CN1187373C - Human resourced monoclone antibody of anti-blood-vessel endothelium growth factor as well as its preparing method and medicine composition - Google Patents

Abstract

The present invention provides a monoclonal antibody of an anti-human VEGF. The variable region of a light chain has an amino acid sequence shown in the SEQ ID NO : 3, and the variable region of a heavy chain has an amino acid sequence shown in the SEQ ID NO : 1. The bioactivity of the monoclonal antibody and the expression amount of the monoclonal antibody in a host cell are obviously enhanced. The present invention also provides DNA molecules for encoding the monoclonal antibody, a preparation method of the monoclonal antibody and a medical composition containing the monoclonal antibody.

Description

Human resourced monoclone antibody of anti-blood-vessel endothelium growth factor and method for making thereof and pharmaceutical composition

Invention field

The present invention relates to biological technical field.Specifically, the present invention relates to a kind of new humanization anti-vascular endothelial growth factor (VEGF) monoclonal antibody.In addition, the pharmaceutical composition that the invention still further relates to this MONOCLONAL ANTIBODIES SPECIFIC FOR method and contain this monoclonal antibody.

Background of invention

People just find before more than 100 year, tumor tissues is more normally organized and is rich in blood vessel, but it is to provide nutrition by the blood vessel that has existed that people are arguing tumour always, still provide nutrition by new vessel, and generally believing that this vascular reaction is a kind of inflammatory reaction, is not that tumor growth institute is essential.1971, it was that blood vessel is dependent that Folkman proposes tumor growth the earliest, but this at that time viewpoint is not accepted by the people.Foundation along with capillary endothelial cell culture technique after 8 years, the discovery of first angiogenesis inhibitor after 11 years, a series of activities such as proteic purifying of first angiogenic activity finishes after 13 years, this viewpoint is supported by more and more evidences, and is made this field become the focus of tumor research and the New Policy of oncotherapy.

Growth of tumor has two visibly different stages, and promptly changing into from avascular slow growth phase has the fast breeding of the blood vessel stage, and vasculogenesis makes tumour can obtain enough nutritive substances, is the key link of facilitating above-mentioned transformation.If there is not vasculogenesis, the growth of primary tumo(u)r can not surpass 1～2mm ³

It is the major cause of oncotherapy failure that tumor invasion shifts.It is the multistage process of a complexity that tumor invasion shifts, and may be summarized to be: primary tumor propagation, tumor neogenetic blood vessels growth; Oncocyte invasion and attack basilar membrane; Penetrate blood vessel or lymphatic vessel; In the recycle system, survive, form the knurl bolt and be transported to and be far apart target organ; Be stranded in the tiny blood vessels of target organ; Pass blood vessel and form micro metastasis; Tumor vessel forms, metastatic carcinoma kitchen range propagation.As seen in the rapid process of multistep of tumour generation Invasion and Metastasis, no matter metastases initial or end stage eventually, vasculogenesis is all being brought into play important effect.

The mensuration of microvessel density helps the diagnosis of tumour and prognosis to judge in the tumor tissues, as: breast tumor microvessel density and its transfer performance are proportionate.Patient with breast cancer for the lymphoglandula feminine gender, multiplicity shows, microvessel density is with respect to classification, tumour size, estrogen receptor positive or other marks of tumour, be the better index of judging Metastasis in Breast Cancer potential, this result is further confirmed by perspective study in 5 years.In addition, other kinds of tumors microvessel densitys are also in close relations with transfer, recurrence, prognosis, as neck squamous cell carcinoma, melanoma, prostate cancer, ovarian cancer, bladder cancer, brain tumor, nonsmall-cell lung cancer, the rectum cancer, myelomatosis etc.Existence that it should be noted that new vessel can not be used for discriminating optimum, malignant tumour, belongs to innocent tumour as adenoma,adrenal, but rich blood vessel.This shows that growth of tumor, transfer, recurrence, prognosis and vasculogenesis are closely related, is target spot with the vasculogenesis of tumour, and the exploitation angiogenesis inhibitor might make the result of treatment of solid tumor obtain bigger raising.

It is basis (Folkman J.N Engl J Med, 1971,285 (21): 1182) of tumor growth, transfer that tumor vessel forms.Since (Folkman J.J Biol Chem such as Weidner, 1992,267 (16): 10931) reported that tumor tissues microvessel density (MVD) has been one of the patient with breast cancer independently since the prognostic indicator, in many tumours, comprises that the prognostic value of MVD in the primary hepatocarcinoma has all obtained (Wang Dong certainly, Gao Fengxun, Chen Li etc., the research of osteosarcoma microvessel density and prognosis, Chinese pathology magazine, 1997,26 (5): 266).Tumor neovasculature formation is the essential condition of its growth, infiltration, transfer.The growth and the transfer of solid tumor depend on vasculogenesis, and therefore disturbing and block the confession of tumour blood is effective methods of treatment.Many interior microvessel density (Intratumoral microvessel density of tumour that studies show that, MVD) relevant with prognosis (Wang Dong etc. with osteosarcomatous transfer, the same), in multiple vasculogenesis correlation factor, vascular endothelial growth factor (VEGF) continues high expression level, and this proof VEGF is that human body osteosarcoma important vessel generates one of correlation factor.

Proved already that most of solid malignants can secrete multiple angiogenic growth factor, induction of vascular generates, and supports tumor growth (Folkman J, Science, 1987,235 (4787): 442).The blood vessel that tumour induces all has characteristic on pathology, physiology still are morphological function, these all are that traditional chemotherapeutics has constituted " bloodtumorbarrier ", make escaped (Hori K L.J Cancer Res, 1991,82 (1): 109) of killing and wounding of chemotherapeutics of tumour cell.The difference of identification tumor-blood-vessel growth process and normal blood vessels generative process, selectivity are attacked tumor vessel specific target position, will quicken the oncotherapy process greatly.

VEGF is that the intensive endothelial cell mitogen that purifies and separates is come out from Niu Chuiti folliculus stellate cell culture is former at first, by combining with tyrosine kinase receptor flt1 and KDR/flk1 specificity on the endotheliocyte, make the receptor autophosphorylation phosphorylation and the interior signal transduction pathway of activating cells, produce biological effect such as inducing endothelial cell propagation, migration, regulate endotheliocyte and integrate the expression of plain and the proteinase activated factor, increase microvascular permeability, promote that plasma proteins leaks outside, basement membrane degradation causes new matrix and new vessel chamber to form.In the 20 multiple polypeptides class angiogenesis factors of finding so far, VEGF is the highest at endothelial cell specific, crucial regulatory factor (the Ferrara N.Endocr Rev that the angiogenic growth effect is the strongest, 1997,18 (1): 4), in multiple primary malignant tumor tissue, all be high expression level (Human Pathol such as Brown L F, 1995,26 (1): 86); Recently (Cancer Res, 1995,55 (18): 3964) find the close ties that have that VEGF high expression level and colorectal carcinoma shift, it is very important to point out VEGF all to play a part in the formation of the formation of tumour and growth and metastatic tumor in position for Takahashi.Verified other multiple angiogenesis factor plays a role by directly or indirectly inducing, stimulate vegf expression, all can raise expression (the Li J of VEGF as TGF-α, bFGF, TGF-β, TNF-α, KGF etc., Hampton T etc., J Clin Invest, 1997,100 (1): 18; Brown L F etc., Detmar M, Claffey K, et al.Vascular permeabilityfactor/vascular endothelial growth factor:a multifunctional angiogenic cytokine.Exs, 1997,79:233).Therefore, VEGF is the desirable target spot of blocking-up tumour blood confession.Kim KJ utilizes the VEGF monoclonal antibody successfully to suppress rhabdosarcoma, glioblastoma, leiomyosarcoma in the intravital growth of nude mice; Asano (Asano M etc., Cancer Res, 1995,55 (22): 5196) utilize VEGF ₁₂₁Monoclonal antibody MV ₃₀₃Not only suppress the Human umbilical vein endothelial cells growth of vitro culture and the growth of the interior human fibrosarcoma cell's strain HT-1080 of BALB/C mouse body, and suppressed the formation of BALB/C mouse metastatic tumor.Our experiment confirm VEGF polyclonal antibody also has restraining effect to the vasculogenesis of osteosarcoma cell OS-732 and the growth of tumour cell, and the effect of VEGF polyclonal antibody may be more comprehensively than the VEGF monoclonal antibody, the effect of blocking VEGF more fully during smaller dose.

Angiogenesis relates to a series of morphology and biochemical change.Morphological change comprises that orientation movement, generation mitotic division, the lumen of vessels of the venular basilar membrane of endotheliocyte biodegradable carrier, endotheliocyte form, the bud formula is grown and form the basilar membrane that blood vessel is fastened with a rope, string, etc., generation is new, the series of steps such as formation of adventitial cell.Generally, endotheliocyte is in dormant state, is extreme immobilized cell in the body.The renewal of endotheliocyte needs hundreds of days, only needs 5 days and the renewal of medullary cell is average, division speed about 6 * 10 ⁹Cell/hour.In the vasculogenesis, the rate of propagation of capillary endothelium is identical with medullary cell, and the adjusting that its biochemical mechanism relates between angiogenesis factor and the Angiostatin is unbalance.

More than 20 kind of angiogenesis factor and correlation factor have been separated with purifying at present, at least 15 kinds of Angiostatins.Angiogenesis factor comprise vascular endothelial growth factor (VPF/VEGF), acidity and Prostatropin (aFGF, bFGF), angiogenin, placenta growth factor (PIGF), Urogastron (EGF), interleukin-8, tumor necrosis factor alpha (TNF α) etc.In all angiogenesis factors, the most deep to the research of VEGF and bFGF.Endothelial cell division propagation and the blood vessel that VEGF has increases microvascular permeability, promote different sources makes up, impels the multiple effects such as migration of endotheliocyte, is penetrating dose of the strongest known blood vessel.VEGF is two kinds of type tyrosine kinase acceptor flt-1 on the vasoactive endothelial cell membrane and KDR optionally, by phosphoinositide specificity Phospholipase C IP3 concentration is raise and plays a role.

Each link and the biochemical change in the generating process thereof with vasculogenesis are target spot, and the development angiogenesis inhibitor is controlled tumor growth and transfer, will become an important channel of treatment and prevention of tumour.On the whole, the research of angiogenesis inhibitor mainly contains following 4 kinds of strategies; (1) ability of matrix around the degraded of blocking-up endotheliocyte; (2) directly suppress the function of endotheliocyte; (3) the synthetic and release of blocking-up angiogenesis factor, its effect of antagonism; (4) the blocking-up endothelial cell surface is integrated plain effect.

At present the factor of finding with angiogenesis inhibitor ability mainly contains acceptor and the anti-VEGF monoclonal antibody of VEGF etc.Extensive studies proves that anti-VEGF monoclonal antibody has the function of obvious suppression tumor-blood-vessel growth.The monoclonal anti physical efficiency suppresses the propagation of human vascular endothelial, can suppress tumor neogenetic blood vessels in vivo and form, thereby suppress growth of tumor and transfer, and its inhibiting rate can reach 49%-70%.

In sum, anti-VEGF monoclonal antibody has demonstrated great application prospect in oncotherapy.

Yet the expression level of producing the cell strain of this VEGF monoclonal antibody in the art only is the 50-100 mg/litre, and this far can not satisfy the needs of scale operation monoclonal antibody.And the activity of at present commercially available VEGF monoclonal antibody goods is not high yet.Therefore, still press for the VEGF monoclonal antibody that provides a kind of activity and expression amount to make moderate progress in this area.

Goal of the invention

The purpose of this invention is to provide a kind of recombinant anti human VEGF monoclonal antibody.

Another object of the present invention provides the dna molecular of the above-mentioned anti-VEGF monoclonal antibody of coding.

Another object of the present invention provides a kind of pharmaceutical composition that contains said monoclonal antibody.

A further object of the invention provides the method for preparing said monoclonal antibody.

To achieve the above object of the invention, one aspect of the present invention provides a kind of anti-people VEGF monoclonal antibody, this antibody contains variable region of heavy chain and variable region of light chain, wherein variable region of light chain has the aminoacid sequence shown in the SEQ ID NO:3, and variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO:1.

The present invention provides the dna molecular of coding said monoclonal antibody on the other hand.

In a preferable example, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the SEQ ID NO:4, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in the SEQ ID NO:2.

Third aspect present invention provides a kind of expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna sequence dna and links to each other with this series of operations.

Fourth aspect present invention provides a kind of host cell, and wherein this host cell is transformed by above-mentioned expression vector.This host cell preferred mammal cell, more preferably Chinese hamster ovary celI.

Fifth aspect present invention provides a kind of pharmaceutical composition for the treatment of tumour, and this pharmaceutical composition contains the pharmaceutically above-mentioned human monoclonal antibodies and the pharmaceutically acceptable carrier of significant quantity.

Sixth aspect present invention provides a kind of method for preparing said monoclonal antibody, and this method comprises:

A) provide an expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna encoding sequence and links to each other with this series of operations;

B) with the described expression vector transformed host cell of step a);

C) host cell of gained culturing step b under the condition that is fit to described monoclonal antibody expression); With

D) separation and purification obtains described monoclonal antibody.

The physiologically active and the expression amount in host cell (as Chinese hamster ovary celI) that the invention has the advantages that monoclonal antibody of the present invention are significantly increased than existing monoclonal antibody.Other purpose of the present invention and advantage can be learnt by following detailed.

Description of drawings

Fig. 1 has shown the restriction enzyme mapping of the used pMG18 carrier of the present invention.Ck represents the constant region gene of antibody kappa light chain among the figure; The IgG1 constant region is represented the weight chain constant area gene of people's IgG antibody 1; PA represents that SV40 adds poly A site.

Detailed Description Of The Invention

The present invention relates to a kind of anti-human VEGF monoclonal antibody of restructuring, this antibody comprises variable region of heavy chain and variable region of light chain, wherein variable region of light chain has the amino acid sequence shown in the SEQ ID NO:3, and variable region of heavy chain has the amino acid sequence shown in the SEQ ID NO:1.

Term used herein " monoclonal antibody (monoclonal antibody) " refers to the antibody that obtains from the colony of the basic homogeneous of a class, and the single antibody that namely comprises in this colony is identical, the sudden change of the natural generation that may exist except minority. Monoclonal antibody is with high specificity for single antigen site. And from conventional Anti-TNF-α body preparation (normally having the different antibodies for different determinants) difference, each monoclonal antibody is for the single determinant on the antigen. Except their specificity, the benefit of monoclonal antibody is that also they are next synthetic by the hybridoma cultivation, can not polluted by other immunoglobulin (Ig). Modifier " monoclonal " has represented the characteristic of antibody, is to obtain from the antibody population of basic homogeneous, and this should not be interpreted into and need to produce antibody with any specific process.

Term used herein " antibody " and " immunoglobulin (Ig) " are the about 150000 daltonian different four glycan albumen that the same structure feature is arranged, and it is comprised of with two identical heavy chains (H) two identical light chains (L). Every light chain links to each other with heavy chain by a covalent disulfide bonds, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different. Every heavy chain and light chain be the intrachain disulfide bond at regular interval also. One end of every heavy chain has variable region (VH), is thereafter a plurality of constant regions. One end of every light chain has variable region (VL), and the other end has constant region; The constant region of light chain is relative with first constant region of heavy chain, and the variable region of light chain is relative with the variable region of heavy chain. Special amino acid residue forms the interface between the variable region of light chain and heavy chain.

Some part of variable region is different on sequence in term used herein " variable " the expression antibody, and it has formed various specific antibodies to combination and the specificity of its specific antigen. Yet changeability is not evenly distributed in the whole antibody variable region. It concentrates in three fragments that are called in light chain and the variable region of heavy chain in complementary determining region (CDR) or the hypervariable region. Part conservative in the variable region is called framework region (FR). Each self-contained four FR district in the variable region of natural heavy chain and light chain, they are the beta sheet configuration haply, are linked to each other by three CDR that form connecting ring, but forming section β-pleated sheet structure in some cases. CDR in every chain closely is close together by the FR district and has formed the antigen-binding site (referring to Kabat etc., NIH Publ.No. 91-3242, volume I, 647-669 page or leaf (1991)) of antibody with the CDR of another chain. Constant region is not participated in the combination of antibody and antigen directly, but they show different effector functions, for example participates in the cytotoxicity that depends on antibody of antibody.

" light chain " of vertebrate antibody (immunoglobulin (Ig)) can be classified as according to the amino acid sequence of its constant region the class in visibly different two classes (being called κ and λ). According to the amino acid sequence of its CH, immunoglobulin (Ig) can be divided into different kinds. Mainly contain 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them also can further be divided into subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. CH corresponding to the inhomogeneity immunoglobulin (Ig) is called α, δ, ε, γ and μ. The subunit structure of inhomogeneity immunoglobulin (Ig) and 3-d modelling are well-known.

Monoclonal antibody can make with the whole bag of tricks well known to those skilled in the art. For example, monoclonal antibody can use hybridoma method (by Kohler etc., Nature, 256:495 (1975) at first proposes) to make, or makes with recombinant DNA method (U.S. Patent No. 4,816,567). Monoclonal antibody is also available such as Clackson etc., Nature, and 352:624-628 (1991) and Marks etc., J.Mol.Biol., the described technology of 222:581-597 (1991) is separated acquisition from phage antibody library.

The present invention also provides the dna molecular of the anti-VEGF monoclonal antibody of code book invention humanization. In a better example, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the SEQ ID NO:4, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in the SEQ ID NO:2.

Behind the nucleotide sequence that obtains code book invention humanization anti-VEGF monoclonal antibody variable region of heavy chain and variable region of light chain, usually can prepare by the following method monoclonal antibody of the present invention.

At first, provide the nucleotide sequence that contains code book invention monoclonal antibody and the expression vector of the expression regulation sequence that links to each other with this series of operations. But those of ordinary skills also can expect, and the nucleotide sequence of code book invention monoclonal antibody variable region of heavy chain and variable region of light chain is inserted respectively carry out coexpression in the different expression vectors and also can obtain anti-VEGF monoclonal antibody of the present invention.

Term used herein " expression regulation sequence " is often referred to and participates in the sequence that the control nucleotide sequence is expressed. Expression regulation sequence comprises promoter and the termination signal that links to each other with target nucleotide sequence operability. They also comprise the suitably required sequence of translation of nucleotide sequence usually. " operability links to each other " refers to that some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence. For example, if promoter or enhancer have increased transcribing of coded sequence, then it is that operability links to each other with coded sequence.

The dna sequence dna of code book invention monoclonal antibody can make with conventional means well known to those skilled in the art. For example, can be manually synthetic or increase with the PCR method and to obtain encoding the nucleotide sequence of this monoclonal antibody variable region of heavy chain and variable region of light chain according to sequence disclosed by the invention. Then, these nucleotide sequences are inserted in the suitable expression vector by the suitable restriction enzyme site of selection with the whole bag of tricks well known in the art, make them respectively before expression vector entrained CH coded sequence and constant region of light chain coded sequence, and make them in same frame. Used expression vector is various commercially available expression vector well known by persons skilled in the art among the present invention, for example available from the expression vector of Qiagen and Promega company, and commercially available expression vector pMG18 (" too development that carries out environmental monitoring according to INCP-9 plasmid sequence " (DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORING BASED ON INCP-9 PLASMIDS SEQUENCES), A.Greated, R.Krasowiak, M.Titok, C.M.Thomas School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK and Faculty of Biology, Dept of Microbiology, Belarus State University Scorina Av.4, Minsk 220080 Belarus).

Subsequently, the expression vector with above-mentioned acquisition transforms suitable host cell. " host cell " generally comprises prokaryotic and eukaryotic. The example of prokaryotic host cell commonly used comprises Escherichia coli, hay bacillus etc. Eukaryotic host cell commonly used comprises yeast cells, insect cell and mammalian cell. In the present invention, preferred mammal cell. Usually be used as expressing the derive host cell of polypeptide of eukaryotic with mammal cell line. The breeding of mammalian cell in culture is well known in the art. See " tissue cultivation ", Academic Press, Kruse and Patterson edits (1973), and this article is included this paper in as a reference. Better mammalian cell is many commercially available immortal cell lines. These immortal cell lines including, but not limited to, Chinese hamster ovary (CHO) cell, Vero cell, HeLa cell, young hamster kidney (BHK) cell, MK cells (COS), human liver cell cancer cell (such as Hep G2) and other many clones. They provide posttranslational modification for protein molecule, comprise that correct folding, correct disulfide bond forms and the glycosylation in correct site. Although among the embodiment, the present invention has only enumerated with the example of Chinese hamster ovary celI as host cell hereinafter, those skilled in the art can know that the present invention also can adopt above-mentioned these clones having read detailed description of the present invention and specific embodiment.

Method with the expression vector transformed host cell has a variety of, used conversion programs to depend on host to be transformed. The method that heterologous polynucleotide is imported in the mammalian cell is known in the art, it comprises transfection, calcium phosphate precipitation, the Polybrene (1 of glucan mediation, 5-dimethyl-1,5-phenodiazine 11 methylene gather Methobromide) mediation transfection, protoplast fusion, electroporation, liposome-mediated transfection and with the direct microinjection of DNA in karyon. In the present invention, better method is electroporation or liposome mediated-method etc. Come transfection such as host cells such as Chinese hamster ovary celIs such as the liposome method kit that can adopt Invitrogen company.

Then, under the condition that is fit to monoclonal antibody expression of the present invention, cultivate the host cell that transforms gained. Then use conventional immunoglobulin purification step, obtain the anti-VEGF monoclonal antibody of humanization of the present invention such as conventional separation and purification means purifying well known to those skilled in the art such as albumin A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion-exchange chromatography, hydrophobic chromatography, sieve chromatography or affinity chromatographys.

The gained monoclonal antibody can be identified with conventional means. The binding specificity of monoclonal antibody can be measured with immunoprecipitation or external combination test (such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) (ELISA)). The binding affinity of monoclonal antibody such as available Munson etc., Anal.Biochem., the Scatchard of 107:220 (1980) analyzes to measure.

The present invention also provides a kind of pharmaceutical composition for the treatment of tumour, and said composition contains pharmaceutically monoclonal antibody of the present invention and the pharmaceutically acceptable carrier of effective dose. For example, pharmaceutical composition of the present invention can be used to treat cancer of the stomach, liver cancer. Term used herein " pharmaceutically acceptable " refers to when molecule body and composition suitably give the animal or human, and they can not produce disadvantageous, irritated or other bad reaction. " pharmaceutically acceptable carrier " used herein should be compatible with mutain of the present invention, can be with its blend the effect of decrease pharmaceutical composition under normal conditions not. The object lesson that can be used as some materials of pharmaceutically acceptable carrier or its component is carbohydrate, such as lactose, dextrose plus saccharose; Starch is such as cornstarch and potato starch; Cellulose and derivative thereof are such as sodium carboxymethylcellulose, ethyl cellulose and methylcellulose; The tragacanth powder; Fructus Hordei Germinatus; Gelatin; Talcum; Kollag is such as stearic acid and dolomol; Calcium sulfate; Vegetable oil is such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cupu oil; Polyalcohol is such as propane diols, glycerine, D-sorbite, mannitol and polyethylene glycol; Alginic acid: emulsifying agent, such as Tween ; Wetting agent is such as NaLS; Colouring agent; Flavor enhancement; Tablet agent, stabilizing agent; Antioxidant; Anticorrisive agent; Apirogen water; Deng oozing salting liquid; With phosphate buffer etc.

Pharmaceutical composition of the present invention can be made various formulations as required, and can by the doctor according to patient's kind, age, body weight and roughly factor such as disease condition, administering mode determine the useful dosage of patient is used.Administering mode for example can adopt perfusion and other therapeutic modality.

Below in conjunction with embodiment the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment, and be not to be used for limiting the present invention just for an illustration.

Embodiment 1 screens the antibody variable gene of VEGF from antibody library

1) structure of human antibody library

According to people J.Mol.Biol. such as Marks, 222,581-597; Hoogenboom and Winte, J.Mol.Biol., 227,381-388; Haidaris CG etc., J Immunol Methods.2001 Nov 1; 257 (1-2): 185-202; Griffiths, EMBO J. such as A.D., 13,3245-3260 (1994); Nissim, EMBO J. such as A., 13, the method for describing among the 692-698 (1994) makes up human antibody library.

2) screening:

The antibody library bacterial strain of recovery is added 14 milliliters of fresh LB substratum for 1 milliliter, in 50 milliliters of triangular flasks, cultivated 16 hours for 37 ℃.

12000rpm high speed centrifugation 10 minutes shifts in supernatant to one 50 milliliters of aseptic centrifuge tubes, preserves standby.Its titre should be 2 * 10 ¹¹More than.Vegf protein (available from Shenzhen brilliant U.S. company) with purifying is an antigen, and bag is by 25 ml cells culturing bottles.Add in the cell bottle behind the bag quilt and be no less than 3 * 10 ¹⁰Phage particle, 37 ℃ of incubations 1 hour.Then, outwell the liquid in the bottle, wash culturing bottle 10 times with 10 milliliters of PBS that added 1%Tween-20.The TG1 cell that adds 1 milliliter of logarithmic phase in culturing bottle, 37 ℃ of incubation concussions were cultivated 16 hours.

Repeat the described step of epimere totally 4 times.

With cell dilution to 100000 cells/ml of above-mentioned acquisition, on 1.5% agar plate that adds 0.1% penbritin, cultivate to obtain mono-clonal then.Get being cloned on the 96 hole depth orifice plates on the above-mentioned flat board and cultivate, the clone in every hole does 960 clones (10 96 orifice plates) altogether.With above-mentioned deep-well plates centrifugal 20 minutes of 5000RPM on 96 orifice plate whizzers, supernatant is transferred to new aseptic deep-well plates, it is standby to be preserved in 4 degree after sealing.

Get 10 of 96 orifice plates, add in every hole the conventional bag of VEGF (10 mcg/ml) 10 microlitres by after, add supernatant 10 microlitres of above-mentioned preservation respectively, 37 ℃ of incubations after 1 hour with the PBS washing that contains 1%Tween-20 20 times.The goat-anti M13 monoclonal antibody that adds 1 microlitre HRP mark, 37 ℃ of incubations use the PBS of 1%Tween-20 to wash 10 times after 30 minutes.

Add and contain PBS 200 microlitres of 0.025%DAB developer and the H of 1 microlitre 1% ₂O ₂, the 37 ℃ of incubation colour developing was read 595 nanometers after 20 minutes on plate reading machine photoabsorption.Determine the hole that color reaction is strong according to the photoabsorption reading, the corresponding clone in these holes is the stronger antibody variable region clone of avidity.

Filter out 415 positive colonies altogether by said process, determine wherein 5 the strongest clones of avidity according to reading.These 5 clones are seeded in respectively in 100 milliliters of LB substratum, after concussion under 37 ℃ of 260rpm conditions is cultivated 9 hours, add final concentration and be isopropylthiogalactoside (IPTG) inducing culture 10 hours of 1mM." the recombinant human scFv purification system " of using Pharmacia then is with reference to shop instruction separation and purification reorganization scFv antibody protein.The reorganization scFv protein of extracting and purifying is used for avidity research.Avidity studies have shown that have 3 to have higher avidity in these 415 positive colonies, they are called after 5H4,9D2 and 3G6 respectively.Wherein, 3G6 promptly has the highest expression amount, has the highest avidity again, and this clone will be used in follow-up research.

3) the antibody variable region encoding sequence is to the clone of expression vector

Above-mentioned 3G6 clone's bacterial strain is increased in 100 milliliters of LB substratum, with the plasmid DNA extracting and purifying test kit plasmid DNA purification of Promega company.

With XbaI with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after the NheI enzyme is cut above-mentioned plasmid DNA, get band about 350bp and carry out glue and reclaim, the gained fragment is the variable region of heavy chain encoding sequence.

With HindIII with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after Bsi WI enzyme is cut above-mentioned plasmid DNA, get band about 320bp and carry out glue and reclaim, the gained fragment is the variable region of light chain encoding sequence.

At first above-mentioned variable region of heavy chain encoding sequence is inserted into expression vector pMG18-3K then (referring to DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORING BASED ONINCP-9 PLASMIDS SEQUENCES.A.Greated, R.Krasowiak, M.Titok, C.M.ThomasSchool of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B152TT, UK and Faculty of Biology, Dept of Microbiology, Belarus State University ScorinaAv.4, Minsk 220080 Belarus) in the XbaI/NheI site, with HindIII and Bsi WI above-mentioned antibody chain variable region encoding sequence is inserted in the HindIII/BsiWI site of the pMG18-3K that is inserted with the variable region of heavy chain encoding sequence again, is built into humanization VEGF antibody expression carrier.

4) screening of the transfection of Chinese hamster ovary celI and recombinant clone

Above-mentioned steps 3) expression vector that has antibody gene that makes up in increases in 100 milliliters of LB substratum in the escherichia coli DH5a inoculation, with ultrapure plasmid DNA purification kit (UltrapurePlasmid DNA Purification Kit) the extracting and purifying plasmid DNA of Qiagen company.The plasmid DNA of above-mentioned purifying is adopted the liposome method test kit transfection CHO cell of Invitrogen company, and operation is carried out with reference to the specification sheets of producer.

The Chinese hamster ovary celI that transforms carries out the extreme dilution at last and cultivates in the selection of selecting to carry out on the substratum continuous 9 weeks on 96 orifice plates, carry out continuously 3 times, carry out mono-clonalization.

The monoclonal cell of choosing ties up on RPMI 1641 substratum and cultivates, and supernatant is carried out the Western Blot experiment, judges expression intensity according to staining reaction, picks out the stronger clone of 10 expression as candidate cell strain (seeing Table 1).

5) Purification of Monoclonal Antibodies

The purifying of above-mentioned monoclonal antibody adopts the directly separation and purification from cells and supernatant of Protein A (Pharmacia company) affinity column, and proves that with the SDA-PAGE electrophoresis products therefrom purity is greater than 90%.The product of above affinity chromatography passes through sieve chromatography once more, has obtained the sample of purity＞98%.These samples are used for following further analysis and research.

The research of embodiment 2 antibody genes expression intensity in Chinese hamster ovary celI

The high expression level candidate clone that above-mentioned screening is obtained is incubated in the tissue culture ware of 10cm, adopt the ELISA method to measure the expression amount of antibody: goat anti-human igg (Fc) bag quilt is in elisa plate, 4 ℃ are spent the night, in 37 ℃ of sealings 2 hours, add culture supernatant to be measured and standard substance (human IgG1) through 2%BSA, hatched 2 hours for 37 ℃, add HRP-goat anti-human igg (κ) and carry out association reaction, hatched 1 hour for 37 ℃, add TMB, use H at last in 37 ℃ of effects 10 minutes ₂SO ₄Termination reaction is surveyed A ₄₅₀Value.The expression amount that records above-mentioned 10 candidate clones is as follows:

Table 1 antibody gene expression intensity in Chinese hamster ovary celI

Cell strain numbering 3E9 5D7 4C6 5H8 6A6 3B4 4D8 4F6 5G7 6G2

Expression amount (ug/ milliliter) 116.7 128.9 366.7 343.2 408.6 372.1 332.1 176.4 231.4 146.9

As can be seen from the above table, the expression level that is numbered the cell strain of 4C6,5H8,6A6 and 3B4 has very high expression level (340-410 mg/ml), head and shoulders above the expression level of domestic and international monoclonal antibody class (50～100mg/ milliliter).

Embodiment 3 monoclonal antibody physiologically actives and avidity research

1) physiological function research

Owing to only find that vascular endothelial cell has special proliferative response to VEGF so far, the method for this experimental evidence Gospodarowicz D etc. (PNAS, 1989, the 86:7311) biologic activity of the anti-VEGF monoclonal antibody of detection.

Concrete grammar is as follows:

Get one of 96 porocyte culture plate, add 0.5 milliliter in every hole and contain 1 * 10 ⁴The VEGF of bovine adrenal vascular endothelial cell (can obtain from Shanghai RESEARCH ON CELL-BIOLOGY institute of Chinese Academy of Sciences cell bank) nutrient solution and 200IU (available from Shenzhen brilliant U.S. company), the VEGF that added same amount at the 4th day separately is to continue to stimulate the bovine adrenal endothelial cell proliferation.

Testing sample and positive control storage liquid concentration are 500 mcg/ml.Positive control is the RhuFab V2 of Genentech company.Behind PBS diluent serial dilution testing sample that contains 0.2% gelatin and positive control, add 0.5 milliliter of testing sample and positive control in every hole.Negative control hole adds 0.5 ml cells nutrient solution.3 repetitions are established in each processing.At 37 ℃ of 5%CO ₂Cultivated in the incubator 4～5 days.

Learnt from else's experience 0.2 milliliter in the cell of above-mentioned processing, adding 1/10 volumetric concentration is the MTT solution of 5 mg/ml, cultivates the photoabsorption of reading the 570nm place after 30 minutes on microplate reader for 37 ℃.Reference wavelength is 630nm.

Test-results is presented in the following table 2.

Table 2VEGF biological activity is analyzed

Content of the test	Final concentration (mcg/ml)
									0	200	400	800	1600	3200	6400	12800
	Negative control (anti-CD20 monoclonal antibody)	0.34	0.71	0.67	0.33	0.46	0.53	0.73									0.29
Positive control (RhuFab, V2)									3341.2	3432.9	2891.3	1983.4	1508.2	935.6	300.3	6.67
	6A6 of the present invention	3548.7	2083.2	1564.9	210.0	5.71	6.44	0.33									0.32

As can be seen from Table 2, human-derived anti-human VEGF monoclonal antibody of the present invention has very high biological activity, exceeds about 8 times than positive control RhuFab V2.

B) rat liver cancer perfusion therapy effect research

The foundation of rat implantation liver cancer model

After the disconnected neck of subcutaneous tumor-bearing rat put to death, the subcutaneous tumors piece was got by partly sterilised, dials and removes fibrous tissue, select no hemorrhage, do not have downright bad tumor tissues, be cut into 0.2mm with the scraper sheet ³The fritter of size is standby.

Other gets the Wistar rat, press 40 mg/kg intraperitoneal injection of anesthesia with vetanarcol, get dorsal position, four limbs are fixed on the operation plate, field of operation with 75% alcohol disinfecting after, in abdomen medisection skin 10 millimeter, with the microstructure tweezers Glisson's capsule is stabbed an osculum, tumor mass is embedded along the tunnel.The transplanting back layer-by-layer suture stomach wall that finishes is raised stand-byly, and whole process is carried out under aseptic condition.

Methods of treatment

Arterial cannulation (is quoted from Zhou Meifang with reference to Lindell etc., cover hard: 1999, the nursing of hepatic artery catheterization chemotherapy and lak/il-2 combination therapy advanced liver cancer, nursing experiences, V18 (6)) method, behind the liver inoculation knurl piece the 7th day, rat abdominal cavity anesthesia back was fixing, cut open the belly once more (about 2～3 centimetres of opening), expose liver, measure tumor surface maximum diameter (a) and path (b), separation gastroduodenal artery, hepatic artery and proper hepatic artery, the far-end of ligation gastroduodenal artery.With silver brain clip temporary interruption arteria hepatica communis, on gastroduodenal artery, cutting an osculum under the operating microscope, thus with the up insertion proper hepatic artery of the special conduit of the about 0.3mm of external diameter.After treating blood back, pour into salt solution and above-mentioned monoclonal antibody preparation of the present invention respectively by following grouping and dosage (table 3).Perfusion back tube drawing, ligation gastroduodenal artery near-end are decontroled the silver brain clip on the arteria hepatica communis, clean wound behind the layer-by-layer suture otch, be coated with duomycin ointment outward, divide cage constant temperature, automatic water-supply to raise for food.

The tumor growth rate is measured

Every group of part rat put to death in the treatment back on the 7th day, measured the maximum diameter (a) and the path (b) of tumour once more, by formula calculated gross tumor volume: V=ab ²/ 2, calculate tumor growth rate (GR): GR=(gross tumor volume before the gross tumor volume/treatment after the treatment) according to the gross tumor volume before and after the treatment again.

The gross tumor volume and the tumor growth rate The results of analysis of variance of 5 groups of rat perfusion front and back see Table 3.The difference of organizing respectively before the treatment that there are no significant between the gross tumor volume, behind the hepatic arterial infusion physiological saline (negative control), the tumour of all rats all obviously increases, and mean tumour volume is significantly greater than before the perfusion.After pouring into anti-VEGF monoclonal antibody, gross tumor volume also obviously increases before the treatment, but the tumor growth rate is lower than control group.The growth that anti-VEGF monoclonal antibody can obviously suppress liver tumor is poured in this explanation.

Table 3 rat artery pour into liver tumor volume and tumor propagation rate behind the anti-VEGF monoclonal antibody (x ± s, n=10)

Group	Gross tumor volume (cm 3)		With the perfusion before compare tumor propagation rate (multiple)	The percentage ratio that reduces of rate of increase compared with the control
					Before the perfusion	After the perfusion
	Negative control group	0.061±0.012					0.937±0.331	15.36	---
The treatment group			0.059±0.027	0.277±0.161	4.69	69.46

C) research of vena gastrica perfusion therapy rat cancer of the stomach

With above-mentioned similar approach the rat model of gastric carcinoma is studied, the result is presented in the table 4.

Table 4 rat artery pour into gross tumor volume and average tumor growth rate before and after the anti-VEGF mab treatment (x ± s, N=12)

Group	Volume (cm 3)		With the perfusion before compare tumor propagation rate (multiple)	The percentage ratio that reduces of rate of increase compared with the control
					Before the treatment	After the treatment
	Negative control	0.086±0.032					2.612±0.871	30.37	---
The treatment group			0.089±0.037	0.132±0.052	1.48	95.13

As can be seen from Table 4, this pours into the growth that this monoclonal antibody can obviously suppress cancer of the stomach, and effect comparison liver cancer also will be got well.

D) avidity is identified

Avidity measure to adopt the Scatchard analytical method (people such as Munson, 1980, Anal.BioChem. 107:220) carries out.The result shows that the avidity of 4C6,5H8,6A6 and four kinds of monoclonal antibodies of 3B4 is respectively 6.8 * 10 ^-9, 3.21 * 10 ^-8, 7.1 * 10 ^-9With 6.64 * 10 ^-6Wherein the avidity of 4C6 and 6A6 two strain monoclonal antibodies has all reached the level of 0.1nM.Compare (seeing Table 2) with positive control, 6A6 is than the obvious height of positive control.This is likely because the molecular structure of positive control is Fab (Fab), and 6A6 of the present invention is the monoclonal antibody with complete molecular structure.

The dna sequencing of embodiment 4.VEGF monoclonal antibody gene

According to pedigree, the anti-VEGF monoclonal antibody gene of above-mentioned cell strain 6A6 is carried out dna sequencing.The result is presented in the sequence table, and wherein SEQ ID NO:2 and SEQ ID NO:4 are respectively the variable region of heavy chain of the high reactivity high expression level VEGF monoclonal antibody that obtains of the present invention and the dna encoding sequence of variable region of light chain; SEQ ID NO:1 and SEQ ID NO:3 are respectively according to the variable region of heavy chain of above-mentioned dna encoding sequence supposition and the aminoacid sequence of variable region of light chain.

Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.

Sequence table

＜110〉Shanghai CP Guojian Pharmaceutical Co.,Ltd

＜120〉human resourced monoclone antibody of anti-blood-vessel endothelium growth factor and method for making thereof and pharmaceutical composition

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Claims (13)

1. human vessel endothelium growth factor resisting monoclonal antibody, it comprises variable region of heavy chain and variable region of light chain, it is characterized in that variable region of light chain has the aminoacid sequence shown in the SEQ ID NO:3, variable region of heavy chain has the aminoacid sequence shown in the SEQID NO:1.

2. a dna molecular is characterized in that, the aminoacid sequence shown in its coding SEQ ID NO:3.

3. dna molecular according to claim 2 is characterized in that, this dna molecular contains the nucleotide sequence shown in the SEQ ID NO:4.

4. a dna molecular is characterized in that, the aminoacid sequence shown in its coding SEQ ID NO:1.

5. dna molecular according to claim 4 is characterized in that this dna molecular contains the nucleotide sequence shown in the SEQ IDNO:2.

6. an expression vector is characterized in that, the expression regulation sequence that it contains claim 2 and 4 described dna sequence dnas and links to each other with described series of operations.

7. a host cell is characterized in that, it is transformed by the described expression vector of claim 6.

8. host cell according to claim 7 is characterized in that described host cell is a mammalian cell.

9. host cell according to claim 8 is characterized in that described host cell is a Chinese hamster ovary celI.

10. a pharmaceutical composition for the treatment of tumour is characterized in that, it contains the pharmaceutically described human monoclonal antibodies of claim 1 and the pharmaceutically acceptable carrier of significant quantity.

11. the application of the described human monoclonal antibodies of claim 1 in the medicine of preparation treatment tumour.

12. application according to claim 11, wherein said tumour is selected from cancer of the stomach and liver cancer.

13. one kind prepares the described monoclonal antibody method of claim 1, it is characterized in that this method comprises:

A) provide an expression vector, the expression regulation sequence that this expression vector contains claim 2 and 4 described dna sequence dnas and links to each other with this series of operations;

B) with the described expression vector transformed host cell of step a);

C) host cell of gained culturing step b under the condition that is fit to described monoclonal antibody expression); With

D) separation and purification obtains described monoclonal antibody.