CN101948540A - Preparation of novel anti-EGFR human source antibody MIL27 and application thereof - Google Patents

Abstract

The invention discloses a novel anti-EGFR human source antibody MIL27 based on computer aided design and application thereof. The antibody can be used for obviously inhibiting and killing human cancer cell expressing EGFR. The invention is based on the interaction structure characteristic of EGFR protein extracellular region and functional antibody, computer virtual screening and design are adopted to obtain the novel anti-EGFR functional human source antibody MIL27, PCR method is applied to complete synthesis of antibody gene, eukaryotic expression vector is constructed, and MIL27 antibody capable of specifically identifying EGFR is expressed. The antibody MIL27 can effectively kill EGFR positive tumour cells, including blaster cancer cell and liver cancer cell. The antibody identifies EGFR new epitope, and competitive experiment verifies that the new epitope is different from EGFR epitope identified by Erbitux.

Description

Preparation and the application thereof of novel anti-EGFR human antibody MIL27

Technical field

The present invention relates to novel antibody and uses thereof.Based on EGFR and functional antibodies interaction space structure, utilize epi-position constitutional features, the physico-chemical property of area of computer aided rational evaluation EGFR albumen and functional antibodies effect dynamic mode and functional antibodies specific recognition EGFR extracellular region functional domain, by the design of virtual screening and accessory molecule combine appropriate design, obtain novel anti-EGFR human antibody MIL27.The applied molecular biology technology is carried out the complete synthesis and protein expression of gene, and by immunological experiment and Cell Biology Experiment the function of new antibodies is estimated.

Background technology

Human epidermal growth factor receptor (EGFR) gene is positioned at karyomit(e) p 13～q22 district No. 7, and total length 200kb is made up of 28 exons, 1186 amino acid of encoding, the about 170kDa of EGFR strand glycoprotein molecule amount of formation.EGFR is one of the important molecule of cell signaling and cytodifferentiation [N.Prenzel, O.M.Fischer, S.Streit, S.Hart, A.Ullrich, The epidermalgrouth factor receptor family as a central element for cellularsignaltransduction and diversification.Endocrine-related cancer 8 (2001) 11-31].621 amino-acid residues of EGFR extracellular region (ECD) N-end are ligand binding domain, are made of I, II, III, four subprovinces of IV (or corresponding L1, S1/CR1, L2, S2/CR2 subprovince of being called).Leucine is rich in I and III district; Halfcystine is rich in II and IV district, and whole 50 cysteine residues participate in forming 25 intramolecular disulfide bonds.There is the glycosylation site that 12 potential N-glycosidic links connect in extracellular region in addition, and wherein I, II district respectively have 2, and III, IV district respectively have 4.Studies show that the glycosylation of EGFR is relevant with its participation binding partner and self homotype dimerization.EGFR strides film (TM) district by the hydrophobic region that 23 amino-acid residues constitute, and is strand α spiral.The EGFR intracellular region is totally 542 amino-acid residues, is made of membrane-proximal region (JM), Tyrosylprotein kinase (TK) district, three subprovinces of C-end region.Preceding 13 amino acid (645～657) of membrane-proximal region are that dimerization effect institute is essential in the mediation born of the same parents.The autophosphorylation site is positioned at the C-end, and wherein Tyr1068,1148,1173 is main site, and Tyr992,1086 is less important site, transmits closely related with signal.The overexpression of EGFR or abnormal activation usually cause malignant transformation of cells, thus with the generation of tumour, develop into pernicious and tumor operation prognosis etc. closely related.EGFR crosses in polytype human entity knurl and expresses, comprise [D.C.Moditahedi H such as lung cancer, colorectal carcinoma, mammary cancer, cancer of the stomach, the cancer of the brain, bladder cancer, tumor of head and neck, ovarian cancer, cancer of the stomach, prostate cancer and liver cancer, The receptor for EGF and its ligands:Expression, prognostic value and target for therapy in cancer.Int J Oncol (1994) 277-296].

The drug main of EGFR magnetic target therapy tumour will be divided into two classes at present: EGFR monoclonal antibody and micromolecular compound Tyrosylprotein kinase antagonist.The Tyrosylprotein kinase antagonist is mainly the small molecules quinolines, can competitive inhibition ATP with EGFR born of the same parents in the combining of tyrosine kinase domain, and then influence the tyrosine residues phosphorylation, the signal transduction in inhibition EGFR downstream.The EGFR monoclonal antibody is to combine EGFR with the endogenic ligand competition, activation by suppressing Tyrosylprotein kinase, promote effect such as EGFR internalization to produce anti-tumour effect, comprise the cytotoxicity (antibody-dependent cell-mediated cytotoxicity, ADC C) of antibody dependent cellular mediation etc.Introduce 3 kinds of monoclonal antibody against EGFR that gone on the market below, compare with other chemotherapeutic, these antibody effect high specificities, side effect is little, has obtained curative effect preferably clinically.

Cetuximab (Erbitux) is the anti-EGFR people/mouse chimeric mAb of in February, 2004 FDA approval listing.Erbitux is made of the Fv district of mouse-anti EGFR antibody and people IGg1 heavy chain and κ constant region of light chain, and molecular weight is about 152kD.Erbitux and radiotherapy combined are used for the treatment of the early stage squamous cell carcinoma of the head and neck of regional area, share the treatment EGFR positive, transitivity colorectal cancer that the irinotecan chemotherapy is invalid with irinotecan.Erbitux combines with tumor cell membrane surface EGFR specifically, and parts such as competitive inhibition EGF and TGF-α combine with EGFR's.Though Erbitux in vivo anti-tumour effect mechanism and indeterminate, but analyzed in vitro and animal vivo test show, Erbitux combine with EGFR the blocking-up phosphorylation and with the activation of receptor-associated kinase, thereby cell growth inhibiting, apoptosis-induced, reduce generation [the Ciardiello F of matrix metalloproteinase and VEGF121, TortoraG.A novel approach in the treatment of cancer:targeting the epidermal growthfactor receptor.Clin Cancer Res, 2001; 7:2958-2970].Erbitux can also make the EGFR internalization of surface of cell membrane, expression amount downward modulation [Kim ES, Khuri FR, Herbst RS, et al.Epidermal growth factor receptor biology.Cur Opin Oncol, 2001; 13:506-513]; Strengthen the susceptibility of tumour cell to chemotherapeutics such as irinotecan and radiotherapy, antitumous effect strengthens [Baselga J to some extent than independent chemotherapy or radiotherapy, Trigo JM, Bourhis J, et al.Phase IImulticenter study of the antiepidermal growth factor receptor monoclonalantibody cetuximab in combination with platinum based chemotherapy inpatients with platinum refractory metastatic and/or recurrent squamous cellcarcinoma of the head and neck.J Clin Oncol, 2005; 23:5568-5577].

Nimotuzomab (Tai Xinsheng) popular name obtains the SFDA approval in April, 2006 for reorganization human-derived anti-human EGF-R ELISA monoclonal antibody, is first human monoclonal antibody medicine of China, unites with radiotherapy and is used for the treatment of EGFR male III/IV nasopharyngeal carcinoma.Nimotuzomab belongs to the IgG1 hypotype, molecular weight is 150kD, be the complementary determining region of mouse monoclonal antibody (ioregf/r3) to be transplanted to monoclonal antibody against EGFR [the Crombet T that people's antibody skeleton constitutes by genetic engineering technique, Osorio M, Cruz T, et al.Use of the humanized antiepidermal growth factorreceptor monoclonal antibody hr3 in combination with radiotherapy in thetreatment of locally advanced head and neck cancer patients.J Clin Onco l, 2004; 22:1646654].Nimotuzomab has and EGFR bonded high-affinity (Kd=10 ^-9Mol/L), experiment in vivo and vitro proof nimotuzomab has inhibition of cell proliferation and blood vessel formation against function, and can promote natural death of cerebral cells, the retardance cell cycle rests on G1/S phase [CrombetRamos T, Rak J, Perez R, et al.Antiproliferative, antiangiogenic andproapoptotic activity of hR3:A humanized anti EGFR antibody.Int J Cancer, 2002; 101:567-575].It is found when the intravital distribution of people with the nimotuzomab of 99mTc marker research, and nimotuzomab has at liver, the heart, kidney, bladder and spleen and accumulates, wherein liver accumulate the most remarkable.Except that kidney, bladder and gi tract, each organ reduces along with the prolongation of time the intake of nimotuzomab, and this is because kidney, bladder and gi tract are Excretory organs of nimotuzomab.Tumour to the removing of nimotuzomab less than of the removing of other healthy tissuess to nimotuzomab, and tumour to the intake of nimotuzomab with respect to more constant [the Crombet T of other organs, TorresL, Neninger E, et al.Pharmacological evaluation of humanized anti epidermalgrowth factor receptor, monoclonal antibody hr3, in patients with advancedepithelial derived cancer.J Immunother, 2003; 26:13948], nimotuzomab is strong to the target of tumour in this explanation, can reach high density at tumor locus and produce the effect that suppresses tumour.Security and the clinical efficacy of nimotuzomab estimated in I/II phase clinical study that nimotuzomab and chemotherapy combined are used.24 patients 16.7% have complete reaction, 20.8% stable disease.The mean value of all patients' survival time and median are respectively 16.76 and 14.77 months.What deserves to be mentioned is that nimotuzomab also has certain curative effect to children and teenager's neurospongioma, and there is not a serious toxic side effect [Bode U, Buchen S, et al.Results of a phase II trial of hR3monoclonalantibody (nimotuzumab) in the treatment of resistant or relapsed high gradegliomas in children and adolescents.J Clin Oncol, 2006; 24 (18S): 1522].The general side effect of nimotuzomab is light moderate fever, ypotension, chatter, sore muscle and headache in I/II phase clinical study.Different with clinical other anti-egfr antibodies is that nimotuzomab has the little advantages of dermal toxicity probability such as the fash of generation, dermatitis.。

Panitumumab (VEXTIBIX) is a kind of complete humanized IgG 2 monoclonal antibody against EGFR of being produced by the XenoMouse technology, no mouse source property [Yang XD, Jia XC, Corvalan JR, et al.Development of ABX EGF, a fully human anti EGF receptor monoclonalantibody for cancer therapy.Crit Rev Oncol Hematol, 2001; 38:17-23].Panitumumab in September, 2006 by FDA approval listing, share with fluorine pyrimidine, oxaliplatin and irinotecan or after chemotherapy, be used for the treatment of the straight colorectal carcinoma of EGFR male transitivity.The same with other anti-egfr antibodies, the mechanism of action of panitumumab also is by the combining of EGFR on blocking-up EGF and TGF-α and the tumour cell, and induces the internalization of EGFR, and then eliminates the cytological effect that EGFR mediates.In animal body in the test, compare with methods of treatment with other anti-egfr antibodies, tumor recurrence [Ciardiello F does not all appear in the animal major part after 8 months that only gives panitumumab, Caputo R, Bianco R, et al.Antitumor effect and potentiation of cytotoxic drugsactivity in human cancer cells by ZD 1839 (Iressa), an epidermal growth factorreceptor selective tyrosine kinase inhibitor.Clin Cancer Res, 2000; 6:2053-2063].No matter there are some researches show, be the tumour of high expression level or the low EGFR of expression, and panitumumab can both have its growth of inhibition.The normally modal untoward reaction of clinical application panitumumab of fash, relevant [the Foon KA of the incidence of fash with the dosage of panitumumab, Yang XD, Weiner LM, et al.Preclinical and clinical evaluations of ABX EGF, a fullyhuman anti epidermal growth factor receptor antibody.Int J Radiat Oncol B iolPhys, 2004; 58:984-990].Symptom such as also occur slight weakness, diarrhoea among the patient, feel sick, but and dosage indifference.Different with other EGFR target small-molecule drugs with the chimeric monoclonal antibody cetuximab of people mouse is even that at high dosage 2.5mg/ (kgwk), supersensitivity untoward reaction and the anti-people's antibody of people do not appear in the panitumumab in people source fully.

These three kinds of monoclonal antibody against EGFR of Cetuximab, panitumumab and nimotuzomab have all that target is strong, the relative features of smaller of toxic side effect.These monoclonal antibodies and chemicotherapy are combined in and are used for the treatment of the EGFR positive tumor clinically and have obtained certain curative effect.At present extensive not enough in the clinical application of China's monoclonal antibody against EGFR, expense is also higher relatively.Monoclonal antibody against EGFR fails to be used for separately tumor treatment, also needs and radiotherapy or chemotherapy combined are used; And clinical to be used for the treatment of tumor type also less, these have limited monoclonal antibody against EGFR use clinically to a certain extent.

The present invention is according to the research background of present proteic structure and properties of EGFR and antibody thereof, utilize the special constitutional features of EGFR, EGFR extracellular region and functional antibodies Erbitux interaction space conformation and ad hoc structure characteristic of field, by computer virtual screening and molecular designing conceptual design novel anti-EGFR human antibody MIL27, and its function is verified and estimated by immunological experiment, Cell Biology Experiment and experimentation on animals.

Summary of the invention

The present invention is based on computer aided molecular design, and obtained novel anti-EGFR human antibody MIL27 by the molecular biology total synthesis method.

The novel human-derived antibody that anti-egfr antibodies MIL27 disclosed by the invention is based on EGFR structural domain, EGFR and functional antibodies interaction space conformation and physicochemical characteristics utilizes computer aided design (CAD) to obtain.

The invention discloses a kind of aminoacid sequence of novel anti-EGFR human antibody, it is characterized in that having as the described sequence of sequence table.The aminoacid sequence of the variable region of light chain of described EGFR humanized antibody MIL27 is selected from one of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, and the aminoacid sequence of variable region of heavy chain is selected from and is one of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

A feature of antibody disclosed by the invention is that described antibody MIL27 identified epitope is different with Erbitux.Competition shows that in conjunction with experiment MIL27-1, MIL27-2 and MIL27-3 all do not combine with the Erbitux competition, and they do not possess the same antigen epi-position.Wherein the aminoacid sequence of MIL27-1 variable region of light chain is SEQ ID NO:1, and the aminoacid sequence of variable region of heavy chain is SEQ ID NO:2; The aminoacid sequence of MIL27-2 variable region of light chain is SEQ ID NO:3, and the aminoacid sequence of variable region of heavy chain is SEQ ID NO:4; The aminoacid sequence of MIL27-3 variable region of light chain is SEQ ID NO:5, and the aminoacid sequence of variable region of heavy chain is SEQ ID NO:6.

The invention discloses EGFR human antibody MIL27 can combine with target antigen positive cell surface EGFR specificity; Disclosing antibody MIL27 simultaneously suppresses effectively and kills and wounds EGFR male tumour cell.

Described antibody MIL27 can specificity mediate the ADCC function, kills and wounds two kinds of target cells of EGFR male SKVO3 and HepG2.

The invention also discloses the preparation method of novel anti-EGFR human antibody MIL27.

Specific implementation process is as follows:

1) the people source anti-egfr antibodies that utilizes computer aided molecular design to obtain;

2) utilize complete synthesis round pcr to synthesize the anti-egfr antibodies gene;

3) expression of antibody and evaluation;

4) the anti-egfr antibodies biologic activity is identified.

Describe the design and the preparation process of anti-EGFR human antibody in detail below with reference to above-mentioned steps.Design and the method that prepare antibody of the present invention only are that methods involving is described, are not to be restrictive; Also can adopt other known methods, perhaps adopt the method for revising.

1) the people source anti-egfr antibodies that utilizes computer aided molecular design to obtain

Feature according to EGFR extracellular region structural domain functional epitope, utilize the small peptide (length is 10～15 amino acid) of computer virtual screening and computer aided molecular design method design antagonism EGFR function, and a series of function small peptides that will obtain are as the CDR district deposit of antibody variable region; The human antibody sequence information data storehouse that the antibody sequence that provides by Protein Data Banks such as being derived from TrEMBL, SwissProt, Kabat, IMGT makes up; By the homology mould build, the mechanics optimization technology carries out rational joint with human antibody variable region framework in the human antibody sequence information data storehouse and the CDR that lays in previously and makes up rational antibody variable region.

2) utilize complete synthesis round pcr to synthesize the anti-egfr antibodies gene;

According to the amino acid sequence of designerantibodies, be chosen in codon commonly used in the mammalian cell and carry out reverse translation, obtain the corresponding antibody gene.Utilize the complete synthesis round pcr of gene, design corresponding primer, by the method acquisition full-length gene of overlapping PCR; Be cloned into cloning vector, carry out sequencing.

3) expression of antibody and evaluation

A) expression of antibody: antibody gene is built up in the carrier for expression of eukaryon,, gather in the crops supernatant after in the time of the cultivation sufficiently long, wherein contain the purpose antibody of expression by liposome transfection, method transient transfection eukaryotic expression cell such as electricity commentariess on classics etc.

B) double fastener heart enzyme-linked immunosorbent assay (ELISA): with behind the suitable antibody sandwich as capture antibody, after sealing, add testing sample and standard model and carry out capture reaction; Utilize suitable ELIAS secondary antibody to carry out color reaction subsequently, the content of target protein in the working sample.

C) SDS-PAGE:, carry out polyacrylamide gel electrophoresis, protein isolate according to experiment needs preparation reduction electrophoresis sample or non-reduced electrophoresis sample according to the polyacrylamide gel of the size of target protein preparation suitable concn; Through behind the coomassie brilliant blue staining, determine the target protein molecular weight.

4) the anti-egfr antibodies biologic activity is identified

A) flow cytometry: collect the purpose cell; After damping fluid (PBS+2% foetal calf serum) washing, add one and resist, 4 ℃ of reaction 30mins; Damping fluid washing 2 times adds corresponding two anti-ly, and 4 ℃ of lucifuges reaction 30mins are resuspended in after the damping fluid washing 2 times and directly carry out flow cytometry in the sheath fluid or fix with 1% Paraformaldehyde 96, and 4 ℃ keep in Dark Place.

B) antibody competition conjugated antigen experiment: behind the Erbitux antibody labeling isothiocyanate fluorescein (FITC), utilize the critical saturation concentration of flow cytometry technical measurement FITC-Erbitux identification target cell; With this critical saturation concentration and target cell reaction, the competition antibody that adds different concns simultaneously is at war with in conjunction with experiment, behind 4 ℃ of reaction 40mins, when Flow Cytometry analysis competition antibody existed, FITC-Erbitux combined positive rate and fluorescence intensity with target cell.

C) cytotoxicity test (ADCC) of antibody dependence: mark EGFR positive cell is isolated peripheral blood mononuclear cell action effect cell as target cell with lymphocyte separation medium from peripheral blood; After the corresponding proportion mixing, add the antibody of inequality, on round bottom 96 well culture plates, carry out killing experiments, set up experimental port, the spontaneous release aperture of target cell, maximum release aperture and background hole; Hatch 2h for 37 ℃.Utilize multi-functional microplate reader to detect, calculate kill rate.

Description of drawings

Fig. 1 SDS-PAGE analyzes the MIL27 antibody that eukaryotic expression obtains.The antibody weight chain variable region gene that random pair combined computer aided design obtains carries out eukaryotic expression, and 9 antibody molecule amounts of acquisition are 155KDa; 9 antibody of 1～9 expression, M represents the molecular weight standard product.

Fig. 2 MIL27 antibodies specific is discerned by the goat anti-human igg.9 antibody that the antibody weight chain variable region gene that random pair combined computer aided design obtains obtains all can be discerned by the goat anti-human igg; 9 antibody of 1～9 expression; IgG represents that human IgG and Erbitux are together as positive control.

Fig. 3 MIL27 antibodies specific identification target antigen EGFR.Carry out Western Blottig with EGFR positive cell lysate and detect, 9 antibody that the antibody weight chain variable region gene that random pair combined computer aided design obtains obtains all can be discerned the target antigen that molecular weight is 185KDa; 9 antibody of 1～9 expression, Erbitux is as positive control.

Fig. 4 MIL27 identification epitope is different with Erbitux.A:Erbitux antibody and target cell SKOV3 binding reactive; B:MIL27-1 does not combine target cell with the Erbitux competition; C:MIL27-2 does not combine target cell with the Erbitux competition; D:MIL27-3 does not combine target cell with the Erbitux competition.

The external mediation of Fig. 5 MIL27 antibody ADCC effect is killed and wounded the EGFR positive cell.A: antibody-mediated ADCC effect is killed and wounded the SKOV3 cell, B: antibody-mediated ADCC effect is killed and wounded the HepG2 cell.

The people source anti-egfr antibodies that embodiment one, computer aided molecular design obtain

One, material:

InsightII 2005 routine packages (MSI molecular simulation, San Diego), this routine package comprises:

Hornology homology mould is built;

The Discover mechanics optimization;

Discover 3 normal temperature dynamics simulations;

The Docking molecular docking;

The apparent static potential energy of Delphi is analyzed.

Ludi molecular designing program (1995);

The IBM graphics workstation;

PDB 2008 databases (network www.rcsb.org download);

SwissProt database (2008, network download);

Kabat database (2001, network download);

IMGT database (2008, network download).

Two, methods and results:

Utilize the constitutional features of the EGFR extracellular region functional epitope of determining, can discern the short peptide sequence of this feature epi-position by the Ludi program through the design of fragment growth method; Utilize the space conformation of from the beginning building with secondary structure prediction logotype simulation antagonism small peptide.

Human antibody variable region skeleton construction database by the antibody sequence information that is derived from network data base PDB, SwissProt, TrEMBL, Kabat, IMGT acquisition constitutes is spliced to form novel human-derived antibody variable region with above-mentioned antagonism small peptide reasonable combination.Utilize the antibody variable region sequence of design to build its space conformation through the homology mould technology of building.

Utilize the molecular docking method to make up antibody variable region and the interactional composite structure of antigen EGFR.Under the prerequisite of considering solvent effect,, determine to act on the antibody variable plot structure of antigen EGFR by interaction energy by the mixture model of normal temperature dynamics simulation acquisition antibody and AI.Further optimize the novel human-derived antibody variable region amino acid sequence that obtains, finally obtain heavy chain amino acid sequence (SEQ_ID NO:1, SEQ_ID NO:2, SEQ ID NO:3) and 3 light chain variable region amino acid sequences (SEQ_ID NO:4, SEQ_ID NO:5 and SEQ_ID NO:6) of 3 targeting EGFRs.

Expression and the evaluation of embodiment two, anti-EGFR human antibody MIL27

One, material:

Primer-design software is a biosun software, and primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd; Pyrobest DNA polymerase is a TAKARA company product; DNTP is a TAKARA company product; PGEM-T Easy vector system is an Invitrogen company product; The T4DNA ligase enzyme is a NEB company product; Gene sequencing is finished by Sinogenomax Co., Ltd.; Antibody carrier for expression of eukaryon pTGS-FRT-DHFR is made up by our company and the (license number: ZL200510064335.0) of application national patent; Liposome, MTT are Invitrogen company product, and goat anti-human igg, the goat anti-human igg who reaches the horseradish enzyme labelling and human IgG are our company's preparation; Restriction endonuclease is a NEB company product; Other reagent are commercial product.

Two, methods and results

1.EGFR antibody gene is synthetic

Antibody gene sequence according to the computer simulation acquisition, gene order SEQ ID NO:7, the SEQ ID NO:8 and the SEQ ID NO:9 that comprise variable region of heavy chain, the gene order SEQID NO:10 of variable region of light chain, SEQ ID NO:11 and SEQ ID NO:12.Be chosen in the higher codon of frequency of utilization in the mammalian cell,, obtain the variable region nucleotide sequence of antibody the antibody weight chain variable region aminoacid sequence reverse translation that the Computer Design screening obtains; The principle complete synthesis according to gene utilized computer aided design software, design primer, correlation parameters such as the secondary structure of consideration primer, GC content; Adopt the complete synthesis antibody weight of overlapping pcr variable region gene.

2. the expression of antibody

2.1 antibody Construction of eukaryotic

That selects that our company obtains contains human IgG1's antibody constant region expression carrier pTGS-FRT-DHFR (license number: ZL200510064335.0) as the expression vector of antibody of the present invention.3 antibody chain variable region genes of the described acquisition of present embodiment step 1,3 heavy chain variable region genes are carried out the random pair combination, be cloned in the pTGS-FRT-DHFR carrier.Through transforming equimolecular biology common technology, obtain the carrier for expression of eukaryon of 9 antibody altogether.

2.2 antibody expression

With the carrier for expression of eukaryon that obtains, to Chinese hamster ovary celI, gather in the crops supernatant by liposome-mediated method transient transfection behind the 48h, by the expression of experimental analysis purpose antibody such as double fastener heart ELISA method and SDS-PAGE.

Utilize the goat anti-human igg of goat anti-human igg and horseradish enzyme labelling to carry out the content that double fastener heart ELISA method detects antibody in the supernatant, as negative control, the pure product of human IgG are as standard substance with the untransfected supernatant.Experimental result shows, the antibody weight chain variable region gene random pair combination that computer aided design (CAD) obtains is expressed, and has target protein in the supernatant of results and expresses.Carry out the SDS-PAGE experiment with non-reducing form, the result shows that the target protein of expression is about 155KDa, meets the molecular weight (Fig. 1) of antibody; Goat anti-human igg with the horseradish enzyme labelling carries out Western Blotting detection, and expressing the target protein that obtains can be had human IgG antibody's feature (Fig. 2) by goat anti-human igg's specific recognition; But gene successful expression in eukaryotic cell that the antibody sequence that prompting obtains obtains through reverse translation, expressing the albumen that obtains is antibody molecule, has typical IgG antibody feature.

2.3 antibody is identified

Collect EGFR positive cells SKOV3, get lysate behind the M2 damping fluid lysing cell and carry out Western Blotting experiment, the antibody weight chain variable region gene random pair combination that computer aided design (CAD) is obtained is expressed 9 antibody obtaining as detecting antibody, detects with the goat anti-human igg of horseradish enzyme labelling.Experimental result shows, but the equal specific recognition target protein of each antibody, its molecular weight is about 185KDa (Fig. 3), prompting, and the antibody MIL27 of acquisition can specific recognition target antigen EGFR.

The functional trial of embodiment three, anti-egfr antibodies MIL27

EGFR human antibody MIL27-1, MIL27-2 and MIL27-3 are example, further verify the biological function of antibody.

One, material

Breast cancer cell line SKVO3 and hepatoma cell line HepG2 are available from ATCC; DELFIAEuTDA cytotoxicity test kit is a PE company product; Other related reagents are referring to embodiment two.

Two, methods and results

1. flow cytometry MIL27 and cell surface EGFR's combines

With the positive breast cancer cell line SKVO3 of EGFR, three kinds of cells of hepatoma cell line HepG2 and lung cancer cell line A549 detect the antigenic ability of MIL27 antibodies cell surface EGFR of different concns as target cell; With Erbitux antibody as positive control.The result shows MIL27 energy specific recognition target cell, and it is in conjunction with positive rate similar to control antibodies Erbitux (table 1, table 2, table 3), and the identification of prompting antibody MIL27 is active consistent with control antibodies Erbitux.

It is active with combining of cell surface EGFR that table 1FACS detects MIL27-1

It is active with combining of cell surface EGFR that table 2FACS detects MIL27-2

It is active with combining of cell surface EGFR that table 3FACS detects MIL27-3

2. the competitive experiment of antigen-antibody bonded

Behind Erbitux antibody labeling isothiocyanate fluorescein (FITC), utilize the critical saturation concentration of flow cytometry technical measurement FITC-Erbitux identification target cell SKOV3; The result shows that the FITC-Erbitux of 0.5 μ g/mL combines with target cell SKOV3 and is critical state of saturation.With FITC-Erbitux and the target cell SKOV3 reaction of 0.5 μ g/mL, the competition antibody MIL27 that adds different concns simultaneously is at war with in conjunction with experiment, behind 4 ℃ of reaction 40mins, and Flow Cytometry analysis competition result; Fig. 4 result shows that antibody MIL27-1, MIL27-2, MIL27-3 can not compete FITC-Erbitux in conjunction with target cell; Even when the concentration of competition antibody when being 100 times of FITC-Erbitux antibody concentration (the competition antibody concentration is 50 μ g/mL), the change not yet that combines of FITC-Erbitux and target cell; Prompting, though Erbitux and MIL27 discern EGFR, its epitope difference.

3. the cytotoxicity test (ADCC) that relies on of antibody

DELFIA EuTDA cytotoxicity test kit with PE company carries out the ADCC experiment, the positive breast cancer cell line SKVO3 of mark EGFR, hepatoma cell line HepG2 and lung cancer cell line A549 isolate peripheral blood mononuclear cell action effect cell as target cell with lymphocyte separation medium from peripheral blood; After the corresponding proportion mixing, add the antibody of inequality, on round bottom 96 well culture plates, carry out killing experiments, set up experimental port, the spontaneous release aperture of target cell, maximum release aperture and background hole; Hatch 2h for 37 ℃.Utilize multi-functional microplate reader to detect, calculate kill rate.The result shows that antibody MIL27 can effectively mediate the ADCC activity and kill and wound three kinds of cells, and its activity is antibody concentration and relies on; And under the same concentrations, MIL27 all is higher than Erbitux to the kill rate of three kinds of cells, prompting MIL27 mediation ADCC function kill and wound cell activity be significantly higher than Erbitux (Fig. 5, A: antibody-mediated ADCC effect is killed and wounded SKOV3 cell, B; Antibody-mediated ADCC effect is killed and wounded the HepG2 cell).

Sequence table

Claims (8)

1. the novel anti-EGFR human antibody MIL27 that computer aided molecular design obtains comprises humanized variable region of heavy chain, variable region of light chain and other zones.The aminoacid sequence of the variable region of light chain of described EGFR humanized antibody MIL27 is selected from one of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, and the aminoacid sequence of variable region of heavy chain is selected from and is one of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

2. the method for the anti-EGFR human antibody of computer aided molecular design according to claim 1 MIL27 is characterized in that described computer aided design (CAD) is based on EGFR extracellular region structural domain space conformation and physicochemical characteristics carries out.

3. the described antibody MIL27 of claim 1, the aminoacid sequence of its variable region of light chain is SEQ ID NO:1, the aminoacid sequence of variable region of heavy chain is SEQ ID NO:2.

4. the described antibody MIL27 of claim 1, the aminoacid sequence of its variable region of light chain is SEQ ID NO:3, the aminoacid sequence of variable region of heavy chain is SEQ ID NO:4.

5. the described antibody MIL27 of claim 1, the aminoacid sequence of its variable region of light chain is SEQ ID NO:5, the aminoacid sequence of variable region of heavy chain is SEQ ID NO:6.

6. according to the described anti-EGFR human antibody MIL27 of claim 3-5, it is characterized in that antibody can be applied to suppress or killing tumor cell.

7. the described tumour cell of claim 6 comprises breast cancer cell and liver cancer cell.

8. the described anti-EGFR human antibody MIL27 of claim 3-5 has the purposes at preparation treatment tumour relative disease medicine.

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