti-thrombin antibody as described above or its antigen-binding fragment or as described above Pharmaceutical composition, is preparing the purposes in being used to treat the disease of thrombin-mediated or the medicine of illness, wherein described disease Or illness is preferably thrombotic diseases (it includes thrombosis and thromboembolism)；More preferably venous thronbosis and lung bolt Plug, Arterial thrombosis, apoplexy caused by thrombosis and distal aorta formation, atherosclerosis disease, EEG source location or Distal aorta disease；Most preferably venous thronbosis, apoplexy and atherosclerosis caused by thrombosis.

Can use the present invention antibody diagnosis Exemplary diseases include thrombotic diseases (it includes thrombosis and blood Bolt embolism), it includes following any one or more of：Venous thronbosis and pulmonary embolism, Arterial thrombosis, thrombosis Caused apoplexy and distal aorta formation, atherosclerosis disease, EEG source location or distal aorta disease；.

In one aspect, the hypercholesterolemia and/or at least one that the present invention is provided in treatment or prevention individual are following The method of symptom：Apoplexy and atherosclerosis caused by venous thronbosis, thrombosis, methods described are included to described Body applies the antithrombase antibody of effective dose.The present invention be also provided with effect amount antagonism it is extracellular or circulation fibrin ferment antithrombase Purposes of the antibody in medicine is prepared, the medicine is used for the thrombosis for treating or preventing individual and/or at least one following Symptom：Apoplexy and atherosclerosis caused by pulmonary embolism, thrombosis.

In one aspect, the present invention is used to detecting or determine the reagent of human thrombin, the reagent comprising above-mentioned antibody or Its antigen-binding fragment.

Brief description of the drawings

Fig. 1：Influences of the antithrombase antibody CH-1512 to human thrombin enzymatic activity；Data result shows anti-thrombin antibody CH-1512 has no effect on its enzymolysis activity to substrate S2228 after being combined with fibrin ferment；

Fig. 2：Influences of the antithrombase antibody H1512-12 to human thrombin enzymatic activity；Data result shows anti-thrombin antibody H1512-12 has no effect on its enzymolysis activity to substrate S2228 after being combined with fibrin ferment；

Fig. 3：Influence of the various concentrations anti-thrombin antibody to normal person's plasma A PTT values；Data result is shown with antibody The increase of H1512-12 concentration, the APTT values of human normal plasma have also extended；H1512-12 in maximum concentration 3200nM, The increase of APTT values also reaches the peak value of 33.6 seconds；

Fig. 4：Efficacy testing of the antibody of the present invention in rat body, antibody H1512-12 intravenous injections induce abdomen to iron chloride The thrombotic influence of cardinal vein；Data result shows tail vein injection H1512-12, in 6mg/kg and 12mg/kg dosage Under, it can significantly suppress the generation of thrombus；The average thrombus weight of H1512-12-6mg/kg groups and H1512-12-12mg/kg groups Amount, is reduced to 2.91 milligrams and 0.18 milligram respectively, the milli of average thrombus weight 10.75 with Isotype antibody group (IgG-6mg/kg) Gram compare, with pole significant difference (* * * p statistically<0.001)；

Fig. 5：Thrombus weight after machin arteriovenous anastomosis model surgery, is shown under 6mg/kg dosage, H1512-12 The generation of macaque thrombus can significantly be suppressed, compared with the average thrombus weight of Isotype antibody group (IgG-6mg/kg), had Pole significant difference (* * * p statistically<0.001).

Embodiment

One, terms

In order to be easier to understand the present invention, some technologies and scientific terminology are defined in detail below.Unless another herein Explicitly define, all other technology used herein and scientific terminology all have those skilled in the art of the art The implication being generally understood that.

In amino acid three-letter codes used and single letter code such as J.biol.chem of the invention, 243, p3558 (1968) It is described.

" antibody " of the present invention refers to immunoglobulin, is passed through by two identical heavy chains and two identical light chains Four peptide chain structures that interchain disulfide bond is formed by connecting.The amino acid of immunoglobulin heavy chain constant region is constituted and put in order not Together, thus its antigenicity is also different.Accordingly, immunoglobulin can be divided into five classes, or be the isotype of immunoglobulin, i.e., IgM, IgD, IgG, IgA and IgE, its corresponding heavy chain are respectively μ chains, δ chains, γ chains, α chains and ε chains.Same class Ig is according to it Hinge region amino acid constitutes the difference with the number and location of heavy chain disulfide bond, different subclass can be divided into again, such as IgG can be divided into IgG1、IgG2、IgG3、IgG4.Light chain is divided into κ chains or λ chains by the difference of constant region.There can be κ per class Ig in five class Ig Chain or λ chains.

In the present invention, antibody light chain of the present invention can further include constant region of light chain, described chain constant κ, λ chain or its variant of the area comprising people source or rabbit source.

In the present invention, heavy chain of antibody of the present invention can further include heavy chain constant region, described light chain constant Area IgG1, IgG2, IgG3, IgG4 or its variant comprising people source or rabbit source.

Heavy chain of antibody and light chain are very big close to the sequence variation of about 110 amino acid of N-terminal, are variable region (Fv areas)；Lean on Remaining amino acid sequence of nearly C-terminal is stablized relatively, is constant region.Variable region includes 3 hypervariable regions (HVR) and 4 sequences are relative Conservative skeleton area (FR).3 hypervariable regions determine the specificity of antibody, also known as complementarity-determining region (CDR).Every light chain can Become area (LCVR) and weight chain variable district (HCVR) by 3 CDR regions, 4 FR district's groups into being arranged in order from aminoterminal to c-terminus Sequentially it is：FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.3 CDR regions of light chain refer to LCDR1, LCDR2 and LCDR3；Weight 3 CDR regions of chain refer to HCDR1, HCDR2 and HCDR3.The LCVR areas of antibody or antigen-binding fragment of the present invention and HCVR The cdr amino acid residue in area meets known Kabat coding rules (LCDR1-3, HCDE2-3), Huo Zhefu in quantity and position Close kabat and chothia coding rule (HCDR1).

The antibody of the present invention includes rabbit source antibody, chimeric antibody, preferably humanized antibody, humanized antibody.

Term " lagomorph " refers to taxology Lagomorpha (Lagomorpha) member, including rabbit section (Leporidae) (such as hare and rabbit) and Ochotonidae (Ochotonidae) (pika).In the most preferred embodiment, lagomorph is rabbit.This Term " rabbit " used herein refers to the animal for belonging to rabbit section (leporidae).

Term " rabbit source antibody " is the Dan Ke to human thrombin prepared according to this area knowledge and skills in the present invention Grand antibody.With fibrin ferment antigen injection subjects during preparation, then separation expression is anti-with required sequence or functional characteristic The monoclonal antibody of body.In yet other embodiments, described rabbit source thrombase antibody or its antigen binding Fragment, can further include rabbit source κ, λ chain or the constant region of light chain of its variant, or further comprising rabbit IgG or its variant Heavy chain constant region.

Term " chimeric antibody (chimeric antibody) ", is the perseverance by the variable region of rabbit endogenous antibody and human antibody Determine the antibody of area's fusion.Chimeric antibody is set up, display technique of bacteriophage can be used to obtain rabbit-anti variable region gene, then by rabbit Variable region gene is connected into after mosaic gene with human constant region gene and inserted in expression vector, finally in eukaryotic system or protokaryon system Chimeric antibody expression molecule in system.In yet other embodiments, the antibody of described fibrin ferment chimeric antibody Light chain further includes people source κ, λ chain or the constant region of light chain of its variant.The heavy chain of antibody of described fibrin ferment chimeric antibody enters One step includes the heavy chain constant region of humanized IgG 1, IgG2, IgG3, IgG4 or its variant, preferably comprise humanized IgG 1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutation (such as YTE mutation) afterwards the IgG1 of half-life period of the extension antibody in serum, IgG2 or IgG4 variants.

Term " humanized antibody (humanized antibody) ", also referred to as CDR grafted antibody (CDR-grafted Antibody), the antibody variable region framework that the CDR sequence of rabbit is transplanted to people, i.e., different types of human germline antibody's structure are referred to The antibody produced in frame sequence.Chimeric antibody can be overcome due to carrying a large amount of rabbit protein ingredients, so that the heterologous of induction is anti- Should.Such frame sequence can be obtained from the public DNA database including germline antibody gene sequences or disclosed bibliography. Germline DNA sequence dna such as people's heavy chain and chain variable region gene can be in " VBase " human germ line sequences' database (in internetwww.mrccpe.com.ac.uk/vbaseCan obtain), and in Kabat, E.A. et al., 1991Sequences of Proteins of Immunological Interest, find in the 5th edition.While to avoid immunogenicity from declining, cause Activity decrease, minimum inverse transition or back mutation can be carried out to described human antibody variable region framework sequence, to keep living Property.The humanized antibody of the present invention also includes further resisting the CDR humanizations carried out after affinity maturation by phage display Body.In yet other embodiments, the CDR sequence of rabbit is selected from SEQ ID in described fibrin ferment humanized antibody NO:7,8,9,10,11 or 12；The antibody variable region framework of people passes through design alternative, wherein on the heavy chain of antibody variable region Heavy chain FR region sequences, from human germline heavy IGHV3-66*01 and hjh4.1 composite sequence；Wherein described antibody light chain can The light chain FR region sequences become in area, from human germline heavy IGLV4-60*01 and hjk4.1 composite sequence.To avoid being immunized While originality declines, caused activity decrease can carry out minimum inverse transition to described human antibody variable region, to keep Activity.

CDR transplanting can cause anti-thrombin antibody or its antigen binding produced due to the Framework residues with antigen contact Fragment weakens to the affinity of antigen.Such interaction can be the result of somatic hypermutation.Accordingly, it is possible to still need Such donor framework amino acid is migrated to the framework of humanized antibody.From inhuman anti-thrombin antibody or its antigen binding fragment The amino acid residue of the participation antigen binding of section can be identified by checking rabbit monoclonal antibodies variable region sequences and structure.CDR Each residue different from germline can be considered as related in donor framework.If not can determine that immediate germline, then Can be by sequence with hypotype consensus sequence or the consensus sequence of the rabbit sequence with high percentage similarity compares.Rare framework is residual Base be deemed likely to be somatic hypermutation result, so as to be played an important role in combination.

" antigen-binding fragment " or " function fragment " of term antibody refer to keep antibody specificity combination antigen (for example, Fibrin ferment) ability one or more fragments.The antigen binding of antibody can be carried out using the fragment of full length antibody by having shown Function.The example of the binding fragment included in " antigen-binding fragment " of term antibody includes (i) Fab fragments, by VL, VH, CL The monovalent fragment constituted with CH1 domains；(ii)F(ab')₂Fragment, includes two connected by the disulphide bridges on hinge area The bivalent fragment of Fab fragments, the Fd fragments that (iii) is made up of VH and CH1 domains；(iv) tied by the VH and VL of the single armed of antibody The Fv fragments of structure domain composition；(v) single domain or dAb fragments (Ward et al., (1989) Nature341：544-546), its by VH domains are constituted；The complementary determining region (CDR) or (vii) of (vi) separation optionally connected by the joint of synthesis two The CDR of individual or more separation combination.In addition, although two domain VL and VH of Fv fragments by the gene code that separates, But recombination method can be used, they are connected by the joint of synthesis, so that it can be produced as wherein VL and VH areas pairing The single protein chain for forming monovalent molecule (is referred to as scFv (scFv)；See, e.g., Bird et al. (1988) Science242:423-426；With Huston et al. (1988) Proc.Natl.Acad.Sci USA85:5879-5883).It is such Single-chain antibody is also intended in " antigen-binding fragment " for being included in term antibody.Use conventional skill well known by persons skilled in the art Art obtains such antigen-binding fragment, and by with for complete antibody in the way of identical mode fragment is screened with regard to instrumentality. Antigen-binding portion thereof can be produced by recombinant DNA technology or by enzymatic or chemical disruption intact immunoglobulins.Antibody can To be the antibody of different isotypes, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM antibody.

Term " single-chain antibody ", " scFv " or " scFv " means comprising the heavy chain of antibody varistructure connected by joint Domain (or region；) and antibody light chain variable domains (or region VH；VL molecule).Such scFv molecules can have general knot Structure：NH₂- VL- joint-VH-COOH or NH₂- VH- joints-VL-COOH.Suitable prior art joint is by GGGGS ammonia repeatedly Base acid sequence or its variant composition, for example using 1-4 repeatedly variant (Holliger et al. (1993), Proc.Natl.Acad.Sci.USA90:6444-6448).Other joints available for the present invention are by Alfthan et al. (1995), Protein Eng.8:725-731, Choi et al. (2001), Eur.J.Immuno is l.31:94-106, Hu et al. (1996), Cancer Res.56:3055-3061, Kipriyanov et al. (1999), J.Mol.Biol.293:41-56 and Roovers et al. (2001), Cancer Immunol. are described.

Term " CDR " refers to mainly facilitate one of 6 hypervariable regions of antigen binding in the variable domains of antibody.Described 6 One of individual CDR the most frequently used definition is by Kabat E.A. et al., (1991) Sequences of proteins of Immunological interest.NIH Publication91-3242) provide.As used in this article, CDR Kabat Definition is only applied to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of light variable domains, And the CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of heavy-chain variable domains.

Term " Antibody framework " used herein, refers to a variable domains VL or VH part, and it is variable that it is used as this The support of the antigen binding loops (CDR) of domain.In essence, it is the variable domains without CDR.

Term " epitope " or " antigenic determinant " refer to the position (example that immunoglobulin or antibody specificity are combined on antigen Such as, the privileged site on prothrombin molecule).Epitope generally includes at least 3,4,5,6,7,8,9,10 with unique space conformation, 11,12,13,14 or 15 amino acid continuously or discontinuously.See, e.g., Epitope Mapping Protocols in Methods in Molecular B iology, volume 66, G.E.Morris, Ed. (1996).

Term " specific binding ", " selective binding ", " optionally with reference to " and " specifically with reference to " refer to antibody Combination to the epitope on predetermined antigen.Generally, antibody is to approximately be less than 10^-7M, such as approximately less than 10^-8M、10^-9M Or 10^-10M or smaller affinity (KD) is combined.

Term " KD " or " Kd " refer to the Dissociation equilibrium constant of specific antibodies-antigen interactions.Generally, it is of the invention anti- Body is less than about 10-7M, to be, for example, less than about 10^-8M、10^-9M or 10^-10M or smaller Dissociation equilibrium constant (KD) combines solidifying Hemase, for example, as determined using surface plasma body resonant vibration (SPR) technology in BIACORE instrument.

Term " nucleic acid molecules " used herein refers to DNA molecular and RNA molecule.Nucleic acid molecules can be single-stranded or double Chain, but preferably double-stranded DNA.When nucleic acid and another nucleotide sequence are placed in functional relationship, nucleic acid is " effectively connection ".If for example, promoter or enhancer influence the transcription of coded sequence, then promoter or enhancer are operatively connected to The coded sequence.

Term " carrier " is the nucleic acid molecules for referring to transport another connected nucleic acid.In an embodiment In, carrier is " plasmid ", and it refers to other DNA section can be connected to circular double stranded DNA ring therein.In another implementation In scheme, carrier is viral vector, wherein other DNA section can be connected in viral genome.It is disclosed herein to carry Body can in the host cell for having been introduced into them autonomous replication (for example, having the bacteria carrier of germy replication orgin and attached Plus type mammalian vector) or the genome of host cell can be integrated into after host cell is introduced, so that with host genome Replicate (for example, non-add type mammalian vector) together.

The method of production and antibody purification and antigen-binding fragment, the antibody assay skill of such as Cold SpringHarbor are known in the prior art Art guide, 5-8 chapters and 15 chapters.For example, rabbit can be immune with human thrombin or its fragment, resulting antibody can be by renaturation, pure Change, and amino acid sequencing can be carried out with conventional method.Antigen-binding fragment can equally be prepared with conventional method.Invention CDR region of the described antibody or antigen-binding fragment gene engineering method in inhuman source adds one or more Ren Yuan FR areas. People FR Germline sequences can by comparing IMGT human antibody variable domains germ line genes database and MOE softwares, from ImMunoGeneTics (IMGT) website http://imgt.cines.fr is obtained, or from immunoglobulin magazine, Obtained on 2001ISBN012441351.

Term " host cell " refers to the cell for introducing expression vector thereto.Host cell may include bacterium, micro- Biological, plant or zooblast.Being easy to the bacterium of conversion includes the member of enterobacteriaceae (enterobacteriaceae), for example Escherichia coli (Escherichia coli) or the bacterial strain of salmonella (Salmonella)；Bacillaceae (Bacillaceae) such as bacillus subtilis (Bacillus subtilis)；Pneumococcus (Pneumococcus)；Hammer Bacterium (Streptococcus) and Hemophilus influenzae (Haemophilus influenzae).Appropriate microorganism includes wine brewing ferment Female (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris).Appropriate animal host cell system Including CHO (Chinese hamster ovary line) and NS0 cells.

The antibody or antigen-binding fragment that the present invention is engineered can be prepared and purified with conventional method.Such as, encoding heavy chain With the cDNA sequence of light chain, it can clone and recombinate to GS expression vectors.The immunoglobulin expression carrier of restructuring can be stablized Ground transfection CHO cell.As a kind of prior art more recommended, mammal expression system can cause the glycosylation of antibody, Particularly in the highly conserved N-terminal site in Fc areas.By expressing gram that the antibody specifically bound with human thrombin is stablized It is grand.Positive being cloned in the serum free medium of bioreactor expands culture to produce antibody.The culture of antibody is secreted Liquid can be purified with routine techniques.Such as, purified with A the or G Sepharose FF posts containing adjusted buffer solution.Wash Go the component of non-specific binding.The antibody of PH gradient method elution of bound is used again, antigen-binding fragment is detected with SDS-PAGE, is received Collection.Antibody can carry out filtering and concentrating with conventional method.Solvable mixture and polymer, can also be removed with conventional method, than Such as molecular sieve, ion exchange.Obtained product need to be freezed immediately, such as -70 DEG C, or lyophilized.

" giving " and " processing " when applied to animal, people, experimental subjects, cell, tissue, organ or biofluid, Refer to exogenous drugs, therapeutic agent, diagnosticum or composition and animal, people, subject, cell, tissue, organ or biofluid Contact." giving " and " processing " can refer to such as treatment, pharmacokinetics, diagnosis, research and experimental method.Cell Processing includes contact of the reagent with cell, and contact of the reagent with fluid, wherein the fluid and cells contacting." giving " and " processing " is still meant that by reagent, diagnosis, binding compositions or by another cells in vitro and ex vivo treatment such as cell. " processing " refers to treatment processing, prevention or preventive measure, studies and examine when applied to people, animal medical science or study subject Disconnected application.

" treatment " means to give patient interior or topical therapeutic agent, such as any binding compounds comprising the present invention Composition, the patient has one or more disease symptomses, and the known therapeutic agent has therapeutic action to these symptoms. Generally, therapeutic agent is given with the amount for effectively alleviating one or more disease symptomses in subject or colony, to induce this Class symptom is degenerated or suppresses this kind of symptom development to the degree of any clinical right measurement.Effectively alleviate any disease specific symptom The amount (also referred to as " therapeutically effective amount ") of therapeutic agent can change, morbid state, age and the body of such as patient according to many factors Weight, and medicine need the ability of curative effect in patient's generation.It is generally used for commenting by doctor or other professional health care personages Whether the seriousness of the valency symptom or any clinical testing procedure of development situation, evaluable disease symptomses have been mitigated.Although Embodiment of the present invention (such as treatment method or product) may be invalid in terms of each target disease symptom is alleviated, but root According to any statistical test method known in the art such as Student t inspections, Chi-square Test, the U according to Mann and Whitney Examine, Kruskal-Wallis examine (H inspection), Jonckheere-Terpstra examine and Wilcoxon examine determination, its Target disease symptom should be mitigated in the patient of statistically significant number.

" conservative modification " or " conservative substitution or substitution " refers to similar characteristics (such as electric charge, side chain size, hydrophobic Property/hydrophily, Conformation of the main chain and rigidity etc.) other amino acid replacement albumen in amino acid so that can frequently be changed Biological activity without changing albumen.Those skilled in the art know, it is however generally that, it is single in the non-essential region of polypeptide Amino acid replacement not substantially changes biological activity (see, for example, Watson etc. (1987) Molecular Biology of The Gene, The Benjamin/Cummings Pub.Co., page 224, (the 4th edition)).In addition, structure or function is similar The unlikely broken ring biological activity of displacement of amino acid.

" effective dose " include be enough to improve or preventive medicine disease symptom or the amount of illness.Effective dose, which is still meant that, to be enough to permit Perhaps or promote diagnosis amount.It can change for particular patient or the effective dose of veterinary science subject according to following factor：For example, Illness to be treated, the general health of patient, the method and approach of administration and dosage and side effect seriousness.Effective dose can To be the maximum dose or dosage regimen for avoiding notable side effect or toxic action.

" exogenous " refers to the material according to circumstances produced outside biological, cell or human body." endogenous ", which refers to, according to circumstances to exist The material produced in cell, biology or human body.

" homology " refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.Compare when two When position in sequence is occupied by identical base or amino acid monomer subunit, if each position of such as two DNA moleculars Put when all being occupied by adenine, then the molecule is homologous in the position.Percent homology between two sequences is The shared matching of two sequences or homologous position number divided by the function for positional number × 100 compared.For example, in sequence optimal comparison When, if there are 6 matchings or homologous 10 positions in two sequences, then two sequences are 60% homologous；If two sequences There are 95 matchings or homologous 100 positions in row, then two sequences are 95% homologous.In general, when two sequences of comparison Arrange and obtain being compared during the Percent homology of maximum.

Statement " cell ", " cell line " and " cell culture " used herein is used interchangeably, and all such names Claim all to include offspring.Therefore, word " transformant " and " transformed cells " include primary subject cell and by its derivative culture, Without considering transfer number.It is to be further understood that due to mutation deliberately or unintentionally, all offsprings are in terms of DNA content Can not possibly be accurate identical.After with the mutation with the identical function or biological activity screened in initial transformed cells Generation.In the case where meaning different names, it is clearly visible by context.

" PCR " used herein or " PCR " refer to the nucleic acid of wherein micro specific part, RNA and/ Or DNA such as the program or technology that are expanded described in such as U.S. Patent number 4,683,195.It is generally desirable to be come from Target area end or outside sequence information so that can be with design oligonucleotides primer；These primers are in terms of sequence with treating The corresponding chain for expanding template is same or similar.5 ' terminal nucleotides of 2 primers can be consistent with the end of material to be amplified. PCR can be used for expanding specific RNA sequence, the specific dna sequence from total genomic dna and by total cell rna transcription CDNA, bacteriophage or plasmid sequence etc..Referring generally to Mullis etc. (1987) Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich is edited, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR used herein is considered as an example of the nucleic acid polymerase reaction method for amplification of nucleic acid test sample, but is not only One example, methods described is including the use of the known nucleic acid and nucleic acid polymerase as primer, to expand or produce the spy of nucleic acid Determine part.

" optional " or " optionally " mean ground described later event or environment can with but need not occur, the explanation includes The occasion that the event or environment occur or do not occurred.For example, " optionally comprising 1-3 heavy chain of antibody variable region " means specific sequence The heavy chain of antibody variable region of row can with but necessarily exist.

" pharmaceutical composition " represent containing one or more compounds described herein or its physiologically/pharmaceutically useful salt or Pro-drug and the mixture of other chemical constituents, the other components such as physiology/pharmaceutically useful carrier and excipient.Medicine The purpose of compositions is to promote the administration to organism, the absorption beneficial to active component and then performance bioactivity.

Two, embodiments and test case

The present invention is further described with reference to embodiments, but these embodiments not limit the scope of the present invention.This The experimental method of unreceipted actual conditions in inventive embodiments, generally according to normal condition, the antibody technique experiment of such as Cold SpringHarbor Handbook, molecular cloning handbook；Or according to the condition proposed by raw material or commodity manufacturer.The unreceipted reagent specifically originated, The conventional reagent bought for market.

The preparation of the fibrin ferment antigen of embodiment 1. and detection albumen

1st, associated coagulation enzyme and factor sequence

Encoding human factor (h-prothrombin) sequence, mouse factor (m-prothrombin) sequence and food Crab macaque factor (cyno-prothrombin) sequence is synthesized by Shanghai Xu Guan biotechnologies Development Co., Ltd of CRO companies (above factor albumen is by stencil plate sequence of the present invention), His or Flag code sequences are taken by One_step PCR at 3 ' ends After row, the human thrombin with His labels is former (h-prothrombin-his), the human thrombin original (h- with Flag labels Prothrombin-flag), the mouse factor (m-prothrombin-his) with His labels and the food crab with his labels Macaque factor (cyno-prothrombin-his) is cloned into (Biovector, Cat# on pTT5 carriers respectively: 102762), transient expression is realized HEK293E is intracellular.Obtained factor (prothrombin) is recombinantly expressed by first Step purifying and Activation In Vitro obtain fibrin ferment (thrombin), and by being further purified, the people of obtained band Flag labels coagulates Hemase (h-thrombin-flag) is used to be immunized, the human thrombin (h-thrombin-His) with His labels, with His labels Mouse fibrin ferment (m-thrombin-his) and Macaca inus fibrin ferment (cyno-thrombin-his) with his labels are used for The antibody test screened in vitro.

SEQ ID NO:1

Human thrombin with Flag labels is former

MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRANTFLEEVRKGNLERECVEETCSYEEAFE ALESSTATDVFWAKYTACETARTPRDKLAACLEGNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADL QENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVCGQDQVTVAMTPRSEGSSVNLSPPLEQCVPDRGQQYQGRLAVTT HGLPCLAWASAQAKALSKHQDFNSAVQLVENFCRNPDGDEEGVWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDEDSDR AIEGRTATSEYQTFFNPR

SEQ ID NO:2

Human thrombin with His labels is former

MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRANTFLEEVRKGNLERECVEETCSYEE AFEALESSTATDVFWAKYTACETARTPRDKLAACLEGNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTT HPGADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVCGQDQVTVAMTPRSEGSSVNLSPPLEQCVPDRGQQYQGRLA VTTHGLPCLAWASAQAKALSKHQDFNSAVQLVENFCRNPDGDEEGVWCYVAGKPGDFGYCDLNYCEEAVEEETGDGLDED SDRAIEGRTATSEYQTFFNPR

SEQ ID NO:3

Macaca inus factor with his labels

MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQALSLLQRVRRASSGFLEEVFKGNLERECVEETCSYE EAFEALESSTATDAFWAKYTACETARTSRDTLAACLEGNCAEDLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHP GADLQENFCRNPDSSTTGPWCYTTDPTVRREECSIPVCGQDQVTVVMTPRSSVNLSLPSEECVPDRGRQYQGHLAVTTHG LPCLAWASAQAKALSKHQDFDSAVQLVENFCRNPDGDEEGVWCYVAGKPGDFEYCDLNYCEEAVDEETGDGLGEDPD RAIEGRTATSEYQTFFDPR

SEQ ID NO:4

Mouse factor with his labels

MSHVRGLGLPGCLALAALVSLVHSQHVFLAPQQALSLLQRVRRANSGFLEELRKGNLERECVEEQCSYE EAFEALESPQDTDVFWAKYTVCDSVRKPRETFMDCLEGRCAMDLGVNYLGTVNVTHTGIQCQLWRSRYPHKPEINST THPGADLKENFCRNPDSSTTGPWCYTTDPTVRREECSVPVCGQEGRTTVVMTPRSGGSKDNLSPPLGQCLTERGRLYQGN LAVTTLGSPCLPWNSLPAKTLSKYQDFDPEVKLVENFCRNPDWDEEGAWCYVAGQPGDFEYCNLNYCEEAVGEENYDVDE SIAGRTTDAEFHTFFNEK

Note：Underscore part is the blood coagulation enzyme sequence with respective labels in sequence.

2nd, the purifying of factor relevant recom-binant protein, Activation In Vitro, fibrin ferment relevant recom-binant protein and recombinant antibodies Purifying

1) human thrombin original, mouse factor with His label and Macaca inus with His label of the with His labels The purification step of factor：

Cell expression Supernatant samples high speed centrifugation is gone into the removal of impurity.With PBS (pH 7.4) balance nickel post, 2- is rinsed 5 times of column volumes, by Supernatant samples with Ni Sepharose excel posts in certain flow rate.Pillar is rinsed with PBS, extremely A₂₈₀Reading is down to baseline, then rinses chromatographic column with PBS+10mM imidazoles, removes the foreign protein of non-specific binding, and collect outflow Liquid, finally elutes destination protein, and collect eluting peak with the PBS solution containing 300mM imidazoles.Used after the eluent concentration of collection Desalting column changes sample buffer into PBS solution, in case subsequent in vitro is activated and is further purified.

2) purification step of human thrombin original weight histones of the with Flag labels：

Sample high speed centrifugation is gone into the removal of impurity, and is concentrated into proper volume.Flag is balanced using 0.5 × PBS (pH 7.4) Affinity column, rinses 2-5 times of column volume.Cell after removal of impurities is expressed into Supernatant samples upper prop.Pillar is rinsed with 0.5 × PBS, extremely A₂₈₀Reading is down to baseline.Pillar is rinsed with PBS, foreign protein is rinsed, and collect.With containing 100 μ g/ml 3 × Flag polypeptides TBS buffer solutions elute destination protein, and collect, in case subsequent in vitro is activated and is further purified.

3) after the Activation In Vitro of factors relevant recom-binant protein and activation fibrin ferment GAP-associated protein GAP it is further pure Change：

The factor obtained by above-mentioned preliminary affinity chromatography (nickel post or Flag affinity columns), with mass ratio 100：1 with Ecarin enzymes are well mixed to be stayed overnight after incubation at room temperature.Sample high speed centrifugation after activation is removed after precipitation, uses ion exchange column It is further purified with molecular exclusion chromatography SEC.

3.1) ion exchange

Human thrombin with his labels, the mouse fibrin ferment with his labels, the Macaca inus fibrin ferment with his labels are used Cation exchange column SP HP posts are purified, and the human thrombin with flag labels is further purified with anion-exchange column QHP posts. Cation exchange is 10mM PB, pH6.8 buffer solutions from buffer A, and buffer B buffers for 10mM PB, pH6.8,1M NaCl Liquid, anion exchange is 10mM Tris-HCl, pH8.5 buffer solutions from buffer A, and buffer B is 10mM Tris-HCl, PH8.5,1M NaCl buffer solutions.

Ion exchange column is balanced with the buffer A buffer solution of 5 column volumes in advance, will shift to swashing for buffer A buffer solution Thrombin samples balance pillar to A280 absorption values with certain flow rate loading with buffer A buffer solution again after end of the sample after work To baseline.Eluted with buffer B buffer solution within 20 column volume times from 0% concentration gradient for rising to 80%, collect each Eluting peak, component where identifying destination protein by SDS-PAGE, and further verified by mass spectrum and peptide figure.

3.2) molecular-exclusion chromatography

SEC posts (superdex75) are balanced with PBS (pH 7.4) in advance, are eluted after sample loading with PBS as mobile phase, Each eluting peak is collected, component where identifying destination protein by SDS-PAGE, and further verified by mass spectrum and peptide figure.

4) purifying of recombinant antibodies

Cell expression Supernatant samples high speed centrifugation is gone into the removal of impurity, hybridoma expression supernatant Protein G post, recombinant antibodies table Purified up to supernatant with albumin A post.Pillar is rinsed with PBS (pH 7.4), baseline is down to A280 readings.Use 100mM acetic acid PH3.0 elutes destination protein, uses 1M Tris-HCl, pH8.0 to neutralize.After elution samples suitably concentration, balanced using PBS Gel chromatography Superdex200 (GE) is further purified, and to remove aggressiveness, collects monomer peak, dispenses standby.

The preparation of the anti-human fibrin ferment monoclonal antibody of embodiment 2.

1. it is immune

Anti-human anti-thrombin antibody is produced by immune rabbit.Experiment new zealand white rabbit, female, 2-3kg.(Shang Haijia Dry bio tech ltd, animal productiong licensing number：SCXK (Shanghai) 2010-0028).Feeding environment：SPF grades.Rabbit is purchased After entering, laboratory environment is raised 1 week, light dark cycles regulation in 12/12 hour, 20-25 DEG C of temperature；Humidity 40-60%.It will fit The rabbit of environment is answered to be immunized.Immunizing antigen is human thrombin (the SEQ ID NO with Flag labels:Shown in 1 double underline).

Immunization protocol：Antigen is emulsified with Freund's adjuvant (Sigma Cat No.F5881/F5506)：Head exempts from complete with Freund Adjuvant (CFA), booster immunization is with incomplete Freund's adjuvant (IFA).Antigen and adjuvant ratio are 1:1500 μ g/ (head exempts from), 250 μ g/ are only (booster immunization).Antigen after the emulsifications of the 0th day subcutaneous μ g/ of multi-point injection 500 only, head strengthens exempting from once every two weeks after exempting from Epidemic disease, common 6-8 weeks.

Blood was taken in the 22nd, 36,50,64 day, the antibody titer in rabbit anteserum is determined with ELISA method.After 4 exempt from, choosing Select Serum Antibody titre height and titre tends to the rabbit of platform, take spleen and bone marrow cell to carry out RNA extractions and build storehouse.

2. rabbit source phage antibody library is built

Rabbit spleen and marrow are taken, total tissue RNA is extracted with Trizol (Invitrogen Cat No.15596-018).Make Use PrimeScript^TMII 1st Strand cDNA Synthesis Kit kits (Takara Cat No.6210A) enter Row reverse transcription obtains cDNA.According to Christoph Rader et al. report (J.Biol.Chem.2000,275:13668- 13676) primer (Jin Weizhi) for building library is designed and synthesized.Reacted by three-wheel PCR, obtain single chain antigen binding fragment. PCR reactions use LA Tag (Takara Cat No.RR02MB).First round PCR is using cDNA as template, and amplification respectively is obtained Weight chain variable district and light-chain variable sequence；Second wheel PCR is using first round product as template, in the end of weight chain variable district 5 ' and light chain The end of variable region 3 ' introduces Sfi1 restriction enzyme site sequences, and catenation sequence is introduced at the end of weight chain variable district 3 ' and the end of light chain variable district 5 '； Third round PCR is common for template with the weight chain variable district and light chain variable district of the second wheel PCR primer, carries out bridging PCR, is weighed Chain variable region is in preceding, the posterior single chain antigen binding fragment of light chain variable district.

Storehouse carrier pCantab5E (Amersham Biosciences/ are built by single chain antigen binding fragment and by transformation GE Cat No.27-9400-01) carry out digestion, electrophoresis with Sfi1 (NEB Cat No.#R0123L) after useGel Extraction Kit (Omega Cat No.D2500-02) carry out purifying recovery.Then T4DNA ligases (NEB Cat are used No.#M0202L) 16 degree connect 16-18 hours, then carry out purifying recovery with above-mentioned kit, are finally washed with deionized water de-.Take 1 μ g connection products are mixed with 1 electricity transformed competence colibacillus TG1 (Lucigen Cat No.60502-2), electric conversion instrument (Bio Rad Micropulser) parameter is set to 2.5kV, 200 Ω, 25uF, carries out electric conversion.Conversion 10 times is repeated, flat board, 37 DEG C of inversions is applied Culture 16-18 hours.All bacterium colonies are scraped mixing together, final concentration of 15% glycerine is added, -80 DEG C of preservations are standby With.The storage capacity in the storehouse is more than 1 × 10⁸。

3. screening phage antibody library obtains the positive monoclonal of anti-human fibrin ferment

Phage antibody library is carried out after packaging concentration, and three-wheel elutriation is carried out first.By phage library (10¹²~10¹³/ Pfu) it is suspended in 1ml 2%MPBS (PBS containing 2% skimmed milk power), and adds 100 μ lM- 280Streptavidin (Invitrogen Cat No.11206D), is placed on turntable and overturns repeatedly, and room temperature is closed 1 hour.Will Pipe is placed on magnetic frame 2 minutes, removes Dynabeads, and phage library is transferred in new pipe.To the bacteriophage after closing Human thrombin (the SEQ ID NO of the band his labels of 2 μ g/ml biotin labelings are added in storehouse:Shown in 2 double underlines), it is placed in and turns Overturn repeatedly on platform 1 hour.Take 100 μ l Dynabeads to be suspended in 1ml 2%MPBS simultaneously, be placed on turntable and turn over repeatedly Turn, room temperature is closed 1 hour.Pipe is placed on magnetic frame 2 minutes, confining liquid is sucked.Dynabeads after closing is added to In phage library and blood coagulation enzyme mixation with his labels, it is placed on turntable and overturns repeatedly 15 minutes.Pipe is placed in magnetic frame Upper 2 minute, suck mixed liquor.Washed Dynabeads10 times, added with 1ml PBST (PBS containing 0.1%Tween-20) 0.5ml 1mg/ml trypsase (Sigma Cat No.T1426-250MG), is placed on turntable upset repeatedly and is incubated 15 minutes, Eluted.The e. coli tg1 of the bacteriophage direct infection logarithmic phase eluted, determines titre and carries out amplification concentration, Eluriated for next round.Second wheel eluriates phage library (10¹¹~10¹²/ pfu), the people of the band his labels of biotin labeling coagulates Hemase concentration is reduced to 0.5 μ g/ml, and binding time is shorten to 30 minutes, and PBST washing times are increased to 15 times.Third round is eluriated and bitten Thalline storehouse (10¹⁰~10¹¹/ pfu), the human thrombin concentration of the band his labels of biotin labeling is reduced to 0.1 μ g/ml, with reference to when Between shorten to 30 minutes, PBST washing times are increased to 20 times.It is after the completion of three-wheel is eluriated, the phage-infect eluted is big Enterobacteria TG1 applies flat board, random picking monoclonal, for Phage-ELISA.

Clone is inoculated in the degree culture 16~18 hours of 37 DEG C of 96 hole depth orifice plate.Take and be seeded to another 96 hole depth hole on a small quantity Plate, 0.5 or so is reached to OD600, is added M13K07 helper phages (NEB Cat No.N0315S) and is packed.4000g from The heart removes thalline in 10 minutes, draws nutrient solution and carries out human thrombin combination ELISA (see test case 1).Screening obtains positive colony MAB-1512 carries out follow-up humanization as emphasis molecule.Positive colony strain freezes conservation and send sequencing company to survey in time Sequence, measures the corresponding amino acid sequence of positive colony MAB-1512DNA sequences as follows：

>MAB-1512VH

QSLEESGGRLITPGGSLTLTCTASGFSLSNYDMSWVRQAPGKGLEWIGMIETDGSIFHASWVNGRFTIS KTATTVDLKMTSLTTEDTATYFCVRGSNDYDAYGYPYFTLWGQGTLVTVSS

SEQ ID NO：5

>MAB-1512VL

ELVLTQSPSASATLGASAKLTCTLSSAHETYTIDWYQQHQGEAPQYLMELKSDGSYNKGAGVPDRFSGS SSGADRYLMIPSVQADDEATYHCGADFSDGYVFGGGTQLTVTG

SEQ ID NO：6

Note：It is sequentially FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, italic is FR sequences in sequence, underscore is CDR sequence.

Each heavy chain of table 1. and light chain CDR region sequence

Molecular cloning and expression and purification are carried out to positive colony MAB-1512 by embodiment 4, chimeric antibody CH- is obtained 1512, it is combined activity identification (test case 2 and test case 3) for human thrombin, factor.Meanwhile, detect inosculating antibody Body CH-1512 and the fibrin ferment of different genera intersect binding activity (see test case 4), affine with the fibrin ferment of different genera Power (see test case 5), and the influence to human thrombin enzymatic activity (see test case 6).Testing result is as shown in table 2 below and Fig. 1.

The external activity of the fibrin ferment and factor of the chimeric antibody of table 2. and different genera

The result of table 2 shows, the fibrin ferment of CH-1512 and different genera, including people, mouse, monkey fibrin ferment have strong combination Power, and and the former combination of human thrombin it is weaker.Fig. 1 results show that CH-1512 has no effect on it to substrate after being combined with fibrin ferment S2228 enzymolysis activity.

The humanization of the rabbit-anti human thrombin monoclonal antibody of embodiment 3.

1. anti-human anti-thrombin antibody CH-1512 humanized frameworks selection

By comparing IMGT human antibodies weight light chain variable district germ line genes database and MOE softwares, select respectively with it is embedding The high heavy light chain variable district germ line genes of antibody CH-1512 homologys are closed as template, the CDR of this rabbit source antibody is transplanted respectively Into corresponding people source template, the variable region sequences that order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are formed.Wherein Amino acid residue is determined and annotated by Kabat numbering systems.

Chimeric antibody CH-1512 humanized heavy chain's template is IGHV3-66*01 and hjh4.1, and humanization light chain template is IGLV4-60*01 and hjk4.1, humanization variable region sequences are as follows：

>H1512-1VH(CDR graft)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYDMSWVRQAPGKGLEWVSMIETDGSIFHASWVNGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARGSNDYDAYGYPYFTLWGQGTLVTVSS

SEQ ID NO：13

>H1512-1VL(CDR graft)

QPVLTQSSSASASLGSSVKLTCTLSSAHETYTIDWHQQQPGKAPRYLMKLKSDGSYNKGAGVPDRFSGS SSGADRYLTISNLQLEDEADYYCGADFSDGYVFGGGTKVEIK

SEQ ID NO：14

Note：It is sequentially FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, italic is FR sequences in sequence, underscore is CDR sequence.

2. the design of back mutation

CH-1512 back mutation design, see the table below 3：

Table 3.CH-1512 back mutations are designed

Note：As P2L is represented according to Kabat numbering systems, 2 P are mutated back L.

Grafted, transplanting, represent rabbit antibody CDR implantation people's germline FR region sequences.

Particular sequence after mutation is exemplified below：

H1512_VL.1D：

ELVLTQSSSASASLGSSVKLTCTLSSAHETYTIDWYQQQPGKAPRYLMELKSDGSYNKGAGVPDRFSGS SSGADRYLTISNLQLEDEATYHCGADFSDGYVFGGGTKVEIK

SEQ ID NO：15

H1512_VH.1F：

EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYDMSWVRQAPGKGLEWIGMIETDGSIFHASWVNGRFTI SKDNSKNTVYLQMNSLRAEDTAVYFCVRGSNDYDAYGYPYFTLWGQGTLVTVSS

SEQ ID NO：16

H1512_VL.1E：

QLVLTQSSSASASLGSSVKLTCTLSSAHETYTIDWYQQQPGKAPRYLMELKSDGSYNKGAGVPDRFSGS SSGADRYLTISNLQLEDEATYHCGADFSDGYVFGGGTKVEIK

SEQ ID NO：17

Light and weight chain variable region after back mutation is reconfigured, and the antibody of acquisition is entered by the preparation method of embodiment 4 Row clone and expression and purification, and its binding activity with human thrombin albumen is detected by the ELISA experiments of test case 3.According to The following antibody of testing result (table 5) final choice (table 4) carries out the internal external activity detection of next step.

The humanized antibody weight chain variable region sequence of table 4.

Antibody is numbered	Weight chain variable district	Light chain variable district
			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 activity identification (test case 2 and test case 3) of human thrombin, factor is combined for antibody of the present invention, Meanwhile, detection antibody of the present invention intersects binding activity (see test case 4) with the fibrin ferment of different genera, solidifying with different genera The affinity (see test case 5) of hemase, and the influence to human thrombin enzymatic activity (see test case 6).

Test result shows that humanized antibody H1512-12 is as CH-1512, the fibrin ferment with different genera, including People, mouse, monkey fibrin ferment have strong adhesion.Fig. 2 results show that H1512-12 is as CH-1512, after being combined with fibrin ferment Have no effect on its enzymolysis activity to substrate S2228.

Embodiment 4. recombinate and humanized antibody preparation

Antibody has been S228P at Fc sections and dashed forward from people's heavy chain IgG4/ light chains kappa constant region and each variable region combination Become to increase the stability of IgG4 antibody, also can select the other known mutation in this area to increase its performance.

Heavy chain constant region：

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO：18

Constant region of light chain：

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：19

1. the molecular cloning of recombinant antibodies

Phage display screens obtained candidate antibodies molecule after sequencing, obtains variable region coding gene sequence. So that gained primers head and the tail primer is sequenced, so that gene is sequenced as template, each antibody VH/VK gene pieces are built by PCR Section, then homologous recombination is carried out with expression vector pHr (band signal peptide and hIgG4 constant region genes (CH1-FC/CL) fragment), build The total length expressed plasmid VH-CH1-FC-pHr/VK-CL-pHr of recombinant antibodies.

2. the molecular cloning of humanized antibody

Antibody sequence after the design of people source, produces the encoding gene sequence of people's codon preference after codon optimization Row, design primer PCR builds each antibody VH/VK genetic fragments, then with expression vector pHr (band signal peptide and constant region gene (CH1-FC/CL) fragment) homologous recombination is carried out, build the total length expressed plasmid VH-CH1-FC-pHr/VK-CL- of humanized antibody pHr。

3. restructuring and the expression and purification of humanized antibody

The plasmid of expression antibody light and weight chain is with 1 respectively:1.5 ratio transfection HEK293E cells, are collected in expression after 6 days Clearly, high speed centrifugation goes the removal of impurity, is purified with albumin A post.Pillar is rinsed with PBS, baseline is down to A280 readings.With 100mM acetic acid pH3.0 elutes destination protein, uses 1M Tris-HCl, pH8.0 to neutralize.After elution samples suitably concentration, PBS is utilized The gel chromatography Superdex200 (GE) balanced is further purified, and to remove aggressiveness, collects monomer peak, dispenses standby.

Obtain humanized antibody H1512-12.

Biochemical test method validation antibody performance of the present invention and beneficial effect are used below.

The ELISA experiments of the phage display single-chain antibody combination human thrombin albumen of test case 1.

With pH7.4 PBS (Sigma, Cat No.P4417-100TAB) buffer solutions by Streptavidin (Sigma, Cat No.S4762-5MG 5 μ g/ml concentration) are diluted to, are added with the volume in 50 μ l/ holes in 96 hole elisa Plates, in 37 DEG C of incubators Place 2 hours.Discard after liquid, add the μ l/ holes of 5% skim milk (bright skimmed milk power) confining liquid 200 diluted with PBS, 37 DEG C of incubators are incubated to be closed for 2.5 hours.After closing terminates, confining liquid is discarded, and (pH7.4PBS contains with PBST buffer solutions 0.05%tweeen-20) after board-washing 3 times, add 50 μ l/ holes and be diluted to sample diluting liquid (pH7.4PBS contains 1%BSA) Human thrombin albumen (internal pair production, SEQ ID NO of the band his labels of 0.125 μ g/ml biotin labeling:2 double underlines It is shown), put 4 DEG C 16-18 hours.The reaction solution in ELISA Plate is discarded, after PBST board-washings 5 times, 100 μ l/ holes samples are added Dilution 1:The bacteriophage nutrient solution of 1 dilution, is put in 37 DEG C of incubators and is incubated 1 hour.Incubation uses PBST board-washings 5 times after terminating, Add Mouse HRP/anti-M13conjugated secondary antibodies (GE, the Cat No.27- that 100 μ l/ holes sample diluting liquids dilute 9421-01), 37 DEG C are incubated 1 hour.After PBST board-washings 5 times, 50 μ l/ holes TMB chromogenic substrates (KPL, Cat No.52- are added 00-03), in incubation at room temperature 5-10min, 50 μ l/ holes 1M H are added₂SO₄Terminating reaction, with NOVOStar ELIASAs in wavelength Absorption value is read at 450nm.

The ELISA experiments of the anti-thrombin antibody combination human thrombin albumen of test case 2.

The adhesion of antithrombase antibody tests to detect by the ELISA of antibody and human thrombin.Use biotin labeling The fibrin ferment of the band His labels of kit (eastern Renhua, Cat No.LK03) mark passes through the strepto- with being coated in ELISA Plate Avidin is combined so as to fixed in 96 hole elisa Plates, and the power of signal be used to judging antibody and fibrin ferment after antibody is added Binding activity, specific experiment method is as follows.

With pH7.4 PBS (Sigma, Cat No.P4417-100TAB) buffer solutions by Streptavidin (Sigma, Cat No.S4762-5MG 5 μ g/ml concentration) are diluted to, are added with the volume in 50 μ l/ holes in 96 hole elisa Plates, in 37 DEG C of incubators Place 2 hours.Discard after liquid, add the μ l/ holes of 5% skim milk (bright skimmed milk power) confining liquid 200 diluted with PBS, 37 DEG C of incubators incubations stand overnight (16-18 hours) for 2.5 hours or 4 DEG C and closed.After closing terminates, confining liquid is discarded, And with after PBST buffer solutions (pH7.4PBS contains 0.05%tweeen-20) board-washing 5 times, add 50 μ l/ holes sample diluting liquids (pH7.4PBS contains 1%BSA) is diluted to human thrombin albumen (the SEQ ID of the band his labels of 0.5 μ g/ml biotin labeling NO:Shown in 2 double underlines), put 37 DEG C of incubators and be incubated 2 hours.After incubation terminates, the reaction solution in ELISA Plate is discarded, is used After PBST board-washings 6 times, the fibrin ferment test antibodies of the present invention for the various concentrations that 50 μ l/ holes sample diluting liquids dilute are added, are put It is incubated 2 hours in 37 DEG C of incubators.Incubation uses PBST board-washings 5 times after terminating, and adds what 100 μ l/ holes sample diluting liquids diluted The goat-anti people secondary antibody (Jackson Immuno Research, Cat No.109-035-003) of HRP marks, 37 DEG C of incubations 1 are small When.After PBST board-washings 5 times, 50 μ l/ holes TMB chromogenic substrates (KPL, Cat No.52-00-03) are added, in incubation at room temperature 10- 15min, adds 50 μ l/ holes 1M H₂SO₄Terminating reaction, absorption value is read with NOVOStar ELIASAs at wavelength 450nm, is calculated Anti-thrombin antibody of the present invention the results are shown in Table 5 to the combination EC50 values of human thrombin.

The binding activity of the humanized antibody of table 5. and antigen

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

As a result show, the fibrin ferment humanized antibody that present invention screening is obtained has higher knot with human thrombin proteantigen Close activity.

The ELISA experiments of the anti-thrombin antibody combination human thrombin of test case 3. original albumen

Factor is the precursor that enzymolysis activation obtains fibrin ferment.The adhesion of antiprothrombin antibody passes through antibody and people The ELISA of factor tests to detect, specific experiment method is as follows.

It is with pH7.4 PBS (Sigma, Cat No.P4417-100TAB) buffer solutions that the human thrombin with his labels is former (SEQ ID NO:2) 10 μ g/ml concentration are diluted to, are added with the volume in 100 μ l/ holes in 96 hole elisa Plates, 4 DEG C stand overnight (16-18 hours).Discard after liquid, add the μ l/ of 5% skim milk (bright skimmed milk power) confining liquid 200 diluted with PBS Hole, 37 DEG C of incubators are incubated to be closed for 2.5 hours.Closing discards confining liquid after terminating, and with PBST buffer solutions (pH7.4PBS Containing 0.05%tweeen-20) after board-washing 5 times, add 50 μ l/ holes sample diluting liquid (pH7.4PBS contains 1%BSA) gradient dilution Fibrin ferment test antibodies of the present invention, be put in 37 DEG C of incubators and be incubated 1 hour.Incubation uses PBST board-washings 5 times after terminating, and adds Goat-anti people secondary antibody (Jackson the Immuno Research, Cat for the HRP marks that 100 μ l/ holes sample diluting liquids dilute No.109-035-003), 37 DEG C are incubated 1 hour.After PBST board-washings 5 times, 50 μ l/ holes TMB chromogenic substrates (KPL, Cat are added No.52-00-03), in incubation at room temperature 10-15min, 50 μ l/ holes 1M H are added₂SO₄Terminating reaction, is existed with NOVOStar ELIASAs Absorption value is read at wavelength 450nm, combination EC50 value of the anti-thrombin antibody to people's Pro fibrin ferments is calculated.

The antithrombase antibody of test case 4. intersects Binding experiment with different genera fibrin ferment

To detect the external binding ability for the fibrin ferment that the anti-thrombin antibody that screens originate for different genera, mouse with Macaca inus fibrin ferment be used to carry out external combination detection.

With pH7.4 PBS (Sigma, Cat No.P4417-100TAB) buffer solutions by His-tag antibody (GenScript, Cat No.A00174) is diluted to 0.5 μ g/ml concentration, is added with the volume in 100 μ l/ holes in 96 hole elisa Plates, Placed 2 hours in 37 DEG C of incubators.Discard after liquid, add 5% skim milk (bright skimmed milk power) envelope diluted with PBS The μ l/ holes of liquid 200 are closed, 37 DEG C of incubators incubations stand overnight (16-18 hours) for 2.5 hours or 4 DEG C and closed.Closing terminates Afterwards, confining liquid is discarded, and with after PBST buffer solutions (pH7.4PBS contains 0.05%tweeen-20) board-washing 5 times, adds 50 μ l/ holes Mouse thrombin proteins (the SEQ of 0.5 μ g/ml band his labels is diluted to (pH7.4PBS contains 1%BSA) with sample diluting liquid ID NO:Shown in 3 double underlines) or Macaca inus thrombin proteins (SEQ ID NO with his labels:Shown in 4 double underlines), 37 DEG C of incubators are put to be incubated 2 hours.After incubation terminates, the reaction solution in ELISA Plate is discarded, after PBST board-washings 6 times, 50 μ are added The various concentrations fibrin ferment test antibodies of the present invention that l/ holes sample diluting liquid dilutes, are put in 37 DEG C of incubators and are incubated 2 hours.Incubate Educate and PBST board-washings are used after end 5 times, add the goat-anti people's secondary antibody for the HRP marks that 100 μ l/ holes sample diluting liquids dilute (Jackson Immuno Research, Cat No.109-035-003), 37 DEG C are incubated 1 hour.After PBST board-washings 5 times, plus Enter 50 μ l/ holes TMB chromogenic substrates (KPL, Cat No.52-00-03), in incubation at room temperature 10-15min, add 50 μ l/ hole 1M H₂SO₄Terminating reaction, absorption value is read with NOVOStar ELIASAs at wavelength 450nm, calculates anti-thrombin antibody to mouse and food The combination EC50 values of crab macaque fibrin ferment.It the results are shown in Table 6.

The external activity that the humanized antibody of table 6. is combined with the fibrin ferment of different genera

The result of table 6 shows that humanized antibody H1512-12 is as CH-1512, the fibrin ferment with different genera, including Mouse, monkey fibrin ferment have strong adhesion.

Test case 5.BIAcore detection anti-thrombin antibody affinity experiments

It is according to the method described in anti-capture agent box (Cat.#BR-1008-39, the GE) specification of people that the anti-capture of people is anti- Body covalent coupling is on CM5 bio-sensing chips (Cat.#BR-1000-12, GE), so that affinity capture test antibodies, Ran Houyu Chip surface flows through fibrin ferment or factor (human thrombin or factor with his labels), real using Biacore instruments When detection reaction signal so as to obtaining association and dissociation curve, affinity numerical value is obtained by fitting, Y is see the table below.In an experiment After the completion of each circulation dissociation, biochip is cleaned regeneration by the actified solution configured in the anti-capture agent box of employment.As a result see Table 7.

The humanized antibody of table 7. and human thrombin and the former affinity of human thrombin

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

As a result show, humanized antibody H1512-12 of the present invention has strong affinity with human thrombin, and former with human thrombin Affinity is weaker.

Influence of the anti-thrombin antibody of test case 6. to fibrin ferment enzyme activity

This experiment detects antibody of the present invention to fibrin ferment enzyme by testing enzymolysis activity of the fibrin ferment to its substrate S2228 Influence living.

It is 100nM with pH7.4 human thrombin of the PBS gradient dilutions with his labels to concentration, 25 μ l/ holes add black wall In the orifice plate of clear bottom 96.It is 2000nM-62.5nM (1 with PBS fibrin ferment test antibodies gradient dilution of the present invention to concentration:2 ladders Degree dilution), 25 μ l/ holes are also added in this plate.After incubation at room temperature 30-60 minutes, diluting S2228 with PBS (is used to detect blood coagulation The substrate of enzymatic activity, sequence：

H-D-Phe-Pip-Arg-pNA.2HCl, gill biochemistry (Shanghai) Co., Ltd. synthesis) to concentration be 4mM, 50 μ l/ Hole, is added in the plate of previous step.Negative control is only to add fibrin ferment, or only adds S2228 control wells.Incubation at room temperature After 30 minutes, absorption value is read at wavelength 405nm with NOVOStar ELIASAs.As a result Fig. 2, wherein fibrin ferment and S2228 are seen Represent the OD values that negative control hole is measured, 0.625:1,1.25:1,2.5:1,5:1,10:1,20:1 represents test antibodies and blood coagulation The OD values measured during the different mol ratio of enzyme.

Fig. 2 results show, after H1512-12 is combined with fibrin ferment, have no effect on its enzymolysis activity to substrate S2228.

The human normal plasma APTT of test case 7. is tested

This experiment is by the way that human normal plasma and the anti-thrombin antibody of IgG or various concentrations are incubated altogether, and the test present invention is anti- Influence of the body to APTT (activated partial thromboplastin time, activated partial thromboplastin time) value.

IgG is negative control, i.e., the people's immune globulin obtained using traditional affinity chromatography method such as ProteinA purifying In vain；H1512-12 is various concentrations test antibodies of the present invention.

Experimental result is as shown in figure 3, with the increase of antibody H1512-12 concentration, and the APTT values of human normal plasma are also Extension.H1512-12 is in maximum concentration 3200nM, and the increase of APTT values also reaches the peak value (table 8) of 33.6 seconds.

The incrementss of the APTT values to human normal plasma of the various concentrations anti-thrombin antibody of table 8.

The venous thrombosis models of the iron chloride of test case 8. induction

Tail vein injection anti-thrombin antibody, to evaluate the antibody of the present invention to suppressing the effect that thrombus is generated.

SD rats (being purchased from the western pul Bi Kai experimental animals Co., Ltd in Shanghai) adapt to 7 in laboratory environment My god.It is injected intraperitoneally after the anesthesia of 20% urethane, tail vein injection administration after being administered 15 minutes, will infiltrate 10%FeCl₃2 × 5 millimeters of filter paper bars are attached on exposed vein tube wall, and filter paper is removed after 3 minutes, with appropriate normal saline flushing iron chloride Sticking portion, intercepts the posterior vena cava blood vessel of following 10 millimeters of the iliolumbar vein above length of renal vein after 27 minutes, peel off tube chamber In thrombus, weighed on a ten thousandth balance.Record thrombus weight.

Experimental result is as shown in Fig. 4 and table 9, tail vein injection H1512-12, under 6mg/kg and 12mg/kg dosage, The generation of thrombus can significantly be suppressed.The average thrombus weight of H1512-12 6mg/kg groups and H1512-12 12mg/kg groups, point 2.91 milligrams and 0.18 milligram are not reduced to, 10.75 milligrams of phases of average thrombus weight with Isotype antibody group (IgG-6mg/kg) Compare, with pole significant difference statistically.

The average weight and standard error of each group thrombus of table 9.

The machin arteriovenous anastomosis model of test case 9.

8 healthy machins (male and female half and half, Hainan Jin Gang Biotechnology Ltd. provides) are in laboratory environment It is middle to adapt to two weeks, weigh, be divided into 2 groups, every group 4 according to body weight equilibrium.Anaesthetized with free from worries, add amobarbital again afterwards Carry out deep anaesthesia.It is preoperative the not slow veins of lower extremity of different component is injected antibody drug of the present invention and control drug (medicine and 10) dosage be shown in Table.Following steps are operated on 37 DEG C of constant operation tables.A burst arteriovenous is isolated in groin menisectomy, first Do left side and do right side again.Operation venous incubation.Intubation fix after, decontrol artery clamp (decontrol left arterial folder the time be 15 minutes after administration, the time on right side is 25 minutes after administration) and timing is started simultaneously at, blood flow continues 15 minutes.Use haemostatic clamp Or blood vessel clip clamps the two ends of intubation and prevents embolus from coming off, the culture dish that silicone tube is put into physiological saline, Ran Houyong are taken out Operating scissors take out the rush synezis with thrombus, and two ends are respectively absorbed water 3 seconds on tissue, and rear precise electronic is weighed, and calculates blood The net weight of bolt.

The machin arteriovenous anastomosis model of table 10. and postoperative thrombus weight

Experimental result is as shown in figure 5, under 6mg/kg dosage, H1512-12 can significantly suppress the life of macaque thrombus Into compared with the average thrombus weight of negative antibodyome (IgG-6mg/kg), with pole significant difference statistically.

Statistical method is One-way ANOVA and student t test.Phase is organized with Isotype antibody group (IgG-6mg/kg) Than * p ＜ 0.05, * * p ＜ 0.01, * * * p ＜ 0.001.

Embodiment 10：The pharmacokinetics test of anti-thrombin antibody of the present invention

Using machin as animal subject, the blood medicine after administration in machin serum is detected with ELISA method (see test case 4) Concentration, the T1/2 and its major parameter of test medicine are calculated with Winnolin softwares and EXCEL.Study the compound of the present invention Pharmacokinetics behavior in cynomolgus monkey, evaluates its characteristics of pharmacokinetics.

Machin, regular grade, male, 3-5 Sui, 3.5-4.5 kilograms, the limited public affairs of Chengdu West China sea boundary medical sci-tech are used in experiment Take charge of autotrophy.Feeding environment：General area room.Laboratory environment adaptability, which is raised, to be not less than 3 days, and light dark cycles are adjusted within 12/12 hour Section, 16-26 DEG C of temperature, relative humidity 40-70%.Machin is grouped, every group 3.Experimental day, every machin point Test medicine is not injected intravenously, and dosage is 3mg/kg, 10mg/kg.Intravenous injection volume is 10ml/kg, injection speed 2- 4ml/ minutes.

Blood sampling time point is 0.25h, 2h, 4h, 8h, 1d, 2d, 3d, 4d, 7d, 10d, 14d, 21d, 28d, 35d after administration. Blood 1mL is about taken every time, liquaemin anti-freezing, ice chest is kept in, 2-8 DEG C of 1800g is centrifuged 10 minutes and dispensed, -70 DEG C of preservations.

The blood concentration in serum is detected with ELISA method, test medicine is calculated with Winnolin softwares and EXCEL T1/2 and its major parameter, it is as a result as follows：

The pharmacokinetic parameter of antibody of the present invention see the table below 11：

Pharmacokinetics is detected in the anti-thrombin antibody cynomolgus monkey of table 11

Conclusion：In the medicine generation of antibody of the present invention, absorbs good, with obvious medicine for assimilation effect.

Sequence table

<110>Hengrui Medicine Co., Ltd., Jiangsu Prov., Hengrui Pharmaceutical Co., Ltd., Shanghai

<120>Anti-thrombin antibody, its antigen-binding fragment and medical usage

<130> 370019CG-360046

<160> 19

<170> PatentIn version 3.3

<210> 1

<211> 630

<212> PRT

<213>Artificial sequence

<220>

<223>The former sequence of human thrombin with Flag labels

<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>The former sequence of human thrombin with His labels

<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>Macaca inus factor sequence with His labels

<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>Mouse factor sequence with His labels

<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>Mouse

<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>Mouse

<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>Mouse

<400> 7

Asn Tyr Asp Met Ser

1 5

<210> 8

<211> 16

<212> PRT

<213>Mouse

<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>Mouse

<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>Mouse

<400> 10

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

1 5 10

<210> 11

<211> 11

<212> PRT

<213>Mouse

<400> 11

Leu Lys Ser Asp Gly Ser Tyr Asn Lys Gly Ala

1 5 10

<210> 12

<211> 9

<212> PRT

<213>Mouse

<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 grafting), 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 grafting), humanization light chain variable district.

<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, the humanization light-chain variable 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, the humanized heavy chain's variable region sequences 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>H1512-12 light chain variable district

<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 constant region of light chain

<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 (27)

1. a kind of anti-thrombin antibody or its antigen-binding fragment, its comprising one or more be selected from following CDR region sequence or and its Amino acid sequence with least 95% sequence identity：

Heavy chain of antibody variable region HCDR region sequences：SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9；With

Antibody light chain variable region LCDR region sequences：SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

2. anti-thrombin antibody according to claim 1 or its antigen-binding fragment, wherein described anti-thrombin antibody or its Antigen-binding fragment is comprising respectively such as SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:HCDR1, HCDR2 shown in 9 and HCDR3, or with SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 have the amino acid of at least 95% sequence identity Sequence.

3. anti-thrombin antibody according to claim 1 or its antigen-binding fragment, wherein described antibody light chain variable region Comprising respectively such as SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:LCDR1, LCDR2 and LCDR3 shown in 12, or With SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 have the amino acid sequence of at least 95% sequence identity.

4. anti-thrombin antibody according to claim 1 or its antigen-binding fragment, wherein described anti-thrombin antibody or its Antigen-binding fragment is rabbit source antibody or its function fragment.

5. anti-thrombin antibody or its antigen-binding fragment according to claim any one of 1-4, wherein described fibrin ferment Antibody or its antigen-binding fragment further include the light chain FR areas of rabbit source κ chains or rabbit source κ chain variants or further include rabbit The light chain FR areas of source λ chains or rabbit source λ chain variants；And/or wherein described anti-thrombin antibody or its antigen-binding fragment are further Heavy chain FR areas comprising rabbit IgG or its variant.

6. anti-thrombin antibody or its antigen-binding fragment according to claim any one of 1-5, wherein described fibrin ferment Antibody includes SEQ ID NO:5 weight chain variabl area sequence and SEQ ID NO:6 light-chain variable sequence.

7. anti-thrombin antibody or its antigen-binding fragment according to claim any one of 1-6, wherein described fibrin ferment Antibody further includes rabbit source κ chains or the constant region of light chain or the constant region of light chain of rabbit source λ chains or its variant of its variant；With/ Or the wherein further heavy chain constant region comprising rabbit IgG or its variant of described anti-thrombin antibody.

8. anti-thrombin antibody according to claim 1 or its antigen-binding fragment, wherein described anti-thrombin antibody or its Antigen-binding fragment is chimeric antibody or its function fragment.

9. anti-thrombin antibody according to claim 1 or its antigen-binding fragment, wherein described anti-thrombin antibody or its Antigen-binding fragment is humanized antibody or its function fragment.

10. anti-thrombin antibody according to claim 9 or its antigen-binding fragment, wherein described humanized antibody or its Composite sequence and its mutation sequence of the heavy chain FR region sequences of function fragment from human germline heavy IGHV3-66*01 and hjh4.1 Row；FR1, FR2, FR3 area of the wherein described humanized antibody comprising human germline heavy IGHV3-66*01 and hjh4.1 FR4 Area and its mutant nucleotide sequence.

11. anti-thrombin antibody according to claim 10 or its antigen-binding fragment, wherein the humanized antibody or its Function fragment includes SEQ ID NO:Weight chain variable district shown in 13, or with SEQ ID NO:13 have at least 95% sequence same Weight chain variable district shown in the amino acid sequence of property；It is preferred that with SEQ ID NO:13 amino acid sequence has 1-10 amino acid Change.

12. anti-thrombin antibody according to claim 10 or its antigen-binding fragment, wherein described humanized antibody or The heavy chain FR region sequences of its function fragment have the back mutation of 1-10 amino acid, and preferably described back mutation is selected from V47I, S48G, R70K, L75V, A93V and Y91F amino acid back mutation.

13. anti-thrombin antibody or its antigen-binding fragment according to claim 10 or 12, wherein the humanized antibody Or its function fragment includes SEQ ID NO:Weight chain variable district shown in 16, or with SEQ ID NO:16 have at least 95% sequence Weight chain variable district shown in the amino acid sequence of homogeneity.

14. anti-thrombin antibody according to claim 9 or its antigen-binding fragment, wherein described humanized antibody or its The light chain FR region sequences of function fragment derive from people's germline light chain template IGLV4-60*01 and hjk4.1 composite sequence and its dashed forward Become sequence；FR1, FR2, FR3 area of the wherein described humanized antibody comprising people's germline light chain IGLV4-60*01 and hjk4.1 FR4 areas and its mutant nucleotide sequence.

15. anti-thrombin antibody according to claim 14 or its antigen-binding fragment, wherein the humanized antibody or its Function fragment includes SEQ ID NO:Light-chain variable sequence shown in 14, or with SEQ ID NO:14 have at least 95% sequence Light-chain variable sequence shown in the amino acid sequence of homogeneity；It is preferred that with described SEQ ID NO:14 amino acid sequences have 1-10 amino acid change.

16. anti-thrombin antibody according to claim 14 or its antigen-binding fragment, wherein described humanized antibody or The light chain FR region sequences of its function fragment have the back mutation of 1-10 amino acid, and preferably described back mutation is selected from K49E, P2L, H36Y, Y91H, Q1E and D89T amino acid back mutation.

17. anti-thrombin antibody or its antigen-binding fragment according to claim 14 or 16, wherein the humanized antibody Or its function fragment includes SEQ ID NO:15 or SEQ ID NO:Light chain variable district shown in 17, or with SEQ ID NO:15 or SEQ ID NO:17 have the light chain variable district at least shown in the amino acid sequence of 95% sequence identity.

18. anti-thrombin antibody according to claim 9 or its antigen-binding fragment, wherein the humanized antibody or its work( Energy fragment includes (a) weight chain variable district, and the sequence of the weight chain variable district is selected from SEQ ID NO:13、SEQ ID NO:16 and With SEQ ID NO:13 or SEQ ID NO:16 have the amino acid sequence of at least 95% sequence identity；Or/and (b) light chain can Become area, the sequence of the light chain variable district is selected from SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:17 and and SEQ ID NO:14 or SEQ ID NO:15、SEQ ID NO:17 have the amino acid sequence of at least 95% sequence identity.

19. anti-thrombin antibody according to claim 9 or its antigen-binding fragment, wherein described anti-thrombin antibody or its Function fragment, which is included, to be selected from the weight chain variable district and light chain variable district of the following group：

1)SEQ ID NO:13 weight chain variable district and SEQ ID NO:14 light chain variable district；

2)SEQ ID NO:13 weight chain variable district and SEQ ID NO:15 light chain variable district；

3)SEQ ID NO:16 weight chain variable district and SEQ ID NO:15 light chain variable district；

4)SEQ ID NO:16 weight chain variable district and SEQ ID NO:14 light chain variable district；

5)SEQ ID NO:13 weight chain variable district and SEQ ID NO:17 light chain variable district；With

6)SEQ ID NO:16 weight chain variable district and SEQ ID NO:17 light chain variable district.

20. anti-thrombin antibody or its antigen-binding fragment according to claim any one of 8-19, wherein the fibrin ferment Antibody further includes the heavy chain constant region selected from humanized IgG 1, IgG2, IgG3, IgG4 and its variant, preferably comprises people source IgG4 or its variant heavy chain constant region, particularly preferably include such as SEQ ID NO:Heavy chain constant region shown in 18；With

The anti-thrombin antibody further includes the constant region of light chain selected from people source κ, λ chain and its variant, preferably comprises such as SEQ ID NO:Constant region of light chain shown in 19.

21. the anti-thrombin antibody or its antigen-binding fragment of any one of claim 1 to 20, wherein the antigen-binding fragment is Selected from Fab, Fab', F (ab') 2, single-chain antibody (scFv), the V areas (double antibody) of dimerization, disulfide-stabilized V areas (dsFv) and the peptide comprising CDR antigen-binding fragment.

22. a kind of pharmaceutical composition, its fibrin ferment according to any one of claim 1 to 21 for containing therapeutically effective amount resists Body or its antigen-binding fragment, and one or more pharmaceutically acceptable carriers, diluent or excipient.

23. a kind of DNA molecular, it encodes the anti-thrombin antibody or its antigen binding fragment according to claim any one of 1-21 Section.

24. a kind of expression vector, it contains DNA molecular according to claim 23.

25. a kind of host cell, it is the host cell converted with expression vector according to claim 24, the host Cell is selected from prokaryotic and eukaryotic, preferably eukaryotic, more preferably mammalian cell.

26. anti-thrombin antibody or its antigen-binding fragment according to any one of claim 1 to 21 or according to claim The DNA molecular described in pharmaceutical composition or claim 23 described in 22, prepare be used for treat thrombin-mediated disease or Purposes in the medicine of illness, wherein described disease or illness is preferably thrombotic diseases；More preferably venous thronbosis Moved with apoplexy caused by pulmonary embolism, Arterial thrombosis, thrombosis and distal aorta formation, atherosclerosis disease, brain Arteries and veins disease or distal aorta disease；Most preferably venous thronbosis, apoplexy and atherosclerosis caused by thrombosis.

27. for detecting or determining the reagent of human thrombin, antibody of the reagent comprising any one of claim 1 to 21 or its Antigen-binding fragment.