CN103599527A - Pharmaceutical composition containing modified type human coagulation factor FVII-Fc fusion protein - Google Patents
Pharmaceutical composition containing modified type human coagulation factor FVII-Fc fusion protein Download PDFInfo
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Abstract
The invention discloses a recombined human coagulation factor FVII-Fc fusion protein, a preparation method thereof and applications thereof. The fusion protein successively comprises, from the N end to the C end, human FVII, a flexible peptide joint and an IgG2Fc mutant. The Fc mutant has no lysis property and exhibits very low bad Fc-mediated side effect. The fusion protein has similar or higher bioactivity than the human FVII, and prolonged the serum half time, thus improving pharmacokinetics and efficacy.
Description
Technical field
The present invention relates to Fc fusion rotein of a kind of modified form human blood coagulation FVII and its production and use, particularly treat the purposes of multiple blood coagulation relevant disease.
Background technology
The process that causes gradually fibrin clot to form that blood coagulation is comprised of interaction complicated between multiple blood constitutent (or factor), the blood constitutent that common participation is called as blood coagulation " cascade reaction " is protoenzyme or proenzyme, do not there is the albumen of enzymatic activity, under the effect of activator, make it change organized enzyme into.Thrombin FVII is exactly a kind of in these thrombins.
FVII is a kind of plasma glycoprotein of vitamin k-dependent, synthetic in liver, and with the single chain protein zymogen forms that molecular weight is about 53KDa be secreted in blood (Broze etc., J Biol Chem, 1980,255:1242-1247).FVII proenzyme, is produced the two strands being connected by a disulfide bond, thereby changes its activity form FVIIa into by protease hydrolysis at Arg152-Ile153 place, single site.Strand thrombin FVII can by Factor Xa, thrombin FXIIa, thrombin FIXa, thrombin FVIIa or thrombin in vitro hydrolysis be double-stranded thrombin FVIIa.The FVIIa of activation comprises NH
2light chain (～20KDa) the heavy chain that via single disulfide bond (Cys135 to Cys262) be connected (～30KDa) derivative with COOH-end that-end is derivative.Light chain contains cell membrane associativity Gla domain and two EGF binding structural domains, and heavy chain contains catalyst structure domain.When activated form, FVIIa, as serine protease, participates in the extrinsic pathway (extrinsic pathway) of coagulation cascade reaction.When the vascular lumen damaged of blood vessel, extrinsic coagulation path is activated, glycoprotein tissue factor (Tissue Factor, TF) be exposed, in conjunction with circulative FVII and the already present FVIIa that activates in a small amount, this combination promotes that FVII is all converted into FVIIa, subsequently under the synergism of calcium ion and phospholipid, make FIX be converted into FIXa, FX is converted into FXa, then further thrombinogen is converted into thrombin.Thrombin is playing a crucial role aspect blood coagulation and wound healing, in the initial period of blood vessel injury, its induced platelet is assembled and is induced fibrin to form, thereby stimulating cellular growth promotes the reparation (Osterud etc. of damaged blood vessels afterwards, Proc Natl Acad Sci USA, 1977,74:5260-5264).
The gene of encoding human FVII (hFVII) is positioned at chromosomal q34-qter9 No. 13, and 9 exons form, length be 12.8Kb (O ' Hara etc., Proc Natl Acad Sci USA, 1987,84:5158-5162).HFVII comprises four domains: aminoterminal Gla (Gla) domain, two " skins somatomedin (EGF) " domains and c-terminus serine protease domain (catalyst structure domain).Exon Ia and Ib coded signal peptide sequence; Exon 2 coding Gla domain; One section of short hydrophobic region of exon 3 coding; Exon 4 and 5 coding skins somatomedin domains; Exon 6 to 8 encoding serine protease catalyst structure domain (Yoshitake etc., Biochemistry, 1985,24:3736-3750).Ripe FVII albumen experiences multiple post translational modification, comprises the carboxylated of vitamin k-dependent, causes this molecule NH2-end to produce 10 glutaminic acid residues that γ is carboxylated.Other post translational modifications comprise hydroxylating (Asp63), N-type glycosylation (Asn145 and Asn322) and O-type glycosylation (Ser52 and Ser60).
Hemophilia A and B are hereditary, and the defect of thrombin FVIII and thrombin FIX causes respectively.Albumen alternative medicine is the traditional therapy of hemophilia A and B, comprises restructuring FVIII or FIX that intravenous gives to prepare from human plasma or that gene engineering method obtains.Yet in the treatment of hemophilia A and B, having the most serious medical problem is for the generation that substitutes the isoantibody (alloantibody) of thrombin, this causes treating effect and reduces or make to fail to respond to any medical treatment.In all hemophilia A patients, produce for thrombin FVIII antibody up to 30%, and it is less for hemophilia B, to produce thrombin FIX antibody occurrence probability, but there is more serious consequence, because they are more insensitive to immunologic tolerance antilepsis.In Treatment of Hemophilia, using FVIIa is the low affinity combination for the platelet surface of activated by thrombin based on FVIIa, by giving the exogenous FVIIa of materia medica dosage, thrombin generation on place, damage position platelet surface is enhanced, the existence of this and FVIII/FIX is irrelevant, and the demand getting around for blood coagulation factor VIII a and factor IXa reaches hemostasis.Therefore, FVIIa can be used for having the treatment of hemorrhagic situation of the hemophiliac of mortifier.FVIIa is also used to treat the patient of congenital FVII defect.In addition, FVIIa is used as more and more indication and uses (off-lable use) outward, and as congenital or acquired hemorrhage in treatment and non-hemophiliac, it is hemorrhage that wound or operation are correlated with.
In view of the source of plasma F VII limited, and with the risk of viral infection.Restructuring FVII has become one of Research Emphasis.There is the effective expression (WO9215686, WO9111514, WO8810295) of report FVII in bhk cell or other mammalian cell, and in purge process, be converted into the FVIIa of activation.Patent FR0604872 has also described the method for utilizing transgenic animal to prepare FVIIa.Commercially available recombinant blood coagulation factor FVIIa only has at present
(Novo Nordisk, Denmark).This medicine has been approved for treatment in worldwide to be had because producing hemophilia A or the B patient of FVIII or FIX antibody (inhibitor), the patient of congenital FVII defect and termination and wound and/or the relevant bleeding episode or prevent hemorrhage of performing the operation.
Therapeutic blood coagulating protein medicine comprises that FVIIa meeting is by proteolytic enzyme fast degradation, and easily by antibody, is neutralized, and this can reduce their half-life and body-internal-circulation time, has limited thus their curative effect.At " Summary Basis for Approval
in (FDA reference number 96-0597), reported that restructuring FVIIa is 2.3 hours in people's body-internal-circulation half-life, and relatively high dosage and frequent drug administration are necessary for the curative effect and the preventive effect that reach and maintain expectation, this causes the inconvenience of high treatment cost and patient treatment.So far, also there is no the commercial restructuring FVIIa that extends plasma half-life that has.Because thrombin FVII/VIIa has the potential as general hemorrhage, therefore still exist exploitation to there is the clinical demand of function half-life (functional half-life) FVII in longer body.There is at present multiple application of policies in improving FVII pharmaco-kinetic properties, extend its Half-life in vivo.As being combined with lipid or PEG.Sugared PVOH two alcoholization FVIIa (referring to patent US20050113565 and US8053410) and PCT application WO2008074032 that Novo Nordisk company is developing also mention a kind of FVIIa-Polysialic acid conjugate that extends Half-life in vivo that has.In addition, published other method increases glycosylation site in addition, such as the high-glycosylation FVIIa that Bayer company is developing, the FVIIa-albumin fusion protein (referring to patent WO2007090584) that T106N and V253N point mutation (referring to patent US20060252128, EP1549677B and US20100260741) HuoCSL Behring company is developing.A kind of long-acting thrombin FVII that U.S. Prolor Biotech company is developing, the c-terminus that the carboxyl peptide (Carboxy-terminal, CTP) of hCG is connected to thrombin FVII has obtained the FVII (referring to patent US20100317585) with prolong half-life.
The immunoglobulin of IgG class is rich in protein in human blood.Their half-life can be up to 21 days, and Fc fragment be in IgG holder compared with long half-lift main cause, there is the effect of stabilize proteins simultaneously.Large quantity research has confirmed that Fc fragment and the activated protein of IgG connect and compose fusion rotein, can improve the Half-life in vivo of activated protein, this method be used to some clinically very important cytokine (as EPO-Fc, GCSF-Fc, IL2-Fc, and IFN α 2a-Fc) and soluble recepter (as TNFR-Fc, VEGFR-Fc, LFA3-Fc and CTLA4-Fc), there is prolongation (U.S. Patent No. 5349053 and 6224867) in various degree the half-life to Fc fusion rotein in vivo.The double end homodimer Fc fusion rotein of natural prototype or transformation is that formation that the cysteine residues by IgG Fc hinge region connects is similar to IgG molecule but without CH1 region and light chain.Due to structural homology, Fc fusion rotein shows the external pharmacokinetic properties suitable with similar same hypotype human IgG, and the Fc fusion rotein having gone on the market is at present all such.In addition, the single head dimeric Fc fusion rotein of Biogen Idec company exploitation recent years is the target protein that fusion is contained in Fc dimer one end, the other end is not contain the blank of any target protein, this method is mainly for following two kinds of situations: the one, and the target protein that those are to be expressed recombinant expressed very difficult, it is correct folding and secernment efficiency is low that double end homodimer Fc merges; The 2nd, target molecule is very large, and double end homodimer Fc merges the Function that may produce the sterically hindered of three dimensional structure and affect target protein.Single head dimeric Fc fusion method has been applied to thrombin FVIII, FIX and FVII at present, to realizing high efficiency recombinant expressed in Chinese hamster ovary celI, the Half-life in vivo simultaneously with prolongation, wherein single head dimer FVIII-Fc and FIX-Fc fusion rotein have all entered the clinical research stage, and in WO2011069164A2 and patent EP1624891B1, disclose respectively, and while European patent EP 1624891B1 also discloses the fusion form of single head dimer FVII and Fc.
For the difficulty of the preparation of relevant homodimer Fc fusion FVII described in above prior art and the limitation existing in process of clinical application thereof, as expression is not high, the half-life is short and poor stability etc., this area is long-acting in the urgent need to developing, good stability and the FVII derivant that can produce with rational cost.Due to its difficulty in essence that is built with of hFVII-L-vFc fusion rotein, still there is no up to now to obtain the FVII-Fc fusion rotein there is half-life significant prolongation and can stability and high efficiency to express.
Summary of the invention
The present invention relates to a kind of hFVII-L-vFc fusion rotein of greatly extending Half-life in vivo and its production and use that has.
The human IgG2 Fc mutant that hFVII-L-vFc fusion rotein of the present invention holds C end to contain successively people FVII, contain a plurality of amino acid whose flexible peptide linkers and contain Pro331Ser sudden change from N.Flexible peptide linker is pliable and tough and non-immunogenic preferably, and between FVII and Fc, produces enough distances, makes the potential interference of these two fusion rotein be decreased to bottom line.Preferably, use an about 6-21 amino acid length to contain the flexible peptide linker that following 2 kinds or several amino acids form: glycine, serine, alanine and threonine.If disclosed preferred sequence in one embodiment of the invention is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
The aminoacid sequence of fusion rotein of the present invention is as shown in SEQ ID NO:2, its maturation protein is for having excised hFVII leader peptide (1 to the 38 amino acids residue) aminoacid sequence shown in SEQ ID NO:2 afterwards, it is characterized in that, human IgG2 Fc mutant contains hinge region, CH2 and CH3 region.Amino acid mutation is contained 331 (positions of being determined by EU number system) in its CH2 region, thereby has eliminated the effector function of Fc.
The present invention discloses a kind of pharmaceutical composition, it is characterized in that, comprises pharmaceutically acceptable carrier or auxiliary shape agent or diluent, and the hFVII-L-vFc fusion rotein of the present invention of effective dose.
Fusion rotein of the present invention is general is applied to the spontaneous of the congenital or acquired deficiency disease patient's of FVII the prevention of hemorrhage and treatment and hemophilia A or B patient or prevention and treatment or other relevant hemorrhages that operation property is hemorrhage.
In another embodiment of the present invention, disclose a kind of method of preparing or producing this recombination fusion protein from mammal cell line cell line as derivative in CHO, caused recombination fusion protein to be produced for every 24 hours in its growth medium and surpass (being preferably 3-4) μ g/10
6the cell line of the stable transfection of cell.These hFVII-L-vFc fusion rotein have the serum half-life greatly extending and without adverse side effect, have improved pharmacokinetics and drug effect, thereby reduced, realize original similar drug effect required dosage and frequency injection.
In addition, the preparation method of fusion rotein disclosed in this invention, expresses output fusion rotein high and IgG2Fc and can obtain efficiently purification easily by Protein A affinity chromatograph.
To sum up, the advantage of disclosed and/or described fusion rotein of the present invention and preparation method thereof is summarized as follows:
The body-internal-circulation half-life of 1.FVII extends greatly, and serum Chinese medicine fluctuation of concentration reduces, and safety improves, and toleration improves, and reduces frequency of injection and improves quality of life of patient.
2. fusion protein expression is high and purification step is efficiently convenient, can reduce production costs.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.
Below specifically introduce content of the present invention:
Non-cracking performance Fc variant
Fc element derives from the constant region fc fragment of immunoglobulin IgG, and it plays an important role in the immune defence of eliminating pathogen.Two kinds of mechanism are passed through in the effector function performance of the IgG of Fc mediation: (1) and cell surface Fc receptor (Fc γ Rs) combination, by phagocytosis or splitting action or killer cell by antibody dependent cellular cytotoxicity (ADCC) approach digestion pathogen, or be combined with the C1q of the first complement component C1 (2), cause CDC (CDC) approach, thus cracking pathogen.In four kinds of human IgG hypotypes, IgG1 and IgG3 can be effectively in conjunction with Fc γ Rs, and the binding affinity of IgG4 and Fc γ Rs is lower, and the combination of IgG2 and Fc γ Rs is so low that to be difficult to measure, so human IgG2 does not almost have ADCC effect.In addition the effective activating complement cascade reaction in conjunction with C1q of human IgG1 and IgG3.Human IgG2 and C1q be in conjunction with a little less than relative, and IgG4 be not combined with C1q (Jefferis R etc., Immunol Rev, 1998,163:59-76), so human IgG2 CDC effect also a little less than.For target protein hFVII of the present invention, it is mainly in order to increase its body-internal-circulation half-life and to reduce production costs that Fc merges, and the effector effect of Fc mediation and cell lysis function is unnecessary even may produce harmful side effect.Obviously, do not have a kind of natural IgG hypotype to be applicable to very much producing hFVII-Fc fusion rotein.In order to obtain not having the non-cracking performance Fc of effector function, most effectual way is the amino acid mutation that carries out in natural Fc fragment, to reduce or to remove effector function.In view of human IgG2's natural characteristic, by its Fc fragment, carrying out point mutation is the wisest selection.In natural IgG2, Pro331 is replaced with Ser331 (position of being determined by EU number system), just makes IgG2 lose the binding affinity with C1q.
Flexible peptide linker
Connection peptides length is extremely important to fusion rotein activity.Report erythropoietin (EPO) derivant (as dimer), compare with EPO monomer, the fusion rotein that contains 2 complete EPO regions (3 to 7 the amino acid peptide joints of being separated by) shows activity (the Qiu H etc. that weaken, J Biol Chem, 1998,273:11173-11176).Yet when the length of the interregional peptide linker of these two EPO is 17 aminoacid, the in vitro and in vivo biological activity of dimer EPO molecule obviously improves (Sytkowski AJ etc., J Biol Chem, 1999,274:24773-24778; U.S. Patent No. 6187564).This possible explanation is the connection peptides increasing between fusion rotein two parts, make two parts of this molecule can exercise respectively its function (Ashkenazi A etc., Curr Opin in Immunol, 1997,9:195-200).Disclosed a kind of FVII/FVIIa-Albumin fusion polypeptide in WO2007090584 patent, between FVII/FVIIa and albumin, insert the connection peptides of different length, discovery demonstrates the biologic activity of remarkable minimizing without the fusion rotein of the II/FVIIa of connection peptides, and the FVII/FVIIa-albumin fusion protein that contains connection peptides, show that the increase of its biologic activity depends on the length of connection peptides, this possible explanation is the connection peptides increasing between fusion rotein two parts, make two parts of this molecule can exercise respectively its function, be conducive to form the more conformation of high molar ratio activity.
Contain glycine and serine that first the inventor has designed 5 kinds of different lengths flexibly connect the fusion rotein that C end that peptide linker makes hFVII is connected with Fc, transient expression experiment shows, the fusion rotein that 2 amino acid whose small peptide joint GlySer connect also has activity, and showing to maintain the bioactive critical function of FVII region, affected by C terminal sequence less.When connection peptides joint is increased to 16 aminoacid GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer, FVII biological activity has fully displayed.In addition, the inventor also finds, the peptide linker adding between hFVII and human IgG2 Fc variant improves the Bioactivity of hFVII-L-Fc in two ways: (1) makes Fc region away from the domain on hFVII, (2) make a hFVII away from the domain of another hFVII, thereby reduce space steric effect.And people's IgG2Fc variant contains point mutation in CH2 region in 331 sites, thereby reduced the effector function of Fc.
Fusion rotein and preparation method thereof
Antigen-4 fusion protein gene of the present invention is being prepared by artificial synthesis of codon optimized mistake.According to nucleotide sequence of the present invention, those skilled in the art can make code nucleic acid of the present invention with various known methods easily.These methods are not limited to synthetic or traditional sub-clone etc., and concrete grammar can be referring to J. Pehanorm Brooker, < < molecular cloning experiment guide > >.As one embodiment of the present invention, the method for carrying out again sub-clone by salvage nucleotide sequence builds nucleic acid sequence encoding of the present invention.
The present invention also provides a kind of expression vector of mammalian cell, comprises coding fusion rotein sequence of the present invention and the connected expression regulation sequence of operability with it.Described " operability is connected " or " being operationally connected in " refer to a kind of like this situation, and some part of linear DNA sequence can regulate or control the activity of same linear DNA sequence other parts.For example, if the transcribing of promoter control sequence, it is exactly to be operationally connected in coded sequence so.
Mammalian cell expression vector can adopt commercially available such as but not limited to: pcDNA3, pIRES, pDR, pBK, pSPORT etc. can be used for the carrier that eukaryotic cell system is expressed.Those skilled in the art can also select suitable expression vector according to host cell.
According to the restriction enzyme mapping of known unloaded expression vector, those skilled in the art can shear and splicing by Restriction Enzyme according to conventional method, and the coded sequence of fusion rotein of the present invention is inserted to suitable restriction site, make recombinant expression carrier of the present invention.
The present invention also provides the host cell of expressing fusion rotein of the present invention, wherein contains the coded sequence of fusion rotein of the present invention.Described host cell is eukaryotic cell preferably, such as but not limited to Chinese hamster ovary celI, and COS cell, 293 cells, RSF cell etc.As optimal way of the present invention, described cell is Chinese hamster ovary celI, and it can preferably express fusion rotein of the present invention, can obtain activity good, the fusion rotein having good stability.
The present invention also provides a kind of method of preparing fusion rotein of the present invention by recombinant DNA technology, and its step comprises:
1) provide the nucleotide sequence of encoding fusion protein;
2) by 1) nucleotide sequence be inserted into suitable expression vector, obtain recombinant expression carrier;
3) by 2) recombinant expression carrier import suitable host cell;
4) cultivate under conditions suitable for the expression transfection host cell;
5) collect supernatant, and purified fusion protein product.
Described coded sequence is imported to the multiple known technology that host cell can adopt this area, such as but not limited to: calcium phosphate precipitation, liposome transfection, electroporation, microinjection, viral infection method, alkali metal ion method.
About the cultivation of host cell with express can be referring to Olander RM etc., Dev Biol Stand1996,86:338.Can, by cell and residue in centrifugal removal suspension, collect supernatant.
The character that can be basic homogeneous by the above-mentioned fusion protein purification preparing, for example, be single band on SDS-PAGE electrophoresis.First will express supernatant concentrated, the method that concentrated solution can adopt gel chromatography is purification in addition further, or adopts the method purification of ion-exchange chromatography.For example anion-exchange chromatography or cation-exchange chromatography.Gel-type vehicle can be the medium that agarose, glucosan, polyamide etc. are usually used in protein purification.Q-or SP-group are comparatively desirable ion-exchange groups.Finally, available hydroxyapatite adsorption chromatography also, metal chelate chromatography, the methods such as hydrophobic interaction chromatography and reversed-phase high-performance liquid chromatography are to the further refining purification of above-mentioned purified product.Above-mentioned all purification steps can utilize different combinations, finally make purity of protein reach basic homogeneous.Also can utilize the affinity column of the specific antibody, receptor or the part that contain described fusion rotein to carry out purification to the fusion rotein of expressing.According to the characteristic of used affinity column, can utilize conventional method, as the amalgamation polypeptide of method elution of bound on affinity column such as high-salt buffer, change pH.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition, and it contains effective dose (as 0.000001-90wt%; 0.1-50wt% preferably; Better, fusion rotein of the present invention 5-40wt%), and pharmaceutically acceptable carrier.Conventionally, the fusion rotein of the present invention of effective dose can be formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, preferably, pH is about 6-8.Term " effective dose " or " effective dose " refer to and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal.The composition of " pharmaceutically acceptable " is applicable to people and/or mammal and without excessive bad side reaction (as toxicity, stimulation and allergy), has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various auxiliary shape agents and diluent.
Pharmaceutically acceptable carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Conventionally pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, for example, with normal saline or contain glucose and the aqueous solution of other adjuvant is prepared by conventional method.Described pharmaceutical composition should be manufactured under aseptic condition.The dosage of active component is treatment effective dose.Pharmaceutical preparation of the present invention also can be made into slow releasing preparation.
The effective dose of fusion rotein of the present invention can change with order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be determined (for example, by clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization rate of described fusion rotein, metabolism, half-life etc.; The order of severity of the disease that patient will treat, weight in patients, patient's immune state, route of administration etc.
Accompanying drawing explanation
Fig. 1 has shown the nucleotide sequence of the hFVII-L-vFc of SpeI-EcoRI fragment in PCDNA3 expression vector and the aminoacid sequence of derivation according to the embodiment of the present invention.People FVII consists of signal peptide (1-38) and ripe FVII albumen (39-444).Ripe fusion rotein contains hFVII (39-444), flexible peptide linker (445-460) and Fc variant (461-683).
Fig. 2 has shown the gene mapping of the eukaryon expression plasmid of constructed hFVII-L-vFc antigen-4 fusion protein gene.This expression plasmid total length 9647bp, contains 10 major gene segments, comprises 1.hCMV promoter; 2. target gene hFVII-L-vFc; 3.EMCV IRES; 4.mDHFR screening-gene; 5.bGH pause sequence; 6.SV40 promoter; 7. kalamycin resistant gene; 8.SV40 pause sequence; 9.ColE1 replicon; 10. ampicillin resistance gene.
Fig. 3 has shown the growth of rotating and culturing bottle inner cell strain and the concentration trend curve of secretion hFVII-L-vFc fusion rotein thereof.
Fig. 4 has shown the plasma half-life of hFVII-L-vFc purifying protein in SD rat.
The specific embodiment
Embodiment 1. builds the expression plasmid of coding hFVII-L-vFc fusion rotein
The target gene sequence of coding hFVII leader peptide and maturation protein is the Chinese hamster ovary celI preference codon that artificial optimization crosses, and through synthetic way, obtains.For the ease of by the specific site of genes of interest fragment inserting expressioning carrier, in the fragment 5 ' and 3 ' of synthesized, hold and respectively have a Restriction Enzyme inscribe site, be respectively SpeI and BamHI.The DNA fragmentation of total length 1351bp is inserted into transfer vector as the EcoRV restriction enzyme site of pUC57, has obtained middle interstitial granules, and its contained hFVII gene order is verified by DNA sequencing.The fusion gene containing 2 amino acid whose flexible peptide linker GlySer and human IgG2 vFc variant (Pro331Ser sudden change) of first-selection of the present invention is also the Chinese hamster ovary celI preference codon that artificial optimization crosses, the fragment 5 ' of synthesized and 3 ' end respectively have a Restriction Enzyme inscribe site, be respectively BamHI and EcoRI, and be provided with EcoRV site in the middle of vFc.The DNA fragmentation application BamHI of total length 682bp and EcoRI restriction enzyme site are inserted into the pUC57 transfer vector of the above-mentioned hFVII of containing gene, obtain second middle interstitial granules, by DNA sequencing checking L-vFc sequence.
Adopt the EMCV IRES fragment of artificial synthesis acquisition and dihydrofolate reductase (DHFR) gene of mice to be inserted in the EcoRI (5 ') of mammalian cell expression vector PCDNA3 (Invitrogen) and the site of XhoI (3 '), obtain carrier PCDNA3-DHFR, its contained fresh target fragment is verified by DNA sequencing.And then the 2069bp DNA fragmentation containing total length hFVII-L-vFc gene of above-mentioned acquisition is transferred to SpeI (5 ') and EcoRI (the 3 ') site of PCDNA-DHFR from intermediate carrier, obtain PCDNA3-hFVII-L-vFc expression plasmid, be referred to as again PFVII-A.This plasmid contains the cytomegalovirus promoter CMV that can guarantee that hFVII-L-vFc can stability and high efficiency expresses.This plasmid also contains selected marker thing, thereby can have amicillin resistance in antibacterial, and can have G418 resistance in mammalian cell.In addition, when host cell is DHFR gene defection type, the PCDNA3 expression vector of above-mentioned transformation contains mice DHFR gene, thereby can coamplification hFVII-L-vFc fusion gene and DHFR gene (U.S. Patent No. 4399216) while there is methotrexate (MTX).Based on PFVII-A, by method for synthesizing gene, produce longer flexible peptide linker sequence and check the impact of peptide linker length on FVII activity.Concrete, the BamHI setting in advance (5 ') and the single site of EcoRV (3 ') are as the flexible peptide linker and the part vFc gene (453bp) that insert different length, thereby obtain other 4 expression plasmids, be called again PFVII-B, PFVII-C, PFVII-D, PFVII-E, corresponding respectively to flexible peptide linker amino acid number is 6 (sequence is GlySerGlyGlyGlySer), 11 (sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySer), 16 (sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) and 21 (sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGl yGlySer).
Fusion rotein and the determination of activity of the flexible peptide linker of embodiment 2. transient expression different lengths
5 kinds of expression plasmids that embodiment 1 obtains are used DNAFect LT reagent (ATGCell) transfection 3X10 in the shaking flask of 30ml
7cHO-K1 cell, grows 5 day in the serum-free growth medium that is containing 100ng/ml vitamin K1 through the cell of transfection, by the method describing in detail in embodiment 8, measures the fusion rotein concentration in supernatant, and measures its activity by the method for description in embodiment 7.ELISA result shows that the FVII transient expression amount of 5 kinds of plasmids under this condition is similar, but their blood coagulation activities but demonstrate bigger difference.The FVII supernatant activity of the PFVII-A plasmid expression that contains 2 aminoacid joints is minimum, PFVII-B, PFVII-C, the FVII supernatant activity of PFVII-D and PFVII-E plasmid expression is respectively 113% of PFVII-A, 134%, 147% and 155%, show that the effect length FVII of peptide linker is active.The aminoacid sequence of the fusion rotein being obtained by PFVII-E expression plasmid is shown in SEQ ID NO:3.
The stably transfected cell line of embodiment 3. screening high expressed fusion rotein
Above-mentioned PFVII-D (containing sequence is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer peptide linker) expression plasmid is transfected into mammalian host cell line, to express hFVII-L-vFc fusion rotein.In order to maintain stable high level expression, preferred host cell is the Chinese hamster ovary celI (U.S. Patent No. 4,818,679) of DHFR deficiency.Preferred transfection method is an electroporation, also can use other method, comprises calcium phosphate cosedimentation, liposome transfection and microinjection etc.Electroporation method application is set to the Gene Pulser Electroporator (Bio-Rad Laboratories) of 250V voltage and 1050 μ Fd electric capacity, is placed on 2～3 * 10 in cuvette
7in individual cell, add the linearizing expression plasmid of 20 μ g PvuI, the cell after electroporation is transferred in the shaking flask containing 30ml growth medium.Transfection two days later, changes culture medium into growth medium containing 0.6mg/mLG418, and cell is planted in 96 well culture plates with finite concentration, cultivates 10-12 days until large discrete cell clone occurs.Elisa assay method with anti-human IgG Fc, screening is to selecting medication to have the transfectant of resistance, also the elisa assay method of available anti-FVII is carried out the quantitative assay of expressing fusion protein, then by Method of Limited Dilution method sub-clone, produces the hole of high level expression fusion rotein.
In order to realize fusion rotein higher level, express, should suppress the coamplification that DHFR gene is realized DHFR and antigen-4 fusion protein gene with MTX medicine.In the MTX growth medium that contains progressive concentration, the antigen-4 fusion protein gene of coamplification transfection.The sub-clone that Method of Limited Dilution method obtains can be survived and slowly growth in up to 6 μ g/mL MTX culture medium, finally obtains transfectant.Measure transfectant secretion rate, and further observe its cell growth characteristics.Secretion rate surpasses 1 (being preferably 3-4) μ g/10
6the cell line of (1,000,000) cell/24 hour is for next step serum-free growth medium suspension culture domestication.
First the high yield cell strain that embodiment 3 preferably obtains carries out serum-free domestication in culture dish cultivates, and then transfers in shaking flask and suspends and tame cultivation.After cell adapted these condition of culture, then in 300ml shaking flask, carry out feed supplement feeding culture or cultivate by changing the way simulation perfusion of culture medium every day.The derivative cell strain of above-mentioned CHO feed supplement feeding culture 14 days in the shaking flask of 100ml volume, the recombination fusion protein cumulative production of its expression is 392mg/L (seeing Fig. 3), viable cell density can reach 11 * 10
6individual/mL.In order to obtain more hFVII-L-vFc recombiant proteins, also can select 1000ml shake-flask culture.Another kind of cultural method, the derivative cell strain of above-mentioned CHO is changed culture medium every day in the shaking flask of 100ml volume, and recombination fusion protein cumulative production every day of its expression is about 40-60mg/L, and in shaking flask, viable cell density can reach 25 * 10
6individual/mL.The biologic activity of the mensuration of the recombination fusion protein that above two kinds of methods are produced is suitable.
With 1N NaOH, the conditioned medium of the fusion rotein that contains embodiment 4 acquisitions is titrated to pH7.0 and adds 5mMEDTA, 0.1%Triton X-100, then filters with the celluloid filter of 0.22 micron.Filtrate is loaded onto to the MabSelect of phosphate buffer saline (PBS) balance
tMon Protein A post (GE Healthcare).After fusion rotein is incorporated into ProteinA post, discard the component of outflow, with PBS, wash this post, until the OD value at 280nm place is lower than 0.01.Then use 20mM Tris, 20mM CaCl
2, 300mM NaCl, 900mM Arginine, 45%Propylene Glycol (v/v), 0.05%Tween80 (v/v), the fusion rotein of the buffer solution elution combination of pH6.8.Merge the component that contains purifying protein, open with PBS and dialyse.Then with the celluloid filter of 0.22 micron, filter, and be stored in immediately-70 ℃.Under non-reduced condition, by SDS-PAGE, recorded the molecular weight ranges of hFVII-L-vFc protein of purification at 80-85kDa.Under reducing condition, the albumen of purification migrates to about 160-170kDa.With BSA as standard, by quantitative this fusion rotein of BCA protein analysis.
The biologic activity of embodiment 6. chromogenic substrate method indirect determination fusion rotein
The present invention adopts BIOPHEN FVII that HYPHEN BioMed company produces to add lustre to, and to measure the vitro enzyme of FVII active for test kit (Ref:A221304).This kit measurement principle is chromogenic substrate method, and FVII is a kind of serine protease working at exogenous cruor pathway, after FVII is combined with tissue factor, at phospholipid and Ca
2+under existence condition, can activate thrombin FX, make it change activity form FXa into.First FVII albumen to be determined form multienzyme complex with the tissue factor that derives from rabbit Thromboplastin, then certain density (excessive) thrombin FX in activating reaction system, make it be converted into activity form FXa, FXa acts on specificity chromogenic substrate SXa-11 in reaction system, make substrate cracking and produce pNA, the pNA that produces amount directly become positive correlation with the activity of FXa.At 405nm place, with tintometer, measure the pNA concentration discharge, the corresponding relation between FVII and FXa activity in known test sample book, calculates the activity size of FVII with this, with normal person's blood plasma as standard substance.Suitable with the direct activity of measuring of blood coagulation method of embodiment 7 use by the hFVII-L-vFc purification of samples activity of above-described embodiment 5 of the method indirect determination that adds lustre to, calculate and be about 1100-1300IU/mg.
It is to obtain by correcting the ability of FVII factor disappearance setting time that blood plasma causes prolongation that blood coagulation method is measured FVII biologic activity.The test kit that the present invention adopts French STAGO company to produce
-Deficient FVII (Cat.No.00743).Detection method is normal person's frozen dry blood plasma (Unicalibrator, Cat.No.00625) of the known FVII activity of dilution to be done to standard measure prothrombin time (PT) together with testing sample, and instrument is STAGO company
series coagulo meter, model standard curve, again FVII sample appropriateness to be measured dilution is mixed with the matrix plasma that lacks the FVII factor, measure its PT value, by the active percentage ratio C (%) of standard curve institute matching and the logarithmic equation of PT time t (s), the activity that can record sample to be tested FVII is how many, its result represents (%) with the percentage ratio of normal plasma, the standard substance percentage ratio active (%) and the corresponding relation of the international IU of Mei Huo unit that in test kit, provide are 100%=1IU, can ask accordingly the specific enzyme activity size of calculating testing sample FVII, unit is IU/mg.The average biologic activity of hFVII-L-vFc of above-described embodiment 5 of directly measuring by blood coagulation method is 1300-1500IU/mg, considers the molecular size average out to 160kD of hFVII-L-vFc, is equivalent to 208-240IU/nM.The restructuring hFVII biologic activity of the HEK cells produce of CSL Behring company report is 2874IU/mg, be equivalent to 144IU/nM, and restructuring hFVII fusion albumin (hFVII-FP) biologic activity that it is produced with HEK or Chinese hamster ovary celI is 620-770IU/mg, be equivalent to 69-75IU/nM (Weimer T etc., Thromb Haemost, 2008,99:659-667).So the hFVII-L-vFc fusion rotein biologic activity in the present invention is better than hFVII and hFVII-FP in identical mole of situation.
SD rat Three doses (20,80,180 μ g/Kg) the hFVII-L-vFc protein sample of the purification of the tail vein injection embodiment of the present invention 5, choose the blood sample that different time points extracts SD rat, standing 1 hour, the supernatant after centrifugal was measured the fusion rotein content in blood plasma by ELISA method.When ELISA measures, multi-resistance 0.2 μ g/ml (Jackson Laboratories with goat-anti people's Fc, Cat No:109-005-008) carry out the anti-human FVII multi-resistance of the rabbit (Cedarlane of embedding, horseradish peroxidase-labeled, Cat No:CL20030HP) 1:2000 detects, and actual recording the results are shown in Figure 4.In embodiment 5, the high, normal, basic dosage of purification hFVII-L-vFc sample plasma half-life in rat is respectively 18.8 ± 1.5 hours, 17.2 ± 0.8 hours and 16.0 ± 0.5 hours.The similar dosage plasma half-life of restructuring FVII of report is about 40-45 minute, and restructuring hFVII-FP plasma half-life be 4.5 hours (Weimer T etc., Thromb Haemost, 2008,99:659-667).Restructuring hFVII-L-vFc fusion rotein plasma half-life in the present invention reaches 16-18 hour, is 20 times and 4 times of hFVII-FP of restructuring FVII, so hFVII-L-vFc fusion rotein has the Half-life in vivo significantly extending.
Get 8 of the SD rats of the about 150g of body weight, the hFVII-L-vFc fusion rotein of the embodiment of the present invention 5 of tail vein injection 180 μ g/Kg or contrast PBS solution, after rat administration 30 minutes, 10% chloral hydrate is injected in intraperitoneal anesthesia, heart extracting blood 1.8ml, add rapidly in the mensuration pipe that has 1:9 sodium citrate solution 0.2ml, centrifugal 10 minutes of 3000rpm, draw supernatant, after getting blood, in 2 hours, with Start4 coagulo meter (France), measure prothrombin time (PT) and partial thromboplastin time (APTT).Experimental result shows that the PT time of fusion rotein and matched group is respectively 14.8 seconds and 19.1 seconds, and the APTT time is respectively 8.5 seconds and 11.8 seconds, illustrates that the hFVII-L-vFc in the present invention has body intravascular coagulation activity.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (5)
1. a pharmaceutical composition, it is characterized in that, comprise pharmaceutically acceptable carrier, auxiliary shape agent or diluent, and the restructuring hFVII-L-vFc fusion rotein of effective dose, this fusion rotein holds C end to contain successively people FVII, flexible peptide linker and human IgG Fc from N, wherein, described human IgG Fc comprises natural human IgG Fc and human IgG Fc variant.
2. pharmaceutical composition as claimed in claim 1, it is characterized in that, described human IgG Fc is the Fc variant in human IgG2 hinge region, CH2 and the CH3 region of containing Pro331Ser sudden change, described people FVII is natural FVII or the FVII variant with identity function, described peptide linker contains 2-20 aminoacid, is present between hFVII and human IgG Fc; And described peptide linker contains two or more aminoacid that are selected from glycine, serine, alanine and threonine, preferred GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer or GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGl yGlySer.
3. pharmaceutical composition as claimed in claim 1, is characterized in that, the aminoacid sequence of described fusion rotein is the aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:3.
4. pharmaceutical composition as claimed in claim 1, is characterized in that, the aminoacid sequence of described fusion rotein is the aminoacid sequence shown in the SEQ ID NO:2 removing after the people FVII leader peptide of 1 to 38 amino acids residue.
5. pharmaceutical composition as claimed in claim 1, wherein the DNA sequence of the described restructuring hFVII-L-vFc fusion rotein of coding has the DNA sequence shown in SEQ ID NO:1.
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