CN105801695A - Recombinant anti-VEGF monoclonal antibody and use thereof - Google Patents
Recombinant anti-VEGF monoclonal antibody and use thereof Download PDFInfo
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Abstract
The invention relates to the field of gene engineering products and discloses a recombinant anti-VEGF monoclonal antibody and its use in preparation of antitumor drugs. In the antibody, amino acids at 35th and 90th sites of a heavy chain variable range shown in the formula of SEQ ID NO: 3 and/or an amino acid at 75th site of a light chain variable range shown in the formula of SEQ ID NO: 4 is replaced. Compared with the existing anti-VEGF monoclonal antibody, the mutational antibody has higher affinity activity and has better antitumor effects.
Description
Technical field
The present invention relates to biomedicine technical field, more specifically, the present invention relates to a kind of humanization anti-vascular endothelial growth factor (VEGF) monoclonal antibody.
Background technology
Tumor angiogenesis is the basis (FolkmanJ.NEnglJMed, 1971,285 (21): 1182) of tumor growth, transfer.Since (FolkmanJ.JBiolChem such as Weidner, 1992,267 (16): 10931) since reporting the independent prognostic indicator that tumor tissues microvessel density (MVD) is patient with breast cancer, in many tumors, it has been obtained for (Wang Dong certainly including the prognostic value of MVD in primary hepatocarcinoma, Gao Fengxun, the research of Chen Li etc., osteosarcoma microvessel density and prognosis, China's pathology magazine, 1997,26 (5): 266).Tumor neovasculature formation is the essential condition of its growth, infiltration, transfer.Growth and the transfer of solid tumor are to rely on angiogenesis, and therefore interference and blocking-up tumor blood supply to be effective Therapeutic Method.Many studies have shown that swollen intratumoral microvascular density (Intratumoralmicrovesseldensity, MVD) (Wang Dong etc. relevant to osteosarcomatous transfer and prognosis, ibid), in multiple Angiogenesis Stimulators in Human, VEGF (VEGF) continues high expressed, and this proves that VEGF is one of important Angiogenesis Stimulators in Human of human bone sarcoma.
It has proved that most of solid malignants can secrete multiple angiogenic growth factor, induction of vascular generates, and supports tumor growth (FolkmanJ, Science, 1987,235 (4787): 442).No matter the blood vessel that tumor induces suffers from characteristic on pathology, physiology or morphological function, these chemotherapeutics being all traditional constitute " bloodtumorbarrier ", tumor cell is made to be able to the killing (HoriKL.JCancerRes of escape chemotherapeutics, 1991,82 (1): 109).Identify that tumor-blood-vessel growth process generates the difference of process, targeted attack tumor vessel specific target position with normal blood vessels, oncotherapy process will be greatly accelerated.
VEGF is that purifies and separates strong endothelial cell mitogen out is former from Niu Chuiti folliculus sternzellen culture at first, by specific binding with tyrosine kinase receptor fltl and KDR/flkl on endotheliocyte, make receptor auto-phosphorylation and signal transduction pathway in active cell, produce biological effect such as inducing cell proliferation, migration, regulate the expression of endotheliocyte integrin and the proteinase activated factor, increase microvascular permeability, promote that plasma protein leaks outside, basement membrane degradation, causes that new substrate and new vessels chamber are formed.In the 20 multiple polypeptides class angiogenesis factors found so far, VEGF is the highest for endothelial cell specific, key regulator (the FerraraN.EndocrRev that angiogenic growth effect is the strongest, 1997,18 (1): 4), in multiple primary malignant tumor tissue all in high expressed (BrownLF etc. HumanPathol, 1995,26 (1): 86)；Recently Takahashi (CancerRes, 1995,55 (18): 3964) find that VEGF high expressed has close ties with colon cancer transfer, point out in the formation of VEGF tumor in position and the formation of growth and metastatic tumor and all play very important effect.It turned out other multiple angiogenesis factor to be played a role by directly or indirectly induction, stimulation vegf expression, as TGF-α, bFGF, TGF-β, TNF-α, KGF etc. all can raise the expression (LiJ of VEGF, HamptonT etc., JClinInvest, 1997,100 (1): 18；BrownLF etc., DetmarM, ClaffeyK, etal.Vascularpermeabilityfactor/vascularendothelialgrowt hfactor:amultifunctionalangiogeniccytokine.Exs, 1997,79:233).Therefore, VEGF is the promising target blocking tumor blood confession.KimKJ utilizes VEGF monoclonal antibody successfully to inhibit the growth in nude mouse of rhabdomyosarcoma, glioblastoma, leiomyosarcoma；Asano (AsanoM etc., CancerRes, 1995,55 (22): 5196) utilize VEGF121 monoclonal antibody MV303 not only to inhibit the Human umbilical vein endothelial cells of In vitro culture to grow and the growth of human fibrosarcoma cell strain HT-1080 in BALB/C Mus body, and inhibit the formation of BALB/C Mus metastatic tumor.Our experiment confirms that the angiogenesis of osteosarcoma cell OS-732 and the growth of tumor cell are also had inhibitory action by VEGF polyclonal antibody, and VEGF polyclonal antibody effect relatively VEGF monoclonal antibody is likely to more comprehensively, the effect of fully blocking VEGF during smaller dose, can be compared.
Angiogenesis relates to a series of morphology and biochemical change.Morphological change include the venular basement membrane of endotheliocyte biodegradable carrier, endotheliocyte directed movement, occur mitosis, lumen of vessels to be formed, bud formula grow and forms blood vessel and fastens with a rope, string, etc., produces the series of steps such as formation of new basement membrane, adventitial cell.Under normal circumstances, endotheliocyte in a dormant state, is the static cell of internal extreme.The renewal of endotheliocyte needs hundreds of skies, and the renewal of medullary cell on average only needs 5 days, and division speed about 6 × 109 cell/hour.In angiogenesis, the growth rate of capillary endothelium is identical with medullary cell, and its biochemical mechanism relates to the Imbalance between angiogenesis factor and Angiostatin.
Separated and purification more than 20 kind of angiogenesis factor and correlation factor, at least 15 kinds of Angiostatins at present.Angiogenesis factor includes vascular endothelial cell growth factor (VPF/VEGF), acid and basic fibroblast growth factor (aFGF, bFGF), angiogenin, placental growth factor (PIGF), epidermal growth factor (EGF), interleukin-8, tumor necrosis factor α (TNF α) etc..In all of angiogenesis factor, the research of VEGF and bFGF is the most deep.VEGF has increases microvascular permeability, promote the endothelial cell division propagation of separate sources and vascular engineering, promote the multiple effect such as migration of endotheliocyte, is the strongest known vascular permeability agent.VEGF is selectively applied to two kinds of type tyrosine kinase receptor flt-1 and KDR on endothelial cellular membrane, makes IP3 concentration in born of the same parents raise by phosphoinositide specificity Phospholipase C and plays a role.
With the biochemical change in the links of angiogenesis and generating process thereof for target spot, develop angiogenesis inhibitor, control growth and metastasis of tumours, an important channel for the treatment of and prevention of tumour will be become.On the whole, the research of angiogenesis inhibitor mainly has following 4 kinds of strategies: (1) blocks the ability of endotheliocyte degraded surrounding substrate；(2) function of endotheliocyte is directly suppressed；(3) synthesis and the release of angiogenesis factor are blocked, its effect of antagonism；(4) effect of endothelial cell surface integrin is blocked.
The factor with angiogenesis inhibitor ability having now been found that mainly has receptor and the Anti-X activity of VEGF etc..Studying proof widely, anti-VEGF mAb has the function significantly suppressing tumor-blood-vessel growth.Monoclonal anti physical ability suppresses the propagation of human vascular endothelial, can suppress tumor angiogenesis in vivo, thus suppressing growth and the transfer of tumor, its suppression ratio is up to 49%-70%.
In sum, Anti-X activity demonstrates huge application prospect on oncotherapy.
Although having had relevant Anti-X activity to be applied to clinic at present, in order to strengthen the specificity of antibody, improve detection sensitivity, the function strengthening antibody reduces the consumption of antibody simultaneously, and then improve fragmentation effect to tumor, more the Anti-X activity of high-affinity is still and is badly in need of at present.
Summary of the invention
The applicant of the present invention, through substantial amounts of research experiment, constructs a kind of restructuring Anti-X activity with high-affinity, and it has higher affinity than existing VEGF antibody, it is possible to significantly more efficient identification VEGF antigen.
Specifically, the invention discloses:
1. a restructuring Anti-X activity, it is characterized in that, the aminoacid that described antibody includes on the 35th of the variable region of heavy chain as shown in SEQIDNO:2 the, 90 sites is replaced, and/or the aminoacid on the 75th of the variable region of light chain as shown in SEQIDNO:4 the site is replaced.
2. the restructuring Anti-X activity described in above-mentioned 1, it is characterised in that the aminoacid on described site is replaced by glutamic acid, lysine, arginine, serine or threonine.
3. the restructuring Anti-X activity described in above-mentioned 2, it is characterized in that, shown in shown in the aminoacid sequence of the variable region of heavy chain of described antibody such as SEQIDNO:5-10 or SEQIDNO:13 is arbitrary, and/or the aminoacid sequence of variable region of light chain is such as shown in SEQIDNO:11 or SEQIDNO:12.
4. the restructuring Anti-X activity described in above-mentioned 3, it is characterised in that shown in shown in the aminoacid sequence of the variable region of heavy chain of described antibody such as SEQIDNO:5-10 or SEQIDNO:13 is arbitrary, the aminoacid sequence of variable region of light chain such as SEQIDNO:4；Or the aminoacid sequence of the variable region of light chain of described antibody is such as shown in SEQIDNO:11 or SEQIDNO:12, the aminoacid sequence of variable region of heavy chain such as SEQIDNO:3；Or the aminoacid sequence of the variable region of heavy chain of described antibody is such as shown in SEQIDNO:13, the aminoacid sequence of variable region of light chain is such as shown in SEQIDNO:11.
5. the restructuring Anti-X activity described in above-mentioned 4, it is characterised in that the aminoacid sequence of the variable region of heavy chain of described antibody is such as shown in SEQIDNO:13, and the aminoacid sequence of variable region of light chain is such as shown in SEQIDNO:11.
6. the arbitrary described restructuring Anti-X activity of above-mentioned 1-5, it is characterised in that the aminoacid sequence of the CH of described antibody is such as shown in SEQIDNO:15, and the aminoacid sequence of constant region of light chain is such as shown in SEQIDNO:17.
7. a preparation, containing the arbitrary described restructuring Anti-X activity of the claims 1 to 6 and pharmaceutically useful carrier.
8. preparation purposes in the medicine of preparation treatment tumor described in above-mentioned 1 to 6 arbitrary described Anti-X activity or claim 7.
9. the purposes described in above-mentioned 8, it is characterised in that described tumor is colorectal cancer.
10. the purposes described in above-mentioned 8, described treatment also includes the antineoplastic agent combined administration with other.
More specifically, the applicant of the present invention first anti-VEGF mAb data and sequence disclosed in Chinese patent CN02111093.X, construct light, the heavy chain variable region gene of VEGF antibody 6A6, then pass through expression, purification, final acquisition VEGF antibody 6A6.Amino acid mutation site according to Fig. 1, adopts the mode of overlapPCR that the method antagonist of series jump is built, and it builds, expresses, the method for purification is identical with the 6A6 not suddenlyd change.Described sudden change includes the aminoacid replacement on any one or more amino acid sites following of 6A6 variable region of heavy chain and/or variable region of light chain, described site is the 35th, 90, variable region of heavy chain, the position of variable region of light chain the 75th amino acids, substituted amino acid is glutamic acid (E), lysine (K), arginine (R), serine (S) or threonine (T).
For the present invention, any suitable expression vector can use, these carriers can be pcDNA3.1 (+), pDR1, the carrier for expression of eukaryon such as pDHFF.
The mutant antibodies of above-mentioned 6A6 has been carried out affinity detection by the applicant of the present invention.Test result indicate that, the affinity of the 6A6 mutant antibodies that applicant builds is greatly improved than commercially available anti-VEGF mAb, the mutant antibodies (H35HT or H35HR) that wherein the 35th, variable region of heavy chain threonine or arginine replace, with the mutant antibodies (L75FK) that the 75th, variable region of light chain arginine replaces, affinity improves at least 3 times；The mutant antibodies (H90DR or H90DS) that the 90th, variable region of heavy chain arginine or serine replace, affinity improves more than 7 times；Its affinity improves 6 times；Carry out the combinatorial mutagenesis of or at 2 at 3 for the catastrophe point of above-mentioned variable region of heavy chain and variable region of light chain, wherein H35HT, H90DR and L75FK three point mutation, its affinity improves nearly 15 times.
Applicant is also directed to the function of 6A6 mutant and studies.By the ADCC Function detection to 6A6 mutant, raising along with 6A6 mutant affinity, ADCC function is significantly enhanced, tested by mouse survival in high-affinity 6A6 mutant body, result shows, H35HT, H90DR and L75FK three the more existing 6A6 of point mutation antibody on A431 multicellular animal model, it is shown that good oncotherapy effect (P < 0.01).
The 6A6 mutant antibodies of the above-mentioned high-affinity of disclosure, pharmaceutical preparations composition can be formed thus more stably playing curative effect together with pharmaceutically acceptable adjuvant, the suspendible that preparation can be commonly used for pharmaceutical field, liquid drugs injection, the preparations such as lyophilizing, preferred liquid drugs injection or lyophilized formulations, liquid drugs injection or lyophilized formulations for said mutation antibody disclosed by the invention, pharmaceutically acceptable adjuvant includes surfactant, solution stabilizer, isoosmotic adjusting agent and buffer one or a combination set of, wherein surfactant includes nonionic surfactant such as Polyoxyethylene Sorbitol Fatty Acid Esters (polysorbas20 or 80)；Poloxamer (such as poloxamer188)；Triton；Sodium lauryl sulphate (SDS)；Sodium laurylsulfate；Myristyl, sub-oil base or octadecyl sarcosine；Pluronics；MONAQUATTM etc., its addition should make C2B8 mutant antibodies granulating trend minimum, solution stabilizer can be saccharide, including reducing sugar and nonreducing sugar, amino acids includes monosodium glutamate or histidine, alcohols includes trihydroxylic alcohol, senior sugar alcohol, propylene glycol, Polyethylene Glycol one or a combination set of, the addition of solution stabilizer should make the preparation those skilled in the art eventually formed think to reach to remain stable for state in the stable time, isoosmotic adjusting agent can be sodium chloride, one of mannitol, buffer can be TRIS, histidine buffering liquid, one of phosphate buffer.
Above-mentioned preparation is the compositions of the 6A6 mutant antibodies comprising high-affinity, and after the animal including people is administered, antitumous effect is obvious.Specifically, to the prevention of tumor and/or therapeutically effective, it is possible to as the drug use of prevention and/or treatment tumor.
6A6 mutant antibodies disclosed by the invention and compositions thereof can also with other antineoplastic agent administering drug combinations, for the treatment of tumor, these antineoplastic agents include 1, cytotoxic drug (1) acts on the medicine of DNA chemical constitution: alkylating agent such as nitrogen mustards, nitrous urine class, pyrovinic acid esters；Platinum-like compounds is cisplatin, carboplatin and JM-216 etc. such as；Mitomycin (MMC)；(2) medicine of nucleic acid synthesis is affected: dihydrofolate reductase inhibitor such as methotrexate (MTX) and Alimta etc.；Thymus nucleoside synthetase inhibitors such as fluorouracil (5FU, FT-207, capecitabine) etc.；Purine nucleosides synthetase inhibitors such as Ismipur (6-MP) and 6-TG etc.；Ribonucleotide reductase inhibitor such as hydroxyurea (HU) etc.；DNA polymerase inhibitor such as cytosine arabinoside (Ara-C) and gemzar (Gemz) etc.；(3) medicine of transcribed nucleic acid is acted on: selectively acting is in DNA profiling, it is suppressed that DNA dependenc RNA polymerase, thus suppressing the RNA medicine synthesized such as actinomycin D, daunorubicin, amycin, epirubicin, aklavine, mithramycin etc.；(4) medicine of tubulin synthesis is mainly acted on: paclitaxel, taxotere, vincaleucoblastine, vinorelbine, Rhizoma Dysosmae Versipellis alkali class, homoharringtonine；(5) other Cytotoxic drugs: asparaginase mainly suppresses the synthesis of protein；2, hormones estrogen antagonist: tamoxifen, droloxifene, exemestane etc.；Arimedex: aminoglutethimide, Lan Telong, letrozole, auspicious Ningde etc.；Androgen antagonist: its ammonia RH-LH agonist/antagonist of fluorine: Zoladex, enatone etc.；3, biological response modifier: suppress tumor interferon mainly through body's immunity；Interleukin II；Thymic；4, monoclonal antibody: Cetuximab (6A6)；Trastuzumab (Trastuzumab) Bevacizumab (Avastin)；5, other draw together some machine-processed medicines failed to understand and need research further at present；Cell-differentiation inducers is retinoids such as；Cell death inducer.6A6 mutant antibodies disclosed by the invention and compositions thereof can with above-mentioned antitumor drug one or a combination set of drug combinations.
The tumor of indication of the present invention includes the epithelial origin tumor that the VEGF such as rectal cancer, non-squamous nonsmall-cell lung cancer, glioblastoma multiforme, metastatic renal cell cancer, breast carcinoma are positive, will not enumerate here.
Accompanying drawing explanation
The aminoacid sequence (in figure the local mutational site representing the mutant built of wire) of Fig. 1 .6A6 heavy chain of antibody and variable region of light chain；
Fig. 2 .6A6 mutant antibodies antitumous effect contrast and experiment figure.
Detailed description of the invention
The present invention is only further detailed by following example, should not be construed as limitation of the present invention.Embodiment does not include detailed descriptions of conventional methods, such as those methods drawn for carrier construction and matter, the method that the gene of encoding proteins is inserted into such carrier and plasmid or the method that plasmid introduces host cell.Such method is well-known for person having ordinary skill in the art, and described by having in many publications, including Sambrook, J., Fritsch, E.F.andManiais, T. (1989) MolecularCloning:ALaboratoryManual, 2ndedition, ColdspringHarborLaboratoryPress.
Embodiment 1. people's antibody is light, the clone of weight chain constant area gene
With lymphocyte separation medium (purchased from Ding Guo biotech development company) separating health human lymphocyte, total serum IgE is extracted with Trizol reagent (Invitrogen company), according to document (ClonedhumanandmousekappaimmunoglobulinconstantandJregion genesconservehomologyinfunctionalsegments.HieterPA, MaxEE, SeidmanJG, MaizelJVJr, LederP.Cell.1980Nov；22 (1Pt1): 197-207.) and document (ThenucleotidesequenceofahumanimmunoglobulinCgamma1gene.E llisonJW, BersonBJ, HoodLE.NucleicAcidsRes.1982Jul10；10 (13): 4071-9.) sequence reported separately designs primer and adopts RT-PCR reaction amplification heavy chain constant region and light chain constant region gene.PCR primer reclaims and is cloned in pGEM-T carrier (purchased from promega company) through agarose gel electrophoresis purification, confirm after sequence verification to obtain correct clone, wherein, SEQIDNO:14 and SEQIDNO:15 respectively illustrates nucleotide sequence and the aminoacid sequence of CH (CH), and SEQIDNO:16 and SEQIDNO:17 respectively illustrates nucleotide sequence and the aminoacid sequence of constant region of light chain (CL).The correct clone of the CH in this example is denoted as pGEM-T/CH, and the correct clone of constant region of light chain is denoted as pGEM-T/CL.
The expression vector establishment of embodiment 2. VEGF antibody 6A6
Anti-VEGF mAb data and sequence disclosed in reference Chinese patent CN02111093.X, entrust Shanghai Sheng Gong biological engineering company limited full genome synthesis Anti-X activity 6A6 heavy chain variable region gene (6A6VH, as shown in SEQIDNO:1) and chain variable region gene (6A6VL, as shown in SEQIDNO:2).Fig. 1 shows the aminoacid sequence (also seeing SEQIDNO:3 and SEQIDNO:4) of 6A6 heavy chain and variable region of light chain.
With 6A6VH gene and pGEM-T/CH carrier for template by over-lap PCR synthetic antibody heavy chain gene, reaction condition is: 95 DEG C 15 minutes；94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 50 seconds, 30 circulations；72 DEG C 10 minutes.Obtaining PCR primer 6A6VHCH, its 5' end contains restriction enzyme sites Hind III and signal peptide gene sequence, and 3' end contains translation stop codon TAA and restriction enzyme sites EcoR I, and signal peptide gene sequence is such as shown in SEQIDNO:18.Last agarose gel electrophoresis separation pcr amplification product, reclaims purpose band and is cloned in pGEMT carrier, and screening positive clone checks order.Select the correct clone Hind III of order-checking and EcoR I enzyme action, heavy chain of antibody fragment 6A6VHCH is reclaimed through agarose gel electrophoresis purification, with with the plasmid pcDNA3.1 of Hind III and EcoR I enzyme action (+) (purchased from American Invitrogen company) be attached, be built into heavy chain eukaryotic expression vector pcDNA3.1 (+) (6A6VHCH).
With 6A6VL gene and pGEM-T/CL carrier for template by over-lap PCR synthetic antibody light chain gene, reaction condition is: 95 DEG C 15 minutes；94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 50 seconds, 30 circulations；72 DEG C 10 minutes.Obtaining PCR primer 6A6VLCL, its 5' end contains restriction enzyme sites Hind III and signal peptide gene sequence, and 3' end contains translation stop codon TAA and restriction enzyme sites EcoR I, and signal peptide gene sequence is such as shown in SEQIDNO:19.Select the correct clone Hind III of order-checking and EcoR I enzyme action, antibody light chain fragment 6A6VLCL is reclaimed through agarose gel electrophoresis purification, it is attached with the plasmid pcDNA3.1 carrier of Hind III and EcoR I enzyme action (purchased from American Invitrogen company), is built into light chain eukaryotic expression vector pcDNA3.1 (6A6VLCL).
The stably express of embodiment 3. VEGF antibody 6A6 and purification
3 × 10 are inoculated in added with the 3.5cm tissue culture dishes containing blood serum medium5CHO-K1 cell (ATCCCRL-9618), cell is cultured to when 90%-95% merges and transfects, and concrete transfection process is as follows:
Take plasmid 10 μ g (plasmid pcDNA3.1 (+) (6A6VHCH) 4 μ g and plasmid pcDNA3.1 (6A6VLCL) 6 μ g) and 20 μ lLipofectamine2000Reagent (purchased from Invitrogen company) be dissolved in 500 μ l plasma-free DMEM medium respectively, room temperature stands 5 minutes, by above 2 kinds of liquid mixing, incubated at room 20 minutes is so that DNA-liposome complex is formed, therebetween with the DMEM culture medium of 3ml serum-free replace in culture dish containing blood serum medium, then the DNA-liposome complex of formation is joined in culture dish, it is placed in CO2Incubator adds the 2ml DMEM complete medium containing 10% serum after cultivating 4 hours, is placed in CO2Incubator continues cultivate.
After transfection carries out 24h, screening resistance clone with containing 600 μ g/mlG418 Selective agar medium, wherein, screening technique is for taking cells and supernatant ELISA detection screening high-expression clone, and screening process is as follows:
Goat anti-human igg (Fc) is coated in elisa plate, 4 DEG C overnight, 2h is closed in 37 DEG C with 2%BSA-PBS, add resistance clone culture supernatant to be measured or standard substance HumanmyelomaIgG1, κ (available from Sigma), 37 DEG C of incubation 2h, add HRP-goat anti-human igg (κ) (purchased from SouthernBiotechnologyAssociates company) and carry out association reaction, 37 DEG C of incubation 1h, add TMB (tetramethyl benzidine) to act on 5 minutes in 37 DEG C, finally use H2SO4Terminate reaction, survey A450Value.
High-expression clone serum-free medium amplification culture screening obtained, separates antibody purification 6A6 with ProteinA affinity column (purchased from GE company).
Antibody purification PBS is dialysed, finally quantitatively determines the concentration of purified antibodies with ultraviolet absorption method.
The structure of embodiment 4.A6A antibody mutants and expression
nullThe mode adopting overlapPCR carries out the structure of A6A antibody mutants，It builds、Express、The method of purification is identical with A6A antibody，The antibody mutants totally 10 built，It is respectively designated as Cmut1～Cmut10，The mutational site of each mutant antibodies is respectively as shown in table 1，The aminoacid sequence of variable region of heavy chain (VH) and variable region of light chain (VL) is shown in SEQIDNO:5～13 respectively，Wherein，SEQIDNO:5 is the mutant nucleotide sequence that 35 amino acids residues of A6A heavy chain variable amino acid sequence are sported T by H，SEQIDNO:6 is the mutant nucleotide sequence that 35 amino acids residues of A6A heavy chain variable amino acid sequence are sported R by H，SEQIDNO:7 is the mutant nucleotide sequence that 35 amino acids residues of A6A heavy chain variable amino acid sequence are sported S by H，SEQIDNO:8 is the mutant nucleotide sequence that 90 amino acids residues of A6A heavy chain variable amino acid sequence are sported R by D，SEQIDNO:9 is the mutant nucleotide sequence that 90 amino acids residues of CA6A heavy chain variable amino acid sequence are sported S by D，SEQIDNO:10 is the mutant nucleotide sequence that 90 amino acids residues of A6A heavy chain variable amino acid sequence are sported E by D，SEQIDNO:11 is the mutant nucleotide sequence that 75 amino acids residues of A6A chain variable region amino acid sequence are sported K by F，SEQIDNO:12 is the mutant nucleotide sequence that 95 amino acids residues of A6A chain variable region amino acid sequence are sported R by F,The 35 amino acids residues that SEQIDNO:13 is A6A heavy chain variable amino acid sequence are sported T by H，95 amino acids residues are sported the mutant nucleotide sequence of R by D.
The biacore of embodiment 5.A6A and mutant identifies
By SA chip (purchased from GE company) 25 DEG C of balance 30min in the PBS solution of 50 μ l/min, then activate 3 times with the activating solution of 1MNaCl and 50mMNaOH, each 1min；People's VEGF molecule (purchased from R&D company) of biotin labelling is diluted to final concentration of 1 μ g/ml, carries out being coated (Δ Ru=1000) with the flow velocity of 10 μ l/min；Then 10min is balanced with PBS 50 μ l/min.Chip after balance is closed chip by the biotin solution of 0.04%.By antibody to wait five concentration of two doubling dilutions, 50 μ l/min loadings 75 seconds, then dissociate 10 minutes with PBS.Under same sample concentration, the relative value of the affinity of mutant antibodies Cmut1-10 is as shown in table 1.
Table 1.biacore detects the affinity of antibody mutants
WT represents the A6A antibody not suddenlyd change；Mutant represents the mutant antibodies of A6A.
Test result indicate that, relative to the existing A6A antibody not suddenlyd change, simple point mutation antibody Cmut6 affinity improves more than 1 times, and Cmut1, Cmut2, Cmut7 affinity all improves more than 3 times, and Cmut4 and Cmut5 affinity improves more than 7 times；Two point mutation antibody Cmut9 affinitys all improve more than 2 times；And three Point mutont antibody Cmut10, its affinity improves 15 times especially.
Embodiment 6.A6A mutant antibodies antitumous effect is tested
Take 30 SCID mice (purchased from Bi Kai company), 3.5 × 105People's above-mentioned SCID mice of colon-cancer cell Caco-2 (ATCC, HTB-37) subcutaneous injection；After 5 days, mice is randomly divided into 3 groups, often group 10；The Cmut10 mutant antibodies of the intravenous injection 100 μ g present invention, existing A6A antibody and PBS respectively, observes the life span of mice.Observe mice situation every day, when gross tumor volume is more than 2000mm3Time, put to death this mice.Survival curve is figure to carry out with reference to the method for Kaplan-Meier, compares employing log-rank inspection and be analyzed [BlandJM, AltmanDG.Survivalprobabilities (theKaplan-Meiermethod) .BMJ.1998 between group；317:1572.], experimental result is as in figure 2 it is shown, Caco-2 cell tumour mouse model is demonstrated stronger antitumous effect (P < 0.001) by the mutant antibodies of the present invention.Other A6A mutant antibodies is also carried out identical experiment, test result indicate that, except Cmut3 and Cmut8, other A6A mutant antibodies all demonstrates the antitumous effect more higher than existing A6A monoclonal antibody.
More than test result indicate that, the restructuring VEGF antibody of the present invention shows higher affinity than existing A6A monoclonal antibody, has stronger antitumous effect, can be used for preparation treatment tumor, particularly prepares the medicine of anti-colorectal carcinoma and metastatic carcinoma.
Claims (10)
1. a restructuring Anti-X activity, it is characterized in that, the aminoacid that described antibody includes on the 35th of the variable region of heavy chain as shown in SEQIDNO:2 the, 90 sites is replaced, and/or the aminoacid on the 75th of the variable region of light chain as shown in SEQIDNO:4 the site is replaced.
2. restructuring Anti-X activity according to claim 1, it is characterised in that the aminoacid on described site is replaced by glutamic acid, lysine, arginine, serine or threonine.
3. restructuring Anti-X activity according to claim 2, it is characterized in that, shown in shown in the aminoacid sequence of the variable region of heavy chain of described antibody such as SEQIDNO:5-10 or SEQIDNO:13 is arbitrary, and/or the aminoacid sequence of variable region of light chain is such as shown in SEQIDNO:11 or SEQIDNO:12.
4. restructuring Anti-X activity according to claim 3, it is characterised in that shown in shown in the aminoacid sequence of the variable region of heavy chain of described antibody such as SEQIDNO:5-10 or SEQIDNO:13 is arbitrary, the aminoacid sequence of variable region of light chain such as SEQIDNO:4；Or the aminoacid sequence of the variable region of light chain of described antibody is such as shown in SEQIDNO:11 or SEQIDNO:12, the aminoacid sequence of variable region of heavy chain such as SEQIDNO:3；Or the aminoacid sequence of the variable region of heavy chain of described antibody is such as shown in SEQIDNO:13, the aminoacid sequence of variable region of light chain is such as shown in SEQIDNO:11.
5. restructuring Anti-X activity according to claim 4, it is characterised in that the aminoacid sequence of the variable region of heavy chain of described antibody is such as shown in SEQIDNO:13, and the aminoacid sequence of variable region of light chain is such as shown in SEQIDNO:11.
6. according to the arbitrary described restructuring Anti-X activity of claim 1-5, it is characterised in that the aminoacid sequence of the CH of described antibody is such as shown in SEQIDNO:15, and the aminoacid sequence of constant region of light chain is such as shown in SEQIDNO:17.
7. a preparation, containing the arbitrary described restructuring Anti-X activity of the claims 1 to 6 and pharmaceutically useful carrier.
8. preparation purposes in the medicine of preparation treatment tumor according to the arbitrary described Anti-X activity of claim 1 to 6 or claim 7.
9. purposes according to claim 8, it is characterised in that described tumor is colorectal cancer.
10. purposes according to claim 8, described treatment also includes the antineoplastic agent combined administration with other.
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| CN201410850230.7A Pending CN105801695A (en) | 2014-12-30 | 2014-12-30 | Recombinant anti-VEGF monoclonal antibody and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110003328A (en) * | 2018-01-05 | 2019-07-12 | 百奥泰生物制药股份有限公司 | A kind of the recombination anti-vegf Humanized monoclonal antibodies and its production method of long-acting low toxicity |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110003328A (en) * | 2018-01-05 | 2019-07-12 | 百奥泰生物制药股份有限公司 | A kind of the recombination anti-vegf Humanized monoclonal antibodies and its production method of long-acting low toxicity |
| CN110283248A (en) * | 2018-01-05 | 2019-09-27 | 百奥泰生物制药股份有限公司 | A kind of the recombination anti-vegf Humanized monoclonal antibodies and its production method of long-acting low toxicity |
| CN110283248B (en) * | 2018-01-05 | 2020-07-28 | 百奥泰生物制药股份有限公司 | Long-acting low-toxicity recombinant anti-VEGF humanized monoclonal antibody and production method thereof |
| CN110003328B (en) * | 2018-01-05 | 2022-04-19 | 百奥泰生物制药股份有限公司 | Long-acting low-toxicity recombinant anti-VEGF humanized monoclonal antibody and production method thereof |
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