CN104974260A - Anti-HGF/VEGF bispecific antibody as well as preparation method and application thereof - Google Patents
Anti-HGF/VEGF bispecific antibody as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and particularly discloses an anti-HGF/VEGF bispecific antibody as well as a preparation method and application thereof. The anti-HGF/VEGF bispecific antibody disclosed by the invention can be combined with HGF as well as VEGF and can effectively inhibit the tumor growth and realizes a good anti-tumor effect.
Description
Technical field
The invention belongs to biological technical field, more specifically, the invention discloses class bi-specific antibody and an its preparation method, and it prepares the application in antitumor drug.
Background technology
Tumour especially malignant tumour is the disease of world today's serious harm human health, in death caused by various disease, be in second.And in recent years, its sickness rate is obvious ascendant trend.Therapeutic effect of malignant tumour is poor, and late period, the rate of transform was high, and prognosis is how not good.Although conventional treatments adopted clinically alleviates slight illness to a great extent as Radiotherapy chemotherapy and operative treatment at present, extending survival time, all there is significant limitation in these methods, and its curative effect is difficult to further raising.
The growth of tumour has two visibly different stages, namely the fast breeding phase having blood vessel is transferred to from avascular slow vegetative period, the generation of blood vessel makes tumour obtain enough nutrition and completes the blood vessel switching phase, if there is no the generation of blood vessel, the growth of primary tumo(u)r is no more than 1 ~ 2mm, and transfer then cannot realize.Vascular endothelial growth factor (VEGF) is that a class can promote vascular endothelial cell division growth, promote that the formation of new vessel also can improve the somatomedin of vascular permeability, after the vascular endothelial growth factor receptor (VEGFR) of they and cell surface combines, play function by activating tyrosine kinase signal Signal Transduction Pathways.VEGFR belongs to tyrosine kinase receptor superfamily, and they have similar structure, by extracellular region, cross-film district, intracellular region composition.The VEGFR of current discovery mainly contains 3 kinds: VEGFR1(Flt-1), VEGFR2(KDR), VEGFR3.Mainly VEGF-A, VEGF-B of being combined with VEGFR1, the mainly VEGF-A, the VEGF-C that are combined with VEGFR2,
VEGF-D, VEGF-E, mainly VEGF-C, VEGF-D of being combined with VEGFR3.VEGFR1 and VEGFR2 is mainly positioned at tumor vascular endothelial cell surface, and the main mediate cytoskeleton of VEGFR1 is reset and causes cell migration, and VEGFR2 mediates propagation, the differentiation of endotheliocyte; VEGFR3 specifically expressing, in lymphatic endothelium, is not substantially expressed in artery, vein, capillary endothelium.VEGF-C and VEGF-D plays a role by VEGF-C/VEGF-D/VEGFR3 signalling system, thus causes the hyperplasia of lymphangiogenesis and the transfer (Neufeld G etc., FASEB J, 1999,13:9-22) of lymphoglandula.In tumor tissues,
The VEGF of the energy such as tumour cell, tumor-infiltrated scavenger cell and mastocyte secreting high levels, stimulate tumor vascular endothelial cell propagation, migration with the form of paracrine, induction of vascular is formed, promotion tumour continued propagation; In addition, high-caliber VEGF can also improve vascular permeability, causes surrounding tissue scleroproein calm, and promotion monocyte, inoblast and endotheliocyte infiltration, be conducive to tumor stroma formation and tumour cell enters new vessel, promotes metastases.Therefore, Tumor suppression vasculogenesis is considered to one of promising tumor therapeuticing method of current most.The PTS Bevacizumab(anti-vegf humanized antibody of Genentech company) be approved listing by U.S. FDA in February, 2004, current Bevacizumab and conventional chemotherapy medicine combined utilization can be used for the first-line treatment of the nonsmall-cell lung cancer for the treatment of metastatic colorectal carcinoma and non-squamous.
PHGF (hepatocyte growth factor, HGF) is present in the protein factor in acute liver damage animal plasma, the DNA synthesis of its energy cell cultured supernatant, and play an important role in liver regeneration.HGF is by 728 Amino acid profiles, and its activity form is by a 69kd heavy chain a and the heterodimer that 34kd light chain b forms, and a chain N end has a hairpin structure; B chain has serine protease spline structure.Receptor tyrosine kinase (c-Met) is the specific cell surface receptor of HGF, is expressed in the film surface of multiple normal tissue cell (as myocardial cell, vascular endothelial cell etc.) and tumour cell, the biological action that mediation HGF is all.More and more many reports show, HGF is that one acts on the factor widely, except to except liver cell effect, also has regulating and controlling effect to Various Tissues and cell, is wherein short cell fission and short cell movement effect the most significantly.HGF dose-dependently can promote the propagation of vascular endothelial cell, and the ability of HGF stimulating endothelial cell propagation is obviously better than Prostatropin (bFGF) and VEGF, and prompting HGF has powerful promotion angiogenic action.Confirm at present, no matter be in vitro or at body, HGF can both promote the mrna expression of VEGF significantly, and these two kinds of cell growth factors have synergy in Angiogensis.In tumor tissues, the division of the HGF energy stimulating endothelial cell produced by autocrine or paracrine mode and propagation, also can promote growth and the transfer of tumour cell self simultaneously.
Therefore, work out can with the VEGFR competitive binding VEGF of Surface of Vascular Endothelial Cells, simultaneously again can with the bi-specific antibody of c-Met competitive binding HGF, thus more effectively block new vessel formation, reach again the object of inhibition tumor cell growth and transfer, this kind of high efficiency anti-tumor medicine is those skilled in the art's problem anxious to be resolved always simultaneously.
Summary of the invention
In order to solve the problem, the present inventor has carried out lot of experiments, and finally obtaining one can be combined with HGF, the bi-specific antibody that can also combine with VEGF, thus complete the present invention i.e. " anti-HGF/VEGF bi-specific antibody " of the present invention.
The invention discloses:
1. anti-HGF/VEGF bi-specific antibody, it can be combined with HGF, can also combine with VEGF.
2. above-mentioned anti-HGF/VEGF bi-specific antibody, it comprises anti-HGF variable region of mab and Anti-X activity variable region.
3. the anti-HGF/VEGF bi-specific antibody described in above-mentioned 1 or 2, its light-chain amino acid sequence is as shown in SEQ IDNO:4, and the aminoacid sequence of heavy chain is as shown in SEQ ID NO:2.
4. the nucleic acid molecule be separated, the arbitrary described anti-HGF/VEGF bi-specific antibody of its above-mentioned 1-3 that encodes, preferably, the nucleotide sequence of encoding said antibody light chain is as shown in SEQ ID NO:3, and the nucleotide sequence of encoding said antibody heavy chain is as shown in SEQ ID NO:1.
5. a carrier, containing the expression regulation sequence that the nucleic acid molecule described in above-mentioned 4 is connected with the sequence being operational of described nucleic acid molecule, preferably, described carrier can be pCHO1.0, pBudCE4.1 or pCGS3, and most preferably, described carrier is pCHO1.0.
6. a host cell, it contains the carrier described in above-mentioned 5.Preferably, described cell is eukaryotic cell, more preferably, is mammalian cell, can be COS, CHO, NS0 cell; Most preferably, described host cell is
or
gS
-/-cell.
7. prepare a method for above-mentioned 1 ~ 3 arbitrary described anti-HGF/VEGF bi-specific antibody, the method comprising the steps of:
A) under expression condition, cultivate the host cell described in above-mentioned 6, express above-mentioned anti-HGF/VEGF bi-specific antibody;
B) isolated or purified a) described in anti-HGF/VEGF bi-specific antibody.
Preferably, described preparation method comprises the following steps:
A) heavy chain fusion gene and the light chain fusion gene of anti-HGF/VEGF bi-specific antibody is built;
B) by above-mentioned a) fusion gene cloning to expression vector pCHO1.0, construction of expression vector pCHO1.0(DVD-HGF/VEGF);
C) by above-mentioned b) expression vector pCHO1.0(DVD-HGF/VEGF) transfection CHO cell, screening, carries out subclone, carries out enlarged culturing by screening the high-expression clone obtained;
D) isolated or purified c) the anti-HGF/VEGF bi-specific antibody of gained.
8. a composition, containing above-mentioned 1 ~ 3 arbitrary described anti-HGF/VEGF bi-specific antibody and pharmaceutically acceptable carrier.
9. described in the anti-HGF/VEGF bi-specific antibody or 8 described in above-mentioned 1 or 2, composition is preparing the purposes in antitumor drug.
10. the purposes described in above-mentioned 9, also comprises the antitumor drug conbined usage with other.
The suitable DNA of any coding anti-HGF antibody variable region and VEGF antibody variable region is suitable for the present invention.
In the present invention, any suitable carrier can use, and can be one of pCHO1.0, pBudCE4.1 or pCGS3, and expression vector comprises and is connected with the suitable fusion dna sequence with translational regulation sequence of transcribing.
The expression of Mammals or insect host cell culture systems fusion rotein used in the present invention, the zooblasts such as COS, CHO, NS0, sf9 and sf21 are all applicable to the present invention, preferred COS, CHO, NS0 cell, most preferably Chinese hamster ovary celI; Also can be the prokaryotic cell prokaryocyte containing above-mentioned carrier, can be one of DH5 α, BL21 (DE3) and TG1.
Disclosed in the present invention, the preparation method of anti-HGF/VEGF bi-specific antibody is cultivate above-mentioned host cell under expression condition, thus expresses bi-specific antibody, and then the bi-specific antibody described in isolated or purified.
The method of affinity chromatography can be utilized to carry out separation and purification to bi-specific antibody disclosed by the invention, according to the characteristic of utilized affinity column, the conventional method such as fusion rotein polypeptide of method elution of bound on affinity column such as high-salt buffer, change PH can be used.
Utilize aforesaid method, can be substantially homogeneous material by fusion protein purification, such as, on SDS-PAGE electrophoresis, be shown as single band.
Above-mentioned anti-HGF/VEGF bi-specific antibody disclosed by the invention obtains by the following method: the aminoacid sequence (Accession Number:DB00112) that full-length gene (light chain and the heavy chain) sequence of anti-VEGF mAb Bevacizumab is announced according to Drug Bank is synthesized by Suzhou Jin Weizhi Technology Service Co., Ltd full genome, and full-length gene (light chain and the heavy chain) sequence of the anti-HGF monoclonal antibody in total man source is according to (the variable region of light chain: SEQ ID NO38 of sequence in US20050118643A1; Variable region of heavy chain: SEQ ID NO39; Constant region of light chain: SEQ ID NO44; CH: SEQ ID NO45) synthesized by Shanghai biotechnology Services Co., Ltd full genome.Recombinate in rear clone to pCHO1.0 carrier with over-lap PCR method with the heavy chain of Bevacizumab and light chain gene respectively and are built into carrier for expression of eukaryon pCHO1.0(DVD-HGF/VEGF in the heavy chain of anti-HGF antibody and variable region of light chain); Above-mentioned plasmid liposome transfection CHO-S cell, and with screening the cell clone of the anti-HGF/VEGF bi-specific antibody of stably express containing the methotrexate (MTX) of proper concn and the Selective agar medium of tetracycline.Utilize the anti-HGF/VEGF bi-specific antibody of Protein A post affinity chromatography purifying from cell culture supernatant.Gained anti-CTLA-4/PD-1 bi-specific antibody variable region comprises above-mentioned anti-VEGF mAb variable region and above-mentioned anti-HGF monoclonal antibody variable region, its constant region is above-mentioned anti-VEGF mAb constant region, its light chain full length amino acid sequence is as shown in SEQ ID NO:4, and heavy chain full length amino acid sequence is for shown in SEQ ID NO:2.
Applicant of the present invention carries out avidity detection, the experiment of protein drug dynamic metabolism, the experiment such as Tumor suppression growth in body to above-mentioned anti-HGF/VEGF bi-specific antibody, experimental result shows, anti-HGF/VEGF bi-specific antibody disclosed by the invention both can combine with one of HGF or VEGF, also can be combined with HGF and VEGF simultaneously, block its intracellular signaling function, thus produce stronger antineoplaston curative effect.In addition, this anti-HGF/VEGF bi-specific antibody has excellent pharmacokinetic property.Experiment shows, under equal dose, anti-HGF/VEGF bi-specific antibody has than the independent anti-HGF antibody of application and the better antitumous effect of VEGF antibody, reaches object of the present invention.
The present invention discloses above-mentioned anti-HGF/VEGF bi-specific antibody; can pharmaceutical preparations composition be formed thus more play consistently curative effect together with pharmaceutically acceptable auxiliary material; these preparations can ensure the Conformational Integrity of fusion receptors amino acid core sequence disclosed by the invention, also want the polyfunctional group of protected protein matter to prevent its degraded (including but not limited to cohesion, deamination or oxidation) simultaneously.Generally, for liquid preparation, usually can stablize under 2-8 DEG C of condition and preserve at least one year; For freeze-dried preparation, within least six months, keep stable at 30 DEG C.Here preparation can be the conventional preparation such as suspendible, liquid drugs injection, freeze-drying of pharmacy field, preferred liquid drugs injection or freeze-dried preparation.For liquid drugs injection or the freeze-dried preparation of above-mentioned anti-HGF/VEGF bi-specific antibody disclosed by the invention, pharmaceutically acceptable auxiliary material includes but not limited to: tensio-active agent, solution stabilizer, isotonic regulator and damping fluid one or a combination set of, wherein tensio-active agent includes but not limited to: nonionic surface active agent is as Polyoxyethylene Sorbitol Fatty Acid Esters (polysorbas20 or 80), poloxamer(is as poloxamer188), Triton, sodium lauryl sulphate (SDS), Sulfuric acid,monododecyl ester, sodium salt, tetradecyl, sub-oil base or octadecyl sarkosine, Pluronics, MONAQUAT
tMdeng, its add-on should make the granulating trend of anti-HGF/VEGF bi-specific antibody minimum, solution stabilizer includes but not limited to: carbohydrate, amino acids or alcohols one or a combination set of, wherein carbohydrate can be reducing sugar and nonreducing sugar, amino acids can be msg powder type or Histidine, alcohols can be trivalent alcohol, senior sugar alcohol, propylene glycol, polyoxyethylene glycol etc., keeps steady state in the time that the add-on of solution stabilizer should make last preparation those skilled in the art formed think to reach stable, isotonic regulator can be one of sodium-chlor, N.F,USP MANNITOL, damping fluid can be one of Tris, histidine buffering liquid, phosphate buffered saline buffer.
Above-mentioned preparation is the composition comprising anti-HGF/VEGF bi-specific antibody, and after to the animals administer comprising people, antitumous effect is obvious.Specifically, preventing and/or treating effectively tumour, can use as antitumor drug.
The tumour of indication of the present invention, include but not limited to: gland cancer, leukemia, lymphoma, melanoma, sarcoma, the source of tumor tissues includes but not limited to suprarenal gland, gall-bladder, bone, marrow, brain, mammary gland, bile duct, gi tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate gland, skin, sialisterium, spleen, testis, thymus gland, Tiroidina and uterus.Except above-mentioned tumour, also can be used for the tumour of central nervous system as spongiocyte diversity knurl, astrocytoma etc., in addition the tumour of eye comprises rodent cancer, squamous cell carcinoma, melanoma etc., also comprise endocrine disrupting effects, neuroendocrine system tumour, gi tract pancreatic endocrine system tumor, genital system tumor and tumor of head and neck etc.Here will not enumerate.
Antitumor drug alleged by the present invention, refer to that there is the medicine suppressing and/or treat tumour, can postpone with the related indication development of tumor growth and/or the severity reducing these symptoms, it can also alleviate the already present symptom that accompanies with tumor growth further and prevent the appearance of other symptoms, also reduces or prevents transfer.
In the present invention, anti-HGF/VEGF bi-specific antibody and composition thereof are to when comprising the animals administer of people, dosage is because of age of patient and body weight, disease traits and seriousness, and route of administration and different, can with reference to zooperal result and all situations, total dosage can not exceed certain limit.Specifically intravenous dosage is 1 ~ 1800mg/ days.
Anti-HGF/VEGF bi-specific antibody disclosed by the invention and composition thereof can also with other antitumour drug Combined Preparation to reach the object of more effectively treating tumour, and these antitumour drugs include but not limited to: 1, cytotoxic drug: the medicine 1) acting on nucleic acid chemistry structure: alkylating agent is as nitrogen mustards, nitrosourea, methanesulfonate ester class; Platinum compound is as cis-platinum (Cisplatin), carboplatin (Carboplatin) and RP-54780 (Oxaliplatin) etc.; Antibiotics is as Zorubicin (Adriamycin/Doxorubicin), dactinomycin (Dactinomycin D), daunorubicin (Daunorubicin), pidorubicin (Epirubicin), aclacinomycin (Aclarubicin), Plicamycin (Mithramycin) etc.; 2) medicine of nucleic acid metabolism is affected: dihydrofolate reductase inhibitor is as Rheumatrex (MTX) and pemetrexed (Pemetrexed) etc.; Thymus nucleoside synthetase inhibitors is as fluorouracil (5-FU, capecitabine) etc.; Purine nucleoside synthetase inhibitors is as Ismipur etc.; Ribonucleotide reductase inhibitor is as hydroxyurea (Hydroxycarbamide) etc.; DNA polymerase inhibitor is as cytosine arabinoside (Cytosine arabinoside) and gemcitabine (Gemcitabine) etc.; 3) medicine of tubulin is acted on: docetaxel (Docetaxel), vincaleucoblastine (Vincristine), vinorelbine (Vinorelbine), Podophyllum emodi var chinense alkali class, homoharringtonine etc.; 2, hormone medicine: estrogen antagonist is as tamoxifen (Tamoxifen), droloxifene (Droloxifene), Exemestane (Exemestane) etc.; Arimedex is as aminoglutethimide (Aminoglutethimide), Formestane (Formestane), letrozole (Letrozle), Anastrozole (Anastrozole) etc.; Androgen antagonist: its ammonia RH-LH agonist/antagonist of fluorine: Zoladex, enatone etc.; 3, biological response modifier class medicine: this type of medicine mainly through conditioner body immunity function with to antineoplastic effect, as interferons (Interferon); Interleukin II (Interleukin-2); Thymic (Thymosins) etc.; 4, monoclonal antibodies medicine: bent appropriate former times monoclonal antibody (Trastuzumab), Rituximab (Rituximab), Cetuximab (Cetuximab), rhuMAb-VEGF (Bevacizumab) etc.; 5, other series antineoplastic medicaments: comprise some current mechanism and be still not clear, need the medicine etc. of further research.Anti-HGF/VEGF bi-specific antibody disclosed by the invention and composition thereof can with above-mentioned antitumor drug one or a combination set of drug combination.
Accompanying drawing explanation
Fig. 1. anti-HGF/VEGF bi-specific antibody and VEGF-A bonding force are tested;
Fig. 2. anti-HGF/VEGF bi-specific antibody and HGF bonding force are tested;
Fig. 3. anti-HGF/VEGF bi-specific antibody time front of blood concentration
Fig. 4. Tumor suppression growth curve in anti-HGF/VEGF bi-specific antibody body
Embodiment
Following examples, experimental example further illustrate of the present invention, should not be construed as limitation of the present invention.Embodiment does not comprise the detailed description to ordinary method, as the method that those draw for carrier construction and matter, the gene of proteins encoded is inserted into the method that such carrier and matter are drawn or method plasmid being introduced host cell.Such method is well-known to person having ordinary skill in the art, and described by having in many publications, such as: Sambrook, J., Fritsch, E.F.and Maniais, T.(1989) MolecularCloning:A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.The structure of embodiment 1. anti-HGF/VEGF bi-specific antibody expression vector
The sequence (comprising light chain and heavy chain) of the full-length gene of VEGF antibody Bevacizumab is according to DrugBank(Accession Number:DB00112) aminoacid sequence announced synthesizes by Suzhou Jin Weizhi biotechnology Services Co., Ltd full genome, full-length gene (light chain and heavy chain) sequence aminoacid sequence (variable region of light chain: SEQ ID NO38 disclosed in US Patent No. 20050118643A1 of the anti-HGF monoclonal antibody in total man source; Variable region of heavy chain: SEQ ID NO39; Constant region of light chain: SEQ ID NO44; CH: SEQ ID NO45) also synthesize by by Suzhou Jin Weizhi biotechnology Services Co., Ltd full genome.Design the light chain of primer polymerase chain reaction (PCR) amplification Bevacizumab gene respectively according to DNA sequence dna and reconnect, and the light chain of anti-HGF monoclonal antibody and reconnect variable region; The complementary overhangs district of certain length is introduced, so that on the light chain variable region gene of anti-HGF monoclonal antibody being connected to Bevacizumab by recombinant PCR method and heavy chain gene segment during design primer; , there is certain space operation degree of freedom the link zone (aminoacid sequence is: GGGGSGGGGS) of complementary overhangs district encoding exogenous in order to make two adjacent Variable domain.
PCR all adopts high-fidelity DNA polymerase (purchased from Takara company
hS DNAPolymerase).Amplification Bevacizumab weight chain full-length gene and the reaction conditions of anti-HGF weight chain variable region fragment are rationally arranged according to archaeal dna polymerase manufacturer specification sheets: 95 DEG C 20 seconds; 55 DEG C 10 seconds, 72 DEG C of 1 point/kb; 30 circulations.Above PCR primer is for the template of recombinant PCR after agarose gel electrophoresis purifying reclaims, and recombinant PCR reaction conditions is: 95 DEG C 20 seconds; 55 DEG C 10 seconds, 72 DEG C 2 points; Corresponding primer is added again after 6 circulations; 95 DEG C 20 seconds; 55 DEG C 10 seconds, 72 DEG C of 1 point/kb.PCR primer reclaims through agarose gel electrophoresis purifying and is cloned in pCHO1.0 carrier (purchased from Life Technologies company), confirms to obtain correct clone after sequence verification.SEQ ID NO:1 and SEQ ID NO:2 respectively illustrates Nucleotide and the aminoacid sequence of heavy chain.SEQ ID NO:3 and SEQ ID NO:4 respectively illustrates Nucleotide and the aminoacid sequence of light chain.Correct clone in this example is denoted as pCHO1.0(DVD-HGF/VEGF).The expression of the anti-HGF/VEGF bi-specific antibody of embodiment 2. in Chinese hamster ovary cell (CHO)
In 125ml cell cultures shaking flask, inoculate the CHO-S cell of 30ml density 5 × 105, second day cell density increase to 1 × 106 and Cell viability higher than 95% time carry out transfection: the plasmid pCHO1.0(DVD-HGF/VEGF by 50 μ l concentration being the coding anti-HGF/VEGF bi-specific antibody weight chain of 1 μ g/ μ l) be diluted to 1.45mlOptiPRO
tMsFM(is purchased from Life Technologies company) in, mix gently; By 50 μ l transfection reagent FreeStyle
tMmAX(is purchased from Life Technologies company) be diluted to 1.45mlOptiPRO
tMin SFM, mix gently; Immediately by the FreeStyle after dilution
tMmAX solution slowly joins in the DNA solution after dilution, adds fashionable rifle head and to be dipped under liquid level and slowly expel liquid; Turning upside down immediately makes solution mix for several times, and incubated at room temperature about 10 minutes is formed to make DNA-liposome complex; By 3mlDNA-FreeStyle
tMmAX complexes drop-wise joins in the shaking flask of above-mentioned culturing cell, limit edged jog culturing bottle; Cell culture after transfection be placed in 37 DEG C, 8%CO2, rotating speed 130rpm cell culture table on cultivate.Transfection is after 48 hours, centrifugal collecting cell, nutrient solution changed into the resistance clone of the Selective agar medium screening stable transfection containing 20 μ g/ml tetracyclines (Puromycin) and 200nM Rheumatrex (MTX); After about two weeks, when Cell viability and density recover, centrifugal collecting cell, changes the Selective agar medium containing 50 μ g/ml tetracyclines (Puromycin) and 1000nM Rheumatrex (MTX) into by nutrient solution; After about one week, when Cell viability and density recover, centrifugal collecting cell, changes into not containing antibiotic substratum by nutrient solution, be placed in 37 DEG C, 8%CO2, rotating speed 130rpm shaking table on suspension culture 10 days, period added glucose at the 3rd, 5,7,9 day with the final concentration of 4g/L; After 10 days, cell culture is after low-speed centrifugal (300g) removes most cells and cell debris in 5 minutes, within 10 minutes, the solid substance still suspended is removed again through high speed centrifugation (10000g), then suction filtration (filter sizes 0.45 μm), finally uses the anti-HGF/VEGF bi-specific antibody of Protein A affinity column (GE Products) separation and purification from the clarified liq obtained.Purified product is through order-checking, and aminoacid sequence and SEQ ID NO:2, SEQID NO:4 sequence are consistent, and being dialysed by purified fusion acceptor PBS, is finally standard substance BCA(Bicinchoninic acid with human normal immunoglobulin) method carries out quantitatively.
Embodiment 3. anti-HGF/VEGF bi-specific antibody avidity test experience-ELISA method
Experimental procedure: recombinant protein HGF(is purchased from R & D company) and VEGF165(purchased from R & D company) be diluted to 2 μ g/ml with 0.05mol/L carbonate buffer solution (pH9.6), the protein solution after the above-mentioned dilution of 100 μ l is added in the 96 every holes of hole enzyme plate, and 4 DEG C are spent the night in wet box; Next day, discard solution in hole, wash 3 times with lavation buffer solution (phosphate buffered saline buffer containing 0.05%Tween-20), each 3 minutes; Dilution for difference anti-HGF antibody (to be prepared according to embodiment 1 and embodiment 2 method according to HGF antibody sequence anti-described in embodiment 1, as follows), VEGF antibody is (according to VEGF antibody sequence described in embodiment 1, prepare according to embodiment 1 and embodiment 2 method, as follows) and the anti-HGF/VEGF bi-specific antibody 100 μ l for preparing of above-described embodiment 2 be added on above-mentioned bag in the reacting hole of quilt, put 37 DEG C and hatch 1 hour; Then with lavation buffer solution washing, goat anti-human igg 1 antibody (Fc is special, Sigma Products) adding horseradish peroxidase-labeled, in each reacting hole, hatches 1 hour for 37 DEG C, washing; The tmb substrate solution 0.1ml adding Extemporaneous in each reacting hole develops the color, and places 10 ~ 30 minutes for 37 DEG C; 2mol/L sulfuric acid 0.05ml termination reaction is added in each reacting hole; Put (SpectraMax i3Multi-Mode Platform, purchased from American BIO-TEK company) in microplate reader and measure 450nm wavelength place mensuration light absorption value.
Experimental result as shown in Figures 1 and 2.Result shows: anti-HGF/VEGF bi-specific antibody both in conjunction with HGF, also in conjunction with VEGF, and can be compared with VEGF antibody with anti-HGF antibody, and the bonding force of anti-HGF/VEGF bi-specific antibody to two kinds of target spots does not all have noticeable change.
The pharmacokinetic studies of the anti-HGF/VEGF bi-specific antibody of experimental example 4.
Experimental procedure: 10 week age, no-special pathogen (Specific-pathogen free, SPF) Thirty male rats (purchased from Shanghai Slac Experimental Animal Co., Ltd.), body weight 350-400g, be divided into eight groups at random, often organize four, give drug solvent, anti-HGF antibody, VEGF antibody and anti-HGF/VEGF bi-specific antibody respectively by intravenous injection, the dosage of antibody is set to 4mg/kg.All rats get blood (angular vein gets blood), every rat extracting blood about 100 μ l after 0.5h, 1h, 2h, 6h, 12h, 24h, 48h, 5d, 10d, 15d, 20d, 25d, 30d after injection, and add the heparin mixing of 0.1%, the centrifugal 20min of 4000rpm, gets supernatant, abandon precipitation.The concentration detecting antibody in blood plasma adopts ELISA method: recombinant protein HGF and VEGF165(R & D Products) be diluted to 2 μ g/ml with 0.05mol/L carbonate buffer solution (pH9.6), the protein solution after the above-mentioned dilution of 100 μ l is added in the 96 every holes of hole enzyme plate, and 4 DEG C are spent the night in wet box; Next day, discard solution in hole, wash 3 times with lavation buffer solution (phosphate buffered saline buffer containing 0.05%Tween-20), each 3 minutes; Dilution for difference rat plasma is added on above-mentioned bag in the reacting hole of quilt, puts 37 DEG C and hatch 1 hour; Then with lavation buffer solution washing, the rat anti-human IgG1 antibody (Fc is special, Sigma Products) adding horseradish peroxidase-labeled, in each reacting hole, hatches 1 hour for 37 DEG C, washing; The tmb substrate solution 0.1ml adding Extemporaneous in each reacting hole develops the color, and places 10 ~ 30 minutes for 37 DEG C; 2mol/L sulfuric acid 0.05ml termination reaction is added in each reacting hole; Put (SpectraMax i3Multi-Mode Platform, BIO-TEK company of the U.S.) in microplate reader and measure 450nm wavelength place mensuration light absorption value.As shown in Figure 3, the Half-life in vivo of anti-HGF antibody, VEGF antibody and anti-HGF/VEGF bi-specific antibody is all longer, does not have significant difference for detected result.
Tumor suppression growth experiment in the body of the anti-HGF/VEGF bi-specific antibody of experimental example 5.
For the anti-tumor in vivo detecting anti-HGF/VEGF bi-specific antibody is active, adopt with Balb/c-nu/nu is people's Replanting model mice of host herein.First by people glioblastoma cell line U87MG(purchased from the type culture classical collection council of Chinese Academy of Sciences cell bank) be inoculated in the Male athymic nude mice Balb/c-nu/nu(Shanghai Slac Experimental Animal Co., Ltd. in 5-6 age in week) right side is subcutaneous, every only inoculation 5 × 106 tumour cells; Inoculate second day mouse random packet, often organize 8, anti-HGF antibody, VEGF antibody and anti-HGF/VEGF bi-specific antibody and irrelevant protein control human IgG1 (available from Sigma) and drug solvent PBS(available from Sigma is given) respectively by abdominal injection, anti-HGF antibody, VEGF antibody dosage are set to 4mg/kg, antagonism HGF/VEGF bi-specific antibody arranges low middle high three dosage groups: 2,4,8mg/kg; After this Per-Hop behavior twice, the every 3 days length (a) with vernier caliper measurement diameter of tumor and wide (b), with following formulae discovery gross tumor volume: V=a × b
2/ 2; Finally map and analysis design mothod result with statistical software GraphPad Prism5.Experimental result as shown in Figure 4.Result shows: between irrelevant protein control human IgG1 and drug solvent PBS group, tumor size is basically identical, anti-HGF/VEGF bi-specific antibody (especially high dose group) than anti-HGF antibody and VEGF antibody group gross tumor volume all obvious much little, have significant difference (P<0.05).
In addition, applicant of the present invention also carries out similar experiment to other tumour (such as: BGC-823 stomach cancer cell line mouse becomes knurl model, MDA-MB-231 breast carcinoma cell strain mouse becomes knurl model etc.), experimental result all shows, anti-HGF/VEGF bi-specific antibody of the present invention can the growth of Tumor suppression effectively, has good antitumous effect.
Claims (10)
1. anti-HGF/VEGF bi-specific antibody, it can be combined with HGF, can also combine with VEGF.
2. antibody according to claim 1, it is characterized in that, described antibody comprises anti-HGF variable region of mab and Anti-X activity variable region.
3. antibody according to claim 1 and 2, is characterized in that, described light chain of antibody aminoacid sequence is as shown in SEQ ID NO:4, and the aminoacid sequence of heavy chain is as shown in SEQ ID NO:2.
4. the nucleic acid molecule be separated, the arbitrary described antibody of its above-mentioned 1-3 that encodes, the nucleotide sequence of encoding said antibody light chain is as shown in SEQ ID NO:3, and the nucleotide sequence of encoding said antibody heavy chain is as shown in SEQ IDNO:1.
5. a carrier, it contains the expression regulation sequence that nucleic acid molecule according to claim 4 is connected with the sequence being operational of described nucleic acid molecule, and described carrier can be pCHO1.0, pBudCE4.1 or pCGS3.
6. a host cell, it contains carrier described in claim 5, and described cell can be COS, CHO or NS0 cell.
7. prepare a method for the arbitrary described anti-HGF/VEGF bi-specific antibody of claims 1 to 3, described method comprises step:
A) under expression condition, cultivate the host cell described in above-mentioned 6, express above-mentioned anti-HGF/VEGF bi-specific antibody;
B) isolated or purified a) described in anti-HGF/VEGF bi-specific antibody.
8. a composition, containing above-mentioned 1 ~ 3 arbitrary described anti-HGF/VEGF bi-specific antibody and pharmaceutically acceptable carrier.
9. described in the anti-HGF/VEGF bi-specific antibody described in claim 1 or 2 or claim 8, composition is preparing the purposes in antitumor drug.
10. purposes according to claim 9, also comprises the antitumor drug conbined usage with other.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105175545A (en) * | 2015-10-20 | 2015-12-23 | 安徽瀚海博兴生物技术有限公司 | VEGF-resistant and PD-1-resistant difunctional antibody and application thereof |
| CN109806393A (en) * | 2017-11-17 | 2019-05-28 | 江苏恒瑞医药股份有限公司 | The purposes of anti-PD-1 antibody, pemetrexed and platinum medicine combination therapy non-small cell lung cancer |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102372778A (en) * | 2010-08-19 | 2012-03-14 | 上海抗体药物国家工程研究中心有限公司 | Anti-human VEGF/anti-OPN bispecific antibody, its preparation method and application |
| WO2012061558A2 (en) * | 2010-11-04 | 2012-05-10 | Abbott Laboratories | Dual variable domain immunoglobulins and uses thereof |
| CN102741423A (en) * | 2009-10-23 | 2012-10-17 | 雅培制药有限公司 | Dual variable domain immunoglobulins and uses thereof |
| CN102946902A (en) * | 2010-03-26 | 2013-02-27 | 罗切格利卡特公司 | Bispecific antibodies |
| CN103167879A (en) * | 2010-07-09 | 2013-06-19 | Abbvie公司 | Dual variable domain immunoglobulins and uses thereof |
| CN103459422A (en) * | 2010-11-24 | 2013-12-18 | 葛兰素集团有限公司 | Multispecific antigen binding proteins targeting HGF |
-
2014
- 2014-04-01 CN CN201410128483.3A patent/CN104974260B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102741423A (en) * | 2009-10-23 | 2012-10-17 | 雅培制药有限公司 | Dual variable domain immunoglobulins and uses thereof |
| CN102946902A (en) * | 2010-03-26 | 2013-02-27 | 罗切格利卡特公司 | Bispecific antibodies |
| CN103167879A (en) * | 2010-07-09 | 2013-06-19 | Abbvie公司 | Dual variable domain immunoglobulins and uses thereof |
| CN102372778A (en) * | 2010-08-19 | 2012-03-14 | 上海抗体药物国家工程研究中心有限公司 | Anti-human VEGF/anti-OPN bispecific antibody, its preparation method and application |
| WO2012061558A2 (en) * | 2010-11-04 | 2012-05-10 | Abbott Laboratories | Dual variable domain immunoglobulins and uses thereof |
| CN103459422A (en) * | 2010-11-24 | 2013-12-18 | 葛兰素集团有限公司 | Multispecific antigen binding proteins targeting HGF |
Non-Patent Citations (1)
| Title |
|---|
| BIOGEN IDEC 等: "Emerging antibody combinations in oncology", 《MABS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105175545A (en) * | 2015-10-20 | 2015-12-23 | 安徽瀚海博兴生物技术有限公司 | VEGF-resistant and PD-1-resistant difunctional antibody and application thereof |
| CN105175545B (en) * | 2015-10-20 | 2019-01-25 | 安徽瀚海博兴生物技术有限公司 | A kind of anti-PD-1 bifunctional antibody of anti-vegf-and its application |
| CN109806393A (en) * | 2017-11-17 | 2019-05-28 | 江苏恒瑞医药股份有限公司 | The purposes of anti-PD-1 antibody, pemetrexed and platinum medicine combination therapy non-small cell lung cancer |
| CN109806393B (en) * | 2017-11-17 | 2022-07-26 | 江苏恒瑞医药股份有限公司 | Application of anti-PD-1 antibody, pemetrexed and platinum drugs in combined treatment of non-small cell lung cancer |
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