CN110343181A

CN110343181A - For the single domain antibody of plasma thromboplastin component (FIX)

Info

Publication number: CN110343181A
Application number: CN201810305368.7A
Authority: CN
Inventors: 徐霆; 王玲; 恽丽红; 白玉; 汪皛皛
Original assignee: Suzhou Alphamab Co Ltd
Current assignee: Suzhou Alphamab Co Ltd
Priority date: 2018-04-08
Filing date: 2018-04-08
Publication date: 2019-10-18
Anticipated expiration: 2038-04-08
Also published as: CN110343181B

Abstract

The present invention relates to field of pharmaceutical biology, the single domain antibody for plasma thromboplastin component (FIX) is disclosed.Specifically, the invention discloses a kind of FIX binding molecule and application thereof derived from the single domain antibody, especially for detecting FIX, preparing the purposes in weary FIX blood plasma.

Description

For the single domain antibody of plasma thromboplastin component (FIX)

Technical field

The present invention relates to field of pharmaceutical biology, the single domain antibody for plasma thromboplastin component (FIX) is disclosed.Specifically, The invention discloses a kind of FIX binding molecule and application thereof derived from the single domain antibody, especially for detecting FIX, system Purposes in standby weary FIX blood plasma.

Background technique

Coagulation factor is the various protein components for participating in Blood Coagulation Process.Its physiological action is, in angiorrbagia When be activated together with platelet adhesion and mend plug blood vessel on leak.This process is referred to as blood coagulation.They partially by Liver generates.It can be inhibited by cumarin.The precedence Rome number being found for Uniform Name, the World Health Organization by it Word number, there is factor I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII etc..

The genetic coagulation disorders as caused by deficiency of coagulation factors are generally divided into A type and B-mode, Belong to sex-linked inheritance.The illness rate of newborn man is about 1:5000.The usual state of an illness is more serious, and hemostasis and bleeding just go out Now it is more early.It is calculated by disease incidence, global about 400,000 patients.The patient in China should have 100,000, but regrettably mostly not Diagnosis is obtained, patient receiving treatment's estimation at present is only~8% (about more than 8000 people).

Hemophilia B be as lack nine factor of blood coagulation (FIX) in vivo and caused by blood coagulation disorders disease, with spontaneous Property or the relevant bleeding of wound are characterized, and bleeding part is mainly in joint, soft tissue and muscle.According to Chinese Academy of Medical Sciences's blood The information at sick hospital's (hematology research institute) thrombus hemostasis diagnosis and treatment center, the haemophiliac that the current whole nation registers on the books have 9804 People, but actual patient quantity is far above in this, and wherein hemophilia B patient accounts for about 15% to 20%.

Currently, hemophilia there is no radical cure method.Best treatment is replacement therapy, that is, is inputted accordingly to patient in time Coagulation factor.Otherwise once there is wound in patient, will because coagulation function lack due to blood loss it is dead.If patient's bleeding , the frequency of medication is just higher.Most of pharmaceutical nine factor of blood coagulation relies on blood plasma product at present in China, i.e., by source of supply Limitation, and there are security risks；And unique recombinant product is then the BeneFIX of Wyeth, the U.S., it is expensive. BeneFIX price Wyeth 0.93 dollar of every international unit of quotation in 2009, patient report per unit actual medical expense up to 4 Dollar, ampoule 250-2000 international unit.Temporary treatment is carried out for bleeding, an annual haemophiliac expends big Ten thousand dollars of about 15-20, and if carrying out prophylactic treatment, then it to double.It is uniquely effective in view of current treatment hemophilia B The drug market effect that only has FIX and FIX to generate, have in the world more companies' research and development and production FIX, FIX imitation medicine and Long-acting FIX.

For recombinating the exploitation of FIX, the key link is just to try to maintain its biological activity and bioavilability. Include a variety of posttranslational modifications on FIX, and has very important influence to its biological activity.Current analysis means are often Posttranslational modification can only be carried out to highly purified recombination FIX and than analysis living in development late stage.If can have a kind of simpler Just detection method compares the recombination FIX's that purity is lower, even in cell feed liquid in the simple and quick analysis of early stage energy Activity, for project development, craft screening of early stage etc. can all have huge help.

In the Activity determination of recombinant blood coagulation factor, need to use weary nine factors blood plasma.The diagnosis of coagulation disorders simultaneously It is also required to the coagulation factor activity of detection patient.This field also need it is efficient, inexpensive prepare weary coagulation factor (such as blood coagulation because Sub- IX) blood plasma method.

Summary of the invention

In a first aspect, the present invention provides a kind of plasma thromboplastin component (FIX) binding molecule, in conjunction with FIX and can include The single variable domains of at least one immunoglobulin, the single variable domains of at least one immunoglobulin include to be selected from CDR1, CDR2 and CDR3 below:

(1) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3；

(2) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6；

(3) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9；

(4) shown in CDR2 shown in CDR1 shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 CDR3；

(5) shown in CDR2 shown in CDR1 shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 CDR3；With

(6) shown in CDR2 shown in CDR1 shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 CDR3。

In some embodiments, the single variable domains of the immunoglobulin are VHH.

In some specific embodiments, the VHH includes any amino acid sequence in SEQ ID NO:19-24.

In some embodiments, the FIX binding molecule also includes immunoglobulin fc region.In some embodiments In, the immunoglobulin fc region is the area human immunoglobulin(HIg) Fc.In some specific embodiments, wherein the immune globulin The amino acid sequence in the white area Fc is shown in SEQ ID NO:25.

In some specific embodiments, the KD value of the FIX binding molecule combination FIX is less than 1 × 10^-7M, preferably smaller than 1×10^-8M, more preferably less than 1 × 10^-9M, more preferably less than 1 × 10^-10M, particularly more preferably less than 1 × 10^-11M。

In second aspect, the present invention provides a kind of nucleic acid molecules, encodes FIX binding molecule of the invention.

In the third aspect, the present invention provides a kind of expression vector, and it includes what is be operably connected with expression regulation element The nucleic acid molecules of second aspect of the present invention.

In fourth aspect, the present invention provides a kind of host cell, it includes nucleic acid molecules of the invention or with of the invention Expression vector conversion, and the FIX binding molecule can be expressed.

At the 5th aspect, the present invention provides a kind of method for generating FIX binding molecule of the invention, comprising:

A) host cell of the invention is cultivated under conditions of allowing the FIX binding molecule to express；

B) from the culture recycling derived from step a) by the FIX binding molecule of the host cell expression；And

C) optionally it is further purified and/or modifies the FIX binding molecule derived from step b).

At the 6th aspect, the present invention provides in a kind of detection target sample the presence of FIX and/or quantitative to FIX in target sample And/or the kit of the carboxylated level of FIX in target sample is detected, include FIX binding molecule of the invention.In some embodiment party In case, the kit also includes the control sample of the FIX containing predetermined amount or the FIX containing predetermined carboxylated level.

At the 7th aspect, the present invention provide in a kind of detection target sample the presence of FIX and/or to FIX in sample it is quantitative and/ Or in test sample the carboxylated level of FIX method, which comprises

A) under conditions of being capable of forming compound between FIX binding molecule and FIX, make the target sample and control sample It is contacted respectively with FIX binding molecule of the invention；

B) formation of compound is detected,

Wherein between the target sample and control sample compound formed difference instruction target sample in FIX presence and/ Or amount and/or its carboxylated it is horizontal, it is preferable that the control sample contains the FIX of predetermined amount or horizontal containing predetermined carboxylated FIX.

In eighth aspect, the present invention provides a kind of method for preparing weary FIX blood sample, including

Contact blood sample with FIX binding molecule of the invention, thus the FIX in the blood sample with it is described FIX binding molecule forms compound,

Separate the compound with the blood sample, and

C) weary FIX blood sample is harvested.

In some embodiments, wherein the FIX binding molecule is fixed on solid support.In some embodiment party In case, wherein the blood sample is blood plasma.

At the 9th aspect, the present invention provides a kind of FIX affinity chromatography medium, and it includes be fixed with FIX of the invention thereon The solid support of binding molecule.In some embodiments, wherein the solid support is made of material selected from the following: Polyethylene, polystyrene, polypropylene, polysulfones, polyacrylonitrile, polycarbonate, polyurethane, silica, latex, glass, fiber Element, cellulose ethanoate, the glucan of crosslinking, the agarose of crosslinking, chitin, chitosan, the glucan of crosslinking, crosslinking sea Alginic acid, silicone resin, fluoropolymer, magnetic medium and other synthetic polymers.In some embodiments, FIX affinity chromatography Medium is the agarose medium or magnetic bead that immobilization has FIX binding molecule of the invention.

At the tenth aspect, the present invention provides a kind of device for being used to prepare weary FIX blood sample, includes FIX of the invention Affinity chromatography medium.

Detailed description of the invention

Fig. 1 shows the sequence of anti-FIX single domain antibody.

Fig. 2 shows the binding curve of anti-FIX single domain antibody-Fc fusion protein and antigen FIX.

Fig. 3 shows the western blot result of FIX and FVII Yu anti-FIX single domain antibody-Fc fusion protein.

Fig. 4 shows the western blot result of recombined human FIX Yu anti-FIX single domain antibody-Fc fusion protein.

Fig. 5 shows the recombined human FIX albumen of different carboxylated and the western blot of anti-FIX single domain antibody-Fc fusion protein As a result.

Detailed description of the invention

Definition

It unless otherwise directed or defines, otherwise all terms used all have the common meaning in this field, which will Understood by those skilled in the art.See, for example standard manual, such as Sambrook et al., " Molecular Cloning: A Laboratory Manual"；Lewin,"Genes VIII"；And Roitt et al., " Immunology " (the 8th edition), with And the general prior art cited herein；In addition, unless otherwise stated, all methods, step, the skill that are not described in detail specifically Art and operation can with and carry out in a way known, which will be understood by those skilled in the art.Also join Examine such as standard manual, the above-mentioned general prior art and other bibliography cited therein.

Unless otherwise stated, no matter the term " antibody " being used interchangeably or " immunoglobulin " refer to herein Heavy chain antibody also refers to conventional 4 chain antibodies, is both used as general terms to include full length antibody, its single chain and its all portion Divide, (including but not limited to antigen-binding domains or segment distinguish such as VHH structural domain or VH/VL structure for structural domain or segment Domain).In addition, the term as used herein " sequence " (such as in " immunoglobulin sequences ", " antibody sequence ", " single varistructure In the term of domain sequence ", " VHH sequence " or " protein sequence " etc.) it is generally understood that not only include related amino acid sequence, but also packet The nucleic acid sequence or nucleotide sequence for encoding the sequence are included, unless the explanation for needing more to limit herein.

As used herein, term (more peptide or proteins) " structural domain " refers to folded protein structure, can be independently of egg White rest part maintains its tertiary structure.In general, structural domain is responsible for the single functional character of albumen, and in many feelings Other albumen can be added, remove or are transferred under condition without losing the rest part of albumen and/or the function of structural domain.

Term " immunoglobulin domains " as used herein refers to antibody chain (such as the chain or again of conventional 4 chain antibodies The chain of chain antibody) spheric region, or refer to the polypeptide being substantially made of this kind of spheric region.Immunoglobulin domains It is characterized in that it maintains the immunoglobulin folding feature of antibody molecule, by being arranged in two β-pleated sheets optionally by guarding two 2 layer interlayers of about 7 stable antiparallel β-pleated sheet stocks of sulfide linkage form.

Term " immunoglobulin variable domain domain " as used herein refers to substantially by this field and separately below Referred to as the four of " framework region 1 " or " FR1 ", " framework region 2 " or " FR2 ", " framework region 3 " or " FR3 " and " framework region 4 " or " FR4 " The immunoglobulin domains of a " framework region " composition, wherein the framework region is known as " complementation by this field and separately below Determine area 1 " or three " complementation decisions of " CDR1 ", " complementary determining region 2 " or " CDR2 " and " complementary determining region 3 " or " CDR3 " Area " or " CDR " are spaced apart.Therefore, the general structure in immunoglobulin variable domain domain or sequence can indicate as follows are as follows: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4.Immunoglobulin variable domain domain assigns antibody pair due to antigen binding site The specificity of antigen.

Term " the single variable domains of immunoglobulin " as used herein be refer to not with other immune globulins The immunoglobulin variable domain domain of molecule of the antigen binding epitope in the case where white variable domains pairing.In meaning of the present invention An example of the single variable domains of immunoglobulin be " domain antibodies ", such as the single varistructure of immunoglobulin Domain VH and VL (VH structural domain and VL structural domain).Another example of the single variable domains of immunoglobulin is as defined below " VHH structural domain " (or being referred to as " VHH ") of Camelidae.

" VHH structural domain ", also known as single domain heavy chain antibody, VHH, V_HH structure domain, VHH antibody fragment and VHH antibody are Referred to as variable domains (the Hamers- of the antigen binding immunoglobulin of " heavy chain antibody " (i.e. " antibody for lacking light chain ") Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.:"Naturally occurring antibodies devoid of light chains"；Nature 363,446-448(1993)).Using term " VHH structural domain " by the variable domains and to be present in conventional 4 chain antibodies Heavy-chain variable domains (its referred to herein as " VH structural domain ") and the light chain variable knot that is present in conventional 4 chain antibodies Structure domain (it is referred to herein as " VL structural domain ") distinguishes.VHH structural domain specific binding epitope is without other antigens (this is with VH the or VL structural domain in conventional 4 chain antibodies on the contrary, epitope is tied by VL structural domain and VH in this case for binding structural domain Structure domain identifies together).VHH structural domain is the compact stabilized and efficient antigen recognizing formed by single immunoglobulin domains Unit.

In the context of the present invention, term " single domain heavy chain antibody ", " VHH structural domain ", " VHH ", " V_HH structure domain ", " VHH antibody fragment ", " VHH antibody " andAnd "(" Nanobody " be structural domain " Ablynx N.V. company, the trade mark of Ghent, Belgium) it is used interchangeably.

Such as institute in Fig. 2 of Riechmann and Muyldermans, J.Immunol.Methods 231,25-38 (1999) Show, amino acid residue applied by the VHH structural domain for Camelidae, according to the general of Kabat et al. VH structural domain provided Numerical system numbers (" Sequence of proteins of immunological interest ", US Public Health Services, NIH Bethesda, MD, publication the 91st).According to the numerical system,

- FR1 is included in the amino acid residue at the 1-30 of position,

- CDR1 is included in the amino acid residue at the 31-35 of position,

- FR2 is included in the amino acid at the 36-49 of position,

- CDR2 is included in the amino acid residue at the 50-65 of position,

- FR3 is included in the amino acid residue at the 66-94 of position,

- CDR3 is included in the amino acid residue at the 95-102 of position, and

- FR4 is included in the amino acid residue at the 103-113 of position.

It is noted, however, that as in this field for the amino acid well known to VH structural domain and VHH structural domain, in each CDR The sum of residue may be different, thereby increases and it is possible to not correspond to sum (the i.e. basis of the amino acid residue by Kabat number instruction One or more positions of Kabat number may not be occupied in actual sequence or actual sequence may be containing more than Kabat Number the amino acid residue of allowed number).This means that in general, may be corresponded to according to the number of Kabat or may not Actual number corresponding to amino acid residue in actual sequence.

There are also the other systems that the amino acid residue of VH structural domain is numbered, such as Chothia to compile as is generally known in the art Number system.The amino acid number of Chothia is identical as Kabat, but it is based in antibody variable plot structure that it, which will divide CDR region, The ring area (loop) carry out, therefore on the amino acid region that CDR region is included can be different with Kabat.Furthermore There are also AbM coded systems etc..Other described coded systems can also be applied similarly to VHH structural domain.However, unless otherwise saying It is bright, otherwise in this specification, claims and attached drawing, it will comply with as described above according to Kabat and suitable for VHH structural domain Number, or the number of comprehensive Kabat and Chothia.

The sum of amino acid residue in VHH structural domain will be usually in 110 to 120 ranges, usually between 112 and 115 Between.It should however be noted that smaller and longer sequence is also suitable for purpose as described herein.

VHH structural domain and containing its polypeptide other structures characteristic and functional character may be summarized as follows:

VHH structural domain (its naturally " design " with there is no light variable domains and not with light chain variable domain In the case where the interaction of domain in conjunction with antigen function) it can be used as single and relatively small functional antigen integrated structure list Member, structural domain or polypeptide.This distinguishes VH the and VL structural domain of VHH structural domain and conventional 4 chain antibodies, these VH and VL structural domains are certainly Body is normally unsuitable for carrying out practical application as single antigen-binding proteins or the single variable domains of immunoglobulin, but needs With some form or another form is combined to provide functional antigen combining unit (such as with such as Fab segment conventional antibody piece The form of section；Or in the form of the scFv that the VH structural domain by being covalently attached with VL structural domain forms).

It is many individually or as a part-offer of larger polypeptide using VHH structural domain-due to these peculiar properties Better than using conventional VH and VL structural domain, scFv or conventional antibody segment (such as Fab- or F (ab ')₂Segment) it is significant excellent Gesture: only needing single structure domain with high-affinity and highly selective combination antigen, so that both having needed not exist for two individually Structural domain also has no need to ensure that two structural domains have that (such as scFv generally requires use with appropriate space conformation and configuration Specially designed connector)；VHH structural domain can express from term single gene and fold or modify after not need translation；VHH structural domain Multivalence and polyspecific format (formatting) can be easily transformed into；VHH structural domain high soluble and without aggregation tendency；VHH knot Structure domain is highly stable to heat, pH, protease and other denaturants or condition, and therefore can not make in preparation, storage or transport With freezing equipment, to reach save the cost, time and environment；VHH structural domain is easily prepared and relatively inexpensive, or even is producing In required scale also so；VHH structural domain compared with conventional 4 chain antibodies and its antigen-binding fragment it is relatively small (about 15kDa or size are the 1/10 of routine IgG), therefore compared to conventional 4 chain antibodies and its antigen-binding fragment, it shows higher It tissue permeability and can be administered with higher dosage；VHH structural domain can show so-called chamber binding property (especially because tying with conventional VH Compare its extended CDR3 ring in structure domain), thus reachable conventional 4 chain antibodies and its not accessibility target of antigen-binding fragment and table Position.

The method for combining the VHH of specific antigen or epitope is obtained, was previously had been disclosed in following documents: R.van der Linden et al.,Journal of Immunological Methods,240(2000)185–195；Li et al.,J Biol Chem.,287(2012)13713–13721；Deffar et al.,African Journal of Biotechnology Vol.8(12),pp.2645-2652,17June,2009；WO94/04678；US 7790367,2006- 09-14, METHOD FOR SCREENING A LIBRARY OF VHH POLYPEPTIDES, Casterman, Cecile, Hamers,Raymond；With US 7786047,2006-02-10, IMMUNOGLOBULINS DEVOID OF LIGHT CHAINS, Casterman, Cecile, Hamers, Raymond.

In addition, those skilled in the art will also be appreciated that, it is possible to by one or more above-mentioned CDR " transplanting " in other " branch On frame " (including but not limited to people's bracket or non-immunoglobulin support).Bracket and technology suitable for CDR transplanting is in ability It is known in domain.

As used herein, term " epitope " or the term " antigenic determinant " being used interchangeably refer to that the paratope of antibody is tied Any antigenic determinant on the antigen of conjunction.Antigenic determinant generally comprises the chemically active surface group of molecule, such as amino Acid or carbohydrate side chain, and usually there is specific three-dimensional structural feature and specific charge characteristic.For example, epitope is usually with only Special space conformation includes at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 amino acid continuously or discontinuously, It can be " linear " epitope or " conformation " epitope.See, e.g., Epitope Mapping Protocols in Methods In Molecular Biology, volume 66, G.E.Morris, Ed. (1996).In linear epitope, protein and phase interaction Linearly exist with the point of all interactions between molecule (such as antibody) along the primary amino acid sequences of protein.In structure As in epitope, the point of interaction exists across gal4 amino acid residue separated from each other.

Many epitope mapping techniques well known in the art can be used to identify the epitope of given antigen.See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, volume 66, G.E.Morris, Ed.(1996).For example, linear epitope can be determined for example, by following methods: simultaneously synthesizing big on solid support Peptide is measured, wherein these peptides correspond to each section of protein molecule, and make these peptides in the case where still connecting with support With antibody response.These technologies are known and are described in such as U.S. Patent No. 4,708,871 in the art；Geysen Et al. (1984) Proc.Natl.Acad.Sci.USA 81:3998-4002；Geysen et al. (1986) In Molec.Immunol.23:709-715.Similarly, comformational epitope can be by such as tieing up for example, by x-ray crystallography and 2 Nuclear magnetic resonance determines that the space configuration of amino acid is identified.See, for example, Epitope Mapping Protocols (ibid).

Routine techniques well known by persons skilled in the art can be used, just with the binding competition screening antibodies of same epitope. For example, can be at war with and cross competition research, to be contended with one other or the antibody of cross competition and antigen binding.Based on it Cross competition be described in international patent application WO03/48731 to obtain the high throughput method of the antibody in conjunction with same epitope In.Therefore, routine techniques well known by persons skilled in the art can be used, obtain and antibody molecule competitive binding FIX of the invention On same epitope antibody and its antigen-binding fragment.

In general, term " specificity " refers to that specific antigen binding molecule or antigen-binding proteins are (such as of the invention The single variable domains of immunoglobulin) the combinative different types of antigens of molecule or epitope number.It can be based on antigen binding The affinity and/or affinity of molecule determine its specificity.By Dissociation equilibrium constant (KD) institute of antigen and antigen-binding proteins The affinity of expression, be the measurement of bond strength between antigen binding site on epitope and antigen-binding proteins: KD value is smaller, table Stronger bond strength between position and antigen binding molecules (is 1/ alternatively, affinity is also referred to as association constant (KA) KD).As it will be apparent to those skilled in the art that affinity can be measured in a known way depending on specific interested antigen.Parent Resultant force is that antigen binding molecules (such as the single variable domains of immunoglobulin, antibody, immunoglobulin or contain the more of its Peptide) and related antigen between bond strength measurement.Affinity and following the two in relation to: with the antigen on its antigen binding molecules Affinity between binding site, and the number of relevant binding site being present on antigen binding molecules.

As used herein, term " FIX binding molecule " mean it is any can be with the molecule of high-affinity combination FIX.FIX knot Closing molecule may include the antibody as defined herein or its conjugate for FIX.FIX binding molecule also covers immunoglobulin Superfamily antibody (IgSF) or CDR transplanted molecule.

" FIX binding molecule " or can refer in conjunction with FIX monovalent molecule (i.e. in conjunction with an epitope of FIX point Son) and divalent or multivalent binding molecule (combining the binding molecule of more than one epitope)." FIX combination point of the invention Son " may include at least one the single variable domains of immunoglobulin such as VHH for combining FIX.In some embodiments, originally " the FIX binding molecule " of invention may include the single variable domains of the immunoglobulin such as VHH of two basic change FIX.

In general, FIX binding molecule of the invention will be such as to pass through Biacore or KinExA or biomembrane interference technique Preferably the 10 of (Biolayerinterferometry, BLI) measurement^-7To 10^-11Mol/L (M), more preferable 10^-8To 10^-11It rubs You/liter, even more preferably 10^-9To 10^-11, even more preferably 10^-10To 10^-11Or lower dissociation constant (KD), and/or so that Few 10⁷M^-1, preferably at least 10⁸M^-1, more preferably at least 10⁹M^-1, more preferably at least 10¹⁰M^-1, for example, at least 10¹¹M^-1Association it is normal Number (KA) antigen to be combined (i.e. FIX).Antigen-binding proteins can be with known to the combination of antigen or epitope What is suitble to mode to measure, including such as surface plasma body resonant vibration art (SPR) measurement, Scatchard measurement, biomembrane interference (Bio-Layer Interferometry, BLI) detection, and/or competitive binding assay (such as radiommunoassay (RIA), Enzyme immunoassay (EIA) (EIA) and sandwich competitive assay.

Compared to its natural biological sources and/or the reaction medium or culture medium of the polypeptide or nucleic acid molecules are obtained, when it With at least one in the source or medium (culture medium) usually associated other components (such as another protein/polypeptide, Another nucleic acid, another biological components or macromolecular or at least one pollutant, impurity or microcomponent) separation when, polypeptide or core Acid molecule is considered as " substantially separate ".Particularly, polypeptide or nucleic acid molecules have purified at least 2 times, particularly at least 10 at it Times, more particularly at least 100 times and up to 1000 times or 1000 times or more whens be considered as " substantially separate ".Through suitable skill Art (such as being suitble to chromatographic technique, such as polyacrylamide gel electrophoresis) determines that " substantially separate " polypeptide or nucleic acid molecules are excellent Choosing is substantially homogeneous.

The anti-FIX antibody of " affinity maturation ", especially VHH or domain antibodies have one in one or more CDR A or multiple variations, the variation cause to increased the affinity of FIX compared to its respective anti-FIX antibody of parent.Parent Anti- FIX antibody with power maturation can be for example, by being prepared by method as known in the art as described below: Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc.Nat.Acad.Sci, USA 91:3809- 3813.；Shier et al., 1995, Gene 169:147-155；Yelton et al., 1995, Immunol.155:1994-2004； Jackson et al., 1995, J.Immunol.154 (7): 3310-9；And Hawkins et al., 1992, J.MoI.Biol.226 (3):889896；KS Johnson and RE Hawkins, " Affinity maturation of antibodies using phage display”,Oxford University Press 1996。

FIX binding molecule of the invention

In a first aspect, it includes at least one to exempt from conjunction with FIX the present invention provides a kind of FIX binding molecule The single variable domains of epidemic disease globulin.In some embodiments, the FIX binding molecule includes the immune of a combination FIX The single variable domains of globulin.In some embodiments, the FIX binding molecule includes that two or more combine FIX The single variable domains of immunoglobulin.In some embodiments, the amino acid sequence of FIX of the present invention is shown in SEQ ID NO:32, for the full length amino acid sequence of mature people FIX.

In some embodiments, the single variable domains of at least one immunoglobulin include selected from the following CDR1, CDR2 and CDR3:

(1) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 (CDR corresponding to antibody strain nFN50)；

(2) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 (CDR corresponding to antibody strain nFN52)；

(3) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 (CDR corresponding to antibody strain nFN62)；

(4) shown in CDR2 shown in CDR1 shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 CDR3 (CDR corresponding to antibody strain nFN64)；

(5) shown in CDR2 shown in CDR1 shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 CDR3 (CDR corresponding to antibody strain nFN65)；With

(6) shown in CDR2 shown in CDR1 shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 CDR3 (CDR corresponding to antibody strain nFN69).

In some embodiments, the single variable knot of at least one immunoglobulin in FIX binding molecule of the invention Structure domain is VHH.In some specific embodiments, the VHH includes amino acid sequence any in SEQ ID NO:19-24. In other embodiments, the VHH in FIX binding molecule of the invention includes and has with any in SEQ ID NO:19-24 The ammonia of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity Base acid sequence.Alternatively, the amino acid sequence of the VHH includes one or more amino compared with any in SEQ ID NO:19-24 Acid replaces, and preferably conserved amino acid replaces.For example, replacing comprising 1,2,3,4,5,6,7,8,9 or 10 conserved amino acid.

In some embodiments, FIX binding molecule of the invention is obtained by affinity maturation.Through affinity at Ripe FIX binding molecule can have one or more variations in one or more CDR, and the variation leads to the parent to FIX It increased with power compared to parent's FIX binding molecule.

In some embodiments, FIX binding molecule of the invention, in addition at least one can specifically bind FIX's The single varistructure of immunoglobulin is overseas, also includes immunoglobulin fc region.Comprising exempting from FIX binding molecule of the invention The area epidemic disease globulin Fc can make the binding molecule form dimer.The area Fc for use in the present invention can come from different subtype Immunoglobulin, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM.

The immunoglobulin fc region is preferably the area human immunoglobulin(HIg) Fc, the more preferably area Fc of human IgG1.Some In specific embodiment, the amino acid sequence of the immunoglobulin fc region is shown in SEQ ID NO:25.

In some specific embodiments, in FIX binding molecule of the invention, the immunoglobulin fc region (such as people The area Fc of IgG1) directly or by connector (such as peptide linker) it is indirectly connected to the single variable domains of the immunoglobulin (such as VHH C-terminal).

On the other hand, FIX binding molecule of the invention also cover can with by ammonia any in SEQ ID NO:19-24 The anti-FIX antibody molecule of same epitope on the VHH combination FIX of base acid sequence composition.

The KD value of the FIX binding molecule combination FIX of the invention can be less than 1 × 10^-7M, preferably smaller than 1 × 10^-8M、 More preferably less than 1 × 10^-9M, more preferably less than 1 × 10^-10M, it is particularly more preferably less than 1 × 10^-11M。

In some embodiments, FIX binding molecule of the invention specifically binds FIX, without combining or with lower Affinity combines other coagulation factors such as FVII, FVIII.

In some embodiments, FIX binding molecule of the invention and gamma carboxylated degree are higher, active better FIX combines stronger.

In addition, FIX binding molecule of the invention has tolerance to acid, alkali and/or salt treatment processing.For example, acid is (such as 0.1M acetic acid) processing about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 8 hours, about 16 hours, it is about 24 small When, about 32 hours or more long and/or alkali (such as 0.1M sodium bicarbonate) handle about 1 hour, about 2 hours, about 3 hours, about 4 hours, About 5 hours, about 8 hours, about 16 hours, about 24 hours, about 32 hours or more long and/or salt (such as 0.5M sodium chloride) processing about 1 Hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 8 hours, about 16 hours, about 24 hours, about 32 hours are more long Afterwards, FIX binding molecule activity of the invention is kept essentially constant.

Nucleic acid, carrier, host cell

In another aspect, the present invention relates to the nucleic acid molecules for encoding FIX binding molecule of the invention.Nucleic acid of the invention It can be RNA, DNA or cDNA.An embodiment according to the present invention, nucleic acid of the invention are substantially separate nucleic acid.? In some specific embodiments, it includes any in SEQ ID NO:26-31 for encoding the nucleic acid molecules of FIX binding molecule of the invention Nucleotide sequence.

Nucleic acid of the invention can also be in carrier format, may be present in carrier and/or can be a part of carrier, the carrier Such as plasmid, cosmid or YAC.Carrier can in particular expression vector, that is, it is external and/or in vivo to can provide FIX binding molecule The carrier of (i.e. in being suitble to host cell, HOST ORGANISMS and/or expression system) expression.The expression vector generally comprises at least A kind of nucleic acid of the invention is operably coupled to one or more suitable expression regulation elements (such as promoter, enhancing Son, terminator etc.).The element and its sequence are carried out being selected as those skilled in the art for the expression in specific host Common sense.The specific example of useful to the expression of FIX binding molecule of the invention or required controlling element and other elements, example Such as promoter, enhancer, terminator, integration factor, selectable marker, leader sequence, reporter gene.

Nucleic acid of the invention can be based on the information of the amino acid sequence about polypeptide of present invention given herein by Mode (such as by the automated DNA synthesis and/or recombinant DNA technology) preparation known obtains, and/or can be from suitable natural Source is separated.

In another aspect, the present invention relates to expression or can express one or more FIX binding molecules of the invention and/ Or the host cell containing nucleic acid or carrier of the invention.Preferred host cell of the invention be bacterial cell, fungal cell or Mammalian cell.

Suitable bacterial cell includes gramnegative bacterium bacterial strain (such as Escherichia coli (Escherichia coli) bacterium Strain, Proteus (Proteus) bacterial strain and pseudomonas (Pseudomonas) bacterial strain) and Gram positive bacteria strain (such as bacillus (Bacillus) bacterial strain, streptomyces (Streptomyces) bacterial strain, staphylococcus (Staphylococcus) bacterial strain and lactococcus (Lactococcus) bacterial strain) cell.

Suitable fungal cell includes trichoderma (Trichoderma), Neurospora (Neurospora) and aspergillus (Aspergillus) cell of species；Or including saccharomyces (Saccharomyces) (such as saccharomyces cerevisiae (Saccharomyces cerevisiae)), Schizosaccharomyces (Schizosaccharomyces) (such as schizosaccharomyces pombe (Schizosaccharomyces pombe)), pichia (Pichia) (such as pichia pastoris yeast (Pichia Pastoris) and thermophilic pichia methanolica (Pichia methanolica)) and Hansenula (Hansenula) species Cell.

Suitable mammalian cell includes such as HEK293 cell, Chinese hamster ovary celI, bhk cell, HeLa cell, COS cell Deng.

However, the present invention can also be used in amphibian animal cell, insect cell, plant cell and this field for express it is heterologous Any other cell of albumen.

The present invention also provides the method for generating FIX binding molecule of the invention, the method generally comprises following steps:

Host cell of the invention is cultivated under conditions of allowing to express FIX binding molecule of the invention；And

From culture recycling by the FIX binding molecule of the host cell expression；And

Optionally it is further purified and/or modifies FIX binding molecule of the invention.

In a preferred embodiment, FIX binding molecule of the invention is generated using mammalian cell.The present invention FIX binding molecule can obtain high expression in mammalian cells.For example, expression is up to about 100mg/L, preferably About 150mg/L, preferably approximately 200mg/L, preferably approximately 300mg/L, more preferably from about 400mg/L or more preferably from about 500mg/L is higher.

FIX binding molecule of the invention can in cell as described above with intracellular mode (such as in cytoplasm, in week In matter or in inclusion body) it generates, it then separates from host cell and is optionally further purified；Or it can be with extracellular mode (such as in culture medium of culture host cell) generates, and then separates from culture medium and is optionally further purified.

For recombinating the method and reagent that generate polypeptide, such as specific suitable expression vector, conversion or transfection method, selection Marker, method, the condition of culture of inducible protein expression etc. are well known in the art.Similarly, it is suitable for manufacturing this hair Protein Separation and purification technique in the method for bright FIX binding molecule is known to those skilled in the art.

However, FIX binding molecule of the invention can also be obtained by other protedogenous methods of production known in the art , such as chemical synthesis, including solid phase or liquid phase synthesis.

FIX detection

On the other hand, quantitative the present invention provides the presence of FIX in detection target sample a kind of and/or to FIX in sample And/or in test sample the carboxylated level of FIX method, which comprises

B) formation of compound is detected,

In some embodiments, the carboxylated is gamma carboxylated.In some embodiments, the life of the FIX Object activity is positively correlated with its carboxylated level.Therefore, the carboxylated level of FIX can be used for assessing in sample in test sample The biological activity of FIX.Present invention also contemplates that assessing the FIX's in sample by the carboxylated level of FIX in test sample The method of biological activity.

In some embodiments, the method also includes by the FIX containing predetermined amount or contain predetermined carboxylated water The serial dilutions of the control sample of flat FIX obtain the step of standard curve.

In some embodiments, FIX binding molecule of the invention can be conjugated with can be used for detect or can be by other reagents Fluorescent dye, chemical substance, polypeptide, enzyme, isotope, label for detecting etc..In one embodiment, the detection passes through Method known in the art for immune detection carries out, such as western blot or ELISA.In some embodiments, of the invention FIX binding molecule can be with the area someone Fc, therefore can be matched with the secondary antibody of commercialization, for the detection of FIX content, with commodity Change FIX detection kit greatly reduces compared to cost.

In some embodiments, the target sample is the FIX product prepared by distinct methods.

The preparation of weary plasma thromboplastin component blood plasma

On the other hand, the present invention provides a kind of method for preparing weary FIX blood sample, including

Separate the compound with the blood sample, and

C) weary FIX blood sample is harvested.

In some embodiments, wherein the FIX binding molecule is fixed on solid support.In some embodiment party In case, the solid support is selected from: polyethylene, polystyrene, polypropylene, polysulfones, polyacrylonitrile, polycarbonate, polyurethane, Silica, latex, glass, cellulose, cellulose ethanoate, the glucan of crosslinking, the agarose of crosslinking, chitin, shell are poly- Sugar, crosslinking glucan, crosslinking alginic acid, silicone resin, fluoropolymer and other synthetic polymers or magnetic medium (such as magnetic bead).

The surface of the solid support can be functionalized or with functional group, as described herein to allow The covalently or non-covalently attachment of FIX binding molecule.In some embodiments, the solid support is the fine jade activated with CNBr Lipolysaccharide.In other embodiments, the solid support is the magnetic bead of Streptavidin (SA) label.

In some embodiments, wherein the blood sample is blood plasma.Weary FIX blood prepared by the method for the present invention Slurry is particularly suitable for clinically detecting FIX activity.It the use of the weary FIX blood plasma detection active method of FIX is well known in the art.

On the other hand, the present invention provides a kind of FIX affinity chromatography medium, and it includes be fixed with FIX of the invention thereon The solid support of binding molecule.In some embodiments, wherein the solid support is made of material selected from the following: Polyethylene, polystyrene, polypropylene, polysulfones, polyacrylonitrile, polycarbonate, polyurethane, silica, latex, glass, fiber Element, cellulose ethanoate, the glucan of crosslinking, the agarose of crosslinking, chitin, chitosan, the glucan of crosslinking, crosslinking sea Alginic acid, silicone resin, fluoropolymer and other synthetic polymers or magnetic medium (such as magnetic bead).The solid support can In the form of being filter, film, solid fiber, doughnut or pearl.The FIX affinity chromatography medium can be used for separating and making Standby FIX, or be particularly useful for preparing for example weary FIX blood plasma of weary FIX blood sample.

On the other hand, the present invention also provides a kind of devices for being used to prepare weary FIX blood sample, comprising of the invention FIX affinity chromatography medium.Described device for example can be chromatographic column.

Kit

It further include kit in the scope of the present invention, which includes FIX binding molecule of the invention, of the invention FIX affinity chromatography medium or the device of the invention.Kit generally comprise show Kit Contents desired use (for example, For detect FIX or its activity, or be used to prepare weary FIX blood plasma) label.Term tag includes on kit or and reagent Box is provided together or otherwise with any written or record the material of kit offer.

Embodiment

The present invention will be further illustrated by way of embodiment below, but is not therefore limited the present invention to described Scope of embodiments in.

Embodiment 1: the screening of anti-FIX single domain heavy chain antibody

1.1. the building in library:

Choose and separate its lymphocyte without 14 camels being immunized, and won 5 camels spleen and 8 camels Lymph node, the RNA extracts kit provided using QIAGEN company extracted lymphocyte and total tissue RNA, uses Super- The RNA whole reverse transcription of extraction is to specifications cDNA by Script III FIRST STRANDSUPERMIX kit, is used The nucleic acid fragment of the variable region of nested PCR amplification encoding heavy chain antibody.

Target single domain heavy chain antibody nucleic acid fragment is recycled, and will using restriction enzyme (being purchased from NEB) PstI and NotI It, which is cloned, enters phage display in carrier pMECS.The subsequent electrotransformation of product to Escherichia coli electricity turns competent cell TG1 In, it constructs nonimmune single domain antibody phage display library and library is examined and determine.By gradient dilution bed board, storage capacity is calculated Size be 1.4 × 10⁹.For the insertion rate for detecting library, 100 cloning and sequencing detections are selected at random, there is correct external source The clone of segment insertion is 99, accuracy 99%.Ratio is analyzed respectively by the DNA and amino acid sequence that clone to sequencing It is right, it was demonstrated that all sequences are entirely different, are expected camel VHH sequence, diversity 100%.

1.2 anti-FIX single domain heavy chain antibody elutriations:

It is coated with plate with the amount in 0.5 hole μ g/ recombination FIX, 4 DEG C stand overnight.It is closed with 2% defatted milk room temperature within second day After 2 hours, 100 μ l bacteriophages (~10 are added in every hole¹⁰×10¹³Pfu shows text from the nonimmune single domain antibody of 1.1 two-humped camels Library), it acts on 2 hours at room temperature.It is washed 25 times with PBST20 (containing 0.05% polysorbas20 in PBS), is not combined with washing off later Bacteriophage.Finally the bacteriophage specifically bound with FIX is dissociated with glycine (100mM, pH 2.06), and infects and is in The e. coli tg1 of logarithmic phase is grown, the screening that simultaneously purified phage is used for next round is generated.Above-mentioned screening process repeats 3 wheels. It is enriched with positive clone as a result, to utilize FIX specific antibody in display technique of bacteriophage screening antibodies library.

1.3 screen the single positive colony of specificity with enzyme-linked immunoassay method (ELISA):

After 3 wheel elutriations, the FIX of acquisition combines positive phage-infect blank Escherichia coli and bed board.Then select 95 A single colonie is seeded to 2TY-AG, when cultivated respectively to about 0.8 OD600, is added IPTG final concentration about 1mM inducing expression, and 25 DEG C Overnight.Single domain antibody is expressed in colibacillus periplasm, and next day harvests thallus, and TES is added and cracks bacterium with permeability evolution Body, supernatant are detected for ELISA.4 DEG C of plate are coated with overnight with FIX, and by the supernatant, (control group is blank Escherichia coli Crack supernatant) it is added, it reacts 2 hours at room temperature.Secondary antibody Goat anti-HA tag HRP is added after washing (to be purchased from Abcam), react at room temperature 2 hours.TMB developing solution is added after washing, reads 405nm wavelength absorption value.When sample well OD value is big When control wells OD value 2 times or more, it is accredited as positive colony hole.By positive colony sequencing.

The amino acid sequence of each clone is analyzed according to sequence alignment program Vector NTI.CDR1, CDR2, CDR3 sequence The clone of column homology > 90% is considered as same antibody strain.6 plants of different antibody are finally obtained altogether, sequence is shown in Fig. 1, and according to The regular collimation mark of Kabat and Chothia goes out CDR region.

Embodiment 2: anti-FIX single domain antibody-Fc fusion protein is prepared by expressing in mammalian cells

2.1. the carrier of expression FIX single domain antibody-Fc fusion protein is prepared

Design primer carries out PCR amplification FIX single domain antibody VHH segment, the DNA fragmentation (amino acid with encoding human IgG1-Fc Sequence is shown in Fig. 1) fusion, and it is cloned into conventional mammalian expression vector, it obtains anti-for expression FIX single domain in mammals The recombinant plasmid of body-Fc fusion protein.Different VHH segments are wherein expanded using universal primer, universal primer is as follows:

Upstream primer cccACCGGTCAGGTGCAGCTGCAGGAGTC (SEQ ID NO:33)

Downstream primer cccGGATCCTGAGGAGACGGTGACCTGG (SEQ ID NO:34)

2.2. the Fc fusion protein of FIX single domain antibody is prepared

2.1 buildings are obtained carrier to transfect to the transient expression of HEK293 cell progress antibody.Recombinant expression plasmid is used Polyethyleneimine (PEI) solution needed for Freestyle293 culture medium dilutes and conversion is added, by every group of plasmid/PEI mixture It is separately added into HEK293 cell suspension, is placed on 37 DEG C, 5%CO2,130rpm culture.EX-CELL293 is added after four hours Culture medium, 130rpm, which suspends, to be cultivated.Add 3.8mM VPA after 24 hours, 4g/L glucose is added after 72 hours.Culture 5~6 days Afterwards, transient expression culture supernatant is collected, Protein A affinity chromatography, purification of target FIX single domain antibody-Fc fusion are passed through Albumen.The purity of protein obtained is detected by SDS-PAGE and SEC-HPLC.It detects and finds through SDS-PAGE NFN50-FC exists in the form of aggressiveness mostly.Therefore, nFN50-FC is excluded from follow-up test.The wink of remaining each protein Turn the results are shown in Table 1.

Table 1: anti-FIX single domain antibody-Fc fusion protein wink obtained turns latter step purification result.

Antibody	Expression quantity (mg/L)	SDS-PAGE purity	Monomer ratio %
				nFN 65-FC	318	> 95%	98.751
nFN 62-FC	402	> 95%	99.713
				nFN 52-FC	351	> 95%	99.710
nFN 64-FC	409	> 95%	95.242
				nFN 69-FC	428	> 95%	95.319

FIX single domain antibody-Fc fusion protein expression passes through Protein A affinity column in 300mg/L or more One step after purification, obtains the target protein with steady concentration and high-purity.

Embodiment 3: the binding curve of anti-FIX single domain antibody-Fc fusion protein and FIX

The binding curve of anti-FIX single domain antibody-Fc fusion protein and FIX is studied by ELISA.

4 DEG C of plate are coated with overnight with the amount in 0.2 hole μ g/ recombined human FIX albumen.By the resulting anti-FIX single domain of embodiment 2 Antibody-Fc fusion protein carries out gradient dilution (10 ×), and reacts 2 hours with coated plate at room temperature.Add after washing Enter secondary antibody (Goat anti-Human IgG-Fc antibody (Sigma) of horseradish peroxidase-labeled), reacts at room temperature 2 hours.After washing Developing solution is added, reads the absorption value of 450nm and 650nm wavelength, the absorption value of 450nm wavelength subtracts the absorption of 650nm wavelength Value is final absorption value.

Application software SotfMax Pro v5.4 carries out data processing and mapping analysis.By four parameter fittings, resisted FIX single domain antibody-Fc fusion protein and the protein bound binding curve of recombined human FIX and EC50 value (the EC50 value of all antibody It is about 50ng/mL).Reflect antibody to the affinity of FIX.

As a result see Fig. 2, wherein ordinate is OD value, and abscissa is that the concentration of anti-FIX single domain antibody-Fc fusion protein is (single Position ng/mL)；Round, rectangular, positive triangle, diamond shape, circle respectively represent anti-FIX single domain antibody-Fc fusion protein: nFN52-FC, nFN64-FC,nFN69-FC,nFN62-FC,nFN65-FC.Five kinds of albumen have very high affinity, and their parent to FIX It is suitable with power.

Embodiment 4: the selection specificity of anti-FIX single domain antibody-Fc fusion protein

The selection specificity of anti-FIX single domain antibody-Fc fusion protein is studied by ELISA.

4 DEG C of plate are coated with overnight with amount difference employment FIX, FVII, FVIII, TFPI albumen in 0.5 hole μ g/, and every hole is added The anti-FIX single domain antibody-Fc fusion protein (control group is plain buffer) obtained of 0.5 μ g embodiment 2, reacts 2 at room temperature Hour.Secondary antibody (Goat anti-Human IgG-Fc antibody (Sigma) of horseradish peroxidase-labeled) is added after washing, room temperature reaction 2 hours.Developing solution is added after washing, reads the absorption value of 405nm wavelength.The results are shown in Table 2.

Table 2: the combination of anti-FIX single domain antibody-Fc fusion protein and people FIX and its albumen of the same clan.

Antibody	OD (to people FIX)	OD (to people FVII)	OD (to people FVIII)	OD (to people TFPI)
					nFN52-FC	2.272	1.045	0.046	0.034
nFN62-FC	2.285	0.798	0.04	0.028
					nFN64-FC	2.379	2.048	0.113	0.077
nFN65-FC	2.312	0.083	0.046	0.036
					nFN69-FC	1.975	0.881	0.054	0.032
Blank	0.042	0.037	0.031	0.022

As it can be seen that the FIX single domain antibody Fc fusion protein of all acquisitions does not combine people's FVIII and TFPI albumen, wherein For nFN52/62/64/69-FC in combination with people's FIX and FVII albumen, nFN65-FC only combines people FIX albumen without combining people FVII albumen.

Embodiment 5: the non-specific binding of detection FIX single domain antibody-Fc fusion protein and ghost

CHOK1 ghost and 293F ghost are resuspended in 3%BSA-PBS, adjustment cell number to 6 × 10⁶Cell/mL, point Not Jia Ru the FIX single domain antibody-Fc fusion protein obtained of embodiment 2, final concentration of 20 μ g/mL, while negative control is set And blank control, ice bath 60min.Secondary antibody APC anti-human igg-Fc antibody (Biolegend) is added after washing, ice bath 30min.It washes Cell is resuspended in 500 μ L after washing to contain in the PBS buffer solution of 1%BSA, is detected with flow cytometer.As a result such as 3 institute of table Show.

Table 3: the combination of anti-FIX single domain antibody-Fc fusion protein and CHOK1 and 293F ghost.

Antibody	MFI (to CHOK1 cell)	MFI (to 293F cell)
			nFN52-FC	5.86	5.16
nFN64-FC	6.86	5.82
			nFN69-FC	4.57	4.6
nFN62-FC	4.88	4.36
			nFN65-FC	5.12	5.16
Blank control	4.28	4.01
			Negative control	4.25	4.12

As it can be seen that average fluorescent strength value of the anti-FIX single domain antibody-Fc fusion protein in conjunction with CHOK1 and 293F ghost There is no significant difference with blank control and negative control, shows anti-FIX single domain antibody-Fc fusion protein and CHOK1 and 293F Ghost is without non-specific binding.

Embodiment 6: the affinity of anti-FIX single domain antibody-Fc fusion protein is detected

Interfere (Biolayerinterferometry, BLI) technology by biomembrane, detection embodiment 2 is obtained anti- The binding kinetics of FIX single domain antibody-Fc fusion protein and recombined human FIX.The detection is carried out using interaction of molecules instrument.

Anti- FIX single domain antibody-Fc fusion protein nFN52-FC, nFN64-FC, nFN69-FC are diluted to 20 μ g/ of final concentration ML is immobilized on AHC biosensor, measures to dynamics.By people FIX, FVII albumen with 0.02%PBST20 points It is not diluted to 5 concentration: 1000nM, 500nM, 250nM, 125nM, 62.5nM.Sample introduction 120s, Dissociation time 300s.With 10mM glycine-HCl (pH1.7) regenerates 5s.Using simple one-to-one Languir binding model, (Octet K2 data are analyzed soft Part 9.0 editions (Data analysis 9.0)), calculations incorporated rate (kon) and dissociation rate (kdis).With kdis/kon ratio Calculated equilibrium dissociation constant (kD).

Affinity of the measured fusion protein in conjunction with FIX is shown in table 4, and the affinity in conjunction with FVII is shown in table 5.Knot Fruit show anti-FIX single domain antibody-Fc fusion protein nFN52-FC, nFN62-FC, nFN64-FC, nFN69-FC in conjunction with FIX than Affinity in conjunction with FVII is higher, and nFN65-FC is only in conjunction with FIX.

Table 4: affinity of the anti-FIX single domain antibody-Fc fusion protein in conjunction with FIX.

Antibody	KD(M)	kon(1/Ms)	kdis(1/s)
				nFN52-FC	3.02E-09	8.55E+04	2.58E-04
nFN64-FC	5.73E-09	7.18E+04	4.11E-04
				nFN69-FC	7.91E-10	6.03E+04	4.77E-05
nFN62-FC	4.77E-09	1.08E+05	5.16E-04
				nFN65-FC	6.24E-09	6.94E+04	4.33E-04

Table 5: affinity of the anti-FIX single domain antibody-Fc fusion protein in conjunction with FVII.

Antibody	KD(M)	kon(1/Ms)	kdis(1/s)
				nFN52-FC	7.62E-09	8.31E+04	6.33E-04
nFN64-FC	1.58E-08	6.71E+04	1.06E-03
				nFN69-FC	7.94E-09	2.63E+04	2.09E-04
nFN62-FC	1.50E-08	3.58E+04	5.38E-04
				nFN65-FC	NA	NA	NA

Anti- FIX single domain antibody-Fc fusion protein is detected to the identification situation of FIX or FVII by western blot.

Recombined human FIX and FVII are separately added into irreducibility sample-loading buffer, then boiling water bath processing fills protein Divide denaturation.Carry out SDS-PAGE, 0.5 μ g protein of applied sample amount.Albumen is transferred to methanol activation by half-dried turn of method On pvdf membrane.After washing film with 0.05%TBST20, film is closed with 5% skimmed milk power, room temperature 2h.With 0.05% After TBST20 washes film.Respectively by anti-FIX single domain antibody-Fc fusion protein nFN52-FC, nFN62- obtained in embodiment 2 FC, nFN64-FC, nFN65-FC, nFN69-FC dilute in 0.05%TBST20, final concentration of 2.5 μ g/mL.By film with through dilute The secondary antibody incubation at room temperature 2h that HRP is marked is added after washing film in the fusion protein incubation at room temperature 2h released.Addition ECL substrate is aobvious after washing film The colour developing of color liquid, gel imaging system exposure image, as a result as shown in Figure 3.

Fig. 3 shows, antibody nFN65-FC only in conjunction with FIX, remaining antibody nFN52-FC, nFN64-FC, nFN69-FC, NFN62-FC is consistent with aforementioned testing result in combination with FVII and FIX.

FVII and FIX amino acid sequence be compared the result shows that, carboxylated region homology is high.In view of FIX is living Property is related to carboxylated degree, and western blot further verifies antibody situation in conjunction with different activities FIX.

By APTT (activated partial thromboplastin time) method, that BeneFIX drug and inventor are voluntarily produced, Recombined human FIX protein 29 3DS-1, CH0DS-1 of different modifying degree carry out survey work.Obtain result: to sighting target BeneFIX activity For standard items 150% when, the activity of recombined human FIX protein 29 3DS-1, CH0DS-1 are respectively 89.3%, 138.6%.It will weigh Group people FIX protein 29 3DS-1, CH0DS-1, after irreducibility sample-loading buffer is added, boiling water bath processing becomes protein sufficiently Property.Carry out SDS-PAGE, 0.5 μ g of applied sample amount.Albumen is transferred on the pvdf membrane of methanol activation by half-dried turn of method.With After 0.05%TBST20 washes film, film is closed with 5% skimmed milk power, room temperature 2h.After washing film with 0.05%TBST20.Point Not by anti-FIX single domain antibody-Fc fusion protein nFN52-FC, nFN62-FC, nFN64-FC obtained in embodiment 2, NFN65-FC, nFN69-FC dilute in 0.05%TBST20, final concentration of 2.5 μ g/mL.By film and diluted fusion protein It is incubated at room temperature 2h, the secondary antibody incubation at room temperature 2h that HRP is marked is added after washing film.The colour developing of ECL substrate developing solution, gel is added after washing film Imaging system exposure image, as a result as shown in Figure 4.

Fig. 4 shows that the FIX of nFN69-FC and different activities is combined with apparent difference, with degree of modification and activity compared with Good FIX CH0DS-1 is combined preferably, poor in conjunction with the FIX 293DS-1 poor with degree of modification and activity.

Then nFN69-FC situation in conjunction with the FIX of different carboxylated is further verified.

When control BeneFIX carboxylated is modified to 12.451/mol, other recombined humans FIX albumen KN020Ko-A14, The carboxylated modification of KN020Ko-A21, KN020BAP150619 are respectively 12.046/mol, 10.785/mol, 7.488/mol. Non- go back is added in recombined human FIX albumen KN020Ko-A14, KN020Ko-A21, KN020BAP150619 of different carboxylated levels Boiling water bath is handled after originality sample-loading buffer, is denaturalized protein sufficiently.Carry out SDS-PAGE, 0.5 μ g of applied sample amount.By half-dried Albumen is transferred on the pvdf membrane of methanol activation by the method turned.After washing film with 0.05%TBST20, with 5% skimmed milk power pair Film is closed, room temperature 2h.After washing film with 0.05%TBST20.Respectively by anti-FIX single domain antibody-obtained in embodiment 2 Fc fusion protein nFN52-FC, nFN62-FC, nFN64-FC, nFN65-FC, nFN69-FC dilute in 0.05%TBST20, eventually Concentration is 2.5 μ g/mL.Film and diluted fusion protein are incubated at room temperature 2h, is washed after film and the secondary antibody room temperature of HRP label is added is incubated Educate 2h.The colour developing of ECL substrate developing solution, gel imaging system exposure image, as a result such as Fig. 5 is added after washing film.

Fig. 5 shows that nFN69-FC is combined with apparent difference from the FIX of different carboxylated, and carboxylated degree is higher, knot It is better to close, and develops the color deeper.

Embodiment 8: the ph stability Journal of Sex Research of anti-FIX single domain antibody-Fc fusion protein

The resulting anti-FIX single domain antibody-Fc fusion protein of Processing Example 2 under the conditions of following three kinds respectively, investigates it To the tolerance and stability of soda acid: (1) 0.1M NaHCO3+0.5M NaCl, pH8.3,4 DEG C of processing is stayed overnight, (2) 0.1M acetic acid + 0.5M NaCl, pH4.0, room temperature processing 3h, (3) 0.1M acetic acid+0.5M NaCl, pH4.0 room temperature handle 3h, then 0.1M NaCl, pH8.3,4 DEG C of NaHCO3+0.5M processing are overnight.

Processed fusion protein is changed into liquid into 1 × PBS, with the concentration of the fusion protein in Nanodrop measurement PBS. The purity of processed fusion protein is detected by SEC.

Its activity in conjunction with antigen FIX is detected by ELISA.Specifically, with the amount recombined human FIX packet in 0.2 hole μ g/ Overnight by 4 DEG C of plate.By processed fusion protein gradient dilution, reacted at room temperature with coated plate 2 hours.Wash it Secondary antibody (Goat anti-Human IgG-Fc antibody (Sigma) of horseradish peroxidase-labeled) is added afterwards, reacts at room temperature 2 hours.Washing Developing solution is added later, reads the absorption value of 450nm and 650nm wavelength, the absorption value of 450nm wavelength subtracts 650nm wavelength Absorption value is final absorption value.Application software SotfMax Pro v5.4 carries out data processing and mapping analysis, passes through four ginsengs Number fitting, obtains binding curve and EC50 value of the processed anti-FIX single domain antibody-Fc fusion protein in conjunction with FIX.Reflection is anti- Affinity of the body to FIX.The results are shown in Table 6.

Table 6: the combination of processed anti-FIX single domain antibody-Fc fusion protein and FIX.

Antibody	EC50 (original sample)	EC50(pH 4.0 3h)	EC50 (pH 8.3 is stayed overnight)	EC50 (pH 8.3 is stayed overnight after 4.0 3h of pH)
					nFN52-Fc	71ng/mL	62.9ng/mL	66ng/mL	65.5ng/mL
nFN64-Fc	66.6ng/mL	73.2ng/mL	74.8ng/mL	75.4ng/mL
					nFN69-Fc	95ng/mL	78.5ng/mL	78.9ng/mL	72.4ng/mL
nFN62-Fc	67ng/mL	68.5ng/mL	69.3ng/mL	67.8ng/mL
					nFN65-Fc	69.5ng/mL	72ng/mL	70ng/mL	70.3ng/mL

As it can be seen that through acid and/or alkali process after antibody nFN52-Fc, nFN64-Fc, nFN69-Fc, nFN62-Fc, Combination activity in conjunction with nFN65-Fc and FIX is fine, is not significantly different compared with before processing.

Processed anti-FIX single domain antibody-Fc fusion protein is used into 0.22 μm of water film filtering respectively.By filtered fusion Albumen carries out SEC-HPLC analysis.SEC-HPLC parameter is as follows: 50 μ g of sample volume, and chromatographic column is TOSOH G3000SWXL 7.8 × 300mm, 5 μm and guard column are 6.0 × 40mm of TOSOH TSK gel guardcolumn SWXL, and 7 μm, mobile phase is 20mM Na₂HPO₄+ 300mM NaCl pH7.4, flow velocity 0.5mL/min, elution time 45min, Detection wavelength 280nm.As a result As shown in table 7.

Table 7: the SEC-HPLC analysis of processed anti-FIX single domain antibody-Fc fusion protein.

By SEC result it is found that there is no significant change before the antibody handled through acid-base condition, Percent main peak and processing, Show that the acidproof alkali ability of fusion antibody is strong, stability is good.

Embodiment 9: weary FIX blood plasma is prepared

9.1. anti-FIX single domain antibody-Fc fusion protein affinity purification medium is prepared

Inventor has attempted two different affinity purification media.

I. FIX single domain antibody-Fc fusion protein is coupled using agarose medium.

By the anti-FIX single domain antibody-Fc fusion protein nFN52-Fc, nFN62-FC obtained of embodiment 2, nFN64-FC, NFN65-FC, nFN69-FC change liquid into the solution of 0.2M NaHCO3 0.5M NaCl, pH8.3 using the super filter tube of 3KDa.

The fusion protein is coupled with through the preactivated agarose medium of CNBr with the carrying capacity of 3mg/mL, 4 DEG C overnight.So Afterwards, the medium through being coupled alternately is eluted with the Tris-HCl of 0.05M, pH8.0 and the acetate buffer solution of 0.2M, pH4.0, then used PBS balance, obtains anti-FIX single domain antibody-Fc fusion protein affinity chromatography medium.

II. the FIX single domain using the fixed biotin labeling of micro- magnetic bead of Streptavidin (streptavidin) label is anti- Body-Fc fusion protein.

By the anti-FIX single domain antibody-Fc fusion protein nFN52-Fc, nFN62-FC obtained of embodiment 2, nFN64-FC, NFN65-FC, nFN69-FC use EZ-Link^TMSulfo-NHS-Biotin kit (Thermo) marks biotin.

With the carrying capacity of 0.1mg/mL by the fusion protein of above-mentioned biotin labeling and the processed Dynabeads of PBS^TM M- 280Streptavidin magnetic bead (Thermo) is incubated at room temperature half an hour, and removed in supernatant with magnetic frame does not have in coupling later Albumen obtains the magnetic bead of FIX single domain antibody-Fc fusion protein label.

9.2. with FIX single domain antibody-Fc fusion protein-agarose medium or FIX single domain antibody-Fc fusion protein-magnetic Pearl processing whole human blood slurry prepares weary FIX blood plasma, and verifies

People's standard plasma is loaded on to the fusion protein-agarose medium as above obtained or magnetic bead, room temperature mildly vibrate It is incubated for 2h.Simultaneously using the agarose medium or magnetic bead that are not coupled handle blood plasma as negative control (be respectively labeled as negative control 1, Negative control 2).Medium or magnetic bead are removed by centrifugation or magnetic frame later, obtain treated plasma supernatant.

The activity of FIX in the plasma supernatant crossed using the method detection processing of APTT.By processed plasma supernatant loading To full automatic blood-coagulation instrument, with Dade Actin Activated Cephaloplastin Reagent, Calcium Chloride After Solution and FACTOR IX DEFICIENT reagent is mixed automatically, is incubated for, its light absorption value is detected at wavelength 660nm, together When with standard items be arranged standard curve (the log clotting time is to log percentage).The results are shown in Table 8.

Table 8:

Sample	Clotting time (s)	Relative standard's product activity %
			Negative control 1	68.3	91.2
The weary FIX blood plasma handled through nFN52-FC agarose	103.2	4.6
			The weary FIX blood plasma handled through nFN62-FC agarose	99.7	6.3
The weary FIX blood plasma handled through nFN64-FC agarose	102.6	4.8
			The weary FIX blood plasma handled through nFN65-FC agarose	102.8	4.8
The weary FIX blood plasma handled through nFN69-FC agarose	104.5	4.1
			Negative control 2	66.5	97.1
The weary FIX blood plasma handled through nFN52-FC magnetic bead	100.1	5.8
			The weary FIX blood plasma handled through nFN62-FC magnetic bead	101.3	5.1
The weary FIX blood plasma handled through nFN64-FC magnetic bead	98.3	7.5
			The weary FIX blood plasma handled through nFN65-FC magnetic bead	99.1	6.8
The weary FIX blood plasma handled through nFN69-FC magnetic bead	102.8	4.8

The results show that compared with the control group, with nFN52-FC, nFN62-FC, nFN64-FC, nFN65-FC, nFN69-FC After marking affinity chromatography medium or magnetic bead processing blood plasma, the APTT time greatly extends, and illustrates the endogenous FIX base in blood plasma This is by medium or magnetic capture and removes.As a result, present invention demonstrates that being able to use above-mentioned anti-FIX single domain antibody-Fc fusion egg It is white to prepare weary plasma thromboplastin component blood plasma.

Sequence table:

>SEQ ID NO:1

GYTYSRYCMG

>SEQ ID NO:2

AICTGGGSTYYADSVKG

>SEQ ID NO:3

YVGSDCGNAGRAY

>SEQ ID NO:4

GDISKVASMA

>SEQ ID NO:5

GLSIGTGRTYYADSVKG

>SEQ ID NO:6

VVAGVKY

SEQ ID NO:7

GDISKVASMA

>SEQ ID NO:8

GLSIGTGRTYYADSVKG

>SEQ ID NO:9

KGDQRFGGYLD

>SEQ ID NO:10

GDISKVASMA

>SEQ ID NO:11

GLSIGTGRTYYADSVKG

>SEQ ID NO:12

VTRWPPLAGGNWPAKY

>SEQ ID NO:13

LHNTNINAMA

>SEQ ID NO:14

ALLTRGGNTWYDDSVKG

>SEQ ID NO:15

VRRRDGYKY

>SEQ ID NO:16

GDISKVASMA

>SEQ ID NO:17

GLSIGTGRTYYADSVKG

>SEQ ID NO:18

NDQRRARY

>SEQ ID NO:19 nFN50

>SEQ ID NO:20 nFN52

>SEQ ID NO:21 nFN62

>SEQ ID NO:22 nFN64

>SEQ ID NO:23 nFN65

>SEQ ID NO:24 nFN69

>SEQ ID NO:25 IgG1-FC

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK

>SEQ ID NO:26 nFN50

CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGTA GGCTCTGGATACACCTACAGTAGGTACTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGC AGCTATTTGTACTGGCGGTGGTAGTACGTACTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAAGT CGAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCCTGTATTACTGTAAATTGTACGTT GGTAGTGATTGTGGAAACGCTGGCCGTGCTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC

>SEQ ID NO:27nFN52

CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACA GGCTCTGGAGACATCAGTAAGGTAGCTTCAATGGCCTGGTTCCGCCAGGCTCCATTGAAGGAGCGCGAGGGGGTCGC CGGTTTAAGTATTGGTACAGGTAGGACATACTACGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACG CCAAGAATACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTATATTACTGCGCCACAGTGGTA GCTGGCGTGAAGTACCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC

>SEQ ID NO:28nFN62

CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACA GGCTCTGGAGACATCAGTAAGGTAGCTTCAATGGCCTGGTTCCGCCAGGCTCCATTGAAGGAGCGCGAGGGGGTCGC CGGTTTAAGTATTGGTACAGGTAGGACATACTACGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACG CCAAGAATACGCTGTACCTGCAGATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAAAAGGC GACCAGCGTTTTGGAGGATATCTTGACTGGGGTCAGGGGACCCAGGTCACCGTCTCCTCATC

>SEQ ID NO:29nFN64

CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACA GGCTCTGGAGACATCAGTAAGGTAGCTTCAATGGCCTGGTTCCGCCAGGCTCCATTGAAGGAGCGCGAGGGGGTCGC CGGTTTAAGTATTGGTACAGGTAGGACATACTACGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACG CCAAGAATACGCTGTACCTGCAGATGAACAGCCTGAAAACTGAGGACACTGCCATATACTACTGTGCGCTCGTGACG AGGTGGCCTCCTCTCGCCGGTGGCAACTGGCCAGCTAAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC

>SEQ ID NO:30nFN65

CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATCGGTGCAGGCTGGAGGATCTCTGACGCTCTCCTGTGCA TACTCTCTGCACAACACCAATATCAACGCCATGGCCTGGTTCCGCCAGACTCCAGGCAAGGAGCGCGAGGGGGTCGC TGCTCTTCTCACTCGTGGTGGCAATACATGGTATGACGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACG CCAAGAACACGGTGTATCTACAAATGGATGACCTGAAACCTGAGGACACTGCCGTGTACTACTGTGCGGCAGTCCGG AGAAGGGATGGATATAAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC

>SEQ ID NO:31nFN69

CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACA GGCTCTGGAGACATCAGTAAGGTAGCTTCAATGGCCTGGTTCCGCCAGGCTCCATTGAAGGAGCGCGAGGGGGTCGC CGGTTTAAGTATTGGTACAGGTAGGACATACTACGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACG CCAAGAATACGCTGTACCTGCAGATGAACAGCCTGAAAACTGAGGACACTGCCATATACTACTGTGCGTATAATGAT CAGCGTCGTGCACGGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCATC

>SEQ ID NO:32mature FIX

YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSY ECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRA ETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCV ETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFL KFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTS FLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT

> SEQ ID NO:33 upstream primer

cccACCGGTCAGGTGCAGCTGCAGGAGTC

> SEQ ID NO:34 downstream primer

cccGGATCCTGAGGAGACGGTGACCTGG

Claims

1. plasma thromboplastin component (FIX) binding molecule in conjunction with FIX and can include the single variable knot of at least one immunoglobulin Structure domain, the single variable domains of at least one immunoglobulin include CDR1, CDR2 and CDR3 selected from the following:

(4) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12；

(5) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15； With

(6) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18.

2. the FIX binding molecule of claim 1, wherein the single variable domains of the immunoglobulin are VHH.

3. the FIX binding molecule of claim 2, wherein the VHH includes any amino acid sequence in SEQ ID NO:19-24.

4. the FIX binding molecule of any one of claim 1-3, also includes immunoglobulin fc region.

5. the FIX binding molecule of claim 4, wherein the immunoglobulin fc region is the area human immunoglobulin(HIg) Fc.

6. the FIX binding molecule of claim 5, wherein the amino acid sequence of the immunoglobulin fc region is shown in SEQ ID NO: 25。

7. the FIX binding molecule of any one of claim 1-6, in conjunction with FIX KD value less than 1 × 10^-7M, preferably smaller than 1 × 10^-8M, more preferably less than 1 × 10^-9M, more preferably less than 1 × 10^-10M, particularly more preferably less than 1 × 10^-11M。

8. nucleic acid molecules, the FIX binding molecule of any one of coding claim 1-7.

9. expression vector, it includes the nucleic acid molecules for the claim 8 being operably connected with expression regulation element.

10. host cell, it includes the nucleic acid molecules of claim 8 or with the expression vector conversion of claim 9, and being capable of table Up to the FIX binding molecule.

11. the method for generating the FIX binding molecule of any one of claim 1-7, comprising:

A) host cell of claim 10 is cultivated under conditions of allowing the FIX binding molecule to express；

12. a kind of detect the presence of FIX in target sample and/or FIX in target sample quantified and/or detected FIX in target sample The kit of carboxylated level, the FIX binding molecule comprising any one of claim 1-7.

13. the kit of claim 12 also includes the FIX containing the predetermined amount or FIX containing predetermined carboxylated level Control sample.

14. in a kind of detection target sample the presence of FIX and/or to FIX in sample is quantitative and/or test sample in FIX carboxyl Change horizontal method, which comprises

A) under conditions of being capable of forming compound between FIX binding molecule and FIX, distinguish the target sample and control sample It is contacted with the FIX binding molecule of any one of claim 1-7；

B) formation of compound is detected,

The presence and/or amount of FIX in the difference instruction target sample that wherein compound is formed between the target sample and control sample And/or its carboxylated is horizontal, it is preferable that the control sample contains the FIX of predetermined amount or containing predetermined carboxylated level FIX。

15. a kind of method for preparing weary FIX blood sample, including

A) contact the FIX binding molecule of any one of blood sample and claim 1-7, thus in the blood sample FIX and the FIX binding molecule form compound,

Separate the compound with the blood sample, and

C) weary FIX blood sample is harvested.

16. the method for claim 15, wherein the FIX binding molecule is fixed on solid support.

17. the method for claim 15 or 16, wherein the blood sample is blood plasma.

18. a kind of FIX affinity chromatography medium, the FIX binding molecule comprising being fixed with any one of claim 1-7 thereon is consolidated Body support.

19. the FIX affinity chromatography medium of claim 18, wherein the solid support is made of material selected from the following: poly- Ethylene, polystyrene, polypropylene, polysulfones, polyacrylonitrile, polycarbonate, polyurethane, silica, latex, glass, cellulose, Cellulose ethanoate, the glucan of crosslinking, the agarose of crosslinking, chitin, chitosan, the glucan of crosslinking, crosslinking seaweed Acid, silicone resin, fluoropolymer, magnetic medium and other synthetic polymers.

20. the FIX affinity chromatography medium of claim 18 is that immobilization has the right to require the FIX combination point of any one of 1-7 The agarose medium or magnetic bead of son.

21. being used to prepare the device of weary FIX blood sample, the FIX affinity chromatography comprising any one of claim 18-20 is situated between Matter.