CN103623396A

CN103623396A - Pharmaceutical composition containing recombinant tissue plasminogen activator

Info

Publication number: CN103623396A
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: 2014-03-12
Anticipated expiration: 2033-04-07
Also published as: CN103159860B; CN103623396B; CN103159860A

Abstract

The invention discloses a pharmaceutical composition containing a recombinant tissue plasminogen activator. The pharmaceutical composition consists of a pharmaceutically acceptable carrier, excipient or thinner, as well as an effective amount of recombinant TNK-tPA-L-Fc fusion protein; from N terminal to C terminal, the protein sequentially includes TNK-tPA, a flexible peptide joint and human IgG natural or variant Fc. The fusion protein is similar to human TNK-tPA in both in vitro and in vivo bioactivities, and can greatly prolong plasma half-life.

Description

The pharmaceutical composition that comprises 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 the preparation technology of described fusion rotein and the application in medical treatment thereof, particularly dissolve fast relevant disease and it with human blood or organize the new purposes aspect the coating medicine on the medical apparatus and instruments that contacts or biomedical material surface with needing thrombosis treatment acute myocardial infarction, acute pulmonary embolism, acute ischemic cerebral apoplexy etc. are multiple.

Background technology

Tissue-type plasminogen activator (Tissue plasminogen activator, tPA) is a kind of of plasminogen activator, mainly by vascular endothelial cell, is synthesized and discharges, and it is a kind of serine protease, is mainly present in mammalian plasma.The efficient special fibrin in thrombosis of tPA is combined, the tPA-fibrin complex producing can activate the plasminogen in blood clot quickly and efficiently, produce fibrinolysin, make fibrin degradation become soluble product, the blood vessel that reaches thromboembolism and thromboembolism is unimpeded object again.TPA was put on market as the genetically engineered drug for the treatment of acute myocardial infarction by U.S. FDA approval in 1987, 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 at present apoplexy.

Ripe tPA molecule is that molecular weight 67～72KD, comprises 17 pairs of disulfide bond containing 527 amino acid whose strand glycoproteins.In molten fine process, tPA molecule is cut by protease in Arg275-Ile276 site, forms active higher duplex molecule, and 275 amino acid residues of N end form heavy chains, and 252 aminoacid of C end form light chains, between light, heavy chain, by a disulfide bond, is connected.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, containing cell-membrane receptor binding site ，Gai district mediation t-PA, is combined with hepatocyte and vascular endothelial cell, and there is to material impact the half-life; K1 district is positioned at 87-176 amino acids sequence, is receptor binding site or the structure relevant with the low affine combination of fibrin; K2 district is positioned at 176-262 amino acids sequence ，Qi Yu K1 district functional similarity, fibre-bearing protein binding site; Light chain, containing serine protease domain (serine protease ，Ser Pr district), forms active center by His322, Asp371 and Ser478, and 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 of being 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 state occurs.Just because of efficient, the special thrombolytic effect of tPA, be one of first batch of biological medicament utilizing in early days technique for gene engineering exploitation, be also the ideal medicament of up to the present treating embolism class diseases.But tPA also has certain limitation for molten fine treatment.It can form complex with inhibitors of plasminogen activator inhibitor (Plasminogen activator inhibitor, PAI) after entering blood on the one hand, causes it to lose activity rapidly; The tPA specific receptor being present on the other hand on liver plasma membrane can be combined with tPA fast, causes the half-life of tPA in blood very short, is only 4～6 minutes.Therefore, the therapeutic dose of tPA, up to 100～150mg, has so just increased the risk being caused bleeding by systemic fibrinolytic.

In recent years, research worker is started with from the structure of tPA, take structure activity relationship as principle, utilize DNA restructuring and protein engineering to build a series of tPA variants, as reteplase (reteplase), Monteplase (monteplase), lanoplase (1anoteplase), pamiteplase (pamiteplase) etc., they all have larger improvement at its Half-life in vivo of prolongation, increase and fibrinous binding affinity and raising to the aspects such as catalytic activity of plasminogen, than natural tPA, have more wide application prospect.Wherein, the tPA multipoint mutation body that TNK-tPA (TNKase) Shi You U.S. Genentech company develops, was used for the treatment of acute myocardial infarction in 2000 by U.S. FDA approval.This mutant combines to improve tPA and fibrin binding specificity and reduce is combined the KHRR296-299AAAA of sensitivity suddenly change (K) to PAI-1, reduce the Thr103Asn sudden change (T) of clearance rate, prolong half-life in body and Asn117G1n sudden change (N) (Keyt B A of shortage high mannose side chain, Proc Natl Acad Sci USA, 1994,91:3670-3674).TNK-tPA has many good characteristics as Increased Plasma Half-life (approximately 15～19 minutes), only needs single bullet formula intravenous administration, is combined increases by 14 times with fibrin-specific; Reduce by 80 times with PAI-1 specific binding, affect hardly body blood coagulation system, the effect of inherent hyperamization bolt be less than other plasminogen activators etc. (Cohen M, Am J Emerg Med, 2004,22:14-23).This determined its revascularization more rapidly, more lasting, thrombolytic effect more by force, more complete, more obvious to being rich in hematoblastic thrombosis effect.

It has been generally acknowledged that, extend the half-life of thrombolytic drug, for the patient who has bleeding tendency, may cause its bleeding risk to increase.Yet the safety of TNKase has obtained confirmation by ASSENT2 (new thrombolytics safety and efficiency assessment).This is an international research, 16919 routine patients, chest pain is occurring accepts in 6 hours the acceleration input of unitary agent TNKase or tPA, relatively 30 days mortality rates.Result of study shows, 30 days mortality rates similar (TNKase group and tPA group are 6.2%) of two groups of patients.The incidence rate of cerebral hemorrhage is similar (intracranial hemorrhage is 0.9%) also, apoplexy (tPA group 1.7%; TNKase group 1.8%).With patient's non-intracranial massive hemorrhage complication and the tPA of TNKase treatment, be respectively 4.7% to 5.9%.The half-life that this explanation TNKase extends does not increase patient's cerebral hemorrhage and apoplexy incidence rate.That the tPA variant of Increased Plasma Half-life is applied is 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 thromboembolism treatment time window is that cerebral ischemia starts the time limit of falling ill to begin treatment, surpasses this time limit, and the thrombolytic drugs such as tPA are not only invalid, can increase hemorrhage risk on the contrary.Surpass 3 hours (especially involve artery of cerebral hemorrhage---because artery of cerebral hemorrhage is branched ending, lack collateral circulation, cerebrovascular wall because of ischemia easily damaged) cerebral ischemia re-pouring after, the danger of cerebral hemorrhage increases greatly.Representative as second filial generation thrombolytic drug, in Cerebral Infarction 3 hours, with tPA, through intravenous thrombolytic, obtained international generally approval, and a nearest analysis confirms to occur in 4.5 hours using tPA to be still safety and effective at apoplexy onset symptoms, thereby make tPA thrombolytic time window extend to 4.5 hours from 3 hours, but the small sample RCT that also has more challenging message: TNKase to be used for Ischemic Stroke 6 hours windows studies show that with standard dose tPA (0.9mg/kg) and compares, the patient of TNKase treatment (0.1mg/kg or 0.25mg/kg) is filling rate higher (P=0.004) again, clinical improvements is (P<0.001) significantly, the effective in cure terminal index of heavy dose of TNKase group is all better than low dosage TNKase group and tPA group, and do not increase the incidence rate of symptomatic intracranial hemorrhage and other adverse events.If the curative effect of TNKase is further confirmed, thereby by probably replacing tPA, the time window for the treatment of of acute stroke is further extended to 6 hours in the RCT of large sample research.

In addition, other thrombolytic drugs are with respect 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 extracting from vampire saliva has the half-life that reaches approximately 4 hours, and a series of research of Germany scientist is expected to make its thrombolytic time window broadening to 9 hour.The medicine of long half time, after single administration, drug level can be brought into play curative effect maintaining for a long time within the scope of valid density.Can know by inference from existing experience, the Increased Plasma Half-life of thrombolytic drug, its corresponding therapeutic time window is corresponding widening also.Yet in the clinical position of ， China, only have a few patients to accept thromboembolism treatment in several hours in morbidity, even if developed country is also no more than 10% of patients with cerebral ischemic.Thereby development long half time, the wider novel thrombolytic drug of therapeutic time window are very necessary.

The immunoglobulin of IgG class is rich in protein in human blood.Their half-life can be up to 21 days, and Fc fragment is the main reason very long half-lift in IgG holder, also has the effect of stabilize proteins simultaneously.Lot of documents report after destination protein and Fc section are merged, do not affect its biologic activity and obtained the half-life of prolongation (referring to, as people such as Capon, Nature, 337:525-531,1989; The people such as Chamow, Trends Biotechno1., 14:52-60,1996; U.S. Patent No. 5,116,964 and 5,541,087).Yet, this is not natural result for tPA, Huang (the Military Medical Science Institute such as celebrate one's birthday, calendar year 2001, post-doctor's paper) tPA and IgG1Fc section are merged, expection raising tPA is the 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 affected tPA after Fc section and tPA merge correct folding, thereby it is lost activity.TPA is a serine protease that has 17 pairs of disulfide bond, five Structure and function domains, and the serine protease domain (Ser Pr) that is positioned at light chain is connected by a pair of disulfide bond with 4 domains of heavy chain.Serine protease domain (Ser Pr) 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 subject to the impact in F1 domain and C-end K2 district, and these five domains are to be mutually related for biological activity and the specific binding of tPA as seen.Early stage result of study confirms, can the space structure of tPA comparatively complexity be also more fragile, after protein translation, correctly fold, and forming activated space conformation is very crucial for the enzymatic activity of tPA.The ser structure territory of the C-end of tPA and other domain RuF1He K2 district relevant to its activity keep enzymatic activity all to hold the balance to it.In other words, aminoacid and its function of the C of tPA end are closely related, therefore tPA is carried out to conventionally avoid C when genetic engineering modified, hold, and also the C-end of tPA and other albumen should not be merged.Thereby, tPA is as a kind of serine protease, the catalytic active center that is positioned at C-end is necessary to maintaining its biological activity, therefore when building the Fc fusion rotein of TNK-tPA, must consider in advance how to overcome the consequence that the space steric effect bringing because of C-end connection Fc section causes its loss of activity.

Because three aminoacid that are positioned at active center on primary structure are relatively near the C-end, particularly the serine site of 478 of total length TNKase sequence, this make TNK-tPA Fc fusion rotein be built with its difficulty in essence.Up to now still not about the research report of TNK-tPA fusion rotein, and the Fc fusant of other protease also not yet has and is approved for clinical precedent.Current two the fastest protease Fc fusion rotein of progress, proconvertin and factor Ⅸ are yet in the clinical research stage.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 further to extend half-life in vivo of tPA variant and may obtain the therapeutic time window of widening.

Although as far back as 20th century the mid-80, gene engineering method exploitation recombinant natural tPA for China Jiu You research institution, does not have the recombinant natural tPA of animal cell expression production or the domestic marketing drugs of TNK-tPA 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 adopt the second filial generation tPA series products of escherichia coli expression, i.e. reteplase, " Easthome is vertical " being produced by Shandong A Hua and Beijing like that " Pai Tongxin " of the exploitation of moral Pharmaceutical got permission to go on the market respectively at 2007 and 2010.Yet the price of tPA series products is high, according to disclosed drug price, known alteplase and reteplase medical expense is once about 2750 dollars, and 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 complicated (containing the glycosylation molecule of 17 pairs of disulfide bond), and vivoexpression difficulty, expression productive rate is low; The 2nd, 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 and use the Thrombolytic Drugs of most convenient up to now, and have broad application prospects, thereby become the focus that various countries' pharmacy corporation is fallen over each other research and development.TNKase is developed by U.S. Genentech company the earliest, i.e. tenecteplase.The TNK-tPA of the U.S. is both at China's patent protection, and product also fails to enter Chinese market.Only there is at home Guangzhou inscription Kanggong department developing third generation tPA series products TNK-tPA, it is reported and complete clinical research.Guangzhou inscription Kanggong department adopts mammalian cell CHO to express TNK-tPA, continuously perfused culture technique, and culture density reaches 2 * 10 ⁷/ ml, output is about 50-150mg/L.With regard to production technology, the batch culture that the continuously perfused culture technology of Guangzhou inscription Kanggong department employing adopts compared with Genentech has unrivaled advantage.With additive method, compare, 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.Yet continuous perfusion culture need constantly add fresh culture medium in results culture fluid, cost is very high, and makes downstream protein purification work increase a lot of difficulties.At present, published tPA recombination classes product is all to adopt continuous perfusion culture technology, mainly comes from tPA albumen self stability poor, and 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 adopting increases production cost, 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 extending.Restructuring TNK-tPA-L-Fc fusion rotein refers to the Fc fusion rotein of tissue-type plasminogen activator mutant TNK-tPA, wherein, between TNK-tPA and Fc, by flexible joint, is connected.

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

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

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 is as shown in SEQ ID NO:7 (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer).

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 SEQ ID NO:2,4 or 6.

Further, the aminoacid sequence of TNK-tPA-L-Fc fusion rotein is the aminoacid sequence shown in the SEQ ID NO:2,4 or 6 having removed after the TNK-tPA leader peptide of 1 to 35 amino acids residue.

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

According to a further aspect of the invention, provide a kind of DNA sequence, this DNA sequence has the DNA sequence shown in SEQ ID NO:1,3 or 5.

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

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

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

According to a further aspect of the invention, provide the application of a kind of above-mentioned restructuring TNK-tPA-L-Fc fusion rotein in the medicine for the preparation of the prevention disease relevant to needing the quick dissolving of thrombosis with treatment.

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

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

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

Below specifically introduce content of the present invention:

Fusion rotein and preparation method thereof

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

(i) human IgG2 hinge region, CH2 and the CH3 region of containing Pro331Ser sudden change;

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

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

Wherein, peptide linker, preferably, use an about 2-20 amino acid length, contain the flexible peptide linker that following 2 kinds or several amino acids form: glycine, serine, alanine and threonine, if disclosed preferred sequence in the embodiment of the present invention is SEQ ID NO.:7 (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer).

The aminoacid sequence of TNK-tPA-L-Fc fusion rotein of the present invention is as shown in SEQ ID NO:2,4 or 6, its maturation protein is for to have removed TNK-tPA leader peptide (1 to the 35 amino acids residue) aminoacid sequence shown in SEQ ID NO: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 of being determined by EU number system) contain amino acid mutation, thereby 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 rich in protein in human blood.Their half-life can be up to 21 days, its high stability in blood plasma mainly have benefited from its up to the molecular weight of 150kD and with the combination of its receptor FcRn, be combined with FcRn and can avoid antibody enter lysosome and be degraded.Albumen and antibody Fc are merged to the structure that obtains antibody-like, not only can extend the Half-life in vivo of protein drug, but also become bivalent molecule by monovalent molecule, improved the adhesion of itself and target protein.FcRn has activity in the epithelial tissue of growing up, and expresses in the epithelial cell of enteric cavity, lung qi pipe, nasal cavity, vagina, coton and rectal.The transcytosis that the fusion rotein being comprised of IgG or Fc section can mediate by FcRn, epithelium barrier effectively shuttles back and forth.In addition, the fusion rotein that comprises Fc section is expressed cell endocytic and the protection of FcRn.These fusion rotein are not marked as degraded, but again enter blood circulation, thereby have increased the Half-life in vivo of these albumen.This method has been used to some very important cytokine (as IL-2 and IFN-α 2a) and soluble recepters (as TNF-Rc and VEGF-Rc) clinically, 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).Medicine Enbrel by the development of American I mmunex company is exactly recombinant human P75 Tumor Necrosis Factor Receptors and the human IgG l Fc fusion rotein dimer with expressing cho cell, this medicine was in application listing in 1998, in 1999, by FDA approval, for alleviating severe activeness, tired out the treatment of rheumatic arthritis patient symptom, and by FDA approval, be used for alleviating psoriasis arthropathica patient's symptom in 2002.

Peptide linker

The length of connection peptides is extremely important to the activity of fusion rotein.Existing people has reported 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, JBiol Chem, 1998,273:11173-6).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 (SytkowskiAJ etc., JBiol Chem, 1999,274:24773-8; U.S. Patent No. 6,187,564).This possible explanation is the connection peptides that fusion rotein two parts are asked increase, make two parts of this molecule can exercise respectively its function (Ashkenazi A etc., Curr Opin in Immunol, 1997,9:195-200).

The present invention is through long-term and deep research, design first a kind of original hinge region peptide linker and reduced space steric effect, the C that can make TNK-tPA holds the fusion rotein being connected with Fc, there is soft peptide linker centre, if disclosed preferred sequence 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 on the contrary, even improve the biological activity of TNK-tPA-L-Fc fusion rotein.

In addition, the present invention also finds, the peptide linker adding 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, thereby reduce space steric effect.

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

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

(b) the derivative cell line of this CHO of feeding culture, thus restructuring TNK-tPA-L-Fc fusion rotein expressed; With

(c) the restructuring TNK-tPA-L-Fc fusion rotein that purification step (b) is expressed.

The DNA of described coding restructuring TNK-tPA-L-Fc fusion rotein has the nucleotide sequence shown in SEQ ID NO:1,3 or 5.

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

In step (b), restructuring TNK-tPA-L-vFc fusion rotein in every 24 hours, is being expressed over 20 (being preferably 30) μ g/10 in its growth medium ⁶under the condition of (1,000,000) individual cell, cultivate the derivative cell line of CHO of transfection.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 derivative cell strain of high yield CHO is cultivated 13 days in 100mL shaking flask, and the recombination fusion protein cumulative production of its expression is 1.90g/L (Fig. 6).Between the 6th day to the 10th day of cell culture, living cells number is about at most 22 * 10 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 form 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, thereby host cell can adopt feeding culture technique, compared with perfusion, cultivate the consumption that has greatly reduced culture medium, and reduced culture fluid results volume, purification workload greatly reduces, and has significantly reduced material cost and human cost.

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

4. restructuring TNK-tPA-L-vFc fusion rotein has the In vitro biological activity higher with Recomposed tPA (mol ratio is active).

5. restructuring TNK-tPA-L-vFc fusion rotein has the serum half-life greatly extending and does not increase patient's bleeding risk, realizes the required dosage of similar drug effect, and can make to widen for the thromboembolism treatment time window of acute stroke patients thereby reduced.

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 of the present invention TNK-tPA-L-vFc fusion rotein is usually applied to the spontaneous of the congenital or acquired deficiency disease patient's of TNK-tPA 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.

Further, the invention provides a kind of pharmaceutical composition, 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 excipient 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, patient's body weight, patient's immune state, the approach of administration etc.

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.As space is limited, at this, tire out and state no longer one by one.

Coating medicine

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

Accompanying drawing explanation

Fig. 1 has shown 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 consists 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 has shown 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 consists 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 has shown 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 consists 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 has shown according to the gene mapping of the eukaryon expression plasmid of the constructed TNK-tPA-L-vFc antigen-4 fusion protein gene of the embodiment of the present invention.This expression plasmid total length 9997bp, contains 10 major gene segments, comprises 1.hCMV promoter; 2. target gene TNK-tPA-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. 5 has shown according to the concentration trend curve figure of 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 has shown according to the external activity of the TNK-tPA-L-vFc purifying protein of the embodiment of the present invention.

The specific embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

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 through chemosynthesis way, obtains.For the ease of by the specific site of object fragment inserting expressioning carrier, in the fragment 5 of synthesized, and 3 ' end respectively have a Restriction Enzyme inscribe site, be respectively SpeI and BamHI.The DNA fragmentation of total length 1705bp inserts transfer vector as between the EcoRV restriction enzyme site in pUC57, has obtained middle interstitial granules, and its contained 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 sudden change) is also the Chinese hamster ovary celI preference codon that artificial optimization crosses, and the fragment 5 ' of synthesized and 3 ' end respectively have a Restriction Enzyme inscribe site, are respectively BamHI and EcoRI.By DNA sequencing checking L-vFc _γthree kinds of gene orders.The DNA fragmentation of acquisition, through being inserted into BamHI and the EcoRI site of mammalian cell expression vector PCDNA3 (Invitrogen), is obtained to tri-plasmids of PCDNA3-L-vFC γ.Again by the TNK-tPA fragment obtaining after NheI/BamHI double digestion, be inserted between the corresponding restriction enzyme site of plasmid PCDNA3-L-vFc γ, obtained three fusion gene expression plasmid pCDNA3-TNK-tPA-L-vFc _γ, this plasmid is containing cytomegalovirus early promoter, and it is the required enhancer of mammalian cell high level expression exogenous gene.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 expression deficiency, the dihydrofolate reductase that PCDNA3 expression vector contains mice (DHFR) gene, thus can coamplification TNK-tPA-L-vFc while there is methotrexate (MTX) _γfusion gene and DHFR gene (United States Patent (USP) 4,399,216).

Between people TNK-tPA and Fc part, exist flexible peptide linker increased the flexible of TNK-tPA region and improved its biological activity.For the purpose of the present invention, preferably length is approximately 20 or (but can not be less than 2) amino acid whose peptide linker still less.Should use contain or by 2 or more multiselect from the peptide linker of following Amino acid profile: glycine, serine, alanine and threonine.The example of peptide linker contains a Gly-Ser peptide member, as GlyGlyGlyGlySer.Fig. 1, Fig. 2, Fig. 3 have shown respectively containing three kinds of Fc _γthe nucleotide sequence of variant fusion gene and the aminoacid sequence of derivation, they jointly contain encoding human TNK-tPA, 16-amino acid peptide joint (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) and contain separately 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-tPA vFc 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 are chosen TNK-tPA-L-vFc γ ₂for explaining, TNK-tPA-L-vFc γ ₁, TNK-tPA-L-vFc γ ₄with and to have mutant that thrombosis the dissolves function fast result in following experiment suitable, do not repeat them here.

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

The expression vector plasmid transfection of restructuring is entered to mammalian host cell line, to express TNK-tPA-L-vFc _γfusion rotein.In order to stablize high-caliber expression, preferred host cell is to be DHFR deficient CHO-cell (U.S. Patent No. 4,818,679).Preferred transfection method is an electroporation, also can use other method, comprises calcium phosphate cosedimentation, fat transfection and Protoplast fusion.In electroporation, with the Gene Pulser Electroporator (Bio-Rad Laboratories, Hercules, CA) that is set to 210V electric field and 1050 μ Fd electric capacity, 2～3 * 1O in cuvette ⁷in individual cell, add the plasmid after PvuI linearisation for 20 μ g.In transfection two days later, culture medium is made into the growth medium containing 0.6mg/mL G418.By the elisa assay method of anti-human IgG Fc, screening is to selecting medication to have the transfectant of resistance.Also the ELISA of available anti-human tPA carries out the quantitative of fusion protein expression.By Method of Limited Dilution 96 well culture plates, sub-clone produces the hole of high-level Fc fusion rotein.

In order to realize the expression of fusion rotein higher level, should carry out coamplification with the DHFR gene that suppressed by MTX medicine.In the growth medium that contains progressive concentration MTX, with the antigen-4 fusion protein gene of DHFR gene coamplification transfection.The transfectant that can grow in up to 5 μ g/mL MTX culture medium with the sub-clone of Method of Limited Dilution.Measuring secretion rate further analyzes the cell line of sub-clone.Secretion rate horizontal exceeding approximately 20 (preferably approximately 30) μ g/10 ⁶the cell line of (1,000,000) individual cell/24 hour adapts to the suspension culture of using serum-free growth medium.Then use conditioned medium purified fusion protein.

The production of embodiment 3. fusion rotein

First the high yield cell strain that embodiment 2 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 300m1 shaking flask, carry out feed supplement and add cultivation or simulate perfusion cultures by changing the way of culture medium every day.The derivative cell strain feed supplement in the shaking flask of 100ml volume of above-mentioned CHO is cultivated 13 days, and the recombination fusion protein cumulative production of its expression 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 can select 2000ml 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 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 1N NaOH, the conditioned medium of the fusion rotein that contains embodiment 3 is titrated to pH7～8, then with the celluloid filter of 0.22 micron, filters.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 Protein A, discard the component of outflow.With PBS, wash this post, until the OD value at 280nm place is lower than 0.01.Then use 0.1M glycyltryosin, 2M NaCl, 0.04%Tween20, the fusion rotein of pH4.58 buffer solution elution combination, merges the component that contains purifying protein, and dialyses with PBS.Then with the celluloid filter of 0.22 micron, filter, and be stored in-70 ℃.Under reducing condition, strand tPA albumen (alteplase for injection, the Boehringer Ingelheim is produced) molecular weight that is recorded purification by SDS-PAGE is about 65kDa.The TNK-tPA-L-vFc albumen of purification migrates to about 90kDa.With BSA as standard, by quantitative this fusion rotein of BCA protein analysis.

The direct chromophoric substrate method of embodiment 5. is measured TNK-tPA-L-vFc fusion rotein external activity

Directly chromophoric substrate method adopts the tPA determination of activity test kit of DiaPharma company, and the method is measured the activity of purification tPA with chromogen substrate S-2288.Detecting principle is to be 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.Thereby, can calculate by measuring the burst size of paranitroanilinum (Para-Nitroaniline, pNA) activity of tPA.Spectrophotography is measured the variation of OD value under 405nm wavelength, reflects the number of the amount that generates pNA.Fig. 6 has shown the Recomposed tPA albumen (alteplase for injection, Boehringer Ingelheim is produced) of purification or the external activity corresponding relation of the restructuring TNK-tPA-L-vFc albumen of purification under identical molar concentration (nM).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 be measured its In vitro biological activity with said method.

The pharmacokinetics of embodiment 6. fusion rotein is measured

SD rat Three doses (6,0.9,0.18mg/Kg) tail vein injection TNK-tPA-L-vFc sample, chooses 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.While measuring with ELISA, the monoclonal antibody that carries out the mouse-anti buman tPA of embedding, horseradish peroxidase-labeled with the multi-resistance of goat-anti people's Fc detects.In embodiment 4, purification TNK-tPA-L-vFc sample is distinguished 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 the similar dosage plasma half-life of restructuring TNK-tPA medicine is about 19 minutes, so the TNK-tPA-L-vFc Half-life in vivo of recombinating in the present invention significantly extends.

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

1. a pharmaceutical composition, it is characterized in that, comprise pharmaceutically acceptable carrier, excipient or diluent, and the restructuring TNK-tPA-L-Fc fusion rotein of effective dose, the TNK-tPA-L-Fc fusion rotein of wherein recombinating is held the IgG Fc that contains successively TNK-tPA, flexible peptide linker and people to C end from N, described human IgG Fc comprises natural human IgG Fc and human IgG Fc variant.

2. pharmaceutical composition according to claim 1, is characterized in that, described human IgG Fc variant is selected from following group:

3. pharmaceutical composition according to claim 1, is characterized in that, described flexible peptide linker contains 2-20 aminoacid, and described flexible peptide linker contains the aminoacid that two or more are selected from glycine, serine, alanine and threonine.

4. pharmaceutical composition according to claim 3, is characterized in that, the amino acid residue sequence of described flexible peptide linker is as shown in SEQ ID NO:7.

5. pharmaceutical composition according to claim 1, is characterized in that, described TNK-tPA is natural tPA or the tPA variant with identity function.

6. pharmaceutical composition according to claim 1, is characterized in that, the aminoacid sequence of described TNK-tPA-L-Fc fusion rotein is as shown in SEQ ID NO:2,4 or 6.

7. pharmaceutical composition according to claim 1, it is characterized in that, the aminoacid sequence of described TNK-tPA-L-Fc fusion rotein is the aminoacid sequence shown in the SEQ ID NO:2,4 or 6 having removed after the TNK-tPA leader peptide of 1 to 35 amino acids residue.

8. pharmaceutical composition according to claim 1, the DNA sequence of the restructuring TNK-tPA-L-Fc fusion rotein that coding is described has the DNA sequence shown in SEQ ID NO:1,3 or 5.