CN103172745A - Long-acting human endothelium chalone containing immune globulin Fc segment - Google Patents
Long-acting human endothelium chalone containing immune globulin Fc segment Download PDFInfo
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- CN103172745A CN103172745A CN2011104332267A CN201110433226A CN103172745A CN 103172745 A CN103172745 A CN 103172745A CN 2011104332267 A CN2011104332267 A CN 2011104332267A CN 201110433226 A CN201110433226 A CN 201110433226A CN 103172745 A CN103172745 A CN 103172745A
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Abstract
The invention relates to a long-acting human endothelium chalone. The long-acting human endothelium chalone is a human endothelium chalone molecule covalently connected with an immune globulin Fc segment by polyethylene glycol. The invention also discloses a preparation and purification method of the long-acting human endothelium chalone and an application of the long-acting human endothelium chalone in preparation of a drug used for treating cancer.
Description
Technical field
The invention belongs to biological products pharmaceutical technology field, relate to a kind of preparation method of long-acting recombinant human Endostatin.
Background technology
From Folkman, " generation that the growth of tumour and transfer all depend on new vessel " [Folkman J.N Engl J Med 1971 was proposed in 1971; 285:1182-1186] viewpoint since, people are just suppressing tumor-blood-vessel growth as a kind of research object.Endostatin research (Endostatin) is at first found in 1997 by people such as U.S. O ' Reilly.The people such as O ' Reilly find that in test the cell culture fluid of the mouse hemangioendothelioma (EOMA) of vitro culture can suppress external bovine capillary endothelial cell propagation, their a kind of new protein of having purified out from this nutrient solution, and called after Endostatin (Endostatin, ES).Show that through evaluation the mouse Endostatin consists of [O ' Reily M S.et al.US 005854205A] by 184 amino acid fragments in collagen XV III C-terminal district.China's Xu's root is emerging waits the people also to find people's endostatin research in then in the work of research Endostatin, and screening and cloning obtains human vas endostatin gene [Xu Genxing etc., CN1177005A] from people's Liver cDNA Library.The direct effect of Endostatin is to play antitumor action by suppressing angiogenic growth, and can not cause resistance to produce.
RhEndostatin (Endostar) is the Research of Recombinant Human Endostatin (Recombinant Human Endostatin, rhES) of China's independent research, in listing in 2005, is used for the treatment of nonsmall-cell lung cancer.It is compared with natural endostatin research, has added 9 aminoacid sequences at the N end, has improved biological activity and stability.RhEndostatin and chemotherapeutics Vinorelbine and cis-platinum (NP chemotherapy regimen) coupling has certain synergy, can delay patient's tumour progression.But Endostatin is as small molecular protein, and character is very unstable, is easy to occur enzymatic degradation and is eliminated, so the transformation period in vivo is shorter.And rhEndostatin clinical application amount is very large at present, and when associating NP chemotherapy regimen was used for the treatment of the III/IV phase Patients with Non-small-cell Lung of just controlling or controlling again, injected dose was 7.5mg/m
2, intravenous drip every day 3-4 hour, successive administration 14 days was a cycle.Therefore, improve the Half-life in vivo of rhES, thereby reduce therapeutic dose, reduce the patient suffering and become a urgent problem to improve patient compliance.
For stabilizing protein, prevention enzymatic degradation and being removed by kidney, can use the polymkeric substance that has a high resolution such as polyoxyethylene glycol (polyethylene glycol, PEG) etc. to come the surface of chemically modified protein matter medicine.The PEG molecule has amphipathic, both can be water-soluble, can be dissolved in most organic solvent again, and nontoxic, non-immunogenicity is one of polymer that can be used for the medicine preparation.By with specific region or the various regional combination of target protein, PEG can improve plasma half-life, strengthens bioavailability, lowers the protein immunogenicity, improves curative effect of medication and security etc.In addition, can be by preparing diploid at the same pharmaceutical grade protein of PEG two ends connection, to improve the activity of protein drug.Also two kinds of different pharmaceutical grade proteins can be connected in the two ends of PEG, the pharmaceutical composition that obtains having two kinds of activity.
The Half-life in vivo that improves protein drug can also be by use gene recombination technology, the gene of the pharmaceutical protein protein gene with the high serum stability of tool of encoding merged mutually, thus production fusion rotein medicine.For example with pharmaceutical grade protein and albumin or its segment composition, perhaps merge with immunoglobulin Fc segments.Studies show that, medicine and immunoglobulin Fc segments merge, and can obviously improve the Half-life in vivo of medicine.
But, although the stability of PEG energy Enhancin matter, also there are some problems in the PEG coupling, as reducing biological activity of albumen.In addition, along with the raising of PEG molecular weight, its output also can decrease etc.Produce fusion rotein by gene recombination and also have some shortcomings.For example, its active of fusion rotein that causes producing significantly reduces.Protein is that conformation by protein determines as the activity of physiologic function material.When polypeptide drugs merge by recombination method and immunoglobulin Fc segments, utilize prokaryotic cell prokaryocyte to express, fusion rotein usually makes a mistake folding and with the formal representation of inclusion body; And utilize eukaryotic cell expression, immunoglobulin Fc segments usually to understand by glycosylation, thereby may cause the undesirable immune response in body.The connector area that merges also may be to Degradation sensitivity of proteolytic ferment etc.
Korea S S. Korea and the USA medicine company limited has developed the technology platform (WO 2005/047337) of a kind of New-type long-acting recombinant protein or polypeptide drugs, wherein disclose a kind of immunoglobulin Fc segments that comprises and to have extended the transformation period of albumen or polypeptide drugs as the pharmaceutical composition of carrier, and improved the interior bioavailability of body of medicine.Yet, such as in WO 2005/047337 record, its gained recombinant protein or polypeptide drugs at most only keep approximately 50% cell in vitro and learn active.
This area also need to keep its biological activity in the stability that improves human endostatin and Half-life in vivo, thereby improves its bioavailability, reduces therapeutic dose, and improves the long-acting rhES preparation of patient compliance.
Summary of the invention
The purpose of this invention is to provide a kind of long-acting endostatin research preparation, thereby strengthen endostatin research stability in vivo, improve its effective blood drug concentration and bioavailability, and prolong half-life.The present invention also provides the method for the long-acting endostatin research preparation of preparation, separation and purifying, and described long-acting endostatin research preparation for the preparation of the treatment cancer medicine in application.
First aspect the invention provides a kind of long-acting endostatin research preparation, and described long-acting recombinant human Endostatin is by polyoxyethylene glycol and the covalently bound human endostatin molecule of immunoglobulin Fc segments.
Long-acting rhES preparation in the present invention with immunoglobulin Fc segments as carrier.Immunoglobulin Fc segments is in vivo can metabolic biodegradable polypeptide, therefore, is safe with it as pharmaceutical carrier.
Described " immunoglobulin Fc segments " has the implication that those skilled in the art understand usually herein.Particularly, described " immunoglobulin Fc segments " refers to comprise immunoglobulin heavy chain constant region 2 (C
H2) and CH3 (C
H3) and not contain the protein of heavy chain immunoglobulin and variable region of light chain.Immunoglobulin Fc segments can further comprise the hinge area that is positioned at CH.In addition, immunoglobulin Fc segments of the present invention also can comprise CH1 (C
H1) and/or constant region of light chain 1 (C
L1) part or all is as long as it has or better physiologic function substantially similar to native protein.Be not included in the Fab fragment that has the height non-homology between antibody subtype due to immunoglobulin Fc segments, so its antigenicity reduces greatly.
Immunoglobulin Fc segments of the present invention can from the mankind or other animal, comprise ox, goat, pig, mouse, rabbit, hamster, rat and cavy, preferably from the mankind.
Immunoglobulin Fc segments of the present invention can be the natural form or derivatives thereof from the mankind or the separation of other animal.
In this article, can pass through to separate complete immunoglobulin (Ig) and process with proteolytic ferment from human or animal body, thereby obtaining the Fc fragment from native immunoglobulin.For example, can use papoid to process the native immunoglobulin that separates, thereby obtain Fab fragment and Fc fragment, and these fragments are carried out purifying, thereby isolate the Fc fragment.
In addition, if necessary, also can carry out derivatize to the Fc fragment, thereby obtain the Fc derivative.Described " Fc fragment derivative " refers to have the performance identical or higher with Fc fragment of the present invention herein, such as the derivative of structural stability (comprise thermotolerance, pH stability) etc.The method that is obtained Fc fragment derivative by the Fc fragment is known in the art.For example can pass through chemical mode, as phosphorylation, methylate, acetylize etc., perhaps biological mode, modes such as sudden change or restructuring obtains the derivative of Fc fragment.Within the Fc fragment derivative that is obtained by the Fc fragment is also contained in scope of the present invention.
Immunoglobulin Fc segments of the present invention can also be by the cell that transforms, and for example zooblast or microorganism cells are expressed and obtained.For example, immunoglobulin Fc segments of the present invention can be the recombination human source immunoglobulin Fc segments that is obtained from microorganism.
Immunoglobulin Fc segments of the present invention can be the form that has natural sugar chain, compares the sugar chain increase with natural form or reduce, and can be perhaps deglycosylated form.The increase of immunoglobulin Fc sugar chain, minimizing or remove and can complete by the ordinary method of this area such as chemical method, enzymatic method with utilize the genetic engineering method etc. of microorganism, but are not limited to this.The glycosyl of removing immunoglobulin Fc segments can cause it and the C1q binding affinity partly of the first complement component C1 obviously to reduce, the cytotoxicity (ADCC) of antibody dependent cellular mediation or reduction or the forfeiture of CDC (CDC), thus can not cause in vivo unnecessary immune response.Therefore, preferably use in the present invention de-glycosylation or nonglycosylated immunoglobulin Fc segments.
Term herein " de-glycosylation " or " non-glycosylated " refer to remove glycosyl part or the immunoglobulin Fc segments not have glycosylated form to produce from immunoglobulin Fc segments.
In addition, immunoglobulin Fc segments can be the Fc fragment from IgG, IgA, IgD, IgE or IgM, and perhaps combination or the heterozygosis by them prepares the Fc fragment of coming.Preferred Fc fragment is from IgG or IgM, and they are one of richs in protein in human blood.Consider that IgG can extend the transformation period of ligand binding protein, most preferably from the Fc fragment of IgG.
Term herein " combination " dimer or the polymer that the single chain polypeptide of the polypeptide of strand immunoglobulin Fc segments in same source and different sources is connected to form that refer to encode.For example by the formed immunoglobulin Fc segments of two or more fragments in IgG1Fc, IgG2Fc, IgG3Fc and IgG4Fc fragment.
Term herein " heterozygosis " refer to the to encode sequence of two or more immunoglobulin Fc segments of different sources is present in the strand immunoglobulin Fc segments.Various types of heterozygotes are included within the scope of the present invention.In one embodiment, immunoglobulin Fc segments of the present invention can comprise and is selected from C
H1, C
H2, C
H3, C
H1-4 in 4 a structural domains structural domain.For example, described immunoglobulin Fc segments can comprise the C of immunoglobulin Fc segments
H1, C
H2, C
H3, and C
HA kind of or four kinds of structural domains in 4.In addition, described immunoglobulin Fc segments can also comprise hinge area.
On the other hand, IgG is divided into IgG1, IgG2, IgG3 and IgG4 hypotype.Therefore, immunoglobulin Fc segments of the present invention can be selected from the Fc fragment of IgG1, IgG2, IgG3, IgG4 and the group of combination and heterozygote composition thereof.Wherein, preferred IgG2 and IgG4 hypotype, most preferably the Fc fragment of IgG4 in the present invention.It has hardly such as effector functions such as CDC.
That is, as pharmaceutical carrier of the present invention, most preferred immunoglobulin Fc segments is the non-glycosylated Fc fragment in human IgG 4 sources.
Human endostatin molecule described in the present invention refers to by the collagen XV IIIC terminal region fragments of 183 Amino acid profiles (aminoacid sequence is as shown in Fig. 1 SEQ ID NO:1), based on recombinant protein or the bioactive fragment of this fragment.
Recombinant protein based on described human endostatin molecule is well known in the art, and the human endostatin molecule that for example uses in the present invention can be the recombinant human endostatin molecule that contains His-tag.In the present invention, the preferred human endostatin molecule that uses can have the recombinant human endostatin (rhES) of aminoacid sequence (SEQ ID NO:2) as shown in Figure 2.Described rhES can be by escherichia coli expression, and in this case, the Met of its N-terminal is optionally deleted.
With regard to human endostatin, " bioactive fragment " of the present invention refers to any protein fragments of being derived by human endostatin molecule shown in SEQ ID NO:1, prerequisite is that this fragment has kept human endostatin molecule shown in SEQ IDNO:1 more than 70%, the activity of preferred inhibition angiogenic growth more than 90%.
PEG described in the present invention refers to polyoxyethylene glycol, for example the polyoxyethylene glycol of molecular weight 2-5kDa.The preferred 3.4kDa PEG (ALD-PEG-ALD) that uses two ends to have the aldehyde radical functional group connects, with Fc fragment and human endostatin combination.More preferably, a rhES molecule is connected by a part PEG with a Fc fragment, and the connection site of both and PEG is the N end.
Second aspect the invention provides preparation and the purification process of described long-acting human endostatin preparation, said method comprising the steps of:
At first, by covalent attachment and the chromatography purification of PEG and human endostatin N end α amino, the human endostatin (rhES-PEG) of the single PEGization of preparation.
For the preparation of rhES-PEG, can in reaction buffer, human endostatin be mixed with PEG, and the reductive agent sodium cyanoborohydride reacts in the reaction mixture.After finishing, reaction with the product dilution, obtains the rhES of N-terminal list PEGization through chromatography purification.
Particularly, can be under the sodium-acetate reaction buffer of certain pH value (as pH3.5, pH5.5, pH6.5), rhES and PEG are mixed by certain molar ratio (as 1: 5,1: 10,1: 15); And add the reductive agent sodium cyanoborohydride of final concentration 20mM in the reaction mixture; Different durations react, as 1h, and 2h, 4h, 20h.Preferably, making mol ratio is rhES and the PEG of 1: 15, at pH5.5, reacts 1h under the condition of 4 ℃.Reaction is diluted product after finishing with sodium-acetate buffer, obtain the rhES of N-terminal list PEGization through Source S chromatography purification.
Secondly, by with the PEG covalent attachment, with the further coupling of the human endostatin of Fc fragment and PEGization, then pass through chromatography purification, prepare described long-acting human endostatin.
After obtaining rhES-PEG, it can be mixed in damping fluid with the Fc fragment, add the reductive agent sodium cyanoborohydride to react.After finishing, reaction with the product dilution, obtains the rhES and the Fc fragment that connect through PEG through chromatography purification.
Particularly, at pH3.5, in the sodium-acetate buffer of pH5.5 or pH6.5, by 1: 5, the molar ratio of 1: 10 or 1: 15 mixed, and adds the reductive agent sodium cyanoborohydride of final concentration 20mM with rhES-PEG and Fc fragment, and under mild stirring 4 ℃ the reaction 8h, 16h or 24h.Preferably, making mol ratio is rhES-PEG and the Fc fragment of 1: 15, at pH5.5, reacts 16h under the condition of 4 ℃.Reaction is diluted product after finishing with sodium-acetate buffer, obtain the rhES and the Fc fragment that connect through PEG through Source S chromatography purification.
Through SDS-PAGE and HPLC, product is carried out Analysis and Identification, this polymkeric substance purity is the rhES-PEG-Fc product more than 97%.Therefore, the present invention has successfully obtained containing immunoglobulin Fc segments as the recombinant human endostatin polymkeric substance of carrier.
The third aspect, the present invention also provide described long-acting human endostatin preparation for the preparation of the treatment cancer medicine in application.
A fourth aspect of the present invention relates to the method for using described long-acting human endostatin preparation for treating cancer.
The transformation period of human endostatin, the stability that has improved human endostatin and bioavailability have been extended by long-acting human endostatin preparation of the present invention, therefore, can be used for the various cancers of human endostatin treatment, include but not limited to mammary cancer, lung cancer, the rectum cancer, ovarian cancer, colorectal carcinoma, liver cancer, prostate cancer, cancer of the stomach, cervical cancer, carcinoma of the pancreas, the esophageal carcinoma, chorioepithelioma, malignant mole, bladder cancer, skin carcinoma, incidence cancer, lung bronchogenic carcinoma, large bowel cancer and leukemia.Preferably, described cancer is lung cancer, most preferably nonsmall-cell lung cancer.
Pharmaceutical composition of the present invention can be become various formulations with the pharmaceutically acceptable carrier formulated in combination, comprise the various formulations that are suitable for gi tract or parenteral administration.
" pharmaceutically acceptable carrier " that the present invention uses refers to not disturb the physiological action of described long-acting human endostatin, and the experimenter who comprises the mankind is not had virose any material.The present invention's pharmaceutically acceptable carrier used is carrier well known by persons skilled in the art.Those skilled in the art can select the carrier that is fit to according to actual needs, and prepare preparation of the present invention by methods known in the art.Described preparation includes but not limited to tablet, solution, outstanding agent, finish, emulsion, gel, aerosol, inhalation, spraying, capsule, pill, patch and suppository etc.
The dosage of long-acting human endostatin preparation of the present invention in described medicine can be at 1.5-37.5mg/m
2Scope.Because long-acting human endostatin preparation of the present invention has very long acting duration in vivo, so can greatly reduce the administration frequency of described medicine.Long-acting human endostatin preparation of the present invention can be administered once weekly, therefore, has improved patient's compliance.
Carried out in the body and the detection of external biologic activity to this product rhES-PEG-Fc.
Cell in vitro is learned the activity experiment result and is shown, the anti-endothelial cell migration activity of this polymkeric substance rhES-PEG-Fc is active close with the rhES's of unmodified.The connection of Fc fragment does not substantially affect the cell in vitro of rhES and learns active.
The pharmacokinetics experimental result shows, the Half-life in vivo of this polymkeric substance rhES-PEG-Fc is 62.4h, improved 26 times than the Half-life in vivo (2.4h) of the rhES of unmodified.The pharmacodynamics experimental result shows, rhES-PEG-Fc is administration 1.3mg/kg and 4mg/kg group weekly, has the anti-tumor activity similar to the rhES of administration every day 4mg/kg.
Above-mentioned advantage shows, rhES through PEG with after the Fc fragment is connected, transformation period significant prolongation, and kept anti-endothelial cell migration activity and anti-tumor activity, thus can reduce dosage, reduce administration frequency.
Description of drawings
Fig. 1: shown human endostatin aminoacid sequence SEQ ID NO:1.
Fig. 2: shown that the N end has added the aminoacid sequence SEQ ID NO:2 of 9 amino acid whose recombinant human endostatins.
Fig. 3: shown the SDS-PAGE evaluation figure of rhES-PEG-Fc after the purifying.In Fig. 3, swimming lane 1: non-reducing rhES-PEG; Swimming lane 2: non-reducing rhES-PEG-Fc; Swimming lane 3: non-reducing Fc fragment; Swimming lane 4: non-reducing rhES; Swimming lane 5: non-reducing Fc fragment; Swimming lane 6: the rhES-PEG of reduction; Swimming lane 7: the rhES-PEG-Fc of reduction; Swimming lane 8: the Fc fragment of reduction; Swimming lane 9: the rhES of reduction; Swimming lane 10: molecular weight of albumen standard
Fig. 4: shown the RP-HPLC purity detecting result of rhES-PEG-Fc after the purifying, wherein with 99.7% peak area normalizing.
Fig. 5: the interior pharmacokinetic curve of body that has shown rhES and rhES-PEG-Fc.
Fig. 6: the active detection of anti-tumor in vivo that has shown rhES and rhES-PEG-Fc.
Embodiment
The present invention is further illustrated by the following examples, but be not limiting the scope of the invention.
The preparation of embodiment 1.rhES-PEG
The 3.4kDa polyoxyethylene glycol (ALD-PEG-ALD) that two ends are had an aldehyde radical with recombinant human endostatin rhES (from Jiangsu first sign Mai Dejin Biology Pharmacy Co., Ltd, lot identification mark 20100305, sequence is as shown in Figure 2) be dissolved in the 2M sodium-acetate buffer with the molar ratios of 15: 1, the reductive agent sodium cyanoborohydride that adds final concentration 20mM in this mixture, and under mild stirring in 4 ℃ of reaction 1h, the N-terminal amino of PEG and rhES is connected.
Adopt Source S (available from GE Healthcare, Source 15S, article No. 17-0944-03) purification reaction end product.With above-mentioned reaction mixture 50mM, loading after 20 times of dilutions of 4-morpholino b acid (MES) damping fluid of pH6.4.Elution buffer is the 50mM MES damping fluid that contains 1M NaCl, adopts the gradient concentration wash-out.Gradient is transitioned into 45% for wash-out buffer concentration in 120min by 15%, and flow velocity is 3ml/min, and the concentration range that obtains product is 30%-40%.Use fraction collector collect sample and identify through SDS-PAGE.Result shows that prepared rhES-PEG is the rhES fragment of single PEGization.
The preparation of embodiment 2.rhES-PEG-Fc
Single PEGization rhES of above-described embodiment 1 purifying acquisition and the N-terminal of immunoglobulin Fc segments are done further coupling.
With the Fc fragment (by the non-glycosylated human IgG4Fc fragment that e. coli bl21 (DE3) is expressed, NCBI Gene ID:3503.Be obtained from the Beijing Hanmei Medicine Co., Ltd) be dissolved in 50mM with described rhES-PEG with the molar ratio of 15: 1, in the pH5.5 sodium-acetate buffer, the reductive agent sodium cyanoborohydride that adds final concentration 20mM in this mixture, and react 16h at 4 ℃ under mild stirring, make the N-terminal coupling of described rhES-PEG and Fc fragment.
Adopt Source S (available from GE Healthcare, Source 15S, article No. 17-0944-03) purification reaction end product.With above-mentioned reaction mixture 50mM, loading after 20 times of dilutions of the sodium-acetate buffer of pH5.5.Elution buffer is the 50mM sodium-acetate buffer that contains 1M NaCl.Adopt the gradient concentration wash-out.Gradient is transitioned into 50% for wash-out buffer concentration in 200min by 20%, and flow velocity is 3ml/min, and the concentration range that obtains product is 35%-45%.Use fraction collector to collect sample, obtain highly purified rhES-PEG-Fc protein composition thereby separate.
Products therefrom identifies with SDS-PAGE, and result as shown in Figure 3.The theoretical molecular of rhES-PEG-Fc is about 74kD.The molecular weight of Fc fragment is about 49kD (swimming lane 3 and swimming lane 5), is connected to form by disulfide linkage by two identical peptide chains.In described rhES-PEG-Fc reduced form electrophoresis, should show two bands after disulfide linkage in the Fc fragment is opened: one of them band is the strand Fc band that has connected rhES-PEG, molecular weight is about 49kD, and another is the approximately band of 25kD (referring to swimming lane 7) of strand Fc.Shown in Fig. 3 that described rhES-PEG-Fc shows as the approximately single band of 74kD (referring to swimming lane 2) of molecular weight in non-reduced type electrophoresis.Therefore, the present invention has successfully prepared rhES-PEG-Fc.
In addition, also gained rhES-PEG-Fc end product has been carried out the reversed-phase HPLC analysis, instrument is Agilent 1200 types, be equipped with C4 chromatographic column (Vydac 214TP), gradient is for to be transitioned into 95% water/5% acetonitrile by 5% water/95% acetonitrile in 50min, flow velocity is 1ml/min, and column temperature is made as 60 ℃.
Gained typical case HPLC spectrogram as shown in Figure 4, the main peak peak area accounts for more than 97%, shows that gained rhES-PEG-Fc has very high purity.
The mensuration of the In Vitro Anti migration of vascular endothelial cells activity of embodiment 3.rhES-PEG-Fc
Detected rhES-PEG-Fc of the present invention in the present embodiment to the anti-migratory activity of human microvascular endothelial cell (mvec) (HMVEC).
After HMVEC cell (available from ATCC, the article No. CRL-4025) hunger that will be in logarithmic phase is spent the night, inoculation 8 * 10
4Individual cell is in the culture dish Transwell (available from Corning company, article No. is 3422) that detects cell migration.The Transwell culture dish is a kind of cultivation cell with the permeability upholder, and the cell bottom is a film that permeability is arranged, i.e. permeability upholder.What adopt in the present embodiment is Transwell culture dish with the PC film of aperture 8.0 μ m.
Establish respectively negative control group (N.C. group, serum free medium DMEM), positive controls (P.C. group is added 2% foetal calf serum FBS and 10ng/ml vascular endothelial growth factor VEGF) and two groups of dosing groups (rhES-PEG-Fc that adds respectively rhES or prepare according to the embodiment of the present invention 2 on the basis of positive controls, adding consistency is 2.5,5,10,20,40,80,160 μ g/ml.The rhES-PEG-Fc group is calculated by the amount of its contained rhES).
The cell of each group is hatched 4h at 37 ℃.Subsequently, take out culture dish, with 4% polyoxymethylene fixed cell 15min, wipe the not migrating cell on Transwell film upper strata off, with Viola crystallina to lower floor's cell dyeing.Utilize at last microscope counting cells under the formed objects visual field, calculation of half inhibitory concentration ED
50
Calculation result shows, the ED of rhES
50Be 36.7 μ g/ml, the ED of rhES-PEG-Fc
50Be 37.3 μ g/ml.Therefore, it is active that rhES-PEG-Fc of the present invention has kept 98.4% the anti-endothelial cell migration of rhES.
The pharmacokinetic of embodiment 4.rhES-PEG-Fc
The present embodiment has detected the transformation period of rhES-PEG-Fc of the present invention in the rat body.
10 male SD rats (available from magnificent Fukang, Beijing biotech inc, in 6-8 week, 180-220g/ only) are divided into 2 groups at random, press 4.5mg/kg body weight subcutaneous administration.The A group is rhES, and the B group is the rhES-PEG-Fc according to the embodiment of the present invention 2 preparations.
5min, 15min, 0.5h, 1h, 1.5h, 2h, 3h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 192h, 288h get blood after administration, blood sample is with anticoagulant heparin, the centrifugal 15min of 3000rpm obtains plasma sample frozen in-80 ℃, and is to be measured.
According to the explanation of manufacturer, end user's Endostatin Quantikine enzyme linked immunological kit is (available from R﹠amp; DSystems, article No. DNST0) the rhES content by sample in ELISA sandwich method for determining blood plasma.The results are shown in Figure 5.The transformation period T of rhES
1/2Be 2.4h, area under the concentration-time curve AUC is 8.61 μ gh/ml, reaches the time T of peak concentration
maxBe 1.63h, peak concentration C
maxBe 2026.15ng/ml.Compare the Half-life in vivo T of rhES-PEG-Fc with rhES
1/2Be 62.4h, AUC is 894.05 μ gh/ml, T
maxBe 24.00h, C
maxBe 6644.33ng/ml.By above test-results as seen, compare with rhES, the Half-life in vivo of rhES-PEG-Fc of the present invention has improved 26 times.And according to the AUC data of measuring, the bioavailability of rhES-PEG-Fc of the present invention has improved more than 100 times than rhES.
The anti-tumor in vivo activity research of embodiment 5.rhES-PEG-Fc
Tested rhES-PEG-Fc of the present invention in the present embodiment to suppressing active in human lung cancer cell A549's body.
Experiment material is selected female BALB/c nude mice (available from Department Of Medicine, Peking University laboratory animal section, in 6-8 week, 18-25g/ only), inoculates respectively 2 * 10
6Individual human lung cancer cell A549 (available from ATCC, article No. CCL-185) is in thoracic cavity, nude mice left side.Treat that the knurl volume grows to 100-300mm
3The time, carry out random packet, 8/group, and the beginning administration.
Set respectively negative control group (sodium-acetate buffer, NaAC), positive controls (rhES 4mg/kg body weight, administration every day, successive administration 14 days), two groups of administration groups are (with rhES-PEG-Fc difference administration 1.3mg/kg and the 4mg/kg body weight according to the embodiment of the present invention 2 preparations, be administered once weekly, two weeks of successive administration.Amount by contained rhES in rhES-PEG-Fc is calculated).Administering mode is subcutaneous injection.Play that measure gross tumor volume every day self administration of medication day until the 28th day, calculation formula is: gross tumor volume (mm
3)=(a * b
2)/2 (a is the major diameter of tumour, and b is the minor axis of tumour).
The results are shown in Figure 6.Experimental result shows: rhES-PEG-Fc is administration 1.3mg/kg and 4mg/kg group weekly, has the anti-tumor activity similar to positive controls (administration every day rhES 4mg/kg).RhES-PEG-Fc of the present invention can reduce dosage greatly, keeps simultaneously same anti-tumour effect.
This shows, the invention provides the anti-endothelial cell migration activity that has kept rhES fully, and have the rhES-PEG-Fc of better stability and bioavailability and longer Half-life in vivo, thereby greatly reduce dosage.
Whole publications that above specification sheets is mentioned are all incorporated this paper by reference into.Although described the present invention with reference to concrete preferred implementation, should be appreciated that the invention of advocating is not limited only to described embodiment.In fact, be all apparent for the various modifications of implementing described pattern of the present invention for biological chemistry and biotechnology or those skilled in the relevant art, all should fall into the scope of claims of the present invention.
Claims (10)
1. a long-acting human endostatin, is characterized in that, described long-acting human endostatin is by polyoxyethylene glycol and the covalently bound human endostatin molecule of immunoglobulin Fc segments.
2. long-acting human endostatin as claimed in claim 1, it is characterized in that: described immunoglobulin Fc segments is nonglycosylated.
3. long-acting human endostatin as claimed in claim 1 or 2, it is characterized in that: described immunoglobulin Fc segments is the Fc fragment of IgG4.
4. long-acting human endostatin as described in any one in claim 1～3, is characterized in that, the molecular weight of described polyoxyethylene glycol is 2～5kDa.
5. as the long-acting human endostatin of any one in claim 1～4, it is characterized in that, described peg molecule two ends have the aldehyde radical functional group.
6. long-acting human endostatin as described in claim 5, is characterized in that, described peg molecule is the 3.4kDa polyoxyethylene glycol that two ends have the aldehyde radical functional group.
7. the described long-acting human endostatin of any one in claim 1-6 is characterized in that: described pharmaceutical composition is by polyoxyethylene glycol, amino the end with the N of immune globulin Fc fragment of the N end α of human endostatin to be connected by covalent linkage.
In claim 1-7 the described long-acting human endostatin of any one for the preparation of the treatment cancer medicine in application.
9. application as claimed in claim 8, wherein said cancer is lung cancer.
10. application as claimed in claim 9, wherein said cancer is nonsmall-cell lung cancer.
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| CN (1) | CN103172745A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106674352A (en) * | 2015-11-10 | 2017-05-17 | 孙嘉琳 | Derivative of anti-tumor protein endostatin and application |
| WO2019219049A1 (en) * | 2018-05-18 | 2019-11-21 | 北京辅仁瑞辉生物医药研究院有限公司 | Improved fviii fusion protein and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1723220A (en) * | 2003-11-13 | 2006-01-18 | 韩美药品工业株式会社 | A pharmaceutical composition comprising an immunoglobulin FC region as a carrier |
| CN1761684A (en) * | 2003-03-13 | 2006-04-19 | 韩美药品株式会社 | Physiologically active polypeptide conjugate having prolonged in vivo half-life |
| CN101596320A (en) * | 2006-01-20 | 2009-12-09 | 清华大学 | A kind of medicine and application thereof for the treatment of tumor |
| CN101646451A (en) * | 2007-01-05 | 2010-02-10 | 韩美药品工业株式会社 | An insulinotropic complex using an immunoglobulin fragment |
-
2011
- 2011-12-21 CN CN2011104332267A patent/CN103172745A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1761684A (en) * | 2003-03-13 | 2006-04-19 | 韩美药品株式会社 | Physiologically active polypeptide conjugate having prolonged in vivo half-life |
| CN1723220A (en) * | 2003-11-13 | 2006-01-18 | 韩美药品工业株式会社 | A pharmaceutical composition comprising an immunoglobulin FC region as a carrier |
| CN101596320A (en) * | 2006-01-20 | 2009-12-09 | 清华大学 | A kind of medicine and application thereof for the treatment of tumor |
| CN101646451A (en) * | 2007-01-05 | 2010-02-10 | 韩美药品工业株式会社 | An insulinotropic complex using an immunoglobulin fragment |
Non-Patent Citations (1)
| Title |
|---|
| LEE TY ET AL.: "Linking antibody Fc domain to endostatin significantly improves endostatin half-life and efficacy", 《CLIN CANCER RES.》, vol. 14, no. 5, 1 March 2008 (2008-03-01), pages 1487 - 1493, XP 055238107, DOI: doi:10.1158/1078-0432.CCR-07-1530 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106674352A (en) * | 2015-11-10 | 2017-05-17 | 孙嘉琳 | Derivative of anti-tumor protein endostatin and application |
| WO2019219049A1 (en) * | 2018-05-18 | 2019-11-21 | 北京辅仁瑞辉生物医药研究院有限公司 | Improved fviii fusion protein and use thereof |
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