CN1930187B

CN1930187B - For antibody and the use thereof of the deletion mutant of EGF-R ELISA

Info

Publication number: CN1930187B
Application number: CN200480018185.3A
Authority: CN
Inventors: 理查德·韦伯; 冯晓; 奥里特·福德; 拉里·格林; 琼·古达斯; 布鲁斯·基特; 刘颖; 帕拉尼·拉塔纳斯瓦米; 罗伯特·拉亚; 杨晓东; 乔斯·克尔瓦兰; 伊恩·福尔茨; 贾小池; 贾斯帕·康; 查德威克·T·金; 斯科特·L·克拉坎普; 巧娟·简·苏
Original assignee: Amgen Fremont Inc
Current assignee: Amgen Fremont Inc
Priority date: 2003-06-27
Filing date: 2004-06-25
Publication date: 2015-08-19
Anticipated expiration: 2024-06-25
Also published as: CN1930187A; CN1816351A

Abstract

The present invention relates to novel antibody, particularly relate to the antibody of the deletion mutant for EGF-R ELISA, and particularly relate to type III deletion mutant EGFRvIII.The present invention also relates to the human monoclonal antibody of the deletion mutant for EGF-R ELISA, and be especially EGFRvIII.The present invention also provides the Diagnosis and Treat formula of these antibody, and immune conjugate.

Description

For antibody and the use thereof of the deletion mutant of EGF-R ELISA

Technical field

The present embodiment relates to novel antibody, particularly relates to the antibody of the deletion mutant for EGF-R ELISA, and particularly relates to type III deletion mutant EGFRvIII.The present embodiment also relates to the human monoclonal antibody of the deletion mutant for EGF-R ELISA, and is especially EGFRvIII.The present embodiment also relates to the variant of these antibody.The present invention also provides the Diagnosis and Treat formula of these antibody, and immune conjugate.

Background technology

Since the last century, the tumor-specific molecule contributing to better Diagnosis and Treat human and animal cancer has been sought.Except those, in the human cancer of most of type, are difficult to provide the conclusive evidence to the tomour specific material based on molecular structure data except the cancer comprising the molecular structure specified by virogene based on virus induction cancer.Rarely about the example of the tumor-specific molecule based on new molecules structure.In malignant human's neurospongioma and other amplification with EGF-R ELISA molecule or when changing potential relevant tumour (such as mammary cancer and other human cancer), there is no the clearly demonstration about the structural changes molecule with unique sequences.

EGF-R ELISA (EGFR) is 170 kilodaltons (kilodalton) the membrane glycoprotein product of proto-oncogene c-erb B.The sequence (people such as Ullrich, 1984) of known EGFR gene.“Human EpidermalGrowth Factor Receptor cDNA Sequence and Aberrant Expression of the AmplifiedGene in A431Epidermoid Carcinoma Cells.”Nature309：418-425)。EGFR gene is cell-isogenic people (1984) such as () Downward of the erb B oncogene identified in avian erythrocytes increase disease virus at first.The people (1984) such as " Close Similarity of Epidermal Growth Factor Receptor and v-erb B OncogeneProtein Sequence. " Nature307:521-527, Ullrich.In various human tumor, observed the activation (people (1987A) such as Haley that this oncogene is amplified by gene." The EpidermalGrowth Factor Receptor Gene in:Oncogenes; Genes, and Growth Factors ", Guroff, G compiles, 12nd edition, the 2nd chapter, 40-76 page, Wiley, and be especially those epidermal growth factor receptor genes (people such as Libermann, (1985) of neuroglia source property N.Y.).Amplification, Enhanced Expressionand Possible Rearrangement of EGF Receptor Gene in Primary Human Brain Tumoursof Glial Origin, Nature313:144-147; The people such as Wong, (1987).Increased Expression of theEpidermal Growth Factor Receptor Gene in Malignant Gliomas is Invariably Associatedwith Gene Amplification.Proc.Natl.Acad.Sci.USA84:6899-6903; The people such as Yamazaki, (1988).Amplification of the Structurally and Functionally Altered Epidermal GrowthFactor Receptor Gene (c-erbB) in Human Brain Tumors.Molecular and Cellular Biology8:1816-1820, the people such as Maiden, (1988).Selective Amplification of the Cytoplasmic Domainof the Epidermal Growth Factor Receptor Gene in Glioblastoma Multiforme.CancerResearch4：2711-2714)。

Confirm EGF-r overexpression in polytype human solid's tumour.Mendelsohn Cancer Cells7：359(1989)，Mendelsohn Cancer Biology1：339-344(1990)，Modjtahedi and Dean Int′lJ.Oncology4：277-296(1994)。Such as, in some lung cancer, mammary cancer, colorectal carcinoma, cancer of the stomach, the cancer of the brain, bladder cancer, head and neck cancer, ovarian cancer, kidney and prostate cancer, EGFR overexpression is observed.Modjtahedi andDean Int′l J.Oncology4：277-296(1994)。Verification table skin growth factor (EGF) and transforminggrowthfactor-α (TGF-α) are all bonded to EGF-r, and cause cell proliferation and tumor growth.

A key distinction between v-erb B oncogene and normal EGFR gene is that viral oncogenes is that normal subject blocks amino modification; They lack most cells matter extracellular portion, but remain cross-film and tyrosine kinase domain (people such as Fung, (1984)).Activation of the Cellular Oncogene c-erb B by LTRInsertion:Molecular Basis for Induction of Erythroblastosis by Avian Leukosis Virus.Cell33:357-368; The people such as Yamamoto, (1983).A New Avain Erythroblastosis Virus, AEV-HCarries erbB Gene Responsible for the Induction of Both Erythroblastosis and Sarcoma.Cell34:225-232, the people such as Nilsen, (1985).C-erbB Activation in ALV-InducedErythroblastosis:Novel RNA Processing and Promoter Insertion Results in Expressionof an Amino-Truncated EGF Receptor.Cell41:719-726; The people such as Gammett, (1986).Differences in Sequences Encoding the Carboxy-Terminal Domain of the EpidermalGrowth Factor Receptor Correlate with Differences in the Disease Potential of ViralerbB Genes.Proc.Natl.Acad.Sci.USA83：6053-6057)。Which results in cannot associative list skin growth factor (EGF), but still can the protein (Gilmore's, (1985)) of other material of phosphorylation.ProteinPhosphorlytion at Tyrosine is Induced by the v-erb B Gene Product in Vivo and In Vitro.Cell40:609-618; The people such as Kris, (1985).Antibodies Against a Synthetic Peptide as a Probefor the Kinase Activity of the Avian EGF Receptor and v-erB Protein.Cell40:619-625), and cause following supposition: v-erb B protein be oncogene be because kinase domain is active people such as (, 1984) Downward without adjustment and in structure.

Various gene variation can betide in viral erb B oncogene, such as, in the C-terminal of gene, amino acid whose replacement and disappearance occurs.But obtainable evidence shows that amino truncation is particularly crucial for carcinogenesis.Amino truncation is a feature of all v-erb B oncogene, comprises those amino truncations (people such as Nilsen, (1985)) caused by the insertion of promotor or retroviral transduction.C-erbB Activation inALV-Induced Erythroblastosis:Novel RNA Processing and Promoter Insertion Resultsin Expression of an Amino-Truncated EGF Receptor.Cell41:719-726; The people such as Gammett, (1986).Differences in Sequences Encoding the Carboxy-Terminal Domain of theEpidermal Growth Factor Receptor Correlate with Differences in the Disease Potentialof Viral erbB Genes.Proc.Natl.Acad.Sci.USA83：6053-6057)。

On the contrary, carboxy-terminal deletion seems only relevant with the tumour caused by retroviral transduction, and seems to determine specificity (people such as Gammett, 1986 of host range and tumor type; The people such as Raines, (1985).c-erbBActivation in Avian Leukosis Virus-Induced Erythroblastosis：Clustered Integration Sitesand the Arrangement of Provirus in the c-erbB Alleles.Proc.Natl.Acad.Sci.USA82：2287-2291)。Show that namely this disappearance is enough to separately to produce transforming protein matter people such as (, (1988)) Pelley with amino-transfection experiment of blocking birds c-erb 1 B gene or infectious virus oncogene-mankind EGF acceptor.Proviral-Activated c-erbB is Leukemogenic but not Sarcomagenic:Characterization of aReplication-Competent Retrovirus Containing the Activated c-erbB.Journal of Virology62:1840-1844; The people such as Wells, (1988).Genetic Determinants of Neoplastic Transformationby the Retroviral Oncogene v-erbB.Proc.Natl.Acad.Sci.USA85：7597-7601)。

The amplification of EGFR gene betides (people such as Libermann, (1985) in malignant human's neurospongioma of 40%.Amplification, Enhanced Expression and Possible Rearrangement of EGFReceptor Gene in Primary Human Brain Tumours of Glial Origin, Nature313:144-147; The people such as Wong, (1987).Increased Expression of the Epidermal Growth Factor ReceptorGene in Malignant Gliomas is Invariably Associated with Gene Amplification.Proc.Natl.Acad.Sci.USA84:6899-6903), acceptor gene be rearranged in many have gene amplify tumour in comparatively obvious.Structural changes seems preferentially to affect N-terminal half (people such as Yamazaki, (1985) of gene.Amplification, Enhanced Expression and Possible Rearrangement of EGF ReceptorGene in Primary Human Brain Tumours of Glial Origin, Nature313:144-147; The people such as Maiden, (1988).Selective Amplification of the Cytoplasmic Domain of the EpidermalGrowth Factor Receptor Gene in Glioblastoma Multiforme.Cancer Research4:2711-2714), but the character of resetting not accurate characterization in any tumour at that time.

The size variant EGFR gene in some human cancers and amplification (people such as Humphrey, (1988)) are reported in.Amplification and Expression of the, Epidermal Growth Factor Receptor Genein Human Glioma Xenografts.Cancer Research48:2231-2238; The people such as Bigner, (1988) J.Neuropathol.Exp.Neurol., 47:191-205; The people such as Wong, (1987).Increased Expression of theEpidermal Growth Factor Receptor Gene in Malignant Gliomas is Invariably Associatedwith Gene Amplification.Proc.Natl.Acad.Sci.USA84:6899-6903; With people such as Humphrey.The amplification of epidermal growth factor receptor gene in human gliomas's xenotransplantation and expression, Cancer Res.48 (8): 2231-8 (1988)).But, there is no about in cell through changing the mensuration of the molecular based of EGFR molecule.1989, the works of Drs.Bigner and Vogelstein illustrated the sequence (also referred to as δ-EGFr or EGFRvIII) being known as the EGF acceptor mutant of type III mutant.This works is described in United States Patent (USP) the 6th, 455, No. 498, the 6th, 127, No. 126, the 5th, 981, No. 725, the 5th, 814, No. 317, the 5th, 710, No. 010, the 5th, 401, No. 828 and the 5th, in 212, No. 290.

EGFR variant is amplified with EGFR gene by gene rearrangement and causes.There are eight kinds of main EGFr variants, be known as: (i) EGFRvI lacks the most cells extracellular portion of EGFR, (ii) EGFRvII is made up of the 83aa in-frame deletion in the extracellular domain of EGFR, (iii) EGFRvIII is made up of the 267aa in-frame deletion in the extracellular domain of EGFR, (iv) EGFRvIV contains the disappearance in the cytoplasmic domain of EGFR, v () EGFRvV contains the disappearance in the cytoplasmic domain of EGFR, (vi) EGFR.TDM/2-7 contains the repetition of the exon 2-7 in the extracellular domain of EGFR, (vii) EGFR.TDM/18-25 contains the repetition of the exons 1 8-26 in the tyrosine kinase domain of EGFR, (viii) EGFR.TDM/18-26 contains the repetition (people such as Kuan of the exons 1 8-26 in the tyrosine kinase domain of EGFR, EGF mutant receptor vIII in Therapeutic cancer as molecular target, Endocr Relat Cancer.8 (2): 83-96 (2001)).In addition, exist the second more rare there is EGFRvIII mutant (EGFRvIII/ Δ the 12-13) (people such as Kuan that the tie point of the second between

exon

11 and 14 introduces the disappearance of novel histidine residues, EGF mutant receptor vIII in Therapeutic cancer as molecular target, Endocr Relat Cancer.8 (2): 83-96 (2001)).

EGFRvIII is the variant (people such as Kuan in the most common generation of human cancer Concentrations of Epidermal Growth Factor (EGF) acceptor, EGF mutant receptor vIII in Therapeutic cancer as molecular target, Endocr Relat Cancer.8 (2): 83-96 (2001)).In the process that gene amplifies, in extracellular domain, there are 267 aminoacid deletion, produce the novel tie point that tumor-specific monoclonal antibody can point to.This variant of EGF acceptor with ligand independently mode contribute to tumour and sent by composing type signal and develop.Known EGFrVIII is not expressed in (people such as Wikstrand, CJ., " Monoclonal antibodies against EGFR in any healthy tissues _viII are tumorspecific and react with breast and lung carcinomas malignant gliomas. ", CancerResearch55 (14): 3140-3148 (1995); Olapade-Olaopa, EO. people is waited, " Evidence for thedifferential expression of a variant EGF receptor protein in human prostate cancer.Br JCancer.82 (1): 186-94 (2000)).But, EGFRvIII is presented at the remarkable expression in tumour cell, such as, EGFRvIII (Wikstrand is expressed in the mammary cancer biopsy of 27-76%, CJ. people is waited, " Monoclonalantibodies against EGFRVIII are tumor specific and react with breast and lungcarcinomas malignant gliomas. ", Cancer Research55 (14): 3140-3148 (1995), the people such as Ge H., " Evidence of high incidence of EGFRvIII expression and coexpression with EGFRin human invasive breast cancer by laser capture microdissection andimmunohistochemical analysis. ", Int J Cancer.98 (3): 357-61 (2002)), 50 ~ 70% neuroglia cancers express EGFRvIII (Wikstrand, CJ. people is waited, " Monoclonal antibodies against EGFR _viII aretumor specific and react with breast and lung carcinomas malignant gliomas. " CancerResearch55 (14): 3140-3148 (1995), Moscatello, G. people is waited, " Frequent expression of amutant epidermal growth factor receptor in multiple human tumors. " Cancer Res.55 (23): 5536-9 (1, 995)), 16%NSCL cancer expresses EGFRvIII (Garcia de Palazzo, IE. people is waited, Expression of mutated epidermal growth factor receptor by non-small cell lungcarcinomas.Cancer Res.53 (14): 3217-20 (1993)), 75% ovarian cancer expresses EGFRvIII (Moscatello, G. people is waited, " Frequent expression of a mutant epidermal growth factorreceptor in multiple human tumors. ", Cancer Res.55 (23): 5536-9 (1995)), EGFRvIII (Olapade-Olaopa is expressed with 68% prostate cancer, EO. people is waited, Evidence for the differential expressionof a variant EGF receptor protein in human prostate cancer.Br J Cancer.82 (1): 186-94 (2000)).

Lack 267 amino acid and replace with glycine and produce the uniqueness connection can determining target antibody.In addition, in view of the expression of EGFRvIII in some tumour and expression in the normal tissue thereof lack, EGFRvIII can be used as the ideal targets for determining target medicine in oncotherapy.Specifically, EGFRvIII seems to can be used as the ideal candidate (such as, with antineoplastic agent or toxin conjugated antibody) of tumour immunity conjugate treatment.The method of the cancer of another kind for the treatment of overexpression EGFRvIII relates to use specificity and determines the tumour-specific ribozyme that target is not extremely separated the variant acceptor of normal EGFR.Find that ribozyme significantly suppresses growth of breast cancers people such as (, Int.J.Cancer.104 (6): 716-21 (2003)) Luo in nude mouse.Universal antibody for whole EGFRvIII protein has been described.

See people such as No. 01/62931, international application WO and Kuan, " EGF mutant receptor vIIIas a molecular target in cancer therapy. " Endocr Relat Cancer.8 (2): 83-96 (2001); The people such as Kuan, " EGFRvIII as a promising target for antibody-based brain tumor therapy. " BrainTumor Pathol.17 (2): 71-78 (2000); The people such as Kuan, " Increased binding affinity enhancestargeting of glioma xenografts by EGFRvIII-specific scFv. " International Journal ofCancer.88 (6): 962-969 (2000); The people such as Landry, " Antibody recognition of a conformationalepitope in a peptide antigen:Fv-peptide complex of an antibody fragment specific forthe mutant EGF receptor, EGFRvIII. " Journal of Molecular Biology.308 (5): 883-893 (2001); The people such as Reist, " Astatine-211labeling of internalizing anti-EGFRvIII monoclonalantibody using N-succinimidyl5-[211At] astato-3-pyridinecarboxylate. " NuclearMedicine and Biology.26 (4): 405-411 (1999); The people such as Reist, " In vitro and in vivo behaviorof radiolabeled chimeric anti-EGFRvIII monoclonal antibody:comparison with itsmurine parent. " Nuclear Medicine and Biology.24 (7): 639-647 (1997); The people such as Wikstrand, " Generation of anti-idiotypic reagents in the EGFRvIII tumor-associated antigensystem. " Cancer Immunology, Immunotherapy.50 (12): 639-652 (2002); The people such as Wikstrand, " Monoclonal antibodies against EGFRvIII are tumor specific and react with breastand lung carcinomas malignant gliomas. " Cancer Research.55 (14): 3140-3148 (1995); The people such as Wikstrand, " The class III variant of the epidermal growth factor receptor (EGFRVIII): characterization and utilization as an immunotherapeutic target. " J.Neurovirol.4 (2): 148-158 (1998); The people such as Wikstrand, " The class III variant of the epidermal growthfactor receptor (EGFRvIII): characterization and utilization as an immunotherapeutictarget. " J.Neurovirol.4 (2): 148-158 (1998); The people such as Jungbluth, " A monoclonal antibodyrecognizing human cancers with amplification/overexpres sion of the human epidermalgrowth factor receptor. " Proc Natl Acad Sci U S A.100 (2): 639-44 (2003); The people such as Mamot, " Epidermal Growth Factor Receptor (EGFR)-targeted Immunoliposomes MediateSpecific and Efficient Drug Delivery to EGFR-and EGFRvIII-overexpres sing TumorCells. " Cancer Research63:3154-3161 (2003)).

But each above-mentioned antibody all has or contains murine sequence in change and/or constant region.

The existence of these Muridae derived proteins can cause the quick removing of antibody maybe can cause resisting the immunoreactive generation of patient's internal antibody.In addition, even if after affinity maturation, these antibody still have 2.2x10 ^-8to 1.5x10 ^-9the relatively low avidity of rank.(people such as Kuan, " EGF mutant receptor vIII as amolecular target in cancer therapy. ", Endocr Relat Cancer.8 (2): 83-96 (2001)).

For avoiding using Muridae or rat-derived antibody, human antibodies function is introduced rodent by investigator can produce complete human antibodies to make rodent, see people such as such as Mendez, " Functional transplant ofmegabase human immunoglobulin loci recapitulates human antibody response in mice. " Nat Genet.15 (2): 146-56 (1997).This method has combined the generation for the successful antibody of Wild type EGFR and has used, see people such as such as Yang X, " Development of ABX-EGF; a fully human anti-EGFreceptor monoclonal antibody, for cancer therapy. " Crit Rev Oncol Hemato38 (1): 17-23 (2001); The people such as Yang X-D, Eradication of Established Tumors by a Fully HumanMonoclonal Antibody to the Epidermal Growth Factor Receptor without ConcomitantChemotherapy, Cancer Research59 (6): 1236-1243 (1999); With United States Patent (USP) the 6th, 235, No. 883.

Summary of the invention

In one embodiment, the present invention comprise specific binding to EGFRvIII through being separated human monoclonal antibody and comprising the peptide (SEQ ID NO:56) of sequence L E E K K G N Y V V T D H C.In one embodiment, therapeutical agent can with antibody coupling.In one embodiment, toxin is used.In another embodiment, the present invention comprise specific binding as comprise the epi-position that contains in the sequence (SEQ ID NO:56) of L E E K K G N Y V V T D H C through being separated human monoclonal antibody, wherein as the Alanine-scanning in being arranged by SPOTs measure, in conjunction with needed for resistates be selected from the group be made up of EEK, KKNYV, LEK, EKNY and EEKGN.

Other embodiment comprise comprise by the heavy chain variable amino acid sequence of VH3-33 genes encoding through be separated human monoclonal antibody.Heavy chain variable amino acid sequence can comprise the aminoacid sequence by JH4b genes encoding, or by being selected from the aminoacid sequence of D genes encoding of the group be made up of D6-13 and D3-9.

Other embodiment comprise comprise by the chain variable region amino acid sequence of A23 (VK2) genes encoding through be separated human monoclonal antibody.Chain variable region amino acid sequence can comprise the aminoacid sequence by JK1 genes encoding.

Other embodiment comprise be bonded to EGFRvIII through separation antibody or its fragment, and its comprise be selected from by be designated (SEQ ID) NO:138,2,4,5,7,9,10,12,13,15, the antibody 13.1.2 of 16 and 17,131,170,150,095,250,139,211,124,318, the heavy chain amino acid sequence of group that forms of the heavy chain amino acid sequence of 342 and 333.Antibody can be monoclonal antibody, chimeric antibody, humanized antibody or human antibodies.Antibody or fragment can be associated with pharmaceutically acceptable supporting agent or thinner, and can be combined with therapeutical agent.Therapeutical agent can be toxin.Therapeutical agent can be the toxin of such as DM-1, AEFP, AURISTATIN E or ZAP.Described medicament can associate through linking agent and antibody.Described toxin can associate through second antibody and antibody.Other embodiment comprises the hybridoma cell strain producing antibody and cell transition comprising encoding antibody genes.Such as, described cell can be Cinese hamster ovary cell.

Other embodiment comprises a kind of method suppressing the cell proliferation be associated with the expression of EGFRvIII, and it comprises the cell of expressing EGFRvIII with the antibody of significant quantity or fragment process.In one embodiment, antibody comprises and is selected from by antibody 13.1.2 (SEQ ID NO:138), 131 (SEQ ID NO:2), 170 (SEQ ID NO:4), 150 (SEQ ID NO:5), 095 (SEQ ID NO:7), 250 (SEQ ID NO:9), 139 (SEQ ID NO:10), 211 (SEQ ID NO:12), 124 (SEQ ID NO:13), 318 (SEQ ID NO:15), the heavy chain amino acid sequence of the group of the heavy chain amino acid sequence composition of 342 (SEQ ID NO:16) and 333 (SEQ ID NO:17).The method is in vivo carried out, and carry out in Mammals (such as the mankind) body, described Mammals stands the cancer relating to epithelial cell proliferation, such as lung cancer, colorectal carcinoma, cancer of the stomach, kidney, prostate cancer, mammary cancer, glioblastoma multiforme or ovarian cancer.

Other embodiment comprises the method for killing target cell.This can by making target cell and reaching with the antibody contacts that toxin associates.Antibodies is to peptide LEEKKGNY (SEQ ID NO:133).In one embodiment, antibody has for peptide and is greater than 1.3*10 ^-9the binding affinity of M.In one embodiment, toxin is selected from AEFP, MMAE, DM-1 and ZAP.In one embodiment, the toxicity of antibody toxin compound to target cell be to without polypeptide cell toxicity more than 10 times.In one embodiment, antibody comprises and is selected from by antibody 13.1.2 (SEQ ID NO:138), 131 (SEQ ID NO:2), 170 (SEQ ID NO:4), 150 (SEQ ID NO:5), 095 (SEQ IDNO:7), 250 (SEQ ID NO:9), 139 (SEQ ID NO:10), 211 (SEQ ID NO:12), 124 (SEQID NO:13), 318 (SEQ ID NO:15), the heavy chain amino acid sequence of the group of the heavy chain amino acid sequence composition of 342 (SEQ ID NO:16) and 333 (SEQ ID NO:17).In another embodiment, antibody associates through peptide linking agent or second antibody and toxin.

The other embodiment of the present invention comprise be bonded to EGFRvIII through separation antibody, and it comprises heavy chain amino acid sequence, and it comprises following complementary determining region (CDR):

(a) CDR1, by be selected from by be designated SEQ ID NO:138,2,4,5,7,9,10,12,13,15, the antibody 13.1.2 of 16 and 17,131,170,150,095,250,139,211,124,318, the sequence of group that forms of the aminoacid sequence in the CDR1 district of 342 and 333 forms;

(b) CDR2, by be selected from by be designated SEQ ID NO:138,2,4,5,7,9,10,12,13,15, the antibody 13.1.2 of 16 and 17,131,170,150,095,250,139,211,124,318, the sequence of group that forms of the aminoacid sequence in the CDR2 district of 342 and 333 forms; With

(c) CDR3, by be selected from by be designated SEQ ID NO:138,2,4,5,7,9,10,12,13,15, the antibody 13.1.2 of 16 and 17,131,170,150,095,250,139,211,124,318, the sequence of group that forms of the aminoacid sequence in the CDR3 district of 342 and 333 forms.

In one embodiment, antibody is monoclonal antibody, chimeric antibody, the mankind or humanized antibody.In one embodiment, antibody and pharmaceutically acceptable supporting agent, thinner and/or therapeutical agent associate.In one embodiment, described therapeutical agent is toxin.

In one embodiment, described toxin is DM-1 or Auristatin E.

Also comprise be bonded to EGFRvIII through separation antibody or its fragment, and its comprise be selected from by be designated (SEQ ID) NO:140,19,20,21,29,23,25,26,28,33, the antibody 13.1.2 of 31 and 32,131,170,150,095,250,139,211,123,318, the light-chain amino acid sequence of group that forms of the light-chain amino acid sequence of 342 and 333.Antibody can be monoclonal antibody, chimeric antibody, humanized antibody or human antibodies.It can associate with pharmaceutically acceptable supporting agent or thinner, or such as, combines with therapeutical agent (such as toxin, DM1 or AURISTATIN E).

In one embodiment, contain the hybridoma cell strain that produces antibody or transition cell, this antibody comprise be selected from by be designated (SEQ ID) NO:140,19,20,21,29,23,25.26,26,28,33, the antibody 13.1.2 of 31 and 32,131,170,150,095,250,139,211,123,318, the light-chain amino acid sequence of group that forms of the light-chain amino acid sequence of 342 and 333.Other embodiment comprises the hybridoma cell strain producing this antibody and cell transition comprising encoding antibody genes, such as Cinese hamster ovary cell.

Another embodiment comprises a kind of method suppressing the cell proliferation be associated with the expression of EGFRvIII, and it comprises the cell of expressing EGFRvIII with the above-mentioned antibody of significant quantity or fragment process.The method is in vivo carried out, and carry out in Mammals (such as the mankind) body, described Mammals stands the cancer relating to epithelial cell proliferation, such as lung cancer, colorectal carcinoma, cancer of the stomach, kidney, prostate cancer, mammary cancer, glioblastoma multiforme or ovarian cancer.

Another embodiment comprise be bonded to EGFRvIII through separation antibody, and it comprises light-chain amino acid sequence, and it comprises following complementary determining region (CDR):

(a) CDR1, by be selected from by be designated SEQ ID NO:140,19,20,21,29,23,25,26,28,33, the antibody 13.1.2 of 31 and 32,131,170,150,123,095,139,250,211,318, the sequence of group that forms of the aminoacid sequence in the CDR1 district of 342 and 333 forms;

(b) CDR2, by be selected from by be designated SEQ ID NO:140,19,20,21,29,23,25,26,28,33, the antibody 13.1.2 of 31 and 32,131,170,150,123,095,139,250,211,318, the sequence of group that forms of the aminoacid sequence in the CDR1 district of 342 and 333 forms; With

(c) CDR3, by be selected from by be designated SEQ ID NO:140,19,20,21,29,23,25,26,28,33, the antibody 13.1.2 of 31 and 32,131,170,150,123,095,139,250,211,318, the sequence of group that forms of the aminoacid sequence in the CDR1 district of 342 and 333 forms.

Antibody described in epimere also can comprise heavy chain amino acid sequence, and it comprises following complementary determining region (CDR):

Other embodiment comprises a kind of method suppressing the cell proliferation be associated with the expression of EGFRvIII, and it comprises the cell of expressing EGFRvIII with the above-mentioned antibody of significant quantity or fragment process.The method is in vivo carried out, and carries out in Mammals (such as the mankind) body, and described Mammals stands the cancer relating to epithelial cell proliferation, such as lung cancer, mammary cancer, head and neck cancer, prostate cancer or glioblastoma multiforme.

Other embodiment comprises separated polynucleotide molecule, it comprises polynucleotide sequence or its fragment of encoding heavy chain aminoacid sequence, it is selected from by being designated SEQ ID NO:138, 2, 4, 5, 7, 9, 10, 12, 13, 15, the antibody 13.1.2 of 16 and 17, 131, 170, 150, 095, 250, 139, 211, 124, 318, 342 and 333 heavy chain amino acid sequence composition group, or comprise polynucleotide sequence or its fragment of encoded light chain amino acid sequence, it is selected from by being designated SEQ ID NO:140, 19, 20, 21, 29, 23, 25, 26, 28, 33, the antibody 13.1.2 of 31 and 33, 131, 170, 150, 095, 139, 250, 211, 318, 342 and 333 light-chain amino acid sequence composition group.

Other embodiment comprises and comprises container, is contained in composition wherein and shows that described composition can be used for treating to be expressed as the package insert of the cancer of feature or the product of label with EGFRvIII, and wherein said composition comprises antibody as described above.Described cancer comprises lung cancer, mammary cancer, head and neck cancer, prostate cancer or glioblastoma multiforme.Also comprise for detecting EGFRvIII in mammalian tissues or cell to screen the calibrating test kit of lung cancer, colorectal carcinoma, cancer of the stomach, kidney, prostate cancer or ovarian cancer, wherein EGFRvIII is the antigen of being expressed by epithelial cancer, and test kit comprises the antibody of conjugated antigen protein and is used to indicate the component (if existence) of reaction of antibody and antigen.Described antibody can be the monoclonal antibody through mark, or described antibody can be the first antibody of un-marked, and the component being used to indicate reaction be included as anti-immunoglobulin through mark second antibody.The antibody of conjugated antigen can be marked by the marker being selected from the group be made up of fluorescent dye, enzyme, radionuclide and radiopaque material.The antibody of conjugated antigen also can be bonded to the wtEGFR of overexpression.This test kit can select Clinical practice for patient.

Another embodiment comprises the antibody that specific recognition contains the EGFRvIII epi-position of novel Gly residue.

Another embodiment comprises the variant of EGFRvIII.Described variant can have pFLAG inset, can be made up of, and can be present in computer simulation SEQID NO:56 amino acid.

Another embodiment comprises the antibody or its variant that are bonded to recognition sequence EEKKGNYVVT (SEQ ID NO:57).

Another embodiment comprises the antibody variants of specific binding to EGFRvIII.Described antibody variants can further combined with to the peptide comprising SEQ ID NO:57.Antibody variants can have and the interactional residue of residue EKNY or EEKGN in peptide.In one embodiment, to be bonded to the EGFR protein that peptide sequence is bonded to wild-type than it tight more than 10 times for antibody variants.In one embodiment, antibody variants specific binding is to EGFRvIII and SEQID NO:56 peptide.In one embodiment, separated antibody or variant have the complementary determining region comprising dark chamber, and wherein said chamber is formed by the sub-fraction of CDR2 and CDR3 of heavy chain, the CDR3 of light chain and light chain CDR1.In one embodiment, separated antibody or variant have in 5 dust binding cavities residue 31,37,95-101,143-147,159,162-166,169-171,211-219,221 and 223.In one embodiment, separated antibody or variant have the complementary determining region comprising shallow slot, and wherein said groove is formed by heavy chain CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3.In one embodiment, separated antibody or variant have in 5 dusts are in conjunction with groove residue 31,33,35-39,51,54-56,58-61,94-101,144-148,160,163-166,172 and 211-221.In one embodiment, separated antibody or variant have in 5 dusts are in conjunction with groove residue 31-33,35,37,55,96-101,148,163,165,170,172,178,217 and 218.In one embodiment, separated antibody or variant have a shaping paratope, to make, when the antibody of peptide EEKKGN (SEQ IDNO 127) determines that base is bonded to the paratope of antibody, to form at least one key between two residues being selected from the group be made up of E2 and Y172, K3 and H31, K4 and H31, N6 and D33, N6 and Y37 and N6 and K55.In one embodiment, separated antibody or variant have a shaping paratope, to make, when the antibody of peptide EEKKGNY (SEQ ID NO 131) determines that base is bonded to the paratope of antibody, to form at least one key between two residues being selected from the group be made up of K4 and Q95, K4 and Q95, N6 and Q98, G5 and H31, Y7 and H31 and Y7 and W165.In one embodiment, antibody have a structure or with the structural interaction that measures in computer simulation.

Another embodiment provides a kind of for selecting with particular combination integrate features to the method for the variant of EGFRvIII, described method comprises use molecular structure to form paratope, use molecular structure to form epi-position, calculate interaction energy therebetween and described energy level is compared with the energy level of the second paratope with the epi-position of mAb variant, and selecting variant based on energy level difference.Described method can comprise further and uses the second variant and the interaction energy between epi-position of paratope to measure third phase interaction energy and to compare third phase interaction energy and second-phase interaction energy decides to select which variant.Variant is formed and carries out combination test in one embodiment.

Another embodiment provides a kind of selection with particular combination integrate features to the method for the variant of EGFRvIII, the residue of described method inspection and the interactional epi-position of paratope, select important residue to form recognition sequence, use described sequence to form EGFRvIII variant, and use described EGFRvIII variant to select mAb variant.

Another embodiment provides a kind of method making antibody variants form EGFRvIII, described method comprises the residue analyzed with the interactional epi-position of paratope, select the more important residue of epi-position to form recognition sequence, use described recognition sequence to form EGFRvIII variant, and use described EGFRvIII variant to select antibody variants.In one embodiment, in computer simulation, reach the selection of antibody.In one embodiment, the antibody by producing opposing EGFRvIII variant reaches through using EGFRvIII variant to select antibody.

In one embodiment, wherein separated antibody variants is bonded to EGFRvIII and SEQ ID NO:57 peptide, and antibody can comprise following point mutation further: Tyr172Arg, Leu99Glu, Arg101Glu, Leu217Glu, Leu99Asn, Leu99His, L99T, Arg101Asp or its some combination.In one embodiment, antibody is monoclonal antibody, chimeric antibody, humanized antibody or human antibodies.

In one embodiment, antibody or its variant are bonded to sequence EEKKGNYVVT (SEQ ID NO:57), and described antibody or variant have secondary nmole binding ability.

In yet another embodiment, antibodies is to EGFRvIII and antibody has the paratope being bonded to epi-position, and epi-position has one group of residue interactional with the paratope comprising E, K, N and Y.In one embodiment, described antibody is antibody 131.

In yet another embodiment, antibodies to EGFRvIII and antibody have be bonded to have one group with the paratope of the epi-position of the interactional residue of the paratope comprising E, E, K, G and N.In one embodiment, the primary structure of epi-position is EEKKGNY (SEQ ID NO:131).In one embodiment, antibody is 13.1.2.

In yet another embodiment, antibodies has be less than 1.3*10 to EGFRvIII ^-9m, be less than 1.0*10 ^-9m or be less than the K of 500pM _d.In one embodiment, compared with the EGFR peptide of wild-type, antibody has specificity for SEQ ID NO:56.In one embodiment, the non-specific binding of the EGFR peptide (SEQ ID NO:134) of antibodies of wild-type lower than antibody to 10% of the specific binding of EGFRVIII (SEQ ID NO:135).In one embodiment, antibody is selected from the group be made up of 131,139 and 13.1.2.In one embodiment, antibody is through internalization.In one embodiment, at least about 70% or all internalization occurs at least about the antibody of 80%.

In one embodiment, with compared with the EGFR protein of wild-type or its variant (SEQ ID NO:134), variant human monoclonal's antibody is preferentially bonded to for EGFRvIII protein epi-position unique in fact.In one embodiment, variant comprises the heavy chain complementary determining region (CDR1) corresponding to canonical class 1.In one embodiment, variant comprises the heavy chain complementary determining region (CDR2) corresponding to canonical class 3.In one embodiment, variant comprises the light chain complementarity determining area (CDR1) corresponding to canonical class 4.In one embodiment, variant comprises the light chain complementarity determining area (CDR2) corresponding to canonical class 1.In one embodiment, variant comprises the light chain complementarity determining area (CDR3) corresponding to canonical class 1.In one embodiment, variant comprises the first heavy chain complementary determining region (CDR1) corresponding to canonical class 1, the second heavy chain complementary determining region (CDR2) corresponding to canonical class 3, the first light chain complementarity determining area (CDR1) corresponding to canonical class 4, the second light chain complementarity determining area (CDR2) corresponding to canonical class 1 and corresponds to the 3rd light chain complementarity determining area (CDR3) of canonical class 1, wherein forming these complementary determining regions can be bonded to compared with EGFR protein to make variant, for the epi-position that EGFRvIII protein is unique in fact.

Accompanying drawing explanation

Fig. 1 is the sequence alignment that display 267 aminoacid deletion between EGFR and EGFRvIII of wild-type and G replace.

Fig. 2 is the schema of EGFRvIII PEP3 14-mer peptide.In fig. 2, there is the N-end sequence of the EGFRvIII of amino acid LEEKK.(SEQ ID NO:58) (1-5) is identical with the N-end sequence of EGFR, is unique glycine residue then, is the amino acid identical with the residue 273 to 280 in EGFR then.Fig. 2 B represents the amino acid of the EGFR of disappearance in EGFRvIII (6-272).

Fig. 3 A-L provides the sequence of antibody of the present invention.For the antibody that each provides, all provide polynucleotide and aminoacid sequence for heavy chain and variable region of light chain.Therefore, four sequences are provided for antibody listed by each.

Fig. 4 is the form that 13.1.2 heavy chain of antibody district compares with specific germline heavy chains district, and "-" represents that the amino-acid residue in hybridoma heavy chain district is identical with the germline of that specific location.Represented by suitable amino-acid residue from the skew of germline.

Fig. 5 is the form that 13.1.2 light chain of antibody district compares with specific germline light chain district, and "-" represents that the amino-acid residue in hybridoma light chain district is identical with the germline of that specific location.Represented by suitable amino-acid residue from the skew of germline.

Fig. 6 is the form that various hybridoma derives heavy chain of antibody district and compares with specific germline heavy chains district, and "-" represents that the amino-acid residue in hybridoma heavy chain district is identical with the germline of that specific location.Represented by suitable amino-acid residue from the skew of germline.

Fig. 7 is the form that various hybridoma derives light chain of antibody district and compares with specific germline light chain district, and "-" represents that the amino-acid residue in hybridoma light chain district is identical with the germline of that specific location.Represented by suitable amino-acid residue from the skew of germline.

Fig. 8 is the representative graph of the combination of display restructuring EGFRvIII mAb and the cell (NR6 cell) of expressing EGFRvIII.Rhombus represents 95, and trilateral represents 133, and square represents 139, and " x " represents 150, and asterisk represents 170, and circle represents 221, and straight line represents 230, and rectangle represents 250.

Fig. 9 A shows for mankind's anti-EGFR-antibodies (ABX-EGF) to the staining analysis of H80.

Fig. 9 B shows the FACS staining analysis for antibody 131 to H80.

Fig. 9 C shows the FACS staining analysis for antibody 139 to H80.

Fig. 9 D shows the FACS staining analysis for antibody 13.1.2 to H80.

Fig. 9 E shows the FACS staining analysis for ABX-EGF to H1477.

Fig. 9 F shows the FACS staining analysis for antibody 131 to H1477.

Fig. 9 G shows the FACS staining analysis for antibody 139 to H1477.

Fig. 9 H shows the FACS staining analysis for antibody 13.1.2 to H1477.

Fig. 9 I shows the FACS staining analysis for ABX-EGF to A549.

Fig. 9 J shows the FACS staining analysis for antibody 131 to A549.

Fig. 9 K shows the FACS staining analysis for antibody 139 to A549.

Fig. 9 L shows the FACS staining analysis for antibody 13.1.2 to A549.

Fig. 9 M is the graphic representation of the combination showing EGFRvIII mAb and glioblastoma cells.

Black triangle representative is bonded to the antibody 131 of H1477.Filled squares representative is bonded to the antibody 13.1.2 of H1477.Hollow triangle representative is bonded to the antibody 131 of H80.Open squares representative is bonded to the antibody 13.1.2 of H80.

Fig. 9 N shows the graphic representation of EGFRvIII mAb to the combination of human epidermoid carcinoma cells's strain A431.Filled squares represents antibody 13.1.2.Black triangle represents antibody 131.

Fig. 9 O is the graphic representation of the combination showing antibody 13.1.2 to NR6 Muridae inoblast cell strain.Square represents NR6.Trilateral representative has the NR6 of Wild type EGFR.Circular representative has the NR6 of EGFRvIII.

Fig. 9 P is the graphic representation of the combination showing antibody 131 to Muridae inoblast cell strain.Square represents NR6.Trilateral representative has the NR6 of Wild type EGFR.Circular representative has the NR6 of EGFRvIII.

Figure 10 A shows FACS staining analysis mankind's anti-EGFR-antibodies (ABX-EGF) being bonded to expression EGFR cell (A431).

Figure 10 B shows for antibody 131 to the FACS staining analysis expressing EGFR cell (A431).

Figure 10 C shows for antibody 139 to the FACS staining analysis expressing EGFR cell (A431).

Figure 10 D shows for antibody 13.1.2 to the FACS staining analysis expressing EGFR cell (A431).

Figure 11 shows the structural models of antibody 131 molecular surface.Six CDR are covered for different shades is to mark their edge.Binding cavity is positioned at close to center.

Figure 12 shows the structural models of antibody 13.1.2 molecular surface.Cover six CDR and identified by numeral.Elongated slot probably distributes along vertical center line.

Figure 13 A is the possible extended model of 13.1.2 antibody and peptide EEKKGN (SEQ ID NO:127) misfit thing.CDR district covers for shade is to indicate border.

Figure 13 B shows the hydrogen bond in 13.1.2 antibody and peptide EEKKGN (SEQ ID NO:127) misfit thing possibility extended model.CDR circle and the shade of residue and identical in Figure 12.Peptide residue is numbered 1 to 6 from the N-end on figure top to C-end.Six hydrogen bonds are indicated by dotted line.The six pairs of amino acid forming hydrogen bond are: E2...Y172, K3...H31, K4...H31, N6...D33, N6...Y37 and N6...K55.

Figure 14 display for one of selected extended model epitope-antibody combine can and the logarithm of Kd between the graphic representation associated.

Figure 15 describes the precise spread model of peptide-13.1.2 antibody misfit thing.Peptide presents with space solid form.

Figure 16 describes the hydrogen bond in precise spread model.

Figure 17 is that the combination of description antibody-antigene can relative to the graphic representation of the linear fit of the logarithm of relative affinity.

Embodiment

As described above, EGFRvIII is the deletion mutant of EGFR, 267 aminoacid deletion in the extracellular domain of wherein EGFr, and carries out single amino acid replacement in junction with glycine.These features are shown in the sequence alignment in Fig. 1 between Wild type EGFR and EGFRvIII.In view of the amino acid of the glycine of the junction in disappearance is replaced, the antibody for being present in EGFRvIII the novel epi-position be not present in the EGFR of wild-type may be produced in theory.Therefore, as shown in Figure 2, be designed for the peptide of immunity and screening, be called the PEP3 (people such as Kuan, EGF mutant receptor vIII as a molecular target in cancer therapy, Endocr Relat Cancer.8 (2): 83-96 (2001)).This 14-mer peptide has for 5 common n-end amino acids of the EGFR of EGFRvIII and wild-type, 8 amino-acid residues contained in the conserved sequence between the EGFR (corresponding to residue 273-280) of unique glycine connection site and wild-type and EGFRvIII (corresponding to residue 7-14).In addition, glioblastoma cells and with the cell of the EGFRvIII transfection of genes encoding (B300.19 cell) also can be used for immunity and screening (being sometimes referred to as B300.19/EGFRvIII transfectant herein).

For producing the human antibodies of opposing EGFRvIII, by transgenosis

mouse carries out immunity with glioblastoma cells/EGFRvIII, B300.19/EGFRvIII cell with the combination of the peptide (PEP3) for the joining region in the novel extracellular domain represented in the EGFRvIII compared with the EGFR of wild-type.The B cell of hanging oneself in the future in immune mouse is separated and for generation of glioblastoma multiforme, then screens be bonded to EGFRvIII or use XenoMax ^tM/ SLAM ^tMtechnology is directly used in screening to be bonded to the EGFRvIII (people such as Babcook, Anovel strategy for generating monoclonal antibodies from single, isolated lymphocytesproducing antibodies of defined specificities, Proc Natl Acad Sci U S A.93 (15): 7843-8 (1996) and United States Patent (USP) the 5th, 627, No. 052).Through identifying that the antibody being bonded to EGFRvIII screens through a series of calibrating with the specific recognition determining EGFRvIII.Produce through this process, be separated and characterize and be bonded to EGFRvIII and to EGFRvIII, there is specific human monoclonal antibody colony.Uniqueness shows epitope mapping subsequently, but overlap specificity.In vitro all antibody of assessment carries out the ability of internalization to assess it in order to cytotoxic drugs is delivered to cell by cell further.Show that the antibody effectively transmitting medicine is directly combined with cytotoxic drugs, and detect the ability that it kills the tumour cell of expressing EGFRvIII in vitro and in vivo.These are studied as the generation next time of the antibody drug conjugates being used for the treatment of patient's cancer provides the foundation, and the tumour of these patients is concealed with specific gene pathology.

The colony of complete mankind's anti-EGFR vIII antibody is produced by said process.Use hybridoma mode, the EGFR produced for wild-type has the reactive several antibody of limited cross, comprises the antibody 13.1,13.2,13.3 and 13.4 be positive to the ELISA for being combined with PEP3.Except these, select antibody 13.1 (with being especially its subclone 13.1.2) for further research and development.Use XenoMax mode, produce antibody population, comprise antibody 131,139,250 and 095, its for pep ³the combination of oligonucleotide has high degree of specificity, and has limited cross reactivity with the EGFR of wild-type.Wherein, 131 antibody have the most interesting characteristic.Show the sequence (SEQ ID NO:1-33 and 141-144) of each antibody in figures 4-7.The sequence of various antibody and binding ability are compared and will be the results are shown in Fig. 4-10.As visible in Fig. 9 A-9L and Fig. 1 OA-10D, compared with ABX-EGF, antibody 131,139 and 13.1.2 show the better selectivity for EGFRvIII express cell (H1477).Some the results are shown in the graphic representation in Fig. 9 M-9P, and it shows simple compared with EGFRvIII cell, and at least two kinds of antibody 13.1.2 and 131 show the better selectivity for EGFRvIII express cell.Finally, based on the structural models of prediction, the variant forming antibody has the antibody of change in conjunction with feature to obtain.

In addition, antibody of the present invention is extremely useful for the screening of other antibody being bonded to identical or similar epi-position.Antibody of the present invention can be used for illustrating in the cross competition research of other antibody, expects that other antibody has identical or through improveing impact for the feature of the Ag-Ab misfit thing formed.

Each has high avidity 131 antibody and 13.1.2 all by the good internalization of cell for EGFRvIII, and shows when being bonded to toxin and efficiently can kill cell.Whether interestingly, no matter result from the difference immunity of XenoMouse mouse, and no matter whether use different technology, two kinds of antibody are all derived from very similar germ line genes.But based on epitope mapping operation, each antibody seems to be bonded to epi-position slightly different on EGFRvIII molecule, and have for combining residue slightly different on necessary EGFRvIII.It is particularly important for the generation of the antibody therapy determining target EGFRvIII that these results show to use germ line genes, and little change is by improveing combination and the impact of antibody based on the further designerantibodies of these topology discoverys and other therapies.

Highly need to be bonded to identical epi-position, or the antibody of competition and 13.1.2 and 131 antibodies.As hereafter more discussed in detail, the Alanine-scanning arranged by SPOTs is illustrated for specific antibodies in conjunction with important residue.Therefore, the shared important antibody in conjunction with residue is also highly needed.

definition

Unless otherwise defined, the Science and Technology term used herein implication that will there are one of ordinary skill in the art usually understand.In addition, unless the context otherwise requires, singular references comprises plural form and plural term comprises singulative.Generally speaking, the term in conjunction with cell and tissue culture, molecular biology and protein and oligonucleotide or polynucleotide chemistry and hybridization as herein described and technology thereof have known in technique and those terms normally used.Standard technique is used for recombinant DNA, oligonucleotide synthesis and tissue culture and transition (such as, electric shock, lipofection).According to that usually realize in the explanation of the producer or technique or as described hereinly carry out enzyme reaction and purification technique.Usually according to the ordinary method known in technique and as this specification sheets in the whole text carry out aforementioned techniques and program described in the various general and more detailed reference quoting and discuss.See, the people such as such as Sambrook, Molecular Cloning:A Laboratory Manual (the 2nd edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989).The term that uses about analytical chemistry, synthetic organic chemistry and medicine and medical chemistry as herein described and testing sequence thereof and technology have been known in the art and have usually used.

Standard technique is used for chemosynthesis, chemical analysis, medicine preparation, allotment and transmission and treatment patient.

As the term is employed herein " through be separated polynucleotide " be meant to genome, cDNA or synthetic source polynucleotide or its certain combination, due to its source, " through being separated polynucleotide " (1) has nothing to do with all or the part polynucleotide that wherein " through being separated polynucleotide " finds at occurring in nature, (2) polynucleotide is connected to operably, do not connect at occurring in nature, or (3) occurring in nature not as larger sequence part and occur.The term " through isolated protein " mentioned herein refer to cDNA, recombinant RNA or synthetic source protein or its certain combination, originate due to it or derive source, the protein that " through isolated protein " (1) and occurring in nature find has nothing to do, (2) not containing the other oroteins from identical sources, such as, not containing Muridae protein, (3) are expressed by the cell from different plant species, or (4) do not occur at occurring in nature.

Term " polypeptide " is used as general terms in this article, refers to the natural protein of peptide sequence, fragment or analogue.Therefore, natural protein, fragment or analogue are polypeptides matters.Better polypeptide according to the present invention comprises human heavy chain immunoglobulin molecules and human kappa light chain's immunoglobulin molecules, and by comprising the antibody molecule be combined to form of heavy chain immunoglobulin molecule and light chain immunoglobulin molecule (such as kappa light chain immunoglobulin molecule or lambda light chain immunoglobulin molecules), and vice versa, and its fragment and analogue.

The term " natural generation " be applied to as used herein on a kind of object refers to this fact that can find a kind of object at occurring in nature.Such as, be present in the organism (comprising virus) that can be separated from natural source, and be natural generation without artificially or otherwise having a mind to the polypeptide of upgrading or polynucleotide sequence in the lab.

As the term is employed herein " operability connection " refer to that the position of described component is in them can be allowed to play in the relation of function in the mode desired by it.Control sequence " is operatively connected " and is connected can reach the mode expressing encoding sequence under the condition compatible with control sequence to encoding sequence.

Term used herein " control sequence " refers to the polynucleotide sequence be necessary for the expression and process that realize the encoding sequence that polynucleotide sequence connects.The essence of these control sequence looks its HOST ORGANISMS and different, and in prokaryotic organism, these control sequence generally include promotor, ribosome bind site and transcription termination sequence; In eukaryote, these control sequence generally include promotor and transcription termination sequence.Term " control sequence " mean comprise at least its exist for such as leader and fusion partners sequence expression and process necessary all components, and comprise it and exist for favourable other component of such as leader and fusion partners sequence.

Term " polynucleotide " is herein meant to the polymerized form that length is at least the Nucleotide of 10 bases, the upgrading form of ribonucleotide or deoxyribonucleotide or any one Nucleotide.Term comprises strand and the double chain form of DNA.

The upgrading Nucleotide that oligonucleotide connexon that is that the term " oligonucleotide " mentioned herein comprises natural generation and that occurred by natural generation and non-natural links together.Oligonucleotide is the polynucleotide subgroup usually comprising 200 bases or less length.Oligonucleotide preferred length is 10 to 60 bases, and optimization length is 12,13,14,15,16,17,18,19 or 20 to 40 bases.Oligonucleotide is generally strand, such as, for detection; Although oligonucleotide can be double-strand, such as, for structural gene mutant.Oligonucleotide of the present invention can be justice or antisense oligonucleotide.

" Nucleotide of natural generation comprises deoxyribonucleotide and ribonucleotide to the term mentioned herein.The term " upgrading Nucleotide " mentioned herein comprises containing upgrading or the Nucleotide and the analogue thereof that replace glycosyl.The term " oligonucleotide connexon " mentioned herein comprises oligonucleotide connexon, such as, and thiophosphatephosphorothioate, phosphorodithioate, phosphoroselenoate, two phosphoroselenoate, aniline thiophosphatephosphorothioate, aniline phosphoric acid ester, phosphoramidate and analogue thereof.See, the people such as such as LaPlanche, Nucl.Acids Res.14:9081 (1986); The people such as Stec, J.Am.Chem.Soc.106:6077 (1984); The people such as Stein, Nucl.Acids Res.16:3209 (1988); The people such as Zon, Anti-Cancer Drug Design6:539 (1991); The people such as Zon, Oligonucleotides and Analogues:APractical Approach, 87-108 page (F.Eckstein compiles, Oxford University Press, OxfordEngland (1991)); The people such as Stec, United States Patent (USP) the 5th, 151, No. 510; Uhlmann and Peyman ChemicalReviews90:543 (1990).If needed, oligonucleotide can comprise the mark for detecting." variant " is for being different from the polypeptide of cited polypeptide and polynucleotide, polynucleotide or molecule as the term is employed herein, but just in order to not cause unfavorable change to protein active.The variant of epi-position can be there is.The variant of antibody can be there is.In a preferred embodiment, protein variant is bonded to the not unfavorable change of ability of epi-position.In one embodiment, protein variant can wild-type mAb ability 10-500% combine.Such as, protein variant can wild-type mAb ability 10%, 50%, 110%, 500% or be greater than 500% combination.In one embodiment, the binding ability between 10-500% scope is included in.Binding ability reflects by various ways, includes, but is not limited to the k of variant to epi-position _a, k _dor K _d.In a preferred embodiment, epi-position is the epi-position described in this specification sheets.

In one embodiment, variant antibodies is different from the sequence of wild-type by replacing, lacking or add 5 or less amino acid.These variants are usually by one of peptide sequence that upgrading discloses, and example representative program evaluates as described herein identifies through the binding characteristic of upgrading polypeptide.In another embodiment, polypeptide variants preferably show with through Recognition polypeptide at least about 70%, more excellent at least about 90% and optimum at least about 95% identity.Preferably, variant is only different in conservative replacement and/or upgrading.Variant proteins comprises the protein structure protein that structurally class Sihe function is suitable described in those and this specification sheets.In another embodiment, if, so this protein can be variant on protein function described in protein and this specification sheets quite, if the paratope of variant and the paratope described in this specification sheets similar.It is in one embodiment, any that to have with the material of the analogous shape of paratope described in Figure 11 be all variant.It is in one embodiment, any that to have with the material of the analogous shape of paratope described in Figure 12 be all variant.It is in one embodiment, any that to have with the material of interactive surfaces analogous shape described in Figure 13 A and 13B be all variant.

In one embodiment, if nucleotide sequence under strict conditions optionally with the sequence hybridization of wild-type, so antibody is variant.In one embodiment, suitable appropriate stringent condition is included in prewashing in 5xSSC solution, 0.5%SDS, 1.0mM EDTA (pH8:0); At 50 DEG C-65 DEG C hybridize, 5xSSC, if overnight or for kind between homologue, then at 45 DEG C, 0.5xSSC; Then wash twice with 2x, 0.5x and 0.2xSSC at every turn containing 0.1%SDS at 65 DEG C, last 20 minutes.These hybrid DNA sequence also belong in category of the present invention, due to the degeneracy of coding, if nucleotide sequence coded antibody polypeptides is also coded by hybrid DNA sequence.The term " selective cross " mentioned herein is meant to detect ground and to combine specifically.Be bonded to non-specific nucleic acid according to polynucleotide of the present invention, oligonucleotide and fragment thereof and be minimised as selectivity and nucleic acid hybridization under the hybridization of appropriate amount and wash conditions can detect.High stringency can be used for reaching as known in the art and in selective cross condition discussed herein.Generally speaking, polynucleotide of the present invention, oligonucleotide and the nucleic acid sequence homology between fragment and interesting nucleotide sequence are at least 80%, and are more generally the homology of the increase of preferably at least 85%, 90%, 95%, 99% and 100%.If there is partially or completely identity between two seed amino acid sequences, so this two seed amino acids sequence is homology.Such as, 85% homology is meant to when two kinds of sequences carry out maximum match comparison, and the amino acid of 85% is consistent.In maximization coupling, allow to there is gap (any one of two kinds of sequences matches); Be preferably 5 or less gap length, 2 or less more excellent.Or or preferably, this term as used herein, if use the ALIGN program containing mutation data matrix and 6 or more gap cost, two kinds of protein sequences (or being peptide sequence derivative at least 30 amino acid whose protein from length) have the comparison score value more than 5, so their homologies.See Dayhoff, M.O., in Atlasof Protein Sequence and Structure, 101-110 page (the 5th volume, National Biomedical ResearchFoundation (1972)) and this 2nd phase supplementary issue rolled up, 1-10 page.If the amino acid of two kinds of sequences or its part is more than or equal to 50% unanimously, so these two kinds of sequences or more excellent homology of its part when using the comparison of ALIGN program the best.Term used herein " corresponds to " and is meant to polynucleotide sequence and reference polynucleotide sequence homology (that is, unanimously, but non-critical develops relevant), or polynucleotide sequence is consistent with reference to polynucleotide sequence.Contrastingly term used herein " complementation " is meant to complementary sequence and all or part of homology with reference to polynucleotide sequence.Such as, nucleotide sequence " TATAC " is with reference sequences " GTATA " complementary corresponding to reference sequences " TATAC ".

Following term is for describing the sequence relation between two or more polynucleotide or aminoacid sequence: " reference sequences ", " comparison window ", " sequence identity ", " sequence identity percentage " and " consistent in fact "." reference sequences " is fixed sequence, as the benchmark of gene comparision; Reference sequences can be the subgroup of larger sequence, such as, as the fragment of full-length cDNA given in sequence list or gene order, maybe can comprise global cDNA or gene order.Generally speaking, reference sequences length is at least 18 Nucleotide or 6 amino acid, and length is often at least 24 Nucleotide or 8 amino acid, and length is generally at least 48 Nucleotide or 16 amino acid.Because (namely two polynucleotides or aminoacid sequence each (1) may be included in sequence similar between two molecules, a part for complete polynucleotide or aminoacid sequence), (2) sequences different between two polynucleotides or aminoacid sequence may be included in further, the general sequence by comparing two molecules through " comparison window " with the regional area identified and comparative sequences is similar carry out two (or more) gene comparision between molecule." comparison window " refers at least 18 adjacent nucleotide positions or 6 amino acid whose conceptual segments as used herein, wherein polynucleotide sequence or aminoacid sequence can compared with the reference sequences of at least 18 adjacent Nucleotide or 6 aminoacid sequences, and wherein compared with the position of polynucleotide sequence in comparison window and reference sequences (its do not comprise add or lack), can comprise 20% or less interpolation, disappearance, replacement and analogue thereof (namely, gap), for the optimal sequence comparison of two sequences.By Smith and Waterman, the local homology algorithm of Adv.Appl.Math.2:482 (1981), by Needleman and Wunsch, the homology alignment algorithm of J.Mol.Biol.48:443 (1970), by for Pearson and Lipman, Proc.Natl.Acad.Sci. the research of the similar approach of (U.S.A.) 85:2444 (1988), by the computer implementation (GAP of these algorithms, BESTFIT, TFASTA in FASTA and Wisconsin Genetics Software Package Release7.0, (GeneticsComputer Group, 575Science Dr., Madison, Wis.), Geneworks or Mac Vector software encapsulation), or the optimal sequence comparison of the sequence for comparison comparison window is carried out by observation, and the optimal sequence comparison that selection is produced by various method (namely, cause the highest per-cent of homology in comparison window).

Term " sequence identity " is meant to two polynucleotides or aminoacid sequence consistent in comparison window (that is, on the basis of Nucleotide × Nucleotide or residue × residue).Term " percentage of sequence identity " is by comparing the sequence of two optimum comparisons in comparison window, measure and consistent nucleic acid base occurs (such as in two sequences, A, T, C, G, U or I) or residue to produce the numbering of position of matched position numbering, by the numbering of matched position divided by the total number of positions in comparison window (namely, window size), and acquired results is multiplied by 100, to produce percentage of sequence identity." consistent in fact " represents the feature of polynucleotide or aminoacid sequence as the term is employed herein, wherein with in the comparison window of at least 18 Nucleotide (6 amino acid) position, reference sequences often on the window of at least 24-48 Nucleotide (8-16 amino acid) position is compared, it is consistent that polynucleotide or amino acid comprise at least 85% sequence, preferably at least 90% to 95% sequence is consistent, the sequence that more generally at least 99% sequence is consistent, wherein by carrying out sequence of calculation consistence per-cent with reference to sequence with may comprise comparison window amounting to compared with 20% or less disappearance of reference sequences or the sequence of interpolation.Reference sequences can be the subgroup of larger sequence.Be the protein of wild-type or the variant of nucleic acid with the protein of wild-type or the consistent in fact amino acid of nucleic acid or nucleic acid.

Common usage is followed in 20 kinds of conventional amino acid as used herein and abbreviation thereof.See Immunology-ASynthesis (the 2nd edition, E.S.Golub and D.R.Gren compiles, Sinauer Associates, Sunderland, Mass. (1991)).20 kinds of conventional amino acid (such as, D-amino acid), alpha-non-natural amino acid (such as a-amino acid, α-bis-substituted amino acid, N-alkyl amino acid, lactic acid) and other unconventional amino acid whose steric isomer also can as the suitable ingredients for polypeptide of the present invention.Unconventional amino acid whose example comprises: 4-oxyproline, Gla, ε-N; N, N-trimethyl lysine, ε-N-acetyllysine, O-phosphoserine, N-acetyl-serine, N-formyl methionine, 3-Methyl histidine, 5-oxylysine, σ-N-methylarginine and other similar amino acid and imino-acid (such as 4-oxyproline).In polypeptide symbol used herein, according to standard usage and routine, left-hand is to being N-terminal direction, and right-hand lay is C-terminal direction.

Similarly, except as otherwise noted, the left hand end of single-stranded polynucleotide sequence is 5 ' end, and the left hand end of double stranded polynucleotide sequence is called 5 ' direction.5 ' to the 3 ' direction of adding of nascent rna transcription is called transcriptional orientation, there is the sequence identical with RNA and for rna transcription 5 ' to 5 ' end DNA chain on sequence area be called " upstream sequence ", there is the sequence identical with RNA and for rna transcription 3 ' to 3 ' end DNA chain on sequence area be called " downstream sequence ".

Term " consistent in fact " as being applied to polypeptide is meant to when the such as comparison optimum by GAP or the BESTFIT program of use default slot weight of two peptide sequences, two peptide sequences share the sequence identity of at least 80%, be preferably the sequence identity of at least 90%, more excellent be at least 95% sequence identity, and the best is the sequence identity of at least 99%.Preferably, incomparable inconsistent resi-dues is replaced by conserved amino acid and different.Conserved amino acid replaces the interchangeability referring to the residue with similar side chain.Such as, the amino acid group with aliphatic lateral chain is glycine, L-Ala, α-amino-isovaleric acid, leucine and Isoleucine; The amino acid group with aliphatic-hydroxy side chains is Serine and Threonine; The amino acid group had containing amide side chain is l-asparagine and glutamine; The amino acid group with beta-branched side is phenylalanine, tyrosine and tryptophane; The amino acid group with basic side chain is Methionin, arginine and Histidine; And the amino acid group with sulfur-containing side chain is halfcystine and methionine(Met).Preferred conserved amino acid replaces group: Val-Leu-Isoleucine, phenylalanine-tyrosine, Lys-Arg, alanine-valine, glutamate-aspartate and asparagine-glutamine.Polypeptide consistent in fact can be variant.

Variant proteins also comprises the protein with subtle change.As discussed herein, take into account the subtle change in the aminoacid sequence of antibody that the present invention comprises or immunoglobulin molecules, as long as the change in aminoacid sequence remains at least 75%, more excellent at least 80%, 90%, 95%, and optimum is 99%.Particularly, contemplate the displacement of conserved amino acid.

Conservative substitution is that those betide the displacement in the amino acid residues relevant to its side chain.Amino acid through genes encoding is divided into following family usually: (1) acidity=aspartic acid, L-glutamic acid; (2) alkalescence=Methionin, arginine, Histidine; (3) nonpolar=L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane and (4) uncharged polar=glycine, l-asparagine, glutamine, halfcystine, Serine, Threonine, tyrosine.More excellent family is: Serine and Threonine are aliphatic-hydroxy family; L-asparagine and glutamine are for containing amide family; L-Ala, α-amino-isovaleric acid, leucine and Isoleucine are aliphatics family, and phenylalanine, tryptophane and tyrosine are aliphatics family.Such as, rational expectation can replace leucine with Isoleucine or α-amino-isovaleric acid through being separated, with L-glutamic acid through being separated displacement aspartic acid, with Serine through being separated displacement Threonine or material impact can not being had to the combination of gained molecule or characteristic, if when especially displacement does not relate to the amino acid of framework position inside with the similar displacement of amino acid through being separated replacement amino acid that structure is correlated with.Whether amino acid change causes functional peptides can measure easily through the activity specific of calibrating polypeptide derivative.Calibrating is described in detail below.One of ordinary skill in the art can be easy to fragment or the analogue of Dispersal risk or immunoglobulin molecules.The amino of preferred fragment or analogue and carboxyl-tenninus betide the adjacent edges of functional domain.By Nucleotide and/or amino acid sequence data are come recognition structure and functional domain compared with open or proprietary sequence library.Preferably, other known structure and/or the sequence motif functionally in protein or predicted protein matter conformation domains is used computer comparative approach to identify to betide.Become known for identifying the method for the protein sequence being folded into known three-dimensional structure.The people such as Bowie, Science253:164 (1991).Therefore, previous examples shows, those skilled in the art's identifiable design can be used for sequence motif and the node configuration that antibody according to the present invention defines structure and function structural domain.

Preferred amino acid replaces with following replacement: (1) reduces proteolysed susceptibility, (2) susceptibility to oxygenizement is reduced, (3) change the binding affinity for forming protein misfit thing, (4) change binding affinity and (5) authorize or other physical chemistry of these analogues of upgrading or functional performance.Analogue can comprise the various muteins of the sequence except the peptide sequence of natural generation.Such as, single or Multiple amino acid replacement (being preferably conserved amino acid to replace) can be carried out in the sequence of natural generation (being preferably the polypeptide portion of the structural domain outside forming intermolecular contacts).Conservative heavy amino acid replaces the constitutional features (such as, replacement amino acid should not destroy the spirochete betided in parental array, or interference characterizes other type of the secondary structure of parental array) that should not change in fact parental array.At Proteins, (Creighton compiles Structures and Molecular Principles, W.H.Freeman and Company, New York (1984)), Introduction to Protein Structure (C.Branden and J.Tooze compile, Garland Publishing, New York, N.Y. (1991)) and the people such as Thornton, the polypeptide secondary of technique identification and the example of tertiary structure are described in Nature354:105 (1991).

" polypeptide fragment " refers to the polypeptide with amino terminals and/or carboxy-terminal deletion as the term is employed herein, but wherein residual aminoacid sequence is consistent with the correspondence position in the sequence of the natural generation of drawing from (such as) full length cDNA sequence.Fragment is generally at least 5,6,8 or 10 amino acid longs, be preferably 14 amino acid longs, more excellent is at least 20 amino acid longs, is generally at least 50 amino acid longs, and even more excellent be at least 70 amino acid longs." analogue " refers to the polypeptide comprising at least 25 amino acid fragments consistent in fact with a part for the aminoacid sequence of drawing as the term is employed herein.Analogue is generally at least 20 amino acid longs, is preferably at least 50 amino acid longs and often can grows to the natural generation polypeptide of total length.Fragment and analogue are all variant forms.

Peptide analogs usually using be similar to template peptide characteristic and as non-peptide drugs in medicine industry.The non-peptide compound of these types is called " stand-in of peptide " and " peptide mimics ", Fauchere, J.Adv.Drug Res.15:29 (1986); Veber and Freidinger TINS, the 392nd page of people such as (1985) and Evans, J.Med.Chetn.30:1229 (1987).Usually these compounds are researched and developed by means of computer molecular model.Similar can be used for producing suitable treatment or preventive effect in the peptide mimics of therapeutic peptide.Generally speaking, peptide mimics is structurally similar (namely with pattern polypeptide, there is the polypeptide of biochemical characteristic or pharmacological activity), such as human antibodies, but one of them or more than one peptide connexon by the method known in technique optionally by being selected from by-CH ₂nH--,--CH ₂s--,-CH ₂-CH ₂--,--CH=CH--(cis and trans),--COCH ₂--,--CH (OH) CH ₂--with-CH ₂the connexon of the group of SO--composition replaced.The amino acid (such as, replacing 1B with D-Lys) replacing one or more Unified Sequences with the D-amino acid systems of identical type can be used for producing more stable peptide.In addition, can by method known in technique produce comprise that Unified Sequences or Unified Sequences consistent in fact change limit peptide (Rizo and Gierasch Ann.Rev.Biochem.61:387 (1992)); Such as, by add can form cyclisation peptide molecule in the internal cysteine residues of two sulphur bridge.The stand-in of peptide and peptide mimics are all variant forms.

" antibody " or " antibody peptide " refers to complete antibody or itself and the binding fragment competed for the complete antibody of specific binding.By recombinant DNA technology or to be decomposed by the enzyme of complete antibody or decomposition produces binding fragment.Binding fragment comprise Fab, Fab ', F (ab ') ₂, Fv and single-chain antibody.It is consistent that antibody except " dual specific " or " difunctional " antibody is interpreted as its each binding site.When excessive antibodies is reduced by least about 20%, 40%, 60% or 80% by what be bonded to counter receptor by the scale of construction, and be more generally greater than about 85% (as examined and determine measured by vitro competitive binding) time, antibody suppresses in fact receptor adhesive to counter receptor.

Term " epi-position " comprise any can specific binding to immunoglobulin (Ig) or T-cell receptors or otherwise with the protein determinant of interaction of molecules.Epi-position bunch is made up of the chemically reactive surface group of such as amino acid or carbohydrate or sugared side chain molecule usually, and usually has specific three dimensional constitutional features and specific charge characteristics.Epi-position can be " linearly " or " configuration ".In linear epitope, all interaction points between protein and interactional molecule (such as antibody) linearly occur along the first aminoacid sequence of protein.At conformation epitopes, the amino acid of interaction point on protein separated from one another intersects and occurs.It is said when dissociation constant≤1 μM, preferably≤100nM and more excellent≤10nM, and even more excellent≤lnM time, antibodies specific conjugated antigen.Once determine the epi-position that antigen needs, just may such as use technology of the present invention generation for the antibody of that epi-position.Or in discovery procedure, the generation of antibody and feature can illustrate the information of required epi-position.Based on these information, the antibody for being bonded to identical epi-position may be screened subsequently competitively.A kind of mode reaching this object carries out finding the cross competition research of the antibody (such as, competition binding is to the antibody of antigen) that contending with one other property combines.A kind of high yield process for " combination " antibody based on its cross competition is described in No. 03/48731, international application WO.As by being appreciated by one of skill in the art that, antibody can specific binding any predetermined substance extremely can be epi-position.Epi-position can comprise antibodies those residues extremely.In one embodiment, epi-position is EGFRvIII epi-position.In a more excellent embodiment, epi-position is the epi-position described in this specification sheets example 4.In one embodiment, epi-position is the epi-position described in example 14.In one embodiment, epi-position comprises sequence LEEKKGNYVVTD (SEQID NO:59).In one embodiment, epi-position comprises sequence EEKKGNYVVT (SEQ ID NO:57).In one embodiment, epi-position comprises sequence EKNY (SEQ ID NO:60).In one embodiment, epi-position comprises sequence EEKGN (SEQ ID NO:61).Be understood by those skilled in the art that these in fact need not with this sequential combination on single peptide, and these form the residue with the interactional epi-position of paratope.If those skilled in the art institute is by understanding, the space occupied by the residue or side chain that produce shape of molecule contributes to why measuring epi-position.Equally, the degree of excursion etc. of relevant to epi-position any functional group, Van der Waals interaction, side chain all can measure epi-position and what is actually.Therefore, epi-position also can comprise energy interactions.

Term " paratope " means the universal architecture describing and measure for the land of the combination of epi-position.Whether this structure influence land is bonded to the mode of epi-position and combination thereof.Paratope can refer to that responsible antibody or its fragment are bonded to the antigen site of the antibody of epi-position bunch.Paratope also refers to the idiotope of antibody, and is bonded to the complementary determining region (CDR) of epi-position.In one embodiment, paratope is the antibody district of L1 10, L2 30 in Figure 11, L3 50, H1 20, H240 and H3 60.In one embodiment, paratope comprises the antibody district for the CDR sequence of L1, L2, L3, H1, H2 and H3 in example 16.In one embodiment, paratope is the L1 in Figure 12,110, L2,130, L3,150, the antibody district of H1 120, H2 140 and H3 160.In one embodiment, paratope comprises the antibody district for the CDR sequence of L1, L2, L3, H1, H2 and H3 in example 18.In one embodiment, paratope comprises sequence listed in example 18.In one embodiment, paratope comprises the interactional residue with epi-position, as shown in Figure 13 A and Figure 13 B.Solid black structure is peptide structure.In one embodiment, paratope comprises the residue Tyr172Arg of 13.1.2mAb.In one embodiment, the paratope of 13.1.2mAb comprises at least one residue being selected from the group be made up of following residue: Tyr172Arg, Arg101Glu, Leu99Asn, Leu99His, Arg101Asp, Leu217Gln, Leu99Thr, Leu217Asn, Arg101Gln and Asn35Gly.If those skilled in the art institute is by understanding, the mode that the paratope of any antibody or its variant all can be stated by subject application measures.If residue prediction can contribute 10% in conjunction with energy, just think residue " important ".In one embodiment, if residue prediction can contribute 2% in conjunction with energy, just think residue " important ".In one embodiment, if residue prediction can contribute 50% in conjunction with energy, just think residue " important ".In one embodiment, if residue prediction and surface of epitopes or paratope surface interaction, residue " important " is just thought.In one embodiment, cause combining loss if change residue, just think residue " important ".

Term " specificity " or " preferentially " are bonded to, or similar wording and do not mean that represent antibody be bonded to that epi-position only.On the contrary, be meant to antibody or its variant can be bonded to that epi-position, its degree is higher than the degree of other material of at least one that antibodies to antibody exposes.In one embodiment, specific binding antibody is by (tighter, or lower K to be bonded to EGFRvIII protein than it to the avidity that EGFR protein is larger _d).Such as, the combination of specific binding antibody tightlier at least will increase by 1%, 1-2%, 2-5%, 5-10%, 10-20%, 20-30%, 30-50%, 50-70%, 70-90%, 90-120%, 120-150%, 150-200%, 200-300%, 300-500%, 500-1000% or more.

Amino acid, numbering, amino acid whose abbreviated form, such as Leu217Gln represents that numbering amino acid is from the first amino acid to the amino acid whose sudden change of the second.Therefore, Tyr172Arg is meant to when the protein of wild-type has the tyrosine of 172 positions, and sudden change has the arginine of 172 positions.

The extract that term used herein " medicament " represents the mixture of compound, compound, biopolymer or formed from biological substance.

" Mammals " used herein refers to think mammiferous any animal.The preferred mankind of Mammals.

The digestion of the antibody containing enzyme (papoid) causes two kinds of consistent Fabs, and also referred to as " Fab " fragment and " Fc " fragment, it does not have antigen-binding activity, but has crystallizing power.The digestion of the antibody containing enzyme (stomach en-) causes F (ab ') ₂fragment, wherein the two-arm of antibody molecule keeps connecting, and comprises two antigen binding sites.F (ab ') ₂fragment has the ability of crosslinking antigen.

" Fv " used herein refers to the minimal segment of the antibody keeping antigen recognition and antigen binding site.These fragments also can think the variant of antibody.

" Fab " used herein refers to the antibody fragment comprising the constant domain of light chain and the CH1 structural domain of heavy chain.

Term " mAb " refers to monoclonal antibody.

Description encoding for the antibody sequence of XenoMax method generation is as follows: " AB "-refer to antibody, the binding specificity of " EGFRvIII "-refer to antibody, " X " refers to the XenoMouse derived from mouse, " G1 "-refer to IgG1 isotype or " G2 "-refer to IgG2 isotype, last three numerals refer to that antibody is from its derivative unicellular numbering, such as: AB-EGFRvIII-XG1-095 has IgG1 isotype XenoMouse mouse to the antibody of the binding specificity of EGFRvIII, and cell is numbered 95.

Term " SC " refers to unicellular, and the antibody that specific XenoMax method derives can be described as SC, thereafter immediately following three numerals, or only has three numerals, refers to the unicellular numbering that antibody is derivative in this article.

The description encoding of the antibody sequence derived for hybridoma is as follows: " AB "-refer to antibody, the binding specificity of " EGFRvIII "-refer to antibody, " X " refers to the XenoMouse derived from mouse, " G1 "-refer to IgG1 isotype or " G2 "-refer to IgG2 isotype, " K " refers to κ, and " L " refers to λ.Last three numerals refer to the pure lines that antibody is derivative, such as: AB-EGFRvIII-XGlK-13.1.2.

" mark " used herein or " through mark " refer to polypeptide add can test section, such as, labelled with radioisotope, fluorescent labelling, enzyme labelling, chemiluminescent labeling or biotinyl.Radio isotope or radionuclide can comprise ³h, ¹⁴c, ¹⁴n, ³⁵s, ⁹⁰y, ⁹⁹tc, ¹¹¹in, ¹²⁵i, ¹³¹i, fluorescent labelling can comprise rhodamine (rhodamine), group of the lanthanides phosphorus or FITC, and enzyme labelling can comprise horseradish peroxidase, beta-galactosidase enzymes, luciferase, alkaline phosphatase.

" medical agent or medicine " refers to compound or the composition that can produce ideal treatment when suitably granting patient as the term is employed herein.As by The McGraw-Hill Dictionary of Chemical Terms (Parker, S. compile, McGraw-Hill, San Francisco (1985)) illustrated in, use other technical term of chemistry in this article according to the common usage in technique.

As used herein " pure in fact " be meant to target material be the main material existed (namely, with molar amount, exist all in a large number than other individual substance any in composition), and preferably, part purified is in fact the composition of all polymer substances at least about 50% (with molar amount) that its target substances accounts for existence.Generally speaking, composition pure in fact by account for all polymer substances of existing in composition more than about 80%, more excellent in about 85%, 90%, 95%, 99% and 99.9%.Optimally, be purified to by target material and be essentially homogeneous (being can't detect the pollution substance in composition by common detection methods), wherein composition is made up of single polymer substance substantially.

Term " patient " comprises the mankind and animal doctor person under inspection.

Term "

technology " refer to " Selected Lymphocyte Antibody Method " (people such as Babcook; Proc.Natl.Acad.Sci.USA, i93:7843-7848 (1996) and Schrader, United States Patent (USP) the 5th; 627, No. 052).

Term " XenoMax ^tM" refer to SLAM Technology with the use (as mentioned below) of mouse.

antibody structure

Known basic antibody structural unit comprises tetramer.Each tetramer forms consistent polypeptide chain by two, and every have " light chain " (about 25kDa) and " heavy chain " (about a 50-70kDa) for a pair.The amino terminals part of each chain comprises an amino acid whose variable region, about 100 to 110 of primary responsibility antigen recognition or more.The carboxy-terminal part of each chain defines the constant region of primary responsibility effector function.Human light chains is categorized as κ and lambda light chain.Heavy chain is categorized as μ, δ, γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.Be connected by about 12 or more amino acid whose " J " districts in light chain and heavy chain kind variable region and constant region, wherein heavy chain also comprises about 10 or more amino acid whose " D " districts.Generally see, FundamentalImmunology the 7th chapter (Paul, W. compile, the 2nd edition, Raven Press, N.Y. (1989)).The variable region that each light/heavy chain is right forms antibody combining site.

Therefore, complete antibody has two basic change site.Except difunctional or bi-specific antibody, two basic change site is identical.

These chains are all shown by the identical universal architecture of the relatively conservative framework region (FR) of three hypervariable regions connections, also referred to as complementary determining region or CDR.The CDR of every a pair two chains, by framework region comparison, makes to be bonded to specificity epitope.From N-end to C-end, light chain and heavy chain all comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 structural domain.It is according to Kabat Sequences ofProteins of Immunological Interest (National Institutes of Health that the amino acid of each structural domain distributes, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J.Mol.Biol196:901-917 (1987); The people such as Chothia, the definition in Nature342:878-883 (1989).

Dual specific or bifunctional antibody have the artificial hybrid antibody of two different heavy chains/light chains to binding site different from two.Bi-specific antibody produces by the multiple method comprising the fusion of hybridoma and the connection of Fab ' fragment.See such as Songsivilai & Lachmann Clin.Exp.Immunol.79:315-321 (1990), the people such as Kostelny, J.Immunol.148:1547-1553 (1992).The generation of bi-specific antibody can be relatively labor-intensive technique compared with the generation of conventional antibody, and the output of bi-specific antibody and purity are usually lower.Bi-specific antibody is not to have form existence (such as, Fab, Fab ' and Fv) that unijunction closes site fragment.Except the universal architecture aspect of antibody, the more special interaction between paratope and epi-position is checked by frame mode.In one embodiment, CDR structure forms paratope, and antibody can be bonded to epi-position through it.The structure of this paratope measures by various ways.Traditional structural survey method can be used, such as NMR or x-radiocrystallography.These methods can check independent paratope, or the structure when it is bonded to epi-position.Or molecular model can produce in computer simulation.Structure can by means of commercially available encapsulation, and such as the InsightII model encapsulation of Accelrys (San Diego, CA), is produced by homology model.In brief, inspection antibody sequence can be utilized to seek the database of known structure protein, such as, Protein structure databases (Protein Data Bank).After identification has the homologous protein of known structure, these homologous proteins can be used as model template.Each template through comparison, can may produce the sequence alignment of structure based in these templates thus.Can will have the antibody sequence of unknown structure and these template matchings to produce the molecular model with unknown structure antibody subsequently.As those skilled in the art will understand, there is the multiple alternative method producing described structure in computer simulation, wherein any one can be used.Such as, people such as being similar to Hardman can be used, employing QUANTA (PolygenCorp., the Waltham of promulgation, and CHARM (Brooks, B.R., Bruccoleri, R.E. Mass.), Olafson, B.D., States, D.J., Swaminathan, S. and Karplus, M., 1983, J.Comp.Chem, .4:187) United States Patent (USP) U.S.Pat.No.5, the technique of technique described in 958, No. 708.

Not only whether the shape of paratope may be bonded to epi-position and good degree is very important by paratope for mensuration, and the interaction itself between epi-position and paratope is the bulk information source of design variable antibody.As those skilled in the art will understand, there is this interactional mode of multiple research.A kind of mode uses the structural models that may produce as described above, and use such as InsightII (Accelrys subsequently, San Diego, CA) program, it has one can be carried out Monte Carlo research expansion module to the configuration between paratope and its epi-position and located space.Result to assess epi-position and the interactional place of paratope and mode.In one embodiment, the fragment of epi-position or variant is only had to measure relevant interaction for assisting.In one embodiment, whole epi-position is used for interactional modeling between paratope and epi-position.As those skilled in the art will understand, these two kinds of different methods have different advantages and disadvantages.Such as, epitope Fragments is only used to make to carry out more detailed inspection to the possible variant of each side chain, and without the need to the at substantial time.On the other hand, by only using epitope Fragments, or only use epi-position to replace whole protein, the feature of epitope Fragments may be different from the feature of whole epi-position, therefore may be increased in the risk misled in computation modeling process.In one embodiment, use two kinds of methods with cross-check result with limited extent.In a preferred embodiment, if use epitopic variants, can optimization so that epitopic variants comprises the most important residue of epi-position.The consistence of most important residue measures by various ways, such as, as described in example 4 and 14 of the present invention.

The structure produced by using these, can measure in the interaction of which residue between epi-position and paratope most important.Therefore, in one embodiment, can be easy to select change which residue to change antibody in conjunction with feature.Such as, apparent from extended model, in epi-position, the side chain of some residue spatially can stop the combination of epi-position, is changed into by these residues thus and has the consistent residue of smaller side chain.

This can measure in many ways.Such as, only need observe two models and assess interaction based on functional group and adjacent group.Or, as described above, the repeating groups pair of epi-position and paratope can be carried out, to obtain more favorably energy interaction.These interactions that also can measure Multiple Antibodies variant measure wherein antibody and can be bonded to the alternative of epi-position.Also how incompatible for various model group mensuration can be changed antibody structure to obtain the antibody with required special characteristic.

The model of said determination is tested by various technology.Such as, interaction energy can be measured to determine to need which kind of variant of inspection further by program discussed above.Again, use coulomb and Van der Waals to interact and measure the interaction energy of epi-position and variant paratope.Also the site for sudden change is used in fact whether can to cause changing needed for feature to the predetermined variation of observing in antibody structure.Or, can change epi-position to confirm model correct or for measure may occur between paratope and epi-position generally in conjunction with theme.

The above-mentioned method for modeling structure can be used for which kind of change measured in protein structure will cause the specific required feature of antibody.These methods can be used for the required feature which kind of change measured in protein structure can not cause antibody.

As those skilled in the art will understand, although these models are by for forming the guide that antibody of the present invention and variant thereof provide necessary, but still need to carry out the conventionally test in computer simulation model, can by In vitro study.In addition, as those skilled in the art will understand, any upgrading also antagonist activity can have other side effect.Such as, although expection causes the change of larger combination can cause larger combination, other also can be caused to reduce or change the structural changes of antibody activity.Very common in the art for the mensuration being whether this situation, and measure by various ways.Such as, as in example 21, active test by ELISA is tested.Or, sample can be tested by using surface plasma body resonant vibration device.

antibodies and the variant antibodies for excellent combination

In one embodiment, above-mentioned model is for increasing the binding ability of antibody to its epi-position.Antibody can be easier to be bonded to epi-position, and therefore has higher association constant (k _a).Or antibody can be slower in epi-position dissociation, and therefore have lower dissociation constant (K _d), or the K of epi-position-paratope _dbe worth less, therefore make the combination degree between epi-position and paratope higher.

In certain embodiments, design variable antibody combines with inverse features.That is, antibody is not combined closely or may not be combined fast.

In other embodiments, the K of variant antibodies _ddifferent from the antibody of wild-type, but variant antibodies has more specificity for defined epitope.This means, through the paratope of designerantibodies, there is the lower risk being bonded to other epi-position.Antibody can have the further feature changed.Such as, variant to non-specific antibody combine have more immunity, even if or when antibody exists with high density, still keep solvation in the solution.This variant can be present in discussed antibody.Such as, although the higher concentration of some variant antibodies hereafter checked result in Biacore experiment slower in conjunction with component (such as, 13.1.2mAb), even if but some variant still do not show under same concentrations described in comparatively slow component, such as L217N-2.1.Can be formed by the variant of the model prediction measured above, and measure it subsequently after tested in fact whether with required integrate features.Can select to have with epi-position the mutant of comparatively large total interaction energy for further test.Interaction energy can measure in many ways, and one of them as described above.

These variants can be tested in many ways.Exemplary selection comprises, and be not limited to KinExA (such as, the patent the 5th that Lackie promulgates, 372, No. 783, on December 13rd, 1994) (Sapidyne Instruments Inc., ID, Boise), surface plasma body resonant vibration (SPR) (e.g., BIACORE ^tMbiacore, Inc., Pistcataway, N.J.), arrhea fluorescent, resonant mirror and fluorescent polarization.Many these are selected not only can record data, and can be provided for being various theoretical curve by data fitting and measuring k thus _a, k _dand K _dand the fabricated parts of other characteristic.Importantly, it should be noted that these fitting of a curve are that result data does not get rid of the possibility that there are some deviations.Therefore, relevant association, dissociation and the equilibrium constant not only can be observed by these fitting of a curve mechanism, also directly mutually can compare according to the knowledge of those skilled in the art.

the humanization of human antibodies and antibodyhuman antibodies avoids and has Muridae or rat is variable and/or the antibody of constant region is relevant problem.The existence of these Muridaes or rat-derived protein can cause the quick removing of antibody maybe can cause resisting the immunoreactive generation of patient's internal antibody.For avoiding using Muridae or rat-derived antibody, by human antibodies function is introduced rodent so that rodent produces complete human antibodies to produce complete human antibodies.

The Human genome seat cloning and reconstruct megabasse size in YAC and the ability being introduced into mouse germline are illustrate greatly or provide strong approach through the function ingredients of the locus of original mappings and the useful model of generation human diseases.In addition, use this technology mouse loci is replaced with its mankind's equivalent can be the expression and regulation of the human gene product between the growth period, their information transmission with other system and disease bring out be in progress in associate particular views be provided.

The important practical application of this strategy is mouse humoral immune system " humanization ".The sequencing expression and combination of studying antibody by offering an opportunity in the mouse of human immunoglobulin (Ig) locus introducing endogenous Ig gene inactivation and the active potential mechanism in B-cell development thereof.In addition, this strategy can be and produces the important milestone that complete human monoclonal antibody (mAb)-a kind of realizes Antybody therapy prospect in human diseases kind and provide ideal source.Expect that immunity and anaphylaxis essence can be minimised as mouse or mouse-derived mAb by complete human antibodies, and because this increasing effect through casting antibody and security.Use the expection of universe's antibody to can be the chronic and recurring human diseases for the treatment of and provide substantial advantage, such as inflammation, autoimmunity and cancer, these all need repeatedly to cast antibody.

Method for this object is the mouse species that design is not enough to mouse antibodies for having human Ig loci's large fragment and produces, to expect that these mouse can when without the large order set producing human antibodies when mouse antibodies.The suitable adjustable that large mankind Ig fragment can keep large variable gene diversity and antagonist to produce and express.Be used for Antibody diversification and the mouse instrument of selection and the shortage for human protein's immunotolerance by adopting, the human antibodies order set reproduced in these mouse species should produce the antibody any interested antigen all (comprising human antigen) to high-affinity.Use hybridoma technology, can be easy to production and selection has required specific antigen-specific human class mAb.Disclosed in 1994, show this general strategy (see people such as Green, Nature Genetics7:13-21 (1994)) in conjunction with first-generation XenoMouse strain.XenoMouse strain is designed to have the yeast artificial chromosome (YAC) of the germline configuration fragments of 245kb and the 190kb size containing human heavy chain loci and κ light chain gene seat respectively, and it contains core variable core constant-region sequences Id.The verified mouse system with resetting and express for antibody of mankind Ig containing YAC is compatible and can be replaced the mouse Ig genes of inactivation.This can bring out B-cell development by it, produces the human commands system of the similar adult of complete human antibodies and produce antigen-specific human class mAb ability and confirm.These results also show, introducing the major part of the human Ig loci containing larger numbering V gene, other adjustment element and mankind Ig constant region can summarize in fact complete instruction system, and it reacts for feature with the Human Fluids for infection and immunity.The works of the people such as Green extends to the human heavy chain loci of introducing megabasse size and the germline configuration YAC fragment of κ light chain gene seat respectively by introducing the human antibodies order set being greater than about 80% recently.See people such as Mendez, NatureGenetics15:146-156 (1997) and U.S. patent application case the 08/759th, apply on December 3rd, 1996 by No. 620.

Resulting from following patent of XenoMouse mouse is discussed further and is described: U.S. patent application case the 07/466th, applies for January 12 nineteen ninety by No. 008; 07/610th, No. 515, apply for November 8 nineteen ninety; 07/919th, No. 297, apply on July 24th, 1992, the 07/922nd, No. 649, apply on July 30th, 1992; 08/031st, No. 801, apply on March 15th, 1993; 08/112nd, No. 848, apply on August 27th, 1993; 08/234th, No. 145, apply on April 28th, 1994; 08/376th, No. 279, apply for January 20 nineteen ninety-five; 08/430th, No. 938, apply for April 27 nineteen ninety-five; 08/464th, No. 584, apply for June 5 nineteen ninety-five; 08/464th, No. 582, apply for June 5 nineteen ninety-five; 08/463rd, No. 191, apply for June 5 nineteen ninety-five; 08/462nd, No. 837, apply for June 5 nineteen ninety-five; 08/486th, No. 853, apply for June 5 nineteen ninety-five; 08/486th, No. 857, apply for June 5 nineteen ninety-five; 08/486th, No. 859, apply for June 5 nineteen ninety-five; 08/462nd, No. 513, apply for June 5 nineteen ninety-five; 08/724th, No. 752, apply on October 2nd, 1996 and the 08/759th, No. 620, apply on December 3rd, 1996 and United States Patent (USP) the 6th, 162, No. 963, the 6th, 150, No. 584, the 6th, 114, No. 598, the 6th, 075, No. 181 and the 5th, 939, No. 598 and Japanese Patent the 3 068 180 No. B2, the 3 068 506 No. B2 and the 3 068 507 No. B2.Also can see people such as Mendez, Nature Genetics15:146-156 (1997) and Green and Jakobovits J.Exp.Med.188:483-495 (1998).

Also can see No. 0 463 151 B1, European patent EP, authorize open on June 12nd, 1996, No. 94/02602, international application WO, open on February 3rd, 1994, No. WO96/34096th, international application, open on October 31st, 1996, No. 98/24893, WO, open on June 11st, 1998, No. 00/76310, WO, open on December 21st, 2000, No. 03/47336, WO.In another approach, other company, comprises GenPharm International company and uses " micro-locus " method.In micro-minilocus approach, exogenous Ig locus is imitated by the material (Individual genes) comprised from Ig locus.Therefore, one or more V _hgene, one or more D _hgene, one or more J _hgene, μ constant region and the second constant region (being preferably γ constant region) are formed as the structure for inserting in animal body.This method is described in following patent: the United States Patent (USP) the 5th, 545 of the people such as Surani, and No. 807 all belong to No. the 5th, 545,806, the United States Patent (USP) of Lonberg and Kay with each patent, 5th, 625, No. 825, 5th, 625, No. 126, 5th, 633, No. 425, 5th, 661, No. 016, 5th, 770, No. 429, 5th, 789, No. 650, 5th, 814, No. 318, 5th, 877, No. 397, 5th, 874, No. 299 and the 6th, 255, No. 458, No. the 5th, 591,669, the United States Patent (USP) of Krimpenfort and Berns and the 6th, No. 023.010, the United States Patent (USP) the 5th, 612 of the people such as Berns, No. 205, 5th, 721, No. 367 and 5,789, No. 215, and No. the 5th, 643,763, the United States Patent (USP) of Choi and Dunnand, and GenPharm International U.S. patent application case the 07/574th, No. 748, apply for August 29 nineteen ninety, 07/575th, No. 962, apply for August 31 nineteen ninety, 07/810th, No. 279, apply on December 17th, 1991, 07/853rd, No. 408, apply on March 18th, 1992, 07/904th, No. 068, apply on June 23rd, 1992, 07/990th, No. 860, apply on December 16th, 1992, 08/053rd, No. 131, apply on April 26th, 1993, 08/096th, No. 762, apply on July 22nd, 1993, 08/155th, No. 301, apply on November 18th, 1993, 08/161st, No. 739, apply on December 3rd, 1993, 08/165th, No. 699, apply on December 10th, 1993, 08/209th, No. 741, apply on March 9th, 1994.Also can see No. 0 546 073 B1, European patent, international application WO 92/03918, WO92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852 and WO 98/24884 and United States Patent (USP) the 5th, 981, No. 175.With further reference to people such as Taylor, 1992 years; The people such as Chen, 1993; The people such as Tuaillon, 1993; The people such as Choi, 1993; The people such as Lonberg, (1994); The people such as Taylor, the people such as (1994) and Tuaillon, (1995); The people such as Fishwild, (1996).

Kirin has also shown to produce human antibodies in mouse, wherein introduces large stretch of karyomit(e) or whole karyomit(e) by microcell fusion.See European patent application the 773 No. 288 and the 843 No. 961.XenerexBiosciences is is researching and developing a kind of technology for potential generation human antibodies.In this technology, reconstitute SCID mouse with Human Lymphocytes (such as, B and/or T cell).Mouse with after through antigen immune, and the immune response of the antigen that can create antagonism.See United States Patent (USP) the 5th, 476, No. 996, the 5th, 698, No. 767 and the 5th, 958, No. 765.

Mankind's anti-mouse antibody (HAMA) reaction is by chimeric for the preparation of industrial production guiding or other humanized antibody.Although chimeric antibody has human constant regions and murine variable region, still wish to can be observed the anti-chimeric antibody of some mankind (HACA) reaction, especially antibody chronic or many-during dosage uses.Therefore, the consideration of reacting to make HAMA or HACA and/or impact were lost efficacy, still desirable to provide whole person's antibody-like of antagonism EGFRvIII. antybody therapy

As described herein, it is very important at least partially that the function of EGFRvIII antibody seems to its operator scheme.Such as, functionally, the activity of EGFRvIII antibody in operating with EGFRvIII is meant to.Therefore, in some aspects, wish the generation of the antibody of the treatment material standed for being combined as antagonism EGFRvIII, antibody can be fixed fill-in and supplement cytotoxic lymphocyte, participates in thus in CDC and ADCC.There is the multiple antibody isotypes with identical function, include, but is not limited to following: Muridae IgM, Muridae IgG2a, Muridae IgG2b, Muridae IgG3, mankind IgM, IgG 1, IgG 3 and human IgA.Again, wish the generation of the antibody of the treatment material standed for being combined as antagonism EGFRvIII, the effector cell that antibody is engaged in such as natural killer (NK) cell by Fc acceptor activates the antibody-mediated cytotoxic reaction (ADCC) of dependence.Exist multiple can the antibody isotypes of ADCC, include, but is not limited to following: Muridae IgG2a, Muridae IgG2b, Muridae IgG3, IgG 1 and IgG 3.Should be appreciated that, the antibody of generation need not have this isotype at the very start, but on the contrary, the antibody of generation can have any isotype and antibody can be the isotype using the routine techniques conversion known in technique subsequently.These technology comprise and use direct recombinant technology (such as, see United States Patent (USP) the 4th, 816, No. 397) and cell-cell fusion techniques (such as, see United States Patent (USP) the 5th, 916, No. 771 and the 6th, 207, No. 418).

In cell-ECM technology, myelomatosis or other cell strain of preparing the heavy chain had containing any required isotype have myelomatosis or other cell strain of light chain with another kind.These cells can through merging subsequently, and the complete antibody of separable expression cell line.

Such as, some anti-EGFR vIII antibody discussed herein is mankind's anti-EGFR vIII IgG1 antibody.Combine needed for EGFRvIII molecule if this antibody has, isotype should be easy to conversion to produce mankind IgM, IgG 3 or IgG A, although still have identical variable region (which defining specificity and some avidity thereof of antibody).These molecules (comprising IgG1) can be fixed fill-in subsequently and participate in CDC, if and comprise IgG1 or IgG3 constant region, these molecules also participate in relying on antibody-mediated cytotoxic reaction (ADCC) by by supplementary cytotoxic lymphocyte.

Therefore, owing to creating the Ab candidates reaching required " structure " attribute mentioned above, therefore, it can have at least " function " attribute needed for some by isotype conversion usually.The design of other treatment and generation

Based on herein about the antibody activity that EGFRvIII produces and characterizes, facilitate the design of other treatment form except antibody moiety.

These forms include, but is not limited to advanced antibody therapeutics (such as the therapy of bi-specific antibody, immunotoxin and labelled with radioisotope), the generation of peptide therapy, gene therapy, are especially interior antibody, anti-sensitization therapy and small molecules.

Such as, in conjunction with the generation of advanced antibody therapeutics, wherein fill-in fix with cytotoxic lymphocyte supplement as desirable attributes, cell can be strengthened kill by using dual specific, immunotoxin or labelled with radioisotope.

Such as, in conjunction with bi-specific antibody, can produce and comprise following bi-specific antibody: (i) two kinds of antibody, a kind of have the specificity of EGFRvIII and another kind has specificity to the second molecule combined; (ii) chain has specificity to EGFRvIII and Article 2 chain has specific monoclonal antibody body to the second molecule; Or (iii) has specific single-chain antibody for EGFRvIII and another kind of molecule.These bi-specific antibodies can use knows technology generation, such as, in conjunction with (i) and (ii), for example, see Immunol Methods4:72-81 (1994) and above-mentioned Wright and Harris, and combine (iii), for example, see people such as Traunecker, Int.J.Cancer (Suppl.) 7:51-52 (1992).At each occurrence, the second specificity can give Fc chain activated receptor, include, but is not limited to CD16 or CD64 (for example, see, the people such as Deo, 18:127 (1997)), CD3 (Micromet ' sBiTE technology) or CD89 (for example, see, the people such as Valerius, Blood90:4485-4492 (1997)).May kill according to the bi-specific antibody of aforementioned preparation and express the cell of EGFRvIII, and especially wherein EGFRvIII antibody of the present invention those cells effective.

Binding immunoassay toxin, antibody can use the technology upgrading known in technique and as immunotoxin.

For example, see Vitetta Immunol Today14:252 (1993).Also for example, see No. the 5th, 194,594, United States Patent (USP).In conjunction with the antibody preparing labelled with radioisotope, these also can be easy to use the technology known in technique to prepare through upgrading antibody.For example, see people such as Junghans, in Cancer Chemotherapy andBiotherapy655-686 (the 2nd edition, Chafner and Longo compiles, Lippincott Raven (1996)).Also see United States Patent (USP) the 4th, 681, No. 581, the 4th, 735, No. 210, the 5th, 101, No. 827, the 5th, 102, No. 990 (RE35,500), the 5th, 648, No. 471 and the 5th, 697, No. 902.The molecule of each immunotoxin and labelled with radioisotope all may kill the cell of expressing EGFRvIII, and especially wherein antibody described herein those cells effective.

Can combine more fast by designerantibodies, or slower in epi-position dissociation, and therefore antibody itself can be designed to therapeutical agent.Such as, antibody through change feature can be used for casting therapeutical agent to patient.Treatment immune conjugate

As understood, the antibody combined with medicine, toxin or other molecule (immune conjugate or immunotoxin) kills in the cell of expressing by the molecule of specific binding molecules (such as antibody) specific binding extremely useful determining target.As described above, not yet knownly in any healthy tissues, EGFRvIII was expressed.In addition, EGFRvIII display is significantly expressed in multiple human tumor.Therefore, EGFRvIII is the molecule attracted most attention for determining target with immunotoxin.

Occur manyly determining target about attempting specificity there is the report of the tumour cell of monoclonal antibody-drug conjugates (people such as Sela, in Immunoconjugates189-216 (C.Vogel, ed.1987); The people such as Ghose, in TargetedDrugs1-22 (E.Goldberg compiles, nineteen eighty-three); The people such as Diener, in Antibody Mediated DeliverySystems1-23 (J.Rodwell compiles, 1988); The people such as Pietersz, in Antibody Mediated DeliverySystems25-53 (J.Rodwell compiles, 1988); The people such as Bumol, in Antibody Mediated DeliverySystems55-79 (J.Rodwell compiles, 1988)).Cytotoxic drugs, such as with multiple murine monoclonal, methotrexate, daunorubicin, Zorubicin, vincristine(VCR), vinealeucoblastine(VLB), melphalan (melphalan), ametycin and Chlorambucil see that body is combined.In some cases, by intermediate supporting agent molecule, drug molecule is connected to antibody molecule, these intermediate supporting agent molecules are seralbumin (people such as Garnett, Cancer Res.46:2407-2412 (1986) such as; The people such as Ohkawa, Cancer Immumol.Immunother.23:81-86 (1986); The people such as Endo, Cancer Res.47:1076-1080 (1980)), dextran (people such as Hurwitz, Appl.Biochem.2:25-35 (1980); The people such as Manabi, Biochem.Pharmacol.34:289-291 (1985); The people such as Dillman, Cancer Res.46:4886-4891 (1986); The people such as Shoval, Proc.Natl.Acad.Sci.85:8276-8280 (1988)) or polyglutamic acid (people such as Tsukada, J.Natl.Canc.Inst.73:721-729 (1984); The people such as Kato, J.Med.Chem.27:1602-1607 (1984); The people such as Tsukada, Br.J.Cancer52:111-116 (1985)).

Adopt multiple connexon technology for these immune conjugates of preparation, and have studied dissociable and not dissociable connexon.But, in most cases, if drug molecule from being that the conjugate of non-upgrading form discharges determining target site, just can only observe the complete cytotoxicity potential of medicine.

For the preparation of acid-unstable connexon that one of the dissociable connexon of antibody-drug conjugates is based on cis-aconitic acid, it has utilized the sour environment of different intracellular compartment, the endosome such as run in the endocytosis of regulation and lysosome.Shen and Ryser introduces this method to prepare the conjugate (Biochem.Biophys.Res.Commun.102:1048-1054 (1981)) of daunorubicin and macromole supporting agent.Yang and Reisfeld use is constructed is coupled to anti-melanoma antigens (J.Natl.Canc.Inst.80:1154-1159 (1988)) by daunorubicin.Recently, the people such as Dillman also uses sour unstable connexon to prepare the conjugate (Cancer Res.48:6097-6102 (1988)) of daunorubicin and anti-T cell antibody in a similar manner.

Related to by the another kind of method of people's researchs such as Trouet and daunorubicin is connected to antibody (Proc.Natl.Acad.Sci.79:626-629 (1982)) through peptide space arm.This carries out under the prerequisite discharged from this conjugate without medicine in the effect by N,O-Diacetylmuramidase peptase.

But disclose in cytotoxicity test in vitro, antibody-drug conjugates seldom reaches the cytotoxicity usefulness identical with free non-coupled medicine.This shows, the mechanism poor efficiency that drug molecule discharges from antibody.In the scope of immunotoxin, the conjugate formed through the two sulphur bridges between monoclonal antibody and catalytic activity archon shows and has more cytotoxicity than the conjugate containing other connexon.See, the people such as Lambert, J.Biol.Chem.260:12035-12041 (1985); The people such as Lambert, in Immunotoxins175-209 (A.Frankel compiles, 1988); The people such as Ghetie, Cancer Res.48:2610-2617 (1988).This is owing to the high IC of the favourable gsh of effective dissociation of the cystine linkage between antagonist molecule and toxin.However, only have on a small quantity about using two sulphur bridge to prepare the report example of the conjugate between medicine and macromole.The people such as Shen describe methotrexate and are converted into mercapto buserelin derivative, then through cystine linkage and poly-D-Lys coupling (J.Biol.Chem.260:10905-10908 (1985)).In addition, one report describes the preparation (people such as Menendez containing the toxin pharmaceutical calicheamycin of trisulphide and the conjugate of antibody, Fourth International Conference on MonoclonalAntibody Immunoconjugates for Cancer, San Diego, Abstract81 (1989)).Another report describes preparation people such as (, 53Cancer Res.3336-3342 (1993)) Hinman containing the toxin pharmaceutical calicheamycin of trisulphide and the conjugate of antibody.

The reason lacking the antibody-drug conjugates that disulphide connects is the unavailability of the cytotoxic drugs with the sulphur atom containing the part that can be easy to for medicine to be connected to antibody through disulphide bridges.In addition, existing drugs is in the nondecreasing situation of its cytotoxicity potential, and it is difficult to carry out chemical modification.

Another main deficiency of existing antibody-drug conjugates is because a limited number of relative appropriate cytotoxicity of determining target antigen and static cancer drug (as methotrexate, daunorubicin and vincristine(VCR)), and it can not by the useful for drug delivery of enough concentration to determining target site.In order to reach significant cytotoxicity, be necessary a large amount of drug molecules directly or by polymerization supporting agent molecule to be connected to antibody.But the antibody of these height upgradings shows usually for determining combination that target antigen weakens and in vivo removing from blood flow fast.

Maytansinoid is high cell toxin pharmaceutical.First maytenin is separated in the shrub tingia Caulis Mayteni of East Africa by people such as Kupchan, and its display is than the cytotoxicity (United States Patent (USP) the 3rd of large 100 to 1000 times of common cancers chemotherapeutic (as methotrexate, daunorubicin and vincristine(VCR)), 896, No. 111).Found that certain micro-organisms also produced maytansinoid, the C-3 ester (United States Patent (USP) the 4th, 151, No. 042) of such as isovaleric acid maytenin ester and isovaleric acid maytenin ester afterwards.Also the synthesis C-3 ester of isovaleric acid maytenin ester and analogue (people such as Kupchan, the J.Med.Chem.21:31-37 (1978) of isovaleric acid maytenin ester has been reported; The people such as Higashide, Nature270:721-722 (1977); The people such as Kawai, Chem.Pharm.Bull.32:3441-3451 (1984)).The example of the analogue of C-3 ester isovaleric acid maytenin prepared therefrom ester to comprise on aromatic ring (such as, dechlorination) or at C-9, C-14 (such as, hydroxylated methyl), C-15, C-18, C-20 and C-4,5 places are through the isovaleric acid maytenin ester of upgrading.

Natural generation and synthesis C-3 ester can be divided into Liang Ge group:

A () has C-3 ester (United States Patent (USP) the 4th, 248, No. 870, the 4th, 265, No. 814, the 4th, 308 of simple carboxylic, No. 268, the 4th, 308, No. 269, the 4th, 309, No. 428, the 4th, 317, No. 821, the 4th, 322, No. 348 and the 4th, 331, No. 598) and

B () has the C-3 ester (United States Patent (USP) the 4th, 137, No. 230, the 4th, 260, No. 608, the 5th, 208, No. 020 and Chem.Pharm.Bull.12:3441 (1984)) of the derivative of N-methyl-L-alanine.

Find that the cytotoxicity of the ester of group (b) is more much bigger than the cytotoxicity of the ester of group (a).

Maytenin is mitotic inhibitor.Report and in vivo processed L1210 cell with maytenin and cause the cell aggregation of 67% in mitotic division.Report mitotic index people such as (, 43Comparative Leukemia Research1975, Bibl.Haemat.495-500 (1976)) Sieber between undressed comparison cell display 3.2% to 5.8%.The experiment of sea urchin egg and clam ovum shows, maytenin suppresses mitotic division people such as (, Science189:1002-1005 (1975)) Remillard by suppressing the polymerization of microtubular protein (tubulin) interference microtubule to be formed.

In vitro, found with 10 ^-3to 10 ^-1the maytenin of μ g/ μ l dosage can suppress P388, L1210 and LY5178 murine leukemia cell suspending liquid, and wherein P388 cell strain is the most responsive.Also shown the activity inhibitor that maytenin is Human NPC cytokines outgrowth, and report acute human Lymphoblastic Leukemia Cells strain CEM can by being low to moderate 10 ^-7the control of the concentration (people such as Wolpert-DeFillippes, Biochem.Pharmacol.24:1735-1738 (1975)) of mg/ml.

In vivo, maytenin also shows and has activity.Be presented at the tumor growth suppressing P388 Iymphoblastic leukemia system within the scope of 50 to 100 multiple doses, this shows to have high therapeutic index; L1210 mouse leukemia system, mankind's Lewis lung cancer system and mankind B-16 melanotic cancer system also show remarkable inhibiting activity (Kupchan, Ped.Proc.33:2288-2295 (1974)).

The method of current coupling maytansinoids and Cell binding agent (such as antibody) comprises two reactions steps.First by Cell binding agent (such as antibody) with cross-linking reagent (such as N-succinimido pyridyl dithiopropionic acid ester (SPDP)) upgrading so that dithiopyridines base is introduced antibody (people such as Carlsson, Biochem.J.173:723-737 (1978); United States Patent (USP) the 5th, 208, No. 020).In second step, by the reactive maytansinoid with thiol group, (such as DM1 (is called N in form ^{2 '}-deacetylate-N ^{2 '}-(3-sulfydryl-1-oxopropyl))-maytenin) be added in upgrading antibody as intitation reagents, cause the displacement of thiopyridine base in upgrading antibody, and the generation (United States Patent (USP) the 5th of the cytotoxicity maytansinoid/ antibody coupling matter to be connected by disulphide, 208, No. 020).At United States Patent (USP) the 6th, the process being used for coupling maytansinoids is described in 441, No. 163.

The immunotoxin technology based on Maytansinoid can be obtained by ImmUnogen Corporation (Cambridge, MA).Another kind of important toxin technology is based on auristatin toxin.The aplysiatoxin Dolastatin10 that Auristatins obtains derived from the sea hare by Indian Ocean, as effective cell growth inhibition material.See United States Patent (USP) the 4th, 816, No. 444 and the 4th, 978, No. 744.About other aplysiatoxin, also see United States Patent (USP) the 4th, 414, No. 205 (Dolastatin-1,2 and 3), the 5th, 076, No. 973 (Dolastatin-3), the 4th, 486, No. 414 (Dolastatin-A and B), the 4th, 986, No. 988 (Dolastatin-13), the 5th, 138, No. 036 (Dolastatin-14) and the 4th, 879, No. 278 (dolastatin-15).The various auristatine derivatives be separated with the colleague of Arizona State university by Dr.Pettit and synthesize after tested and the high toxicity demonstrated cell.See people such as Pettit, antineoplastic agent 337.The synthesis of aplysiatoxin 10 structure upgrading.Anticancer Drug Des.10 (7): 529-44 (1995), the people such as Woyke.The extracellular activity of effective aplysiatoxin 10 structure upgrading auristatin PHE and rear antifungic action.Antimicrobial Agents and Chemotherapy.45:3580-3584 (2001), the people such as Pettit.The activity specific of aplysiatoxin 10 and peptide derivant antagonism cryptococcus neoformans.Antimicrobial Agents andChemotherapy.42：2961-2965(1998)，Woyke。During the cryptococcus neoformans cell cycle, the dimensional visualization of microtubule and auristatin PHE are on the impact of microtubule globality and nuclear location.Submitted，Antimicrobial Agentsand Chemotherapy。

Recently, researched and developed and to have seemed other auristatin derivative quite effective when transmitting on antibody as useful load.Such as, shown monomethyl auristatin E (MMAE) with during tumor specific antibody coupling as the effective toxin for tumour cell.The people such as Doronina, Development of potent monoclonal antibodyauristatin conjugates for cancer therapy.Nature Biotechnology. (2003) (can obtain online); The people such as Francisco, cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugatewith potent and selective antitumor activity.Blood. (2003) May8 [open before E printing] .Epub2003Apr24 (can obtain online).Except the toxicity of auristatin molecule, it is more stable that research has shown the conjugate connected through peptide, and therefore in buffer reagent and blood plasma than the specificity of other connexon technology normal tissue more greatly and toxicity is less.The people such as Doronina, Development of potent monoclonal antibody auristatinconjugates for cancer therapy.Nature Biotechnology. (2003) (can obtain online); The people such as Francisco, cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate with potent andselective antitumor activity.Blood. (2003) May8 [open before E printing]. ' Epub2003Apr24 (can obtain online).

These connexons design based on branched chain peptide and comprise such as mAb-valine-citrulline-MMAE and mAb-Phe-Lys-MMAE conjugate.The people such as Doronina, Development of potent monoclonalantibody auristatin conjugates for cancer therapy.Nature Biotechnology. (2003) (can obtain online); The people such as Francisco, cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugatewith potent and selective antitumor activity.Blood. (2003) May8 [open before E printing]. ' Epub2003Apr24 (can obtain online).These designs and coupling technology are by the following stated, Cathepsin B-sensitive dipeptide prodrugs.2.Models of anticancer drugs paclitaxel (Taxol) of the people such as Monoclonal antibody conjugates of doxorubicin prepared with branched peptide linkers:inhibition of aggregation by rnethoxytriethyleneglycol chains.J Med Chem.45 (19): 4336-43 (2002) of the people such as such as King and Dubowchik, mitomycin C and doxorubicin.BioorgMed Chem Lett.8 (23): 3347-52 (1998).Can be obtained by Seattle Genetics Corporation (Seattle, WA) based on aforesaid Auristatin E-base immunotoxin technology.

Exist in a large number by natural source extract and the compounds affecting microtubule that is semi-synthetic and synthetic analogues acquisition, it seems to have the potential as the toxin for generation of immune conjugate.(see newmedinc.com website).These molecules and to use the exemplary drugs of these molecules to comprise following: colchicine-site bonding agent (Curacin), windmill element (AVE806, combretastatin A-4 prodrug (CA4P), Oxi-4503), hidden algae element (LY355703), Discodermolide, Dolastatin and Analogs (Auristatin PHE, Dolastatin10, ILX-651, Symplostatin1, TZT-1027), ebormycine (BMS-247550, BMS-310705, EP0906, KOS-862, ZK-EPO), pomegranate plug Lip river element (Eleutherobin), FR182877, Halichondrin B (E7389), moon benzyl three Matsubain (Halimide) (NPI-2352 and NPI-2358), Hemiasterlins (HTI-286), Laulimalide, Maytansinoids (" DM1 ") (Bivatuzumab maytenin, Cantuzumab maytenin, huN901-DMl/BB-10901TAP, MLN591DM1, My9-6-DM1, Herceptin (Trastuzumab)-DM1), PC-SPES, Peloruside A, trans-resveratrol (Resveratrol), S-SAMC (SAMC), Spongistatin (Spongistatin), Wei Ti island acid amides (Vitilevuamide), molecular moter-kinesin (Molecular Motor-Kinesins) (SB-715992), colchicine-site the bonding agent (A-289099, A-293620/A-318315, ABT-751/E7010, D-24851/D-64131, ZD6126) of design, other novel spindle poison (methoxyestradiol (2-ME2), benzimidazole carbamate (ANG600 series, Vermox), CP248/CP461, HMN-214, R440, SDX-103, T67/T607).In addition, the Muridae having commented on other in the Mayer of 1998, A.M.S.Marine Pharmacology:Antitumor and Cytotoxic Compounds.ThePharmacologist.41 (4): 159-164 (1999) derives toxin.

treatment casts and fills a prescription

Can make feed process more infrequently by parenteral approach (such as, intravenously, subcutaneous or intramuscular injection) alternately the action time extended and more convenient.

When for administered in vivo, antibody formulation as herein described should be aseptic.Such as, this can realize through sterile filtration membrane filtration easily through before or after freeze-drying and restructuring.Antibody usually stores with lyophilized form or stores in the solution.Treatment antibody compositions is placed in the container with aseptic access entrance usually, such as, has intravenous solution bag or the bottle of the shifting coupling (stopper that such as hypodermic needle can be passed) of recyclable formula.

The path that antibody casts is according to currently known methods, such as, pass through in intravenously, intraperitoneal, brain, in intramuscular, intraocular, intra-arterial, sheath, suck or intralesional routes, or injected by slow-released system as mentioned below or inculcate.Antibody is preferably by inculcating or being cast continuously by bolus injection.

The significant quantity of the antibody therapeutically adopted depends on (such as) treatment target, the patient's condition of dosing way and patient.Therefore, for therapist, preferably optionally titration dosage improve administration routes to obtain optimum result for the treatment of.In theory, clinicist will cast antibody, until reach the dosage of ideal effect.The process of this therapy is easily through the calibrating of routine or controlled by calibrating as herein described.

Antibody as described herein can be prepared in the mixture containing pharmaceutically acceptable supporting agent.Therapeutic composition can preferably cast through intravenously or intranasal or lung with the form of liquid or Powder aerosol (through freeze-drying).Composition also can optionally parenteral or through subcutaneous administration.When carrying out general and casting, therapeutic composition should be aseptic, has in the solution that should have pH value, isotonicity and stability without thermal source and parenteral is acceptable.These conditions are that those skilled in the art is known.In brief, compound dosage formulation is prepared for storing and casting by being mixed with supporting agent acceptable on physiology, vehicle or stablizer by the compound with required purity.These materials are avirulent when adopted dosage and concentration for recipient, and it comprises buffer reagent, such as TRIS HCl, phosphoric acid salt, Citrate trianion, acetate and other organic acid salt; Antioxidant, such as xitix; Lower molecular weight (being less than about ten residues) peptide, such as poly arginine; Protein, such as seralbumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, such as polyvinylpyrrolidone; Amino acid, such as glycine, L-glutamic acid, aspartic acid or arginine; Monose, disaccharides and other carbohydrate, comprise Mierocrystalline cellulose or derivatives thereof, glucose, seminose or dextrin; Sequestrant, such as EDTA; Sugar alcohol, such as mannitol or Sorbitol Powder; Counterion, such as sodium and/or nonionogenic tenside, such as TWEENPLURONICS or polyoxyethylene glycol.

Aseptic composite for injecting can be allocated according to the conventional pharmaceutical practice described in Remington ' s Pharmaceutical Sciences (the 18th edition, Mack Publishing Company, Easton, PA, 1990).Such as, active compound may be needed at the vegetables oil of such as water or natural generation, as the vehicle of sesame oil, peanut oil or Oleum Gossypii semen, or as the lysate in the synthetic fat vehicle of ethyl oleate or its analogue or suspension.Buffer reagent, sanitas, antioxidant and analogue thereof can be incorporated to according to acceptable medicine practice.

The suitable example of sustained release preparation comprises the semipermeable matrices of the solid hydrophobic polymers containing polypeptide, and described matrix is the form of shaping article, film or microcapsule.The example of sustained-release matrix comprises polyester, hydrogel (such as, by the J.Biomed Mater.Res. of the people such as Langer, (1981) Chem.Tech. of 15:167-277 and Langer, (1982) poly-(2-hydroxyethyl-methacrylic ester) or poly-(vinyl alcohol) described in 12:98-105), poly(lactic acid) (United States Patent (USP) the 3rd, 773, No. 919, EP 58, 481), multipolymer (the Biopolymers of the people such as Sidman of Pidolidone and γ ethyl-L-glutamate ester, (1983) 22:547-556), non-degradable stretches the ethyl vinyl acetate (people such as Langer, above-mentioned), degraded lactic acid-ethanol copolymer, such as LUPRON Depot ^tM(the Injectable microspheres body be made up of lactic acid-ethanol copolymer and TAP-144) and poly-D-(-)-3-hydroxybutyrate (EP 133,988).

Although the polymkeric substance such as stretching ethyl vinyl acetate and lactic acid-ethanol makes can discharge molecule 1 00 day, some hydrogel still continues short period release protein.When encapsulate protein retains in vivo for a long time, they because sex change or gathering under being exposed to the moisture of 37 DEG C, may cause bioactive forfeiture and may change immunogenicity.Can be reasonably tactful in stabilizing protein according to involved mechanismic design.Such as, if find that Accumulation Mechanism is for form intermolecular S-S key through disulfide exchange, just can by upgrading sulfhedryl residue, freeze-drying in acidic solution, control moisture content, use suitable additive and research and develop specific polymer matrix composition to reach stable.

Slow releasing composition also comprises the preparation of antibody crystals in the Suitable formulations being suspended in and crystal can being remained in suspension.When by subcutaneous or peritoneal injection, these preparations can produce slowly releasing effect.Other composition also comprises the antibody that liposome retains.Liposome containing these antibody is prepared by known method itself: United States Patent (USP) DE3,218, No. 121, the Proc.Natl.Acad.Sci.USA of the people such as Epstein, (1985) Proc.Natl.Acad.Sci.USA of the people such as 82:3688-3692, Hwang, (1980) 77:4030-4034, EP 52,322, EP 36,676, EP88,046, EP 143,949,142,641, No. 83-118008th, Japanese patent application case, United States Patent (USP) the 4th, 485, No. 045 and the 4th, 544, No. 545 and EP 102,324.

Dosage for the antibody formulation of given patient can be taken into consideration known various factors to measure with the effect of upgrading medicine by attending doctor, comprises severity and type, body weight, sex, diet, time of administration and path, other medicament and other correlated clinical factors of disease.Treatment effective dose by vitro or ex vivo methods measure.

The significant quantity of the antibody therapeutically adopted depends on (such as) treatment target, the patient's condition of dosing way and patient.Therefore, therapist's preferably optionally titration dosage improve administration routes to obtain optimum result for the treatment of.According to factor mentioned above, general per daily dose can between about 0.001mg/kg to as many as 100mg/kg or more.In theory, clinicist will cast treatment antibody, until reach the dosage of ideal effect.The process of this therapy easily through routine calibrating or as described hereinly to control.

Should be appreciated that, casting of the treatment entity carried out according to composition herein and method should cast with being incorporated to together with the Suitable carriers in formula, vehicle and other reagent, to provide through the transfer of improvement, transmission, tolerance and similarity thereof.Multiple Suitable formulations can be found: Remington ' sPharmaceutical Sciences (the 18th edition in the formula that all pharmaceutical chemistry teachers are known, Mack Publishing Company, Easton, PA (1990)), especially be the 87th chapter of the Block in its literary composition, Lawrence.Such as, these formula comprise powder, slurry, ointment, colloid, wax, oil, lipid, containing lipid (positively charged ion or negatively charged ion) bubble (such as Lipofectin ^tM), DNA conjugate, anhydrous absorption slurry, oil-in-water and W/O emulsion, emulsion polyoxyethylene glycol (polyoxyethylene glycol of various molecular weight), half gel and half solid mixture containing polyoxyethylene glycol admittedly.According to the present invention, any aforementioned mixture can be suitable in treatment and therapy, as long as the activeconstituents in formula can not by passivation of filling a prescription, and administration routes described in compatible and tolerable on described formula physiology.Also see " the Pharmaceutical excipientdevelopment:the need for preclinical guidance. " of Baldrick P., Regul.Toxicol.Pharmacol.32 (2): 210-8 (2000); " the Lyophilization and development of solid proteinPharmaceuticals. " of Wang W., Int.J.Pharm.203 (1-2): 1-60 (2000); " Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts. " J PharmSci.89 (8): 967-78 (2000) of Charman WN; " the Compendium of excipients for parenteralformulations " of the people such as Powell, PDA J Pharm Sci Technol.52:238-311 (1998) and wherein quote to formula, the vehicle extraneous information relevant with supporting agent known by pharmaceutical chemistry teacher.

the preparation of antibody

As described herein, antibody is by using as mentioned below

prepared by technology.Described mouse can produce human immunoglobulin molecules and antibody subsequently, but is not enough to produce murine immunoglobulin molecule and antibody.Technology for reaching identical object is disclosed in patent disclosed herein, application case and reference case.But, especially for producing mouse about transgenosis and being disclosed in by an embodiment of the antibody of its generation the U.S. patent application case the 08/759th applying on December 3rd, 1996, in WO 00/76310 disclosed in international application WO 98/24893 disclosed in No. 620 and 11 days June in 1998 and 21 days December in 2000, its disclosure is incorporated to herein in the mode introduced.Also see the Nature Genetics15:146-156 (1997) of the people such as Mendez.

By using this technology, the complete human monoclonal antibody for Multiple Antibodies can be produced.In one embodiment, by mouse

cell strain with interesting antigen (such as, EGFRvIII) immunity, lymphocyte (such as B-cell) is reclaimed in the mouse of expressing antibody, and described cell and BM form cell strain are merged to prepare immortal hybridoma cells strain, and screen and select these hybridoma cell strains to identify the hybridoma cell strain producing and interesting antigen is had to specific antibody.Be provided for herein producing the method that can produce multiple hybridoma cell strain EGFRvIII to specific antibody.In addition, the invention provides the sign of the antibody produced by these cell strains, comprise the heavy chain of these antibody and the Nucleotide of light chain and aminoacid sequence.

Or, replace with myeloma cell fusion to produce hybridoma, screen further by the generation of reclaimed cell, immune mouse of hanging oneself according to the reactive behavior of initiating antigen (being preferably EGFRvIII protein)

the antibody that cell strain is separated.Described screening comprises carries out ELISA with EGFRvIII protein, be in vitro bonded to stably express length EGF RvIII NR6M cell and by the antibody internalization EGFRvIII acceptor in NR6M cell.EGFRvIII-specific hemolytic plate test is used to be separated single B cell people such as (, Proc.Natl.Acad.Sci.USA, i93:7843-7848 (1996)) Babcook of the interesting antibody of secretion subsequently.Determine target and be used for the cell of bacteriolyze preferably with the sheep red blood cell (SRBC) (SRBC) of EGFRvIII antigen coating.Under the interesting B cell substratum of immunoglobulin (Ig) of secretion and the existence of fill-in, dull and stereotyped formation shows that the specificity EGFRvIII determining target cell mediates bacteriolysis.The monoclonal antibody at separable plate center place is former-specific plasma cells, and the specific genetic information of encoding antibody can be separated from single plasma cell.Use ThermoScript II PCR can the DNA of variable region of antibody secreted by clones coding.Describedly can insert in Suitable expression vectors further subsequently through cloned DNA, be preferably carrier box, such as pcDNA, the more excellent pcDNA carrier for the constant domain containing heavy chain immunoglobulin and light chain.Subsequently, the carrier produced can transfection be host cell, is preferably Chinese hamster ovary celI, and is incubated in the conventional nutrient culture of suitable upgrading for bringing out promotor, selecting transformant or amplify sequence needed for encoding gene.The separation producing multiple single plasma cell EGFRvIII to specific antibody is described herein.In addition, being separated, introducing transfection subsequently by the genetic material of coding anti-EGFR vIII antibodies specific is in the Suitable expression vectors of host cell.

B cell from XenoMouse mouse also can be used as the source of genetic material, can produce antibody display libraries from it.Described library can use the general technology in described field via ribosomal display at phage, yeast or in vitro occur.A kind of rich source is can be used as, from its separable antigen-reactive antibody with high-affinity through hyperimmune XenoMouse mouse.Therefore, can be used for producing antibody display libraries, from the high-affinity antibody of its separable antagonism EGFRvIII through the hyperimmune XenoMouse mouse of antagonism EGFRvIII.Described library can be screened for pep3 oligopeptides, and gained derives antibody for the specificity expressed the cell of EGFRvIII and carry out screening determining for displaying antigen naturally.Complete IgG antibody can use recombinant DNA technology to express subsequently.For example, see WO 99/53049.

Generally speaking, the antibody produced by above-mentioned cell strain has complete IgG 1 or IgG2 heavy chain and human kappa light chain.In one embodiment, antibody has high-affinity, and when by solid phase and molten liquid phase measuring, it generally has about 10 ^-9m is to about 10 ^-13the Kd value of M.In other embodiments, antibody has lower avidity, from about 10 ^-6m is to about 10 ^-8m.

As an understanding of the skilled in the art, can express in the cell strain except hybridoma cell strain according to the antibody of the present embodiment.The sequence of encoding particular antibodies can be used for the transition of Suitable mammalian host cell.Undertaken making the transition that polynucleotide is introduced host cell by any currently known methods, such as comprise and polynucleotide to be packaged in virus (or virus vector) and with virus (or carrier) or by transfection procedures transformed host cells known in technique, as by United States Patent (USP) the 4th, 399, No. 216, the 4th, 912, No. 040, the 4th, 740, No. 461 and the 4th, illustrated in 959, No. 455.Program transition used depends on host to be made the transition.Known the method introduced by xenogenesis polynucleotide in mammalian cell in technique, and it comprises dextran regulates transfection, calcium phosphate precipitation, polybrene regulate transfection, protoplast fusion, electric shock, polynucleotide are packaged in liposome and DNA direct microinjection in karyon.

The mammalian cell strain that can be used as expressive host has been known in technique, and it comprises by the multiple immortalized cell line obtained by American Type Culture Collecti (American Type Culture Collection (ATCC)), include, but is not limited to Chinese hamster ovary cell (CHO), HeLa cell, baby hamster kidney cell (BHK), monkey kidney cell (COS), hepatocellular carcinoma cells (such as, Hep G2) and other cell strains many.Especially preferably cell strain is selected by measuring which cell strain there is high expression level and producing the antibody with composing type EGFRvIII binding characteristic.

example

Following instance is provided, comprises the experiment carried out and the result obtained only in order to reach the object of explanation and and be not interpreted as limitation of the present invention.

The strategy of initial generation EGFRvIII-specific antibody relates to complex antigen (peptide, multiple soluble protein and antigen-expressing cells) immune XenoMouse mouse, to be hybridized cell or pass through XenoMax subsequently by fusion ^tM/ SLAM ^tMtechnology separation B cell and separation antibody produce cell.Antibody produced cell is that specificity is presented to one-level screening by ELISA, is that cell surface combination is presented to secondary screening by FMAT and/or FACS.Carry out the antibody that internalization calibrating may be useful to drug conveying with identification subsequently.Measure the avidity of these antibody.Specific antibodies is selected to carry out epitope mapping.In addition, select specific antibodies to carry out in body and vitro test to analyze these antibody effect to Therapeutic cancer.

prepared by example 1 antigen

A. prepared by EGFRvIII PEP3-KLH antigen

Together with example 2,14-mer human EGFR vIII PEP3 (L E E K K G N Y V V T D H C (SEQID NO:56)) peptide by the synthesis of R & d system routine.PEP3 peptide and keyhole limpet hemocyanin (KLH) combine subsequently, as follows: the keyhole limpet hemocyanin (KLH of EGFRvIII PEP3 (200mcg) (R & D) and 50mcg; Pierce, Rockford, IL) mix and use distilled water constant volume to 165mcl.Add 250mcl binding buffer liquid (0.1M MES, 0.9M NaCl, storage liquid (the EDC of 1-ethyl-3-[3-dimethyl aminopropyl] the carbonization two imide hydrochloride pH4.7) and by the 10mg/ml that adds 25mcl, Pierce, Rockford, IL) EGFRvIII PEP3 and KLH is cross-linked.At room temperature hatch in conjunction with 2 hours, use the PBS of pH7.4 by a 1kDa filter membrane (centrifugal filter membrane, Millipore, Bedford, MA) by centrifugal for unreacted EDC removing.

Together with example 3,14-mer human EGFR vIII PEP3 (L E E K K G N Y V V T D H C (SEQID NO:56)) peptide by the synthesis of R & d system routine.PEP3 peptide is combined with KLH subsequently, as follows: the keyhole limpet hemocyanin (KLH of EGFRvIIIPEP3 (200mcg) and 50mcg; Pierce, Rockford, IL) mix and use distilled water constant volume to 165mcl.Add 250mcl binding buffer liquid (0.1M MES, 0.9M NaCl, storage liquid (the EDC of 1-ethyl-3-[3-dimethyl aminopropyl] the carbonization two imide hydrochloride pH4.7) and by the 10mg/ml that adds 25mcl, Pierce, Rockford, IL) EGFRvIII PEP3 and KLH is cross-linked.At room temperature hatch in conjunction with 2 hours, use the PBS of pH7.4 by a 1kDa filter membrane (centrifugal filter membrane, Millipore, Bedford, MA) by centrifugal for unreacted EDC removing.

B. b300.19/EGFRvIII transfectant

In order to prepare B300.19/EGFRvIII transfectant, Wild type EGFR is gone out from A431 cell initial clones, and be modified for the EGFR gene of the EGFRvIII that encodes thus lack the codon of coding residue 6-273, and the codon of encodes glycine residue is produced in the junction of disappearance.This disappearance occurs in disappearance GTT (α-amino-isovaleric acid) and CGT (arginine) codon around, and after the disappearance obtained, codon is GGT (glycine).(Wikstrandet al.J Neurovirol.4(2)：148-58(1998))

1. the clone of Wild type EGFR structure:

Use Micro-fast RNA test kit (Invitrogen, Burlington, ON) from A431 (ATCC), extract poly-A+mRNA.Total cDNA is synthesized from polyA+mRNA with random pdN6 primer and M-MuLV reversed transcriptive enzyme (NEB, New EnglandBiolabs, Beverly, Mass.).To be increased the PCR primer of 2.3kb by A431cDNA with following primer:

Justice 5 '-GGATCTCGAGCCAGACCGGAACGACAGGCCACCTC-3 '; (SEQ IDNO:62)

Antisense 5 '-CGGATCTCGAGCCGGAGCCCAGCACTTTGATCTT-3 ' (SEQ ID NO:63)

Use Pfu archaeal dna polymerase.

PCR primer XhoI is digested, gel-purified, and be connected to by the linearized plasmid pWBFNP (see No. 99/45031, international application WO) of XhoI, to produce plasmid Wt-EGFR/pWBFNP.

The generation that 2.EGFRvIII builds:

PCR primer is amplified by plasmid Wt-EGFR/pWBFNP with primer pair C13659/C29538 and C29539/C14288 (BioSource International), wherein C29538 and C29539 is by T4 polynucleotide kinase (NEB, New England Biolabs, Beverly, Mass.) phosphorylation:

C13659:5 '-CGGATGAATTCCCAGACCGGACGACAGGCCACCTC-3 ' (justice) (SEQ ID NO:64)

C29538:5 '-CTTTCTTTTCCTCCAGAGCC-3 ' (antisense) (SEQ ID NO:65);

C29539:5 '-GTAATTATGTGGTGACAGATC-3 ' (justice) (SEQ ID NO:66);

C14288:5 '-CGGATCTCGAGCTCAAGAGAGCTTGGTTGGGAGCT-3 ' (antisense) (SEQ ID NO:67).

Be connected to introduce disappearance in 6 to 273 amino acid whose sequences of coding EGFR extracellular domain, and be subcloned into (see No. 99/45031, international application WO) in expression vector pWBDHFR2.

Produced the fragment of the 232bp of 5 ' end of representative disappearance by Wt-EGFR/pWBFNP template Pfu polysaccharase (NEB, NewEngland Biolabs, Beverly, Mass.) amplification with primer pair C13659/C29538.By EcoRl (NEB, New England Biolabs, Beverly, Mass.) digestion also gel-purified PCR primer.Produce the fragment of 1273bp of 3 ' end of representative disappearance from Wt-EGFR/pWBFNP with primer pair C29539/C14288, and by Pfu polymeric enzymatic amplification template.PCR primer is digested with Xhol (NEB, New England Biolabs, Beverly, Mass.).With T4DNA ligase enzyme (NEB, New England Biolabs, Beverly, Mass.), fragment is connected to the pWBDHFR2 digested with EcoRl/Xhol and builds EGFRvIII/pWBDHFR to produce.

The intracellular domain of EGFR is introduced in the structure of following acquisition: isolate from plasmid Wt-EGFR/pWBFNP 1566bp fragment and be connected to DraIII/XhoI digestion EGFRvIII/pWBDHFR to produce EGFRvIII-FL/pWBDHFR.

3. use eGFRvIII-FL/pWBDHFR transfection B300.19 cell:

B300.19 cell (8x10 ⁶) be used in every 700 μ l DMEM/HI media transfection.Add 20 μ gEGFRvIII-FL/pWBDHFR and 2 μ g CMV-Puro plasmid DNA.Cell is shocked by electricity under 300volts/960uF with Bio-Rad Gene Pulser.After electric shock, in cooled on ice cell 10 minutes, add 10 non-selective media (DMEM/HI glucose, 10%FBS, 50 μMs of BME, 2mM L-glutaminate, 100 units of Penicillin-G/ml, 100 unit MCG Streptomycin sulphate/ml) subsequently.At 37 DEG C, 7.5%CO ₂lower incubated cell 48 hours.

After hatching, cell is by split selectable medium (DMEM/HI glucose, 10%FBS, 2mM L-glutaminate, 50 μMs of BME, 100 units of Penicillin-G/ml, 100 unit MCG Streptomycin sulphate/ml, 2ug/ml tetracyclines) in, with 2x10 in 96 orifice plates ⁴, 0.4x10 ⁴' and 0.08x10 ⁴the concentration of cells/well, in elective medium by selection 14 days to produce stable clone.By Puro resistance clone E752mAb (anti-EGFR-antibodies, at Yang et al., Crit Rev Oncol Hematol., have described in 38 (1): 17-23 (2001)) and Goat anti human class IgG PE dye, go up analysis at FACS Vantage (Becton Dickinson) subsequently.

C. build EGFRvIII-RbFc and express structure

For producing EGFRvIII rabbit fusion rotein, first we build the carrier of the DNA containing coding rabbit Fc.This is connected with the DNA of coding EGFRvIII.To this method be described in detail below:

The structure of 1.RbFc/pcDNA3.1Hygro:

(following) primer 1322/867 is for the fragment of the 721bp of the Hinge-CH2-CH3 structural domain of amplification coding rabbit igg.

#1322 (justice): 5 '-GGTGGCGGTACCTGGACAAGACCGTTGCG-3 ' (SEQ IDNO:68)

#867 (antisense): 5 '-ATAAGAATGCGGCCGCTCATTTACCCGGAGAGCGGGA-3 ' (SEQ ID NO:69)

Digested by the PCR primer KpnI obtained and NotI, gel-purified is also connected to pcDNA3.1 (+)/Hygro (Invitrogen, Burlington, ON) with KpnI/NotI digestion, to produce plasmid RbFc/pcDNA3.1Hygro.

The structure of 2.EGFRvIII-RbFc/pCEP4:

(following) primer 1290/1293 is for the product of Pfu polymeric enzymatic amplification from the 1165bp of EGFRvIII-FL/pWBDHFR plasmid template.

#1290 (justice): 5 '-CTACTAGCTAGCCACCATGCGACCCTCCGGGA-3 ' (SEQ IDNO:70)

#1293 (antisense): 5 '-CGGGGTACCCGGCGATGGACGGGATC-3 ' (SEQ ID NO:71)

By PCR primer NheI and KpnI digestion, gel-purified and be connected to NheI/KpnI digestion RbFc/pcDNA3.1 to produce plasmid EGFRvIII-RbFc/pcDNA3.1Hygro.

The SnaBI/XhoI fragment of 2170bp is separated from EGFRvIII-RbFc/pcDNA3.1Hygro and subclone enters the pCEP4 (Invitrogen that SnaBI/XhoI digests, Burlington, ON) in, to produce plasmid EGFRvIII-RbFc/pCEP4.

3. produce 293F EGFRvIII-RbFc stable cell lines:

With the following method plasmid EGFRvIH-RbFc/pCEP4 is introduced 293F cell (Gibco, Grand Island, NY) by calcium phosphate transfection: day before transfection, 1x10 ⁶in the 100mm tissue culture medium (TCM) culture dish that 293F cell is covered to gelatin by plating, and at 5%CO ₂, hatch at 37 DEG C.Before transfection, with the fresh non-selective medium of 10ml (DMEM/F12,10%FBS, 2mM L-glutaminate, 100U/ml penicillin G, 100U/ml MCG Streptomycin sulphate) culturing cell 2-3 hour.Transfection reagent is prepared in micro-centrifuge tube, as follows: the DNA (EGFRvIII-RbFc/pCEP4) of 10 μ g mixes with the 2M calcium phosphate of 62 μ l, and with deionized water constant volume to 500 μ l.With the 2XHBS of another pipette, extract 500ul for shifting described transfection reagent.

Dropwise the solution in transfer pipet being added cell, simultaneously for keeping suitable pH value, cell being placed in 5%CO ₂until carry out transfection in incubator.15-20 hour after transfection, with PBS washed cell also with the non-selective medium feeder cell of the fresh 293F of 10ml.With the cell that transfection results after trypsinase 48-72 are expressed, and by cell with 0.08x10 ⁴cells/well plating, to 96 orifice plates, to be placed in 293F elective medium (DMEM/F12,10%FBS, 2mM L-glutaminate, 100U/ml penicillin G, 100U/ml MCG Streptomycin sulphate, 250ug/ml hydromycin B) 14 days.

Use the anti-EGFR-antibodies E763 (U.S. Patent number 6,235,883) of 1ug/ml to make capture antibodies and detect with the anti-rabbit igg HRPO (CalTag) of goat of 1: 100 dilution, screening hydromycin B resistance clone by ELISA.

example 2

produced by hybrid cell and manufacture anti-EgfrViii antibody

By producing, eight mouse (XenoMouse G1 mouse) with the antibody of γ-1 constant region are immune at the 0th day, then will strengthen at the 11st, 21,32,44 and 54 day for this scheme, then merge at the 58th day.All immunity are all by injecting in the mode of root of the tail portion subcutaneous administration and intraperitoneal administration.1.5x10 is used in immunity in 0th day ⁷the cell (Example1A) of B300.19/EGFRvIH transfection be suspended in mix with complete Freunds adjuvant (CFA) (Sigma, St.Louis, MO) 1: 1v/v without completing in the DPBS of pyrogeneous substance.11st, 21 and 32 days strengthen is use 1.5x10 ⁷the cell of B300.19/EGFRvIII transfection completes in the DPBS mixed with incomplete Freunds adjuvant (IFA) (Sigma, St.Louis, MO) 1: 1v/v.Within 44th day, strengthen being complete with being combined (example 1) with PEP3 (EGFRvIII the peptide)-KLH of IFA1: the 1v/v 5 μ g mixed, and last reinforcement is with not having 5 μ gPEP3 (EGFRvIII peptide)-KLH in the DPBS of adjuvant to combine.

At the 58th day, euthanasia is implemented to mouse, reclaims its inguinal region and lumbar vertebrae lymphoglandula subsequently.Using-system pulverizer discharges lymphocyte by physical disturbance from lymphoglandula, subsequently by CD90 Solid phase removing T cell.By the B cell by washing enrichment with purchased from ATCC, the nonsecreting type myelomatosis P3X63Ag8.653 cell of cat#CRL1580 (Kearney et al, J.Immunol.123:1548-1550 (1979)) with 1: 1 ratio mixing and merge.This cell mixture is by being precipitated lightly so that 800g is centrifugal.After thorough removal supernatant liquor, be no more than 2 minutes with 2-4ml pronase solution (CalBiochem, cat.#53702,0.5mg/ml are in PBS) process.Subsequently, add 3-5ml FBS and stop enzymic activity, electricity consumption cytogamy solution E CFS (0.3M sucrose, Sigma, Cat#S7903,0.1mM magnesium acetate, Sigma, Cat#M2545,0.1mM calcium acetate, Sigma, Cat#C4705 (St.Louis, MO)) be settled to 40ml.

Centrifugal rear removing supernatant liquor, by the washed cell that again suspends in 40ml ECFS.Repeated washing step, again with ECFS suspension cell to reach 2x10 ⁶the concentration of cell/ml.Fusion generator (model ECM2001, Genetronic, Inc., San Diego, CA) is used to carry out electricity-cytogamy.Fusion room size used is 2.0ml, and uses following apparatus to arrange: comparison condition: voltage: 50V, the time: 50s, film rupture condition: voltage: 3000V, the time: 30 μ s, set time: 3s after merging.After fusion, at DMEM (JRH Biosciences), 15%FCS (Hyclone) and containing suspension cell in the solution of HAT, and add L-glutaminate, Pen .-Strep, OPI (oxaloacetic acid, pyruvic acid, Sigma I8405) (all purchased from Sigma, St.Louis, MO) and IL-6 (BoehringerMannheim) at 37 DEG C and 10%CO ²cultivate in air.

By cell with 4x10 ⁴cells/well plating is in flat 96 hole tissue culturing plates.Before transferring to HT (xanthoglobulin and Thymine deoxyriboside) feed supplement medium, cultivate and be stored in 2 weeks in HAT (xanthoglobulin, aminopterinum and Thymine deoxyriboside) feed supplement medium.Select hybrid cell by the survival at HAT medium, and screen the antigenic activity of supernatant liquor with ELISA.ELISA form need antigen Coated Flat Plate (as counting screening EGFRvIII peptide-OVA Coated Flat Plate and wild-type EGFr peptide-OVA Coated Flat Plate) on hatch supernatant liquor and the mouse anti human class IgG using horseradish peroxidase (HRP) to mark detect EGFRvIII specific combination (see table 2.1).

Table 2.1

It should be noted that, at least four antigen-specific hybrid cells detected: 13.1,13.2,13.3 and 13.4.These these hybrid cells that EGFRvIII specificity is positive in ELISA detects are by being verified at 300.19 cells of stable transfection and the contrast of 300.19 untransfected parent cells of expressing EGFRvIII.

Limiting dilution plating is used to clone on the antigen negative hole selected.By the existence of access panel checklist clonal antibody growth, screen the supernatant liquor from mono-clonal hole by antigen-specific ELISA and verify with above-described FACS.Use Luminex instrument to examine and determine high reactivity clone by polynary ELISA thus inspection mankind γ and

the purity of chain.Based on the specificity of EGFRvIII in ELISA and FACS calibrating, select clone 13.1.2 for screening further and analyzing most promising candidate.Illustrate the heavy chain of 13.1.2 antibody and the nucleotide sequence of light chain and aminoacid sequence in Fig. 3 L, and SEQ ID NO 137 and 139 is heavy chain and light chain nucleic acid and 138 and 140 is heavy chain and light-chain amino acid sequence.In addition, show in figures 4 and 5 13.1.2 heavy chain and sequence of light chain and its from of sequence of germ cell line compare.

example 3

antibody is produced by using XENOMAX technology

The immunity of XenoMouse animal

The human monoclonal antibody of anti-human EGFRvIII be by progressively immunity produce there is γ-1 constant region antibody XenoMouse mouse (XenoMouse G1 mouse), produce (XenoMouse G4 mouse) that the XenoMouse mouse (XenoMouse XMG2 mouse) with γ-2 constant region antibody and generation have γ-4 constant region antibody.

For producing mAb by XenoMax technology, XenoMouse G1 group and XMG2 group mouse are by 300.19 cells (example 1B) with EGFRvIII PEP3 (example 1A) and expression EGFRvIII, or with EGFRvIII albumen (EGFRvIII-ECD) (Dr.Bigner, Duke University) the extracellular domain of bacterial expression and the EGFRvIII300.19 cell of expression, or with EGFRvIII rabbit Fc fusion rotein (EGFRvIII-RbFc) (example 1C) with express 300.19 cells of EGFRvIII, or only use EGFRvIII-RbFc by palmula (FP), or by subcutaneous injection in tail base portion and abdominal cavity (BIP) immunity.

For palmula immunity, primary immune every mouse uses or does not use 10x10 ⁶express 300.19 cells of EGFRvIII and use or do not use with Titermax gold (Sigma, Oakville, ON) with EGFRvIII PEP3 or EGFRvIII-ECD of 1: the 1 10 μ g mixed or EGFRvIII-RbFc.Reinforcement subsequently uses the half of primary immune immunogenic dose used.Front 4 reinforcements inject mouse, absorption of spending the night by immunogen and alum (Sigma, Oakville, ON) being mixed, and every mouse is as shown in following table 3.1.Then the respective reaction injecting Titermax is former, and reinject alum, and the immunogenic last reinforcement subsequently in PBS is as shown in following table 3.1.Especially, the 0th, 3,7,10,14,17,21 and 24 day immune animal.The 19th day to animal get blood with adaptive immune serum and determine gather in the crops select tire.The 28th day results animal.

Table 3.1 palmula immunization schedule

As described in palmula immunity, initial BIP immunity every corresponding immunogen of mouse mixes with 1: 1v/v and complete Freund ' s adjuvant (CFA, Sigma, Oakville, ON).Reinforcement subsequently first every mouse uses corresponding immunogen 1: 1 to mix with incomplete Freund ' s adjuvant (IFA, Sigma, Oakville, ON) to carry out, be the last reinforcement of every mouse PBS subsequently.As shown in following table 3.2, the 0th, 14,28,42,56 and 75 (finally strengthening) sky immune animal.The 63rd day to animal get blood with adaptive immune serum and determine gather in the crops select tire.The 78th day results animal.

Table 3.2 Bip immunization schedule

the animal selecting results is determined by usefulness

By ELISA determine anti-hEGFRvIII antibody tire by EGFRvIII-RbFc (2.5 μ g/ml) or contrast RbFc (2 μ g/ml) EGFRvIII peptide-OVA (2 μ g/ml) (example 1) or contrast OVA (4 μ g/ml) be coated onto CostarLabcoat Universal Binding Polystyrene96 orifice plate (Corning, Acton, MA) upper four spend night.Removing comprises the solution of free antigen and processes 4 minutes (4000 micro-joule) to described plate UV-light (365nm).With plate described in distilled water wash five times.Double 1: 100 at first dilution from EGFRvIII immunity

animal or first for test

the serum 1: 2 that animal obtains uses 2%milk/PBS titration after diluting.Leave a blank in last hole.With plate described in distilled water wash five times.Add with the final concentration of 1 μ g/ml the antibody 1 hour that the special horseradish peroxidase of Goat anti human class IgG Fc (HRP, Pierce, Rockford, EL) combines under room temperature.With plate described in distilled water wash five times.Add TMB chromogenic substrate (Gaithersburg, MD) plate to be developed the color in 30 minutes, add 1M phosphoric acid and stop ELISA.

animal individual special is tired and to be determined by optical density(OD) during 450nm and to show in table 3.3 and 3.4.This inverse dilution representing immune serum of tiring, therefore the reaction of the larger then humoral immunization of this numerical value to hEGFRvIII is stronger.

For by the mouse at the subcutaneous injection of tail base portion and abdominal injection, the determination of tiring as mentioned previously, except scribbling the flat board of EGFRvIII-RbFc (2.0 μ g/ml) or contrast RbFc (2.5 μ g/ml).

Table 3.3

Based on serology, in table 3.3 all 5th group XenoMouse animal and done XenoMax results from the animal 0743-5 of the XenoMouse of the 6th group by selection.

Table 3.4

Based on the Serology data in table 3.4, XenoMouse animal (0695-1,0695-3 and 0695-4) is selected gathers in the crops.

the selection of B cell

B cell from above-mentioned animal is gathered in the crops and is cultivated.Isolate secretor type EGFRvIII-peptide-specific antibody, as people such as Babcook, the description in Proc.Natl.Acad.Sci.USA, 93:7843-7848 (1996).ELISA is used to the special hole of identification the one EGFRvIII-peptide-OVA-.In 24596 orifice plates, turned out about 500 ten thousand B cell from XenoMouse animal with the density of 500 or 150 or 50 cells/well, these cells are screened to identify antigen-specific well on EGFRvIII-peptide-OVA.Nearly 515 holes demonstrate obvious OD in background, illustrate a representative sample in table 3.5.

Table 3.5

The EGFRvIII-specificity verifying them is screened again in the positive holes of 244 EGFRvIII-peptide-OVA-Elisa of OD > 0.5 on ofEGFRvIII-peptide-OVA on OVA.A representative example of these results is shown in table 3.6.

Table 3.6

limiting antigen calibrating and analysis

Limiting antigen analysis is a kind of by the method for the antibody of antigen-specific in B cell medium supernatant and other antigen specific antibodies avidity classifications all.When be coated with antigen is little, only the antibody of most high-affinity can combine by detection level with any in the state of the equilibrium.(see (such as) international application No.WO 03/48730)

EGFRvIII peptide-OVA is coated onto on flat board by with three concentration 7.5ng/ml, 1.5ng/ml and 0.03ng/ml, and on 96 orifice plates 4 DEG C spend the night.Before 1% milk adding the 50ul containing 0.05% sodiumazide in flat board is dissolved in the solution of PBS, each flat board distilled water wash 5 times, then adds the B cell supernatant liquor of 4 μ l in each hole.Be placed in shaking table under room temperature after 18 hours, again use distilled water wash dull and stereotyped 5 times.Goat anti human's class (the Fc)-HRP of the 1 μ g/ml of 50ul is added in every hole.Room temperature, after lower 1 hour, is again used distilled water wash dull and stereotyped 5 times, and in every hole, is added the TMB of 50 μ l.By adding the phosphoric acid termination reaction of 50uL 1M in every hole, and under wavelength 451nm plate read, result is shown in table 3.7.

Table 3.7

The result produced by Limiting antigen analysis is made comparisons with the total OD value obtained in high antigen is examined and determine.By the OD value that will obtain in Limiting antigen calibrating than on the OD value of acquisition in the calibrating of high antigen, complete the relative classification of avidity.The antibody with height ratio will have high avidity.The sample of the B cell medium supernatant examining and determine the ratio classification of OD value based on Limiting antigen calibrating OD value with high antigen shown by table 3.7.

calibrating is combined by the juvenile cell of FMAT

The ability that analysis EGFRvIII peptide-OVA-Elisa positive hole supernatant liquor is combined with the natural form of the EGFRvIII of the upper stably express of NR6 cell (NR6M cell) is (see people Epidermal growth factorligand-independent such as Batra, unregulated, cell-transforming potential of a naturally occurring humanmutant EGFRvIII gene.Cell Growth Differ.6 (10): 1251-9 (1995)).NR6M cell with the density of every hole 8000 cell sowing, and in 96 hole FMAT plates night incubation.Then removing remain the medium of 15 μ l in hole.In hole, add the B cell medium supernatant of 15 μ l, be then that 1 μ g/ml adds 15 μ l anti-human IgGFc Cy5 with final concentration.Then 2 hours are hatched at placing it in 4 DEG C.With 150 μ lPBS washed cells, and be fixed before reading on FMAT.Result (table 3.8) is represented with total fluorescent density.Mankind's anti-EGFR vIII mAb13.1.2 is used as positive control, and its initial concentration is final concentration 1 μ g/ml, and negative control is the PK16.3.1 of same concentrations.Have 134 to be tested as and NR6M Cell binding in 244 samples, wherein 62 have the total fluorescent value being greater than 8000, have 6 false positives in this 134 combination.

NR6Wt cell (NR6 cell expressing EGF acceptor) does identical natural combination calibrating (see people .Epidermal growth factor ligand-independent such as Batra, unregulated, cell-transforming potential of anaturally occurring human mutant EGFRvIII gene.Cell Growth Differ.6 (10): 1251-9 (1995)) to get rid of the combination (table 3.8) because being attached to Wt acceptor.ABX-EGF is used as positive control and the PK16.3.1 of same concentrations is used as negative control antibody.3 are had to be combined with NR6Wt cell tight in 134 NR6M combinations.190 holes are had to be also coupled to natural form on cell in the hole that 244 EGFRvIII peptides in Elisa are combined.Example is given in table 3.8.

Table 3.8

At table 3.8 kind, be identified as Wt combination from the supernatant liquor in the 187A4 of hole and 141C6 and the NR6M Cell binding false positive that is.Hole 129H6 and 183E11 is the strong peptide combination not having natural combination.

internalization is examined and determine

The ability of front 60 the natural internalization acceptors in conjunction with B cell medium supernatant of further calibrating.NR6M cell to be inoculated into the density of 8000 cells/well on 96 hole FMAT plates and night incubation.Removing medium, and double ground adds 10-15 μ l B cell medium supernatant in cumulative volume 30 μ l medium.Then, add the second antibody (final concentration is the SS Alexa647 anti-human IgG Fab of 1.5 μ g/ml) of 15 μ l and this mixture is hatched 1 hour on ice.Use irrelevant B cell substratum damping fluid to understand the effect of substratum medium.Mankind's anti-EGFR vIII mAb13.2.1 is used as positive control, and its initial concentration is final concentration 1 μ g/ml, and negative control is the PK16.3.1 (mankind anti-KLH IgG2 antibody) of same concentrations.After hatching, with cold PBS washed cell, in institute is porose, add 50 μ l media, one-duplicate copy is hatched 30 minutes at 37 DEG C, and another duplicate is still hatched on ice.After medium is hatched in removing, hatch group at 37 DEG C and add the cold 50mM gsh of 100ul and add the cold medium of 100ul in another group, all place 1 hour on ice for two groups.Then use the PBS washed cell that 100 μ l are cold, mixed being incorporated in FMAT is read with 1% paraformaldehyde subsequently.Result represents with % internalization, with gsh/do not have total fluorescent value x100 during gsh to calculate total fluorescent value.Information representative is provided in table 3.9.

Table 3.9

EGFRvIII-specific hemolytic plaque is tested

Carry out this test and need many special agents.System joins these reagent with the following method.

1. the biotinylation (SRBC) of sheep red blood cell.SRBC is stored in the stock solution as 25% in RPMI medium.By the SRBC of 1.0ml being distributed to the cell pellet obtaining in new Eppendorf tube and fill 250 μ l SRBC.With the pulse Spin precipitate SRBC of 8000rpm (6800rcf) in micro-whizzer, suck supernatant, will precipitate with the PBS of 1.0ml pH8.6 and again hang, then repeated centrifugation.Repeated washing circulates 2 times, then SRBC precipitation is transferred to 15ml plastics tubing and is settled to 5ml with the PBS of pH8.6.In independent 50ml plastics tubing, in the PBS of 45ml pH8.6, add the Sulfo-NHS vitamin H of 2.5mg.Once vitamin H dissolves completely, just add the SRBC of 5ml, and rotate this pipe at normal temperatures 1 hour.With the centrifugal SRBC5 minute of 3000rpm, and sop up supernatant.Biotinylated SRBC is transferred in Eppendorf tube, and washs 3 times with the PBS of aforesaid method pH7.4, then in 15ml plastics tubing, be settled to 5ml (5%B-SRBC stock solution) with immunocyte medium (RPMI1640).Before need using, by stock solution 4 DEG C of preservations.

1. apply B-SRBC with streptavidin (SA).The B-SRBC stock solution of 5% of 1ml is transferred in new Eppendorf tube.The B-SRBC cells were washed3times as above and resuspended in1.0ml of PBS at pH7.4to give a final concentration of5% (v/v). add the streptavidin (CalBiochem of 10 μ l 10mg/ml, San Diego, CA) stock solution, mixing in pipe also rotates 20 minutes at normal temperatures.Repeated washing step also uses the PBS of pH7.4 1ml by SA-SRBC settling flux (5% (v/v)).

3. apply SA-SRBC with EGFRvIII.Apply SA-SRBC with the EGFRvIII peptide-OVA of biotinylated 10 μ g/ml, mixing also rotates 20 minutes at normal temperatures.SRBC is washed twice with the PBS of the pH7.4 of aforesaid method 1.0ml.The SRBC applied with RPMI (+10%FCS) settling flux EGFRvIII is also settled to final concentration 5% (v/v).

4. the quality of EGFRvIII peptide-SRBC is determined by immunofluorescence (IF).The SRBC of the 5%SA-SRBC of 10 μ l and the 5%EGFRvIII peptide coating of 10 μ l is added in the new 1.5ml micro-centrifuge tube of the PBS independently containing 40ul.With the concentration of 45 μ g/ml, anti-for contrast mankind EGFRvIII antibody is added in each SRBC sample.These pipes are rotated 25 minutes at normal temperatures, uses the PBS washed cell three times of 100 μ l subsequently.With the PBS of 50 μ l by cell settling flux, and hatch together with the IgG Fc antibody connected with Alexa488 (Molecular Probes, Eugene, OR) of 40mcg/mL.These pipes rotate 25 minutes at normal temperatures, and wash with the PBS of 100 μ l subsequently, and with the PBS of 10 μ l by cell settling flux.The cell point dyeed by 10 μ l, in clean glass microscope slide, covers with glass cover-slip, observes and count in the scope of any 0-4 under fluorescent.

5. plasmacytic preparation.Results had previously determined the content of the single micro-culture hole of the B cell clone containing the interested immunoglobulin (Ig) of secretion through multiple test.The autospencer of 100-1000 μ l is used to collect the content in described hole by adding 37CRPMI (10%FCS).With transfer pipet by cell settling flux, then transfer to (final volume is about 500-700 μ l) in new 1.5ml Eppendorf tube.Under room temperature in Eppendorf centrifuge with the rotating speed eccentric cell 1 minute of 2500rpm (660rcf), and rotate these pipes with 180 degree subsequently and with the rotating speed recentrifuge of 2500rpm.Draw Frozen Medium also with 100 μ l RPMI (10%FCS) settling flux cells, and centrifugal.Repeat with RPMI (10%FCS) washing, and be also placed in before use with 60 μ l RPMI (10%FCS) settling flux cells and preserve on ice.

6. plasmacytic micrurgy.Prepare glass slide (2x3 inch) with silicone resin edge in advance and allow preservation of spending the night at normal temperatures.Before using, with the surface of the even wiping slide of the SigmaCoat (Sigma, Oakville, ON) of about 5ul, wiping tempestuously after drying.The SRBC (5%v/v stock solution) of the EGFRvIII peptide coating of 60 μ l is added in the cell sample of each 60 μ l, 4x GPC (the Sigma of preparation in RPMI (10%FCS), Oakville, ON) stock solution and 4x strengthen immune serum stock solution (with 10%FCS 1: 150 preparation in RPMI).Mixture (10-15 μ l) to be dripped on ready slide and to cover drop with undiluted paraffin oil.Slide is at least hatched 45 minutes at 37 DEG C.The is from plaque identification EGFRvIII specific plasma cell and by micrurgy recovery (see table 3.10).

Table 3.10

Singe-cell PCR, clone, expression, purifying be anti-EGFRvIII antibody and qualification is recombinated.

The genes encoding the variable regions were rescued by the reclaims the gene in territory, encoding variable regions by carrying out RT-PCR on single micrurgy plasmocyte.Extract mRNA and carry out reverse transcription PCR to produce cDNA.The cDNA of coding variable heavy chain and light chain is increased specifically by with polymerase chain reaction.Mankind's Weight variable sequence is cloned in IgG1 expression vector.This carrier is produced by multiple cloning sites of the constant domain of IgG 1 being cloned into pcDNA3.1+/Hygro (Invitrogen, Burlington, ON).Mankind's Weight variable sequence is cloned in IgK expression vector.This carrier is produced by multiple cloning sites of the constant domain of mankind IgK being cloned into pcDNA3.1+/Neo (Invitrogen, Burlington, ON).The heavy chain and the light chainexpression vectors were then co-lipofected into a60mm dish of70%confluent humanembryonal kidney293cells and the transfected cells were allowed to secrete a recombinantantibody with the identical specificity as the original plasma cell for24-72hours. is from HEK293 cell harvesting supernatant liquor (3mL) and prove the secretion (table 3.11) of complete antibody by the method that sandwich ELISA detects IgG specifically.ELISA is used to evaluate specificity (table 3.11) by the combination of recombinant antibodies and EGFRvIII.

Table 3.11

Secretion ELISA test is carried out as follows.For antibody-secreting, by the Goat anti human class IgG H+L of 2 μ g/mL with to be coated on Costar Labcoat UniversalBinding Polystyrene96 orifice plate for the EGFRvIII-Rab Ig Fc fusion rotein of 1.5 μ g/ml that antigen combines and to remain on four and spend night.With plate described in distilled water wash five times.From undiluted small-sized lipofection supernatant liquor to 7 hole 1: 2 titration recombinant antibodies.With plate described in distilled water wash five times.Add Goat anti human class IgG Fc special HRP binding antibody normal temperature to the secretion flat board detected at room temperature 1 hour with 1ug/ml Rb anti-Hu Fc and combination flat board with the final concentration of 1 μ g/mL and keep 1 hour.With plate described in distilled water wash five times.Add and plate was developed the color in TMB30 minute, add 1M phosphoric acid and stop ELISA.Analyze each ELISA dull and stereotyped to determine the optical density(OD) of every hole when 450nm.

order-checking and sequential analysis

Check order at the heavy chain of both direction to clone, and analyze to determine that the germline sequence of antibody is originated and identifies the change with germline sequence.Provide these sequences in the accompanying drawings.3A-3K and (SEQ ID NO:34-55). in accompanying drawing 4-7, provide comparing of the germline sequence in each heavy chain and sequence of light chain and its source.In addition, in the accompanying drawings the sequence of 13.1.2 antibody and its germline sequence that derive from hybrid cell are compared.4 and 5.

Should be appreciated that from discussion herein, each 131 antibody and 13.1.2 antibody all have the affinity very high to EGFRvIII, and it is by cell internalization, and when with toxin in conjunction with time show very high cell killing efficiency.What is interesting is, each antibody, even if it produces from the different immunity of XenoMouse mouse or uses different technology, all derive from closely similar germline genes.But based on the location (herein to some extent discuss) of epi-position, it seems that each antibody to combine from visibly different epi-position on EGFRvIII molecule and have significantly different residues on the EGFRvIII combining necessity.These results indicate, the utilization of germline genes is very important to the antibody therapeutics for EGFRvIII, and the fact of the small change combination and effect that can change antibody allow for the further design of antagonist and other antibody therapeuticses based on these topology discoverys. the natural EGFRvIII that anti-EGFR vIII mAbs expresses on cell is combined

In this example, measure the combination of anti-EGFR vIII antibody and NR6M cell.Especially, calibrating derives from the not quantitative IgG1 supernatant liquor of XenoMax and the binding ability of NR6M and NR6WT.Overnight incubation in FMAT96 orifice plate with the density inoculating cell in 10000/ hole and at 37 DEG C.Removing medium also adds the titration of 40 μ l small-sized fat supernatant liquor, and cell is hatched 1 hour on ice.Mankind 13.1.2EGFRvIII antibody and ABX EGF (E7.6.3, U.S. Patent number 6,235,883) antibody add as positive control.PK16.3.1 antibody is used as negative control.With cold PBS washed cell, add second antibody (SS Alexa anti-human IgG Fc) with the density in 1 μ g/ml, 40 μ l/ holes and hatch 1 hour on ice.Then with cold PBS washed cell, more fixing then to read with FMAT.By the binding specificity of counting to all antibody of the selective mechanisms of NR6WT.

the purifying of restructuring anti-EGFR vIII antibody.

In order to large-scale production, heavy chain and light chain expression vector (the every chain/plate of 2.5 μ g) are by the 70% HEK293 cell be paved with in lipofection to ten 100mm plates.Transfectional cell hatches 4 days at 37 DEG C, and results supernatant liquor (6mL) also substitute by the fresh medium of 6ml.At the 7th day, removing supernatant liquor was also with initially gathering in the crops merging (from 10 flat boards 120ml altogether).Use each antibody of protein-A agarose (Amersham Biosciences, Piscataway, NJ) affinity chromatography (1mL) purifying from supernatant liquor.With the 0.1M glycine of 500mcL pH2.5, antibody is washed from protein-A post.Washings is dialysed in the PBS of pH7.4, and carries out filter membrane sterilizing.Antibody is analyzed to evaluate its purity and output with non-reduced SDS-PAGE.Also concentration is measured with the ultraviolet analysis of OD250.

by the EGFRvIII receptor internalization of restructuring anti-EGFR vIII mAb

As previously mentioned, express, purifying the IgG1 recombinant antibodies in quantitative XenoMax source.Further calibrating antibody is in the ability of NR6M cell internalization EGFRvIII acceptor.250,000NR6M cell hatches 7 minute on ice with the concentration of 0.25 μ g/ml together with first antibody (SC95, SC131, SC133, SC139, SC150, SC170, SC211, SC230, SC250 and mankind 13.1.2 in contrast) on 96 hole v base plates, has three such duplicates.Add the second antibody (SS Alexa anti-human IgG Fab) of 3 μ g/ml Fab and hatch 7 minutes on ice with the PBS washed cell of cold 10%FCS.With the cold PBS washed cell containing 10%FCS once and subsequently with cold medium settling flux.Then, to hatch and remaining one group hatches 1 hour at 4 DEG C at 37 DEG C for two groups in three duplicates.After this, 4 DEG C of cells of hatching and at 37 DEG C of one group of cells of hatching on ice with gsh process (as mentioned before) 1 hour.Then with the washing of the PBS containing the 1%FCS also settling flux cell that 100 μ l are cold, and facs analysis is used.% internalization [(37 DEG C of mean number with mean number-4 DEG C gsh process of gsh process)/(37 DEG C of mean number without mean number-4 DEG C gsh process of gsh process)] is calculated from the geometric mean obtained from facs analysis.NA means and has carried out facs analysis but data do not provide in table 3.12.

Table 3.12

13.1.2 be one by the hybrid cell previously for EGFRvIII epi-position produces (example 2) and generation antibody and as the negative control of in this test.Table 3.12 these results show two subgroup antibody: effectively internalization those (70-80%) and there is no internalization those (22% or less).

example 4

the location of the epi-position of the anti-EgfrViii antibody of the mankind

In order to determine the epi-position that specific antibodies of the present invention combines, 6 mankind of anti-EGFRvIII use the synthetic peptide from the peptide sequence of special EGFRvIII to position with 3 murine monoclonal antibodies (mAb).The antibody of location be derive from mankind's hybrid cell anti-EGFRvIII13.1.2 antibody, derive from mankind XenoMax anti-EGFRvIII131,139,250,095 and 211 antibody and anti-EGFRvIII H10, Y10 and B9 antibody of murine.

The method used is for conventional SPOT peptide array (Sigma Genosys) is to study the interaction of molecules between the anti-EGFrVIII antibody of the mankind and its peptide epitopes.SPOTs technology is based on the peptide solid phase synthesis of applicable antibody epitope systems analysis.The synthesis of conventional arrays oligopeptides can be buied from Sigma-Genosys.The peptide array from the overlapping oligo-peptides of EGFRVIII variable amino acid sequence is ordered from Sigma-Genosys.

A series of nine 12-mer peptides are as the plaque synthesis on polypropylene screen.Peptide array, across the residue 1-20 of EGFrVIII sequence, represents the disappearance of the amino acid 6-273 of the outer wtEGFr structural domain of born of the same parents, and in the generation of tie point glycine (G).Each continuous peptide derived from previous peptide, produces nested, the overlapping storehouse of an array oligopeptides with a residue.Film with 9 peptides anti-EGFrVIII antibody different from 9 (1 μ g/ml) reacts.The HRP second antibody combined is used then to evaluate the combination of mAb and film binding peptide with the chemoluminescence (ECL) strengthened by enzyme link hybridization.Array used is shown in table 4.1.

Table 4.1 plaque array sequence:

In addition, by group and L-Ala screening position location functionality epi-position.I in this course, use in a combination L-Ala screening method identification EGFrVIII peptide to anti-EGFRvIII mAb interaction essential amino acid.For reaching this target, ordering second group of SPOT array and screening for L-Ala.As the panel of the variable peptide having L-Ala to replace in above-mentioned scanning 12 residues.Plaque #1, the sequence do not made a variation is a positive control of antibodies.Array used is shown in table 4.2.

Table4.2 L-Ala screening array:

All 9 mAb pass through SPOT program fixation and recognition to the epi-position of mankind EGFrVIII.All 9 antibody all can with described reactive polypeptide.The result obtained with the human antibodies that 3 murine antibodies and 6 derive from XenoMouse mouse is shown in table 4.3.The residue highlighted is that we make a variation into L-Ala and cancel by the antibody of test the residue combined.Therefore these are the relevant residue be attached on antibody.

Table 4.3

The hypographous amino acid shown in table 4.3 is the residue of the maximally related antigen recognition site of antagonist identification.The peptide of overlap is used accurately to locate the shortest length of the epi-position of all ten mAb, and by determining with each residue that L-Ala systematically replaces in epi-position the permission that mAb is combined with variant epitope.

In table 4.4, summarize the supplementary features of antibody.Especially, in the Western flat board of polyacrylamide gel electrophoresis, test the combination of a subgroup antibodies on tumor cell system lysate under non-reduced or reductive condition.Also comprise the recombinant protein of purifying.The antibody combined under reduction and non reducing conditions shows that epi-position is linear.Sample identification:

EGFRvIII-rabbit Fc fusion rotein

H1477-expresses the H80 human tumour cell line building transfection with EGFRvIII.These cell expressings EGFR and EGFRvIII.

The Wild type EGFR albumen of EGFR-purifying.

A431-only expresses the human tumour cell line of Wild type EGFR

A459-only expresses the human tumour cell line of Wild type EGFR

H80-only expresses the human tumour cell line of Wild type EGFR

EGFR Biacore-, as a pair specific high responsive test, tests the combination of the EGFR of mAb and purifying in Biacore

Table 4.4

Result shows most of mAb and has binding specificity identical in essence, the binding specificity of seven mAb displays and variable EGFrVIII, 2 mAb and wild-type EGFr (muroid H10 and the mankind 211) cross reaction in the western blot of purifying protein and the lysate of A431 cell simultaneously.Note, but although antibody 211 is all combined with naivety and the purifying EGFRvIII that reduces in western blot, the combination of itself and non-reduced albumen is slightly strong.In the test to A431 cell pyrolysis liquid, antibody 211 is strongly combined with a group Wild type EGFR in non-reducing sample, but does not have signal going back in raw sample.This shows that the combination of antibody 211 is due to the conformational epitope in Wild type EGFR, and performance is different in EGFRvIII.The epi-position of 5 mAb is in the residue 2-12 across the variable specificity glycine residue of EGFRvIII, but the residue 7-16 that 4 mAb (comprising H10 and 211) have across EGFRvIII and wild-type EGFr.Antibody 131 and A431 and A549 Cell binding in FACS.These cells are negative to EGFRvIII expression and are positive to EGFR expression.In Western, antibody 131 is not combined with the EGFR of non-reduced or non-reduced purifying or reduction or non-reduced A43 and A549 cell pyrolysis liquid and shows that antibody 131 may be combined with the variable EGFR of the cell surface expression some human tumour cell lines.These variants are responsive to sex change.

example 5

the specific sign of In Vitro Anti EgfrViii antibody

By to the specificity of carrying out facs analysis determination antibody purification with the NR6 cell of wild-type or variation EGFR transfection.With 5 μ g/ml corresponding antibody, cell is hatched 1 hour on ice, use FACs buffer solution, and hatch with the Goat anti human class IgG that PE combines subsequently.

example 6

with the cross reactivity of the EGFR of amplification

With the subgroup cross reaction (Johns et al., Int.J.Cancer.98:398,2002) of wild-type EGF acceptor on the cell that show needle increases at producer to the antibody of variable EGF acceptor.For determining whether human EGFR vIII antibody recognition has similar characteristic, in the medium the ability of the wild-type EGF acceptor that it identifies in various kinds of cell is tested.Hatch at 4 DEG C with designated cell system and antibody.After FACS buffer solution, add the second antibody being combined with phyoerythrin, and continue to hatch.The clone of all analyses all expresses Wild type EGFR.By antibody on A431 and SF-539 cell not on A498 or SKRC-52 cell antibody XG1-131 identify the subgroup of Wild type EGFR.The antibody 13.1.2 of another EGFRvIII the subgroup of nonrecognition Wild type EGFR.Consider these data, show the wild-type EGRF that only can identify cell surface for the subgroup of antibody of variation EGFRvIII.Specifically for the subgroup of the antibody recognition wild-type EGF acceptor of variation EGFRvIII ability and do not rely on total EGFR density but the new conformational epitope special to tumour cell may be represented.Ability for the antibody of EGFRvIII and the subgroup cross reaction of wild-type receptor uniquely may determine that the avidity of the antibody of base determines jointly (result see epitope mapping and avidity determining section herein) by the specific epitopes of the acceptor inter-node that makes a variation with to this.

the specific sign of example 7 In Vitro Anti EgfrViii antibody

the combination of antibody and clone

The specificity of these antibody purifications is determined by carrying out facs analysis on the panel of clone.Clone uses: H80-mankind's glioblastoma cell line, the H1477 (H80-EGFRvIII) expressing high-level EGFRvIII, A431-human epidermal tumor cell line and A549-human lung tumor clone.All cells system all from Dr.Bigner except A431 and A549 from ATCC (Rockville, MD, U.S.A).With 10 μ g/ml corresponding antibody, cell is hatched 30 hours on ice, use FACs buffer solution, and hatch with the Goat anti human class IgG that the PE from Jackson ImmunoResearch (WestGrove, PA, U.S.A.) combines subsequently.In Fig. 9 A-9L and 10A-10D, the dark histogram display cell of irrelevant IgG dyeing, skeleton diagram or white histogram represent the dyeing of associated antibodies.

Anti-EGFRvIII antibody 13.1.2,131 and 139 and the EGFRvIII protein binding fastened of transfectional cell.The figure summing up some results is shown in Fig. 9 M-9P.

With the subgroup cross reaction (Johns et al., Int.J.Cancer.98:398,2002) of wild-type EGF acceptor on the cell that show needle increases at producer to the antibody of variable EGF acceptor.In this example, with XG1-131 and XG1-139, A431 and A549 is dyeed.Figure 10 B and Figure 10 C shows that 131 and 139 have the specific cross reactivity with Wild type EGFR, instead of only identifies the subgroup of H80, A431 and A549 clone.But, 10% of the level of the ABX-EGF (E7.6.3) that this cross reactivity only dyes in these clones.Result provides in Fig. 9 A-9P and 10A-10D.

Antibody for cell-surface antigens can be used as delivery vehicles medicine or toxin being transported to specifically cell.If the internalization of this antibody stimulator antigen, perhaps after medicine or toxin rupture from antibody, medicine or toxin can cause the death of cell.This mechanism to be used in animal or patient body killing tumor cell specifically.The method that selection can conduct drugs to the antibody of cell is by secondary cell toxicity test.In these trials, first antibody is combined with cell surface and adds the second antibody be combined with medicine or toxin.If the internalization of first antibody stimulator antigen, second antibody will be total to internalization, once medicine or toxin fracture just cause cell killing.

example 8

secondary cell toxicity test

This example shows how the toxin combined by secondary antibody can be directed to the cell (target cell) of expressing target epi-position by antibody.The antibody be combined with toxin is cast the cell of expressing target peptide.The death of those cells shows the validity that antibody toxin combines.

Need specific killing the visual specific use of amount and change.In one embodiment, the minimizing of any possible cancer cells is all enough.Such as, the minimizing of 0-1,1-5,5-10,10-20,20-30,30-40,40-50,50-60,60-70,70-80,80-90,90-95,95-99 or 100% target cell will be enough.In another embodiment, the minimizing of the target cell numbers expected or a function of the non-specific damage of Antibody Combination.Such as, if having non-specific target and the destruction of little antibody, the antibody/Toxin Combinations only with 10% target cell reduced number may be just enough.Such as, antibody/toxin complex kills and wounds 10% lower than non-targeted group.Equally, specific amount will depend on specific needs and situation.Useful especially antibody is that those have highly selective and and target cell good combination to target cell.In one embodiment, target is EGFRvIII albumen or its fragment.In one embodiment, illustrate the mankind or humanized, can effectively internalization, to EGFRvIII albumen or its fragment special, be closely connected and the antibody be connected with effective toxin with EGFRvIII albumen or fragment.

example 9

secondary cell toxicity produces clonogenic assay

Except secondary cell toxicity test, also in product clonogenic assay, employ EGFRvIII specific antibody.As mentioned above, the antibody be combined with toxin is cast cell.The multiplication capacity of monitoring cell.The minimizing of propagation shows that antibody toxin combination is effective.

example 10

carefullyborn of the same parents anti-EgfrViii antibody (13.1.2) in toxicity test be binding substances directly

Except the above example indirectly combined, these tests can also be carried out with the antibody directly in conjunction with toxin.Directly in conjunction with the antibody of toxin be with example 8 in use for the mode that the mode described by antibody-toxin conjugate is identical.

example 11

anti-EgfrViii antibody characterization in body

Also antibody-toxin conjugate can be tested in vivo.Antibody-toxin conjugate is cast test organism, described test organism has the target cell of expressing target peptide.The minimizing of target cell numbers in test organism shows, the toxin of binding antibody can play a role in environment in vivo.

example 12

the expression of the EgfrViii in cancer patients/human tumor

The expression of EGFRvIII on human tumor is by 2 murine monoclonal antibody (B9 from multiple cancer patients and known specific combination EGFRvIII, IgG1 and Y10, IgG2 (Dr.Bigner, Duke University)) the dyeing of frozen tissue part of combination determine.The control antibodies dyeing that same section mates by homotype.The summary of the coloration result obtained from Patient Sample A is illustrated in table 12.1.

table 12.1

the summary of the coloration result of Patient Sample A

Tumor type	Sample size (N)	EGFRvIII＞+	EGFRvIII＞++
				Neuroblast glioma	8	100％	100％
Breast cancer
		100	31％	24％
NSCL cancer
		51	47％	39％
Head and neck cancer					21	42％	38％
	Prostate cancer
		22	4.5％	4.5％

EGFRvIII >+: the tumour comprising all expression EGFRvIII

EGFRvIII > ++: the tumour expression comprising at least expression 10% or more EGFRvIII mainly finds on cytolemma and/or tenuigenin.The breast (31%) of remarkable quantity, NSCL (47%) and neck (42%) cancer sample dyeing are that EGFRvIII is positive.In specific examples, dye to obtain high-quality IHC, the use of two antibody may than better with an antibody.Frozen tissue sample is better than fixing organization.

The technician in described field should be appreciated that, before the antibody using treatment, test patient is favourable to guarantee that pending tumour expresses EGFRvIII.

example 13

anti-EgfrViii antibody characterization in body

The method of example 11 will be used to process lung cancer and neurospongioma.This extensively will detect by making animal model.Formed as follows for becoming the animal model of neurospongioma and lung cancer: will the lung carcinoma cell EGRFvIII transfection of wt-EGFR be expressed.Cell is injected the lung of nu/nu mouse, allow tumor development to can compare the stage above.Anti-EGFRvIII binding substances every 1 to 10 day subsequently (optionally) is by subcutaneous injection.By the size to these cancer cells continued growths, obstruction or constrain monitor, to determine the effect of these anti-EGFRvIII antibody and antibody-toxin conjugate.The technician in described field should be appreciated that, any antibody disclosed herein can so do.

example 14

by replacing to analyze, the function of epi-position is characterized

In order to solve these identification to the indispensable amino-acid residue of EGFRvIII epi-position further, carry out the replacement analysis of the amino-acid residue to epitope peptide.Initiation site is the sequence LEEKKGNYVVTD (SEQ IDNO 59) from example 4.In this example, each amino acid of the epi-position of location is by one by one by 20 L-type aminoacid replacement, and therefore, synthesize all possible unit point analogue, row filter of going forward side by side is to provide the details of peptide binding pattern.Discrete substitute mode is used to the identification of mAb131and13.1.2.Result is summed up in table 14.1.

Table 14.1

mAb	Recognition sequence
		131	EEKKGNYVVT(SEQ ID NO：57)
13.1.2	EEKKGNYVVT(SEQ ID NO：57)

Its display, mAb13.1.2,5 residues are very important to combination (runic), although only have 4 residues to be necessary for the combination of mAb131.Remaining residue is replaced by multiple amino acids and the not significant loss combined.Although in sequence and length 131 and 13.1.2 be identical, its binding pattern is different.The combination of MAb131 has very strong dependency to residue EKNY (SEQID NO:60).On the other hand, data presentation residue EEKGN (SEQ ID NO:61) relates to the combination at mAb13.1.2.

example 15

mAb chain is reorganized

The heavy chain of reorganization MAb131 and 13.1.2 and light chain, and by its transient transfection to 293T cell.After 72 hours, collect supernatant liquor and carry out secreting and EGFrVIII binding tests with ELISA.

Result display antibody sources in the expression of 131 heavy chains having 13.1.2 κ chain, otherwise is also expressed well, but have dropped for 75% (data do not show) due to the binding pattern binding activities that the antibody of two kinds of EGFrVIII antigens is different.Which show the difference of 131 and 13.1.2mAb paratope, again imply that the constitutional features of the epi-position selected at two kinds of mAb is different.

Example 16

131 and the molecular modeling of paratope

This example shows the three-dimensional structure that how can produce embodiment albumen.The 3 d structure model of the variable region of antibody 131 is produced by a homology model method, and described method uses the InsightII modeling packaging of Accelrys (San Diego, CA).According to the sequence construct model of the variable region of following described table 16.1.The numbering of residue from light chain amino acid, and extends to heavy chain amino.

Table 16.1

Variable region of light chain

Variable region of heavy chain

Use the search in albumen database (Protein Data Bank) of the sequence of antibody 131, to identify homologous antibody and structure thereof.Similar with on the basis of the homologous antibody sequence of 131 antibody, have selected some structures.The structure for modelled samples chosen from albumen database has albumen database identifier: 1HEZ, 2H1P, 1AQK, 1DQL, 1MF2 and 1FLR.The sequence alignment of the structure based of overlap ratio to these formwork structures and for generation of these templates subsequently.Subsequently by the sequence of antibody 131 variable region and template sequence comparison.Use the molecular model of comparison result for generation of 131 antibody variable regions of structure and sequence.The light chain of CDR1 sequence is: RSSQSLVHSDGNTYLS (SEQ ID NO 103).The light chain of CDR2 sequence is: RISRRFS (SEQ ID NO 103).The light chain of CDR3 sequence is: MQSTHVPWT (SEQ ID NO 105).The heavy chain of CDR1 sequence is: NYGMH (SEQ ID NO 108).The heavy chain of CDR2 sequence is: VIWYDGSDKYYADSVRG (SEQ ID NO 110).The heavy chain of CDR3 sequence is: DGYDILTGNPRDFDY (SEQ ID NO 112).

The mutual work surface of antibody 131 calculates according to structural models and shows in fig. 11.Different CDR identifies as follows: L1 (light CDR1) 10, H1 (heavy CDR1) 20, L2 30, H2 40, L3 50 and H3 60.It is a dark hole that mutual of doing surface of the antibody 131 predicted significantly refers in particular to.Dark hole primarily of heavy chain CDR2, CDR3 and light chain CDR3 institute around, and its sub-fraction is owing to light chain CDR1.Described hole may be in conjunction with depression.5 dusts in conjunction with in hole be

residue

31,37,95-101,143-147,159,162-166,169-171,211-219,221 and 223.These residues may comprise paratope and in conjunction with the effect playing critical contact during EGFRvIII paratope.These residues also may provide the key structural feature of binding site usually.

example 17

confirm that the site directed mutation of the model of antibody 131 occurs

This examples prove method, can be tested by this method and think that residue is important model in combination.This example also draws some antibody variants.By being introduced into the heavy antibody variants creating 131 clones with the independent residue mutations of light chain of mAb131.These variants of subsequent analysis are to determine how institute's replacement side chain of point mutation affects antigen and combine.

Weigh at mAb131 and change with light chain.On heavy chain, L216 is made to change over R by site directed mutation.On light chain, V99 changes over F.The two kinds of sudden changes that have impact on variant antibodies expression and secretion compare with wild-type sequence.Two kinds of sudden changes all cause the loss of mAb variant in conjunction with EGFRvIII antigen.Because these residues replace to R and F respectively and cause active reduction, so this demonstrate that L216 with V99 may contact significantly with EGFRvIII antigen.Certainly, these replace the ordinary construction destroying antibody, and therefore it is a selection always.

example 18

13.1.2 and the molecular modeling of paratope

13.1.2 the 3 d structure model of antibody variable region is produced by a homology model method, and described method uses the InsightII modeling packaging of Accelrys (San Diego, CA).Use the x ray crystal structure delivered as template, the variable region sequences according to following table 18.1 builds model.

Table 18.1

Variable region of light chain (1-113)

Variable region of heavy chain (114-234)

The light chain of CDR1 sequence is: RSSQSLVHSDGNTYLS (SEQ ID NO:101).The light chain of CDR2 sequence is: KISNRFS (SEQ ID NO:116).The light chain of CDR3 sequence is: MQATQLPRT (SEQ ID NO:118).The heavy chain of CDR1 sequence is: SYGMH (SEQ ID NO:121).The heavy chain of CDR2 sequence is: VIWYDGSNKYYVDSVKG (SEQ ID NO:123).The heavy chain of CDR3 sequence is: DGWQQLAPFDY (SEQ ID NO:125).

The sequence of antibody 13.1.2 is used to search in albumen database, to identify homologous antibody.According to the similarity of its sequence and antibody 13.1.2., have selected there is albumen database identifier 1HEZ, 2H1P, 8FAB and 1AQK structure as modeling template.The sequence alignment of the structure based of overlap ratio to these formwork structures and for generation of these templates.Subsequently by the sequence of 13.1.2 antibody variable region and template sequence comparison.Use the molecular model of comparison result for generation of antibody 13.1.2 variable region of structure and sequence.

The mutual work surface of computation model also shows in fig. 12.13.1.2 the surface in model principal character Shi CDR district has a long and narrow groove.Described groove delineated by heavy chain CDR2 140, CDR3 160 and light chain CDR1 110, CDR2 130 and CDR3 150.The remainder of one section of groove contact light chain CDR3 150, and other one section in the wide region opening heavy chain CDR3 160 near heavy chain-light chain interface.Described groove may be antigen in conjunction with depression.5 dusts in conjunction with in hole be

residue

31,33,35-39,51,54-56,58-61,94-101,144-148,160,163-166,172 and 211-221.These residues may comprise the paratope in conjunction with EGFRvIII epi-position.These residues also may provide the key structural feature of binding site usually.

example 19

peptide docks model with antibody

Epitope mapping research in example 14 shows and needs for being arranged in 6-residue peptide EEKKGN (SEQ ID.NO:127) in conjunction with epi-position to the related amino acid of 13.2.1mAb antibody junction.Therefore, what create this 6-residue peptide complex body and 13.1.2 structure C DR district docks model.First, the model of peptide EEKKGN (SEQ ID NO:127) is created.Except using 1I8I X-ray crystalline structure specifically, to aforementioned similar use this as template, described crystalline structure identifies in albumen database.Then, this peptide structure is manual is positioned in long groove to form initial assembling complex body.Docking module in InsightII is used in conformation and directional space, automatically to carry out Monte Carlo search subsequently.By giving each Phi, Psi and Chi angle complete rotary freedom, to allow the flexible of peptide conformation.In docking operation, 5 dusts are allowed to move while other antibody residue is fixed in conjunction with the residue in groove.Monte Carlo searches for the specious structure of discovery through stimulating annealing and energy minimization to reach resulting complex structural models.For each docking model obtained, use and make energy mutually between the Discover_3 module calculating antibody of InsightII packaging and peptide.That assesses all docking models makes energy mutually, and test has model that the strongest all antibody-peptides do mutually and it shows in Figure 13 A and 13B.

In this docking model, as shown in Figure 13 B, there is the hydrogen bond between 6 peptide EEKKGN (SEQ ID NO:127) and antibody 13.1.2.From N-terminal to C-terminal to peptide residue numbering 1 to 6.Green dotted line indicates 6 hydrogen bonds.The 6 pairs of amino acid forming hydrogen bond are: E2...Y172, K3...H31, K4...H31, N6...D33, N6...Y37 and N6...K55.In this docking model, in extension α-chain structure peptide constraint with in groove.In peptide, residue is alternatively towards solvent and antibody surface.Be E2, K4 and N6 towards the residue in conjunction with groove, describedly significantly contact antibody in conjunction with groove.This indicates these 3 residues may combine consistent with epitope mapping results for peptide is important.What use Discover_3 module to calculate each 6 peptide residues and 13.1.2 antibody junction makes energy mutually, and display in table 19.1.What table 19.1 showed each 6 peptide residues and 13.1.2 antibody junction makes energy mutually.All energy are with the unit representation of kcal/mol.

Having the residue making energy is the most mutually N6, K4 and E2 successively, unanimously with experimental data again confirms that these residues are the significant contribution persons on antigen limit in antibody-antigene is done mutually.These data provide support and dock the strong evidence of model.In the present embodiment, antibody junction is defined as the residue in 5 dust docking peptides.20 residues comprising paratope thus defined be residue 31-33,35,37,55,96-101,148,163,165,170,172,178 and 217-218.On the basis of an independent residue, in order to assess antibody-antigene do mutually in the contribution of these residues each of antibody, calculate and make energy mutually between the paratope of each above-mentioned 20 residues and peptide EEKKGN (SEQ ID NO:127).The results are shown in table 19.2.What table 19.2 showed each 20 paratope residues and peptide EEKKGN (SEQ ID NO:127) makes energy mutually.All energy are with the unit representation of kcal/mol.Having with peptide the residue making energy is the most mutually Lys55 and His31, is then Tyr172, Ala96, Asp33, Tyr37, Leu99, Thr97, Gln98, Lys178 and Asn170.

Table 19.1

Peptide residue	Coulomb	VdW	Amount to
				E1	-2.013	-3.738	-5.751
E2	-10.661	-0.617	-11.278
				K3	-9.816	-0.493	-10.309
K4	-11.123	-0.968	-12.091
				G5	-1.241	-1.468	-2.709
N6	-16.504	-0.181	-16.685

Table 19.2

13.1.2 residue	Coulomb	VdW	Amount to
				His31	-12.835	3.033	-9.801
Ser32	2.857	-1.062	1.794
				Asp33	-4.181	-0.698	-4.879
Asn35	0.253	-1.009	-0.756
				Tyr37	-2.058	-2.463	-4.521
Lys55	-14.363	1.568	-12.794
				Ala96	-6.077	0.896	-5.182
Thr97	-2.739	-1.431	-4.171
				Gln98	-2.542	-1.548	-4.09
Leu99	-1.507	-2.779	-4.286
				Pro100	0.439	-0.379	0.061
Arg101	3.992	-0.549	3.443
				His148	0.101	-0.083	0.018
Val163	-0.104	-0.237	-0.342
				Trp165	1.358	-1.122	0.236
Asn170	-2.102	-0.487	-2.589
				Tyr172	-8.7	0.896	-7.804
Lys178	-3.614	-0.03	-3.644
				Leu217	0.761	-1.426	-0.664
Ala218	-0.071	-0.281	-0.352

example 20

improve the appropriate design of affinity antibodies

This examples prove is occurred by site directed mutation, and how docking model can by the basis making improvements affinity antibodies appropriate design.Computer simulation (in silico) makes each 13.1.2 paratope residue mutations become all 19 other amino acid, and middle extraction amounts to 19 × 20 or 380 virtual mutant.Sudden change has been replaced by residue, thereafter then 50 step energy minimization steps, which solves and can change induced any local conformational change by side chain.Calculate whole peptide of each mutant and make energy mutually all between paratope.The mutant with altogether the react to each other energy strong compared with wild-type 13.1.2 can have the higher affinity with peptide EEKKGN (SEQ ID NO:127) potentially, and, may be even and whole EGFRvIII albumen.Compared with wild-type 13.1.2, these mutant major parts have higher coulomb and do mutually, but wherein some have lower van der Waals (VdW) compared with wild-type antibodies to be done mutually.The VdW of wild-type 13.1.2 antibody is-9.689kcal/mol as energy mutually, eliminates and is less than the mutant that-8.5kcal/mol VdW makes energy mutually.List residue in table 20.1 and there is the mutant of altogether mutually making energy strong compared with wild-type 13.1.2.The bottom of table lists wild-type data for comparing.All energy are with the unit representation of kcal/mol.

Table 20.1

Mutant	Coulomb	VdW	Amount to
				Tyr172Arg	-93.004	-8.702	-101.706
Leu99Glu	-79.897	-8.506	-88.403
				Arg101Glu	-77.984	-8.833	-86.817
Leu217Glu	-75.124	-8.998	-84.123
				Leu99Asn	-73.337	-9.894	-83.231
Leu99His	-73.631	-9.008	-82.639
				Arg101Asp	-71.983	′-9.877	-81.861
Leu217Gin	-70.263	-9.795	-80.058
				Leu99Thr	-69.882	-10.153	-80.035
Gln98G！u	-70.651	-9.257	-79.908
				Leu217Asn	-70.989	-8.769	-79.758
Arg101Gln	-69.432	-10.164	-79.596
				Leu217Asp	-69.934	-9.643	-79.578
Asn35Gly	-69.016	-10.191	-79.207
				Tyr172His	-69.312	-9.509	-78.820
Val163Asn	-68.841	-9.944	-78.784
				Tyr172Asn	-68.896	-9.871	-78.767
Aia218Lys	-70.024	-8.570	-78.594
				Asn35Arg	-68.989	-9.604	-78.593
Trp165Lys	-69.578	-8.766	-78.344
				Trp165Arg	-68.814	-9.216	-78.030
Leu99Tyr	-67.052	-10.464	-77.517
				Tyr172Thr	-68.146	-9.225	-77.371
Aia96Thr	-67.534	-9.623	-77.158
				AIa96Ser	-67.222	-9.822	-77.045
Pro100Trp	-67.399	-9.496	-76.894
				Leu217Ser	-66.676	-10.133	-76.810
Ser32lle	-66.700	-10.077	-76.777
				Tyr172Ser	-67.588	-9.146	-76.734
His31Glu	-67.070	-9.461	-76.531
				Leu217Tyr	-65.605	-10.726	-76.331
Val163His	-67.236	-9.064	-76.300
				His148Ser	-66.780	-9.495	-76.274
His148Val	-66.634	-9.629	-76.263
				His148Ala	-66.770	-9.473	-76.243
His148Gly	-66.762	-9.456	-76.217

His148Thr	-66.700	-9.508	-76.209
				Leu99Ser	-66.126	-10.006	-76.132
Pro100Asp	-66.153	-9.787	-75.940
				Trp165Glu	-66.665	-9.267	-75.932
His148Asn	-66.010	-9.889	-75.899
				Pro100GIn	-65.873	-9.871	-75.745
Leu217Thr	-66.045	-9.672	-75.717
				Ser32Val	-65.845	-9.854	-75.699
Ser32Pro	-65.807	-9.813	-75.620
				Pro100Gly	-65.841	-9.774	-75.615
Pro100AIa	-65.889	-9.712	-75.601
				Ser32Ala	-65.497	-10.089	-75.586
Ser32Thr	-65.723	-9.861	-75.584
				Mutant	Coulomb	VdW	Amount to
Ala218Thr	-66.054	-9.505	-75.560
				Pro100Ser	-65.831	-9.699	-75.530
Val163Gly	-65.993	-9.536	-75.529
				Gln98Thr	-66.162	-9.277	-75.438
Pro100Met	-65.811	-9.602	-75.412
				Ser32Met	-66.252	-9.153	-75.406
Ser32Gly	-65.509	-9.891	-75.399
				Pro100Asn	-65.729	-9.655	-75.384
Tyr37Phe	-66.253	-9.020	-75.272
				Val163Ala	-65.713	-9.543	-75.255
Leu217lle	-65.479	-9.759	-75.238
				Wild-type 13.1.2	-65.517	-9.689	-75.205

The mutant that table 20.1 is listed can be the candidate that through engineering approaches improves affinity antibodies.For 14 candidates at top in list, analyze the contribution of antigen limit and the every residue in antibody limit further, to detect proposed impact of modifying.10 mutant in vitro site directed mutation generation are Tyr172Arg, Arg101Glu, Leu99Asn, Leu99His, Arg101Asp, Leu217Gln, Leu99Thr, Leu217Asn, Arg101Gln and Asn35Gly.Result is found in example 21.

example 21

confirm that the site directed mutation of the model of 13.1.2 occurs

This examples prove method, can be tested by this method and think that residue is important above-mentioned model in combination.This example also draws some antibody variants.Create 13.1.2 antibody variants by independent residue mutations, independent residue mutations is weight and the light chain of introducing 13.1.2mAb.Analyze variant to determine the contribution of many side chains in antigen combination.The row sudden change occurring to introduce by site directed mutation is summarised in table 21.1.

Table 21.1

	Chain	Sudden change
			1	Light chain (CDR3)	Arg101Asp
2	Light chain (CDR3)	Arg101Gln

3	Light chain (CDR3)	Arg101Glu
			4	Light chain (CDR1)	Asn35Gly
5	Heavy chain (CDR3)	Leu217Asn
			6	Heavy chain (CDR3)	Leu217Gln
7	Light chain (CDR3)	Leu99Asn
			8	Light chain (CDR3)	Leu99His
9	Light chain (CDR3)	leu99Thr
			10	Heavy chain (CDR2)	Tyr172Arg

Each in 10 sudden changes in table 21.1 is introduced into weight or the light chain of 13.1.2mAb.Each through sudden change chain subsequently with the complementary wild-type chain transfection in 293 cells.Test the expression and secretion of the human IgG antibodies of supernatant liquor subsequently, and in conjunction with EGFrVIII antigen.The result determined by ELISA is summarised in table 21.2.

Table 21.2

	Sudden change	In conjunction with energy	Express	In conjunction with
					1	Arg101Asp	-81.861	Be	No
2	Arg101Gln	-79.596	Be	No
					3	Arg101Glu	-86.817	Be	No
4	Asn35Gly	-79.207	Be	Be
					5	Leu217Asn	-79.758	Be	Be
6	Leu217Gln	-80.058	Be	Be
					7	Leu99Asn	-83.231	Be	Be
8	Leu99His	-82.639	Be	Be
					9	Leu99Thr	-80.035	Be	Be
10	Tyr172Arq	-101.706	Be	Be
					11	WT	-75.205	Be	Be

example 22

the preparation of EGFRVIII/PFLAG variant construct

The effect of this examples prove manufactures the variant of EGFRvIII.Primer pair 9712 and 9713 (Qiagen, Valencia, CA) produces the extracellular domain of the fragment EGFRvIII of one section of 1092bp coding.

Primer#9712:5 '-ataaaagcttctggaggaaaagaaaggtaatta-3 ' (justice) (SEQ ID NO:128); Primer#9713:5 '-TTATTGGTACCTCAGGCGATGGACGGGATCTTA-3 ' (antisense) (SEQ IDNO 129) Pfu archaeal dna polymerase (Stratagene, La Jolle, CA) increase from plasmid template EGFRvIII-rbIgG/pCEP4 (as above-mentioned).Primer #9712 introduces HindIII site and primer #9713 introduces KpnI site.PCR primer is through column purification (Qiagen column purification kit, Valencia, CA), HindIII and KpnI (NEB, NewEngland Biolabs, Beverly, Mass.) digestion also glue purification (Qiagen gel purification kit, Valencia, CA).T4DNA ligase enzyme (NEB, New England Biolabs, Beverly, Mass.) junction fragment is to HindIII and KpnI (NEB, New England Biolabs, Beverly, Mass.) linearizing pFLAG-CMV-1 (Sigma, St.Louis, MO).The carrier of drawing is named as EGFRvIII/pFLAG-CMV-1#1.

example 23

the preparation of EgfrViii/PFLAG recombinant protein

How this examples prove can obtain variant EGFRvIII.First, 500 μ g EGFRvIII/pFLAG-CMV-1#1 plasmid DNA settling flux are in 25ml Opti-MEMI (Invitrogen, Burlington, ON), and with the 500 μ l293fectin (Invitrogens of settling flux in 25mlOpti-MEMI, Burlington, ON) combine.Mixture is incubation 20min at room temperature, and subsequently with prepare 293Tcells (1 × 10 in 1L293FreeStyle substratum (Invitrogen, Burlington, ON) ⁹) mixing, described culture medium supplemented has 2%FBS and 50 μ g/ml G418 (Invitrogen, Burlington, ON).Cell is at 37 DEG C, 8%CO ₂and grow 7 days under 125rpm rotating speed.

According to the experimental program of manufacturer, use Anti-FLAG M2Affinity Chromatography test kit (Sigma, St.Louis, MO) purified fusion protein.

Prepare monomeric fusion protein as follows.First, purifying protein (1508 μ g) was with the DTT55 of final concentration 10mM DEG C of reduction 30 minutes.Then, add IAA (iodoacetic acid) (Sigma, St.Louis, MO) to 22mM, and room temperature dark place incubation 15 minutes, then anti-PBS dialysis in the 7k MWCO dialysis cassette (Pierce, Rockford, Ill) of 4 DEG C.

example 24-30

the binding of antibody variants

Following instance comprises Biacore experiment (surface plasma resonance) and KinExA experiment.How these examples proves test the much antibody and variant thereof that are obtained by above-mentioned example, to determine them needed for whether having in conjunction with feature.All variants through test are all the variants of 13.1.2 background.

laboratory apparatus

All surface plasma resonance experiments are all use Biacore2000 optical biosensor (Biacore, Inc., Piscataway, NJ) to carry out.It is all use KinExA3000 instrument (Sapidyne Instruments, Inc., Boise, ID) to carry out that all power repels mensuration (Kinetic Exclusion Assay). reagent

Synthesize and buy Pep-3 from Anatech, Inc. (San Jose, CA) customization, NH ₂-LEEKKGNYWTDHG-OH (MW=1590Da) (SEQ ID NO:130).The mAbs that indoor preparation is all.Indoor preparation MW39, the antigen EGFRvIIIpflag (reaction iodoacetic acid is to stop the polymerization of free hydroxyl sulfenyl (sulfhdryl) group) of 907.Bovine serum albumin (BSA) part V (#BP1605-100) is buied from FisherScientific (Pittsburgh, PA).Other all general reagent is all purchased from Sigma-Aldrich, Inc (St.Louis, MO).

All antigen for Biacore and KinExA analysis and mAb sample are at (the 0.01M HEPES containing 100 μ g/mL BSA, 0.15M NaCl, 0.005% tensio-active agent P-20, Biacore Inc., Uppsala, Sweden) prepare in vacuum stripping HBS-P damping fluid.The agent of Biacore coupling amine, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and thanomin are purchased from Biacore, Inc..For pep-3/mAb131 experiment, the regeneration of Biacore tensio-active agent has 12 pulse per second (PPS)s of 26mM NaOH.Other mAb all at 20 minutes internal disintegrations to baseline.Research grade CM5 biology sensor chip is purchased from Biacore, Inc.

KinExA detects goat-anti IgG () that antibody is Fc γ special Cy5-mark and dilutes 1000 times from 0.5mg/mL liquid storage (1 × PBS, pH7.4) with HEPES damping fluid (0.01M HEPES, 0.15M NaCl, pH7.2).Solid phase particles for KinExA experiment is the Sepharose4Fast Flow pearl (PharmaciaBiotech AB, Uppsala, Sweden, #17-0906-01) that NHS activates.Before sepharose 4B and antigen-reactive, in micro-centrifuge tube, the pearl liquid storage aliquot of 1.5ml is through centrifugal and with cold deionization H ₂o water at least washes 6 times.Once after rinsing pearl with sodium carbonate buffer (0.05M, pH9.3), the antigen (~ 40 μ g) in sodium carbonate buffer just adds on sepharose 4B.Agarose/antigen pipe 4 DEG C spends the night and rocks.After rocking, shake agarose with 1M Tris damping fluid, pH8.3 rinses twice.Antigen coated pearl is shaken at room temperature 1 hour in the 1M Tris damping fluid containing 2%BSA subsequently.

biacore measures

Use standard EDC/NHS and carbohydrate are coupled fixes mAb to CM5 sensor chip with covalency.In order to minimize a large amount of transmission and crowded, be fixed on by mAb on certain level, the maximum antigen that now gas is no more than 50-100RU combines response (Rmax).Reference wandering cells on each chip, with fixedly activating or stoping without mAb, makes it when comparing.

All Biacore dynamic experiments are carried out at 23 DEG C.For each experiment, prepare one group of continuous print, 6 to 8 antigen concentrations (from 1.01 μMs of pep-3) with 2 times of diluents.Three repeat on the surface of room temperature sensor with the antigen samples that 100 μ L/min inject arbitrarily.Each pep-3 concentration and blank inject 90 seconds.Then dissociate 13 to 180 minutes.To dissociate data by replacing three extra 251nM pep-3 with three extra blank injection and inject and then obtain period of dissociating of 3-4 hour the pep-3 being bonded to mAb131.

Scrubber software (Version l.lf, BioLogic Software, Australia) is used to process all Biacore sensing spectrums.First zeroing sensing spectrum in y-axis, and the x arrangement when injecting beginning subsequently.The change of a large amount of refractive index is removed by the response deducted with reference to wandering cells.The average response of all blank injection is deducted to remove the system artifact between experiment and reference wandering cells from all assays and blank induction spectrum.CLAMP biological sensor data analysis software (Version3.40, BioLogic Software, Australia) is used to determine the k of treated data set _aand k _d.The data of all wandering cellss are all applicable to 1: 1 bimolecular combination model of the condition comprising a large amount of transmission.For some mAb, be excluded outside non-linear dynamic is applicable to corresponding to the injection of the first or second concentration of the continuous concentration of pep-3, in applicable non-linear dynamic, by 1: 1, induction spectrum does not do that model well describes mutually is obvious.From business's calculating K of kd/ka _d.

kinExA balancing a survey

At room temperature (~ 23 DEG C) carry out all KinExA experiments.For all balance tests, antigen is had the solution of constant mAb binding site concentration by being diluted to continuously.For the one 10 titration point, diluent be 2 times and the 11st and the 12nd serial dilution is 10 times.The sample flow speed of all experiments is 0.25mL/min, and traget antibody flow rate is 0.5mL/min.Antigen/antibody sample allows to reach balance subsequently, this process need cost ~ 48-72hr.For pep-3/mAb131KinExA experiment, K _din-control titration, the initial concentration of pep-3 is 352nM, and constant [mAb binding site]=219pM; Titration is controlled for mAb-, initial [pep-3]=251nM, and [mAb binding site]=11nM.At the K of pep-3/mAb131 _dduring-Control release, in wandering cells, each sample proposes 1.25mL.The sample volume of 250 μ L is analyzed in antibody Control release.For all balance tests, measure two or three repetitions of each sample.Use KinExA software (Version2.4, Sapidyne Instruments), equilibrium titration data are applicable to the hyperbolic line analysis of 1: 1 combination model.

Only at K _dthe KinExA of EGFRvIIIpflag/mAb131 complex body is have studied under-control condition.Initial [EGFRvIIIpflag] is 198nM, and [mAb binding site] is 150pM.The sample of 1mL volume is proposed in wandering cells.Collect the repeated test data of all samples.Use KinExA software (Version2.4, SapidyneInstruments), equilibrium titration data are applicable to the hyperbolic line analysis of 1: 1 combination model.Referring to result and the prediction equilibrium constant of following instance 28.

For the KinExA titration of EGFRvIIIpflag/mAb13.1.2 complex body, the initial concentration of EGFRvIII is 5.26 μMs (mAb-controls), 230.1nM (K _d-control), and [mAb binding site]=9.59nM (mAb-control), 498pM (K _d-control).At K _dduring-Control release, in wandering cells, each sample proposes 1.30mL.The sample volume of 250 μ L is analyzed in antibody Control release.For all balance tests, measure two or three repetitions of each sample.Use KinExA software (Version2.4, Sapidyne Instruments), equilibrium titration data are applicable to the hyperbolic line analysis of 1: 1 combination model.

example 24

in vitro determining of antibodies constant

By using the binding kinetics feature of Surface Plasmon Resonance (SPR) the Instrument observation wild-type mAb131 of Biacore.Owing to extremely low k _dwith quick k _a, therefore K _dbe extremely low, be only 380pM.Other Chemical kinetic parameter estimation k be separated to from curve device _a=2.246*10 ⁶and kd=8.502*10 ^-4.

In one embodiment, illustrate improvement or have and improve dynamic (dynamical) variant antibodies.By the kinetics improved, the antibody dynamical element meant in conjunction with epi-position is better than the same element of the antibody of the same epi-position of previous known use.For example, have and be greater than (binding ability) 1.3*10 ^-9m K _dantibody in conjunction with pep-3 can be improvement antibody.Contain the K so having and be less than 500nM, 500-300nM, 300-100nM, 100-lnM, 1.3nM, 1.3nM to 1000pM, 1000pM to 900pM, 900-500pM, 500-400pM, 400-300pM, 300-100pM, 100-50pM, 50-1pM _dor less K _dantibody.

example 25

in vitro determining of antibodies constant

Similar to example 24, test mAb13.1.2 to Pep-3 (EGFRvIII epi-position) in conjunction with power.Estimate K _dbe 67nM, but experiment between have less deviation.Other Chemical kinetic parameter estimation k be separated to from curve device _a=2.835*10 ⁵and kd=0.01922.

example 26

in vitro determining of antibodies constant

Similar to example 24, test mAb095 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K estimated _d66nM.Other Chemical kinetic parameter estimation k be separated to from curve device _a=1.491*10 ⁵and kd=9.927*10 ^-3.

example 27

in vitro determining of antibodies constant

Similar to example 24, test the power of mAb139 in conjunction with Pep-3 (EGFRvIII epi-position).The K estimated _d290nM.Other Chemical kinetic parameter estimation k be separated to from curve device _a=10328 and kd=2.981 × 10 ^-3.

example 28

in vitro determining of antibodies constant

In order to analyze more completely antibody in conjunction with feature, carry out KinExA experiment with determine mAb131 in conjunction with feature.According to the K that hyperbolic line analysis is determined _d1.74*10 ^-10.In a KinExA experiment, the K of EGFRvIIIpflag to mAb131 _d6.266*10 ^-11.

example 29

in vitro determining of variant antibodies binding constant

In order to analyze more completely 13.1.2 antibody in conjunction with feature, carry out KinExA experiment with determine mAb13.1.2 in conjunction with feature.According to the K that hyperbolic line analysis is determined _d7.538*10 ^-10.In addition, the antigen in this example is EGFRvIIIpflag, and itself and iodoacetic acid (IAA) react.

example 30

bIACORE result compares with KINEXA result

The result of previous case and KinExA test is illustrated in following table 30.1.Numeral in table 30.1 bracket is the fiducial interval of 95%." ND " does not determine, and " * " representative is in conjunction with EGFRvIIIpflag (iodoacetic acid of reaction), instead of pep-3.

As shown in rate constant, mAb131 shows have maximum association constant and have minimum dissociation constant, therefore makes mAb131 have minimum K _d.

Table 30.1

example 31

in vitro determining of the binding constant of L99t-5.3 variant antibodies

Test mAb L99T-5.3 to Pep-3 (EGFRvIII epi-position) in conjunction with power.First step is that use standard EDC/NHS is coupled chemistry, by 5,600 resonating member (RU) are fixed on 8 of the mAb L99T-5.3 of CM5 sensor chip two wandering cellss (Fc), on 000 RU, and by 5,600 resonating member (RU) are fixed on 8,000 RU of the mAb13.1.2 of a Fc.This surface density produces the binding signal with pep-3 being less than 100 RU.Altogether employ two CM5 sensor chips to fix two mAb.Use the data of previously collecting, create 5 independent experiments altogether that two antibody allow 95% fiducial interval to be calculated.All research all uses Biacore2000 optical bio sensor.

Then, pep-3 flows through the fixing biological sensor surface of mAb.Pep-3 initial concentration is 1.25 μMs, and this to be connected in any three duplicate injections after 8 twice serial dilutions.For reaching the object of two reference, running through every 6th sample in injection continuously and carrying out blank injection.

Finally, by Scrubber process biological sensor data, and data are applicable to adopting having and comprise the curve that 1: 1 of a large amount of transmission condition make the Clamp of model mutually.The high density injection of 1.25 μMs excludes power and is applicable to, because data significantly do not meet 1: 1 make model mutually.This departs from is most possibly that non-specific mutual work by occurring in high density pep-3 causes.All dynamic dates are applicable to 1: 1 satisfactorily and make model mutually.

Estimated K _dfor 54-70nM.Other kinetic parameter k estimated _a=2.238*10 ⁵and k _d=0.01217, this interstitial fluid in each operation is by Light Difference.

example 32-38

Example 32-38 tests by using Biacore device further, variant mAb in conjunction with power.First step in these examples is that use standard EDC/NHS is coupled chemistry, by 5,600 resonating member (RU) are fixed on 8,000 RU of each mAb L99T-5.3 after tested of a wandering cells (Fc) of CM5 sensor chip.This surface density produces the binding signal with pep-3 being less than 100 RU.3 CM5 sensor chips are altogether used to fix all mutant mAb with the same mAb that is fixed on each wandering cells.MAb13.1.2 is included on a wandering cells of in 3 sensor chips two.All research all uses Biacore2000 optical bio sensor.

Then, pep-3 runs through the fixing biological sensor surface of mAb.Pep-3 initial concentration is 4.98 μMs, and this is connected on any two and repeats or in three duplicate injections after 8 to 11 twice serial dilutions.For reaching the object of two reference, running through every 6th sample in injection continuously and carrying out blank injection.

Finally, by Scrubber process biological sensor data, and data are applicable to adopting having and comprise the Clamp that 1: 1 of a large amount of transmission condition make model mutually.Depend on mAb and avidity thereof, when data significantly do not meet 1: 1 make model mutually time, some high densitys injection (4.98-1.25 μM) exclude power be applicable to.This departs from is most possibly that non-specific mutual work by occurring in high density pep-3 causes.All dynamic dates are applicable to 1: 1 and make model mutually.

example 32

in vitro determining of the binding constant of L217q-10.1 variant antibodies

Test mAb L217Q-10.1 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K estimated _d92nM.Other Chemical kinetic parameter estimation k be separated to from curve device _a=2.04*10 ⁵and kd=0.01885.

example 33

in vitro determining of the binding constant of L217n-2.1 variant antibodies

Similar in appearance to example 32, test mAb L217N-2.1 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K estimated _d185nM.Other Chemical kinetic parameter estimation k be separated to from curve device _a=2.198*10 ⁵and kd=0.04069.

example 34

in vitro determining of the binding constant of N35g-3.1 variant antibodies

Similar in appearance to example 32, test mAb N35G-3.1 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K estimated _d204nM.Other Chemical kinetic parameter estimation k be separated to from curve device _a=1.497*10 ⁵and kd=0.03057.

example 35

in vitro determining of variant antibodies binding constant

Similar to example 32, test mAb L99H-9.2 to Pep-3 (EGFRvIII epi-position) in conjunction with power.Estimate K _dfor 395nM.Other Chemical kinetic parameter estimation k be separated to from curve device _a=83390 and kd=0.03293.

example 36

in vitro determining of variant antibodies binding constant

Similar to example 32, test mAb Y172R-1.2 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K estimated _d927nM.Other Chemical kinetic parameter estimation k be separated to from curve device _a=82237 and kd=0.07622.

example 37

in vitro determining of variant antibodies binding constant

Similar to example 32, test mAb L99N-4.1 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K estimated _dit is 1.4 μMs.In order to determine K _d, be difficult to because power is exceedingly fast be applicable to, therefore MAb L99N-4.1 is applicable to using steady state (balance) combination model.

example 38

13.1.2 be designed comparing of variant

As being found in table 38.1, develop the mAb having and improve in conjunction with feature.The fiducial interval of 95% is shown in bracket.L99T-5.3 shows the k increased _a, the k of minimizing _d, and therefore all lower K _d.If although Pep-3 shows statistically little significant difference in conjunction with between the equilibrium dissociation constant of mAb13.1.2 and L99T-5.3 (fiducial interval 95%) and Kinetic rate constant, seem still to exist Pep-3, in conjunction with the marginal increment of L99T-5.3 avidity, there is intuition deviation.In addition, when using identical biological sensor chip, L99T-5.3 seems always have the avidity high compared with 13.1.2.

Table 38.1

MAb	k _a(M ^-1s ^-1)	k _d(s ^-1)	K _D(nM)
				13.1.2	2.10(0.58)x10 ⁵	0.016(0.003)	75(14)
L99T-5.3	2.16(0.12)x10 ⁵	0.013(0.001)	60(10)
				L217Q-10.1	2.04x10 ⁵	0.019	92
L217N-2.1	2.20x10 ⁵	0.040	185
				N35G-3.1	1.50x10 ⁵	0.030	204
L99H-9.2	8.34x10 ⁴	0.033	395
				Y172R-1.2	8.22x10 ⁴	0.076	927
L99N-4.1	ND	ND	1,400*

Extra docking model and preference pattern level predict the method for binding affinity

In other embodiments, above-mentioned example can carry out together with different lengths peptide and not only time six amino acid length peptide, as long as peptide comprises critical binding residues.For example, replace the six amino acid peptide of EEKKGN (SEQ ID NO:127), 7 amino acid peptides of EEKKGNY (SEQ ID NO:131) can be used.The epi-position of any size peptide can be used.In other embodiments, peptide is selected from following peptide: LEEKKGNYVVTDHC (SEQ ID NO:56), LEEKKGNYVVTD (SEQ ID NO:59), LEEKKGNYVVT (SEQ ID NO:132) and EEKKGNYVVT (SEQ ID NO:57).Peptide or its variant of any size between the section fragment disclosed to full-length peptide can be used here.

As described in those skilled in the art should be appreciated that to there is the mode that additional amino acid can change peptide binding antibody.Not only the existence of additional amino acid allows to be formed between peptide and antibody and replaces and extra bonding, and extra amino acid can change the structure of peptide and the antibody structure of antibodies peptide.Therefore, in one embodiment, different lengths epitope peptide can be test, the binding characteristic of such as EEKKGN (SEQ ID NO:127) and EEKKGNY (SEQ ID NO:131) with in conjunction with optimizing.Not only peptide accurately describe to do mutually the peptide-antibody of peptide compared with long segment compared with long segment, and bonding strength and be included in the residue in combination change test allow about longer peptide, the extraneous information of being derived by data.

In addition, and may be the longer peptide fragment complementary with test, extra filtration step can be carried out to select to dock model accurately.Extra filtration step can allow to filter to find the model with Available experimental data consistent in many docking models.

In one embodiment, filtration is the epi-position cartographic data based on good segregation, and the independent residue of such as test feature is in conjunction with overview, and described cartographic data is in relation to each amino acid whose calculations incorporated energy overview in peptide.For example, 7 amino acid peptides may be used for selecting containing the similar docking model in conjunction with energy overview in conjunction with energy overview.In conjunction with the assignment in conjunction with energy that energy overview is each amino acid and specific amino acids in peptide, amino acid whose combination creates the overview of peptide, and it is in conjunction with energy according to each amino acid in described model.For example, a given peptide comprising amino acid A and B in a docking model, wherein A have-5 in conjunction with energy and B have-20 in conjunction with energy, the overview of A1 (when-5) and B2 (when-20) can be had.This overview can with elect other docking model filtration.For example, use this can cause the selection of other docking model as filtration or " template " in conjunction with energy overview, if the peptide in candidate family has the relatively low value contributing to position A, and there is the relatively high value (larger negative value, higher absolute value) contributing to position B.In an other embodiment, template needs extra restriction, and the value of such as position B is higher than the value of position A 4 times.

Can compare in conjunction with energy overview template with other overview of docking peptide in model in many ways.If similar in conjunction with energy overview to required in conjunction with energy overview template, so subsequent filtration can as selecting favourable docking model for further test.If in conjunction with energy overview template and required dissimilar in conjunction with energy overview, so subsequent filtration can be used as to eliminate unfavorable docking model.In one embodiment, filtration procedure comprises and has the favourable and unfavorable template in conjunction with energy, and filters and be used for selecting and get rid of docking model.As described in those skilled in the art with understand, there is many possible differences in conjunction with energy overview, and therefore depend on different situations, many differences can be used in conjunction with energy overview.In one embodiment, can define in conjunction with energy overview is that specific position has a series of relatively high template in conjunction with energy in peptide.In a preferred embodiment, relative high in conjunction with energy in conjunction with position 2,4 or 6 place at peptide EEKKGNY (SEQ ID NO:131) had in conjunction with energy overview selected by energy height empty template and template.In another embodiment, to have at position 2,4 or 6 place of peptide EEKKGNY (SEQ ID NO:131) in conjunction with energy overview template relatively high in conjunction with energy.In another embodiment, to have at position 3 place of peptide EEKKGNY (SEQ ID NO:131) in conjunction with energy overview template relative to energy.In above discussion, described position is assigned as follows: E1, E2, K3, K4, G5, N6, Y7.

In one embodiment, first filtration treatment is included in the contrast of K3 and K4 place in conjunction with energy.Select to cause K4 in contrast to the relatively high docking model in conjunction with energy of K3, screen out simultaneously and cause K4 in contrast to the relatively low docking model in conjunction with energy of K3.Therefore, what " relatively high " meant K4 is greater than (negative value larger, larger absolute value) K3 in conjunction with energy.Then, in conjunction with energy overview template, filter docking model again, this time, select, at position 2,4 or 6 place, there is relatively high-octane combination model, other model can be removed simultaneously.Therefore, what " relatively high " meant at position 2,4 or 6 place in peptide is minimum in conjunction with energy high (larger negative value, larger absolute value) in conjunction with energy Ratios.Therefore, in this embodiment, can sum up in conjunction with energy feature template: E1 as follows can be any value, E2 should be larger than Schwellenwert, and K3 should be less than K4, and K4 should be greater than Schwellenwert, G5 can be any value, and N6 should be greater than Schwellenwert, and Y7 can be any value.Therefore, as long as at least one (or K3) is less than at least in E2, K4 and N6 one, E1, G5 and Y7 can be any value.In another embodiment, " relatively high " can be set as the standard value that modeling or experiment are determined.In one embodiment, docking model filters better important by the first specific filtration resistance docking model by second.As one of ordinary skill in the art should be appreciated that, do not need one after the other to carry out this two steps, and these two steps can be carried out simultaneously.

In addition, depend on peptide, antibody and conjugation condition, these overview templates of filter result can be different.Give this disclosure, one of ordinary skill in the art are especially referring to after example 14, and it is suitable for energy overview template to determine.For example, as shown in table 14.1, in conjunction with 131 and 13.1.2 antibody, there is the important residue that some are possible in peptide.In 131mAb, position E2, K4, N6 and Y7 are important for specific peptide after tested.In 13.1.2mAb, position E1, E2, K4, G5 and N6 are important for specific peptide after tested.These important residues can be contained in create in conjunction with the residue in energy overview template.As can be clearly known from following discussion, go out to analyze think different from example 14 in conjunction with energy overview template for displaying in example 39.

Example 39 is versions that template preciseness is lower, and it allows more model by screening step.If to the trees being reduced by the model screening step, the needs about E1 and G5 so can be added further.

Following instance proves to use longer peptide how can change the result of above proof and what these changes can mean, and proves to use above-mentioned for selecting the filtration of specific docking model.

example 39

the epitope-antibody docking model of 7 amino acid peptides

This examples prove produces one group of docking model, and described model is for the structural models of 7 residue peptide in the CDR district complexity of 13.1.2.In addition, this examples prove selects the method for a docking model on another docking model.

First, 7 residue peptide EEKKGNY (SEQ ID NO:131) structural models builds up in extended configuration, and the Discover_3 module minimization of energy in packing by InsightII modeling.Then, this peptide structure is manual is positioned in connection site to form initial assembling.Docking module in InsightII is used in translation and revolution space, automatically to carry out Monte Carlo search subsequently.In docking operation, 5 dusts are allowed to move while other antibody residue is fixed in conjunction with the residue in groove.Peptide is limited in 5 dust zero positions.Monte Carlo searches for the specious structure of discovery then through stimulating annealing and energy minimization to reach resulting complex structural models.Obtain 63 docking models altogether.

For each docking model, in peptide, antibody and the energy Discover_3 that does mutually separately between residue calculate.Independent residue contribution overview during epitope-antibody combines is examined to select the docking model consistent with epi-position cartographic data of well emanating, and meaning i.e. " in conjunction with energy overview template ".19 in 63 docking models have passed this and check.A typical residue is separately shown in conjunction with energy overview in table 39.1.Consistent with epi-position cartographic data in example 14, K4's is significant in conjunction with energy, and N6 and E2 is large.This is greater than the practical work of K3 in conjunction with energy overview template lay special stress on K4.This needs E2, K4 relative with N6 large in conjunction with energy overview also lay special stress on.In other words, E2, K4 and N6's is not (minimum negative value or least absolute value) in conjunction with minimum energy in peptide in conjunction with energy.

table 39.1

in 7 residue peptide, residue is consistent with the epi-position cartographic data in conjunction with energy overview and example 14 of antibody 13.1.2 separately.

For based in conjunction with 19 models by filtering in energy overview, there is 7 mutant (Tyr172Arg, the example 36 of affinity data; Leu217Asn, example 33; Leu217Gln, example 32; Asn35Gly, example 34; Leu99Asn, example 37; Leu99His, example 35 and Leu99Thr, example 31) each on carry out epitope-antibody and emulate in conjunction with thermodynamics.Because the sympathetic degree of the electrostatic in this complex body is close, allly in a series of calculating, use many different dielectric constant.Sudden change has been replaced by residue, and then 30-100 walks energy minimization step thereafter, which solves and can change induced any local conformational change by side chain.For each docking model, to parameter selected by each mutant, calculate and make energy mutually between 7 residue peptide and whole antibody.For often organizing 8 in conjunction with energy (7 mutant add wild-type), contrasting with Kd algorithm and carrying out every group and be applicable to process in conjunction with the linear of data.Calculate each linear relation conefficient be applicable to.Obtain the best correlation of a model, described model has the data described in table 39.1, and described best correlation has the energy minimization of dielectric constant 1*r and 50 steps.The epitope-antibody of this model is shown in conjunction with energy in table 39.2.The relation conefficient of all data is 0.80.Referring to above example 37, because do not measure the high precision K of Leu99Asn _d, so carried out being linearly applicable to separately except Leu99Asn data.As shown in figure 14, a fabulous relation conefficient 0.91 is obtained.Therefore above selected model represents well and docks model accurately.Figure 15 shows has the model that peptide is filled in space, and Figure 16 shows hydrogen bond.In Figure 16, L3 150 is comparatively lower part and H3 160 is higher part divides.H2 140 is on the right in territory, binding domain polypeptide.Peptide self is positioned at binding site, and in binding site, E1 is positioned at the page top of bright shading, is K3, K4, G5, N6 and Y7 dimmed successively under it.Figure 16 shows the antibody residue being contained in hydrogen bonding.Model proof existence 7 hydrogen bonds that this example is obtained.K4...Q95, K4...Q95, N6...Q98, G5...H31, Y7...H31 and Y7...W165.

table 39.2.

contrast with KD logarithm, emulation epitope-antibody is in conjunction with thermodynamics

Mutant	Coulomb	vdw	Amount to	Ln(Kd)
					172Arg	-19.103	-27.962	-47.065	-13.891
217Asn	-19.003	-28.715	-47.718	-15.503
					217Gln	-18.977	-28.73	-47.707	-16.201
35Gly	-19.095	-28.431	-47.526	-15.405
					99Asn	-18.719	-28.778	-47.497	(-13.479)
99His	-18.837	-28.719	-47.556	-14.744
					-99Thr	-19.155	-28.704	-47.859	-16.475
WT	-18.981	-28.728	-47.708	-16.269

As being found in selected model in example 39, representing in Figure 15, docking some unpredicted result of models show.Although interesting result is residue E2, K4 and N6 is important being combined as in overall peptide, these not all amino acid profiles are turned to be contained in and form H-key with antibody.Display K4 is contained in and forms two H-keys with Q95, and this and K4 are consistent in conjunction with the importance in energy overview and overview template.Also N6 simulation is shown with bonding Q98; But in this special model, E2 also walks display formation H-key in a model.A consistent interesting trend is that each Key residues (such as E2, K4 and N6) major part derived from conjunction with energy overview template is buried, and therefore with antibodies groove close contact.Therefore, docking Model Selection can illustrate that these Key residues are important practical works, because itself and the tight of antibody are done mutually.In addition, E1 may form hydrogen bond with W214.

Example 39 also proves that aforesaid method causes in conjunction with energy and K _dbetween height be correlated with, this model thinking that present method creates also allows the K of antibody-peptide complex body _doptimizing or at least predict.

As being found in the contrast of example 39 and example 19, there are some is residues important between two models, and some residues only show in 7 amino acid docking models, and the step display in 7 amino acid docking models of some residues is important.For example, 7 peptide epitopes are presented between K4...Q95, K4...Q95, N6...Q98, G5...H31, Y7...H31 and Y7...W165 and create H key.On the other hand, 6 peptide epitopes are presented between E2...Y172, K3...H31, K4...H31, N6...D33, N6...Y37 and N6...K55 and create H key.As found out from above-mentioned data, the importance of H31 all emphasized by 6 and 7 amino acid peptide models, because two models all comprise the H31 forming two hydrogen bonds with peptide.Although there is other possibility trend between two data sets, it also shows many combinations and does mutually to change over 7 amino acid profiles from six amino acid model.But these examples proves can go out change owing to epi-position size with these model inspection, and therefore from being comparatively as short as the convergent-divergent of longer epitope peptide and step should be problem according to this disclosure.There is amino acid and prove that its importance in many combination models allows therefore to depart from the importance of many mutual works continuously, can be therefore the more Typical Representative that longer peptide is done mutually compared with small peptide model.

As art, technician should be appreciated that, also 7 amino acid peptide EEKKGNY (SEQ ID NO:131) can be applied to about the above discussion of six amino acid peptide EEKKGN (SEQ ID NO:127) or example, or any longer peptide.For example, example 20 can repeat the information of example 39, and it is the antibody improved by site directed mutation generation appropriate design avidity.In addition, the result of use-case 39 can repeat example 21, is then to occur to attempt appropriate design avidity by site directed mutation to improve antibody with any new antibody of test from example 20 separation.

In one embodiment, the result of use-case 39 does region to redefine between antibody and peptide mutually.For example, the paratope of EEKKGNY (SEQ ID NO:131) can be defined as other residue comprising and antibody is predicted as and makes (such as, residue 95) with peptide mutually.Or as example 19, paratope can be defined as all residues in 5 dust docking peptides.

the computer simulation affinity maturation of different albumen

Successfully affinity matured antibody is obtained in vitro in much difference research.Usually, any mutated library needs to be built by molecular biology method, and needs exploitation selection/screening assay with the abundant clone with good combination ability.Selected variant needs purified to determine avidity subsequently.This process need carries out a series of length and arduous experiment.Following instance demonstrates the selection possible accuracy prediction affinity maturation by utilizing separately Antibody-antigen complexes structure.

example 40

by the computer simulation affinity maturation that antibody-antigene emulates in conjunction with thermodynamics

This examples prove can use computer simulation antibody-antigene in conjunction with thermodynamics emulation for affinity maturation.This example proves can being predicted by above computer simulation process in conjunction with power of Fab-12 (IgG form is known as rhuMAb VEGF) and VEGF (tieing up endothelial tube somatomedin) especially.

The VEGF-Fab complex body crystalline structure used in PDB database, and has accession number 1BJ1, and it is resolved at 2.4 dusts.What use a series of mutant of anti-vegf Fab delivers experiment affinity data for testing viewpoint.The 3-D of the VEGF-Fab structure thing that matches suddenlys change for the computer simulation of following mutant: H97Y, S100aT, T28D, 28D31H, 28D31H97Y100aT, N31H, Y53W, 71I73K, 71V73V.Described affinity data obtains from the people such as Chen, Y (J Mol Biol., 293 (4): 865-81 (1999)).Between many VEGF-Fab mutant, thermodynamics emulation is carried out described by example 39.The results are shown in table 40.1.The result of this example proves to obtain the significant correlation between arranging in conjunction with energy and avidity by this process.Figure 17 display is in conjunction with the logarithm be applicable in detail relative affinity of energy.The relation conefficient of-0.91 is illustrated computer mould emulation and is accurately caught work in detail mutually at atomic level.

table 40.1

emulate in conjunction with energy with the antibody-antigene that affinity data contrasts

As clearly understood from above example, can infer that emulation qualification does not use the higher affinity of ex vivo experiment to suddenly change.In addition, present method is obviously useful for different antibodies and different peptide.Only use high parsing Antibody-antigen complexes structure, present method usually can be applied to and perform affinity maturation computer simulation.In one embodiment, this uses computer simulation affinity maturation to save time and the resource of huge amount.

example 41

specification antibody-like is determined

The people such as Chothia have described the antibody structure (J.Mol.Biol.1987Aug20 of the basis " specification class " of the hypervariable region of each immunoglobulin chain; 196 (4): 901-17).Analyze the atomic structure of Fab and the VL fragment of panimmunity sphaeroprotein, to determine the relation of the three-dimensional structure of aminoacid sequence and its antigen binding site.The people such as Chothia find that there is relatively few residue of primary responsibility hypervariable region backbone construction, its hydrogen bond or ability present abnormal in its packaging

ψ or ω constructs.Find that these residues occur in site in hypervariable region and in conservative β-thin slice frame.Pass a test and have the immunoglobulin sequences of unknown structure, the people such as Chothia show many immunoglobulin (Ig)s and have hypervariable region, and these hypervariable regions and known structure have similar size, and the site additionally constructed observed by load contains same residue.

It finds that these hypervariable regions of hint have the structure close with known structure.For 5 hypervariable regions, all constituents display of structure is limited to relatively small number object discrete topology class.What backbone construction of hypervariable region occurred usually for these is term " norm structure ".The people such as Chothia (Nature.1989Dec21-28; 342 (6252): 877-83) and other (Martin, waits people J Mol Biol.1996Nov15; 263 (5): 800-15) further work confirms 5 of at least antibody 6 hypervariable regions backbone construction that there is sub-fraction all constituents.

Analyze some above-mentioned antibody to determine the specification class of each complementary antibody determining area (CDR).As known, only specification class is distributed to CDR1 and CDR2 of heavy chain of antibody, and CDR1, CDR2 and CDR3 of light chain of antibody.Following table (41.1) summarizes analytical results.Specification class data are with the form of * HCDR1-HCDR2-LCDR1-LCDR2-LCDR3, and wherein " HCDR " refers to heavy chain CDR and " LCDR " refers to light chain CDR.Therefore, such as, a class specification 1-3-2-1-5 refers to have the antibody that HCDR1 falls into specification class 1, HCDR2 falls into specification class 3, LCDR1 falls into specification class 2, LCDR2 falls into specification class 1, LCDR3 falls into specification class 5.

Table 41.1

If it meets length needs and meet the Key residues defined in specification class, so each CDR (except H3) is dispensed to norm structure.The amino acid that each antibody defines can be found, such as, in the article referring to people such as above Chothia.

equivalent

Previous description and Examples detail certain preferred embodiment of the present invention, and the optimal mode describing that inventor contains.But, should be appreciated that no matter how detailed previous description appears in content, and the present invention can realize in many ways, and the present invention should build according to additional claims and its any equivalent.

Sequence table

<110> Abgenix Inc.

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Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

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Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro

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Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile

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Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Ile Val Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His His Ser Tyr Pro Leu

85 90 95

Thr Ser Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>26

<211>107

<212>PRT

<213> homo sapiens

<400>26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Ser Val Thr Ile Thr Cys Arg Thr Ser Gln Gly Ile Arg Lys Asn

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His His Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Arg Val Glu Ile Arg

100 105

<210>27

<211>111

<212>PRT

<213> homo sapiens

<400>27

Asp Ile Val Met Thr Gln Ser Pro Leu Leu Pro Val Thr Pro Gly Glu

1 5 10 15

Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn

20 25 30

Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu

85 90 95

Gln Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>28

<211>112

<212>PRT

<213> homo sapiens

<400>28

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Arg

20 25 30

Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>29

<211>112

<212>PRT

<213> homo sapiens

<400>29

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Arg

20 25 30

Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly His Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>30

<211>112

<212>PRT

<213> homo sapiens

<400>30

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>31

<211>112

<212>PRT

<213> homo sapiens

<400>31

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Arg

20 25 30

Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>32

<211>112

<212>PRT

<213> homo sapiens

<400>32

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Arg

20 25 30

Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>33

<211>112

<212>PRT

<213> homo sapiens

<400>33

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Arg

20 25 30

Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>34

<211>336

<212>DNA

<213> homo sapiens

<400>34

gatattgtga tgacccagac tccactctcc tcacctgtca cccttggaca gccggcctcc 60

atctcctgca ggtctagtca aagcctcata cacactgatg gaaacatcta tttgagttgg 120

cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taatcggttc 180

tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaagatc 240

agcagggtgg aagctgagga tgtgggggtt tattactgca tgcaaggtac acaatttcct 300

atcaccttcg gccaagggac acgactggag attaaa 336

<210>35

<211>375

<212>DNA

<213> homo sapiens

<400>35

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggttcatct attacagagg gaacacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagttg acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgcgagac 300

ggatattgta gtagaaccgg ctgctatggc ggctggttcg acccctgggg ccagggaacc 360

ctggtcacgt ctcct 375

<210>36

<211>335

<212>DNA

<213> homo sapiens

<400>36

atattgtgat gactcagtct ccactctccc tgcccgtcac ccctggagag ccggcctcca 60

tctcctgcag gtctagtcag agcctcctgt atagaaatgg aaacaactat ttggattggt 120

atctgcagaa gccagggcag tctccacagc tcctgatcta tttgggttct aatcgggcct 180

ccggggtccc tgacaggttc agtggcagtg gatcgggcac agattttaca ctgaacatca 240

gcagagtgga ggctgaggat gttgggcatt attactgcat gcaggctcta caaactcctc 300

ggacgttcgg ccaagggacc aaggtggaaa tcaaa 335

<210>37

<211>384

<212>DNA

<213> homo sapiens

<400>37

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctccggatt caccctcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atgtcatatg atggaagtaa agaagactat 180

gcagactccg tgaagggccg attcaccatc tctagagaca attccgagaa catgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtat attactgtgt gagcgaagga 300

tattgtagta gtcgtagctg ctataagtac tactactacg gcatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ctca 384

<210>38

<211>336

<212>DNA

<213> homo sapiens

<400>38

gatactgtga tgacccagac tccactctcc tcacatgtaa cccttggaca gccggcctcc 60

atctcctgca ggtctagtca aagcctcgta cacagtgatg gaaacaccta cttgagttgg 120

cttcagcaga ggccaggcca acctccaaga ctcctaattt ataggatttc taggcggttc 180

tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actggaaatc 240

agcagggtgg aggctgagga tgtcggggtt tattactgca tgcaatctac acacgttcct 300

cggacgttcg gccaagggac caaggtggag atcaaa 336

<210>39

<211>372

<212>DNA

<213> homo sapiens

<400>39

caggtgcagc tggtggagtc tgggggaggc gtggtccagt ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcaga aactatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtga taaatactat 180

gcagactccg tgaggggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatggc 300

tacgatattt tgactggtaa tcctagggac tttgactact ggggccaggg aaccctggtc 360

accgtctcct ca 372

<210>40

<211>348

<212>DNA

<213> homo sapiens

<400>40

gaggtgcagg tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcggct attagtggta gtggtggtag tacaaactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacactgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtct attactgtgc tgggagcagt 300

ggctggtccg agtactgggg ccagggaacc ctggtcaccg tctcctcg 348

<210>41

<211>321

<212>DNA

<213> homo sapiens

<400>41

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggctagtca gggcattaga aataatttag cctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcctccaatt tgcaaagtgg ggtcccatca 180

aggttcaccg gcagtggatc tgggacagaa ttcactctca tagtcagcag cctgcagcct 240

gaagattttg cgacttatta ctgtctacag catcacagtt acccgctcac ttccggcgga 300

gggaccaagg tggagatcaa a 321

<210>42

<211>336

<212>DNA

<213> homo sapiens

<400>42

gatattgtga tgacccagac tccactctcc tcacctgtca cccttggaca gccggcctcc 60

atctcctgca ggtctagtca aagcctcgta cacagggatg gaaataccta cttgagttgg 120

cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180

tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatt 240

agcagggtgg aagctgagga tgtcgggatt tatttctgca tgcatactac acaatttcct 300

tggacgttcg gccaagggac cagggtggaa atcaaa 336

<210>43

<211>354

<212>DNA

<213> homo sapiens

<400>43

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agctacagtt ctgcttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaagggcat atcacaggtc caggtggtat 180

tacgagtatg cagtatcggt gaaaagtcga ataaacatca ccccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300

agaggcagtc gctttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354

<210>44

<211>354

<212>DNA

<213> homo sapiens

<400>44

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacaatg ctgcttggaa ctggatcagg 120

cagtccccag cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg tagtatctgt gaaaagtcga ataaccatca acccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgta 300

agaggcagtc gctttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 354

<210>45

<211>336

<212>DNA

<213> homo sapiens

<400>45

gctattgtgt tgacccagac tccactctcc tcacctgtca cccttggaca gccggcctcc 60

atctcctgca ggtctagtca aagcctcgtt cacagggatg gaaacaccta cttgagttgg 120

cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180

tctggggtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240

agcagggtgg aacctgacga tgtcggggtt tattactgca tgcatactac acaacttcct 300

tggacgttcg gccaagggac caaggtggaa atcaaa 336

<210>46

<211>336

<212>DNA

<213> homo sapiens

<400>46

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctccta tatagaaatg gaaacaacta tttggattgg 120

tatctgcaga ggccagggca gtctccacaa ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac attgaaaatc 240

ggcagagtgg aggctgagga tgttggggtt tattactgca tgcaggctct acaaactcct 300

cggacgttcg gccaagggac caaggtggaa atcaaa 336

<210>47

<211>384

<212>DNA

<213> homo sapiens

<400>47

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgtag cctctggatt caccctcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtg acatcatatg atggaagtaa aaaagactat 180

gcagactccg cgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgt gagcgaagga 300

tattgtagta gtagtagctg ctataagtac tactattacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ttca 384

<210>48

<211>348

<212>DNA

<213> homo sapiens

<400>48

gaggggcagc tgttggagtc tgggggaggc tgggtacagc ctggggagtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcggct attagtggta gtggtggtag cacaaattac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaagtga acagcctgag agtcgaggac acggccgtat attactgtgc tgggagcagt 300

ggctggtccg agtactgggg ccagggaacc ctggtcaccg tctcctca 348

<210>49

<211>321

<212>DNA

<213> homo sapiens

<400>49

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagcgtcacc 60

atcacttgcc ggacaagtca gggcattaga aaaaatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tacaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatccgcag cctgcagcct 240

gaagattttg caacttatta ctgtctccag catcatagtt acccgctcac tttcggcgga 300

gggaccaggg tggagatcag a 321

<210>50

<211>336

<212>DNA

<213> homo sapiens

<400>50

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctccta tatagaaatg gaaacaacta tttggattgg 120

tatctgcaga ggccagggca gtctccacaa ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgca tgcaggctct acaaactcct 300

cggacgttcg gccaagggac caaggtggaa atcaaa 336

<210>51

<211>384

<212>DNA

<213> homo sapiens

<400>51

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgtag cctctggatt caccctcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtg acatcatatg atggaagtaa aaaagactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgt gagcgaagga 300

tattgtgata gtagtagctg ctataagtac tactactacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ttca 384

<210>52

<211>336

<212>DNA

<213> homo sapiens

<400>52

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctccta tatagaaatg gaaacaacta tttggattgg 120

tatctgcaga ggccagggca gtctccacaa ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgca tgcaggctct acaaactcct 300

cggacgttcg gccaagggac caaggtggaa atcaaa 336

<210>53

<211>384

<212>DNA

<213> homo sapiens

<400>53

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgtag cctctggatt caccctcagt agctatggca tgcactgggt ccgccaggct 120

ctaggcaagg ggctggagtg ggtggcagtg acatcatatg atggaagtaa aaaagactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgt gagcgaagga 300

tattgtgata gtactagttg ctataagtac tactactacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ttca 384

<210>54

<211>336

<212>DNA

<213> homo sapiens

<400>54

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctccta tatagaaatg gaaacaacta tttggattgg 120

tatctgcaga ggccagggca gtctccacaa ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgca tgcaggctct acaaactcct 300

cggacgttcg gccaagggac caaggtggaa atcaaa 336

<210>55

<211>384

<212>DNA

<213> homo sapiens

<400>55

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgtag cctctggatt caccctcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtg acatcatatg atggaagtaa aaaagactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgt gagcgaagga 300

tattgtgata gtactagctg ctataagtac tactactacg gtatggacgt ctggggccaa 360

gggaccacgg tcaccgtctc ttca 384

<210>56

<211>14

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>56

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Cys

1 5 10

<210>57

<211>10

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>57

Glu Glu Lys Lys Gly Asn Tyr Val Val Thr

1 5 10

<210>58

<211>5

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>58

Leu Glu Glu Lys Lys

1 5

<210>59

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>59

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp

1 5 10

<210>60

<211>4

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>60

Glu Lys Asn Tyr

1

<210>61

<211>5

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>61

Glu Glu Lys Gly Asn

1 5

<210>62

<211>35

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>62

ggatctcgag ccagaccgga acgacaggcc acctc 35

<210>63

<211>34

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>63

cggatctcga gccggagccc agcactttgatctt 34

<210>64

<211>35

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>64

cggatgaatt cccagaccgg acgacaggcc acctc 35

<210>65

<211>20

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>65

ctttcttttc ctccagagcc 20

<210>66

<211>21

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>66

gtaattatgt ggtgacagat c 21

<210>67

<211>35

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>67

cggatctcga gctcaagaga gcttggttgg gagct 35

<210>68

<211>29

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>68

ggtggcggta cctggacaag accgttgcg 29

<210>69

<211>37

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>69

ataagaatgc ggccgctcat ttacccggag agcggga 37

<210>70

<211>32

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>70

ctactagcta gccaccatgc gaccctccgg ga 32

<210>71

<211>26

<212>DNA

<213> artificial sequence

<220>

<223> synthetic oligonucleotide primer subsequence

<400>71

cggggtaccc ggcgatggac gggatc 26

<210>72

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>72

Ala Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr

1 5 10

<210>73

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>73

Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His

1 5 10

<210>74

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>74

Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly

1 5 10

<210>75

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>75

Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser

1 5 10

<210>76

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>76

Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys

1 5 10

<210>77

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>77

Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys Val

1 5 10

<210>78

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>78

Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg

1 5 10

<210>79

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>79

Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala

1 5 10

<210>80

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>80

Ala Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp

1 5 10

<210>81

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>81

Leu Ala Glu Lys Lys Gly Asn Tyr Val Val Thr Asp

1 5 10

<210>82

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>82

Leu Glu Ala Lys Lys Gly Asn Tyr Val Val Thr Asp

1 5 10

<210>83

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>83

Leu Glu Glu Ala Lys Gly Asn Tyr Val Val Thr Asp

1 5 10

<210>84

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>84

Leu Glu Glu Lys Ala Gly Asn Tyr Val Val Thr Asp

1 5 10

<210>85

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>85

Leu Glu Glu Lys Lys Ala Asn Tyr Val Val Thr Asp

1 5 10

<210>86

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>86

Leu Glu Glu Lys Lys Gly Ala Tyr Val Val Thr Asp

1 5 10

<210>87

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>87

Leu Glu Glu Lys Lys Gly Asn Ala Val Val Thr Asp

1 5 10

<210>88

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>88

Leu Glu Glu Lys Lys Gly Asn Tyr Ala Val Thr Asp

1 5 10

<210>89

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>89

Leu Glu Glu Lys Lys Gly Asn Tyr Val Ala Thr Asp

1 5 10

<210>90

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>90

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Ala Asp

1 5 10

<210>91

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>91

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Ala

1 5 10

<210>92

<211>22

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>92

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

1 5 10 15

Gly Ser Cys Val Arg Ala

20

<210>93

<211>19

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>93

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys

1 5 10 15

Val Arg Ala

<210>94

<211>10

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>94

Glu Glu Lys Lys Gly Asn Tyr Val Val Thr

1 5 10

<210>95

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>95

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp

1 5 10

<210>96

<211>6

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>96

Tyr Val Val Thr Asp His

1 5

<210>97

<211>5

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>97

Tyr Val Val Thr Asp

1 5

<210>98

<211>10

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>98

Glu Glu Lys Lys Gly Asn Tyr Val Val Thr

1 5 10

<210>99

<211>6

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>99

Gly Asn Tyr Val Val Thr

1 5

<210>100

<211>23

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>100

Asp Thr Val Met Thr Gln Thr Pro Leu Ser Ser His Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys

20

<210>101

<211>16

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>101

Arg Ser Ser Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr Leu Ser

1 5 10 15

<210>102

<211>14

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>102

Trp Leu Gln Gln Arg Pro Gly Pro Pro Arg Leu Leu Ile Tyr

1 5 10

<210>103

<211>7

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>103

Arg Ile Ser Arg Arg Phe Ser

1 5

<210>104

<211>32

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>104

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr

1 5 10 15

Leu Glu Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

20 25 30

<210>105

<400>109

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1 5 10

<210>110

<211>17

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>110

Val Ile Trp Tyr Asp Gly Ser Asp Lys Tyr Tyr Ala Asp Ser Val Arg

1 5 10 15

Gly

<210>111

<211>32

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>111

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>112

<211>15

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>112

Asp Gly Tyr Asp Ile Leu Thr Gly Asn Pro Arg Asp Phe Asp Tyr

1 5 10 15

<210>113

<211>11

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>113

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210>114

<211>23

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>114

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys

20

<210>115

<211>15

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>115

Trp Leu His Gln Arg Pro Gly Gln Pro Pro Arg Leu Leu Ile Tyr

1 5 10 15

<210>116

<211>7

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>116

Lys Ile Ser Asn Arg Phe Ser

1 5

<210>117

<211>32

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>117

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Ala Phe Thr

1 5 10 15

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

20 25 30

<210>118

<211>9

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>118

Met Gln Ala Thr Gln Leu Pro Arg Thr

1 5

<210>119

<211>11

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>119

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

1 5 10

<210>120

<211>30

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>120

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

20 25 30

<210>121

<211>5

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>121

Ser Tyr Gly Met His

1 5

<210>122

<211>14

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>122

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1 5 10

<210>123

<211>17

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>123

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Lys

1 5 10 15

Gly

<210>124

<211>32

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>124

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>125

<211>11

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>125

Asp Gly Trp Gln Gln Leu Ala Pro Phe Asp Tyr

1 5 10

<210>126

<211>12

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>126

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

1 5 10

<210>127

<211>6

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>127

Glu Glu Lys Lys Gly Asn

1 5

<210>128

<211>33

<212>DNA

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>128

ataaaagctt ctggaggaaa agaaaggtaa tta 33

<210>129

<211>33

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>129

Thr Thr Ala Thr Thr Gly Gly Thr Ala Cys Cys Thr Cys Ala Gly Gly

1 5 10 15

Cys Gly Ala Thr Gly Gly Ala Cys Gly Gly Gly Ala Thr Cys Thr Thr

20 25 30

Ala

<210>130

<211>14

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>130

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly

1 5 10

<210>131

<211>7

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>131

Glu Glu Lys Lys Gly Asn Tyr

1 5

<210>132

<211>11

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>132

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr

1 5 10

<210>133

<211>8

<212>PRT

<213> artificial sequence

<220>

<223> synthetic peptide sequence

<400>133

Leu Glu Glu Lys Lys Gly Asn Tyr

1 5

<210>134

<211>1186

<212>PRT

<213> homo sapiens

<400>134

Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln

1 5 10 15

Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn

20 25 30

Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg

35 40 45

Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr

50 55 60

Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu

65 70 75 80

Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala

85 90 95

Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro

100 105 110

Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser Asn

115 120 125

Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val

130 135 140

Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His Leu

145 150 155 160

Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp

165 170 175

Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala

180 185 190

Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys

195 200 205

His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys

210 215 220

Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr Cys

225 230 235 240

Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn

245 250 255

Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro

260 265 270

Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly

275 280 285

Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys

290 295 300

Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu

305 310 315 320

Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys

325 330 335

Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe

340 345 350

Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu

355 360 365

Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln

370 375 380

Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu

385 390 395 400

Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val

405 410 415

Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile

420 425 430

Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala

435 440 445

Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr

450 455 460

Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln

465 470 4754 80

Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro

485 490 495

Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val

500 505 510

Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn

515 520 525

Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn

530 535 540

Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His

545 550 555 560

Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met

565 570 575

Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val

580 585 590

Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly

595 600 605

Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr

610 615 620

Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile

625 630 635 640

Gly Leu Phe Met Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg

645 650 655

Arg Leu Leu Gln Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly

660 665 670

Glu Ala Pro Asn Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe

675 680 685

Lys Lys Ile Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

690 695 700

Gly Leu Trp Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile

705 710 715 720

Lys Glu Leu Arg Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

725 730 735

Asp Glu Ala Tyr Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg

740 745 750

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu

755 760 765

Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn

770 775 780

Ile Gly Ser Gln Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly

785 790 795 800

Met Asn Tyr Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala

805 810 815

Arg Asn Val Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe

820 825 830

Gly Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu

835 840 845

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His

850 855 860

Arg Ile Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

865 870 875 880

Trp Glu Leu Met Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala

885 890 895

Ser Glu Ile Ser Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

900 905 910

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

915 920 925

Ile Asp Ala Asp Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe

930 935 940

Ser Lys Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp

945 950 955 960

Glu Arg Met His Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala

965 970 975

Leu Met Asp Glu Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr

980 985 990

Leu Ile Pro Gln Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr

995 1000 1005

Pro Leu Leu Ser Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala

1010 1015 1020

Cys Ile Asp Arg Asn Gly LeuGln Ser Cys Pro Ile Lys Glu Asp Ser

1025 1030 1035 1040

Phe Leu Gln Arg Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp

1045 1050 1055

Ser Ile Asp Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser

1060 1065 1070

Val Pro Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn

1075 1080 1085

Gln Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro

1090 1095 1100

His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro

1105 1110 1115 1120

Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys

1125 1130 1135

Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe

1140 1145 1150

Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala

1155 1160 1165

Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile

1170 1175 1180

Gly Ala

1185

<210>135

<211>919

<212>PRT

<213> homo sapiens

<400>135

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys

1 5 10 15

Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val

20 25 30

Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly

35 40 45

Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn

50 55 60

Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile

65 70 75 80

Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu

85 90 95

Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly

100 105 110

Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala

115 120 125

Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln

130 135 140

Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg

145 150 155 160

Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys

165 170 175

Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr

180 185 190

Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys

195 200 205

Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys

210 215 220

Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg

225 230 235 240

Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg

245 250 255

Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu

260 265 270

Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys

275 280 285

Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys

290 295 300

Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala

305 310 315 320

Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly

325 330 335

Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile

340 345 350

Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val

355 360 365

Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His Ile Val Arg

370 375 380

Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val Glu Pro

385 390 395 400

Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg Ile Leu

405 410 415

Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly Ala Phe

420 425 430

Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu Lys Val Lys

435 440 445

Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser Pro Lys Ala

450 455 460

Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser Val Asp Asn

465 470 475 480

Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln

485 490 495

Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg

500 505 510

Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn Trp Cys Val

515 520 525

Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg Leu Val His

530 535 540

Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln His Val

545 550 555 560

Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys

565 570 575

Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu

580 585 590

Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp Val Trp Ser

595 600 605

Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser Lys Pro Tyr

610 615 620

Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu Lys Gly Glu

625 630 635 640

Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met

645 650 655

Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys Phe Arg Glu

660 665 670

Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln Arg Tyr Leu

675 680 685

Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro Thr Asp Ser

690 695 700

Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp Asp Val Val

705 710 715 720

Asp Ala Asp Glu Tyr LeuIle Pro Gln Gln Gly Phe Phe Ser Ser Pro

725 730 735

Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala Thr Ser Asn

740 745 750

Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln Ser Cys Pro

755 760 765

Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp Pro Thr Gly

770 775 780

Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro Val Pro Glu

785 790 795 800

Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser Val Gln Asn

805 810 815

Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro

820 825 830

His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu

835 840 845

Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala

850 855 860

His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp

865 870 875 880

Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe

885 890 895

Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln

900 905 910

Ser Ser Glu Phe Ile Gly Ala

915

<210>136

<211>268

<212>PRT

<213> homo sapiens

<400>136

Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu

1 5 10 15

Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val

20 25 30

Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser

35 40 45

Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu

50 55 60

Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly

65 70 75 80

Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr

85 90 95

Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln

100 105 110

Glu Ile Leu His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys

115 120 125

Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu

130 135 140

Ser Asn Met Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys

145 150 155 160

Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu

165 170 175

Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly

180 185 190

Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala

195 200 205

Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys

210 215 220

Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu

225 230 235 240

Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr

245 250 255

Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro Arg

260 265

<210>137

<211>512

<212>DNA

<213> homo sapiens

<400>137

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gtagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatgga 300

tggcagcagc tggccccctt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360

gcctccacca agggcccatc ggtcttcccc ctggcaccct ctagcaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg ca 512

<210>138

<211>170

<212>PRT

<213> homo sapiens

<400>138

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Trp Gln Gln Leu Ala Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

165 170

<210>139

<211>496

<212>DNA

<213> homo sapiens

<400>139

gatattgtga tgacccagac tccactctcc tcacctgtca cccttggaca gccggcctcc 60

atctcctgca ggtctagtca aagcctcgtg catagtgatg gaaacaccta cttgagttgg 120

cttcaccaga ggccaggcca gcctccaaga ctcctaattt ataagatttc taaccggttc 180

tctggggtcc cagacagatt cagtggcagt ggggcaggga cagctttcac actgaaaatc 240

agcagggtgg aagctgagga tgtcggggtt tattactgca tgcaagctac acaacttcct 300

cggacgttcg gccaagggac caaggtggaa atcaaacgaa ctgtggctgc accatctgtc 360

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgctagcgt tgtgtgcctg 420

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480

tcgggtaact cccagg 496

<210>140

<211>165

<212>PRT

<213> homo sapiens

<400>140

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Ser Trp Leu His Gln Arg Pro Gly Gln Pro

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Ala Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Thr Gln Leu Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln

165

<210>141

<211>110

<212>PRT

<213> homo sapiens

<400>141

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

100 105 110

<210>142

<211>121

<212>PRT

<213> homo sapiens

<400>142

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Trp Gln Gln Leu Ala Pro Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210>143

<211>113

<212>PRT

<213> homo sapiens

<400>143

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Thr Gln Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg

<210>144

<211>113

<212>PRT

<213> homo sapiens

<400>144

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Ser Trp Leu His Gln Arg Pro Gly Gln Pro

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Ala Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Thr Gln Leu Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg

Claims

1., through being separated a human monoclonal antibody, it comprises

(a) human antibody heavy chain's polypeptide, it comprises following complementary determining region: heavy chain CDR1, and it is the CDR1 in SEQ ID NO:142; Heavy chain CDR2, it is the CDR2 in SEQ ID NO:142; Heavy chain CDR3, it is the CDR3 in SEQID NO:142; With non-human antibody light chain's polypeptide, it comprises following complementary determining region: light chain CDR1, and it is the CDR1 in SEQ ID NO:144; Light chain CDR2, it is the CDR2 in SEQ ID NO:144; Light chain CDR3, it is the CDR3 in SEQ ID NO:144; Or

(b) human antibody heavy chain's polypeptide, it comprises following complementary determining region: heavy chain CDR1, and it is the CDR1 in SEQ ID NO:2; Heavy chain CDR2, it is the CDR2 in SEQ ID NO:2; Heavy chain CDR3, it is the CDR3 in SEQ IDNO:2; With non-human antibody light chain's polypeptide, it comprises following complementary determining region: light chain CDR1, and it is the CDR1 in SEQ ID NO:19; Light chain CDR2, it is the CDR2 in SEQ ID NO:19; Light chain CDR3, it is the CDR3 in SEQ ID NO:19.

2. according to claim 1 through being separated human monoclonal antibody, wherein said human antibody heavy chain's polypeptide comprises described aminoacid sequence SEQ ID NO:2.

3. according to claim 1 through being separated human monoclonal antibody, wherein said non-human antibody light chain's polypeptide comprises described aminoacid sequence SEQ ID NO:142.

4. according to claim 1 through being separated human monoclonal antibody, wherein said antibodies is to the peptide be made up of sequence LEEKKGNYVVTDHC (SEQ ID NO:56).

5. according to claim 1 through being separated human monoclonal antibody, wherein said human antibody heavy chain's polypeptide comprises the aminoacid sequence shown in SEQ ID NO:2, and described non-human antibody light chain's polypeptide comprises the aminoacid sequence shown in SEQ ID NO:19.

6. according to claim 1 through being separated human monoclonal antibody, wherein said human antibody heavy chain's polypeptide comprises at least one in following aminoacid sequence: SEQ ID NO:108, SEQ ID NO:110 and SEQ ID NO:112.

7. according to claim 1 through being separated human monoclonal antibody, wherein said human antibody heavy chain's polypeptide comprises all less than three aminoacid sequences: SEQ ID NO:108, SEQ ID NO:110 and SEQ ID NO:112.

8. according to claim 1 through being separated human monoclonal antibody, wherein said non-human antibody light chain's polypeptide comprises at least one in following aminoacid sequence: SEQ ID NO:101, SEQ ID NO:103 and SEQ ID NO:105.

9. according to claim 1 through being separated human monoclonal antibody, wherein said non-human antibody light chain's polypeptide comprises all less than three aminoacid sequences: SEQ ID NO:101, SEQ ID NO:103 and SEQ ID NO:105.

10. according to claim 1 through being separated human monoclonal antibody, wherein said human antibody heavy chain's polypeptide comprises the aminoacid sequence shown in SEQ ID NO:142, and described non-human antibody light chain's polypeptide comprises the aminoacid sequence shown in SEQ ID NO:144.

11. is according to claim 1 through being separated human monoclonal antibody, and wherein said human antibody heavy chain's polypeptide comprises at least one in following aminoacid sequence: SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125.

12. is according to claim 1 through being separated human monoclonal antibody, and wherein said human antibody heavy chain's polypeptide comprises all less than three aminoacid sequences: SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125.

13. is according to claim 1 through being separated human monoclonal antibody, and wherein said non-human antibody light chain's polypeptide comprises at least one in following aminoacid sequence: SEQ ID NO:101, SEQ ID NO:116 and SEQ ID NO:118.

14. is according to claim 1 through being separated human monoclonal antibody, and wherein said non-human antibody light chain's polypeptide comprises all less than three aminoacid sequences: SEQ ID NO:101, SEQ ID NO:116 and SEQ ID NO:118.

15. is according to claim 1 through being separated human monoclonal antibody, and wherein said human antibody heavy chain's polypeptide comprises: heavy chain CDR1, and it is the CDR1 in SEQ ID NO:142; Heavy chain CDR2, it is the CDR2 in SEQ ID NO:142; Heavy chain CDR3, it is the CDR3 in SEQ ID NO:142; And described non-human antibody light chain's polypeptide comprises: light chain CDR1, it is the CDR1 in SEQ ID NO:144; Light chain CDR2, it is the CDR2 in SEQID NO:144; Light chain CDR3, it is the CDR3 in SEQ ID NO:144.

16. is according to claim 1 through being separated human monoclonal antibody, and wherein said human antibody heavy chain's polypeptide comprises: heavy chain CDR1, and it is the CDR1 in SEQ ID NO:2; Heavy chain CDR2, it is the CDR2 in SEQ ID NO:2; Heavy chain CDR3, it is the CDR3 in SEQ ID NO:2; And described non-human antibody light chain's polypeptide comprises: light chain CDR1, it is the CDR1 in SEQ ID NO:19; Light chain CDR2, it is the CDR2 in SEQ ID NO:19; Light chain CDR3, it is the CDR3 in SEQ ID NO:19.

17. according to claim 1 through being separated human monoclonal antibody, wherein each CDR is defined according to the CDR definition of Kabat.

18. according to claim 1 through being separated human monoclonal antibody, wherein each CDR is defined according to the CDR definition of Chothia.

19. 1 kinds be bonded to EGFRvIII through be separated human monoclonal antibody, it comprises:

Comprise human antibody heavy chain's polypeptide of following complementary determining region (CDRs): heavy chain CDR1, it is the CDR1 in SEQ IDNO:142; Heavy chain CDR2, it is the CDR2 in SEQ ID NO:142; Heavy chain CDR3, it is the CDR3 in SEQ ID NO:142; With

Comprise non-human antibody light chain's polypeptide of following CDRs: light chain CDR1, it is the CDR1 in SEQ ID NO:144; Light chain CDR2, it is the CDR2 in SEQ ID NO:144; Light chain CDR3, it is the CDR3 in SEQ IDNO:144.

20. is according to claim 19 through being separated human monoclonal antibody, and wherein said human antibody heavy chain's polypeptide comprises all less than three aminoacid sequences: SEQ ID NO:121, SEQ ID NO:123 and SEQ ID NO:125.

21. is according to claim 19 through being separated human monoclonal antibody, and wherein said non-human antibody light chain's polypeptide comprises all less than three aminoacid sequences: SEQ ID NO:101, SEQ ID NO:116 and SEQ ID NO:118.

22. 1 kinds be bonded to EGFRvIII through be separated human monoclonal antibody, it comprises:

Comprise human antibody heavy chain's polypeptide of following complementary determining region (CDRs): heavy chain CDR1, it is the CDR1 in SEQ IDNO:2; Heavy chain CDR2, it is the CDR2 in SEQ ID NO:2; Heavy chain CDR3, it is the CDR3 in SEQ ID NO:2; With

Comprise non-human antibody light chain's polypeptide of following CDRs: light chain CDR1, it is the CDR1 in SEQ ID NO:19; Light chain CDR2, it is the CDR2 in SEQ ID NO:19; With light chain CDR3, it is the CDR3 in SEQ IDNO:19.

23. is according to claim 22 through being separated human monoclonal antibody, and wherein said human antibody heavy chain's polypeptide comprises all less than three aminoacid sequences: SEQ ID NO:108, SEQ ID NO:110 and SEQ ID NO:112.

24. is according to claim 22 through being separated human monoclonal antibody, and wherein said non-human antibody light chain's polypeptide comprises all less than three aminoacid sequences: SEQ ID NO:101, SEQ ID NO:103 and SEQ ID NO:105.

25. 1 kinds for detecting the calibrating test kit of the EGFRvIII in mammalian tissues and cell, if its comprise according at least one in antibody described in claim 1 to 24 and exist time, be used to indicate the component that antibody is combined with EGFRvIII.

26. calibrating test kits according to claim 25, wherein said antibody is monoclonal antibody.

27. calibrating test kits according to claim 26, wherein said antibody is through mark.

28. calibrating test kits according to claim 25, wherein said antibody is the first antibody of un-marked and is used to indicate the component of reaction, described component be included as anti-immunoglobulin (Ig) through mark second antibody.

29. calibrating test kits according to claim 25, wherein said antibody is marked by the marker being selected from the group be made up of fluorescence dye, enzyme, radionuclide and not saturating radiative material.

30. 1 kinds are in vitro suppressed to express the method for relevant cell proliferation with EGFRvIII, and it comprises and is contacted with according at least one in antibody described in claim 1 to 24 by the cell of expression EGFRvIII.

31. 1 kinds of purposes expressed for process in preparation according at least one in antibody described in claim 1 to 24 in the medicine of the cancer cells of EGF-R ELISA vIII (EGFRvIII).

32. purposes according to claim 31, wherein said cancer cells is epithelial cell.

33. purposes according to claim 31, wherein said cancer cells is selected from the group be made up of lung cancer, colorectal carcinoma, cancer of the stomach, kidney, prostate cancer, mammary cancer, glioblastoma multiforme or ovarian cancer cell.