CN103623396B

CN103623396B - Comprise the pharmaceutical composition of rt-PA

Info

Publication number: CN103623396B
Application number: CN201310445879.6A
Authority: CN
Inventors: 李强; 孙乃超; 周若芸; 刘瑞贤; 金宜慧
Original assignee: ANYUAN BIOTECHNOLOGY (SHANGHAI) Co Ltd
Current assignee: Anyuan Pharmaceutical Technology (Shanghai) Co.,Ltd.
Priority date: 2013-04-07
Filing date: 2013-04-07
Publication date: 2016-03-02
Anticipated expiration: 2033-04-07
Also published as: CN103623396A; CN103159860B; CN103159860A

Abstract

The invention discloses a kind of pharmaceutical composition comprising rt-PA; Comprise pharmaceutically acceptable carrier, excipient or diluent, and the restructuring TNK-tPA-L-Fc fusion rotein of effective dose.This albumen is held C to hold from N to comprise the natural or variant Fc of people TNK-tPA, flexible peptide linker and human IgG successively.This fusion rotein has the in vitro and in vivo biologic activity similar with people TNK-tPA, and the plasma half-life greatly extended.

Description

Comprise the pharmaceutical composition of rt-PA

Technical field

The present invention relates to molecular medicine field, more specifically, the present invention relates to the Fc fusion rotein of a kind of recombinant long-acting tissue-type plasminogen activator mutant TNK-tPA, relate on the other hand described fusion rotein preparation technology and in application medically, particularly the multiple diseases relevant to needing thrombosis rapid solution such as treatment acute myocardial infarction, acute pulmonary embolism, acute ischemic cerebral apoplexy and it with the medical apparatus and instruments of human blood or contact tissue or the coating medicine on biomedical material surface in novelty teabag.

Background technology

Tissue-type plasminogen activator (Tissueplasminogenactivator, tPA) be the one of plasminogen activator, primarily of vascular endothelial cell synthesis and release, it is a kind of serine protease, is mainly present in mammalian plasma.The efficiently special fibrin in thrombosis of tPA is combined, the tPA-fibrin complex produced can activate the plasminogen in blood clot quickly and efficiently, produce fibrinolysin, make fibrin degradation become soluble product, reach thromboembolism and the again unimpeded object of the blood vessel of thromboembolism.TPA was ratified to put on market as the genetically engineered drug for the treatment of acute myocardial infarction in 1987 by U.S. FDA, and nineteen ninety FDA ratifies it and is used for the treatment of acute pulmonary embolism.1996, then be approved for acute ischemic cerebral apoplexy, tPA is unique first aid medicine for the treatment of apoplexy at present.

Ripe tPA molecule is that molecular weight 67 ~ 72KD, comprises 17 pairs of disulfide bond containing 527 amino acid whose single chain glycoprotein.In molten fine process, tPA molecule is cut by protease in Arg275-Ile276 site, forms active higher duplex molecule, and N holds 275 amino acid residues to form heavy chains, and C holds 252 aminoacid to form light chains, is connected between light, heavy chain by a disulfide bond.Heavy chain, containing 4 domains, is respectively and refers to district (Finger, F), somatomedin district (EGF), primary area 1 (Kringle1, K1), primary area 2 (Kringle2, K2); F district is positioned at 4-50 amino acids sequence, fibre-bearing protein binding site (high-affinity combined function territory); EGF district is positioned at 51-87 amino acids sequence, and containing cell-membrane receptor binding site, this district mediation t-PA is combined with hepatocyte and vascular endothelial cell, has material impact to the half-life; K1 district is positioned at 87-176 amino acids sequence, is receptor binding site or low affine in conjunction with relevant structure with fibrin; K2 district is positioned at 176-262 amino acids sequence, Qi Yu K1 district functional similarity, fibre-bearing protein binding site; Light chain is containing serine protease domain (serineprotease, SerPr district), and form active center by His322, Asp371 and Ser478, catalysis plasminogen changes into fibrinolysin.

The maximum feature of tPA is that the high-affinity special with fibrin is combined, and the tPA of the ability specific ionization state of its plasminogen activation of tPA be combined with fibrin is high 100 times.Because of rarely fibrin generation in normal human blood, tPA can not make normal person that systemic Fibrinolytic function occurs.Just because of efficient, the special thrombolytic effect of tPA, being one of first batch of biological medicament of early utilization technique for gene engineering exploitation, is also the ideal medicament of up to the present treating embolism class diseases.But tPA is used for molten fibre treatment also certain limitation.Complex can be formed with inhibitors of plasminogen activator inhibitor (Plasminogenactivatorinhibitor, PAI) after it enters blood on the one hand, cause it to lose activity rapidly; The tPA specific receptor be present on liver plasma membrane can be combined with tPA fast on the other hand, causes the tPA half-life in blood very short, is only 4 ~ 6 minutes.Therefore, the therapeutic dose of tPA, up to 100 ~ 150mg, which adds the risk caused bleeding by systemic fibrinolytic.

In recent years, research worker is started with from the structure of tPA, take structure activity relationship as principle, DNA restructuring and protein engineering is utilized to construct a series of tPA variant, as reteplase (reteplase), Monteplase (monteplase), lanoplase (1anoteplase), pamiteplase (pamiteplase) etc., they extending its Half-life in vivo, increase and fibrinous binding affinity and improve and all have larger improvement in the catalytic activity of plasminogen etc., have more wide application prospect than natural tPA.Wherein, TNK-tPA (TNKase) is the tPA multipoint mutation body developed by Genentech company of the U.S., is used for the treatment of acute myocardial infarction in 2000 by U.S. FDA approval.This mutant combines and improves tPA and fibrin binding specificity and suddenly change (K) to PAI-1 in conjunction with the KHRR296-299AAAA of sensitivity with reducing, Thr103Asn sudden change (T) reducing clearance rate, prolong half-life in body and Asn117G1n sudden change (the N) (KeytBA lacking high mannose side chain, ProcNatlAcadSciUSA, 1994,91:3670-3674).TNK-tPA has many good characteristics as Increased Plasma Half-life (about 15 ~ 19 minutes), only needs single bullet formula intravenous administration, with fibrin-specific in conjunction with increase by 14 times; Reduce by 80 times with PAI-1 specific binding, affect body blood coagulation system hardly, the effect of inherent hyperamization bolt is less than (CohenM, AmJEmergMed, 2004,22:14-23) such as other plasminogen activators.Which dictates that its revascularization more rapidly, more lastingly, thrombolytic effect more by force, more completely, to being rich in, hematoblastic thrombosis effect is more obvious.

It has been generally acknowledged that, extending the half-life of thrombolytic drug, its bleeding risk may be caused to increase for there being the patient of bleeding tendency.But the safety of TNKase obtains confirmation by ASSENT2 (new thrombolytics safety and efficiency assessment).This is an international research, 16919 routine patients, there is chest pain and accept in 6 hours the acceleration input of unitary agent TNKase or tPA, compares 30 days mortality rates.Result of study shows, and 30 days mortality rates of two groups of patients are similar (TNKase group and tPA group are 6.2%).The incidence rate of cerebral hemorrhage also similar (intracranial hemorrhage is 0.9%), apoplexy (tPA group 1.7%; TNKase group 1.8%).Patient's non-intracranial massive hemorrhage complication for the treatment of with TNKase and tPA are respectively 4.7% to 5.9%.This illustrates that the half-life of TNKase prolongation does not increase cerebral hemorrhage and the apoplexy incidence rate of patient.The tPA variant of Increased Plasma Half-life apply clinically more convenient, efficient, reduce dosage and do not increase bleeding risk.

In addition, also there is the short problem of therapeutic time window in tPA, and most important reason is that its thrombolytic rate is low.The key issue that restriction acute cerebral ischemia thromboembolism treatment effect and thromboembolism treatment are extensively carried out is cerebral hemorrhage after thrombolytic.

Cerebral ischemia thrombolytic treatment time window is the time limit that cerebral ischemia starts to fall ill to starting to treat, and exceed this time limit, the thrombolytic drugs such as tPA are not only invalid, can increase hemorrhage risk on the contrary.More than 3 hours (especially involve artery of cerebral hemorrhage---because artery of cerebral hemorrhage is branched ending, lack collateral circulation, cerebrovascular wall is easily damaged because of ischemia) cerebral ischemia re-pouring after, the danger of cerebral hemorrhage increases greatly.As the representative of second filial generation thrombolytic drug, at Cerebral Infarction within 3 hours, obtain internationally generally approving through intravenous thrombolytic with tPA, and a nearest analysis confirms to occur using tPA to be still safety with effective in 4.5 hours at apoplexy onset symptoms, thus make tPA thrombolytic time window extend to 4.5 hours from 3 hours, but the small sample RCT also having more challenging message: TNKase to be used for Ischemic Stroke 6 hours window studies display compared with standard dose tPA (0.9mg/kg), patient's Reperfu-sion rate higher (P=0.004) of TNKase treatment (0.1mg/kg or 0.25mg/kg), clinical improvements significantly (P<0.001), the all efficacy endpoint indexs of heavy dose of TNKase group are all better than low dosage TNKase group and tPA group, and do not increase the incidence rate of hemorrhage and other adverse events of symptomatic intracranial.If the curative effect of TNKase is confirmed further in the RCT research of large sample, probably will replace tPA thus the time window for the treatment of of acute stroke is extended to 6 hours further.

In addition, other thrombolytic drugs are relative to tPA, and half-life and therapeutic time window also all have prolongation, and if the urokinase half-life is 13 ~ 20 minutes, the time window of its treatment acute ischemic stroke is 6 hours.A kind of plasminogen activator desmoteplase extracted from vampire saliva has the half-life reaching about 4 hours, and a series of research of Germany scientist is expected to make its thrombolytic time window broadening to 9 hours.The medicine of long half time, after single administration, drug level can maintain in Valid concentration for a long time and play curative effect.Can be able to know by inference from existing experience, the Increased Plasma Half-life of thrombolytic drug, the therapeutic time window of its correspondence is also corresponding to be widened.But, in the clinical position of China, only have a few patients can morbidity several hours in accept thromboembolism treatment, even if developed country is also no more than 10% of patients with cerebral ischemic.Thus the novel thrombolytic drug developing long half time, therapeutic time window wider is very necessary.

The immunoglobulin of IgG class is most rich in protein in human blood.Their half-life can up to 21 days, and Fc fragment be in IgG holder very long half-lift main reason, also there is the effect of stabilize proteins simultaneously.Lot of documents report destination protein and Fc section are merged after do not affect its biologic activity and obtain prolongation half-life (see, as people such as Capon, Nature, 337:525-531,1989; The people such as Chamow, TrendsBiotechno1., 14:52-60,1996; U.S. Patent No. 5,116,964 and 5,541,087).But, this is not natural result for tPA, Huang such as to be celebrated one's birthday at (the Military Medical Science Institute, calendar year 2001, post-doctor's paper) tPA and IgG1Fc section to be merged, expection improves the tPA half-life in vivo, but experimental result is regrettable, the fusion of the two causes the forfeiture of tPA activity, and analyzing reason may be have impact on the correct of tPA after Fc section and tPA merge to fold, thus makes it lose activity.TPA is a serine protease having 17 pairs of disulfide bond, five Structure and function domains, and 4 domains of the serine protease domain (SerPr) and heavy chain that are positioned at light chain are connected by a pair disulfide bond.Serine protease domain (SerPr) near C-end forms active center by His322, Asp371 and Ser478, can specificity cracking plasminogen Arg560-Val561 place peptide bond, be translated into fibrinolysin, after tPA is combined with fibrin, its activity will significantly increase, this is by the impact in F1 domain and C-end K2 district, and these five domains are be mutually related for the biological activity of tPA and specific binding as seen.Early stage result of study confirms, can the space structure of tPA comparatively complexity be also more fragile, correctly fold after protein translation, and it is very crucial for forming activated space conformation for the enzymatic activity of tPA.Ser structure territory and other domain such as F1 and K2 district relevant to its activity of the C-end of tPA keep enzymatic activity to be all very important to it.In other words, aminoacid and its function of the C end of tPA are closely related, therefore usually avoid C end when carrying out genetic engineering modified to tPA, also the C-end of tPA and other albumen should not be merged.Thus, tPA is as a kind of serine protease, the catalytic active center being positioned at C-end is necessary to its biological activity of maintenance, therefore necessarily considers the consequence how overcoming and cause its loss of activity because C-end connects the next space steric effect of Fc length of tape in advance when building the Fc fusion rotein of TNK-tPA.

Owing to being positioned at three aminoacid in active center relatively near the C-end of total length TNKase sequence, the particularly serine sites of 478 on primary structure, what this made the Fc fusion rotein of TNK-tPA is built with its difficulty in essence.Still not do not report about the research of TNK-tPA fusion rotein up to now, and the Fc fusant of other protease also not yet has and is approved for clinical precedent.Two protease Fc fusion rotein that current progress is the fastest, namely proconvertin and factor Ⅸ are yet in clinical investigation phase.Therefore, the construction and expression of the Fc fusion rotein of TNK-tPA and process study thereof are a forward-looking and initiative job, are expected to extend further tPA variant half-life in vivo and may obtain the therapeutic time window widened.

Although as far back as 20th century the mid-80, just there is research institution gene engineering method exploitation recombinant natural tPA in China, the domestic marketing drugs of recombinant natural tPA or TNK-tPA not having animal cell expression to produce so far.The first generation recombinant natural tPA series products of domestic listing, complete dependence on import, trade name " Ai Tongli ", 50mg/ props up, and price is up to 5717 yuan.And adopting the second filial generation tPA series products of escherichia coli expression, i.e. reteplase, " Easthome is stood " of being produced by Shandong A Hua and Beijing like that " Pai Tongxin " of the exploitation of moral Pharmaceutical gets permission listing respectively at 2007 and 2010 years.But the price of tPA series products is high, according to disclosed drug price, known alteplase and reteplase medical expense is once about 2750 dollars, tenecteplase medical expense once, also up to 2196 dollars, is difficult to apply clinically.The high main cause one of price is that development difficulty is large, tPA molecular structure complexity (glycosylated molecule containing 17 pairs of disulfide bond), vivoexpression difficulty, and it is low to express productive rate; Two is that clinical medicine dose is large, and first generation Recomposed tPA clinical medicine dose is that 100mg/ props up, and the third generation still needs 20 ~ 50mg/ to prop up.

Because TNK-tPA is the most safe and effective up to now and the Thrombolytic Drugs of use most convenient, and have broad application prospects, thus become the focus that various countries' pharmacy corporation falls over each other to research and develop.TNKase is developed by Genentech company of the U.S. the earliest, i.e. tenecteplase.The TNK-tPA of the U.S. is not both at China's patent protection, and product also fails to enter Chinese market.Only there is Guangzhou inscription Kanggong department developing third generation tPA series products TNK-tPA at home, it is reported and complete clinical research.Guangzhou inscription Kanggong department adopts mammalian cell CHO to express TNK-tPA, and continuously perfused culture technique, culture density reaches 2 × 10 ⁷/ ml, output is about 50-150mg/L.With regard to production technology, the continuously perfused culture technology of Guangzhou inscription Kanggong department employing has unrivaled advantage compared with the batch culture that Genentech adopts.Compare with additive method, the major advantage of perfusion cultures is that the culture medium of continous pouring can provide sufficient nutritional labeling, and can take away metabolite, and cell is retained in reactor assembly simultaneously, can reach very high cell density, productive rate can improve an order of magnitude.But continuous perfusion culture need constantly add fresh culture medium while results culture fluid, and cost is very high, and makes downstream albumen purification work add a lot of difficulty.At present, published tPA recombination classes product is all adopt continuous perfusion culture technology, and mainly come from tPA albumen self stability poor, cell strain expression is low, and perfusion culture process is selection preferably.

To sum up, TNK-tPA product still has some limitations in clinical practice, as relatively short in the half-life, therapeutic time window is wide not.In addition, the expression cell line output of structure is lower, and the perfusion production technology adopted makes production cost increase, and makes downstream protein purification more complicated and difficult.

Summary of the invention

The present invention aims to provide a kind of restructuring TNK-tPA-L-Fc fusion rotein, and it has the In vitro biological activity similar or higher with natural tPA, and the Half-life in vivo extended.Restructuring TNK-tPA-L-Fc fusion rotein refers to the Fc fusion rotein of tissue-type plasminogen activator mutant TNK-tPA, wherein, is connected between TNK-tPA and Fc by flexible joint.

To achieve these goals, according to an aspect of the present invention, a kind of restructuring TNK-tPA-L-Fc fusion rotein is provided.This restructuring TNK-tPA-L-Fc fusion rotein is from N end to the IgGFc of C end successively containing TNK-tPA, flexible peptide linker and people, and wherein, human IgG Fc comprises natural human IgG Fc and human IgG Fc variant.

Further, human IgG Fc variant is selected from following group: the human IgG2 hinge region that (i) suddenlys change containing Pro331Ser, CH2 and CH3 region; (ii) human IgG 4 hinge region containing Ser228Pro and Leu235Ala sudden change, CH2 and CH3 region; (iii) the human IgG1 hinge region containing Leu234Val, Leu235Ala and Pro331Ser sudden change, CH2 and CH3 region.

Further, flexible peptide linker contains 2-20 aminoacid, and flexible peptide linker contains the aminoacid that two or more are selected from glycine, serine, alanine and threonine.

Further, the amino acid residue sequence of flexible peptide linker (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) as shown in SEQIDNO:7.

Further, TNK-tPA is natural tPA or the tPA variant with identity function.

Further, the aminoacid sequence of TNK-tPA-L-Fc fusion rotein is as shown in SEQIDNO:2,4 or 6.

Further, the aminoacid sequence of TNK-tPA-L-Fc fusion rotein is SEQIDNO:2, the aminoacid sequence shown in 4 or 6 after the TNK-tPA leader peptide eliminating 1 to 35 amino acids residues.

According to another aspect of the present invention, a kind of DNA sequence of above-mentioned restructuring TNK-tPA-L-Fc fusion rotein of encoding is provided.

According to a further aspect of the invention, provide a kind of DNA sequence, this DNA sequence has SEQIDNO:1, the DNA sequence shown in 3 or 5.

According to a further aspect of the invention, a kind of carrier is provided.This carrier comprises above-mentioned DNA sequence.

According to a further aspect of the invention, a kind of host cell is provided.This host cell comprises above-mentioned carrier.

Further, host cell is the derived cell strain of CHO.

According to a further aspect of the invention, a kind of pharmaceutical composition is provided.This pharmaceutical composition comprises pharmaceutically acceptable carrier, excipient or diluent, and the above-mentioned restructuring TNK-tPA-L-Fc fusion rotein of effective dose.

According to a further aspect of the invention, a kind of preparation method of above-mentioned restructuring TNK-tPA-L-Fc fusion rotein is provided.This preparation method adopts above-mentioned host cell to prepare restructuring TNK-tPA-L-Fc fusion rotein.

According to a further aspect of the invention, the application of a kind of above-mentioned restructuring TNK-tPA-L-Fc fusion rotein in the medicine of the disease relevant to needing thrombosis rapid solution for the preparation of prevention and therapy is provided.

Further, disease comprises acute myocardial infarction, acute pulmonary embolism, acute ischemic brain soldier.

According to a further aspect of the invention, a kind of above-mentioned restructuring TNK-tPA-L-Fc fusion rotein is provided to apply in preparation with human blood or the medical apparatus and instruments of contact tissue or the coating medicine on biomedical material surface.

To sum up, in the present invention, inventor creatively transforms further to TNK-tPA, holds its C by one section of general flexible peptide linker (1inker) and IgGFc segment composition, obtains the half-life longer and have the Fc fusion rotein of the TNK-tPA of better biologic activity.

Below content of the present invention is specifically introduced:

Fusion rotein and preparation method thereof

The invention provides a kind of restructuring TNK-tPA-L-Fc fusion rotein, this fusion rotein holds C to hold or variant Fc natural containing people TNK-tPA, peptide linker and human IgG successively from N, and human IgG Fc variant is selected from lower group:

(i) suddenly change containing Pro331Ser human IgG2 hinge region, CH2 and CH3 region;

(ii) human IgG 4 hinge region containing Ser228Pro and Leu235Ala sudden change, CH2 and CH3 region;

(iii) the human IgG1 hinge region containing Leu234Val, Leu235Ala and Pro331Ser sudden change, CH2 and CH3 region.

Wherein, peptide linker, preferably, flexible peptide linker that use an about 2-20 amino acid length, that form containing following 2 kinds or several amino acids: glycine, serine, alanine and threonine, the preferred sequence as disclosed in the embodiment of the present invention is SEQIDNO.:7 (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer).

The aminoacid sequence of TNK-tPA-L-Fc fusion rotein of the present invention is as shown in SEQIDNO:2,4 or 6, its maturation protein is for eliminating TNK-tPA leader peptide (1 to the 35 amino acids residue) aminoacid sequence shown in SEQIDNO:2,4 or 6 afterwards, and human IgG Fc contains hinge region, CH2 and CH3 region.Its CH2 region of variant Fc 228,234,235 and 33l position (position determined by EU number system) containing amino acid mutation, thus reduce the effector function (U.S. Patent No. 6,797,493 and 6,900,292) of Fc.

Fc element

The immunoglobulin of IgG class is most rich in protein in human blood.Their half-life can up to 21 days, and its high stability in blood plasma mainly has benefited from it up to the molecular weight of 150kD and the combination with its receptor FcRn, is combined antibody can be avoided to enter lysosome and be degraded with FcRn.Albumen and antibody Fc are merged the structure obtaining antibody-like, not only can extend the Half-life in vivo of protein drug, but also become bivalent molecule by monovalent molecule, improve the adhesion of itself and target protein.FcRn has activity in adult epithelial tissue, and expresses in the epithelial cell of enteric cavity, lung qi pipe, nasal cavity, vagina, coton and rectal.The transcytosis that the fusion rotein be made up of IgG or Fc section mediates by FcRn, effectively shuttled across epithelial barriers.In addition, the fusion rotein comprising Fc section is expressed cell endocytic and the protection of FcRn.These fusion rotein are not marked as degraded, but again enter blood circulation, thus add the Half-life in vivo of these albumen.This method has been used to some cytokines very important clinically (as IL-2 and IFN-α 2a) and soluble recepter (as TNF-Rc and VEGF-Rc), there is prolongation (U.S. Patent No. 5 in various degree half-life to Fc fusion rotein in vivo, 349,053 and 6,224,867).The medicine Enbrel developed by American I mmunex company is exactly recombined human P75 Tumor Necrosis Factor Receptors and the human IgG lFc fusion rotein dimer of using expressing cho cell, this medicine was in application listing in 1998, tired out the treatment of rheumatoid arthritis patients's symptom for severe activeness in alleviating by FDA approval in 1999, and in 2002 by the symptom of FDA approval for alleviating psoriasis arthropathica patient.

Peptide linker

The activity of length to fusion rotein of connection peptides is extremely important.Existing people reports erythropoietin (EPO) derivant (as dimer), compared with EPO monomer, fusion rotein containing 2 complete EPO regions (3 to 7 amino acid peptide joints of being separated by) shows the activity (QiuH etc. weakened, JBiolChem, 1998,273:11173-6).But 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 significantly improves (SytkowskiAJ etc., JBiolChem, 1999,274:24773-8; U.S. Patent No. 6,187,564).This possible explanation is the connection peptides that fusion rotein two parts ask increase, makes two parts of this molecule can exercise its function (AshkenaziA etc., CurrOpininImmunol, 1997,9:195-200) respectively.

The present invention is through long-term and deep research, devise a kind of original hinge region peptide linker first to reduce space steric effect, the C that can obtain TNK-tPA holds the fusion rotein be connected with Fc, there is soft peptide linker centre, and the preferred sequence as disclosed in the embodiment of the present invention is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.Surprisingly, this fusion rotein does not only cause the biological function of TNK-tPA to be lost, and can maintain, even improve the biological activity of TNK-tPA-L-Fc fusion rotein on the contrary.

In addition, the present invention also finds, the peptide linker added between TNK-tPA and human IgG Fc variant improves the Bioactivity of restructuring TNK-tPA-L-Fc in two ways: (1) makes Fc region away from the domain on TNK-tPA, (2) make a TNK-tPA away from the domain of another TNK-tPA, thus reduce space steric effect.

Provide a kind of cell line preparation derived as CHO from mammal cell line or the method for producing this recombination fusion protein according to a further aspect in the invention, comprise the following steps:

A the DNA of coding restructuring TNK-tPA-L-Fc fusion rotein is introduced CH0 cell by (), generate the cell line that CHO is derivative;

B cell line that this CHO of () feeding culture is derivative, thus express restructuring TNK-tPA-L-Fc fusion rotein; With

C restructuring TNK-tPA-L-Fc fusion rotein that () purification step (b) expresses.

The DNA of described coding restructuring TNK-tPA-L-Fc fusion rotein has SEQIDNO:1, the nucleotide sequence shown in 3 or 5.

In step (a), fusion rotein holds C to hold successively containing TNK-tPA, flexible peptide linker and human IgG Fc variant (can be expressed as TNK-tPA-L-vFc) from N, it shows good Bioactivity, namely on a molar basis, there is the Bioactivity similar or higher with NK-tPA, and longer Half-life in vivo; Wherein exist containing 2-20 amino acid whose flexible peptide linker of having an appointment between TNK-tPA and IgGFc variant; The aminoacid that 2 or multiple aminoacid are selected from glycine, serine, alanine and threonine is contained with flexible peptide linker; Wherein human IgG Fc variant contain be selected from following human IgG hinge region, CH2 and CH3 region: Pro331Ser suddenly change human IgG2; The AIgG4 of Ser228Pro and Leu235Ala sudden change; With the human IgG l of Leu234Val, Leu235Ala and Pro331Ser sudden change.

In step (b), restructuring TNK-tPA-L-vFc fusion rotein in its growth medium in every 24 hours, in expression more than 20 (being preferably 30) μ g/10 ⁶under the condition of (1,000,000) individual cell, cultivate the cell line that the CHO of transfection is derivative.Animal cell culture can select batch culture, perfusion to cultivate or feeding culture technique, in a preferred embodiment of the present invention, select feeding culture technique, the cell strain that high yield CHO derives is cultivated 13 days in 100mL shaking flask, and its recombination fusion protein cumulative production expressed is 1.90g/L (Fig. 6).Between the 6th day to the 10th day of cell culture, number of viable cells is about 22 × 10 at most ⁶individual/mL, with this understanding, secretion rate is determined as 30 μ g/10 ⁶individual cell/24 hour.

In step (c), the preferred ProteinA affinity chromatography of purification process of restructuring TNK-tPA-L-vFc fusion rotein, through single step purification, purity of protein can reach 90% with on t.

Compared with prior art, the advantage of fusion rotein of the present invention and preparation method thereof is summarized as follows:

The restructuring TNK-tPA-L-vFc fusion rotein that 1.Fc and TNK-tPA coupling is formed, has very high expression in Chinese hamster ovary celI, higher more than 10 times than restructuring TNK-tPA expression in Chinese hamster ovary celI.

2., because of TNK-tPA and Fc amalgamation and expression, the stability of albumen is strengthened, and thus host cell can adopt feeding culture technique, the consumption greatly reducing culture medium is cultivated compared with perfusion, and decreasing culture fluid results volume, purification work amount greatly reduces, and significantly reduces material cost and human cost.

3. TNK-tPA-L-vFc fusion rotein of recombinating adopts ProteinA affinity chromatography to carry out purification, and purification step simply, efficiently, significantly reduces the cost of purification.

4. TNK-tPA-L-vFc fusion rotein of recombinating has the In vitro biological activity (molar specific activity) higher with Recomposed tPA.

5. the TNK-tPA-L-vFc fusion rotein of recombinating has the serum half-life that greatly extends and does not increase the bleeding risk of patient, thus reduces the dosage realized needed for similar drug effect, and the thrombolytic treatment time window for acute stroke patients can be made to widen.

Pharmaceutical composition

The invention provides a kind of pharmaceutical composition, comprise pharmaceutically acceptable carrier, excipient or diluent, and the restructuring TNK-tPA-L-Fc fusion rotein of the present invention of effective dose.

Restructuring TNK-tPA-L-vFc fusion rotein of the present invention is usually applied to the prevention and therapy of hemorrhage of the congenital or acquired deficiency disease patient of TNK-tPA and the spontaneous or surgical hemorrhage prevention and therapy of hemophilia A or B patient or other relevant hemorrhages.

Further, the invention provides a kind of pharmaceutical composition, it contains effective dose (as 0.000001-90wt%; Preferably 0.1-50wt%; Better, 5-40wt%) fusion rotein of the present invention, and pharmaceutically acceptable carrier.Usually, the fusion rotein of the present invention of effective dose can be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably, pH is about 6-8.Term " effective dose " or " effective dose " refer to can to people and/or animal produce function or activity and can by people and/or animal the amount that accepts.The composition of " pharmaceutically acceptable " is applicable to people and/or mammal and without excessive bad side reaction (as toxicity, stimulation and allergy), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent administration, comprises various excipient and diluent.

Pharmaceutically acceptable carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usual pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant.Described pharmaceutical composition should aseptically manufacture.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 (such as passing through clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: pharmacokinetic parameter such as bioavailability, metabolism, the half-life etc. of described fusion rotein; The order of severity of the disease that patient will treat, the body weight of patient, the immune state of patient, the approach etc. of administration.

Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Coating medicine

On the other hand, the present invention relates to the novelty teabag of TNK-tPA-L-vFc fusion rotein making coatings medicine, more specifically, relate to the novelty teabag of interventional medical apparatus or the biomedical material face coat medicine contacted with human blood or tissue, as hemodialysis system, extracorporeal circulation system, Cardiac valve prosthesis, cardiac pacemaker, artificial blood vessel, intravascular stent, surgical cable and conduit etc.The coating material being wherein used as the biomedical material of pharmaceutical carrier need have good histocompatibility, optional autohemagglutination compound, nano material etc., the pharmaceutical carrier as common has methacrylate, chondroitin sulfate, polylactide and its copolymer, polycaprolactone, phosphocholine etc.Described fusion rotein is combined in the surface of carrier material by absorption or chemical mode.

Accompanying drawing explanation

Fig. 1 shows the TNK-tPA-L-vFc of SpeI-EcoRI fragment in PCDNA3 expression vector according to the embodiment of the present invention _{γ 2}nucleotide sequence and the aminoacid sequence of derivation.People TNK-tPA is made up of signal peptide (1-35), mature T NK-tPA albumen (36-562).Ripe fusion rotein contains people TNK-tPA (36-562), flexible peptide linker (563-578) and Fc _{γ 2}variant (579-801).

Fig. 2 shows the TNK-tPA-L-vFc of SpeI-EcoRI fragment in PCDNA3 expression vector according to the embodiment of the present invention _{γ 4}nucleotide sequence and the aminoacid sequence of derivation.People TNK-tPA is made up of signal peptide (1-35), mature T NK-tPA albumen (36-562).Ripe fusion rotein contains people TNK-tPA (36-562), flexible peptide linker (563-578) and Fc _{γ 4}variant (579-807).

Fig. 3 shows the TNK-tPA-L-vFc of SpeI-EcoRI fragment in PCDNA3 expression vector according to the embodiment of the present invention _{γ 1}nucleotide sequence and the aminoacid sequence of derivation.People TNK-tPA is made up of signal peptide (1-35), mature T NK-tPA albumen (36-562).Ripe fusion rotein contains people TNK-tPA (36-562), flexible peptide linker (563-578) and Fc _{γ 4}variant (579-805).

Fig. 4 shows the gene mapping of the eukaryon expression plasmid of TNK-tPA-L-vFc antigen-4 fusion protein gene constructed by the embodiment of the present invention.This expression plasmid total length 9997bp, containing 10 major gene segments, comprises 1.hCMV promoter; 2. target gene TNK-tPA-L-vFc; 3.EMCVIRES; 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. 5 shows the concentration trends curve chart according to the growth in the strain of 300ml shaking flask inner cell of the embodiment of the present invention and secretion TNK-tPA-L-vFc fusion rotein thereof.

Fig. 6 shows the external activity of the TNK-tPA-L-vFc purifying protein according to the embodiment of the present invention.

Detailed description of the invention

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.

Embodiment 1. builds the gene of coding TNK-tPA-L-vFc γ fusion rotein

The gene order of coding TNK-tPA leader peptide and maturation protein is the Chinese hamster ovary celI preference codon that artificial optimization crosses, and obtains through chemosynthesis way.For the ease of the specific site by object fragment inserting expressioning carrier, in synthesized fragment 5, and 3 ' end respectively has a Restriction Enzyme inscribe site, is respectively SpeI and BamHI.The DNA fragmentation of total length 1705bp inserts transfer vector as between the EcoRV restriction enzyme site in pUC57, and obtain middle interstitial granules, contained by it, TNK-tPA gene order is verified by DNA sequencing.

Flexible peptide linker and human IgG Fc region Fc _{γ 2}variant vFc _{γ 2}(Pro331Ser sudden change), Fc _{γ 4}variant vFc _{γ 4}(Ser228Pro and Leu235Ala sudden change), Fc _{γ 1}variant vFc _{γ 1}the fusion gene of (Leu234Val, Leu235Ala and Pro331SSer suddenly change) is also the Chinese hamster ovary celI preference codon that artificial optimization crosses, and synthesized fragment 5 ' and 3 ' end respectively has a Restriction Enzyme inscribe site, is respectively BamHI and EcoRI.L-vFc is verified by DNA sequencing _γthree kinds of gene orders.By the DNA fragmentation of acquisition through being inserted into BamHI and the EcoRI site of mammalian cell expression vector PCDNA3 (Invitrogen), obtain PCDNA3-L-vFC γ tri-plasmids.Again by the TNK-tPA fragment that obtains after NheI/BamHI double digestion, between the corresponding restriction enzyme site being inserted into plasmid PCDNA3-L-vFc γ, obtain three fusion gene expression plasmid pCDNA3-TNK-tPA-L-vFc _γ, this plasmid is containing cytomegalovirus early promoter, and it is the enhancer needed for mammalian cell high level expression exogenous gene.This plasmid also containing selected marker thing, thus can have amicillin resistance in antibacterial, and can have G418 resistance in mammalian cell.In addition, when host cell is DHFR gene expression deficiency, PCDNA3 expression vector contains dihydrofolate reductase (DHFR) gene of mice, thus can coamplification TNK-tPA-L-vFc when there is methotrexate (MTX) _γfusion gene and DHFR gene (United States Patent (USP) 4,399,216).

Exist between people TNK-tPA and Fc part flexible peptide linker add the flexibility in TNK-tPA region and improve its biological activity.For the purpose of the present invention, preferably length is about 20 or less (but can not be less than 2) amino acid whose peptide linkers.Should use containing or be selected from by 2 or more the peptide linker of following Amino acid profile: glycine, serine, alanine and threonine.The example of peptide linker contains a Gly-Ser peptide component, as GlyGlyGlyGlySer.Fig. 1, Fig. 2, Fig. 3 respectively illustrate containing three kinds of Fc _γthe nucleotide sequence of variant fusion gene and the aminoacid sequence of derivation, they are jointly containing encoding human TNK-tPA, 16-amino acid peptide joint (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) with separately containing coding Fc _{γ 2}variant vFc _{γ 2}(TNK-tPA-L-vFc _{γ 2}), Fc _{γ 4}variant vFc _{γ 4}(TNK-tPA-L-vFc _{γ 4}) or Fc _{γ 1}variant vFc _{γ 1}(TNK-tPA-L-vFc γ ₁) sequence.The TNK-tPAvFc fusion rotein of three kinds of variants is all expressed in Chinese hamster ovary celI, purification and carry out active determination in vitro, and the correlation properties that they show are very similar.Following examples choose TNK-tPA-L-vFc γ ₂for stating, TNK-tPA-L-vFc γ ₁, TNK-tPA-L-vFc γ ₄and its result of mutant in following experiment with thrombosis rapid solution function is suitable, does not repeat them here.

The expression of embodiment 2. fusion rotein in transfectional cell series

The expression vector plasmid transfection of restructuring is entered mammalian host cell line, to express TNK-tPA-L-vFc _γfusion rotein.In order to stablize high-caliber expression, preferred host cell system is DHFR deficient CHO-cell (U.S. Patent No. 4,818,679).A kind of preferred transfection method is electroporation, also can use other method, comprise calcium phosphate cosedimentation, fat transfection and Protoplast fusion.In electroporation, with the GenePulserElectroporator (Bio-RadLaboratories, Hercules, CA) being set to 210V electric field and 1050 μ Fd electric capacity, 2 ~ 3 × 1O in cuvette ⁷the plasmid after 20 μ g PvuI linearisations is added in individual cell.In transfection two days later, culture medium is made into the growth medium containing 0.6mg/mLG418.By the elisa assay method of anti-human igg Fc, screen the transfectant to selecting medication to have resistance.Also the quantitative of fusion protein expression can be carried out with the ELISA of anti-human tPA.By Method of Limited Dilution 96 well culture plate, sub-clone produces the hole of high-level Fc fusion rotein.

In order to realize the expression of fusion rotein higher level, coamplification should be carried out with the DHFR gene by MTX Drug inhibition.In the growth medium containing progressive concentration MTX, with the antigen-4 fusion protein gene of DHFR gene coamplification transfection.With the transfectant that the sub-clone of Method of Limited Dilution can grow in up to 5 μ g/mLMTX culture medium.Measure the cell line of secretion rate to sub-clone further to analyze.Secretion rate horizontal exceeding about 20 (preferably about 30) μ g/10 ⁶the cell line of (namely 1,000,000) individual cell/24 hour adapts to the suspension culture using serum free growth medium.Then conditioned medium purified fusion protein is used.

The production of embodiment 3. fusion rotein

First the high yield cell strain that embodiment 2 preferably obtains carries out serum-free domestication and cultivates in culture dish, then transfers in shaking flask and carries out suspension domestication cultivation.After these condition of culture cell adapted, in 300m1 shaking flask, then carry out feed supplement add cultivation or the way simulation perfusion cultures by replaced medium every day.The cell strain that above-mentioned CHO derives feed-batch culture 13 days in the shaking flask of 100ml volume, its recombination fusion protein cumulative production expressed is 1.90g/L (Fig. 6).Between the 6th day to the 10th day of cell culture, viable cell density can reach 22 × 10 ⁶individual/mL.In order to obtain more TNK-tPA-L-vFc recombiant protein, also 2000ml shake-flask culture can be selected.Another kind of cultural method, the cell strain that above-mentioned CHO derives in the shaking flask of 100ml volume every day replaced medium, its recombination fusion protein cumulative production every day expressed is about 0.20-0.35g/L, and in shaking flask, viable cell density can reach 31 × 10 ⁶individual/mL.The In vitro biological activity of the mensuration of the recombination fusion protein that above two kinds of methods are produced is suitable.

The purification of embodiment 4. fusion rotein and qualitative

With 1NNaOH, the conditioned medium of the fusion rotein containing embodiment 3 is titrated to pH7 ~ 8, then filters with the nitrocellulose filter of 0.22 micron.Filtrate is loaded onto the MabSelect that phosphate buffer saline (PBS) balances ^tMon ProteinA post (GEHealthcare).Protein binding to be fused, after ProteinA, discards the component of outflow.This post is washed, until the OD value at 280nm place is lower than 0.01 with PBS.Then use 0.1Mglycyltryosin, the fusion rotein that 2MNaCl, 0.04%Tween20, pH4.58 buffer solution elution combines, merge the component containing purifying protein, and dialyse with PBS.Then filter with the nitrocellulose filter of 0.22 micron, and be stored in-70 DEG C.Under the reducing conditions, strand tPA albumen (alteplase for injection, the Boehringer Ingelheim produce) molecular weight recording purification by SDS-PAGE is about 65kDa.The TNK-tPA-L-vFc protein migrates of purification is to about 90kDa.With BSA as standard, by BCA protein analysis quantitatively this fusion rotein.

Embodiment 5. directly Chromogenic assay measures TNK-tPA-L-vFc fusion rotein external activity

Direct Chromogenic assay adopts the tPA Activity Assay Kit of DiaPharma company, and the method chromogen substrate S-2288 measures the activity of purification tPA.Cleaning Principle is a kind of serine protease based on tPA, can make Arg-Val place peptide bond fission in plasminogen single chain molecule.In purification system, this enzyme hydrolyzable tripeptides chromogen substrate.Thus, the burst size by measuring paranitroanilinum (Para-Nitroaniline, pNA) calculates the activity of tPA.Spectrophotography measures the change of OD value under 405nm wavelength, reflect generate pNA amount number.Fig. 6 shows the Recomposed tPA albumen (alteplase for injection, Boehringer Ingelheim is produced) of purification or the external activity corresponding relation of restructuring TNK-tPA-L-vFc albumen under identical molar concentration (nM) of purification.Under these conditions, the EC of tPA ₅₀value is 43.2nM, and TNK-tPA-Fc is 19.5nM, so under same molar ratio condition, the external activity of TNK-tPA-L-vFc is 2.5 times of tPA.Fusion rotein in feeding culture supernatant also can measure its In vitro biological activity with said method.

The pharmacokinetics of embodiment 6. fusion rotein measures

SD rat Three doses (6,0.9,0.18mg/Kg) tail vein injection TNK-tPA-L-vFc sample, choose the blood sample that different time points extracts SD rat, leave standstill 1 hour, the supernatant ELISA method after centrifugal measures the fusion rotein content in blood plasma.When measuring with ELISA, with the multi-resistance of the Fc of goat-anti people carry out embedding, the monoclonal antibody of the mouse-anti buman tPA of horseradish peroxidase-labeled detects.In embodiment 4, purification TNK-tPA-L-vFc sample distinguishes 109.1 ± 3.5 minutes in the rat plasma half-life at high, normal, basic dosage, 112.6 ± 1.5 minutes and 124.8 ± 3.5 minutes, and TNK-tPA medicine comparable amount plasma half-life of recombinating is about 19 minutes, so TNK-tPA-L-vFc Half-life in vivo of recombinating in the present invention significantly extends.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.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

1. a pharmaceutical composition, is characterized in that, comprises the restructuring TNK-tPA-L-Fc fusion rotein of aminoacid sequence as shown in SEQIDNO:2,4 or 6 of normal saline and effective dose.