CN1816351A - Antibodies directed to the deletion mutants of epidermal growth factor receptor and uses thereof - Google Patents

Abstract

The present invention relates to novel antibodies, particularly antibodies directed against deletion mutants of epidermal growth factor receptor and particularly to the type III deletion mutant, EGFRvIII. The invention also relates to human monoclonal antibodies directed against deletion mutants of epidermal growth factor receptor and particularly to EGFRvIII. Diagnostic and therapeutic formulations of such antibodies, and immunoconjugates thereof, are also provided.

Description

Antibody and use thereof at the deletion mutant of EGF-R ELISA

Technical field

Present embodiment relates to novel antibody, relates in particular to the antibody at the deletion mutant of EGF-R ELISA, and relates in particular to III type deletion mutant EGFRvIII.Present embodiment also relates to the human monoclonal antibody at the deletion mutant of EGF-R ELISA, and especially is EGFRvIII.Present embodiment also relates to the variant of these antibody.The present invention also provides the diagnosis and the treatment prescription of these antibody, and immune conjugate.

Background technology

Since last century, sought to help the tomour specific molecule of better diagnosis and treatment human and animal cancer.Except those based on the virus induction cancer and comprise the cancer by the molecular structure of viral gene regulation, in the human cancer of most of types, be difficult to provide to definite evidence based on the tomour specific material of molecular structure data.Little about example based on the tomour specific molecule of novel molecular structure.Pernicious human nerve's glioma with other with the amplification of EGF-R ELISA molecule or change under the situation of potential relevant tumor (such as breast carcinoma and other human cancer), still have nothing to do in the clearly demonstration of structural change molecule with unique sequences.

EGF-R ELISA (EGFR) is 170 kilodaltons (kilodalton) the membrane glycoprotein product of proto-oncogene c-erb B.The sequence of known EGFR gene (people such as Ullrich, 1984).“Human?EpidermalGrowth?Factor?Receptor?cDNA?Sequence?and?Aberrant?Expression?of?the?AmplifiedGene?in?A431?Epidermoid?Carcinoma?Cells.”Nature?309：418-425)。The EGFR gene is cell homologue people (1984) such as () Downward of the erb B oncogene discerned in avian erythrocytes increase disease virus at first." Close Similarity of Epidermal Growth Factor Receptor and v-erb B OncogeneProtein Sequence. " Nature 307:521-527, people such as Ullrich (1984).In various human tumors, observed the activation that this oncogene amplifies by gene people (1987A) such as () Haley." The Epidermal GrowthFactor Receptor Gene in:Oncogenes; Genes, and Growth Factors ", Guroff, G compiles, the 12nd edition, the 2nd chapter, 40-76 page or leaf, Wiley, and especially be those epidermal growth factor receptor genes (people such as Libermann, (1985) of neuroglia source property N.Y.." Amplification, Enhanced Expression andPossible Rearrangement of EGF Receptor Gene in Primary Human Brain Tumours ofGlial Origin ", Nature 313:144-147; 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.USA 84:6899-6903; People such as Yamazaki, (1988)." Amplification of the Structurally and Functionally Altered Epidermal GrowthFactor Receptor Gene (c-erbB) in Human Brain Tumors. " Molecular and CellularBiology 8:1816-l820, people such as Maiden, (1988).“Selective?Amplification?of?the?CytoplasmicDomain?of?the?Epidermal?Growth?Factor?Receptor?Gene?in?Glioblastoma?Multiforme.”Cancer?Research?4：2711-2714)。

Confirmed EGF-r overexpression in polytype human solid tumor.Mendelsohn?Cancer?Cells7：359(1989)，Mendelsohn?Cancer?Biology?1：339-344(1990)，Modjtahedi?and?Dean?Int′lJ.Oncology?4：277-296(1994)。For example, in some pulmonary carcinoma, breast carcinoma, colon cancer, gastric cancer, the brain cancer, bladder cancer, head and neck cancer, ovarian cancer, renal carcinoma and carcinoma of prostate, observed the EGFR overexpression.Modjtahedi?andDean?Int′l?J.Oncology?4：277-296(1994)。Verification table skin growth factor (EGF) and transforminggrowthfactor-(TGF-a) all are bonded to EGF-r, and cause cell proliferation and tumor growth.

A main distinction between v-erb B oncogene and the normal EGFR gene is that viral oncogene is that normal receptor blocks amino modification; They lack most of Cytoplasm external structure territory, stride film and tyrosine kinase domain (people such as Fung, (1984)) but kept.Activation of the Cellular Oncogene c-erb B by LTRInsertion:Molecular Basis for Induction of Erythroblastosis by Avian Leukosis Virus.Cell 33:357-368; People such as Yamamoto, (1983)." A New Avain Erythroblastosis Virus; AEV-H Carries erbB Gene Responsible for the Induction of Both Erythroblastosis andSarcoma. " Cell 34:225-232, 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. " Cell 41:719-726; 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.USA?83：6053-6057)。This has caused cannot associative list skin growth factor (EGF), but still protein (Gilmore's, (1985)) that can other material of phosphorylation." ProteinPhosphorlytion at Tyrosine is Induced by the v-erb B Gene Product in Vivo and In Vitro.Cell 40:609-618; 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 without regulate and structure on be active people such as (, 1984) Downward.

The range gene variation can betide in the viral erb B oncogene, for example, amino acid whose replacement and disappearance takes place in the carboxyl terminal of gene.Yet 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 of being caused by the insertion of promoter or retrovirus transduction people such as (, (1985)) Nilsen." c-erbB Activation inALV-Induced Erythroblastosis:Novel RNA Processing and Promoter Insertion Resultsin Expression of an Amino-Truncated EGF Receptor. " Cell 41:719-726; 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.USA?83：6053-6057)。

As if as if on the contrary, carboxyl-terminal deletion is only relevant with the tumor that is caused by the retrovirus transduction, and determined specificity (people such as Gammett, 1986 of host range and tumor type; People such as Raines, (1985).“c-erbBActivation?in?Avian?Leukosis?Virus-Induced?Erythroblastosis：Clustered?IntegrationSites?and?the?Arrangement?of?Provirus?in?the?c-erbB?Alleles.”Proc.Natl.Acad.Sci.USA?82：2287-2291)。Show that with amino-transfection experiment of blocking birds c-erb B gene or infectious virus oncogene-human EGF receptor this disappearance promptly is enough to produce transforming protein matter people such as (, (1988)) Pelley separately." Proviral-Activated c-erbB is Leukemogenic but not Sarcomagenic:Characterization ofa Replication-Competent Retrovirus Containing the Activated c-erbB. " Journal ofVirology 62:1840-1844; People such as Wells, (1988).“Genetic?Determinants?of?NeoplasticTransformation?by?the?Retroviral?Oncogene?v-erbB.”Proc.Natl.Acad.Sci.USA85：7597-7601)。

The amplification of EGFR gene betides (people such as Libermann, (1985) in pernicious human nerve's glioma of 40%." Amplification, Enhanced Expression and Possible Rearrangement of EGFReceptor Gene in Primary Human Brain Tumours of Glial Origin ", Nature 313:144-147; 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.USA 84:6899-6903), acceptor gene be rearranged in many have in the tumor that gene amplifies comparatively obvious.Preferentially as if structural change influence amino 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 ", Nature 313:144-147; 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 in any tumor, accurately characterize at that time.

Big or small variant EGFR gene and amplification (people such as Humphrey, (1988)) in some human cancers have been reported in." Amplification and Expression of the, Epidermal Growth Factor Receptor Genein Human Glioma Xenografts " .Cancer Research 48:2231-2238; People such as Bigner, (1988) J.Neuropathol.Exp.Neural., 47:191-205; People such as Wong, (1987)." Increased Expression ofthe Epidermal Growth Factor Receptor Gene in Malignant Gliomas is InvariablyAssociated with Gene Amplification " .Proc.Natl.Acad.Sci.USA 84:6899-6903; With people such as Humphrey.Amplification and the expression of epidermal growth factor receptor gene in the xenotransplantation of human nerve's glioma, Cancer Res.48 (8): 2231-8 (1988)).Yet, the mensuration of the molecular based that still has nothing to do in cell through changing the EGFR molecule.1989, the works explanation of Drs.Bigner and Vogelstein was known as the sequence (being also referred to as δ-EGFr or EGFRvIII) of the EGF acceptor mutant of III type mutant.This works is described in United States Patent (USP) the 6th, 455, and 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, 212, in No. 290.

The EGFR variant is followed the EGFR gene to amplify by gene rearrangement and is caused.There are eight kinds of main EGFr variants, be known as: (i) EGFRvI lacks most of extracellular domain 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 Cytoplasm domain of EGFR, (v) EGFRvV contains the disappearance in the Cytoplasm 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 the treatment cancer in as molecular targets, Endocr Relat Cancer.8 (2): 83-96 (2001)).In addition, exist second kind of more rare second kind of junction point between exon 11 and 14 that have to introduce the EGFRvIII mutant of the disappearance of novel histidine residues (EGFRvIII/ Δ 12-13) (people such as Kuan, EGF mutant receptor vIII the treatment cancer in as molecular targets, Endocr Relat Cancer.8 (2): 83-96 (2001)).

EGFRvIII is at human cancer mesocuticle somatomedin (EGF) the receptor variant of common generation (people such as Kuan, EGF mutant receptor vIII in the treatment cancer as molecular target, Endocr Relat Cancer.8 (2): 83-96 (2001)).In the process that gene amplifies, 267 aminoacid deletion take place in extracellular domain, produce the novel junction point that the tomour specific monoclonal antibody can be pointed to.This variant of EGF receptor with ligand independently mode help tumor to send to develop by the composing type signal.Known EGFrVIII is not expressed in (Wikstrand on any normal structure, CJ. wait the people, " Monoclonal antibodies against EGFRvIII are tumorspecific and react with breast and lung carcinomas malignant gliomas. " CancerResearch 55 (14): 3140-3148 (1995); Olapade-Olaopa, people such as EO. " Evidence for thedifferential expression of a variant EGF receptor protein in human prostate cancer.Br J Cancer.82 (1): 186-94 (2000)).Yet, EGFRvIII is presented at the remarkable expression in the tumor cell, for example, EGFRvIII (Wikstrand, people such as CJ. " Monoclonal antibodies against EGFRVIII are tumor specific and react withbreast and lung carcinomas malignant gliomas. " Cancer Research 55 (14): 3140-3148 (1995) are expressed in 27～76% breast carcinoma biopsies; People such as Ge H., " Evidence of high incidence of EGFRvIIIexpression and coexpression with EGFR in human invasive breast cancer by lasercapture microdissection and immunohistochemical analysis. " Int J Cancer.98 (3): 357-61 (2002)), 50～70% neuroglia cancers are expressed EGFRvIII (Wikstrand, CJ. wait the people, " Monoclonal antibodies against EGFRvIII are tumor specific and react with breastand lung carcinomas malignant gliomas. " Cancer Research 55 (14): 3140-3148 (1995); Moscatello, people such as G., " Frequent expression of a mutant epidermal growthfactor receptor in

Multiple human tumors. " Cancer Res.55 (23): 5536-9 (1; 995)); 16% NSCL cancer is expressed EGFRvIII (Garcia de Palazzo; people such as IE.; " Expression of mutated epidermalgrowth factor receptor by non-small cell lung carcinomas. " Cancer Res.53 (14): 3217-20 (1993)); 75% ovarian cancer is expressed EGFRvIII (Moscatello, G. wait the people, " Frequentexpression of a mutant epidermal growth factor receptor in multiple humantumors. " Cancer Res.55 (23): 5536-9 (1995)).

Express EGFRvIII (Olapade-Olaopa with 68% carcinoma of prostate, EO. wait the people, " Evidence for thedifferential expression of a variant EGF receptor protein in human prostate cancer. " Br JCancer.82 (1): 186-94 (2000)).

Lack 267 aminoacid and replace with glycine and produce the uniqueness connection that to decide target antibody.In addition, expression and the expression in normal structure thereof in some tumor lacks in view of EGFRvIII, and EGFRvIII can be used as the desirable target that is used for deciding the target medicine in oncotherapy.Particularly, as if EGFRvIII can be used as the ideal candidate (for example, with antitumor agent or toxin conjugated antibody) of tumour immunity conjugate treatment.The method for cancer of another kind of treatment overexpression EGFRvIII relates to uses specificity to decide target to the tumour-specific ribozyme that does not separate the variant receptor 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.Described and be used for the proteinic universal antibody of whole EGFRvIII.

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); People such as Kuan, " EGFRvIII as a promising target for antibody-based braintumor therapy. " Brain Tumor Pathol.17 (2): 71-78 (2000); People such as Kuan, " Increasedbinding affinity enhances targeting of glioma xenografts by EGFRvIII-specificscFv. " International Journal of Cancer.88 (6): 962-969 (2000); People such as Landry, " Antibody recognition of a conformational epitope in a peptide antigen:Fv-peptide complex of an antibody fragment specific for the mutant EGF receptor, EGFRvIII. " Journal of Molecular Biology.308 (5): 883-893 (2001); People such as Reist, " Astatine-211 labeling of internalizing anti-EGFRvIII monoclonal antibody usingN-succinimidyl 5-[211At] astato-3-pyridinecarboxylate. " Nuclear Medicine andBiology.26 (4): 405-411 (1999); People such as Reist, " In vitro and in vivo behavior ofradiolabeled chimeric anti-EGFRvIII monoclonal antibody:comparison with itsmurine parent. " Nuclear Medicine and Biology.24 (7): 639-647 (1997); People such as Wikstrand, " Generation of anti-idiotypic reagents in the EGFRvIII tumor-associatedantigen system. " Cancer Immunology, Immunotherapy.50 (12): 639-652 (2002); People such as Wikstrand, " Monoclonal antibodies against EGFRvIII are tumor specific andreact with breast and lung carcinomas malignant gliomas. " Cancer Research.55 (14): 3140-3148 (1995); People such as Wikstrand, " The class III variant of the epidermalgrowth factor receptor (EGFRvIII): characterization and utilization as animmunotherapeutic target. " J.Neurovirol.4 (2): 148-158 (1998); 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); People such as Jungbluth, " A monoclonal antibody recognizing humancancers with amplification/overexpression of the human epidermal growth factorreceptor. " Proc Natl Acad Sci U S is (2): 639-44 (2003) A.100; People such as Mamot, " Epidermal Growth Factor Receptor (EGFR)-targeted Immunoliposomes MediateSpecific and Efficient Drug Delivery to EGFR-and EGFRvIII-overexpressingTumor Cells. " Cancer Research 63:3154-3161 (2003)).Yet, each above-mentioned antibody change and/or constant region in all have or contain the Muridae sequence.

The immunoreactive generation that the existence of these Muridae derived proteins can cause the quick removing of antibody maybe can cause resisting patient's internal antibody.In addition, even after affinity maturation, these antibody still have 2.2 * 10 ^-8To 1.5 * 10 ^-9Other low relatively affinity of level.(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, researcher is introduced rodent so that rodent can produce complete human antibodies with the human antibodies function, see for example people such as Mendez, " Functional transplant ofmegabase human immunoglobulin loci recapitulates human antibody response in mice. " Nat Genet.15 (2): 146-56 (1997).This method is in conjunction with using at the successful production of antibodies of Wild type EGFR, see for example people such as Yang X, " Development of ABX-EGF; a fully humananti-EGF receptor monoclonal antibody, for cancer therapy. " Crit Rev OncolHemato 38 (1): 17-23 (2001); People such as Yang X-D, " Eradication of Established Tumors bya Fully Human Monoclonal Antibody to the Epidermal Growth Factor Receptor withoutConcomitant Chemotherapy ", Cancer Research 59 (6): 1236-1243 (1999) and United States Patent (USP) the 6th, 235, No. 883.

Summary of the invention

In one embodiment, the present invention comprise specificity be bonded to EGFRvIII through separating the human monoclonal antibody and comprising the peptide (SEQ ID NO:56) of sequence LEEKKGNYVVTDHC.In another embodiment, the present invention comprise specificity in conjunction with as for the epi-position that contains in the sequence that comprises LEEKKGNYVVTDHC (SEQ ID NO:56) through separating the human monoclonal antibody, wherein such as the alanine scanning in arranging by SPOTs mensuration, be selected from the group that forms by EEK, KKNYV, LEK, EKNY and EEKGN in conjunction with required residue.

Additional embodiments comprise comprise by the weight chain variable region amino acid sequence of VH3-33 gene code through separating the human monoclonal antibody.The weight chain variable region amino acid sequence can comprise the aminoacid sequence by the JH4b gene code, or by the aminoacid sequence of the D gene code that is selected from the group that forms by D6-13 and D3-9.

Other embodiment comprise comprise by the light chain variable region amino acid sequence of A23 (VK2) gene code through separating the human monoclonal antibody.The light chain variable region amino acid sequence can comprise the aminoacid sequence by the JK1 gene code.

Other embodiment comprise be bonded to EGFRvIII through separation antibody or its fragment, and it comprises the heavy chain amino acid sequence that is selected from by being designated the group that (SEQ ID) NO:138,2,4,5,7,9,10,12,13,15,16 and 17 antibody 13.1.2,131,170,150,095,250,139,211,124,318,342 and 333 heavy chain amino acid sequence form.Antibody can be monoclonal antibody, chimeric antibody, humanized antibody or human antibodies.Antibody or fragment can be associated with pharmaceutically acceptable supporting agent or diluent, and can with the therapeutic agent coupling.Therapeutic agent can be toxin.Therapeutic agent can be the toxin such as DM-1, AEFP, AURISTATINE or ZAP.Described medicament can associate through bridging agent and antibody.Described toxin can associate through second antibody and antibody.Additional embodiments comprises the hybridoma cell strain that produces antibody and comprises cell transition of encoding antibody gene.For example, described cell can be Cinese hamster ovary cell.

Additional embodiments comprises the method for the cell proliferation that a kind of inhibition is associated with the expression of EGFRvIII, and it comprises with the antibody of effective dose or fragment handles the cell of expressing EGFRvIII.In one embodiment, antibody comprises the heavy chain amino acid sequence that is selected from the group that is made up of the heavy chain amino acid sequence of 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), 342 (SEQ ID NO:16) and 333 (SEQ ID NO:17).This method is in vivo carried out, and in mammal (such as the mankind) body, carry out, described mammal stands to relate to the cancer of epithelial cell proliferation, such as pulmonary carcinoma, colon cancer, gastric cancer, renal carcinoma, carcinoma of prostate, breast carcinoma, glioblastoma multiforme or ovarian cancer.

Additional embodiments comprises the method for killing target cell.This can reach with contacting with the associating antibody of toxin by making target cell.Antibodies is to peptide LEEKKGNY (SEQ ID NO:133).In one embodiment, antibody has greater than 1.3 * 10 for peptide ^-9The binding affinity of M.In one embodiment, toxin is selected from AEFP, DM-1 and ZAP.In one embodiment, the antibody toxin compound is toxic more than 10 times to no polypeptide cell to the toxicity of target cell.In one embodiment, antibody comprises the heavy chain amino acid sequence that is selected from the group that is made up of the heavy chain amino acid sequence of 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), 342 (SEQ ID NO:16) and 333 (SEQ ID NO:17).In another embodiment, antibody is through peptide bridging agent or second antibody and toxin association.

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

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

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

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

In one embodiment, antibody is monoclonal antibody, chimeric antibody, the mankind or humanized antibody.In one embodiment, antibody and pharmaceutically acceptable supporting agent, diluent and/or therapeutic agent association.In one embodiment, described therapeutic agent is a 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 it comprises the light-chain amino acid sequence that is selected from by being designated the group that (SEQ ID) NO:140,19,20,21,29,23,25,26,28,33,31 and 32 antibody 13.1.2,131,170,150,095,250,139,211,123,318,342 and 333 light-chain amino acid sequence form.Antibody can be monoclonal antibody, chimeric antibody, humanized antibody or human antibodies.It can associate with pharmaceutically acceptable supporting agent or diluent, or with therapeutic agent (such as toxin, for example DM1 or AURISTATIN E) coupling.

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

Another embodiment comprises the method for the cell proliferation that a kind of inhibition is associated with the expression of EGFRvIII, and it comprises with the above-mentioned antibody of effective dose or fragment handles the cell of expressing EGFRvIII.This method is in vivo carried out, and in mammal (such as the mankind) body, carry out, described mammal stands to relate to the cancer of epithelial cell proliferation, such as pulmonary carcinoma, colon cancer, gastric cancer, renal carcinoma, carcinoma of prostate, breast carcinoma, glioblastoma multiforme or ovarian cancer.

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

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

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

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

The antibody that epimere is discerned can further comprise heavy chain amino acid sequence, and it comprises following complementary determining region (CDR):

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

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

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

Additional embodiments comprises the method for the cell proliferation that a kind of inhibition is associated with the expression of EGFRvIII, and it comprises with above-mentioned antibody of effective dose or fragment handles the cell of expressing EGFRvIII.This method is in vivo carried out, and carries out in mammal (such as the mankind) body, and described mammal stands to relate to the cancer of epithelial cell proliferation, such as pulmonary carcinoma, breast carcinoma, head and neck cancer, carcinoma of prostate or glioblastoma multiforme.

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

Additional embodiments comprises and comprises container, is contained in compositions wherein and shows that described compositions can be used for treating with the package insert of the cancer that is expressed as feature of EGFRvIII or the product of label, and wherein said compositions comprises antibody as indicated above.Described cancer comprises pulmonary carcinoma, breast carcinoma, head and neck cancer, carcinoma of prostate or glioblastoma multiforme.Comprise that also the EGFRvIII that is used for detecting mammalian tissues or cell screens the calibrating test kit of pulmonary carcinoma, colon cancer, gastric cancer, renal carcinoma, carcinoma of prostate or ovarian cancer, wherein EGFRvIII is the antigen of being expressed by the epithelium cancer, and test kit comprises the proteinic antibody of conjugated antigen and is used to indicate the member of antibody and antigenic reaction (if existence).Described antibody can be the monoclonal antibody through labelling, or described antibody can be the first antibody of un-marked, and the member that is used for Indicator Reaction be included as anti-immunoglobulin through the labelling second antibody.The antibody of conjugated antigen can come labelling by the label that is selected from the group that is 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 be selected clinical use for the patient.

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

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

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

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

Another embodiment provides a kind of method that is used to select to be bonded to the particular combination feature variant of EGFRvIII, described method comprises uses molecular structure to form paratope, use molecular structure to form epi-position, calculate the interaction energy between the two and the energy level of the epi-position of described energy level and mAb variant and second paratope compared and select variant based on energy level difference.Described method can comprise further that second variant that uses paratope and the interaction energy between the epi-position are measured the third phase interaction energy and relatively third phase interaction energy and second interaction energy decide and select which variant.Variant forms and carries out the combination test in one embodiment.

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

Another embodiment provides a kind of method that makes antibody variants form EGFRvIII, described method comprises the residue of analyzing with the interactional epi-position of paratope, the more important residue of selecting epi-position is to form recognition sequence, use described recognition sequence with formation 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, reach through using the EGFRvIII variant to select antibody by the antibody that produces opposing EGFRvIII variant.

In one embodiment, wherein separated antibody variants is bonded to EGFRvIII and SEQ ID NO:57 peptide, and antibody can further comprise following point mutation: 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 inferior nanomole binding ability.

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

In yet another embodiment, antibodies to EGFRvIII and antibody has to be bonded to and has one group of paratope with the epi-position of the interactional residue of paratope that comprises 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 is to EGFRvIII and have less than 1.3 * 10 ^-9M, less than 1.0 * 10 ^-9M or less than the K of 500pM _DIn one embodiment, compare with the EGFR peptide of wild type, antibody has specificity for SEQ ID NO:56.In one embodiment, antibody is lower than the specificity bonded 10% of antibody to EGFRVIII (SEQ ID NO:135) to the non-specific binding of the EGFR peptide (SEQ ID NO:134) of wild type.In one embodiment, antibody is selected from by 131,139 and the group that forms of 13.1.2.In one embodiment, antibody is through internalization.In one embodiment, at least about 70% or internalization all takes place at least about 80% antibody.

In one embodiment, compare with EGFR protein or its variant (SEQ ID NO:134) for wild type, the variant human monoclonal antibody preferentially is bonded to for the unique in fact epi-position of EGFRvIII protein.

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 complementary determining region (CDR1) corresponding to canonical class 4.

In one embodiment, variant comprises the light chain complementary determining region (CDR2) corresponding to canonical class 1.In one embodiment, variant comprises the light chain complementary determining region (CDR3) corresponding to canonical class 1.In one embodiment, variant comprises the first heavy chain complementary determining region (CDR1) corresponding to canonical class 1, corresponding to the second heavy chain complementary determining region (CDR2) of canonical class 3, corresponding to the first light chain complementary determining region (CDR1) of canonical class 4, corresponding to the second light chain complementary determining region (CDR2) of canonical class 1 with corresponding to the 3rd light chain complementary determining region (CDR3) of canonical class 1, wherein form these complementary determining regions so that variant can be bonded to and compare for EGFR protein, for the unique in fact epi-position of EGFRvIII protein.In yet another embodiment, provide a kind of method that suppresses the cell proliferation be associated with the expression of EGFRvIII.

Described method relates to the antibody of effective dose or its fragment handles the cell of expressing EGFRvIII, wherein said antibody or its fragment are bonded to EGFRvIII, wherein said antibody combines with toxin, and wherein said 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 that the heavy chain amino acid sequence of 342 (SEQ ID NO:16) and 333 (SEQ ID NO:17) is formed.This method is in vivo carried out in mammalian body, described mammal can be the mankind, and can stand to relate to the cancer of epithelial cell proliferation, and described cancer can comprise pulmonary carcinoma, colon cancer, gastric cancer, renal carcinoma, carcinoma of prostate, breast carcinoma, glioblastoma multiforme or ovarian cancer.Described toxin can be DM-1, AEFP, MMAE, AURISTATIN E or ZAP.

In yet another embodiment, provide a kind of method that suppresses to express the cell proliferation of EGFRvIII cell.This method relates to the antibody of effective dose or its fragment handles the cell of expressing EGFRvIII, wherein said antibody combines with toxin, and wherein said antibody has the light-chain amino acid sequence that is selected from by being designated the group that (SEQ ID) NO:19,20,21,29,23,25,26,28,33,31 and 32 antibody 13.1.2,131,170,150,123,095,139,250,211,342,333 and 318 light-chain amino acid sequence form, and wherein said separated polynucleotide molecule will be in conjunction with the peptide that has with the sequence of SEQ ID NO:56 identification.This method is in vivo carried out in mammalian body, described mammal can be the mankind, and can stand to relate to the cancer of epithelial cell proliferation, and described cancer can comprise pulmonary carcinoma, colon cancer, gastric cancer, renal carcinoma, carcinoma of prostate, breast carcinoma, glioblastoma multiforme or ovarian cancer.Described toxin can be DM-1, AEFP, MMAE, AURISTATIN E or ZAP.

Description of drawings

Fig. 1 is the EGFR of wild type and the sequence alignment of 267 aminoacid deletion of the demonstration between the EGFRvIII and G replacement.

Fig. 2 is the design drawing of EGFRvIII PEP3 14-mer peptide.In Fig. 2 A, has the N-end sequence of the EGFRvIII of aminoacid LEEKK.(SEQ ID NO:58) (1-5) the N-end sequence with EGFR is identical, is unique glycine residue then, is then and the identical aminoacid of residue 273 to 280 among the EGFR.Fig. 2 B represents the aminoacid of the EGFR of disappearance among the EGFRvIII (6-272).

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

Fig. 4 is that 13.1.2 heavy chain of antibody district and specific kind are heavy chain district form relatively, and the amino acid residue in "-" expression hybridoma heavy chain district is identical with the kind of that specific location.Skew from kind of system is represented by suitable amino acid residue.

Fig. 5 is that 13.1.2 light chain of antibody district and specific kind are light chain district form relatively, and the amino acid residue in "-" expression hybridoma light chain district is identical with the kind of that specific location.Skew from kind of system is represented by suitable amino acid residue.

Fig. 6 is a heavy chain district form relatively for derive heavy chain of antibody district and specific kind of various hybridomas, and "-" represents that the amino acid residue in hybridoma heavy chain district is identical with the kind of that specific location.Skew from kind of system is represented by suitable amino acid residue.

Fig. 7 is a light chain district form relatively for derive light chain of antibody district and specific kind of various hybridomas, and "-" represents that the amino acid residue in hybridoma light chain district is identical with the kind of that specific location.Skew from kind of system is represented by suitable amino acid residue.

Fig. 8 is the bonded representative graph of demonstration reorganization EGFRvIII mAb with the cell (NR6 cell) of expressing EGFRvIII.Rhombus represents 95, and triangle represents 133, the square represent 139, " x " represents 150, asterisk represents 170, the circle represent 221, straight line represent 230 and rectangle represent 250.

Fig. 9 A shows for the staining analysis of human anti-EGFR-antibodies (ABX-EGF) to 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 for showing the bonded curve chart of EGFRvIII mAb and glioblastoma cells.The black triangle representative is bonded to the antibody 131 of H1477.The filled squares representative is bonded to the antibody 13.1.2 of H1477.Empty triangle representative is bonded to the antibody 131 of H80.Empty square representative is bonded to the antibody 13.1.2 of H80.

Fig. 9 N is for showing the bonded curve chart of EGFRvIII mAb to human epidermal sample JEG-3 A431.Filled squares is represented antibody 13.1.2.Black triangle is represented antibody 131.

Fig. 9 O is for showing the bonded curve chart of antibody 13.1.2 to NR6 Muridae fibroblast cell strain.Square is represented NR6.The triangle representative has the NR6 of Wild type EGFR.Circular representative has the NR6 of EGFRvIII.

Fig. 9 P is for showing the bonded curve chart of antibody 131 to Muridae fibroblast cell strain.Square is represented NR6.The triangle representative has the NR6 of Wild type EGFR.Circular representative has the NR6 of EGFRvIII.

Figure 10 A shows the FACS staining analysis that is bonded to expression EGFR cell (A431) for human anti-EGFR-antibodies (ABX-EGF).

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

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

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

Figure 11 A show indirectly with express in the EGFRvIII cell (H1477, circle) AEFP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody of the cell 13.1.2 that does not express EGFRvIII (H80, square).

Figure 11 B show indirectly with express in the EGFRvIII cell (H1477, circle) DM1 in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody of the cell 13.1.2 that does not express EGFRvIII (H80, square).

Figure 11 C show indirectly with express in the EGFRvIII cell (H1477, circle) ZAP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody of the cell 13.1.2 that does not express EGFRvIII (H80, square).

Figure 11 D show indirectly with express in the EGFRvIII cell (H1477, circle) AEFP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 95 of cell of not expressing EGFRvIII (H80, square).

Figure 11 E show indirectly with express in the EGFRvIII cell (H1477, circle) DM1 in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 95 of cell of not expressing EGFRvIII (H80, square).

Figure 11 F show indirectly with express in the EGFRvIII cell (H1477, circle) ZAP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 95 of cell of not expressing EGFRvIII (H80, square).

Figure 11 G show indirectly with express in the EGFRvIII cell (H1477, circle) AEFP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 131 of cell of not expressing EGFRvIII (H80, square).

Figure 11 H show indirectly with express in the EGFRvIII cell (H1477, circle) DM1 in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 131 of cell of not expressing EGFRvIII (H80, square).

Figure 11 I show indirectly with express in the EGFRvIII cell (H1477, circle) ZAP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 131 of cell of not expressing EGFRvIII (H80, square).

Figure 12 A show indirectly with express in the EGFRvIII cell (H1477, circle) AEFP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 139 of cell of not expressing EGFRvIII (H80, square).

Figure 12 B show indirectly with express in the EGFRvIII cell (H1477, circle) DM1 in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 139 of cell of not expressing EGFRvIII (H80, square).

Figure 12 C show indirectly with express in the EGFRvIII cell (H1477, circle) ZAP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 139 of cell of not expressing EGFRvIII (H80, square).

Figure 12 D show indirectly with express in the EGFRvIII cell (H1477, circle) AEFP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 150 of cell of not expressing EGFRvIII (H80, square).

Figure 12 E show indirectly with express in the EGFRvIII cell (H1477, circle) DM1 in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 150 of cell of not expressing EGFRvIII (H80, square).

Figure 12 F show indirectly with express in the EGFRvIII cell (H1477, circle) ZAP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 150 of cell of not expressing EGFRvIII (H80, square).

Figure 12 G show indirectly with express in the EGFRvIII cell (H1477, circle) AEFP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 170 of cell of not expressing EGFRvIII (H80, square).

Figure 12 H show indirectly with express in the EGFRvIII cell (H1477, circle) DM1 in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 150 of cell of not expressing EGFRvIII (H80, square).

Figure 12 I show indirectly with express in the EGFRvIII cell (H1477, circle) ZAP in conjunction with respect to the in vitro toxicity of the bonded EGFRvIII antibody 150 of cell of not expressing EGFRvIII (H80, square).

Figure 13 A show indirectly with express in the EGFRvIII cell (H1477, circle) AEFP in conjunction with respect to the in vitro toxicity of the bonded antibody 211 of cell of not expressing EGFRvIII (H80, square).

Figure 13 B show indirectly with express in the EGFRvIII cell (H1477, circle) DM1 in conjunction with respect to the in vitro toxicity of the bonded antibody 211 of cell of not expressing EGFRvIII (H80, square).

Figure 13 C show indirectly with express in the EGFRvIII cell (H1477, circle) ZAP in conjunction with respect to the in vitro toxicity of the bonded antibody 211 of cell of not expressing EGFRvIII (H80, square).

Figure 13 D show indirectly with express in the EGFRvIII cell (H1477, circle) AEFP in conjunction with respect to the in vitro toxicity of the bonded antibody 250 of cell of not expressing EGFRvIII (H80, square).

Figure 13 E show indirectly with express in the EGFRvIII cell (H1477, circle) DM1 in conjunction with respect to the in vitro toxicity of the bonded antibody 250 of cell of not expressing EGFRvIII (H80, square).

Figure 13 F show indirectly with express in the EGFRvIII cell (H1477, circle) ZAP in conjunction with respect to the in vitro toxicity of the bonded antibody 250 of cell of not expressing EGFRvIII (H80, square).

Figure 13 G show indirectly with express in the EGFRvIII cell (H1477, circle) AEFP in conjunction with respect to the in vitro toxicity of the bonded IgG antibody 1 of cell of not expressing EGFRvIII (H80, square) (passive contrast).

Figure 13 H show indirectly with express in the EGFRvIII cell (H1477, circle) DM1 in conjunction with respect to the in vitro toxicity of the bonded IgG antibody 1 of cell of not expressing EGFRvIII (H80, square) (passive contrast).

Figure 13 I show indirectly with express in the EGFRvIII cell (H1477, circle) ZAP in conjunction with respect to the in vitro toxicity of the bonded IgG antibody 1 of cell of not expressing EGFRvIII (H80, square) (passive contrast).

Figure 14 A is a bar diagram, and it shows that EGFRvIII antibody (13.1.2,131 and 139) suppresses to duplicate the bacterium colony that generates H1477 cell in the calibrating and forms when being bonded to AEFP.

Figure 14 B is a bar diagram, and it shows that EGFRvIII antibody (13.1.2,131 and 139) suppresses to duplicate the group that generates H1477 cell in the calibrating and forms when being bonded to DM1.

Figure 15 A is expressing in the EGFRvIII cell (H1477, circle) with respect to the in vitro toxic curve chart of not expressing the direct conjugate among the EGFRvIII (H80, square) for showing anti-EGFR vIII antibody (13.1.2) and toxin (MMAE).Figure 15 B is expressing the in vitro toxic curve chart of EGFRvIII cell (H1477, circle) with respect to the direct conjugate in the cell of not expressing GFRvIII (H80, square) for showing anti-EGFR vIII antibody (13.1.2) and toxin (AEFP).

Figure 15 C is expressing the in vitro toxic curve chart of EGFRvIII cell (H1477) with respect to the direct conjugate in the cell of not expressing GFRvIII (H80) for showing anti-EGFR vIII antibody (13.1.2) and toxin (DM1).

Figure 16 shows wherein have the result of the heteroplastic mice of definite tumor through the in vivo animal model of anti-EGFR vIII (or dEGFR) antibody (13.1.2) (directly being bonded to toxin (DM1, MMAE or AEFP)) processing.Filled squares is represented 250 microgram dEGFR-DM1.The triangle that solid drift angle makes progress is represented 75 microgram same substance.The downward triangle of solid drift angle is represented 75 microgram dEGFR-MMAE.Rhombus is represented 250 microgram same substance.More shallow square is represented 75 microgram dEGFR-AEFP.Empty square is represented 250 microgram same substance.The triangle that hollow drift angle makes progress is represented dEGFR and free DM1.The antibody that hollow drift angle makes progress is represented PBS.All antibody that use are 13.1.2.Arrow is represented the prodrug processing.Figure 17 shows the molecular surface of antibody 131 structural models.Six CDR cresteds are that different shade is with their border of labelling.Binding cavity is positioned near the center.Figure 18 shows the structural model of antibody 13.1.2 molecular surface.Six CDR zones are covered for shade and by numeral and are discerned.Elongated slot probably distributes along vertical center line.Figure 19 A is the possible extended model of 13.1.2 antibody and peptide EEKKGN (SEQ ID NO:127) misfit thing.CDR cover in the zone for shade with the indication border.

Hydrogen bond in the possible extended model of Figure 19 B demonstration 13.1.2 antibody and peptide EEKKGN (SEQ ID NO:127) misfit thing.As Figure 18, the shade of CDR circle is identical with residue.Peptide residue is numbered 1 to 6 from the N-at figure top end to C-end.By six hydrogen bonds of dotted line indication.Six pairs of aminoacid that hydrogen bond forms are as follows: E2...Y172, K3...H31, K4...H31, N6...D33, N6...Y37 and N6...K55.

Figure 20 is the related curve chart that shows between the logarithm of the epitope-antibody binding energy of one of selected extended model and Kd.

Figure 21 describes the precise spread model of peptide-13.1.2 antibody misfit thing.Peptide presents with the space solid form.

Figure 22 describes the hydrogen bond in the precise spread model.

Figure 23 describes the curve chart of antibody-antigen binding energy with respect to the logarithmic linear fit of relative affinity.

The specific embodiment

As indicated above, EGFRvIII is the deletion mutant of EGFR, 267 aminoacid deletion in the extracellular domain of EGFr wherein, and carry out single amino acid in the junction with glycine and replace.These features are shown among Fig. 1 in the sequence alignment between the Wild type EGFR and EGFRvIII.Replace in view of aminoacid, may produce the antibody of the novel epi-position among the EGFR that is not present in wild type at being present among the EGFRvIII in theory at the glycine of the junction of disappearance.Therefore, as shown in Figure 2, be designed for the peptide of immunity and screening, be called 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 5 common n-end amino acids for the EGFR of EGFRvIII and wild type, 8 contained amino acid residues in the conserved sequence between EGFR of unique glycine connection site and wild type (corresponding to residue 273-280) and the EGFRvIII (corresponding to residue 7-14).In addition, glioblastoma cells and also can be used for immunity and screen (being sometimes referred to as the B300.19/EGFRvIII transfectant) at this paper with the EGFRvIII cells transfected (B300.19 cell) of gene code.

Be to produce the human antibodies of opposing EGFRvIII, with transgenic XenoMouse_ mice with glioblastoma cells/EGFRvIII, B300.19/EGFRvIII cell with at EGFRvIII that the EGFR of wild type compares in the combination of peptide (PEP3) of bonding pad in the novel extracellular domain represented carry out immunity.Hang oneself in the future in the immune mouse the B cell separation and be used to produce glioblastoma multiforme, screen then to be bonded to EGFRvIII or to use XenoMax ^TM/ SLAM ^TMTechnology is directly used in screening to be bonded to EGFRvIII (people such as Babcook, Anovel strategy for generating monoclonal antibodies from single, isolated lymphocytesproducing antibodies of defined specificities, Proc Natl Acad Sci USA.93 (15): 7843-8 (1996) and United States Patent (USP) the 5th, 627, No. 052).The antibody that is bonded to EGFRvIII through identification is through the specific recognition of a series of calibrating screenings with definite EGFRvIII.Produce, separate and characterize through this process and be bonded to EGFRvIII and EGFRvIII is had specific human monoclonal antibody colony.Uniqueness shows epitope mapping subsequently, but overlapping specificity.Further assess all antibody in vitro to assess it carries out internalization for cytotoxic drugs is delivered to cell by cell ability.The antibody that shows effective transmission medicine directly combines with cytotoxic drugs, and detects the ability that it kills the tumor cell of expressing EGFRvIII in vitro and in vivo.These researchs are that the generation next time that is used for the treatment of the antibody drug conjugates of patient's cancer provides the foundation, and these patients' tumor is concealed with the specific gene pathological changes.

Produce the colony of complete human anti-EGFR vIII antibody by said process.Use the hybridoma mode, the EGFR that produces for wild type has the reactive several antibody of limited cross, comprises the antibody 13.1,13.2,13.3 and 13.4 to being used for being positive with the bonded ELISA of PEP3.Except these, select antibody 13.1 (with especially being its sub-clone 13.1.2) for further research and development.Use the XenoMax mode, produce antibody colony, comprise antibody 131,139,250 and 095, it is for having high degree of specificity with combining of pep3 oligonucleotide, and has limited cross reactivity with the EGFR of wild type.Wherein, 131 antibody have the most interesting characteristic.The sequence (SEQ ID NO:1-33 and 141-144) of in Fig. 4-7, showing each antibody.To the sequence of various antibody with binding ability compares and will the results are shown among Fig. 4-10.As among Fig. 9 A-9L and Figure 10 A-10D as seen, compare with ABX-EGF, antibody 131,139 and 13.1.2 show the preferable selectivity for EGFRvIII express cell (H1477).Some the results are shown in the curve chart among Fig. 9 M-9P, and it shows simply compares with the EGFRvIII cell, at least two kinds of antibody 13.1.2 and the 131 preferable selectivitys that show for the EGFRvIII express cell.In addition, detect several possibility purposes of present embodiment antibody; It the results are shown among Figure 11-16.At last, based on the structural model of prediction, the variant that forms antibody is to obtain to have the antibody of variation in conjunction with feature.

In addition, antibody of the present invention is extremely useful for the screening of other antibody that is 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 influence identical or through improveing for the feature of the Ag-Ab misfit thing that forms.

Each has high affinity 131 antibody for EGFRvIII and 13.1.2 passes through the good internalization of cell, and shows efficiently cell killing when being bonded to toxin.Interesting is whether no matter result from the different immunity of XenoMouse mice, and no matter whether use different technology, it is gene that two kinds of antibody all are derived from very similar the kind.Yet as if based on the epitope mapping operation, each antibody is bonded to epi-position slightly different on the EGFRvIII molecule, and has for going up slightly different residue in conjunction with necessary EGFRvIII.These results show that using kind is that gene is particularly important for the generation of the antibody therapy of deciding target EGFRvIII, and little variation can be by improveing the combination and the influence of antibody based on the further designerantibodies of these topology discoverys and other therapies.

Height need be bonded to identical epi-position, or the antibody of competition and 13.1.2 and 131 antibodies.As hereinafter more going through, the alanine scanning of arranging by SPOTs is illustrated for specific antibodies in conjunction with important residue.Therefore, also highly need to share important antibody in conjunction with residue.

Definition

Unless otherwise defined, Science and Technology term used herein will have the implication that one of ordinary skill in the art understand usually.In addition, unless the other requirement of this paper, singular references comprises that plural form and plural term comprise singulative.Generally speaking, term and the technology thereof in conjunction with cell and tissue culture, molecular biology and protein and oligonucleotide or polynucleotide chemistry and hybridization as herein described is to have known in this technology and normally used those terms.Standard technique is used for recombinant DNA, oligonucleotide is synthetic and tissue culture and transition (for example, electric shock, lipofection).According to that realize usually in the explanation of Producer or this technology or enzyme reaction and the purification technique of carrying out as described herein.Usually carry out aforementioned techniques and program described in the various general and more detailed list of references of quoting and discussing according to the conventional method of knowing in this technology with as this description in the whole text.See people such as Sambrook for example, Molecular Cloning:A Laboratory Manual (the 2nd edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).Term and the test procedure thereof that uses about analytical chemistry, synthetic organic chemistry and medicine and medical chemistry as herein described and technology has been known in this technology and use usually.

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

Term " through the separating polynucleotide " meaning is polynucleotide or its certain combination of chromosome set, cDNA or synthetic source as used herein, because its source, " through separating polynucleotide " (1) and all or a part wherein " through separating polynucleotide " have nothing to do at the polynucleotide of occurring in nature discovery, (2) be connected to operability polynucleotide, do not connect at occurring in nature, or (3) do not take place as the part of big sequence at occurring in nature.The term of mentioning herein " through isolated protein " is meant protein or its certain combination of cDNA, recombinant RNA or synthetic source, because its source or the source of deriving, " through isolated protein " (1) is irrelevant with the protein that occurring in nature is found, (2) do not contain other protein from identical sources, for example, do not contain Muridae protein, (3) are expressed by the cell from different plant species, or (4) do not take place at occurring in nature.

Term " polypeptide " is meant native protein, fragment or the analog of peptide sequence in this article as general terms.Therefore, native protein, fragment or analog are polypeptides matters.Preferred polypeptide according to the present invention comprises human heavy chain immunoglobulin molecules and human kappa light chain immunoglobulin molecule, and by the antibody molecule that is combined to form that comprises 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, with and fragment and analog.

The term " natural generation " that is applied to as used herein on a kind of object is meant this fact that can find a kind of object at occurring in nature.For example, being present in can be in the isolating organism of natural source (comprising virus), and is natural generation without artificially or otherwise have a mind to the polypeptide or the polymerized nucleoside acid sequence of upgrading in laboratory.

Term " operability connection " is meant that the position of described component is in and can allows them to bring into play in the function relationship in its desired mode as used herein.Control sequence " can be operatively connected " to coded sequence be the in addition colligation of mode of expressing coded sequence under the condition compatible, can reach with control sequence.

Term used herein " control sequence " is meant for expression that realizes the coded sequence that the polymerized nucleoside acid sequence is connected and the polymerized nucleoside acid sequence that processing is necessary.The essence of these control sequence is looked its host organisms and difference, and in prokaryote, these control sequence generally include promoter, ribosome binding site and transcription terminator; In eukaryote, these control sequence generally include promoter and transcription terminator.Term " control sequence " means and comprises that at least it exists for the expression of for example leader and fusion partners sequence and handles necessary all components, and comprises that it exists for for example leader and other favourable component of fusion partners sequence.

Mentioned term " polynucleotide " meaning of this paper is the polymerized form that length is at least the nucleotide of 10 bases, the upgrading form of ribonucleotide or deoxyribonucleotide or any nucleotide.Term comprises strand and the double chain form of DNA.

The term that this paper mentions " oligonucleotide " comprises the upgrading nucleotide that oligonucleotide connexon natural generation and that taken place by natural generation and non-natural links together.Oligonucleotide is the polynucleotide subgroup that comprises 200 bases or shorter length usually.The 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, for example as probe; Although oligonucleotide can be two strands, for example be used to make up gene mutation body.Oligonucleotide of the present invention can be justice or antisense oligonucleotide.

" nucleotide of natural generation comprises deoxyribonucleotide and ribonucleotide to the term that this paper mentions.The term that this paper mentions " upgrading nucleotide " comprises nucleotide and the analog thereof that contains upgrading or replace glycosyl.The term that this paper mentions " oligonucleotide connexon " comprises the oligonucleotide connexon, such as, thiophosphate, phosphorodithioate, seleno phosphate ester, two seleno phosphate esters, aniline thiophosphate, aniline phosphate ester, phosphoramidate and analog thereof.See people such as LaPlanche for example, Nucl.Acids Res.14:9081 (1986); People such as Stec, J.Am.Chem.Soc.106:6077 (1984); People such as Stein, Nucl.Acids Res.16:3209 (1988); People such as Zon, Anti-Cancer Drug Design 6:539 (1991); People such as Zon, Oligonucleotides and Analogues:APractical Approach, 87-108 page or leaf (F.Eckstein compiles, Oxford University Press, OxfordEngland (1991)); People such as Stec, United States Patent (USP) the 5th, 151, No. 510; Uhlmann and Peyman ChemicalReviews 90:543 (1990).If desired, oligonucleotide can comprise the label that is used to detect.Term " variant " is polypeptide, polynucleotide or the molecule that is different from cited polypeptide or polynucleotide as used herein, but just in order protein active not to be caused unfavorable change.The variant that can have epi-position.The variant that can have antibody.In a preferred embodiment, protein variants is bonded to the not unfavorable change of ability of epi-position.In one embodiment, protein variants can combine with the 10-500% of wild type mAb ability.For example, protein variants can with wild type mAb ability 10%, 50%, 110%, 500% or combine greater than 500%.In one embodiment, be included in binding ability between the 10-500% scope.Binding ability can reflect in several ways, includes, but is not limited to the k of variant to epi-position _a, k _dOr K _DIn a preferred embodiment, epi-position is the epi-position described in this description.

In one embodiment, variant antibody can by replace, lack or add 5 or still less aminoacid be different from the sequence of wild type.These variants usually can be by one of peptide sequence that upgrading discloses, and uses representative program assessment for example as herein described to discern through the binding characteristic of upgrading polypeptide.In another embodiment, polypeptide variants preferably show with through the identification polypeptide at least about 70%, more excellent in 90% and optimum at least about 95% homogeneity.Preferably, variant is only different on conservative replacement and/or upgrading.Variant proteins comprises the structurally suitable protein of class Sihe function of those and the described protein structure of this description.In another embodiment, if suitable on the protein function described in protein and this description, so this protein can be variant, as long as the paratope and the paratope described in this description of variant are similar.In one embodiment, any material that has with the analogous shape of paratope described in Figure 17 all is a variant.In one embodiment, any material that has with the analogous shape of paratope described in Figure 18 all is a variant.In one embodiment, any material that has with interactive surfaces analogous shape described in Figure 19 A and the 19B all is a variant.

In one embodiment, if nucleotide sequence under stringent condition optionally with the sequence hybridization of wild type, antibody is variant so.In one embodiment, suitable appropriate stringent condition is included in prewashing in the 5xSSC solution, 0.5% SDS, 1.0mM EDTA (pH8: 0); 50 ℃-65 ℃ down hybridization, 5xSSC, if overnight or for kind between congener, then under 45 ℃, 0.5xSSC; Then 2x, 0.5x and the 0.2xSSC eluting twice to contain 0.1% SDS at every turn under 65 ℃ lasts 20 minutes.These hybrid dna sequences also belong in the category of the present invention, because the degeneracy of coding is also coded by the hybrid dna sequence as nucleotide sequence coded antibody polypeptides.The term that this paper mentions " selective cross " meaning is to detect ground and combination specifically.According to the more polynucleotide of the present invention, oligonucleotide and fragment thereof under certain hybridization and elution requirement with the nucleic acid selective cross, described hybridization and elution requirement can reduce to measure with detecting of non-specific nucleic acid is bonded.Highly rigorous condition can be used for reaching as known in the art and in selective cross condition discussed herein.Generally speaking, the more the nucleic acid sequence homology between polynucleotide of the present invention, oligonucleotide and fragment and the interesting nucleotide sequence is at least 80%, and more generally is the homology of preferred at least 85%, 90%, 95%, 99% and 100% increase.If have partially or completely homogeneity between two seed amino acid sequences, this two seed amino acids sequence is homology so.For example, the 85% homology meaning is when two kinds of sequences are carried out the maximum match comparison, 85% aminoacid unanimity.In the maximization coupling, allow to exist gap (any of two kinds of sequences is complementary); Be preferably 5 or gap length still less, 2 or still less for more excellent.Perhaps or preferably, this term as used herein, contain accidental data matrix and 6 or the ALIGN program of more gaps cost if use, two kinds of protein sequences (or from length at least 30 amino acid whose protein in the polypeptides derived sequence) have the comparison score value more than 5, their homologies so.See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, the 2nd phase supplementary issue of 101-110 page or leaf (the 5th volume, National Biomedical Research Foundation (1972)) and this volume, 1-10 page or leaf.If the aminoacid of two kinds of sequences or its part when using the best comparison of ALIGN program more than or equal to 50% unanimity, these two kinds of sequences or the more excellent homology of its part so.Term used herein " corresponding to " meaning be the more polynucleotide sequence with reference to the polynucleotide sequence homology (that is, and unanimity, but non-strict develop relevant), or the more polynucleotide sequence with consistent with reference to the polymerized nucleoside acid sequence.Contrast, term used herein " complementation " meaning are complementary seriess and all or part of homology with reference to the polymerized nucleoside acid sequence.For example, nucleotide sequence " TATAC " corresponding to reference sequences " TATAC " and with reference sequences " GTATA " complementation.

Following term is used to describe the sequence relation between two or more polynucleotide or the aminoacid sequence: " reference sequences ", " comparison window ", " sequence identity ", " sequence identity percentage rate " and " essence concordance "." reference sequences " is fixed sequence, as sequence benchmark relatively; Reference sequences can be the subgroup of bigger sequence, for example, as the full-length cDNA given in the sequence list or the fragment of gene order, maybe can comprise global cDNA or gene order.Generally speaking, reference sequences length is at least 18 nucleotide or 6 aminoacid, and length often is at least 24 nucleotide or 8 aminoacid, and length is generally at least 48 nucleotide or 16 aminoacid.Because two polynucleotides or aminoacid sequence may each (1) be included between two molecules similarly sequence (promptly, the part of complete polynucleotide or aminoacid sequence), (2) may further be included in different sequence between two polynucleotides or the aminoacid sequence, generally carry out two sequences between (or more) molecules relatively with identification and the similar regional area of comparative sequences by the sequence that compares two molecules through " comparison window "." comparison window " is meant at least 18 in abutting connection with nucleotide position or 6 amino acid whose conceptual segments as used herein, wherein polymerized nucleoside acid sequence or aminoacid sequence can be compared with at least 18 reference sequences in abutting connection with nucleotide or 6 aminoacid sequences, and wherein the position of polymerized nucleoside acid sequence in comparison window compared with reference sequences (it does not comprise interpolation or disappearance), can comprise 20% or interpolation still less, disappearance, replacement and analog thereof (promptly, the gap), compare for the optimal sequence of two sequences.Can be by Smith and Waterman, the local homologue algorithm of Adv.Appl.Math.2:482 (1981), by Needleman and Wunsch, the homologue alignment algorithm of J.Mol.Biol.48:443 (1970), by being used for Pearson and Lipman, Proc.Natl.Acad.Sci. the research of the similar approach of (U.S.A.) 85:2444 (1988), computer implementation (GAP by these algorithms, BESTFIT, TFASTA among FASTA and the Wisconsin Genetics Software Package Release 7.0, (GeneticsComputer Group, 575Science Dr., Madison, Wis.), Geneworks or Mac Vector software encapsulation), or be used to compare the optimal sequence comparison of the sequence of comparison window by observation, and the optimal sequence of selecting to be produced by the whole bag of tricks is compared (that is, causing the highest percentage ratio of homologue on comparison window).

Term " sequence identity " meaning is two polynucleotides or aminoacid sequence consistent (that is, on basis of nucleotide * nucleotide or residue * residue) on comparison window.Term " sequence identity percentage ratio " is by comparing the sequences of two optimum comparisons on comparison window, measure in two sequences and consistent nucleic acid base takes place (for example, A, T, C, G, U or I) or residue with the numbering of the position that produces the matched position numbering, with the numbering of matched position (promptly divided by the total number of positions in the comparison window, window size), and the result multiply by 100 with gained, to produce sequence identity percentage ratio.The feature of polynucleotide or aminoacid sequence represented in term " essence concordance " as used herein, wherein with the comparison window of at least 18 nucleotide (6 aminoacid) position on, often the reference sequences on the window of 24-48 nucleotide (8-16 aminoacid) position is at least compared, polynucleotide or aminoacid comprise at least 85% sequence unanimity, preferred at least 90% to 95% sequence unanimity, the sequence of at least 99% sequence unanimity more generally is wherein by amounting to 20% or still less the disappearance of reference sequences or the sequence of interpolation compare sequence of calculation concordance percentage ratio with reference sequences with may comprising on the comparison window.Reference sequences can be the subgroup of bigger sequence.The protein that is wild type with consistent in fact aminoacid of the protein of wild type or nucleic acid or nucleic acid or the variant example of nucleic acid.

Conventional usage is followed in 20 kinds of conventional aminoacid and abbreviation thereof as used herein.See Immunology-ASynthesis (the 2nd edition, E.S.Golub and D.R.Gren compiles, Sinauer Associates, Sunderland, Mass. (1991)).20 kinds of conventional aminoacid (for example, D-aminoacid), artificial aminoacid (such as a-amino acid, α-two substituted amino acids, N-alkyl amino acid, lactic acid) and other unconventional amino acid whose stereoisomer also can be used as the suitable ingredients that is used for polypeptide of the present invention.Unconventional amino acid whose example comprises: 4-hydroxyproline, Gla, ε-N; N, N-trimethyl lysine, ε-N-acetyl group lysine, O-phosphoserine, N-acetyl group serine, N-formoxyl methionine, 3-Methyl histidine, 5-oxylysine, σ-N-methylarginine and other similar aminoacid and imino acid (for example 4-hydroxyproline).In polypeptide symbol used herein, according to standard purposes and routine, left-hand is to being the amino terminal direction, and right-hand lay is the carboxyl terminal direction.

Similarly, except as otherwise noted, the left hand end of strand polymerized nucleoside acid sequence is 5 ' end, and the left hand end of double-stranded polymerized nucleoside acid sequence is called 5 ' direction.The direction of 5 of nascent rna transcription ' to 3 ' add is called transcriptional orientation, have the sequence identical and for the sequence area on the DNA chain of 5 of rna transcription ' to 5 ' end is called " upstream sequence ", have the sequence identical and be called " downstream sequence " for the sequence area of 3 of rna transcription ' to the DNA chain of 3 ' end with RNA with RNA.

As term " essence concordance " meaning that is applied to polypeptide is when two peptide sequences such as GAP that gives tacit consent to gap weight by use or the optimum comparison of BESTFIT program, two peptide sequences are shared at least 80% sequence identity, be preferably at least 90% sequence identity, more excellent is at least 95% sequence identity, and the best is at least 99% sequence identity.Preferably, replace by conserved amino acid and different incomparable inconsistent residue position.Conserved amino acid is replaced the interchangeability that is meant the residue with similar side chain.For example, the aminoacid group with aliphatic lateral chain is glycine, alanine, valine, leucine and isoleucine; Aminoacid group with aliphatic-hydroxyl side chain is serine and threonine; Having the aminoacid group that contains the amide side chain is agedoite and glutamine; Aminoacid group with aromatic series side chain is phenylalanine, tyrosine and tryptophan; Aminoacid group with basic side chain is lysine, arginine and histidine; And the aminoacid group with sulfur-containing side chain is cysteine and methionine.Preferred conserved amino acid is replaced group: Val-Leu-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and agedoite-glutamine.Yi Zhi polypeptide can be variant in fact.

Variant proteins also comprises the protein with minor variations.Discuss as this paper, considered the minor variations in the aminoacid sequence of antibody that the present invention comprised or immunoglobulin molecules, more excellent at least 80%, 90%, 95% as long as the variation in the aminoacid sequence remains at least 75%, and optimum is 99%.Especially, also considered the displacement of conserved amino acid.

Conservative substitution betides displacement in the aminoacid family relevant with its side chain for those.Aminoacid through gene code is divided into following family usually: (1) acidity-aspartic acid, glutamic acid; (2) alkalescence-lysine, arginine, histidine; (3) nonpolar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and (4) uncharged polar-glycine, agedoite, glutamine, cysteine, serine, threonine, tyrosine.More excellent family is: serine and threonine are aliphatic-hydroxyl family; Agedoite and glutamine are for containing amide family; Alanine, valine, leucine and isoleucine are aliphatic family, and phenylalanine, tryptophan and tyrosine are aliphatic family.For example, but rational expectation is replaced leucine with isoleucine or valine through separating, replace aspartic acid with glutamic acid through separating, with serine through separating the displacement threonine or will can not have material impact, if when especially displacement does not relate to the aminoacid of inside, framework site to the combination or the characteristic of gained molecule with the similar displacement of aminoacid through separating replacement amino acid of structurally associated.Whether the aminoacid variation causes Functional Polypeptides can be easy to measure by the activity specific of calibrating polypeptide derivative.Examine and determine in hereinafter being described in detail.One of ordinary skill in the art can be easy to prepare the fragment or the analog of antibody or immunoglobulin molecules.Near the amino of preferred fragment or analog and the edge that carboxyl-end betides functional domain.Can be by nucleotide and/or amino acid sequence data be compared recognition structure and functional domain with open or proprietary sequence library.Preferably, the comparative approach that uses a computer is discerned and is betided other known structure and/or sequence motif or the predicted protein matter configuration domain in the protein functionally.Become known for discerning the method for the protein sequence that is folded into known three dimensional structure.People such as Bowie, Science 253:164 (1991).Therefore, previous examples shows that the those skilled in the art can discern and can be used for sequence motif and the node configuration that antibody according to the present invention defines the 26S Proteasome Structure and Function domain.

Preferred amino acid replaces with following replacement: reduce proteolysed sensitivity (1), (2) minimizing is to the sensitivity of Oxidation, (3) change for the binding affinity that forms protein misfit thing, (4) change that authorize binding affinity and (5) or other physical chemistry or the functional characteristic of these analog of upgrading.Analog can comprise the various muteins of the sequence except that the peptide sequence of natural generation.For example, can in the sequence (being preferably the polypeptide portion that forms the overseas portion of intermolecular contacting structure) of natural generation, carry out single or multiple aminoacid and replace (being preferably conserved amino acid replaces).Conservative heavy aminoacid is replaced the architectural feature (for example, replacement amino acid should not destroy the spirillum that betides in the parental array, or disturbs other type of the secondary structure that characterizes parental array) that should not change parental array in fact.At Proteins, (Creighton compiles Structures and Molecular Principles, W.H.Freeman and Company, New York (1984)), (C.Branden and J.Tooze compile Introduction to Protein Structure, Garland Publishing, New York, N.Y. (1991)) and people such as Thornton, the polypeptide secondary of this technology identification and the example of tertiary structure are described among the Nature 354:105 (1991).

Term " polypeptide fragment " is meant the polypeptide with amino-end and/or carboxyl-terminal deletion as used herein, but the correspondence position in the sequence of wherein residual aminoacid sequence and the natural generation of drawing from (for example) full length cDNA sequence is consistent.Fragment is generally at least 5,6,8 or 10 amino acid longs, is preferably 14 amino acid longs, more excellently is at least 20 amino acid longs, is generally at least 50 amino acid longs, and even more excellently is at least 70 amino acid longs.Term " analog " is meant the polypeptide that comprises with consistent in fact at least 25 amino acid fragments of the part of the aminoacid sequence of drawing as used herein.Analog is generally at least 20 amino acid longs, is preferably at least 50 amino acid longs and can often grows to the natural generation polypeptide of total length.Fragment and analog all are variant forms.

Peptide analogues is used for medical industry with the characteristic that is similar to the template peptide as non-peptide medicine usually.The non-peptide compound of these types is called " analogies of peptide " and " peptide mimics ", Fauchere, J.Adv.Drug Res.15:29 (1986); Veber and Freidinger TINS, people such as the 392nd page (1985) and Evans, J.Med.Chetn.30:1229 (1987).Usually research and develop these chemical compounds by means of the 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 (promptly with the pattern polypeptide, polypeptide with biochemical characteristic or pharmacological activity), such as human antibodies, but one of them or above peptide connexon by the method known in this technology according to circumstances by being selected from by-CH ₂NH--,--CH ₂S--,-CH ₂-CH ₂--,--CH=CH--(cis and trans),--COCH ₂--,--CH (OH) CH ₂--and-CH ₂The connexon of the group that SO--forms is replaced.Replace one or more aminoacid of unifying sequence (for example, replacing L-lysine) with the D-aminoacid system of same type and can be used for producing more stable peptide with D-lysine.In addition, can be thus in the technology known method produce and comprise the unified sequence or the qualification peptide (Rizo and Gierasch Ann.Rev.Biochem.61:387 (1992)) of consistent unified sequence variation in fact; For example, by adding the inside cysteine residues of the two sulphur bridges of intramolecularly that can form the cyclisation peptide.The analogies of peptide and peptide mimics all are variant forms.

" antibody " or " antibody peptide " is meant the binding fragment of the bonded complete antibody competition of complete antibody or itself and confession specificity.Decompose or chemolysis produces binding fragment by recombinant DNA technology or the enzyme by complete antibody.Binding fragment comprises Fab, Fab ', F (ab ') ₂, Fv and single-chain antibody.Antibody except that " bispecific " or " difunctional " antibody is interpreted as its each binding site unanimity.When excessive antibody will be bonded to counter receptor be reduced by at least by the scale of construction about 20%, 40%, 60% or 80%, and during more generally greater than about 85% (as by in vitro competitive measured in conjunction with calibrating), antibody suppresses receptor in fact and is bonded to counter receptor.Term " epi-position " but comprise any specificity be bonded to immunoglobulin 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 such as aminoacid or carbohydrate or sugared side chain molecule, and have specificity Three Dimensions Structure and specificity charge characteristic usually usually.Epi-position can be " linearity " or " configuration ".In linear epitope, all interaction points between protein and the interactional molecule (such as antibody) take place along proteinic one-level aminoacid sequence is linear.In the configuration epi-position, interaction point intersects generation on the aminoacid on the protein separated from one another.It is said as dissociation constant≤1 μ M, preferred≤100nM and more excellent≤10nM, and even more excellent≤during 1nM, the antibody specificity conjugated antigen.In case the epi-position that needs on definite antigen just may for example be used the antibody of technology generation of the present invention for that epi-position.Perhaps, in discovery procedure, production of antibodies and feature can be illustrated the information of required epi-position.Based on these information, may screen the antibody that is used to be bonded to identical epi-position competitively subsequently.A kind of mode that reaches this purpose is to seek the cross competition research of competitive each other bonded antibody (for example, competition is bonded to antigenic antibody).A kind of high yield that is used for " combination " antibody based on its cross competition is handled and is described in No. 03/48731, international application WO.As by being appreciated by one of skill in the art that, but any predetermined substance that the antibody specificity is bonded to can be an epi-position.Epi-position can comprise antibodies those residues extremely.In one embodiment, epi-position is the EGFRvIII epi-position.In a more excellent embodiment, epi-position is the epi-position described in this description example 4.In one embodiment, epi-position is the epi-position described in the example 14.In one embodiment, epi-position comprises sequence LEEKKGNYVVTD (SEQ ID NO:59).In one embodiment, epi-position comprises sequence EEKKGNYWT (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 needn't be with this sequential combination on single peptide, and these are the residues that form with the interactional epi-position of paratope.To understand as the those skilled in the art, and help why to measure epi-position by residue that produces molecular shape or the space that side chain occupies.Equally, in fact why relevant with epi-position any functional group, Van der Waals interact, the degree of excursion of side chain etc. all can measure epi-position.Therefore, epi-position also can comprise the energy interaction.

Term " paratope " means describes the universal architecture of measuring for the bonded land of epi-position.Whether this structure influence land is bonded to epi-position and bonded mode thereof.Paratope can refer to be responsible for the antigen site that antibody or its fragment are bonded to 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, L3 50, H1 20, H2 40 and H3 60 among Figure 17.In one embodiment, paratope is the antibody district that comprises the CDR sequence that is used for L1, L2, L3, H1, H2 and H3 in the example 16.In one embodiment, paratope is the L1 among Figure 18,110, L2,130, L3,150, the antibody district of H1 120, H2 140 and H3 160.In one embodiment, paratope is the antibody district that comprises the CDR sequence that is used for L1, L2, L3, H1, H2 and H3 in the example 18.In one embodiment, paratope comprises sequence listed in the example 18.In one embodiment, paratope comprises and the interactional residue of epi-position, as shown in Figure 19 A and Figure 19 B.The entity black structures is the 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 that is selected from the group that is made up of following each residue: Tyr 172Arg, Arg101Glu, Leu99Asn, Leu99His, Arg101Asp, Leu217Gln, Leu99Thr, Leu217Asn, Arg101Gln and Asn35Gly.To understand as the those skilled in the art, the paratope of any antibody or its variant all can be measured by the mode that the application's case is stated.If residue prediction can be contributed 10% binding energy, just think residue " important ".In one embodiment, if residue prediction can be contributed 2% binding energy, just think residue " important ".In one embodiment, if residue prediction can be contributed 50% binding energy, just think residue " important ".In one embodiment, if residue prediction and surface of epitopes or paratope surface interaction are just thought residue " important ".In one embodiment, cause in conjunction with loss, just think residue " important " if change residue.

Term " specificity " or " preferentially " are bonded to, or similar wording and do not mean that the expression antibody be bonded to that epi-position only.On the contrary, the meaning is that antibody or its variant can be bonded to that epi-position, and its degree is higher to the degree of at least a other material that antibody exposed than antibodies.In one embodiment, the specificity binding antibody will be (tighter, or lower K to be bonded to EGFRvIII protein than it to the bigger affinity of EGFR protein _D).For example, the specificity binding antibody in conjunction with will be tighter increase by 1% at least, 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.

Aminoacid, numbering, amino acid whose abbreviated form, for example Leu217Gln represents to number the sudden change of aminoacid from first seed amino acid to second seed amino acid.Therefore, the Tyr172Arg meaning is when the protein of wild type has the tyrosine of 172 positions, and sudden change has the arginine of 172 positions.

Mixture, the biopolymer of term used herein " medicament " expression chemical compound, chemical compound or the extract that forms from biological substance.

" mammal " used herein refers to think mammiferous any animal.Mammal is preferably human.

The digestion that contains the antibody of enzyme (papain) causes the Fab of two kinds of unanimities, is also referred to as " Fab " fragment and " Fc " fragment, and it does not have antigen-binding activity, but has crystallizing power.The digestion that contains the antibody of enzyme (pepsin) causes F (ab ') ₂Fragment, wherein two arms of antibody molecule keep connecting, and comprise two antigen binding sites.F (ab ') ₂Fragment has crosslinked antigenic ability.

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

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

Term " mAb " refers to monoclonal antibody.

The description coding of the antibody sequence that produces for the XenoMax method is as follows: " AB "-refer to antibody, " EGFRvIII "-refer to binding specificity of antibody, " X " refers to the XenoMouse derived from mice, " G1 "-refer to IgG1 isotype or " G2 "-refer to IgG2 isotype, last three numerals refer to that antibody is from its deutero-unicellular numbering, for example: AB-EGFRvIII-XG1-095 has the antibody of IgG1 isotype XenoMouse mice to the binding specificity of EGFRvIII, and cell is numbered 95.

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

As follows for the description of the deutero-antibody sequence of hybridoma coding: " AB "-refer to antibody, " EGFRvIII "-refer to the binding specificity of antibody, " X " refers to the XenoMouse derived from mice, " 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 deutero-pure lines of antibody, for example: AB-EGFRvIII-XG1K-13.1.2.

The test section that " labelling " used herein or " through labelling " but refer to adds polypeptide, for example, labelled with radioisotope, fluorescent labelling, enzyme labelling, chemiluminescent labeling or biotinyl.Radiosiotope or radionuclide can comprise ³H, ¹⁴C, ¹⁵N, 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, luciferase, alkali phosphatase.

Term " medical agent or medicine " refers to can produce the chemical compound or the compositions of ideal treatment when suitably granting the patient as used herein.As illustrated, use other technical terms of chemistry in this article according to the conventional usage in this technology by The McGraw-Hill Dictionary of Chemical Terms (Parker, S. compiles, McGraw-Hill, San Francisco (1985)).

As used herein " pure in fact " meaning be the target material for the main material that exists (promptly, in mole, all exist in a large number than any other individual substance in the compositions), and preferably, purified in fact part accounts for the compositions of all polymer substances of existence at least about 50% (in mole) for its material that hits.Generally speaking, pure in fact compositions will account for all polymer substances of existing in the compositions more than about 80%, more excellent in about 85%, 90%, 95%, 99% and 99.9%.Optimally, the target material is purified to is essentially homogenizing (detecting less than the polluter in the compositions by the conventional sense method), wherein compositions is made up of single polymer substance basically.

Term " patient " comprises human and veterinary person under inspection.

Term " SLAM_Technology " refers 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 the use (as mentioned below) of SLAM Technology and XenoMouse_ mice.

Antibody structure

Known basic antibody structure unit comprises tetramer.Each tetramer is made up of the polypeptide chain of two pairs of unanimities, and each is to having one " light chain " (about 25kDa) and one " heavy chain " (about 50-70kDa).Amino-the end portion of each chain comprises main about 100 to 110 or more a plurality of amino acid whose variable region of being responsible for antigen recognition.Carboxyl-the end portion of each chain defines the main constant region of being responsible for effector function.Human light chain 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 a plurality of amino acid whose " J " district in light chain and heavy chain kind variable region and constant region, wherein heavy chain also comprises about 10 or more a plurality of amino acid whose " D " district.Generally see, FundamentalImmunology the 7th chapter (Paul, W. compiles, and the 2nd edition, Raven Press, N.Y. (1989)).The right variable region of each light/heavy chain forms antibody combining site.

Therefore, complete antibody has two binding sites.Except difunctional or bi-specific antibody, two binding sites are identical.

These chains are all showed the conservative relatively identical universal architecture of framework region (FR) that is connected by three hypervariable regions, are also referred to as complementary determining region or CDR.Each CDR to two chains compares by framework region, makes to be bonded to specificity epitope.To the C-end, light chain and heavy chain all comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domain from the N-end.It is according to Kabat Sequences ofProteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia ﹠amp that the aminoacid of each domain distributes; Lesk J.Mol.Biol 196:901-917 (1987); People such as Chothia, the definition among the Nature342:878-883 (1989).

Bispecific or bifunctional antibody be have two different heavy chains/light chains to the artificial hybrid antibody of two different binding sites.Bi-specific antibody can comprise that the fusion of hybridoma produces with the segmental method that is connected of Fab ' by multiple.See for example Songsivilai ﹠amp; Lachmann Clin.Exp.Immunol.79:315-321 (1990), people such as Kostelny, J.Immunol.148:1547-1553 (1992).The generation of bi-specific antibody is compared with the generation of conventional antibody and be can be relative labor-intensive technology, and the output of bi-specific antibody and purity are lower usually.Bi-specific antibody does not close the segmental form in site and has (for example, Fab, Fab ' and Fv) to have unijunction.Except the universal architecture aspect of antibody, the more special interaction between paratope and the epi-position can be checked by frame mode.In one embodiment, the CDR structure forms paratope, and antibody can be bonded to epi-position through it.The structure of this paratope can be measured in several ways.Can use traditional structural testing method, such as NMR or x-radiocrystallography.These methods can be checked independent paratope, or the structure when it is bonded to epi-position.Perhaps, molecular model can produce in computer simulation.Structure can be by means of commercially available encapsulation, and (San Diego, InsightII model encapsulation CA) produces by the homology model such as Accelrys.In brief, can utilize the check antibody sequence to seek the proteinic data base of known structure, such as, protein structure data base (Protein Data Bank).After identification had the homology protein of known structure, these homology protein can be used as model template.Each may template can be through comparison, produce in these templates sequence alignment thus based on structure.Can the antibody sequence of unknown structure and the comparison of these templates has unknown structure antibody with generation molecular model will be had subsequently.As be understood by those skilled in the art that, there is the multiple alternative method that in computer simulation, produces described structure, can use wherein any.For example, can use people such as being similar to Hardman, employing QUANTA (PolygenCorp., the Waltham of promulgation, Mass.) and CHARM (Brooks, B.R., Bruccoleri, R.E., 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 technology of technology described in 958, No. 708.

Not only whether the possibility paratope is bonded to epi-position to the shape of paratope and good degree is very important for measuring, and the interaction between epi-position and the paratope itself is the bulk information source of design variable antibody.As be understood by those skilled in the art that, there is this interactional mode of multiple research.A kind of mode is to use the structural model of possibility generation as indicated above, and use (Accelrys subsequently such as InsightII, San Diego, program CA), it has one can carry out the expansion module that Monte Carlo studies to configuration between paratope and its epi-position and space, location.The result can assess epi-position and interactional place of paratope and mode.In one embodiment, have only the fragment of epi-position or variant to be used to assist to measure relevant interaction.In one embodiment, whole epi-position is used for interactional modeling between paratope and the epi-position.As be understood by those skilled in the art that these two kinds of distinct methods have different advantages and deficiency.For example, only use the epi-position fragment to make and to carry out more detailed check to the possible variant of each side chain, and need not to expend the plenty of time.On the other hand, by only using the epi-position fragment, or only use epi-position to replace whole protein, the feature of the segmental feature of epi-position and whole epi-position may be different, therefore may be increased in and calculate the risk that misleads in the modeling process.In one embodiment, use two kinds of methods with the cross-check result with limited extent.In a preferred embodiment, if use the epi-position variant, but optimization is so that the epi-position variant comprises the most important residue of epi-position.The concordance of most important residue can be measured in several ways, for example described in example 4 of the present invention and 14.

Which by using the structure of these generations, can measure in the interaction of residue between epi-position and paratope most important.Therefore, in one embodiment, can be easy to select to change which residue change antibody in conjunction with feature.For example, apparent from extended model, the side chain of some residue can spatially stop the combination of epi-position in the epi-position, thus these residues is changed into the residue with less side chain unanimity.

This can measure in many ways.For example, two models of need observation are assessed the interaction based on functional group and adjacent group.Perhaps, as indicated above, the repeating groups that can carry out epi-position and paratope is right, to obtain more favourable energy interaction.These that also can measure multiple antibody variants interact and measure the alternative that antibody wherein can be bonded to epi-position.Also how the incompatible mensuration of various model group can be changed the antibody that antibody structure obtains to have required special characteristic.

The model of said determination can be tested by various technology.For example, interaction energy can measure with decision to need further which kind of variant of check by program discussed above.Use coulomb and Van der Waals to interact and measure the interaction energy of epi-position and variant paratope again.Also use and observe predetermined variation in the antibody structure at the site of sudden change and in fact whether can cause required variation in conjunction with feature.Perhaps, can change epi-position confirm that model is correct or be used to measure may between paratope and epi-position, take place generally in conjunction with theme.

Which kind of variation that the above-mentioned method that is used for modeling structure can be used for measuring protein structure will cause the specific required feature of antibody.In the protein structure which kind of be these methods can be used for measuring and change the required feature can not cause antibody.

As be understood by those skilled in the art that, although these models will provide necessary guide for forming antibody of the present invention and variant thereof, but still need carry out the conventionally test in the computer simulation model, may be by in vitro research.In addition, as be understood by those skilled in the art that any upgrading also can the antagonist activity have other seondary effect.For example, although expection causes bigger bonded change can cause bigger combination, can cause that also other can reduce or change the structural change of antibody activity.For whether being very common in the field under being determined at of this situation, and can measure in several ways.For example, in example 21, activity can be tested by ELISA.Perhaps, can come specimen by using the surface plasma body resonant vibration device.

The variant antibody of antibodies and confession excellent combination

In one embodiment, above-mentioned model is used to increase 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).Perhaps, antibody can dissociate from epi-position more slowly, and therefore has lower dissociation constant (K _D), or the K of epi-position-paratope _DTherefore be worth lessly, make that the combination degree between epi-position and the paratope is higher.

In certain embodiments, design variable antibody is with opposite feature combination.That is, antibody is not combined closely or not combination fast.

In other embodiments, the K of variant antibody _DDifferent with the antibody of wild type, but variant antibody has more specificity for defined epitope.This means that the paratope through designerantibodies has the low risk that is bonded to other epi-position.Antibody can have the further feature that has changed.For example, variant to non-specific antibody in conjunction with having more immunity, even or when antibody exists with high concentration, in solution, still keep solvation.This variant can be present in the antibody of being discussed.For example, although hereinafter the higher concentration of Jian Yan some variant antibody caused in the Biacore experiment slower in conjunction with component (for example, 13.1.2mAb) described than slow component even some variant is not still showed under same concentrations, L217N-2.1 for example.Can form variant, and measure it subsequently after tested in fact whether with required feature combination by the model prediction of above measuring.Can select to have the mutant of big total interaction energy for further test with epi-position.Interaction energy can be measured in many ways, and one of them is as indicated above.

These variants can be tested in many ways.Exemplary selection comprises, and is not limited to KinExA (for example, the patent the 5th of Lackie promulgation, 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 data fitting be various theoretical curves and measure k thus _a, k _dAnd K _DAnd the fabricated parts of other characteristic.Importantly, it should be noted that these curve fittings are that result data is not got rid of the probability that has some deviations.Therefore, relevant association, disassociation and equilibrium constant not only can be observed by these curve fitting mechanism, also can directly compare mutually according to those skilled in the art's knowledge.

The humanization of human antibodies and antibody

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

Human gene's seat of megabasse size and the ability that is introduced into mice kind system are for illustrating greatly or providing strong approach through the function ingredients of the locus of original mappings and the useful model of generation human diseases among clone and the reconstruct YAC.In addition, use this technology that the expression and adjusting, they and the information transmission of other system and the related particular views that provides in disease is brought out and made progress thereof that its human equivalent can be the human gene's product between the period of development is provided the mice locus.

The important practical application of this strategy is mouse humoral immune system " humanization ".Human immunoglobulin (Ig) locus is introduced the sequencing of studying antibody that offered an opportunity in the mice of inactivation of endogenous Ig gene expresses and combination and the active potential mechanism in the B-cell development thereof.In addition, this strategy can be generation complete human monoclonal antibody (mAb)-a kind of important milestone in human diseases kind realization Antybody therapy prospect desirable source is provided.Expect that complete human antibodies can be minimised as mice or mouse-derived mAb with immunity and anaphylaxis essence, and therefore increased effect and the safety of giving antibody through throwing.Use whole mankind's antibody expection and can be the chronic and recurrent human diseases of treatment substantial advantage is provided, such as inflammation, autoimmunity and cancer, these all need to throw repeatedly and give antibody.

A kind of method at this purpose is that design is not enough to be used to have the mouse species that the big segmental mouse antibodies of human Ig locus is produced, to expect that these mices can produce the big instruction system of human antibodies under the situation of no mouse antibodies.Big human Ig fragment can keep the big variable gene multiformity and the suitable adjustable of antagonist production and expression.Be used for antibody variation and the mice instrument of selecting and for the shortage of human protein's immunologic tolerance, the human antibodies instruction system that reproduces in these mouse species should produce the antibody that all has (comprising human antigen) high-affinity for any interested antigen by employing.Use hybridoma technology, can be easy to production and selection and have the human mAb of required specific antigen-specificity.As 1994 disclosed, show this general strategy (seeing people such as Green, Nature Genetics 7:13-21 (1994)) in conjunction with first generation XenoMouse strain.The XenoMouse strain is designed to have the 245kb that contains human heavy chain locus and κ light chain gene seat respectively and the kind of 190kb size is the segmental yeast artificial chromosome of configuration (YAC), and it contains nuclear variable core constant region sequence Id.Contain the human Ig of YAC verified with reset compatible with the mice system of expressing and the replaceable mice Ig gene of inactivation that is for antibody.This can be confirmed by its human commands system and the human mAb ability of generation antigen-specificity that bring out the similar adult of B-cell development, the complete human antibodies of generation.These results show that also introducing contains the major part of the human Ig locus of bigger numbering V gene, other adjusting element and human Ig constant region can be summarized the complete instruction system in fact, and it is a feature with the human humoral response for infection and immunity.People's such as Green works extends to the human heavy chain locus of introducing the megabasse size respectively and the kind of κ light chain gene seat is a configuration YAC fragment by introducing greater than about 80% human antibodies instruction system recently.See people such as Mendez, No. the 08/759th, 620, Nature Genetics15:146-156 (1997) and U.S. patent application case applied for 1996 on Decembers 3.

Resulting from the following patent of XenoMouse mice further discussed and narration: No. the 07/466th, 008, U.S. patent application case applies for January 12 nineteen ninety; The 07/610th, No. 515, apply for November 8 nineteen ninety; The 07/919th, No. 297, apply on July 24th, 1992, the 07/922nd, No. 649, apply on July 30th, 1992; The 08/031st, No. 801, apply on March 15th, 1993; The 08/112nd, No. 848, apply on August 27th, 1993; The 08/234th, No. 145, apply on April 28th, 1994; The 08/376th, No. 279, apply for January 20 nineteen ninety-five; The 08/430th, No. 938, apply for April 27 nineteen ninety-five; The 08/464th, No. 584, apply for June 5 nineteen ninety-five; The 08/464th, No. 582, apply for June 5 nineteen ninety-five; The 08/463rd, No. 191, apply for June 5 nineteen ninety-five; The 08/462nd, No. 837, apply for June 5 nineteen ninety-five; The 08/486th, No. 853, apply for June 5 nineteen ninety-five; The 08/486th, No. 857, apply for June 5 nineteen ninety-five; The 08/486th, No. 859, apply for June 5 nineteen ninety-five; The 08/462nd, No. 513, apply for June 5 nineteen ninety-five; The 08/724th, No. 752, apply on October 2nd, 1996 and the 08/759th, No. 620, apply for December in 1996 3 days 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 Japan Patent the 3 068 180 B2 number, the 3 068 506 B2 number and the 3 068 507 B2 number.People, Nature Genetics 15:146-156 (1997) and Green and Jakobovits J.Exp.Med.188:483-495 (1998) such as also visible Mendez.

Also visible European patent EP 0 463 151 B1 numbers, authorize open on June 12nd, 1996, No. 94/02602, international application WO, open on February 3rd, 1994, international application WO96/34096 number, open on October 31st, 1996, No. 98/24893, WO, open on June 11st, 1998, No. 00/76310, WO, in December in 2000 No. 03/47336, WO was disclosed in 21.In another approach, other company comprises that GenPharm International company uses " little locus " method.In little locus method, external source Ig locus imitates by the material (indivedual gene) that comprises from the Ig locus.Therefore, one or more V _HGene, one or more D _HGene, one or more J _HGene, μ constant region and second constant region (being preferably the γ constant region) form and are used to insert the intravital structure of animal.This method is described in the following patent: No. the 5th, 545,807, people's such as Surani United States Patent (USP) and each patent all belong to the United States Patent (USP) the 5th of Lonberg and Kay, 545, No. 806, the 5th, 625, No. 825, the 5th, 625, No. 126, the 5th, 633, No. 425, the 5th, 661, No. 016, the 5th, 770, No. 429, the 5th, 789, No. 650, the 5th, 814, No. 318, the 5th, 877, No. 397, the 5th, 874, No. 299 and the 6th, 255, No. 458, the United States Patent (USP) the 5th, 591 of Krimpenfort and Berns, No. 669 and the 6th, No. 023.010, people's such as Berns United States Patent (USP) the 5th, 612, No. 205, the 5th, 721, No. 367 and 5,789, No. 215, and the United States Patent (USP) the 5th of Choi and Dunnand, 643, No. 763, and GenPharm International U.S. patent application case the 07/574th, No. 748, apply for August 29 nineteen ninety, the 07/575th, No. 962, apply for August 31 nineteen ninety, the 07/810th, No. 279, applied for December in 1991 17, the 07/853rd, No. 408, apply on March 18th, 1992, the 07/904th, No. 068, apply on June 23rd, 1992, the 07/990th, No. 860, applied for December in 1992 16, the 08/053rd, No. 131, apply on April 26th, 1993, the 08/096th, No. 762, apply on July 22nd, 1993, the 08/155th, No. 301, apply on November 18th, 1993, the 08/161st, No. 739, applied for December in 1993 3, the 08/165th, No. 699, applied for December in 1993 10, the 08/209th, No. 741, apply on March 9th, 1994.Also visible European patent the 0 546 073 B1 number, 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.Further see people such as Taylor, 1992; People such as Chen, 1993; People such as Tuaillon, 1993; People such as Choi, 1993; People such as Lonberg, (1994); People such as Taylor, people such as (1994) and Tuaillon, (1995); People such as Fishwild, (1996).

Kirin has also shown produced human antibodies in mice, has wherein merged by microcell and has introduced large stretch of chromosome or whole chromosome.See European patent application the 773 No. 288 and the 843 No. 961.Xenerex Biosciences is is researching and developing a kind of technology that is used for potential generation human antibodies.In this technology, reconstitute the SCID mice with people's quasi-lymphocyte (for example, B and/or T cell).Mice is with after antigen immune, and the antigenic immunoreation 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.

Human resisting-mouse antibodies (HAMA) reaction is with chimeric or other humanized antibody of commercial production guiding preparation.Although chimeric antibody has human constant region and Muridae variable region, still wish can be observed some human resisting-chimeric antibody (HACA) reaction, especially in chronic or many-dosage of antibody uses.Therefore, lost efficacy, still be desirable to provide whole person's antibody-like of antagonism EGFRvIII for the consideration and/or the influence that make HAMA or HACA reaction.

Antybody therapy

As if as described herein, the function of EGFRvIII antibody is very important at least a portion of its operator scheme.For example, with regard to function, the meaning is the activity of EGFRvIII antibody in operating with EGFRvIII.Therefore, in some aspects, wish that antibody fixedly fill-in also replenishes cytotoxic lymphocyte, participates in thus among CDC and the ADCC in conjunction with production of antibodies as the treatment material standed for that resists EGFRvIII.There is multiple antibody isotype, includes, but is not limited to following: Muridae IgM, Muridae IgG2a, Muridae IgG2b, Muridae IgG3, human IgM, IgG 1, IgG 3 and human IgA with identical function.Again, wish that antibody can activate the antibody-mediated cytotoxic reaction (ADCC) of dependence by the effector lymphocyte that the Fc receptor is engaged in such as natural killer (NK) cell in conjunction with production of antibodies as the treatment material standed for that resists EGFRvIII.But there is the antibody isotype of multiple ADCC, includes, 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 needn't 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 of using the routine techniques conversion of knowing in this technology subsequently.These technology comprise uses direct recombinant technique (for example, seeing United States Patent (USP) the 4th, 816, No. 397) and cell-cell-fusion techniques (for example, see United States Patent (USP) the 5th, 916, No. 771 and the 6th, 207, No. 418).

In cell-cell technology, preparation has myeloma or other cell strain and another kind of myeloma or other cell strain with light chain of the heavy chain that contains any required isotype.These cells can be through merging subsequently, and the complete antibody of separable expression cell line.

For example, some anti-EGFR vIII antibody of this paper discussion is human anti-EGFR vIII IgG1 antibody.If this antibody has the required combination to the EGFRvIII molecule, isotype should be easy to conversion to produce human IgM, IgG 3 or IgG A, although still have identical variable region (it has defined specificity and some affinity thereof of antibody).These molecules (comprising IgG1) are fixing fill-in and participate in CDC subsequently, and if comprise IgG1 or IgG3 constant region, these molecules also can participate in relying on antibody-mediated cytotoxic reaction (ADCC) by replenishing cytotoxic lymphocyte.

Therefore, owing to produced the antibody material standed for that reaches required " structure " mentioned above attribute, therefore, it can have some required " function " attribute at least by the isotype conversion usually.The design of other treatment and generation

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

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

For example, in conjunction with the generation of senior antibody therapy, wherein fill-in is fixing is desirable attributes with replenishing of cytotoxic lymphocyte, may kill by using bispecific, immunotoxin or labelled with radioisotope strengthen cell.

For example,, can produce and comprise following bi-specific antibody: (i) two kinds of antibody, a kind of specificity and another kind of specificity that has the second kind of molecule that combines that has EGFRvIII in conjunction with bi-specific antibody; (ii) chain has specificity to EGFRvIII and the second chain has specific monoclonal antibody body to second kind of molecule; Or (iii) has specific single-chain antibody for EGFRvIII and another kind of molecule.These bi-specific antibodys can use knows the technology generation, for example, in conjunction with (i) and (ii), for example see Immunol Methods 4:72-81 (1994) and above-mentioned Wright and Harris, and combination (iii), for example see people such as Traunecker, Int.J.Cancer (Suppl.) 7:51-52 (1992).Under each situation, second species specificity can be given Fc chain activated receptor, include, but is not limited to CD16 or CD64 (for example sees, people such as Deo, 18:127 (1997)), CD3 (Micromet ' sBiTE technology) or CD89 (for example see, people such as Valerius, Blood 90:4485-4492 (1997)).Bi-specific antibody according to aforementioned preparation may kill the cell of expressing EGFRvIII, and effective those cells of EGFRvIII antibody especially wherein of the present invention.

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

For example see Vitetta Immunol Today 14:252 (1993).Also for example see United States Patent (USP) the 5th, 194, No. 594.In conjunction with the antibody of preparation labelled with radioisotope, these also can be easy to use the technology of knowing in this technology to prepare through upgrading antibody.For example see people such as Junghans, among the Cancer Chemotherapy and Biotherapy 655-686 (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 (RE 35,500), 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 effective those cells of antibody especially wherein described herein.

But the quicker combination of designerantibodies, or slower in the epi-position disassociation, and therefore antibody itself can be designed to therapeutic agent.For example, antibody can be used for therapeutic agent is given in patient's throwing through changing feature.The treatment immune conjugate

As understand, kill expression and can pass through in the cell of the bonded molecule of specific binding molecules (such as antibody) specificity extremely useful deciding target with the bonded antibody of medicine, toxin or other molecule (immune conjugate or immunotoxin).As indicated above, unknownly as yet on any normal structure, expressed EGFRvIII.In addition, the EGFRvIII demonstration significantly is expressed in the multiple human tumor.Therefore, EGFRvIII is the molecule that attracts most attention that is used for deciding with immunotoxin target.

Occurred many about attempt specificity decide the report that target has the tumor cell of monoclonal antibody-drug conjugates (people such as Sela, among the Immunoconjugates 189-216 (C.Vogel, ed.1987); People such as Ghose are among the TargetedDrugs 1-22 (E.Goldberg compiles, nineteen eighty-three); People such as Diener are among the Antibody Mediated DeliverySystems 1-23 (J.Rodwell compiles, 1988); People such as Pietersz are among the Antibody Mediated DeliverySystems 25-53 (J.Rodwell compiles, 1988); People such as Bumol are among the Antibody Mediated DeliverySystems 55-79 (J.Rodwell compiles, 1988)).Cytotoxic drugs sees that with multiple Muridae monoclonal body combines such as methotrexate, daunorubicin, amycin, vincristine, vinblastine, melphalan (melphalan), ametycin and chlorambucil.In some cases, drug molecule is connected to antibody molecule, these intermediate supporting agent molecule such as blood albumin (people such as Garnett, Cancer Res.46:2407-2412 (1986) by intermediate supporting agent molecule; People such as Ohkawa, Cancer Immumol.Immunother.23:81-86 (1986); People such as Endo, Cancer Res.47:1076-1080 (1980)), dextran (people such as Hurwitz, Appl.Biochem.2:25-35 (1980); People such as Manabi, Biochem.Pharmacol.34:289-291 (1985); People such as Dillman, Cancer Res.46:4886-4891 (1986); 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); People such as Kato, J.Med.Chem.27:1602-1607 (1984); People such as Tsukada, Br.J.Cancer 52:111-116 (1985)).

Adopt multiple connexon technology being used to preparing these immune conjugates, and studied dissociable and not dissociable connexon.Yet, under most of situation, if drug molecule can just can only be observed the complete cytotoxicity potential of medicine from being that the conjugate of non-upgrading form discharges deciding target site.

One of dissociable connexon that has been used to prepare antibody-drug conjugates is the acid-unstable connexon based on suitable-aconitic acid, and it utilizes the sour environment of different intracellular compartments, such as the endosome and the lysosome that run in the endocytosis of regulating at receptor.Shen and Ryser introduce the conjugate (Biochem.Biophys.Res.Commun.102:1048-1054 (1981)) that this method prepares daunorubicin and macromole supporting agent.Yang and Reisfeld use constructed anti--melanoma antibody (J.Natl.Canc.Inst.80:1154-1159 (1988)) that daunorubicin is coupled to.Recently, 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.

Relate to by the another kind of method of people such as Trouet research daunorubicin is connected to antibody (Proc.Natl.Acad.Sci.79:626-629 (1982)) through peptide space arm.This is not carry out have the prerequisite that medicine discharges from this conjugate by the effect of lysozyme peptidase under.

Yet, disclosing in the cytotoxicity test in vitro, antibody-drug conjugates seldom reaches the cytotoxicity usefulness identical with free non-coupling drug.This shows, the mechanism poor efficiency that drug molecule discharges from antibody.In the scope of immunotoxin, the conjugate demonstration that the two sulphur bridges between monoclonal antibody and catalytic activity archon form has more cytotoxicity than the conjugate that contains other connexon.See people such as Lambert, J.Biol.Chem.260:12035-12041 (1985); People such as Lambert are among the Immunotoxins 175-209 (A.Frankel compiles, 1988); People such as Ghetie, Cancer Res.48:2610-2617 (1988).This is owing to the high IC of the favourable glutathion of effective disassociation of the cystine linkage between antagonist molecule and the toxin.However, only have on a small quantity about using two sulphur bridges to prepare the report example of the conjugate between medicine and the macromole.People such as Shen have described methotrexate and have been converted into mercapto buserelin derivant, then through cystine linkage and poly--D-lysine coupling (J.Biol.Chem.260:10905-10908 (1985)).In addition, one report has been described the preparation (people such as Menendez of the conjugate of the toxin medicine calicheamycin that contains trisulfide and antibody, Fourth International Conference on MonoclonalAntibody Immunoconjugates for Cancer, San Diego, Abstract 81 (1989)).Another report has been described the preparation (people such as Hinman, 53Cancer Res.3336-3342 (1993)) of the conjugate of the toxin medicine calicheamycin that contains trisulfide and antibody.

A reason that lacks the antibody-drug conjugates that disulphide connects is to have to contain the unavailability of cytotoxic drugs of sulphur atom that can be easy to be used for medicine is connected to through disulphide bridges the part of antibody.In addition, existing medicine is under the nondecreasing situation of its cytotoxicity potential, and it is difficult to carry out chemical modification.

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

Maytansinoid is the height cytotoxic drugs.Maytansine is at first separated in the shrub tingia Folium Mayteni hookeri of East Africa by people such as Kupchan, and it shows the cytotoxicity (United States Patent (USP) 3rd bigger 100 to 1000 times than conventional cancer chemotherapeutic agents (as methotrexate, daunorubicin and vincristine), 896, No. 111).Found afterwards that certain micro-organisms also produced maytansinoid, such as the C-3 ester (United States Patent (USP) the 4th, 151, No. 042) of isovaleric acid maytansine ester and isovaleric acid maytansine ester.The synthetic C-3 ester of isovaleric acid maytansine ester and the analog of isovaleric acid maytansine ester (people such as Kupchan, J.Med.Chem.21:31-37 (1978) have also been reported; People such as Higashide, Nature270:721-722 (1977); People such as Kawai, Chem.Pharm.Bull.32:3441-3451 (1984)).The example of the analog of C-3 ester isovaleric acid maytansine prepared therefrom ester comprises on the aromatic ring (for example, dechlorination) or at C-9, C-14 (for example, the hydroxylating methyl), C-15, C-18, C-20 and C-4,5 places are through the isovaleric acid maytansine ester of upgrading.

Natural generation and synthetic C-3 ester can be divided into two groups:

(a) have simple carboxylic C-3 ester (United States Patent (USP) the 4th, 248, No. 870, the 4th, 265, No. 814, the 4th, 308, 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 derivant of N-methyl-L-alanine.

The cytotoxicity of the ester of discovery group (b) is more much bigger than the cytotoxicity of the ester of group (a).

Maytansine is a mitotic inhibitor.Reported and in vivo handled the L1210 cell with maytansine and cause 67% cell aggregation in mitosis.Reported undressed and shown mitotic index between 3.2% to 5.8% people such as (, 43 Comparative Leukemia Research 1975, Bibl.Haemat.495-500 (1976)) Sieber than pair cell.The experiment of sea urchin egg and Carnis Mactrae ovum shows, and maytansine disturbs microtubule to form to suppress mitosis people such as (, Science189:1002-1005 (1975)) Remillard by the polymerization that suppresses microtubular protein (tubulin).

In vitro, find with 10 ^-3To 10 ^-1The maytansine of μ g/ μ l dosage can suppress P388, L1210 and LY5178 Muridae leukaemia suspension, and wherein the P388 cell strain is the most responsive.Shown that also maytansine is the activity inhibitor that human nasopharyngeal carcinoma cell is in vitro grown, and report human acute lymphoblastic leukemia cell strain CEM can be by being low to moderate 10 ^-7The concentration of mg/ml suppresses people such as (, Biochem.Pharmacol.24:1735-1738 (1975)) Wolpert-DeFillippes.

In vivo, maytansine also shows to have activity.Be presented at the tumor growth that suppresses P388 lymphoblast leukemia system in 50 to the 100 multiple dose scopes, this shows to have high therapeutic index; L1210 mouse leukemia system, human Lewis lung cancer system and human B-16 melanotic cancer system also show remarkable inhibiting activity (Kupchan, Ped.Proc.33:2288-2295 (1974)).

The method of coupling maytansinoids and cell bonding agent (such as antibody) comprises two reactions steps at present.At first with cell bonding agent (for example antibody) with cross-linking reagent (such as N-succinimido pyridine radicals dithio propionic ester (SPDP)) upgrading the dithio pyridine radicals 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, the reactive maytansinoid that will have mercapto ((is called N such as DMl in form ^{2 '}-deacetylate-N ^{2 '}-(3-sulfydryl-1-oxopropyl))-and maytansine) be added in upgrading antibody as initial reagent, cause the displacement of sulfo-pyridine radicals in the upgrading antibody, reach the generation (United States Patent (USP) the 5th, 208, No. 020) of the cytotoxicity maytansinoid/ antibody coupling matter that connects by disulphide.At United States Patent (USP) the 6th, 441, a process that is used for coupling maytansinoids is described in No. 163.

(Cambridge MA) can obtain immunotoxin technology based on Maytansinoid by ImmUnogen Corporation.Another kind of important toxin technology is based on the auristatin toxin.The aplysiatoxin Dolastatin 10 that Auristatins obtains derived from the sea hare by sea, the Indian Ocean is as effective cell growth inhibitory substance.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).By the colleague of Dr.Pettit and Arizona State university separate and synthetic various auristatine derivants after tested and demonstrate the height toxicity of pair cell.See people such as Pettit, antitumor agent 337.Synthesizing of aplysiatoxin 10 structure upgradings.Anticancer Drug Des.10 (7): 529-44 (1995), people such as Woyke.Active and the back antifungic action in the extracellular of effective aplysiatoxin 10 structure upgrading auristatin PHE.AntimicrobialAgents and Chemotherapy.45:3580-3584 (2001), people such as Pettit.The activity specific of aplysiatoxin 10 and peptide derivant antagonism neogenesis cryptococcus.Antimicrobial?Agents?and?Chemotherapy.42：2961-2965(1998)，Woyke。Development of the three-dimensional of microtubule and auristatinPHE are to the influence of microtubule globality and nuclear location during the neogenesis cryptococcus cell cycle.Submitted，Antimicrobial?Agents?andChemotherapy。

Recently, researched and developed as if quite effective other auristatin derivant when on antibody, transmitting as payload.For example, shown monomethyl auristatin E (MMAE) with the tumor specific antibody coupling time as for effective toxin of tumor cell.People such as Doronina, Development of potent monoclonal antibodyauristatin conjugates for cancer therapy.Nature Biotechnology. (2003) (online getting); People such as Francisco, cAC10-vcMMAE, open before an anti-CD30-monomethyl auristatin E conjugatewith potent and selective antitumor activity.Blood. (2003) the May 8[E printing] .Epub2003 Apr 24 (online getting).Except that the toxicity of auristatin molecule, research has shown that the conjugate that connects through peptide is more stable, and therefore bigger and toxicity is littler to the specificity of normal structure than other connexon technology in buffer agent and blood plasma.People such as Doronina, Development of potent monoclonal antibody auristatinconjugates for cancer therapy.Nature Biotechnology. (2003) (online getting); People such as Francisco, cAC10-vcMMAE, open before an anti-CD30-monomethyl auristatin E conjugate with potent andselective antitumor activity.Blood. (2003) the May 8[E printing]. ' Epub 2003 Apr 24 (online getting).

These connexons are based on the branched chain peptide design and comprise for example mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugate.People such as Doronina, Development of potent monoclonalantibody auristatin conjugates for cancer therapy.Nature Biotechnology. (2003) (online getting); People such as Francisco, cAC10-vcMMAE, open before an anti-CD30-monomethyl auristatin Econjugate with potent and selective antitumor activity.Blood. (2003) the May 8[E printing]. ' Epub 2003 Apr 24 (online getting). these designs and coupling technology are by the following stated; Monoclonal antibody conjugates of doxorubicin prepared with branched peptidelinkers:inhibition of aggregation by rnethoxytriethyleneglycol chains.J Med Chem.45 (19) such as people such as King: the people's such as 4336-43 (2002) and Dubowchik Cathepsin B-sensitive dipeptide prodrugs.2.Models of anticancer drugs paclitaxel (Taxol), mitomycin C and doxorubicin.BioorgMed Chem Lett.8 (23): 3347-52 (1998). (Seattle WA) can get by Seattle Genetics Corporation based on aforesaid Auristatin E-base immunotoxin technology.

As if exist in a large number by natural source extract and the compounds that influences microtubule semi-synthetic and that synthetic analogues obtains, it has the potential as the toxin that is used to produce immune conjugate.(seeing the newmedinc.com website).These molecules and use the exemplary drugs of these molecules to comprise following: colchicine-site bonding agent (Curacin), plain (the AVE806 of windmill, windmill plain A-4 prodrug (CA4P), Oxi-4503), latent algin (LY355703), Discodermolide, Dolastatin and Analogs (Auristatin PHE, Dolastatin 10, ILX-651, Symplostatin 1, TZT-1027), Epothilones (BMS-247550, BMS-310705, EPO906, KOS-862, ZK-EPO), pomegranate plug Lip river element (Eleutherobin), FR182877, Halichondrin B (E7389), month benzyl three Matsubains (Halimide) (NPI-2352 and NPI-2358), Hemiasterlins (HTI-286), Laulimalide, Maytansinoids (" DM1 ") (Bivatuzumab maytansine, the Cantuzumab maytansine, huN901-DMI/BB-10901TAP, MLN591DM1, My9-6-DM1, Herceptin (Trastuzumab)-DM1), PC-SPES, Peloruside A, resveratrol (Resveratrol), S-allyl sulfydryl cysteine (SAMC), sponge element (Spongistatin), dimension is carried island amide (Vitilevuamide), molecular moter-kinesin (Molecular Motor-Kinesins) (SB-715992), colchicine-site bonding agent (the A-289099 of design, A-293620/A-318315, ABT-751/E7010, D-24851/D-64131, ZD6126), other novel spindle poison (2-methoxyestradiol (2-ME2), benzimidazole carbamate (ANG 600 series, mebendazole), CP248/CP461, HMN-214, R440, SDX-103, T67/T607).In addition, the Mayer in 1998, the Muridae of having commented on other among A.M.S.Marine Pharmacology:Antitumor and Cytotoxic Compounds.ThePharmacologist.41 (4): the 159-164 (1999) toxin of deriving.

The treatment throwing is given and is filled a prescription

The action time that prolongs can by alternative non-through the intestinal approach (such as, intravenous, subcutaneous or intramuscular injection) make the feed process more not frequently and more convenient.

When being used in vivo throwing when giving, antibody prescription as herein described should be aseptic.For example, this can be easy to by realizing through the aseptic filtration membrane filtration before or after lyophilizing and reorganization.Antibody stores or is stored in the solution with lyophilized form usually.The treatment antibody compositions is placed in the container with aseptic access inlet usually, for example, has the intravenous solution bag or the bottle of the jointer (stopper that can pass such as hypodermic needle) of recyclable prescription.

It is according to known method that antibody is thrown the path give, for example by in intravenous, intraperitoneal, the brain, in the intramuscular, ophthalmic, intra-arterial, sheath, suction or intralesional approach, or by slow-released system injection as mentioned below or inculcate.Antibody is preferably by inculcating or giving by continuous throwing of bolus injection.

The effective dose of the antibody that adopts on the therapeutics depends on (for example) treatment target, dosing way and patient's the patient's condition.Therefore, for the therapist, preferred titration dosage optionally and improvement dispensing path are to obtain optimum therapeutic effect.In theory, the clinicist gives antibody with throwing, up to the dosage that reaches ideal effect.The process of this therapy is easy to control by the calibrating of routine or by calibrating as herein described.

Antibody as described herein can prepare in the mixture that contains pharmaceutically acceptable supporting agent.Therapeutic combination can be preferably thrown through intravenous or per nasal or lung with the form of liquid or powder aerosol (through lyophilizing) and is given.Compositions also can optionally non-ly be given through intestinal or through subcutaneous throwing.Throw when giving when carrying out general, therapeutic combination should be aseptic, no thermal source and non-through intestinal acceptable have pH value should be arranged, in isotonicity and the stable solution.These conditions are that the those skilled in the art is known.In brief, the chemical compound by will having required purity and physiology go up acceptable supporting agent, excipient or stabilizing agent and mix and prepare chemical compound dosage prescription for storing and throwing is given.These materials are avirulent for the receiver when dosage that is adopted and concentration, and it comprises buffer agent, such as TRIS HCl, phosphate, citrate, acetate and other acylate; Antioxidant is such as ascorbic acid; Low-molecular-weight (less than about ten residues) peptide is such as poly arginine; Protein is such as blood albumin, gelatin or immunoglobulin; Hydrophilic polymer is such as polyvinylpyrrolidone; Aminoacid is such as glycine, glutamic acid, aspartic acid or arginine; Monosaccharide, disaccharide and other carbohydrate comprise cellulose or derivatives thereof, glucose, mannose or dextrin; Chelating agen is such as EDTA; Sugar alcohol is such as mannitol or Sorbitol; Equilibrium ion is such as sodium and/or non-ionic surface active agent, such as TWEENPLURONICS or Polyethylene Glycol.

The aseptic composite that is used to inject can be allocated according to the conventional medicine practice described in the Remington ' s Pharmaceutical Sciences (the 18th edition, Mack Publishing Company, Easton, PA, 1990).For example, may need reactive compound at vegetable oil such as water or natural generation, as the vehicle of Oleum sesami, Oleum Arachidis hypogaeae semen or Oleum Gossypii semen, or as lysate or suspension in the synthctic fat vehicle of ethyl oleate or its analog.Can incorporate buffer agent, antiseptic, antioxidant and analog thereof into according to acceptable medicine practice.

The suitable example of slow releasing preparation comprises the semi permeability substrate of the solid hydrophobic polymer that contains polypeptide, and described substrate is the form of shaping article, film or microcapsule.The example of sustained-release matrix comprises polyester, hydrogel (for example, J.Biomed Mater.Res. by people such as Langer, (1981) Chem.Tech. of 15:167-277 and Langer, (1982) 12:98-105 described poly-(2-ethoxy-methacrylate) or poly-(vinyl alcohol)), polylactic acid (United States Patent (USP) the 3rd, 773, No. 919, EP 58,481), the copolymer of L-glutamic acid and γ ethyl-L-glutamate (people's such as Sidman Biopolymers, (1983) 22:547-556), ethyl-vinyl acetate (people such as Langer is stretched in non-degraded, above-mentioned), the degraded lactic acid-ethanol copolymer is such as LUPRON Depot ^TM(the Injectable microspheres body of forming by lactic acid-ethanol copolymer and acetic acid leuprorelin) and poly--D-(-)-3-hydroxybutyric acid (EP 133,988).

Although can discharge molecule 1 00 day such as the polymer of stretching ethyl-vinyl acetate and lactic acid-ethanol is feasible, some hydrogel still continues short period release protein.When encapsulate protein kept in vivo for a long time, their are possible, and degeneration or gathering caused bioactive forfeiture and may change immunogenicity because the dampness that is exposed to 37 ℃ is following.Can be according to related mechanism strategy reasonable in design for stable protein.For example, if it is to exchange through disulphide to form intermolecular S-S key that mechanism is assembled in discovery, just can reach stable by upgrading sulfydryl residue, lyophilizing in acid solution, control moisture content, suitable additive and the research and development specificity polymer matrix composition of use.

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

The dosage that is used for given patient's antibody prescription can be taken known various factors into consideration by the attending doctor and measure effect with the upgrading medicine, comprises the order of severity and type, body weight, sex, diet, dispensing time and path, other medicament and other relevant clinical factor of disease.The treatment effective dose can be measured by method in vitro or in vitro.

The effective dose of the antibody that adopts on the therapeutics depends on (for example) treatment target, dosing way and patient's the patient's condition.Therefore, the therapist preferably optionally titration dosage and improvement dispensing path to obtain optimum therapeutic effect.According to factor mentioned above, general daily dose can about 0.001mg/kg to as many as 100mg/kg or more between.In theory, the clinicist gives treatment antibody with throwing, up to the dosage that reaches ideal effect.The process of this therapy is easy to by the calibrating of routine or as described herein control.

Should be appreciated that the throwing of the treatment entity that carries out according to compositions and the method for this paper is given should suitable supporting agent, excipient and other reagent in incorporating prescription into throwing and given, so that transfer, transmission, toleration and the similarity thereof through improvement to be provided.In the known prescription of all pharmaceutical chemistry teachers, can find multiple suitable prescription: Remington ' sPharmaceutical Sciences (the 18th edition, Mack Publishing Company, Easton, PA (1990)), especially be the Block in its literary composition, the 87th chapter of Lawrence.For example, these prescriptions comprise powder, slurry, ointment, colloid, wax, oil, lipid, contain lipid (cation or anion) bubble (such as Lipofectin ^TM), DNA conjugate, anhydrous absorption slurry, water medium oil and W/O emulsion, emulsion Polyethylene Glycol (various molecular weight polyethylene glycol), half gel and contain half solid mixture of Polyethylene Glycol admittedly.According to the present invention, any aforementioned mixture can be suitable in treatment and the therapy, as long as the active component in the prescription can be by the prescription passivation, and the last compatible and described dispensing of the tolerable path of described prescription physiology.Also see Baldrick P. " Pharmaceutical excipientdevelopment:the need for preclinical guidance. ", 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; People's such as Powell " Compendium of excipients for parenteralformulations ", PDA J Pharm Sci Technol.52:238-311 (1998) and the extraneous information of wherein quoting relevant with prescription, excipient and supporting agent are known by the pharmaceutical chemistry teacher.

The preparation of antibody

As described herein, antibody can prepare by using XenoMouse_ technology as mentioned below.Described mice can produce human immunoglobulin molecule and antibody subsequently, but is not enough to produce Muridae immunoglobulin molecules and antibody.The technology that is used for reaching identical purpose is disclosed in patent disclosed herein, application case and with reference to case.Yet, especially for produce about transgenic mice and by an embodiment of the antibody of its generation be disclosed in apply for No. the 08/759th, 620,3 days U.S. patent application case of December in 1996 and on June 11st, 1998 disclosed international application WO 98/24893 and December in 2000 disclosed WO 00/76310 on the 21st in.Also see people's such as Mendez Nature Genetics 15:146-156 (1997).

By using this technology, can produce complete human monoclonal antibody at multiple antibody.In one embodiment, with the XenoMouse_ cell strain of mice (for example with interesting antigen, EGFRvIII) immunity, in the mice of expressing antibodies, reclaim lymphocyte (such as the B-cell), and described cell and BM form cell strain are merged with preparation immortalization hybridoma cell strain, and screening and select these hybridoma cell strains to produce the hybridoma cell strain that has specific antibody for interesting antigen with identification.This paper is provided for producing and can produces the method that EGFRvIII is had the multiple hybridoma cell strain of specific antibody.In addition, the invention provides the sign of the antibody that produces by these cell strains, comprise the heavy chain of these antibody and the nucleotide and the aminoacid sequence of light chain.

Perhaps, replacement and myeloma cell are merged to produce hybridoma, further screen by the cell generation of being reclaimed, the immune mouse XenoMouse_ cell strain isolated antibody of hanging oneself according to the reactivity to initial antigen (being preferably EGFRvIII protein).Described screening comprises with EGFRvIII protein carries out ELISA, in vitro is bonded to the NR6M cell of stably express length EGF RvIII and by the antibody internalization EGFRvIII receptor in the NR6M cell.Use EGFRvIII-specificity hemolytic plate test to separate single B cell people such as (, Proc.Natl.Acad.Sci.USA, i93:7843-7848 (1996)) Babcook of the interesting antibody of secretion subsequently.Decide cell that target is used for bacteriolyze and be preferably sheep red blood cell (SRBC) (SRBC) with the coating of EGFRvIII antigen.In the presence of the B cell culture medium of secreting interesting immunoglobulin and fill-in, dull and stereotyped formation shows the specificity EGFRvIII mediation bacteriolysis of target cell.The monoclonal antibody of separable dull and stereotyped center is former-the specificity plasma cell, and the specific hereditary information of encoding antibody can be separated from single plasma cell.But use the DNA of the variable region of the secreted antibody of reverse transcription PCR clones coding.Describedly can further insert in the suitable expression vector subsequently, be preferably carrier box through cloned DNA, such as pcDNA, more excellent pcDNA carrier for the constant domain that contains heavy chain immunoglobulin and light chain.Subsequently, but the carrier transfection that is produced is a host cell, is preferably Chinese hamster ovary celI, and is incubated in the conventional Nutrient medium of suitable upgrading for the required sequence of bringing out promoter, selecting transformant or amplifying encoding gene.Describe herein and produce the separation that EGFRvIII is had a plurality of single plasma cells of specific antibody.In addition, the hereditary material with coding anti-EGFR vIII antibody specificity separates, introduces in the suitable expression vector that transfection subsequently is a host cell.

Also can be used as the source of hereditary material from the B cell of XenoMouse mice, can produce the antibody display libraries from it.The general technology that described field can be used in described library via ribosomal display at phage, yeast or in vitro make.Can be used as a kind of abundant source through hyperimmune XenoMouse mice, from its separable antigen-reactive antibody with high-affinity.Therefore, can be used for producing the antibody display libraries through the hyperimmune XenoMouse mice of antagonism EGFRvIII, from the high-affinity antibody of its separable antagonism EGFRvIII.Described storehouse can be screened at the pep3 oligopeptide, and the gained antibody of deriving screens to determine for the antigenic specificity of displaying naturally at the cell of expressing EGFRvIII.IgG antibody can use recombinant DNA technology to express subsequently fully.For example see WO99/53049.

Generally speaking, the antibody that is produced by above-mentioned cell strain has complete IgG 1 or IgG2 heavy chain and human κ light chain.In one embodiment, antibody has high-affinity, and when measuring mutually with solution by solid phase, it generally has about 10 ^-9M is to about 10 ^-13The Kd value of M.In other embodiments, antibody has lower affinity, and about certainly 10 ^-6M is to about 10 ^-8M.

As be appreciated by one of skill in the art that, can in the cell strain except that the hybridization tumor cell strain, express according to the antibody of present embodiment.The sequence of coding specific antibodies can be used for the transition of suitable mammalian host cell.Can make the transition so that polynucleotide is introduced host cell by any known method, for example comprise and polynucleotide being packaged in the virus (or viral vector) and with virus (or carrier) or by known transfection program alternate host cell in this technology, 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.Employed transition, program depended on host to be made the transition.Known in this technology the xenogenesis polynucleotide introduced method in the mammalian cell, and it comprise that dextran regulates that transfection, protoplast fusion, electric shock are regulated in transfection, calcium phosphate precipitation, polybrene, polynucleotide is packaged in the liposome and the dna direct microinjection in karyon.

Known the mammalian cell strain that can be used as for expressive host in this technology, and it comprises the immortalized cell line that is obtained by American Type Culture Collecti (American Type Culture Collection (ATCC)) by multiple, 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 (for example, Hep G2) and many other cell strains.Have the antibody that high expression level and generation have a composing type EGFRvIII binding characteristic and select especially more excellent cell strain by measuring which cell strain.

Example

Following example is provided, comprises that experiment of carrying out and the result who obtains are only in order to reach illustrative purposes and be not interpreted as limitation of the present invention.

The initial strategy that produces the EGFRvIII-specific antibody relates to the immune XenoMouse mice of complex antigen (peptide, multiple soluble protein and antigen presentation cell), subsequently by hybridize cell or pass through XenoMax of fusion ^TM/ SLAM ^TMTechnical point is from the B cell and separation antibody produces cell.Antibody produced cell is that specificity is presented to the one-level screening by ELISA, is that cell surface is in conjunction with being presented to the secondary screening by FMAT and/or FACS.Carry out subsequently internalization calibrating with identification may be useful to drug conveying antibody.Measure the affinity of these antibody.Select specific antibodies to carry out epitope mapping.In addition, select specific antibodies to carry out reaching in the body testing in vitro to analyze the effect of these antibody to the treatment cancer.

Example 1 ' antigen preparation

A. EGFRvIII PEP3-KLH antigen preparation

Together with example 2,14-mer people EGFRvIII PEP3 (LEEKKGNYVVTDHC (SEQ IDNO:56)) peptide passes through R﹠amp; D system is conventional synthetic.The PEP3 peptide combines with keyhole limpet hemocyanin (KLH) subsequently, and is as follows: EGFRvIII PEP3 (200mcg) (R﹠amp; D) with the keyhole limpet hemocyanin (KLH of 50mcg; Pierce, Rockford, IL) mix and with the distilled water standardize solution to 165mcl.Add 250mcl binding buffer liquid (0.1M MES, 0.9M NaCl, pH4.7) and the 1-ethyl-3-[3-dimethyl aminopropyl of the 10mg/ml by adding 25mcl] storage liquid (EDC, the Pierce of carbonization two imide hydrochlorides, Rockford IL) makes EGFRvIII PEP3 and KLH crosslinked.At room temperature cultivate in conjunction with 2 hours, (Bedford MA) removes unreacted EDC is centrifugal the PBS that uses pH7.4 for centrifugal filter membrane, Millipore by the 1kDa filter membrane.

Together with example 3,14-mer people EGFRvIII PEP3 (LEEKKGNYVVTDHC (SEQ IDNO:56)) peptide passes through R﹠amp; D system is conventional synthetic.The PEP3 peptide combines with KLH subsequently, and is as follows: the keyhole limpet hemocyanin (KLH of EGFRvIIIPEP3 (200mcg) and 50mcg; Pierce, Rockford, IL) mix and with the distilled water standardize solution to 165mcl.Add 250mcl binding buffer liquid (0.1M MES, 0.9M NaCl, pH4.7) and the 1-ethyl-3-[3-dimethyl aminopropyl of the 10mg/ml by adding 25mcl] storage liquid (EDC, the Pierce of carbonization two imide hydrochlorides, Rockford IL) makes EGFRvIII PEP3 and KLH crosslinked.At room temperature cultivate in conjunction with 2 hours, (Bedford MA) removes unreacted EDC is centrifugal the PBS that uses pH7.4 for centrifugal filter membrane, Millipore by the 1kDa filter membrane.

B. The B300.19/EGFRvIII transfectant

In order to prepare the B300.19/EGFRvIII transfectant, initially clone Wild type EGFR from the A431 cell, and thereby the EGFR gene of the EGFRvIII that is used to encode is modified the codon of disappearance coding residue 6-273, and produces the codon of coding glycine residue in the junction of disappearance.This disappearance occurs in disappearance GTT (valine) and CGT (arginine) codon on every side, and codon is GGT (glycine) after the disappearance that obtains.(Wikstrandet?al.J?Neurovirol.4(2)：148-58(1998))

1. the clone that makes up of Wild type EGFR:

(Invitrogen, Burlington ON) extract poly-A+mRNA from A431 (ATCC) to use Micro-fast RNA test kit.(Beverly is Mass.) from the synthetic total cDNA of polyA+mRNA for NEB, New EnglandBiolabs with pdN6 primer and M-MuLV reverse transcriptase at random.With the PCR product of following primer by A431cDNA amplification 2.3kb:

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

Antisense 5 '-CGGATCTCGAGCCGGAGCCCAGCACTTTGATCTT-3 ' (SEQ IDNO:63)

Use the Pfu archaeal dna polymerase.

The PCR product is digested with XhoI, gel-purified, and be connected to by among the plasmid pWBFNP of XhoI line styleization (seeing international application WO No. 99/45031), to produce plasmid Wt-EGFR/pWBFNP.

2.EGFRvIII the generation that makes up:

With primer C13659/C29538 and C29539/C14288 (BioSource International) are amplified the PCR product by plasmid Wt-EGFR/pWBFNP, wherein C29538 and C29539 are 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 with introducing disappearance in 6 to 273 amino acid whose sequences of coding EGFR extracellular domain, and sub-clone (is seen international application WO No. 99/45031) in expression vector pWBDHFR2.

With primer to C13659/C29538 by the Wt-EGFR/pWBFNP template with the Pfu polymerase (NEB, NewEngland Biolabs, Beverly, Mass.) amplification produces the fragment of 232bp of 5 ' end of representative disappearance.With EcoRl (NEB, New England Biolabs, Beverly, Mass.) digestion and gel-purified PCR product.C29539/C14288 is produced the fragment of 1273bp of 3 ' end of representative disappearance with primer from Wt-EGFR/pWBFNP, and with Pfu polymeric enzymatic amplification template.With Xhol (NEB, New England Biolabs, Beverly, Mass.) digestion PCR product.(Beverly Mass.) is connected to fragment the pWBDHFR2 that digests with EcoRl/Xhol and makes up EGFRvIII/pWBDHFR to produce for NEB, New England Biolabs with the T4DNA ligase.

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

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

B300.19 cell (8 * 10 ⁶) be used to the transfection in per 700 μ l DMEM/III media.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 the electric shock,, add 10 non-selective media (DMEM/HI glucose, 10% FBS, 50 μ M BME, 2mM L-glutaminate, the 100 penicillin-G/ml of unit, 100 MCG of unit streptomycins/ml) subsequently in cooled on ice cell 10 minutes.At 37 ℃, 7.5% CO ₂Following cultured cell 48 hours.

After the cultivation, with cell division in selective dielectric (DMEM/HI glucose, 10% FBS, 2mM L-glutaminate, 50 μ M BME, the 100 penicillin-G/ml of unit, the 100 MCG streptomycin/ml of unit, 2ug/ml puromycin), in 96 orifice plates with 2 * 10 ⁴, 0.4 * 10 ⁴' and 0.08 * 10 ⁴The concentration of cells/well, in selective dielectric selected 14 days to produce stable clone.With Puro resistance clone E752mAb (anti-EGFR-antibodies, people such as Yang, Crit Rev Oncol Hematol., 38 (1): describe to some extent among the 17-23 (2001)) and the anti-human IgG PE dyeing of goat, go up at FACS Vantage (Becton Dickinson) subsequently and analyze.

C. Make up EGFRvIII-RbFc and express structure

For producing EGFRvIII rabbit fusion rotein, we at first make up the carrier of the DNA that contains coding rabbit Fc.This DNA with coding EGFRvIII is connected.To describe the method in detail below:

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

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

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

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

With the PCR product that obtains with KpnI and NotI digestion, gel-purified and be connected to pcDNA3.1 (+)/Hygro with KpnI/NotI digestion (Invitrogen, Burlington, ON), to produce plasmid RbFc/pcDNA3.1Hygro.

2.EGFRvIII-RbFc/pCEP4 structure:

(following) primer 1290/1293 is used 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)

With NheI and KpnI digestion, gel-purified also is connected to RbFc/pcDNA3.1 with NheI/KpnI digestion to produce plasmid EGFRvIII-RbFc/pcDNA3.1Hygro with the PCR product.

The SnaBI/XhoI fragment of 2170bp is separated from EGFRvIII-RbFc/pcDNA3.1 Hygro and sub-clone go into SnaBI/XhoI digestion pCEP4 (Invitrogen, Burlington, ON) in, to produce plasmid EGFRvIII-RbFc/pCEP4.

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

With the following method with plasmid EGFRvIH-RbFc/pCEP4 by calcium phosphate transfection introduce the 293F cell (Gibco, Grand Island, NY): transfection the previous day, 1 * 10 ⁶In the 100mm tissue culture medium (TCM) culture dish that the 293F cell is covered to gelatin by plating, and cultivate down 5% CO2,37 ℃.Before the transfection, use the fresh non-selective medium of 10ml (DMEM/F12,10% FBS, 2mM L-glutaminate, 100U/ml benzylpenicillin, 100U/ml MCG streptomycin) cultured cell 2-3 hour.Prepare transfection reagent in micro-centrifuge tube, as follows: the DNA of 10 μ g (EGFRvIII-RbFc/pCEP4) mixes with the 2M calcium phosphate of 62 μ l, and with the deionized water standardize solution to 500 μ l.2XHBS with another pipette, extract 500ul is used to shift described transfection reagent.

Dropwise the solution in the pipet is added cell, for keeping suitable pH value, cell is placed 5%CO simultaneously ₂In the incubator until carrying out transfection.After the transfection 15-20 hour, with the PBS washed cell and with the non-selective medium feeder cell of the fresh 293F of 10ml.The cell of expressing with transfection results behind the trypsin 48-72, and with cell with 0.08 * 10 ⁴The cells/well plating is to 96 orifice plates, places in the 293F selective dielectric (DMEM/F12,10% FBS, 2mM L-glutaminate, 100U/ml benzylpenicillin, 100U/ml MCG streptomycin, 250ug/ml hydromycin B) 14 days.

The anti-EGFR-antibodies E763 (United States Patent (USP) the 6th, 235, No. 883) of use 1ug/ml makes capture antibodies and also resists-rabbit igg HRPO (CalTag) detection with the goat of dilution in 1: 100, screens the hydromycin B resistance clone by ELISA.

Make the EGFRvIII peptide-OVA that is used for antigen titration (example 3) with the following method:

Use from Pierce (#20291) through the DTT of pre-weighing reductase 12 07 μ g EGFRvIII PEP3.Use the DTT of 100 μ L deionized water dissolving bottle 7.7mg through pre-weighing.The DTT stock solution is added EGFRvIIIPEP3.The PBS that uses pH 7.4 with the reactant liquor standardize solution to 600 μ L.The rotation reactant liquor is 30 minutes under the room temperature.

Restrain G10 polydextran gel pearls and add 40mlPBS by weighing 5, mix also and at room temperature left standstill 10 minutes, made the G10 tubing string in 10 minutes with the 1000rpm beads centrifuged then.Remove supernatant, and add the PBS of 20ml again.With pearl under 1000rpm centrifugal 10 minutes.Remove supernatant, the PBS that adds q.s is to produce the suspension of 50%G10 polydextran gel pearl.In the 5ml column spinner, add described 50% suspended mixture of 5ml, subsequently this post is placed the 14ml polypropylene tube.With pillar under 1000rpm centrifugal 3 minutes.The PBS that adds 3ml in addition again, and then with pillar under 1000rpm centrifugal 3 minutes.The polypropylene tube that more renews, described like this pillar just can use.

From phthalin, remove DTT.After reaction in reductive 30 minutes, in each pillar, add the phthalin of 300 μ L with peptide.With pillar under 1000rpm centrifugal 3 minutes.The PBS that in each pillar, adds 250 μ L in addition again, and then under 1000rpm centrifugal 3 minutes.Phthalin is collected in the polypropylene tube of 14ml.

With described phthalin be connected with the activated OVA of maleimide, and collect in the microcentrifugal tube.The activated OVA of the maleimide of 2mg is dissolved (Pierce:77126, Rockford IL) to make the 10mg/ml stock solution with maleimide binding buffer liquid.In the phthalin in the activated OVA adding of the maleimide of the 414 μ g microcentrifugal tube.In reaction, add 500 μ L maleimide binding buffer liquid.Reactant liquor at room temperature cultivated 2 hours and add any active maleimide base group that the cysteine of 2mg may exist with quencher subsequently.Allow described cysteine to react again under the room temperature 30 minutes.Described conjugate used the 1xPBS washing of pH 7.4 and with the centrifugal post of 10K centrifugal for 3 times.This has not removed not and bonded all free peptides of OVA and cysteine freely.Use gel application of sample suction nozzle that described conjugate is taken off and adds the microcentrifugal tube from centrifugal post.At last, use the 1X PBS of pH7.4 that the conjugate standardize solution is arrived expectation concentration.The mol ratio of described conjugate is 14.5: 1 (peptides: OVA).

Example 2

Produce manufacturing anti-EGFR vIII antibody by hybrid cell

Eight mices (XenoMouse G1 mice) that generation had the antibody of γ-1 constant region will be strengthened at the 11st, 21,32,44 and 54 day for this scheme then immunity in the 0th day, merge at the 58th day then.All immunity are all by injecting in the mode of root of the tail portion subcutaneous administration and intraperitoneal administration.Immunity in the 0th day is with 1.5 * 10 ⁷B300.19/EGFRvIH cells transfected (example 1A) is suspended in and complete Freunds adjuvant (CFA) (Sigma, St.Louis, MO) 1: finish among the DPBS of the blended no pyrogen of 1v/v.11st, strengthening in 21 and 32 days is with 1.5 * 10 ⁷The B300.19/EGFRvIII cells transfected with incomplete Freunds adjuvant (IFA) (Sigma, St.Louis, MO) 1: finish among the blended DPBS of 1v/v.Strengthening in the 44th day is to use to combine (example 1) and finish with PEP3 (EGFRvIII peptide)-KLH of the blended 5 μ g of IFA1: 1v/v, and last reinforcement is in conjunction with finishing with 5 μ gPEP3 (EGFRvIII peptide)-KLH among the DPBS that does not have adjuvant.

At the 58th day, mice is implemented euthanasia, reclaim its groin and lumbar vertebra lymph node subsequently.The using-system pulverizer discharges lymphocyte by mechanical damage from lymph node, remove the T cell by the negative selection of CD90 subsequently.By will washing and the B cell of enrichment and available from ATCC, the nonsecreting type myeloma P3X63Ag8.653 cell of cat # CRL 1580 (Kearney et al, J.Immunol.123:1548-1550 (1979)) merges with 1: 1 mixed.This cell mixture is by precipitating lightly with the centrifugal quilt of 800g.After thoroughly removing supernatant, handle to be no more than 2 minutes with 2-4ml pronase solution (CalBiochem, cat.# 53702,0.5mg/ml is in PBS).Subsequently, add 3-5ml FBS and stop enzymatic activity, electricity consumption cell fusion 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)) is settled to 40ml.

Remove supernatant after centrifugal, by the washed cell that in 40ml ECFS, suspends once more.The repeated washing step, once more with the ECFS suspension cell to reach 2 * 10 ⁶The concentration of cell/ml.Use fusion generator (model ECM2001, Genetronic, Inc., San Diego CA) carries out electricity-cell fusion.Used fusion chamber size is 2.0ml, and uses following instrument setting: comparison condition: voltage: 50V, and time: 50s, film rupture condition: voltage: 3000V, the time: 30 μ s, merge back set time: 3s.After the fusion, at DMEM (JRH Biosciences), 15%FCS (Hyclone) and contain suspension cell in the solution of HAT, and add L-glutaminate, penicillin-streptomycin, OPI (oxaloacetic acid, acetone acid, bovine insulin) (all available from Sigma, St.Louis, MO) and IL-6 (Boehringer Mannheim) at 37 ℃ and 10% CO ²Cultivate in the air.

With cell with 4 * 10 ⁴The cells/well plating is to flat 96 hole tissue culturing plates.Before transferring to HT (hypoxanthine and thymidine) feed supplement medium, cultivate and be stored in 2 weeks in HAT (hypoxanthine, aminopterin and thymidine) the feed supplement medium.Select hybrid cell by survival, and screen the antigen active of supernatant with ELISA at the HAT medium.The ELISA form need apply the culture supernatant on the flat board (applying dull and stereotyped and wild type EGFr peptide-OVA coating flat board as the EGFRvIII peptide of counting screening-OVA) and use the human IgG of mouse anti of horseradish peroxidase (HRP) labelling to detect EGFRvIII specific bond (seeing Table 2.1) at antigen.

Table 2.1

Plate/hole	Hybrid cell	The one OD	The 2nd OD
					? 13.2D10 13.3C12 13.3F11 13.6B11	? 13.1 13.2 13.3 13.4	Merge plate 4.034 3.829 3.874 3.322	muEGFr 2.653 2.443 1.081 1.311	EGFr 0.051 0.049 0.049 0.052

The clone	Plate	OD#	1	OD#2
						? 13.1.1 13.1.2	? 0.5c/wD2 0.5c/wD2	Clone's plate 2.614 2.248	muEGFr 2.586 1.272	EGFr 0.042 0.041

It should be noted that, detect at least four antigen-specific hybrid cells: 13.1,13.2,13.3 and 13.4.These hybridomas are positive in ELISA calibrating, by confirming the EGFRvIII specificitys at 300.19 cells through the expression EGFRvIII of stable transfection with respect to 300.19 FACS without the transfection blast cell.

Use the limiting dilution plating on the antigen negative hole of selecting, to clone.By the existence of access panel checklist clonal antibody growth, verify from the supernatant in monoclonal hole and with above-described FACS by antigenic specificity ELISA screening.Thereby use the Luminex instrument to check the purity of human γ and κ chain by polynary ELISA calibrating high activity clone.Based on the specificity of EGFRvIII in ELISA and the FACS calibrating, select clone 13.1.2 for further screening and analyze most promising candidate.Showed the heavy chain of 13.1.2 antibody and the nucleotide sequence and the aminoacid sequence of light chain among Fig. 3 L, and SEQ ID NO 137 and 139 be heavy chain and light chain nucleic acid and 138 and 140 for heavy chain and light-chain amino acid sequence.In addition, in Figure 4 and 5, show 13.1.2 heavy chain and sequence of light chain and its from the comparison of sequence of germ cell line.

Example 3

By using the XenoMax technology to produce antibody

The immunity of XenoMouse animal

The human monoclonal antibody of anti-human EGFR vIII produces XenoMouse mice (XenoMouse G1 mice) with γ-1 constant region antibody, produces (XenoMouse G4 mice) that XenoMouse mice (XenoMouse XMG2 mice) with γ-2 constant region antibody and generation have γ-4 constant region antibody by immunity progressively.

For producing mAb by the XenoMax technology, XenoMouse G1 group and XMG2 group mice are by 300.19 cells (example 1B) with EGFRvIII PEP3 (example 1A) and expression EGFRvIII, or with the extracellular domain of the bacterial expression of EGFRvIII albumen (EGFRvIII-ECD) (Dr.Bigner, Duke University) 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 the palmula immunity, every mice of initial immunity is used or is not used 10 * 10 ⁶Express 300.19 cells of EGFRvIII and use or do not use that (Sigma, Oakville is ON) with EGFRvIII PEP3 or EGFRvIII-ECD or the EGFRvIII-RbFc of 1: 1 blended 10 μ g with the Titermax gold.Half of initial immune used immunogen dosage used in reinforcement subsequently.Preceding strengthen for 4 times be by with immunogen and Alumen (Sigma, Oakville ON) mix the injection mice, the absorption of spending the night, every mice is shown in following table 3.1.The respective reaction of injecting Titermax then is former, the Alumen that reinjects, subsequently in the immunogenic last reinforcement among the PBS shown in following table 3.1.Especially, O, 3,7,10,14,17,21 and 24 days immune animals.At the 19th day animal is got blood to obtain immune serum and to determine the titer that results are selected.The 28th day results animal.

Table 3.1 palmula immunity timetable

Grouping #

	1	2	3	4	5	6	7	8
									The # animal	5	5	5	5	5	5	5	5
Mouse species	XMG2	XM3C-3	XMG2	XM3C-3	XMG2	XM3C-3	XM3C-3	XMG2
									Strengthen #	Adjuvant	Immunogen	Immunogen	Immunogen	Immunogen
For the first time	The Titermax gold	EGFRvIII-300.19 cell+PEP3-KLH	EGFRvIII-300.19 cell+EGFRvIII-ECD	EGFRvIII-300.19 cell+EGFRvIII-RbFc	EGFRvIII-RbFc ? ?
						For the second time	Alumen	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	EGFRvIII-RbFc ? ? ?
For the third time	Alumen	PEP3-KLH ? ? ?	EGFRvIII-ECD ? ? ?	EGFRvIII-ECD ? ? ?	EGFRvIII-RbFc ? ? ?
						The 4th time	Alumen	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	EGFRvIII-RbFc ? ?
The 5th time	Alumen	PEP3-KLH ? ?	EGFRvIII-ECD ? ?	EGFRvIII-ECD ? ?	EGFRvIII-RbFc ? ?
						The 6th time	The Titermax gold	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	EGFRvIII-RbFc ? ? ?
The 7th time	Alumen	PEP3-KLH ? ?	EGFRvIII-ECD ? ?	EGFRvIII-ECD ? ?	EGFRvIII-RbFc ? ?
						The 8th time	PBS ? ?	EGFRvIII-300.19 cell+PEP3-KLH	EGFRvIII-300.19 cell+EGFRvIII-ECD	EGFRvIII-300.19 cell+EGFRvIII-RbFc	EGFRvIII-RbFc ? ?
Results

As in the palmula immunity, describing, every mice of initial BIP immunity with corresponding immunogen with 1: 1v/v and FreundShi adjuvant (CFA, Sigma, Oakville, ON) mixing fully.Reinforcement subsequently at first every mice is used corresponding immunogen 1: 1 and incomplete FreundShi adjuvant (Oakville ON) is mixed and carries out, and is the last reinforcement of every mice with PBS subsequently for IFA, Sigma.Shown in following table 3.2, the 0th, 14,28,42,56 and 75 (strengthening at last) day immune animal.At the 63rd day animal is got blood to obtain immune serum and to determine tiring of results selection.The 78th day results animal.

Table 3.2Bip immunity timetable

Grouping

	9	10	11	12	13	14	15	16
									The # animal	5	5	5	5	5	5	5	5
Mouse species	XMG2	XM3C-3	XMG2	XM3C-3	XMG2	XM3C-3	XMG2	XM3C-3
									Strengthen #	Adjuvant	Immunogen	Immunogen	Immunogen	Immunogen
For the first time	CFA ? ?	EGFRvIII-300.19 cell+PEP3-KLH	EGFRvIII-300.19 cell+EGFRvIII-ECD	EGFRvIII-300.19 cell+EGFRvIII-RbFc	EGFRvIII-RbFc ? ?
						For the second time	IFA ?	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	EGFRvIII-RbFc ?
For the third time	IFA ?	PEP3-KLH ?	EGFRvIII-ECD ?	EGFRvIII-ECD ?	EGFRvIII-RbFc ?
						The 4th time	IFA ?	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	The EGFRvIII-300.19 cell	EGFRvIII-RbFc ?
The 5th time	IFA ?	PEP3-KLH ?	EGFRvIII-ECD ?	EGFRvIII-ECD ?	EGFRvIII-RbFc ?
						The 6th time	PBS ? ?	EGFRvIII-300.19 cell+PEP3-KLH	EGFRvIII-300.19 cell+EGFRvIII-ECD	EGFRvIII-300.19 cell+EGFRvIII-RbFc	EGFRvIII-RbFc ? ?
Results

Determine to select the animal of results by usefulness

Determine that by ELISA tiring of anti--hEGFRvIII antibody is coated onto CostarLabcoat Universal Binding Polystyrene 96 orifice plate (Corning with EGFRvIII-RbFc (2.5 μ g/ml) or contrast RbFc (2 μ g/ml) or EGFRvIII peptide-OVA (2 μ g/ml) (example 1) or contrast OVA (4 μ g/ml), Acton MA) last four spends night.Remove the solution that comprises free antigen and described plate is handled 4 minutes (4000 little joules) with ultraviolet light (365nm).With the described plate of distilled water wash five times.1: 2 dilution back of the serum that obtains from the XenoMouse_ animal of EGFRvIII immunity or the XenoMouse_ animal that is used to test first of the initial dilution of double 1: 100 is with 2% milk/PBS titration.Leave a blank in last hole.With the described plate of distilled water wash five times.Under the room temperature with the final concentration of 1 μ g/ml add the special horseradish peroxidase of the anti-IgG Fc of goat (HRP, Pierce, Rockford, EL) bonded antibody is 1 hour.With the described plate of distilled water wash five times.(Gaithersburg, MD) adding 1M phosphoric acid stopped ELISA with the plate colour developing in 30 minutes to add the TMB chromogenic substrate.Optical density when XenoMouse_ animal individual special tired by 450nm is determined and is showed in table 3.3 and 3.4.The inverse dilution of this expression immune serum of tiring, so the big more then humoral immunization of this numerical value is strong more to the reaction of hEGFRvIII.

For the mice that passes through at tail base portion subcutaneous injection and lumbar injection, that tires determines as mentioned above, except scribbling the flat board of EGFRvIII-RbFc (2.0 μ g/ml) or contrast RbFc (2.5 μ g/ml).

Table 3.3

Grouping #	Immunity (site and immunogen)	Mouse species and sex	Mice I.D	EGFRvIII -RbFc@ 2.5ug/ml. ? ?	Contrast-RbFc@ 2.0ug/ml.	EGFRvIII peptide-OV A applies with 2.0 μ g/ml	OVA applies with 4.0 μ g/ml
								1	FP EGFRvIII-30 0.19 cell+EGFRvIII PEP3-KLH (seeing Imm.Sched.)	XMG2	0748-1	330		13549	＜100
0748-2	237		7635	＜100
					0748-3	109					9824	＜100
0748-4	714		8014	＜100
					0748-5	165					9421	＜100
Be used to first test	＜100 ? ?		n/a ? ?	n/a ? ?
					2	FP EGFRvIII-30 0.19 cell+EGFRvIII PEP3-KLH (seeing Imm.Sched.)	XM3C-3				0741-1	388		347	＜100
0741-2	327		240	＜100
								0741-3	385		330	＜100
0741-4	589		227	＜100
								0741-5	273		626	＜100
Be used to first test	＜100 ? ?		n/a ? ?	n/a ? ?
								? 3	FP EGFRvIII-300. 19 cells+EGFRvIII-EC D (seeing Imm.Sched.)	? XMG2	0749-1 ?	552 ?		＜100 ?	＜100 ?
0749-2	477		＜100	＜100
					0749-3	100					＜100	＜100
0749-4	100		＜100	＜100
					0749-5	1631					＜100	＜100
Be used to first test	100 ? ?		n/a ? ?	n/a ? ?
					? 4	FP EGFRvIII-300. 19 cells+EGFRvIII-EC D (seeing Imm.Sched.)	? XM3C-3				0742-1 ?	372 ?		＜100 ?	＜100 ?
0742-2	745		＜100	＜100
								0742-3	484		＜100	＜100
07424	530		＜100	＜100
								0742-5	270		＜100	＜100
Be used to first test	100 ? ?		n/a ? ?	n/a ? ?
								? 5	FP ? EGFRvIII-300.	XMG2 ?	0750-1 ?	5399 ?	175 ?	＜100 ?	＜100 ?
0750-2	3072	151	＜100	＜100

	19 cells+EGFRvIII-Rb Fc (seeing Imm.Sched.)		0750-3	＞6400	358	＜100	＜100
								0750-4	5845	196	＜100	＜100
			0750-5	5770	196	＜100	＜100
								Be used to first test	100 ? ?	100 ? ?	n/a ? ?	n/a ? ?
			? 6	FP EGFRvIII-300. 19 cells+EGFRvIII-Rb Fc (seeing Inm.Sched.)	? XM3C-3	0743-1 ?	1220 ?						＜100 ?	＜100 ?	＜100 ?
0743-2	1183	＜100						＜100	＜100
						0743-3	645			＜100	＜100	＜100
0743-4	759	＜100						＜100	＜100
						0743-5	1260			＜100	＜100	＜100
Be used to first test	100 ? ?	＜100 ? ?						n/a ? ?	n/a ? ?

Grouping #	Immunity (site and immunogen)	Mouse species and sex	Mice I.D	EGFRvIII -RbFc@ 2.5ug/ml. ? ?	Contrast RbFc@ 2.0ug/ml.	EGFRvIII peptide-O VA applies with 2.0 μ g/ml	OVA applies with 4.0 μ g/ml
								7	FP EGFRvIII-RbF c (seeing Imm.Sched.)	XMG2	0745-1	1897	＜100	＜100	＜100
0745-2	＞6400	323	＜100	＜100
					0745-3	1225	＜100				＜100	＜100
0745-4	4047	＜100	＜100	＜100
					0745-5	852	＜100				＜100	＜100
Be used to first test	100 ? ?	＜100 ? ?	n/a ? ?	n/a ? ?
					8	FP EGFRvIII-Rb Fc (seeing Imm.Sched.)	XM3C-3				0744-1	362	＜100	＜100	＜100
0744-2	807	＜100	＜100	＜100
								0744-3	479	＜100	＜100	＜100
0744-4	631	＜100	＜100	＜100
								0744-5	1112	＜100	＜100	＜100
Be used to first test	100 ? ?	＜100 ? ?	n/a ? ?	n/a ? ?

Based on serology, all XenoMouse animals of the 5th group and from the selected XenoMax results of doing of the animal 0743-5 of the 6th group XenoMouse in the table 3.3.

Table 3.4

Grouping #	Immunity (site and immunogen)	Mouse species and sex	Mice I.D	EGFRvIII -RbFc@ 2.0ug/ml. ? ?	Contrast RbFc@ 2.5ug/ml.	Apply EGFRvIII peptide-OVA with 2.0 μ g/ml	OVA applies with 4.0 μ g/ml
								9	BIP EGFRvIII-300. 19 cells+EGFRvIII PEP3-KLH (seeing Imm.Sched.)	XMG2	0695-1	2921		＞128000	472
0695-2	2219		30504	379
					0695-3	4609					＞128000	608
0695-4	＞6400		＞128000	368
					0695-5	1580					19757	269
Be used to first test	＜100 ? ?		n/a ? ?	242 ? ?
					10	BIP EGFRvIII-300. 19 cells+EGFRvIII PEP3-KLH (sees	XM3C-3				O700-1	＜100
O700-2	＜100
								O700-3	＞6400
O700-4	5342
								O700-5	＞6400

	Imm.Sched.) ? ?		Be used to first test	＜100 ? ?
								11	BIP EGFRvIII-300. 19 cells+EGFRvIII-EC D (seeing Imm.Sched.)	XMG2	0696-1	＜100		561	240
0696-2	＜100		788	326
					0696-3	＜100					604	266
0696-4	143		444	263
					0696-5	＜100					303	254
Be used to first test	＜100 ? ?			242 ? ?
					12	BIP EGFRvIII-300. 19 cells+EGFRvIII-EC D (seeing Imm.Sched.)	XM3C-3				0700-1	358
0702-2	469
								0702-3	401
0702-4	＞6400
								0702-5 ?	＞6400 ?
			Be used to first test	＜100 ? ?

Grouping #	Immunity (site and immunogen)	Mouse species and sex	Mice I.D	EGFRvIII -RbFc@ 2.0ug/ml. ? ?	Contrast RbFc@ 2.5ug/ml.	Apply EGFRvIII peptide-OVA with 2.0 μ g/ml	OVA applies with 4.0 μ g/ml
								13	BIP EGFRvIII-300.19 cell+EGFRvIII-RbFc (seeing Imm.Sched.)	XMG2 ?	0694-1 ?	＞6400 ?	＞6400 ?	250 ?	?243 ?
0694-2	＞6400	＞6400	296	?309
					0694-3	＞6400	＞6400				736	?605
0694-4	＞6400	＞6400	739	?1111
					0694-5	3710	＞6400				517	?465
Be used to first test	＜100 ? ?	＞6400 ? ?		?242 ? ?
					14	BIP EGFRvIII-300.19 cell+EGFRvIII-RbFc (seeing Imm.Sched.)	XM3C-3				0703-1 ?	2740 ?	＞6400 ?
0703-2	408	＞6400
								0703-3	1406	＞6400
0703-4	1017	＞6400
								0703-5	403	＞6400
Be used to first test	＜100 ? ?	＞6400 ? ?
								15	BIP EGFRvIII-RbFc (seeing Imm.Sched.)	XMG2	0697-1 ?	＞6400 ?	＞6400 ?	340 ?	348 ?
0697-2 ?	＞6400 ?	＞6400 ?	642 ?	1793 ?
					0697-3	6242	＞6400				319	246
0697-4	1766	＞6400	133	＜100
					0697-5	＞6400	＞6400				685	448
Be used to first test	＜100 ? ?	＞6400 ? ?	243 ? ?	242 ? ?
					16	BIP EGFRvIII-RbFc (seeing Imm.Sched.)	XM3C-3				0701-1 ?	592 ?	＞6400 ?
0701-2 ?	1118 ?	＞6400 ?
								0701-3	＞6400	＞6400
0701-4	＜100	＜100
								0701-5	n/a	n/a
Be used to first test	＜100 ? ?	＞6400 ? ?

Based on the serology data in the table 3.4, the selected results of doing of XenoMouse animal (0695-1,0695-3 and 0695-4).

The selection of B cell

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

Table 3.5

	Total # flat board	Block on the OD on the occasion of:
																		0.0 ?	0.1 ?	0.2 ?	0.3 ?	0.4 ?	0.5 ?	0.6 ?	0.7 ?	0.8 ?	0.9 ?	1.0 ?	1.5 ?	2.0 ?	2.5 ?	3.0 ?	3.5 ?
		Cansera 500 cells/well	?12 ? ?	11 52 ?	63 4 ?	81 ? ?	56 ? ?	49 ? ?	45 ? ?	38 ? ?	32 ? ?	29 ? ?	26 ? ?	25 ? ?	18 ? ?	11 ? ?	4 ? ?																	1 ? ?	0 ? ?
∑ 500 cells/well	?13 ? ?																	12 48 ?	77 3 ?	19 5 5	13 5 ?	11 7 ?	99 ? ?	80 ? ?	73 ? ?	58 ? ?	53 ? ?	49 ? ?	21 ? ?	9 ? ?	5 ? ?	1 ? ?	0 ? ?
		∑ 500 cells/well	?20 ?	19 20	13 04	47 8	178 ?	91 ?	67 ?	55 ?	47 ?	45 ?	36 ?	33 ?	19 ?	9 ?	5 ?																	2 ?	0 ?
Always	?45 ?																	43 20	27 11	754 ?	37 3	25 7	21 1	17 3	15 2	13 2	11 5	10 7	58 ?	29 ?	14 ?	4 ?	0 ?

244 EGFRvIII-peptides of OD＞0.5-OVA-Elisa is positive, and screen to verify their EGFRvIII-specificity on EGFRvIII-peptide-OVA and on OVA once more in the hole.A representative example of in table 3.6, showing these results.

Table 3.6

Dull and stereotyped	The hole		1′EGFRvIII peptide-OVA?OD	2′EGFRvIII peptide-OVA?OD	OVA?OD ?
						121 ?	?G ?	1 ?	0.7534 ?	1.4065 ?	0.135 5
121 ?	?A ?	7 ?	1.3472 ?	2.1491 ?	0.126 8
						121 ?	?D ?	8 ?	0.6743 ?	0.4179 ?	0.153 1
121 ?	?E ?	8 ?	2.0415 ?	2.6965 ?	0.149 8
						121 ?	?H ?	10 ?	0.8611 ?	0.4288 ?	0.159 5
121 ?	?C ?	12 ?	2.1455 ?	2.6443 ?	0.140 4
						122 ?	?H ?	1 ?	1.8890 ?	2.5987 ?	0.116 4
122 ?	?H ?	5 ?	0.5943 ?	0.8321 ?	0.157 2
						122 ?	?F ?	8 ?	0.6834 ?	0.7715 ?	0.145 0

Limited antigen calibrating and analysis

Limited antigenic analysis is a kind of antibody and fractionated method of all other antigen-specific affinity of antibodies with antigen-specific in the B cell culture medium supernatant.When the antigen that is coated with seldom the time, but only the antibody of high-affinity can combine with any detection level under poised state.(seeing (for example) international application WO 03/48730)

EGFRvIII peptide-OVA is by with three concentration 7.5ng/ml, and 1.5ng/ml and 0.03ng/ml are coated onto on the flat board, and on 96 orifice plates 4 ℃ spend the night.Before 1% milk that adds the 50ul that contains 0.05% Hydrazoic acid,sodium salt in flat board is dissolved in the solution of PBS, each dull and stereotyped distilled water wash 5 times, B cell conditioned medium liquid of adding 4 μ l in each hole then used.Place shaking table after 18 hours under the room temperature, use distilled water wash once more dull and stereotyped 5 times.Anti-human (the Fc)-HRP of goat that adds the 1 μ g/ml of 50ul in every hole.After following 1 hour of the room temperature, use distilled water wash once more dull and stereotyped 5 times, and in every hole, add the TMB of 50 μ l.By the phosphoric acid cessation reaction of adding 50uL 1M in every hole, and read flat board under wavelength 451nm, the result shows in table 3.7.

Table 3.7

Culture medium flat plate	The hole		Limited antigen						High antigen (1.0 μ g/ml)
										?0.03ng/ml		1.5ng/ml		?7.5ng/ml
			133	B	2	?OD ?0.7670	Rank 1	OD 1.189	Rank 54								?OD ?1.871	Rank 95
?2.050
	124	G								12	?0.7400	2	1.895	1	?3.101	1			?3.463
145			C	1	?0.715	3	1.552	7	?2.671								10	?3.194
	129	G								10	?0.6720	4	1.367	22	?2.692	8			?2.977
186			B	6	?0.657	5	1.842	2	?2.859								3	?3.411
	143	F								12	?0.653	6	1.677	3	?2.741	6			?3.156
136			E	3	?0.6340	7	1.468	15	?2.683								9	?3.280
	137	C								11	?0.595	8	1.582	5	?2.94	2			?3.444
139			A	11	?0.582	9	1.374	19	?2.282								47	?2.255
	174	F								1	?0.573	10	1.577	6	?2.775	4			?2.364

Limited antigenic analysis result who produces and the total OD value that obtains in high antigen calibrating are made comparisons.By the OD value that in high antigen calibrating, obtains on the OD value ratio that will in limited antigen calibrating, obtain, finish the relative classification of affinity.Antibody with height ratio will have high affinity.Table 3.7 is showed based on the sample of limited antigen calibrating OD value with the fractionated B cell culture medium of the ratio supernatant of high antigen calibrating OD value.

Juvenile cell by FMAT is in conjunction with calibrating

Analysis EGFRvIII peptide-positive hole supernatant of OVA-Elisa and NR6 cell (NR6 M cell) are gone up the bonded ability of native form of the EGFRvIII of stably express and (are seen people Epidermal growth factorligand-independent such as Batra, unregulated, cell-transforming potential of a naturally occurring humanmutant EGFRvlII gene.Cell Growth Differ.6 (10): 1251-9 (1995)).NR6 M cell is with the sowing of the density of every hole 8000 cells, and in 96 hole FMAT plates incubated overnight.Remove and in the hole, remain the medium of 15 μ l then.Adding the B cell culture medium supernatant of 15 μ l in the hole, is that 1 μ g/ml adds the anti-IgG Fc Cy5 of 15 μ l with final concentration then.Placing it in 4 ℃ then cultivated 2 hours down.With 150 μ lPBS washed cells, and before reading on the FMAT, fix.With total fluorescent density ecbatic (table 3.8).Human anti-EGFR vIII mAb 13.1.2 is used as positive control, and its initial concentration is final concentration 1 μ g/ml, and negative control is the PK 16.3.1 of same concentrations.Have 134 to be tested as with the NR6M cell and to combine in 244 samples, 62 total fluorescent values that have greater than 8000 wherein, this 134 in conjunction with in 6 false positives are arranged.

On NR6Wt cell (NR6 cellular expression EGF receptor), do identical natural combination calibrating and (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 because of being attached to the combination (table 3.8) of Wt receptor.The PK16.3.1 that ABX-EGF is used as positive control and same concentrations is used as negative control antibody.There are 3 to combine closely with the NR6Wt cell in 134 NR6M coalitions.244 with Elisa in the bonded hole of EGFRvIII peptide in have 190 holes to be also coupled to native form on the cell.Provided example in the table 3.8.

Table 3.8

Dull and stereotyped			? ?1′VIII-pep-OVA ? OD ?	? ?2′VIII-pep-OVA ? OD ?	? OVA?OD ? ? ?	The natural FMAT that is attached to the NR6M cell	The natural FMAT that is attached to the NR6Wt cell
								174	F	1	2.4945	3.0308	0.1900	138373	1668
187	A	4	1.5337	1.2085	0.1920	128626	202459.8
								132	D	8	0.8555	1.2070	0.1649	109379	0
142	C	11	2.2889	2.8194	0.2239	94944	0
								129	A	7	2.1501	2.8208	0.1515	84024	0
127	E	1	2.6923	3.1986	0.1219	82031	0
								124	G	12	3.2929	3.5634	0.1455	73080	0
141	C	6	0.7512	1.2567	0.1547	60816	814.5
								173	C	1	2.5728	2.5714	0.2134	58702	2523.4
128	G	9	0.6293	0.7483	0.1520	49631	0
								129	H	6	2.9370	3.0952	0.2582	0	0
183	E	11	2.3450	2.7717	0.1050	0	0

In table 3.8, be identified as the Wt coalition and 141C6 combines the false positive that is with the NR6M cell from the supernatant among the 187A4 of hole.Hole 129H6 and 183E11 do not have natural bonded strong peptide coalition.

The internalization calibrating

The further ability of preceding 60 the natural internalization receptors in conjunction with B cell culture medium supernatant of calibrating.The NR6M cell is inoculated on the 96 hole FMAT plates and incubated overnight by the density with 8000 cells/well.Remove medium, and double ground adds 10-15 μ l B cell culture medium supernatant in cumulative volume 30 μ l media.Then, the second antibody (final concentration is the SS Alexa 647 anti-IgG Fab of 1.5 μ g/ml) that adds 15 μ l was also cultivated this mixture 1 hour on ice.Use irrelevant B cell culture medium buffer to understand the effect of culture medium medium.Human 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 (human anti-KLH IgG2 antibody) of same concentrations.After the cultivation, with cold PBS washed cell, add 50 μ l media in institute is porose, a duplicate was cultivated 30 minutes at 37 ℃, and another duplicate is still being cultivated on ice.After removing culture medium, add the cold 50mM glutathion of 100ul and add the cold medium of 100ul, all placed 1 hour on ice for two groups in another group 37 ℃ of cultivation groups.Use the cold PBS washed cell of 100 μ l then, mix to be incorporated among the FMAT with 1% paraformaldehyde subsequently and read.The result represents with the % internalization, calculates total fluorescent value with total fluorescent value x100 of glutathion/when not having glutathion.In table 3.9, provide information representative.

Table 3.9

Hole number	No glutathion FL1x counting	Glutathion FLIx counting is arranged	The % internalization, (glut+/glut-) X 100
				?124?C9	1877	1394	74.3％
?124?G12	26465	9959	37.6％
				?125?H1	14608	3686	25.2％
?125?D10	2342	1236	52.8％
				?127?E1	15059	1318	8.7％
?127?B9	12444.	7109	57.1％
				?127?E11	6623	0	0.0％
?128?G9	10071	1851	18.4％
				?129?A7	27648	8708	31.5％
?130?B4	4558	4354	95.5％
				?131?H5	9258	2656	28.7％
?132?D8	35820	13293	37.1％
				?133?F9	9773	3621	37.0％
?136?F10	2392	0	0.0％
				?137?G6	5104	1021	20.0％
?137?G10	3451	0	0.0％

EGFRvIII-specificity hemolytic plaque assay

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

1. the biotinylation of sheep red blood cell (SRBC).SRBC is stored in the RPMI medium stock solution as 25%.Be distributed to the cell pellet that obtains to fill 250 μ l SRBC in the new microcentrifugal tube by SRBC with 1.0ml.In little centrifuge,, inhale and remove supernatant, will precipitate again with the PBS of 1.0ml pH8.6 and hang, again repeated centrifugation with the pulse rotation precipitation SRBC of 8000rpm (6800rcf).Repeated washing circulation 2 times is transferred to the 15ml plastic tube with the SRBC precipitation then and also is settled to 5ml with the PBS of pH8.6.In independent 50ml plastic tube, in the PBS of 45ml pH8.6, add the Sulfo-NHS biotin of 2.5mg.In case biotin dissolves fully, just add the SRBC of 5ml, and rotated this pipe at normal temperatures 1 hour.With the centrifugal SRBC of 3000rpm 5 minutes, and sop up supernatant.Biotinylated SRBC is transferred in the microcentrifugal tube, and wash 3 times, in the 15ml plastic tube, be settled to 5ml (5% B-SRBC stock solution) then with immunocyte medium (RPMI 1640) with the PBS of said method with pH7.4.Before need using, with stock solution 4 ℃ of preservations.

2. apply B-SRBC with Succ-PEG-DSPE (SA).5% the B-SRBC stock solution of 1ml is transferred in the new microcentrifugal tube.The B-SRBC cell is washed 3 times as mentioned above, and resuspending is in the PBS of the pH7.4 of 1.0ml, to produce the ultimate density of 5% (v/v).(CA) stock solution is mixed also in pipe and was rotated at normal temperatures 20 minutes for CalBiochem, San Diego to add the Succ-PEG-DSPE of 10 μ l 10mg/ml.The PBS of the 1ml of repeated washing step and usefulness pH7.4 is with SA-SRBC resuspending (5% (v/v)).

3. apply SA-SRBC with EGFRvIII.EGFRvIII peptide-OVA with biotinylated 10 μ g/ml applies SA-SRBC, mixes also and rotates at normal temperatures 20 minutes.Use the PBS of the pH7.4 of 1.0ml to wash SRBC twice with said method.With the RPMI (+10%FCS) SRBC that applies of resuspending EGFRvIII and be settled to final concentration 5% (v/v).

4. determine the quality of EGFRvIII peptide-SRBC by immunofluorescence (IF).The SRBC that the 5%EGFRvIII peptide of the 5%SA-SRBC of 10 μ l and 10 μ l applies is added respectively in the new 1.5ml micro-centrifuge tube of the PBS that independently contains 40ul.To contrast human anti-EGFRvIII antibody with the concentration of 45 μ g/ml adds in each SRBC sample.These pipes were rotated 25 minutes at normal temperatures, use the PBS washed cell three times of 100 μ l subsequently.With the PBS of 50 μ l with the cell resuspending, and with 40mcg/mL (OR) the IgG Fc antibody of Lian Jieing is cultivated together for Molecular Probes, Eugene with Alexa488.These pipes rotate 25 minutes at normal temperatures, and subsequently with the PBS of 100 μ l washing, and with the PBS of 10 μ l with the cell resuspending.The painted cell of 10 μ l is put on the clean glass microscope slide, and lid is observed under fluorescent and is counted in the scope of any 0-4 with the glass cover slide.

5. plasmacytic preparation.Results had before determined to contain the content of the single micro-culture hole of the B cell clone of secreting interested immunoglobulin through multiple test.Use the automatic pipettor of 100-1000 μ l to collect the content in described hole by adding 37CRPMI (10%FCS)., transfer to then (the about 500-700 μ of final volume l) in the new 1.5ml microcentrifugal tube the cell resuspending with pipet.Under the room temperature in microcentrifuge with the rotating speed centrifuge cell of 2500rpm (660rcf) 1 minute, and subsequently with 180 degree these pipes of rotation and with the rotating speed recentrifuge of 2500rpm.Draw freezing medium also with 100 μ l RPMI (10%FCS) resuspending cells, and centrifugal.Repeat washing, and also place preservation on ice before use with 60 μ l RPMI (10%FCS) resuspending cells with RPMI (10%FCS).

6. plasmacytic micrurgy.Prepare glass slide (2 * 3 inches) and allow the preservation of spending the night at normal temperatures with the silicones edge in advance.Before the use, with SigmaCoat (Sigma, Oakville, the ON) surface of even wiping slide, the wiping tempestuously after the drying of about 5ul.The SRBC (5%v/v stock solution) that in the cell sample of each 60 μ l, adds the EGFRvIII peptide coating of 60 μ l, 4x GPC (the Sigma of preparation in RPMI (10%FCS), Oakville, ON) stock solution and 4x enhance immunity serum stock solution (prepare at 1: 150 in RPMI to use 10%FCS).Mixture (10-15 μ l) is dripped on the ready slide also with undiluted paraffin oil covering drop.Slide was cultivated under 37 ℃ 45 minutes at least.Reclaim (seeing Table 3.10) from plaque identification EGFRvIII specificity plasma cell and by micrurgy.

Table 3.10

Hole ID			The individual cells numbering	The total amount of the individual cells of results
					124	G	12	EGFRvIII-SCX-105-116(LL)	12
129	A	7	EGFRvIII-SCX-117-128(DM)	12
					174	F	1	EGFRvIII-SCX-129-137(DM)	9
182 ?	A ?	5 ?	EGFRvIII-SCX-138-149(LL)；162-169 (OP)	20 ?
					125 ?	D ?	10 ?	EGFRvIII-SCX-170-181(DM)；194-201 (LL)	20 ?
127 ?	B ?	9 ?	EGFRvIII-SCX-182-193(LL)；202-209 (OP)	20 ?
					190	D	7	EGFRvIII-SCX-210-229(LL)	20
130	B	4	EGFRvIII-SCX-230-249(LL)	20
					138	D	2	EGFRvIII-SCX-250-269(LL)	20
145	C	1	EGFRvIII-SCX-80-92(DM)	13
					172	B	12	EGFRvIII-SCX-93-104(LL)	12
187	A	4	EGFRvIII-SCX-270-281(LL)	12
					173	C	1	EGFRvllI-SCX-282-293(BC)	12
127	E	1	EGFRvIII-SCX-294-305(LL)	12
					142	C	11	EGFRvIII-SCX-306-317(LL)	12
141	A	10	EGFRvIII-SCX-318-329(BC)	12
					132	D	8	EGFRvIII-SCX-330-341(LL)	12
124	D	4	EGFRvIII-SCX-342-349(BC)	8

Unicellular PCR, clone, expression, purification are also identified the anti-EGFRvIII antibody of reorganization.

The genes encoding the variable regions were rescued by on the is by carrying out the gene of RT-PCR recovery coding Variable Area on single micrurgy plasma cell.Extract mRNA and carry out reverse transcription PCR to produce cDNA.The cDNA of coding variable heavy chain and light chain is increased specifically with polymerase chain reaction.Be cloned in the IgG1 expression vector in the district with human variable heavy chain. (Invitrogen; Burlington produce this carrier in a plurality of cloning sites ON ) by the constant domain of IgG 1 being cloned into pcDNA3.1+/Hygro.Be cloned in the IgK expression vector in the district with human variable heavy chain. ( Invitrogen, Burlington produce this carrier in a plurality of cloning sites ON ) by the constant domain of human IgK being cloned into pcDNA3.1+/Neo.The heavy chain and the light chainexpression vectors were then co-lipofected into a 60mm dish of 70％ confluent humanembryonal kidney 293 cells and the transfected cells were allowed to secrete a recombinantantibody with the identical specificity as the original plasma cell for 24-72 hours.HEK293 ( 3mL ) ELISAIgG ( 3.11 ) 。 Use ELISA to evaluate specificity (table 3.11) with combining of EGFRvIII by recombinant antibodies.

Table 3.11

mAb?ID	Cell #	Titration
				Total antibody	The antigen combination
		129A7	SC-EGFRvIII-XG1-123/124			?＞1∶64	＞1∶64
138D2	SC-EGFRvIII-XG1-250			?＞1∶64	＞1∶64
		174F1	SC-EGFRvIII-XG1-131			?＞1∶64	＞1∶64
182A5	SC-EGFRvIII-XG1-139			?＞1∶64	＞1∶64
		190D7	SC-EGFRvIII-XG1-211			?＞1∶64	＞1∶64
125D10	SC-EGFRvIII-XG2-170			?＞1∶64	＞1∶64
		182D5	SC-EGFRvIII-XG2-150			?＞1∶64	＞1∶64
141A10	SC-EGFRvIII-XG1-318			?1∶64	1∶64
		132D8	SC-EGFRvIII-XG1-333			?＞1∶64	＞1∶64
124D4	SC-EGFRvIII-XG1-342			?＞1∶64	＞1∶64

Following the carrying out of secretion ELISA test.For antibody-secreting, the anti-IgG H+L of the goat of 2 μ g/mL and the EGFRvIII-Rab Ig Fc fusion rotein that is used for the bonded 1.5 μ g/ml of antigen be coated on Costar LabcoatUniversal Binding Polystyrene 96 orifice plates and remain on four spend night.With the described plate of distilled water wash five times.From undiluted small-sized lipofection supernatant to 1: the 2 titration recombinant antibodies in 7 holes.With the described plate of distilled water wash five times.Add the special HRP binding antibody of the anti-IgG Fc of goat room temperature maintenance 1 hour to and the dull and stereotyped final concentration of combination dull and stereotyped with 1 μ g/mL with the secretion of the anti-Hu Fc of 1ug/ml Rb detection at room temperature 1 hour.With the described plate of distilled water wash five times.Adding TMB added the plate colour developing 1M phosphoric acid and stops ELISA in 30 minutes.Analyze each ELISA flat board to determine the optical density of every hole when the 450nm.

Order-checking and sequence analysis

Check order at the heavy chain of both direction, and analyze with the germ cell line sequence source of definite antibody and the change of identification and germ cell line sequence to the clone.These sequences are provided in the accompanying drawings.3A-3K and (SEQ ID NO:34-55). the comparison of the germ cell line sequence in each heavy chain and sequence of light chain and its source is provided in accompanying drawing 4-7.In addition, in the accompanying drawings sequence and its germ cell line sequence of the 13.1.2 antibody that derives from hybrid cell compared 4 and 5.

Should be appreciated that from the discussion of this paper each 131 antibody and 13.1.2 antibody all have the affinity very high to EGFRvIII, it is by cell internalization, and shows very high cell killing efficient when combining with toxin.What is interesting is that each antibody even it produces from the different immunity of XenoMouse mice or uses different technology, all derives from closely similar germ cell line gene.Yet based on the location of epi-position (discussing to some extent herein), each antibody it seems with the EGFRvIII molecule on visibly different epi-position in conjunction with and in conjunction with necessary EGFRvIII, have significantly different residue.These result's indications, the utilization of germ cell line gene is very important to the antibody therapeutics at EGFRvIII, and small change can be changed the combination of antibody and the fact of effect has allowed antagonist and other further designs based on the antibody therapeutics of these topology discoverys.

The natural EGFRvIII that expresses on anti-EGFR vIII mAbs and the cell combines

In this example, measurement anti-EGFR vIII antibody combines with the NR6M cell.Especially, calibrating derives from the not quantitative IgG1 supernatant of XenoMax and the binding ability of NR6M and NR6WT.With the density inoculating cell in 10000/ hole and in 37 ℃ of following FMAT 96 orifice plates overnight incubation.Remove medium and add the small-sized fat supernatant of 40 μ l and come titration, cell was cultivated on ice 1 hour.Human 13.1.2EGFRvIII antibody and ABX EGF (E7.6.3, United States Patent (USP) the 6th, 235, No. 883) antibody add as positive control.PK 16.3.1 antibody is as negative control.With cold PBS washed cell, cultivated 1 hour with the density adding second antibody (the anti-IgG Fc of SS Alexa) in 1 μ g/ml, 40 μ l/ holes and on ice.Use cold PBS washed cell then, fix again and read with FMAT then.The screening of NR6WT is detected the binding specificity of all antibody by counting.

Reorganization anti-EGFR vIII purifying antibody.

For large-scale production, heavy chain and light chain expression vector (the every chain/plate of 2.5 μ g) are by in the 70% HEK293 cell that is paved with in lipofection to the ten 100mm plate.Transfectional cell was cultivated 4 days down at 37 ℃, and results supernatant (6mL) also use the fresh medium of 6ml to substitute.At the 7th day, remove supernatant and also merge (being total to 120ml) from 10 flat boards with initial results.Use protein-A agarose (Amersham Biosciences, Piscataway, NJ) affinity chromatograph (1mL) each antibody of purification from supernatant.With the 0.1M glycine of 500mcL pH2.5 with antibody eluting from protein-A post.Eluent is dialysed in the PBS of pH7.4, and carries out the filter membrane sterilization.Analyze antibody to evaluate its purity and output with non-reduced SDS-PAGE.Also measure concentration with the ultraviolet analysis of OD250.

EGFRvIII receptor internalization by reorganization anti-EGFR vIII mAb

The IgG1 recombinant antibodies of express as previously mentioned,, the also quantitative XenoMax of purification originating.Further calibrating antibody is in the ability of NR6M cell internalization EGFRvIII receptor.250, the 000NR6M cell was cultivated 7 minutes on ice on 96 hole v base plates with the concentration of 0.25 μ g/ml with first antibody (SC95, SC131, SC133, SC139, SC150, SC170, SC211, SC230, SC250 and human 13.1.2 in contrast), and three such duplicates are arranged.With the PBS washed cell of cold 10%FCS and add the second antibody (the anti-IgG Fab of SS Alexa) of 3 μ g/ml Fab and cultivated 7 minutes on ice.Once and subsequently use cold medium resuspending with the cold PBS washed cell that contains 10%FCS.Then, two groups in three duplicates 37 ℃ cultivate and remaining one groups cultivated 1 hour at 4 ℃.After this, handling (as mentioned before) 1 hour with glutathion on ice 4 ℃ of cultured cells with at one group of cell of 37 ℃ of cultivations.Wash and the resuspending cell with the cold PBS that contains 1%FCS of 100 μ l then, and use facs analysis.From calculate % internalization [(37 ℃ of average-4 ℃ averages of handling with glutathion)/(37 ℃ of average-4 ℃ averages)] from the geometric mean of facs analysis acquisition with the glutathion processing without the glutathion processing with the glutathion processing.NA means carried out facs analysis but data do not provide in table 3.12.

Table 3.12

mAb	The FACS geometrical mean			The % internalization
					37 ℃ of glutathion useless	With 37 ℃ of glutathion	With 4 ℃ of glutathion
	13.1.2	22.12	19.19					5.38	82.5％
sc95				22.56	17.75	5.13	72.4％
	sc131	NA	NA					NA	72％
sc133				23.39	18.63	6.24	72.2％
	sc139	22.64	19.23					4.88	80.8％
sc150				20.29	7.78	4.66	20.0％
	sc170	19.97	7.75					4.67	20.1％
sc211				20.76	8.23	4.78	21.6％
	sc230	20.68	7.97					5.02	18.8％
sc250				24.13	8.07	4.84	16.7％

13.1.2 be one by before producing antibody that (example 2) produce and as a negative control in this test at the hybrid cell of EGFRvIII epi-position.These results of table 3.12 have shown two subgroup antibody: those of internalization (70-80%) and do not have internalization those (22% or still less) effectively.

The location of the epi-position of example 4 human anti-EGFRvIII antibody

In order to determine the bonded epi-position of specific antibodies of the present invention, 6 mankind of anti-EGFRvIII position with the synthetic peptide of 3 murine monoclonal antibodies (mAb) use from the peptide sequence of special EGFRvIII.Localized antibody is the anti-EGFRvIII 13.1.2 antibody that derives from human hybrid cell, the anti-EGFRvIII 131,139,250,095 that derives from human XenoMax and 211 antibody and the anti-EGFRvIII H10 of murine, Y10 and B9 antibody.

The method of using for conventional SPOT peptide array (Sigma Genosys) to study the interaction of molecules between human anti-EGFrVIII antibody and its peptide epitopes.The SPOTs technology is based on to be fit to the peptide solid phase synthesis of antibody epitope systematic analysis.The synthetic of conventional arrays oligopeptide can be buied from Sigma-Genosys.From the peptide array of Sigma-Genosys order from the overlapping oligopeptide of EGFRVIII variable amino acid sequence.

A series of nine 12-mer peptides are synthetic as the plaque on the polypropylene screen.The peptide array is striden the residue 1-20 of EGFrVIII sequence, represent the outer wtEGFr domain of born of the same parents aminoacid 6-273 disappearance and in the generation of junction point one glycine (G).Each continuous peptide derived from previous peptide, produces nested, the eclipsed storehouse of an array oligopeptide with a residue.Have the different anti-EGFrVIII antibody of film (the 1 μ g/ml) reaction of 9 peptides with 9.Connecting immunosorbent adsorption test by an enzyme uses bonded HRP second antibody to use combining of enhanced chemiluminescence (ECL) evaluation mAb and film binding peptide then.In table 4.1, show used array.

Table 4.1 plaque array sequence:

1.ALEEKKGNYVVT(SEQ?ID?NO：72)
	2.LEEKKGNYVVTD(SEQ?ID?NO：59)
3.EEKKGNYWTDH(SEQ?ID?NO：73)
	4.EKKGNYWTDHG(SEQ?ID?NO：74)
5.KKGNYWTDHGS(SEQ?ID?NO：75)
	6.KGNYWTDHGSC(SEQ?ID?NO：76)
7.GNYWTDHGSCV(SEQ?ID?NO：77)
	8.NYVVTDHGSCVR(SEQ?ID?NO：78)
9.YVVTDHGSCVRA(SEQ?ID?NO：79)

In addition, by group and alanine screening position location functionality epi-position.I in this course, use in the combination alanine screening technique identification EGFrVIII peptide to anti-EGFRvIII mAb interaction essential amino acid.For reaching this target, order second group of SPOT array and be used for the alanine screening.Panel as the variable peptide that the alanine replacement is arranged in 12 residues of above-mentioned scanning.Plaque #1, Bian Yi sequence is not a positive control of antibodies.In table 4.2, show used array.

Table 4.2 alanine screening array:

1.LEEKKGNYVVTD(SEQ?ID?NO：59)
	2.AEEKKGNYVVTD(SEQ?ID?NO：80)
3.LAEKKGNYVVTD(SEQ?ID?NO：81)
	4.LEAKKGNYVVTD(SEQ?ID?NO：82)
5.LEEAKGNYVVTD(SEQ?ID?NO：83)
	6.LEEKAGNYWTD(SEQ?ID?NO：84)
7.LEEKKANYVVTD(SEQ?ID?NO：85)
	8.LEEKKGAYWTD(SEQ?ID?NO：86)
9.LEEKKGNAVVTD(SEQ?ID?NO：87)
	10.LEEKKGNYAVTD(SEQ?ID?NO：88)
11.LEEKKGNYVATD(SEQ?ID?NO：89)
	12.LEEKKGNYVVAD(SEQ?ID?NO：90)
13.LEEKKGNYVVTA(SEQ?ID?NO：91)

All 9 mAb discern by SPOT program location the epi-position of human EGFrVIII.All 9 antibody all can with described reactive polypeptide.In table 4.3, shown with 3 murine antibody and 6 results that derive from the human antibodies acquisition of XenoMouse mice.The residue that highlights makes a variation into alanine and cancels bonded residue by the antibody of test for us.Therefore these are the relevant residue that is attached on the antibody.

Table 4.3

EGFR	?A	?T	?C	?V	K	?K	?C	?P	?R	?N	?Y	?V	?V	?T	?D	?H	?G	?S	?C	?V	?R	?A	?SEQ?ID?NO：92
																								EGFRvIII				?L	E	?E	?K	?K	?G	?N	?Y	?V	?V	?T	?D	?H	?G	?S	?C	?V	?R	?A	?(SEQ?ID?NO：93)
13.1.2					E	?E	?K	?K	?G	?N	?Y	?V	?V	?T									?(SEQ?ID?NO：94)
																								131					E	?E	?K	?K	?G	?N	?Y	?V	?V	?T									?(SEQ?ID?NO：94)
139				?L	E	?E	?K	?K	?G	?N	?Y	?V	?V	?T	?D								?(SEQ?ID?NO：95)

250				?L	?E	?E	?K	?K	?G	?N	?Y	?V	?V	?T	?D								?(SEQ?ID?NO：95)
																								095											?Y	?V	?V	?T	?D	?H							?(SEQ?ID?NO：96)
211											?Y	?V	?V	?T	?D								?(SEQ?ID?NO：97)
																								H10											?Y	?V	?V	?T	?D								?(SEQ?ID?NO：97)
Y10					?E	?E	?K	?K	?G	?N	?Y	?V	?V	?T									?(SEQ?ID?NO：98)
																								B9									?G	?N	?Y	?V	?V	?T									?(SEQ?ID?NO：99)

The hypographous aminoacid of showing in the table 4.3 is the residue that antagonist is discerned maximally related antigen recognition site.The peptide of use overlapping sequence is accurately located the shortest length of the epi-position of all ten mAb, and determines mAb and the bonded permission of variant epitope by each residue that replaces with alanine in the epi-position systemicly.

In table 4.4, the supplementary features of antibody have been summed up.Especially, under the non-reduced or reducing condition in the Western of polyacrylamide gel electrophoresis flat board test subgroup antibody to the combination of tumor cell line lysate.The recombiant protein that also comprises purification.Bonded antibody shows that epi-position is linear under reduction and non-reduced condition.Sample identification:

EGFRvIII-rabbit Fc fusion rotein

H1477-expresses the H80 human tumour cell line who makes up transfection with EGFRvIII.These cellular expressions EGFR and EGFRvIII.

The Wild type EGFR albumen of EGFR-purification.

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

A549-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-is as to specific high responsive test, and the EGFR's of test mAb and purification combines in Biacore

Table 4.4

mAb ? ?	EGFRvIII Western (naivety)	REGFRvIII Western (reduction)	EGFRvIII FACS ?	H1477 Western (naivety)	H1477 Western (reduction)	pep3 Kin′ExA ?	EGFR Western (naivety)	EGFR Western (reduction)
									13.1.2	+	+	+	?+	?+	25pM	?-	?-
131	+	+	+	?+	?+	0.05pM	?-	?-
									139 ?	？ ?	+ ?	+ ?	?ND ?	?ND ?	ND ?	?ND ?	?ND ?
095	+	+	+	?ND	?ND	ND	?ND	?ND
									211	+	+	+	?ND	?ND	ND	?ND	?ND
250	+	+	+	?ND	?ND	ND	?ND	?ND

MAb ? ?	EGFR Biacore ?	A431 FACS ?	A431 Western (naivety)	A431 Western (reduction)	A549 FACS ?	A549 Western (naivety)	A549 Western (reduction)	H80 FACS ?	H80 Western (naivety)	H80 Western (reduction)
											13.1.2	-	-	-	-	-	-	-	-	-	-

131	-	++	-	-	+	-	-	-	-	-
											139	N.D.	ND	ND	ND	ND	ND	ND	ND	ND	ND
095	-	ND	ND	ND	ND	ND	ND	ND	ND	ND
											211	-	ND	ND	ND	ND	ND	ND	ND	ND	ND
250	-	ND	ND	ND	ND	ND	ND	ND	ND	ND

The result shows that most of mAb have identical in essence binding specificity, seven mAb show the binding specificity with variable EGFrVIII, simultaneously 2 mAb and wild type EGFr (the muroid H10 and the mankind 211) cross reaction in the lysate of the Western of purifying protein trace and A431 cell.Note, yet, although in the Western trace antibody 211 and naivety and equal combination of reductive purification EGFRvIII, its with non-reduced proteic combine strong slightly.In the test to the A431 cell pyrolysis liquid, antibody 211 combines with a group Wild type EGFR is strong in non-reduced sample, but does not have signal in going back raw sample.This shows that the combination of antibody 211 is owing to the comformational epitope in Wild type EGFR, and performance is different in EGFRvIII.The epi-position of 5 mAb is in the residue 2-12 that strides the variable specificity glycine residue of EGFRvIII, yet 4 mAb (comprising H10 and 211) stride EGFRvIII and the total residue 7-16 of wild type EGFr.Antibody 131 combines with A431 and A549 cell in FACS.These cells are expressed EGFRvIII and are negative and EGFR are expressed be positive.Antibody 131 does not combine with the EGFR of non-reduced or non-reduced purification or reduction or non-reduced A43 and A549 cell pyrolysis liquid and shows that antibody 131 may combine with the variable EGFR at some human tumour cell lines' cell surface expression in Western.These variants are to the degeneration sensitivity.

Example 5

The specificity of external anti-EGFRvIII antibody

By the NR6 cell with wild type or variation EGFR transfection is carried out the specificity that facs analysis is determined antibody purification.With the corresponding antibody of 5 μ g/ml cell was cultivated on ice 1 hour,, and cultivated with the anti-IgG of the bonded goat of PE subsequently with the washing of FACs buffer.

Example 6

Cross reactivity with the EGFR that increases

Demonstration at the antibody of variable EGF receptor on the producer expanded cells with the subgroup cross reaction (people such as Johns, Int.J.Cancer.98:398,2002) of wild type EGF receptor.For determining whether human EGFR vIII antibody recognition has similar characteristic, in culture medium, the ability of the wild type EGF receptor on its identification various kinds of cell is tested.Cultivate down at 4 ℃ with antibody with designated cell system.After the washing of FACS buffer, add the second antibody that is combined with phyoerythrin, and continue to cultivate.The cell line of all analyses is all expressed Wild type EGFR.By antibody on A431 and SF-539 cell and not on A498 or SKRC-52 cell the subgroup of antibody XG1-131 identification Wild type EGFR.The antibody 13.1.2 of another EGFRvIII and the subgroup of nonrecognition Wild type EGFR.Take all factors into consideration these data, only show can the recognizing cells surface at the subgroup of the antibody of variation EGFRvIII wild type EGRF.The ability of the subgroup of specific antibody recognition wild type EGF receptor at variation EGFRvIII does not also rely on total EGFR density but may represent the new comformational epitope special to tumor cell.May determine (seeing the result of epitope mapping and affinity determining section herein) jointly by the specific epitopes of variation receptor inter-node with to the affinity of the antibody of this unique decision base at the ability of the subgroup cross reaction of the antibody of EGFRvIII and wild-type receptor.

The specificity of anti-EGFRvIII antibody in example 7 bodies

Antibody combines with cell line

The specificity of these antibody purifications is determined by carry out facs analysis on the panel of cell line.What cell line was used is: the human glioblastoma cell line of H80-, the H1477 (H80-EGFRvIII) that expresses high-level EGFRvIII, A431-human epidermal tumor cell line and the human lung tumor cell of A549-system.All cells system all from Dr.Bigner except from ATCC (Rockville, MD, A431 U.S.A) and A549.With the corresponding antibody of 10 μ g/ml cell was cultivated on ice 30 hours,, and used (WestGrove, PA, the anti-IgG cultivation of the bonded goat of PE U.S.A.) subsequently from Jackson ImmunoResearch with the washing of FACs buffer.In Fig. 9 A-9L and 10A-10D, dark block diagram shows with the irrelevant painted cell of IgG, the dyeing of profile diagram or white histogram graph representation associated antibodies.

Anti-EGFRvIII antibody 13.1.2,131 and 139 and the EGFRvIII protein binding fastened of transfectional cell.In Fig. 9 M-9P, show the figure that sums up some results.

Demonstration at the antibody of variable EGF receptor on the producer expanded cells with the subgroup cross reaction (people such as Johns, Int.J.Cancer.98:398,2002) of wild type EGF receptor.In this example, A431 and A549 are dyeed with XG1-131 and XG1-139.Figure 10 B and Figure 10 C show that 131 and 139 have the cross reactivity specific with Wild type EGFR, rather than only discern the subgroup of H80, A431 and A549 cell line.Yet this cross reactivity is only on these cell lines 10% of the level of painted ABX-EGF (E7.6.3).The result provides in Fig. 9 A-9P and 10A-10D.

Can be used as the delivery vehicles that medicine or toxin is transported to specifically cell at the antibody of cell surface antigen.If the internalization of this antibody stimulator antigen, perhaps at medicine or toxin after the antibody fracture, medicine or toxin can cause the death of cell.This mechanism is used in animal or the patient body killing tumor cell specifically.The method that selection can conduct drugs to the antibody of cell is by the secondary cell toxicity test.In these trials, first antibody combines with cell surface and adding and medicine or the bonded second antibody of toxin.If the internalization of first antibody stimulator antigen, second antibody will be total to internalization, in case just medicine or toxin fracture cause cell killing.

Example 8 secondary cell toxicity tests

In following research, the EGFRvIII-specific antibody be used to with the glioblastoma cell line (H1477) of the bonded toxin of second antibody guiding glioblastoma cell line (H80) and EGFRvIII transfection.From human IgG of Pharmingen (BD Biosciences Pharmingen) mouse anti (cat#555784) and toxin A EFP (Seattle Genetics, Inc.) and maytansine (DM1, Immunogen Inc.) in conjunction with to produce mah-AEFP (anti-IgG of muroid-AEFP) and mah-DM1 (the anti-IgG of muroid-DM1).In conjunction with by the anti-IgG of the proteic goat of saporin, Hum-ZAP (TM, cat # IT-22-250, the anti-IgG of the goat of affinity purification-saporin albumen) be from Advanced Targeting Systems (San Diego, CA, U.S.A.).H80 and H1477 cell with the concentration plating of 1000 cells in the 10 μ l somatomedins of every hole on 96 orifice plates.After 24 hours, first antibody and second antibody are mixed at 1: 3, continuously 6 dilutions in aerial 1: 5.The first antibody of 100 μ l dilution and toxin second antibody mixture add in the hole of cell with the final initial concentration 0.1 μ g/ml of first antibody, the final initial concentration 0.3 μ g/ml of second antibody.Allow these dull and stereotyped continuation to cultivate 3 days.At the 4th day, add that (CellTiter-Glo reagent (cat#G7571) U.S.A.) is also read with fluorescent for Madison, WI available from Promega.Figure 11 A-111,12A-12I and 13A-13I show this result of experiment.In the special mAb test of most of EGFRvIII, relatively kill and wound with the antigen-specific that at the Hum-ZAP of H80 (embankment piece) is medium at HI477 (filling out circle).MAb XG1-131 and XG1-139 produce the antigen-specific secondary with mah-AEFP and kill and wound, and are using mah-DM1 more among a small circle.In the antibody of test, XG1-131 has better performance than 13.1.2, XG1-095, XG1-139, XG1-150, XG1-170, XG1-250 and XG1-211 at least in a record.IgG1 is used as negative control and antigen-positive cell (H1477) and antigen negative cell (H80) is compared.

The visual specific use of amount of the specific killing that needs and changing.In one embodiment, the minimizing of any possible cancerous cell all is enough.For example, 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 desired destination cell quantity still is a non-specific destructive function of antibody combination.For example, if the non-specific target and the destruction of antibody are seldom arranged, only have that the antibody of 10% target cell decreased number/toxin combination may be just enough.For example, antibody/toxin mixture kills and wounds and is lower than 10% of non-target complex.Equally, specific amount will depend on specific needs and situation.Useful especially antibody be those to target cell (for example H1477) have high selectivity and with the target cell good combination.In one embodiment, target is EGFRvIII albumen or its fragment.In one embodiment, showed human or humanized, effectively internalization, to EGFRvIII albumen or its fragment antibody special, that closely link to each other and link to each other with EGFRvIII albumen or fragment with effective toxin.

Example 9 secondary cell toxicity tests

Except cell toxicity test, also in producing clonogenic assay, tested the EGFRvIII specific antibody.Special EGFRvIII antibody will with the bonded second antibody of toxin guiding EGFRvIII transfection glioblastoma cell line (H1477), toxin is released in cell, and has finally reduced the ability that cell proliferation forms the clone.Therefore, when cell when the firsts and seconds toxin antibody is handled the back by plating once more, the application of these EGFRvIII antibody-toxin produces the minimizing of clone's quantity.In this example, H80 and H1477 cell are by with the density of every hole 30,000 cells once more on plating to 6 flat board and incubated overnight.First antibody and secondary toxin antibody were with 1: 3 mixed.The final concentration of this mixtures of antibodies with first antibody 0.5 μ g/ml, second antibody 1.5 μ g/ml added in the suitable hole.37 ℃ of incubated overnight.After the cultivation, handle this toxin mixture suitably, and cell is therefrom separated with the 1x trypsin.Cell be counted and with the density plating of every plate 200 cells in 6 new orifice plates.Three duplicates of corresponding each processed group of plating.Cultivate these dull and stereotyped 2-3 weeks at 37 ℃, until forming clone and can be with the naked eye or microscopically identification.The sucking-off medium also added the 5M methylene blue 1 hour.Wash with water dull and stereotyped and the counting clone.Figure 14 A and Figure 14 B show this result of experiment.As seen, with regard to three kinds of EGFRvAIII antibody of test, mab-AEFP secondary toxin antibody has suppressed the clone and has formed.

Example 10 anti-EGFRvIII antibody (13.1.2)

Direct combination in the cell toxicity test

Combining by antibody and cell toxicity medicament direct provides the antibody can be with the further evidence of drug conveying to a tumor cell.In following example, direct and the auristatin E MMAE of EGRvIII antibody 13.1.2, combine (MMAE and AEFP are all available from Seattle Genetics and description to some extent hereinbefore) with AEFP by peptide bond, and close (DM1 is available from Immunogen and description to some extent hereinbefore) with the mercaptan bond with maytansine (DM1).In case in the cell of expressing EGFRvIII antigen H1477, add conjugate, just observe special cytotoxicity.The cell of antigen expressed H80 being is not is not only just being killed and wounded when being exposed to the antibody of unusual high concentration.The result who in Figure 15 A-15C, shows this test.

EGFRvIII antibody is the method that has superiority especially that is used for the treatment of with direct combination of medicine or toxin.Therefore, initial test shows that such combination causes expressing the specific killing of the cell of EGFRvIII really.

Anti-EGFRvIII antibody characteristic in example 11 bodies.

The optional method whether a kind of definite antibody can be transported to cytotoxic drug cell is an effect of estimating the growth of binding antibody human tumor in vivo.This example provides a kind of like this method.Become neuroglial cytoma at In vitro culture H1477, by the trypsin results, and such as hereinafter explanation be embedded in the matrigel.Subcutaneous injection 5,000,000 cells in female nude mouse, and allow tumor development to reach about 0.5cm until it ³Size.This moment is the animal random packet, and begins not have once hypodermic processing in 4 days with the binding antibody of indicating concentration.Result among Figure 16 shows that antibody 13.1.2 is when causing the degeneration of glioblastoma multiforme when combining with maytansine (dEGFR-DM1) or auristatin E (dEGFR-MMAE).If with the unconjugated medicine that equates together during administration, (group 2),, illustrate that body interior orientation tumor cell needs combining of antibody and toxin to not influence of tumor growth.

Above the animal model of Shi Yonging obtains by H1477 cell xenograft is injected nude mice.Not commensurability cell is together injected 8 all nu/nu female mices separately or with MATRIGEL, and the post analysis tumor was implanted in several days.From this was analyzed, it was 5,000,000 about 22 days in MATRIGEL cells that identification allows the cell quantity of about size tumor.Comprise group G8 in contrast, be used to show that killing and wounding is antibody specificity.Comprise group G7 as negative control.

Therefore develop and a following scheme that is used for toxin study:

The 1st day: implanted 8 weeks of 500 ten thousand tumor cells in MATRIGEL are female mice greatly.The 22nd day: such as table 11.1 displaying, handle with the antibody prodrug by per four days of I.V..

Table 11.1

Group	The mice numbering	Handle
			G1	8	13.1.2-DM1 per 4 days of 250 μ g pass through IV.
G2	8	13.1.2-DM1 per 4 days of 75 μ g pass through IV.
			G3	9	13.1.2-MMAE per 4 days of 75 μ g pass through IV.
G4	8	13.1.2-MMAE per 4 days of 250 μ g pass through IV.
			G5	9	13.1.2-AEFP per 4 days of 75 μ g pass through IV.
G6	8	13.1.2-AEFP per 4 days of 250 μ g pass through IV.
			G7	8	Per 4 days of PBS passes through IV.
G8	9	(13.1.2 not combination) 250 μ g+ maytansines, 4 μ g

The result shows in Figure 16, the interpolation of arrow indication medicine.Group G1, G6 and G4 have showed effectively and have killed and wounded.Group G3 shows more a spot of killing and wounding.Group G8 and G7 do not show and kill and wound.In the vc-AEFP of high dose group-group G8, may observe certain toxicity.The processing of twice 250 μ g of these animals received and the processing of one time 125 μ g.

The EGFRvIII of example 12 in cancer patient/human tumor

The expression of EGFRvIII on human tumor is by 2 muroid monoclonal antibody (B9 from a plurality of cancer patients and known specific bond EGFRvIII, IgG1 and Y10, the dyeing of bonded frozen tissue part IgG2 (Dr.Bigner, Duke University)) is determined.Same section dyes with the control antibodies of homotype coupling.Showed the summary of the coloration result that from patient's sample, obtains in the table 12.1.

Table 12.1

Tumor type	Sample size (N)	?EGFRvIII＞+	EGFRvIII＞++
				The neuroblast glioma	?8	?100％	100％
Breast carcinoma	?100	?31％	24％
				The NSCL cancer	?51	?47％	39％
Head and neck cancer	?21	?42％	38％
				Carcinoma of prostate	?22	?4.5％	4.5％

EGFRvIII＞+: the tumor that comprises all expression EGFRvIII

EGFRvIII＞++: comprise and express 10% or the tumor of more EGFRvIII at least

Express and mainly on cell membrane and/or Cytoplasm, find.Significantly breast (31%), NSCL (47%) and head and neck (42%) the cancer sample dyeing of quantity are the EGFRvIII positive.In specific examples, in order to obtain high-quality IHC dyeing, the use of two antibody may be than better with an antibody.The frozen tissue sample is better than fixing organization.

The technical staff in described field should be appreciated that test patient is favourable to guarantee that pending tumor is expressed EGFRvIII before the antibody that uses treatment.

Anti-EGFRvIII antibody characteristic in example 13 bodies

The method of example 11 will be used to handle pulmonary carcinoma and glioma.This will extensively detect by making animal model.Be used to into the following formation of animal model of glioma and pulmonary carcinoma: will express the lung carcinoma cell EGRFvIII transfection of wt-EGFR.Cell is injected the lung of nu/nu mice, allow tumor development to above can be relatively to the stage.Anti-EGFRvIII conjugate per subsequently 1 to 10 day (optionally) is by subcutaneous injection.Will be to size, obstruction or oppressive monitoring of these cancerous cell continued growths, to determine the effect of these anti-EGFRvIII antibody and antibody-toxin conjugate.The technical staff in described field should be appreciated that any antibody that this paper discloses can so be done.

Example 14 is by replacing the functional characteristic of analyzing peptide

In order further to solve these identifications, carry out replacement analysis to the amino acid residue of epitope peptide to the indispensable amino acid residue of EGFRvIII epi-position.Initiation site is from the sequence LEEKKGNYVVTD of example 4 (SEQ IDNO 59).In this example, each aminoacid of localized epi-position by 20 L type aminoacid replacement, therefore, is synthesized all possible unit point analog by one by one, and the 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 mAb 131 and 13.1.2.The result sums 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 demonstration, mAb 13.1.2,5 residues are necessary although have only 4 residues for the combination of mAb 131 to very important in conjunction with (runic).Remaining residue is replaced by several amino acids and not significant bonded losing.Although 131 is identical with 13.1.2 on sequence and length, its binding pattern difference.The combination of MAb 131 has very strong dependency to residue EKNY (SEQID NO:60).On the other hand, data show residue EEKGN (SEQ ID NO:61) relates to the combination at mAb13.1.2.

The reorganization of example 15mAb chain

Heavy chain and the light chain of reorganization MAb131 and 13.1.2, and with its transient transfection to the 293T cell.After 72 hours, collect supernatant and with ELISA secrete and EGFrVIII in conjunction with test.

The result shows the expression of antibody sources in 131 heavy chains that 13.1.2 κ chain is arranged, otherwise also expresses well, but since the different binding pattern of two kinds of antigenic antibody of EGFrVIII in conjunction with activity 75% (data not shown) that descended.This has shown 131 different with the 13.1.2mAb paratope, hints that once more the architectural feature of the epi-position of selecting at two kinds of mAb is different.

The molecular modelization of example 16131 and paratope thereof

How this example can produce the proteic three dimensional structure of embodiment if showing.The 3 d structure model of the variable region of antibody 131 produces by a homology model method, and described method is used Accelrys (San Diego, InsightII modelling packing CA).Sequence construct model according to the variable region of following described table 16.1.The numbering of residue is from light chain amino acid, and extends to heavy chain aminoacid.

Table 16.1

Variable region of light chain

DTVMTQTPLSSHVTLGQPASISC (SEQ?ID?NO：100)

RSSQSLVHSDGNTYLS(CDR1) (SEQ?ID?NO：101)

WLQQRPGPPRLLIY (SEQ?ID?NO：102)

RISRRFS(CDR2) (SEQ?ID?NO：103)

GVPDRFSGSGAGTDFTLEISRVEAEDVGVYYC (SEQ?ID?NO：104)

MQSTHVPWT (CDR3)(SEQ?ID?NO：105)

FGQTKVEIK (SEQ?ID?NO：106)

Variable region of heavy chain

QVQLVESGGGVVQSGRSLRLSCAASGFTFR (SEQ?ID?NO：107)

NYGMH(CDR1) (SEQ?ID?NO：108)

WVRQAPGKGLEWVA (SEQ?ID?NO：109)

VIWYDGSDKYYADSVRG(CDR2) (SEQ?ID?NO：110)

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ?ID?NO：111)

DGYDILTGNPRDFDY(CDR3) (SEQ?ID?NO：112)

WGQGTLVTVSS (SEQ?ID?NO：113)

Use the sequence of antibody 131 in albumen database (Protein Data Bank), to search for, to identify homologous antibody and structure thereof.On the basis of the homologous antibody sequence of similar and 131 antibody, some structures have been selected.The modelling structures of samples of choosing from albumen database that is used for has albumen database identifier: 1HEZ, 2H1P, 1AQK, 1DQL, 1MF2 and 1FLR.Overlap ratio is to these formwork structures and be used to produce the sequence alignment based on structure of these templates subsequently.Subsequently the sequence and the template sequence of antibody 131 variable regions are compared.The comparison result of utilization structure and sequence is used to produce the molecular model of 131 antibody variable regions.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 be according to structural model calculating and in Figure 17, show.Different CDR identifications are 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 antibody 131 mutual of doing the surface that predicted significantly refer in particular to.Dark hole mainly by 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 the hole in be residue 31,37,95-101,143-147,159,162-166,169-171,211-219,221 and 223.These residues may comprise paratope and play the effect of closing key contacts in conjunction with the EGFRvIII paratope time.These residues also may provide the primary structure feature of binding site usually.

Example 17 confirms that the site directed mutation of the model of antibody 131 takes place

This example has proved a method, can test by this method and think that residue is important model in combination.This example is also drawn some antibody variants.131 antibody variants of cloning have been produced by being introduced into mAb 131 heavy independent residue sudden changes with light chain.How these variants of subsequent analysis influence the antigen combination with institute's replacement side chain of determining point mutation.

Heavily change at mAb 131 with light chain.On heavy chain, make L216 change over R by site directed mutation.On light chain, V99 changes over F.Having influenced variant antibody expression and excretory two kinds of sudden changes and wild-type sequence compares.Two kinds of sudden changes have caused that all the mAb variant is in conjunction with the antigenic loss of EGFRvIII.Because these residues replace to R respectively and F has caused active reduction, so this has proved that L216 may contact with EGFRvIII antigen significantly with V99.Certainly, these replace the ordinary construction that destroys antibody, so it is a selection always.

Example 18

13.1.2 and the molecular modelization of paratope

13.1.2 the 3 d structure model of antibody variable region produces by a homology model method, described method is used Accelrys (San Diego, InsightII modelling packing CA).Use the x luminescent crystal structure of having delivered as template, make up model according to the variable region sequences shown in the following table 18.1.

Table 18.1

Variable region of light chain (1-113)

DIVMTQTPLSSPVTLGQPASISC (SEQ?ID?NO：114)

RSSQSLVHSDGNTYLS(CDR1) (SEQ?ID?NO：101)

WLHQRPGQPPRLLIY (SEQ?ID?NO：115)

KISNRFS(CDR2) (SEQ?ID?NO：116)

GVPDRFSGSGAGTAFTLKISRVEAEDVGVYYC (SEQ?ID?NO：117)

MQATQLPRT(CDR3) (SEQ?ID?NO：118)

FGQGTKVEIKR (SEQ?ID?NO：119)

Variable region of heavy chain (114-234)

QVQLVESGGGVVQPGRSLRLSCAASGFTFS (SEQ?ID?NO：120)

SYGMH(CDR1) (SEQ?ID?NO：121)

WVRQAPGKGLEWVA (SEQ?ID?NO：122)

VIWYDGSNKYYVDSVKG(CDR2) (SEQ?ID?NO：123)

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ?ID?NO：124)

DGWQQLAPFDY(CDR3) (SEQ?ID?NO：125)

WGQGTLVTVSSA (SEQ?ID?NO：126)

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).

Use the sequence of antibody 13.1.2 in albumen database, to search for, to identify homologous antibody.According to the similarity of its sequence and antibody 13.1.2., selected to have the structure of albumen database identifier 1HEZ, 2H1P, 8FAB and 1AQK as the modelling template.Overlap ratio is to these formwork structures and be used to produce the sequence alignment based on structure of these templates.Subsequently the sequence and the template sequence of 13.1.2 antibody variable region are compared.The comparison result of utilization structure and sequence is used to produce the molecular model of antibody 13.1.2 variable region.

The mutual work surface of computation model also shows in Figure 18.13.1.2 a principal character of model is to have a long and narrow groove on the surface in CDR district.Described groove is delineated by heavy chain CDR2 140, CDR3 160 and light chain CDR1 110, CDR2 130 and CDR3 150.The remainder of one section contact light chain CDR3 150 of groove, and other one section wide region of opening heavy chain CDR3 160 at close heavy chain-light chain at the interface.Described groove may be antigenic in conjunction with depression.5 dusts in conjunction with the hole in 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 the EGFRvIII epi-position.These residues also may provide the primary structure feature of binding site usually.

The model that docks of example 19 peptides and antibody

Epitope mapping in the example 14 studies show that and need be used for being positioned at 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, produced the dock model of this 6-residue peptide complex with 13.1.2 structure C DR district.At first, produced the model of peptide EEKKGN (SEQ ID NO:127).Except current use 1I8I X-ray crystal structure, to aforementioned similar use this as template, described crystal structure identifies in albumen database.Then, this peptide structure is manual is positioned in the long groove to form initial assembling complex.Expansion module carries out Monte Carlo search subsequently automatically among the use InsightII in structure picture and directional space.By giving each Phi, Psi and Chi angle complete rotary freedom, to allow the flexible of peptide structure picture.In docking operation, allow 5 dusts when other antibody residue is fixed, to move in conjunction with the residue in the groove.The specious structure that Monte Carlo search is found is through stimulating annealing and energy minimization to reach the resulting complex structural model.For each the butt joint model that is obtained, use and to make energy mutually between the Discover_3 module calculating antibody of InsightII packing and the peptide.That assesses all butt joint models makes energy mutually, and test has model that the strongest whole antibody-peptide does mutually and it shows in Figure 19 A and 19B.In this butt joint model, shown in Figure 19 B, there is the hydrogen bond between 6 peptide EEKKGN (SEQ ID NO:127) and antibody 13.1.2.To C-terminal peptide residue is numbered 1 to 6 from N-terminal.6 hydrogen bonds of green dotted line indication.6 pairs of aminoacid that form hydrogen bond are: E2...Y172, K3...H31, K4...H31, N6...D33, N6...Y37 and N6...K55.In this butt joint model, in extending α-chain structure the peptide constraint with in groove.Residue is alternatively towards solvent and antibody surface in the peptide.Towards the residue in conjunction with groove is E2, K4 and N6, described in conjunction with the most remarkable contact antibody of groove.This indicate these 3 residues may for peptide in conjunction with consistent with the epitope mapping result be important.That uses that the Discover_3 module calculates each 6 peptide residue and 13.1.2 antibody junction does energy and demonstration in table 19.1 mutually.What table 19.1 showed each 6 peptide residue and 13.1.2 antibody junction makes energy mutually.All energy are with the unit representation of kcal/mol.

Having the strongest residue of making energy mutually is N6, K4 and E2 successively, and consistent with experimental data again these residues that confirm are the significant contribution persons on antigen limit in antibody-antigen is done mutually.These data provide support and dock the strong evidence of model.In the present embodiment, the antibody junction is defined as the residue in the 5 dusts butt joint peptide.Comprise thus defined 20 residues of paratope and 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, for assess antibody-antigen do mutually in the contribution of each these residue of antibody, calculated and made energy mutually between the paratope of each above-mentioned 20 residue and peptide EEKKGN (SEQ ID NO:127).The results are shown in the table 19.2.Table 19.2 shows that each 20 paratope residue and peptide EEKKGN's (SEQ ID NO:127) makes energy mutually.All energy are with the unit representation of kcal/mol.Having the strongest residue of making energy mutually with peptide is Lys55 and His31, then is 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 example proof takes place by site directed mutation, and the butt joint model how can be with the basis that makes improvements the affinity antibodies appropriate design.Computer simulation makes each 13.1.2 paratope residue be mutated into all 19 other aminoacid, in drawn and amounted to 19 * 20 or 380 virtual mutons.Sudden change is replaced by residue and is finished, and then 50 goes on foot the energy minimization steps thereafter, and this has solved and can have been changed by the inductive any local structure picture of side chain change institute.Calculate whole peptides of each muton and all make energy mutually between paratope.Muton with altogether react to each other energy strong than wild type 13.1.2 can have the higher affinity with peptide EEKKGN (SEQ ID NO:127) potentially, and, even may be and whole EGFRvIII albumen.13.1.2 compares with wild type, and these muton major parts have higher coulomb mutual the work, and still wherein some are compared with wild type antibody and had lower van der Waals (VdW) work mutually.The VdW of wild type 13.1.2 antibody as energy is-9.689kcal/mol mutually, eliminates less than-8.5kcal/mol VdW and makes the muton of energy mutually.Listed residue in the table 20.1 and had the muton of altogether mutually making energy strong than wild type 13.1.2.The bottom of table has been listed the wild type data and has been used for comparison.All energy are with the unit representation of kcal/mol.

Table 20.1

Muton	Coulomb	VdW	Amount to
				TVr172Arg	-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
Gln98Glu	-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
				Tyr1772Asn	-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
Ser32Ile	-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	-750601
Ser32Ala	-65.497	-10.089	-75.586
				Ser32Thr	-65.723	-9.861	075.584
Muton	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 muton that table 20.1 is listed can be the candidate that through engineering approaches is improved affinity antibodies.14 candidates for top in the tabulation further analyze the contribution of the every residue in antigen limit and antibody limit, are proposed the influence of modifying to detect.10 mutons that are used in vitro site directed mutation generation are Tyr172Arg, Arg101Glu, Leu99Asn, Leu99His, Arg101Asp, Leu217Gln, Leu99Thr, Leu217Asn, Arg101Gln and Asn35Gly.The result is found in example 21.

Example 21 confirms that the site directed mutation of 13.1.2 model takes place

This example has proved a method, can test by this method and think that residue is important above-mentioned model in combination.This example is also drawn some antibody variants.Produced the 13.1.2 antibody variants by independent residue sudden change, the residue sudden change is weight and a light chain of introducing 13.1.2mAb separately.Analyze variant to determine the contribution of many side chains in the antigen combination.The string sudden change that takes place to introduce by site directed mutation is summarised in the 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

In in the table 21.1 10 sudden change each all is introduced into weight or the light chain of 13.1.2mAb.Each through the sudden change chain subsequently with the complementary wild type chain transfection in 293 cells.Test the expression and the secretion of the IgG antibody of supernatant subsequently, and in conjunction with EGFrVIII antigen.The result who determines by ELISA is summarised in the table 21.2.

Table 21.2

	Sudden change	In conjunction with energy	Express	In conjunction with
					1	Arg101Asp	-81.861	Be	Not
2	Arg101Gln	-79.596	Be	Not
					3	Arg101Glu	-86.817	Be	Not
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

This example function of proof is made the variant of EGFRvIII.Primer is to 9712 and 9713 (Qiagen, Valencia, CA) extracellular domains of the fragment EGFRvIII of one section 1092bp coding of generation.

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) increases from plasmid template EGFRvIII-rbIgG/pCEP4 (as above-mentioned).Primer #9712 introduces the HindIII site and primer #9713 introduces the KpnI site.PCR product process column purification (Qiagen column purification kit, Valencia, CA), HindIII and KpnI (NEB, NewEngland Biolabs, Beverly, Mass.) digestion and glue purification (Qiagen gel purification kit, Valencia, CA).The T4DNA ligase (NEB, New England Biolabs, Beverly, Mass.) junction fragment 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 recombiant protein

How this example proof can make variant EGFRvIII.At first, 500 μ g EGFRvIII/pFLAG-CMV-1#1 plasmid DNA resuspending are in 25ml Opti-MEMI (Invitrogen, Burlington, ON), and with 500 μ l 293fectin (Invitrogen, Burlington, ON) combinations of resuspending in 25mlOpti-MEMI.Mixture is incubation 20min at room temperature, and subsequently with prepare at 1L 293 FreeStyle culture medium (Invitrogen, Burlington, ON) the 293T cells (1 * 10 in ⁹) mix, described culture media supplemented have 2% FBS and 50 μ g/ml G418 (Invitrogen, Burlington, ON).Cell is at 37 ℃, 8% CO ₂And the 125rpm rotating speed was grown 7 days down.

According to the experimental program of manufacturer, use Anti-FLAG M2 Affinity Chromatography test kit (Sigma, St.Louis, MO) purified fusion protein.

Be prepared as follows the monomer fusion rotein.At first, purifying protein (1508 μ g) reduced 30 minutes for 55 ℃ with the DTT of final concentration 10mM.Then, add IAA (iodoacetic acid) (Sigma, St.Louis, MO) to 22mM, and room temperature dark place incubation 15 minutes, then 4 ℃ 7k MWCO dialysis cassette (Pierce, Rockford, Ill) in anti-PBS dialysis.

The combination research of Examples 24-30 antibody variants

Following example comprises Biacore experiment (surface plasma resonance) and KinExA experiment.Whether how these example proofs test many antibody and the variant thereof that is made by above-mentioned example, have required in conjunction with feature to determine them.All variants through test all are the variants of 13.1.2 background.

Experimental apparatus

All surface plasma resonance experiments all are to use Biacore 2000 optical biosensors, and (Piscataway NJ) carries out for Biacore, Inc..All power repulsion mensuration (Kinetic Exclusion Assay) all is to use the KinExA3000 instrument, and (Boise ID) carries out for Sapidyne Instruments, Inc..Reagent

From Anatech, (San Jose, CA) customization is synthetic and buy Pep-3, NH2-LEEKKGNYVVTDHG-OH (MW=1590Da) (SEQ ID NO:130) for Inc..The mAbs that indoor preparation is all.The antigen EGFRvIIIpfiag of indoor preparation MW 39,907 (the reaction iodoacetic acid is to stop the polymerization of free sulfhydryl base group).(Pittsburgh PA) buys bovine serum albumin (BSA) part of V (#BP1605-100) from Fisher Scientific.Other all general reagents are all available from Sigma-Aldrich, and Inc (St.Louis, MO).

The antigen analyzed of the Biacore that is useful on and KinExA and mAb sample at (the 0.01M HEPES that contains 100 μ g/mL BSA, 0.15M NaCl, 0.005% surfactant P-20, Biacore Inc., Uppsala Sweden) prepares in the vacuum degassing HBS-P buffer.The agent of Biacore coupling amine, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), N-maloyl imines (NHS) and ethanolamine be available from Biacore, Inc..For pep-3/mAb 131 experiments, the regeneration of Biacore surfactant has 12 pulse per second (PPS)s of 26mM NaOH.All other mAb at 20 minutes internal disintegrations to baseline.Research grade CM5 sensor biology chip is available from Biacore, Inc.

KinExA detects goat-anti IgG () that antibody is the special Cy5-labelling of Fc γ and (1 * PBS, pH7.4) (pH7.2) dilution is 1000 times for 0.01M HEPES, 0.15M NaCl with the HEPES buffer from the 0.5mg/mL liquid storage.The solid phase particles that is used for KinExA experiment be the activated Sepharose 4 Fast Flow pearls of NHS (PharmaciaBiotech AB, Uppsala, Sweden, #17-0906-01).Before sepharose 4B and antigen-reactive, the pearl liquid storage aliquot of 1.5ml is through centrifugal and with cold deionization H in the micro-centrifuge tube ₂O water is washed 6 times at least.In case (0.05M, pH9.3) after the flushing pearl, the antigen in the sodium carbonate buffer (～40 μ g) just adds on the sepharose 4B with sodium carbonate buffer.Agarose/antigen pipe spends the night for 4 ℃ and rocks.After rocking, shake agarose and, twice of pH8.3 flushing with 1M Tris buffer.Antigen coated pearl rocked 1 hour under the room temperature in containing the 1M Tris buffer of 2% BSA subsequently.

Biacore measures

Use standard EDC/NHS and carbohydrate coupling connection are with Covalent Immobilization mAb to CM5 sensor chip.In order to minimize a large amount of transmission with crowded, mAb is fixed on the certain level, this seasonal epidemic pathogens is no more than the maximum antigen of 50-100RU in conjunction with replying (Rmax).On each chip with reference to flow cell with fixedly the activating or stop of no mAb, make it when comparing.

Under 23 ℃, carry out all Biacore dynamic experiments.For each experiment, prepare one group of successive 6 to 8 antigen concentration (since 1.01 μ M pep-3) with 2 times of diluents.The antigen samples of in three repetitions, injecting arbitrarily with 100 μ L/min on the surface of room temperature sensor.Each pep-3 concentration and blank were all injected 90 seconds.Then dissociated 13 to 180 minutes.By the pep-3 that replaces three extra 251nM pep-3 injections with three extra blank injections and then obtained to be bonded to mAb 131 3-4 hour the period of the dissociating data of dissociating.

(Version l.lf, BioLogic Software Australia) handle all Biacore sensing spectrums to use Scrubber software.At first zeroing sensing spectrum on the y axle, and x arrangement when the injection beginning subsequently.By deducting the change that removes a large amount of refractive indexes with reference to replying of flow cell.From all analytes and blank induction spectrum, deduct on average replying of all blank injections to remove experiment and with reference to the system's anthropic factor between flow cell.(Version 3.40, and BioLogic Software Australia) determines the ka and the kd of treated data set to use CLAMP biological sensor data analysis software.The data of all flow cells all are fit to comprise 1: 1 bimolecular combination model of the condition of a large amount of transmission.For some mAb, be excluded outside non-linear dynamic is fit to corresponding to the injection of first or second concentration of the continuous concentration of pep-3, in being fit to non-linear dynamic the induction spectrum can not by mutually do at 1: 1 model well description be tangible.Calculate K from discussing of kd/ka _D

The KinExA balancing a survey

At room temperature (～23 ℃) carry out all KinExA experiments.For all balance tests, antigen is diluted to the solution with constant mAb binding site concentration continuously.For the one 10 titration point, diluent be 2 times and the 11st and the 12nd serial dilution be 1O doubly.The sample flow speed of all experiments is 0.25mL/min, and the traget antibody flow rate is 0.5mL/min.The antigen/antibody sample allows to reach balance subsequently, this process need cost～48-72hr.For pep-3/mAb 131KinExA experiment, K _DThe initial concentration of pep-3 is 352nM in the-control titration, and constant [mAb binding site]=219pM; For mAb-control titration, initial [pep-3]=251nM, and [mAb binding site]=11nM.K at pep-3/mAb 131 _D-control experimental session, each sample proposes 1.25mL in the flow cell.Analyzed the sample volume of 250 μ L in the antibody control experiment.For all balance tests, measure two or three repetitions of each sample.Use KinExA software (Version 2.4, Sapidyne Instruments), the balance titration data is fit to the hyperbola analysis of 1: 1 combination model.

Only at K _DStudied the KinExA of EGFRvIIIpflag/mAb 131 complexs under the-controlled condition.Initial [EGFRvIIIpflag] is 198nM, and [mAb binding site] is 150pM.The sample of 1mL volume is proposed in the flow cell.Collect the retest data of all samples.(Version 2.4, and SapidyneInstruments), the balance titration data is fit to the hyperbola analysis of 1: 1 combination model to use KinExA software.Result and prediction equilibrium constant referring to following example 28.

For the KinExA titration of EGFRvIIIpflag/mAb 13.1.2 complex, the initial concentration of EGFRvIII is 5.26 μ M (mAb-control), 230.1nM (K _D-control), and [mAb binding site]=9.59nM (mAb-control), 498pM (K _D-control).At K _D-control experimental session, each sample proposes 1.30mL in the flow cell.Analyzed the sample volume of 250 μ L in the antibody control experiment.For all balance tests, measure two or three repetitions of each sample.Use KinExA software (Version 2.4, Sapidyne Instruments), the balance titration data is fit to the hyperbola analysis of 1: 1 combination model.

In vitro determining of example round antibody binding constant

The binding kinetics feature of Surface Plasmon Resonance (SPR) Instrument observation wild type mAb 131 by using Biacore.Owing to extremely low k _dWith quick k _a, so K _DBeing extremely low, only is 380pM.Other kinetic parameter that is separated to from the curve device is estimated k _a=2.246 * 10 ⁶And kd=8.502 * 10 ^-4

In one embodiment, showed improved or tool by improving dynamic (dynamical) variant antibody.By improved kinetics, an antibody dynamical element that means in conjunction with epi-position is better than the same element of previously known with the antibody of same epi-position.For example, have greater than (binding ability) 1.3 * 10 ^-9M K _DAntibody in conjunction with pep-3 can be improvement antibody.Contain the K that so has less than 500nM, 500-300nM, 300-100nM, 100-1nM, 1.3nM, 1.3nM to 1000pM, 1000pM to 900pM, 900-500pM, 500-400pM, 400-300pM, 300-100pM, 100-50pM, 50-1pM _DOr littler 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 the experiment between less deviation is arranged.Other kinetic parameter that is separated to from the curve device is estimated k _a=2.835 * 10 ⁵And kd=0.01922.

Example 26

In vitro determining of antibodies constant

Similar to example 24, test mAb 095 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K that estimates _DBe 66nM.Other kinetic parameter that is separated to from the curve device is estimated k _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 mAb 139 in conjunction with Pep-3 (EGFRvIII epi-position).The K that estimates _DBe 290nM.Other kinetic parameter that is separated to from the curve device is estimated k _a=10328 and kd=2.98P10 ^-3

Example 28

In vitro determining of antibodies constant

For analyze more completely antibody in conjunction with feature, carry out KinExA experiment with determine mAb 131 in conjunction with feature.The K definite according to the hyperbola analysis _DBe 1.74 * 10 ^-10In a KinExA experiment, the K of EGFRvIIIpflag to mAb131 _DBe 6.266 * 10 ^-11

Example 29

In vitro determining of variant antibodies constant

For analyze more completely 13.1.2 antibody in conjunction with feature, carry out KinExA experiment with determine mAb 13.1.2 in conjunction with feature.The K definite according to the hyperbola analysis _DBe 7.538 * 10 ^-10In addition, the antigen in this example is EGFRvIIIpflag, and the reaction of itself and iodoacetic acid (IAA).

Example 30

Biacore result and KinExA result's comparison

The result who has showed previous example and KinExA test in the following table 30.1.Numeral in table 30.1 bracket is 95% confidence interval." ND " is not definite, and " * " representative is in conjunction with EGFRvIIIpflag (iodoacetic acid of reaction), rather than pep-3.

As shown in speed constant, mAb 131 shows to have maximum association constant and have minimum dissociation constant, therefore makes mAb 131 that minimum K is arranged _D

Table 30.1

MAb	K a(M -1S -1)	K d(s -1)	K D(nM)	KinExA?K D(nM)
					131 ?	2.25×10 6?	8.50×10 -4?	0.380 ?	0.174(0.0627on EGFRvIIIpflag)
13.1.2 ? ?	2.10(0.58)×10 5? ?	0.016(0.003) ? ?	75(14) ? ?	0.75(on EGFRvIIIpflag (IAA?reacted))
					095	1.49×10 5	9.90×10 -3	66	ND
139	1.03×10 4	2.98×10 -3	290	ND

Example 31

In vitro determining of L99T-5.3 variant antibodies constant

Test mAb L99T-5.3 to Pep-3 (EGFRvIII epi-position) in conjunction with power.First step is to use standard EDC/NHS coupling connection chemistry, with 5,600 resonance unit (RU) be fixed on two flow cells of CM5 sensor chip (Fc) mAb L99T-5.3 8, on 000 RU, and with 5,600 resonance unit (RU) are fixed on 8,000 RU of mAb 13.1.2 of a Fc.This area density produces the binding signal with pep-3 less than 100 RU.Used two CM5 sensor chips to fix two mAb altogether.Use previous data of collecting, produced 5 independent experiments altogether that two antibody allow 95% confidence interval to be calculated.Biacore2000 optical bio sensor is all used in all researchs.

Then, the pep-3 fixed biological sensor of the mAb surface of flowing through.The pep-3 initial concentration is 1.25 μ M, and this is connected on any three and repeats in the injection after 8 twice serial dilutions.For reaching the purpose of Radix Triplostegiae Grandiflorae photograph, run through in the continuous injection per the 6th sample and carry out the blank injection.

At last, handle the biological sensor data, and the data adapting employing has the curve of mutually making the Clamp of model at 1: 1 that comprises a large amount of transmission conditions with Scrubber.Be fit to 1.25 the injection of the high concentration of μ M excludes power, do not make model mutually because data significantly meet 1: 1.This departs from most possibly is that non-special mutual work by occurring in high concentration pep-3 causes.All dynamic dates were made model satisfactorily in suitable 1: 1 mutually.

Estimated K _DBe 54-70nM.Other kinetic parameter k that estimates _a=2.238 * 10 ⁵And k _d=0.01217, this each the operation between liquid by Light Difference.

Example 32-38

Example 32-38 further test by using the Biacore device, variant mAb in conjunction with power.First step in these examples is to use standard EDC/NHS coupling connection chemistry, 5,600 resonance unit (RU) is fixed on 8,000 RU of each mAb L99T-5.3 after tested of a flow cell (Fc) of CM5 sensor chip.This area density produces the binding signal with pep-3 less than 100 RU.Use 3 CM5 sensor chips to fix all muton mAb altogether with a same mAb who is fixed on each flow cell.MAb 13.1.2 is included in 3 sensor chips on two the flow cell.Biacore 2000 optical bio sensors are all used in all researchs.

Then, the pep-3 operation is through the fixed biological sensor of mAb surface.The pep-3 initial concentration is 4.98 μ M, and this is connected on any two and repeats or three repeat in the injection after 8 to 11 twice serial dilutions.For reaching the purpose of Radix Triplostegiae Grandiflorae photograph, run through in the continuous injection per the 6th sample and carry out the blank injection.

At last, handle the biological sensor data, and the data adapting employing has the Clamp that mutually makes model at 1: 1 that comprises a large amount of transmission conditions with Scrubber.Depend on mAb and affinity thereof, when data did not significantly meet 1: 1 and make model mutually, some high concentrations injections (4.98-1.25 μ M) excluded power and are fit to.This departs from most possibly is that non-special mutual work by occurring in high concentration pep-3 causes.All dynamic dates were made model in suitable 1: 1 mutually.

Example 32

L217Q-10.1 variant antibody in vitro binding constant is determinedTest mAb L217Q-10.1 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K that estimates _DBe 92nM.Other kinetic parameter that is separated to from the curve device is estimated k _a=2.04 * 10 ⁵And kd=0.01885.

Example 33

L217N-2.1 variant antibody in vitro binding constant determines similar in appearance to example 32, test mAb L217N-2.1 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K that estimates _DBe 185nM.Other kinetic parameter that is separated to from the curve device is estimated k _a=2.198 * 10 ⁵And kd=0.04069.

Example 34

N35G-3.1 variant antibody in vitro binding constant is determinedSimilar in appearance to example 32, test mAb N35G-3.1 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K that estimates _DBe 204nM.Other kinetic parameter that is separated to from the curve device is estimated k _a=1.497 * 10 ⁵And kd=0.03057.

Example 35

In vitro determining of variant antibodies constant

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

Example 36

In vitro determining of variant antibodies constant

Similar to example 32, test mAb Y172R-1.2 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K that estimates _DBe 927nM.Other kinetic parameter that is separated to from the curve device is estimated k _a=82237 and kd=0.07622.

Example 37

In vitro determining of variant antibodies constant

Similar to example 32, test mAb L99N-4.1 to Pep-3 (EGFRvIII epi-position) in conjunction with power.The K that estimates _DBe 1.4 μ M.In order to determine K _D, be difficult to be fit to because power is exceedingly fast, so MAb L99N-4.1 is fit to use steady statue (balance) combination model.

Example 38

13.1.2 with the comparison that is designed variant

As be found in table 38.1, developed and had the mAb of improvement in conjunction with feature.Shown 95% confidence interval in the bracket.L99T-5.3 shows the k that increases _a, the k of minimizing _d, and therefore whole lower K _DIf although as if Pep-3 is in conjunction with showing between the equilibrium dissociation constant of mAb 13.1.2 and L99T-5.3 (confidence interval 95%) and power speed constant that statistics goes up little significant difference, still exists Pep-3 to have the intuition deviation in conjunction with the marginal increment of L99T-5.3 affinity.In addition, as if when using identical biological sensor chip, L99T-5.3 always has the affinity high than 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)×10 5	0.016(0.003)	75(14)
L99T-5.3	2.16(0.12)×10 5	0.013(0.001)	60(10)
				L217Q-10.1	2.04×10 5	0.019	92
L217N-2.1	2.20×10 5	0.040	185
				N35G-3.1	1.50×10 5	0.030	204
L99H-9.2	8.34×10 4	0.033	395
				Y172R-1.2	8.22×10 4	0.076	927
L99N-4.1	ND	ND	1.400 *

The method of extra butt joint model and preference pattern level prediction binding affinity

In other embodiments, above-mentioned example can carry out and the peptide of six amino acid length not only the time with the different length peptide, as long as comprise crucial in conjunction with residue in the peptide.For example, replace the six amino acid peptide of EEKKGN (SEQ ID NO:127), can use 7 amino acid peptides of EEKKGNY (SEQ ID NO:131).Can use the epi-position of any big or small peptide.In other embodiments, peptide is to be 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).Can use between peptide or its variant of the section fragment that discloses here to any size between the full-length peptide.

Those skilled in the art should be appreciated that as described, exist additional amino acid can change the mode of 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 aminoacid can change the structure of peptide and the antibody structure of antibodies peptide.Therefore, in one embodiment, can test the different length epitope peptide, for example the binding characteristic of EEKKGN (SEQ ID NO:127) and EEKKGNY (SEQ ID NO:131) with combine optimization.Not only peptide accurately describe peptide is done mutually than the peptide-antibody of long segment than long segment, and bond strength and being included in conjunction with the change of interior residue test allows about longer peptide, the extraneous information of being derived by data.

In addition, and may be and the complementary longer fragments of peptides of test, can carry out extra filtration step to select to dock accurately model.Extra filtration step can allow to filter in many butt joint models to seek the model consistent with available experimental data.

In one embodiment, filter and be based on good isolating epi-position cartographic data, for example the independent residue of test feature is in conjunction with overview, and described cartographic data is in relation to each amino acid whose calculations incorporated energy overview in the peptide.For example, 7 amino acid peptides can be used to select to contain similar butt joint model in conjunction with the energy overview in conjunction with the energy overview.In conjunction with the energy overview is the assignment that combines energy of each aminoacid and specific amino acids in the peptide, amino acid whose in conjunction with the overview of creating peptide, and it is in conjunction with energy according to each aminoacid in described model.For example, a given peptide that comprises aminoacid A and B in a butt joint model, wherein A have-5 in conjunction with energy and B have-20 in conjunction with energy, can have the overview of A1 () and B2 () at 5 o'clock at 20 o'clock.This overview can other docks the filtration of model with electing.For example, use this can cause the selection of other butt joint model as filtration or " template " in conjunction with the energy overview, if the peptide in candidate's model has the low relatively value that contributes to position A, and has the high relatively value (big negative value, higher absolute value) that contributes to position B.In an other embodiment, template needs extra restriction, and for example the value of position B is higher 4 times than the value of position A.

Can be in many ways with relatively in conjunction with the overview of energy overview template and peptide in other docks model.If with required to combine the energy overview similar, subsequent filtration can be used for further test as selecting favourable butt joint model so in conjunction with energy overview template.If in conjunction with energy overview template and the required energy overview dissmilarity that combines, subsequent filtration can be as eliminating unfavorable butt joint model so.In one embodiment, filter process comprises having favourable and unfavorable template in conjunction with energy, and filtration is used for selecting and gets rid of the butt joint model.Those skilled in the art exist many possible differences in conjunction with the energy overview with what understand as described, and therefore depend on different situations, can use many differences in conjunction with the energy overview.In one embodiment, can define in conjunction with the energy overview is that ad-hoc location has a series of high relatively templates in conjunction with energy in peptide.In a preferred embodiment, in conjunction with high empty template of energy and template selected in conjunction with the energy overview will be in the position 2,4 of peptide EEKKGNY (SEQ ID NO:131) or 6 places have relative high in conjunction with energy.In another embodiment, in conjunction with energy overview template in the position 2,4 of peptide EEKKGNY (SEQ ID NO:131) or 6 places have high relatively in conjunction with energy.In another embodiment, have at 3 places, position of peptide EEKKGNY (SEQ ID NO:131) in conjunction with energy overview template relative to energy.In above discussion, described position is doubly assigned as follows: E1, E2, K3, K4, G5, N6, Y7.

In one embodiment, filtration treatment at first is included in the contrast in conjunction with energy of K3 and K4 place.Selection causes that K4 in contrast to the higher relatively butt joint model in conjunction with energy of K3, screens out simultaneously to cause that K4 in contrast to the relatively low butt joint model in conjunction with energy of K3.Therefore, " high relatively " mean K4 in conjunction with energy greater than (negative value is bigger, bigger absolute value) K3.Then, in conjunction with energy overview template, filter the butt joint model again, this time, be chosen in the combination model that position 2,4 or 6 places have relative higher-energy, can remove other model simultaneously.Therefore, " high relatively " mean in peptide in the position 2,4 or 6 places in conjunction with energy than minimum in conjunction with energy height (bigger negative value, bigger absolute value).Therefore, in this embodiment, can following summary binding energy measure feature template: E1 can be any value, E2 should be bigger than minimum, and K3 should be less than K4, and K4 should be greater than minimum, G5 can be any value, and N6 should be greater than minimum, and Y7 can be any value.Therefore, as long as at least one (or K3) less than among E2, K4 and the N6 at least one, E1, G5 and Y7 can be any value.In another embodiment, " high relatively " can be set at as modelling or the definite standard value of experiment.In one embodiment, the butt joint model filters better important by first specific filtration resistance butt joint model by second.Should be appreciated that as one of ordinary skill in the art, do not need one after the other to carry out these two steps, and these two steps can be carried out simultaneously.

In addition, depend on peptide, antibody and in conjunction with condition, these overview templates of filter result can be different.Give this disclosure, one of ordinary skill in the art are especially referring to behind the example 14, and it is suitable for energy overview template to determine.For example, shown in table 14.1, there are some possible important residues in peptide in conjunction with 131 and 13.1.2 antibody.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 to be contained in establishment in conjunction with the residue in the energy overview template.As can clearly knowing, go out with example 14 to analyze thought different in conjunction with energy overview template for displaying in the example 39 from following discussion.

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

What result and these changes that following example proof uses longer peptide how can change above proof can mean, and proof is used the above-mentioned filtration that is used to select specific butt joint model.

The epitope-antibody butt joint model of example 397 amino acid peptides

This example proof produces one group of butt joint model, and described model is at the complicated structural model in the CDR district of 13.1.2 for 7 residue peptide.In addition, this example proof is selected the method for a butt joint model on another butt joint model.At first, 7 residue peptide EEKKGNY (SEQ ID NO:131) structural model builds up in extended configuration, and packs interior Discover_3 module minimization of energy with the InsightII modelling.Then, this peptide structure is manual is positioned in the connection site to form initial assembling.The Docking module is carried out the MonteCarlo search subsequently automatically among the use InsightII in translation and revolution space.In docking operation, allow 5 dusts when other antibody residue is fixed, to move in conjunction with the residue in the groove.Peptide is limited in the 5 dust original positions.The specious structure that Monte Carlo search is found is then through stimulating annealing and energy minimization to reach the resulting complex structural model.Obtain 63 butt joint models altogether.

For each butt joint model, the energy of doing mutually in the peptide between antibody and independent residue calculates with Discover_3.Independent residue contribution overview in the epitope-antibody combination is examined to select and the consistent butt joint model of good segregation epi-position cartographic data anticipate i.e. " in conjunction with energy overview template ".63 19 of docking in the model have passed through this inspection.Shown in the table 39.1 that a typical residue separately is in conjunction with the energy overview.Consistent with epi-position cartographic data in the example 14, K4's is significant in conjunction with energy, and N6 and E2 are big.This is in conjunction with the practical work of energy overview template lay special stress on K4 greater than K3.This needs E2, K4 relative with N6 big in conjunction with energy overview also lay special stress on.In other words, E2, K4 and N6's is not in conjunction with energy minimum (minimum negative value or least absolute value) in the peptide in conjunction with energy.Residue is consistent with the epi-position cartographic data in conjunction with energy overview and example 14 of antibody 13.1.2 separately in table 39.17 residue peptide.

E1	E2	K3	K4	G5	N6	Y7	Amount to
								-10.97	-19.34	-13.46	-24.26	-10.1	-18.19	-15.15	-111.45

For based in conjunction with passing through filtering 19 models on the energy overview, have 7 mutons (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 in conjunction with thermodynamics emulation.Because the sympathetic degree of this compound intravital static is approaching, all use many different static constants in a series of calculating.Sudden change is replaced by residue and is finished, and then 30-100 goes on foot the energy minimization step thereafter, and this has solved and can have been changed by the inductive any local structure picture of side chain change institute.For each butt joint model,, calculate and make energy mutually between 7 residue peptide and whole antibody the selected parameter of each muton.For 8 every group in conjunction with energy (7 mutons add wild type), the linearity of carrying out every group of binding data with Kd algorithm contrast is fit to process.Calculate each linear correlation coefficient that is fit to.Obtain the best correlation of a model, described model has the data described in the table 39.1, and described best correlation has the energy minimization in static constant l*r and 50 steps.The epitope-antibody that has shown this model in the table 39.2 is in conjunction with energy.The correlation coefficient of all data is 0.80.Referring to above example 37, because do not measure the high accuracy K of Leu99Asn _DSo the independent linearity of having carried out except that the Leu99Asn data is fit to.As shown in figure 20, obtained a fabulous correlation coefficient 0.91.Therefore above selected model is represented accurate butt joint model well.Figure 21 has shown to have the model that peptide is filled in the space, and Figure 22 has shown hydrogen bond.L3 150 is to be higher parts than lower part and H3 160 among Figure 22.H2 140 is on the right in binding domain polypeptide territory.Self is positioned at binding site peptide, and E1 is positioned at the page or leaf top of bright shading in the binding site, and it is K3, K4, G5, N6 and the Y7 of deepening successively down.Figure 22 has shown the antibody residue that is contained in hydrogen bonding.There are 7 hydrogen bonds in the model proof that this example makes.K4...Q95, K4...Q95, N6...Q98, G5...H31, Y7...H31 and Y7...W165.

Table 39.2.

With the contrast of Kd logarithm, the emulation epitope-antibody is in conjunction with thermodynamics

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

As be found in selected model in the example 39, represent among Figure 21, the butt joint models show some non-anticipating results.Although an interesting result is that residue E2, K4 and N6 are important in combination peptide as a whole, these not all amino acid profiles turn into to being contained in antibody and form the H-key.Showing that K4 is contained in forms two H-keys with Q95, this with K4 consistent in conjunction with the importance in energy overview and the overview template.Show that also the N6 simulation is with bonding Q98; Yet in this special model, E2 and step are presented at and form the H-key in the model.The interesting trend of a unanimity is most of being buried of each Key residues (for example E2, K4 and N6) that derives from conjunction with energy overview template, and therefore closely contacts with the antibodies groove.Therefore, the butt joint Model Selection can illustrate that these Key residues are important practical works, because itself and the closely mutually work of antibody.In addition, E1 may form hydrogen bond with W214.

Example 39 proves that also said method causes in conjunction with energy and K _DBetween height relevant, this model of thinking that this method is created also allows the K of antibody-peptide complex _DOptimization or at least the prediction.

As be found in the contrast of example 39 and example 19, and having some is important residues between two models, some residues only show in 7 aminoacid butt joint model, and some residues step in 7 aminoacid butt joint model shows it is important.For example, 7 peptide epitopes are presented at and create the H key between K4...Q95, K4...Q95, N6...Q98, G5...H31, Y7...H31 and Y7...W165.On the other hand, 6 peptide epitopes are presented at and create the H key between E2...Y172, K3...H31, K4...H31, N6...D33, N6...Y37 and N6...K55.As finding out from above-mentioned data, 6 and 7 amino acid peptide models are all emphasized the importance of H31, because two models all comprise the H31 that forms two hydrogen bonds with peptide.Although there is other possibility trend between two data sets, it shows that also mutual work of many combinations changes over 7 amino acid profiles from the six amino acid model.Yet these examples proof can detect variation owing to the epi-position size with these models, and therefore from the convergent-divergent that is as short as longer epitope peptide and step should be problem according to this disclosure.Existing aminoacid to prove that continuously its importance in many combination models allows therefore to depart from the importance of many mutual works, can be the more typical representative that longer peptide is done mutually than the small peptide model therefore.

Should be appreciated that as affiliated technical field technical staff, also can be applied to 7 amino acid peptide EEKKGNY (SEQ ID NO:131) about above discussion or the example of six amino acid peptide EEKKGN (SEQ ID NO:127), or any longer peptide.For example, example 20 can repeat the information of example 39, and it is by the improved antibody of site directed mutation generation appropriate design affinity.In addition, the result of use-case 39 can repeat example 21, then is by site directed mutation the appropriate design affinity to take place to attempt to improve antibody to test from example 20 isolating any new antibody.

In one embodiment, the result of use-case 39 is to redefine the mutual work zone between antibody and the peptide.For example, the paratope of EEKKGNY (SEQ ID NO:131) can be defined as and comprise and be predicted as with peptide other residue of doing (for example, residue 95) mutually on the antibody.Perhaps, as example 19, paratope can be defined as all residues in the 5 dusts butt joint peptide.Different proteic computer simulation affinity maturations.

Success in vitro obtains affinity matured antibody in many different researchs.Usually, the library that suddenlys change arbitrarily need make up by molecular biology method, and needs exploitation selection/Screening test with abundant clone with good combination ability.Selected variant needs purified to determine affinity subsequently.This process need carries out a series of length and arduous experiment.Following example has proved by utilizing the selection possible accuracy prediction affinity maturation of antibody-antigenic complex structure separately.

Example 40 is by the computer simulation affinity maturation of antibody-antigen in conjunction with thermodynamics emulation

This example proves that the analog antibody-antigen that can use a computer is used for affinity maturation in conjunction with thermodynamics emulation.This example proves that especially Fab-12 (the IgG form is known as rhuMAb VEGF) and the power that combines of VEGF (microtubule endothelial cell growth factor (ECGF)) can be by the predictions of aforementioned calculation machine simulation process.

The VEGF-Fab complex crystal structure that uses and has accession number 1BJ1 in the PDB data base, it is resolved at 2.4 dusts.Use the experiment affinity data of delivering of a series of mutons of anti-VEGF Fab to be used to test viewpoint.The 3-D of the VEGF-Fab structure thing that matches is used for the computer simulation sudden change of following muton: H97Y, S100aT, T28D, 28D31H, 28D31H97Y100aT, N31H, Y53W, 71I73K, 71V73V.Described affinity data is from Chen, people such as Y (J Mol Biol., 293 (4): 865-81 (1999)) obtain.Between many VEGF-Fab mutons, carry out thermodynamics emulation as example 39 is described.The results are shown in the table 40.1.Result's proof of this example has obtained in conjunction with the significant correlation between energy and affinity row by this process.Figure 23 shows the suitable in detail logarithm to relative affinity in conjunction with energy.-0.91 correlation coefficient is illustrated computer mould emulation and is swashed accurately to catch in detail mutually at atomic level and do.

Table 40.1 antibody-antigen is in conjunction with energy emulation contrast affinity data.

The Kabat number	Sequence relative number affinity	Ln (relative affinity)	In conjunction with energy
					H97Y S100aT T28D 28D31H 28D31H97Y100a T N31H Y53W 71I73K 71V73V WT	101Y 105T 28D 28D31H ? 28D31H101Y105T 31H 54W 72I74K 72V74V WT	14 1.9 1.4 3.1 ? 20 3.6 1.3 0.9 0.3 1	2.639 0.642 0.336 1.131 ? 2.996 1.281 0.262 -0.105 -1.204 0.000	-59.065 -57.465 -57.647 -57.699 ? -59.518 -57.724 -57.504 -57.158 -57.314 -57.404

As can clearly understanding, can infer that emulation identifies the higher affinity sudden change do not use ex vivo experiment from above example.In addition, this method obviously is useful for different antibodies and different peptides.Only use the high antibody-antigenic complex structure of resolving, this method can be applied to carry out the affinity maturation computer simulation usually.In one embodiment, this simulation affinity maturation that uses a computer will be saved the time and the resource of huge amount.

Example 41 standard antibody-likes are determined

People such as Chothia have described antibody structure (the J.Mol.Biol.1987 Aug 20 of the basis " standard class " of the hypervariable region of each immunoglobulin chain; 196 (4): 901-17).Analyze the Fab and the segmental atomic structure of VL of panimmunity globulin, with the relation of the three dimensional structure of determining aminoacid sequence and its antigen binding site.In present its packing of Crinis Carbonisatus such as Chothia, have the main few relatively residue that the hypervariable region main chain is constructed of being responsible for, its hydrogen bond or ability present abnormal phi, psi or omega structure.Find these residues occur in the hypervariable region the site and in conservative beta-thin slice frame.Passing a test has the immunoglobulin sequences of unknown structure, and people such as Chothia show that many immunoglobulins have hypervariable region, and these hypervariable regions have similar size to known structure, and additionally contains same residue in the site of the observed structure of load.

It finds that these hypervariable regions of hint have the structure close with known structure.For 5 hypervariable regions, the demonstration of all constituents of structure is limited to relatively small number purpose discrete topology class.What these main chains that hypervariable region takes place were usually constructed is term " norm structure ".People such as Chothia (Nature.1989 Dec 21-28; 342 (6252): 877-83) and other (Martin waits people J Mol Biol.1996 Nov 15; 263 (5): further work 800-15) confirms 5 main chain structures that have the sub-fraction all constituents of 6 hypervariable regions of antibody at least.

Analyze some above-mentioned antibody to determine the standard class of each complementary antibody determining area (CDR).As known, only the standard class is distributed to the CDR1 and the CDR2 of heavy chain of antibody, and the CDR1 of light chain of antibody, CDR2 and CDR3.Following table (41.1) has been summed up analysis result.Standard class data are the forms with * HCDR1-HCDR2-LCDR1-LCDR2-LCDR3, and wherein " HCDR " refers to that heavy chain CDR and " LCDR " refer to light chain CDR.Therefore, for example, a class standard 1-3-2-1-5 refers to have HCDR1 and falls into standard class 1, HCDR2 and fall into standard class 3, LCDR1 and fall into standard class 2, LCDR2 and fall into the antibody that standard class 1, LCDR3 fall into standard class 5.

Table 41.1

H1-H2-L1-

mAb L2-L3

139 1-3-2-1-1

250 1-3-2-1-1

123 1-3-4-1-1

131 1-3-4-1-1

13_1_2?1-3-4-1-1

211 1-3-4-1-1

318 1-3-4-1-1

333 1-3-4-1-1

342 1-3-4-1-1

95 3-1-4-1-1

150 3-Y-4-1-1

170 3-Y-4-1-1

If it satisfies length needs and the defined Key residues of compliant apoplexy due to endogenous wind, each CDR (except H3) is dispensed to norm structure so.Can find the defined aminoacid of each antibody, for example in article referring to people such as above Chothia.

Equivalent

Previous description and example have described certain preferred embodiment of the present invention in detail, and describe the optimal mode that the inventor is contained.Yet, should be appreciated that no matter how detailed previous description appears in content the present invention can realize in many ways, and the present invention should make up according to accessory claim book and its any equivalent.

Sequence table

Sequence table

＜110〉Abgenix Inc.

＜120〉at the antibody and the use thereof of the deletion mutant of EGF-R ELISA

<130>ABGENIX.087VPC1

<150>US?60/483,145

<151>2003-06-27

<150>US?60/525,570

<151>2003-11-26

<150>US?60/562,453

<151>2004-04-15

<160>144

<170>FastSEQ?for?Windows?Version?4.0

<210>1

<211>109

<212>PRT

＜213〉homo sapiens

<400>1

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

100 105

<210>2

<211>124

<212>PRT

＜213〉homo sapiens

<400>2

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Ser?Gly?Arg

1 5 10 15

Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Arg?Asn?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?Asp?Lys?Tyr?Tyr?Ala?Asp?Ser?Val

50 55 60

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

100 105 110

Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>3

<211>112

<212>PRT

＜213〉homo sapiens

<400>3

Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Ala?Ile?Ser?Gly?Asp?Ser?Val?Ser?Ser?Asn

20 25 30

Ser?Ala?Ala?Trp?Asn?Trp?Ile?Arg?Gln?Ser?Pro?Ser?Arg?Gly?Leu?Glu

35 40 45

Trp?Leu?Gly?Arg?Thr?Tyr?Tyr?Arg?Ser?Lys?Trp?Tyr?Asn?Asp?Tyr?Ala

50 55 60

Val?Ser?Val?Lys?Ser?Arg?Ile?Thr?Ile?Asn?Pro?Asp?Thr?Ser?Lys?Asn

65 70 75 80

Gln?Phe?Ser?Leu?Gln?Leu?Asn?Ser?Val?Thr?Pro?Glu?Asp?Thr?Ala?Val

85 90 95

Tyr?Tyr?Cys?Ala?Arg?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

100 105 110

<210>4

<211>118

<212>PRT

＜213〉homo sapiens

<400>4

Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Ala?Ile?Ser?Gly?Asp?Ser?Val?Ser?Ser?Asn

20 25 30

Asn?Ala?Ala?Trp?Asn?Trp?Ile?Arg?Gln?Ser?Pro?Ala?Arg?Gly?Leu?Glu

35 40 45

Trp?Leu?Gly?Arg?Thr?Tyr?Tyr?Arg?Ser?Lys?Trp?Tyr?Asn?Asp?Tyr?Val

50 55 60

Val?Ser?Val?Lys?Ser?Arg?Ile?Thr?Ile?Asn?Pro?Asp?Thr?Ser?Lys?Asn

65 70 75 80

Gln?Phe?Ser?Leu?Gln?Leu?Asn?Ser?Val?Thr?Pro?Glu?Asp?Thr?Ala?Val

85 90 95

Tyr?Tyr?Cys?Val?Arg?Ala?Thr?Ala?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr

100 105 110

Leu?Val?Thr?Val?Ser?Ser

115

<210>5

<211>118

<212>PRT

＜213〉homo sapiens

<400>5

Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Gln

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Ala?Ile?Ser?Gly?Asp?Ser?Val?Ser?Ser?Tyr

20 25 30

Ser?Ser?Ala?Trp?Asn?Trp?Ile?Arg?Gln?Ser?Pro?Ser?Arg?Gly?Leu?Glu

35 40 45

Trp?Leu?Gly?Arg?Ala?Tyr?His?Arg?Ser?Arg?Trp?Tyr?Tyr?Glu?Tyr?Ala

50 55 60

Val?Ser?Val?Lys?Ser?Arg?Ile?Asn?Ile?Thr?Pro?Asp?Thr?Ser?Lys?Asn

65 70 75 80

Gln?Phe?Ser?Leu?Gln?Leu?Asn?Ser?Val?Thr?Pro?Glu?Asp?Thr?Ala?Val

85 90 95

Tyr?Tyr?Cys?Ala?Arg?Gly?Ser?Arg?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr

100 105 110

Leu?Val?Thr?Val?Ser?Ser

115

<210>6

<211>110

<212>PRT

＜213〉homo sapiens

<400>6

Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Phe?Leu?Val?Lys?Pro?Ser?Gln

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly

20 25 30

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

35 40 45

Trp?Ile?Gly?Tyr?Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Tyr?Tyr?Asn?Pro?Ser

50 55 60

Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?nys?Asn?Gln?Phe

65 70 75 80

Ser?Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr

85 90 95

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

100 105 110

<210>7

<211>126

<212>PRT

＜213〉homo sapiens

<400>7

Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Phe?Leu?Val?Lys?Pro?Ser?Gln

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly

20 25 30

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

35 40 45

Trp?Ile?Gly?Phe?Ile?Tyr?Tyr?Arg?Gly?Asn?Thr?Tyr?Tyr?Asn?Pro?Ser

50 55 60

Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe

65 70 75 80

Ser?Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr

85 90 95

Cys?Ala?Arg?Asp?Gly?Tyr?Cys?Ser?Arg?Thr?Gly?Cys?Tyr?Gly?Gly?Trp

100 105 110

Phe?Asp?Pro?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Pro

115 120 125

<210>8

<211>109

<212>PRT

＜213〉homo sapiens

<400>8

Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?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?Lys?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

100 105

<210>9

<211>116

<212>PRT

＜213〉homo sapiens

<400>9

Glu?Gly?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Trp?Val?Gln?Pro?Gly?Glu

1 5 10 15

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

20 25 30

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

35 40 45

Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Asn?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?Val?Asn?Ser?Leu?Arg?Val?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Gly?Ser?Ser?Gly?Trp?Ser?Glu?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val

100 105 110

Thr?Val?Ser?Ser

115

<210>10

<211>116

<212>PRT

＜213〉homo sapiens

<400>10

Glu?Val?Gln?Val?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Asn?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?Gly?Ser?Ser?Gly?Trp?Ser?Glu?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val

100 105 110

Thr?Val?Ser?Ser

115

<210>11

<211>109

<212>PRT

＜213〉homo sapiens

<400>11

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?Ser?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?Lys?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser

100 105

<210>12

<211>128

<212>PRT

＜213〉homo sapiens

<400>12

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?Val?Ala?Ser?Gly?Phe?Thr?Leu?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?Thr?Ser?Tyr?Asp?Gly?Ser?Lys?Lys?Asp?Tyr?Ala?Asp?Ser?Ala

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

Val?Ser?Glu?Gly?Tyr?Cys?Ser?Ser?Ser?Ser?Cys?Tyr?Lys?Tyr?Tyr?Tyr

100 105 110

Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser

115 120 125

<210>13

<211>128

<212>PRT

＜213〉homo sapiens

<400>13

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?Leu?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?Met?Ser?Tyr?Asp?Gly?Ser?Lys?Glu?Asp?Tyr?Ala?Asp?Ser?Val

50 55 60

Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Glu?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

Val?Ser?Glu?Gly?Tyr?Cys?Ser?Ser?Arg?Ser?Cys?Tyr?Lys?Tyr?Tyr?Tyr

100 105 110

Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser

115 120 125

<210>14

<211>109

<212>PRT

＜213〉homo sapiens

<400>14

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?Ser?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?Lys?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser

100 105

<210>15

<211>128

<212>PRT

＜213〉homo sapiens

<400>15

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?Val?Ala?Ser?Gly?Phe?Thr?Leu?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?Thr?Ser?Tyr?Asp?Gly?Ser?Lys?Lys?Asp?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

Val?Ser?Glu?Gly?Tyr?Cys?Asp?Ser?Ser?Ser?Cys?Tyr?Lys?Tyr?Tyr?Tyr

100 105 110

Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser

115 120 125

<210>16

<211>128

<212>PRT

＜213〉homo sapiens

<400>16

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?Val?Ala?Ser?Gly?Phe?Thr?Leu?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?Thr?Ser?Tyr?Asp?Gly?Ser?Lys?Lys?Asp?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

Val?Ser?Glu?Gly?Tyr?Cys?Asp?Ser?Thr?Ser?Cys?Tyr?Lys?Tyr?Tyr?Tyr

100 105 110

Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser

115 120 125

<210>17

<211>128

<212>PRT

＜213〉homo sapiens

<400>17

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?Val?Ala?Ser?Gly?Phe?Thr?Leu?Ser?Ser?Tyr

20 25 30

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

35 40 45

Ala?Val?Thr?Ser?Tyr?Asp?Gly?Ser?Lys?Lys?Asp?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

Val?Ser?Glu?Gly?Tyr?Cys?Asp?Ser?Thr?Ser?Cys?Tyr?Lys?Tyr?Tyr?Tyr

100 105 110

Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser

115 120 125

<210>18

<211>112

<212>PRT

＜213〉homo sapiens

<400>18

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

100 105 110

<210>19

<211>112

<212>PRT

＜213〉homo sapiens

<400>19

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?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?Arg?Ile?Ser?Arg?Arg?Phe?Ser?Gly?Val?Pro

50 55 60

Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ala?Gly?Thr?Asp?Phe?Thr?Leu?Glu?Ile

65 70 75 80

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

85 90 95

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

100 105 110

<210>20

<211>112

<212>PRT

＜213〉homo sapiens

<400>20

Ala?Ile?Val?Leu?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?Arg

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?Pro?Asp?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Met?His?Thr

85 90 95

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

100 105 110

<210>21

<211>112

<212>PRT

＜213〉homo sapiens

<400>21

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?Arg

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?Ile?Tyr?Phe?Cys?Met?His?Thr

85 90 95

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

100 105 110

<210>22

<211>112

<212>PRT

＜213〉homo sapiens

<400>22

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?Ile?Thr?Phe?Gly?Gln?Gly?Thr?Arg?Leu?Glu?Ile?Lys

100 105 110

<210>23

<211>112

<212>PRT

＜213〉homo sapiens

<400>23

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?Ile?His?Thr

20 25 30

Asp?Gly?Asn?Ile?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?Gly

85 90 95

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

100 105 110

<210>24

<211>107

<212>PRT

＜213〉homo sapiens

<400>24

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Arg?Asn?Asp

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?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys

100 105

<210>25

<211>107

<212>PRT

＜213〉homo sapiens

<400>25

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Arg?Asn?Asn

20 25 30

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>DMA

＜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?G1u?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?agcactttga?tctt 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 introduction sequence

<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>DMA

＜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

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>105

Met?Gln?Ser?Thr?His?Val?Pro?Trp?Thr

1 5

<210>106

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>106

Phe?Gly?Gln?Thr?Lys?Val?Glu?Ile?Lys

1 5

<210>107

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>107

Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Ser?Gly?Arg

1 5 10 15

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

20 25 30

<210>108

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<400>108

Asn?Tyr?Gly?Met?His

1 5

<210>109

<211>14

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic peptide sequence

<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

<21l>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

<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

465 470 475 480

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?Leu?Gln?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

<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?Leu?Ile?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 (22)

1. method of killing target cell, described method comprises:

Make target cell contact and the associating antibody of toxin, wherein said antibodies is to peptide LEEKKGNY (SEQ IDNO:133), and wherein said target cell is expressed the peptide of the sequence that comprises LEEKKGNY, and wherein said toxin is to be selected from the group that is made up of AEFP, MMAE, DM-1 and ZAP.

2. method according to claim 1, wherein said antibody have with described peptide greater than 1.3 * 10 ^-9The binding affinity of M.

3. method according to claim 1, wherein said antibody toxin compositions is at least 10 times greatly of the toxicity of the cell of the no described peptide of the toxicity of target cell comparison.

4. method according to claim 1, wherein said antibody is to be selected from the group that is made up of antibody, described antibody comprises heavy chain amino acid sequence, and described heavy chain amino acid sequence is to be 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 EDNO: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 group that the heavy chain amino acid sequence of 342 (SEQ ID NO:16) and 333 (SEQ IDNO:17) is formed.

5. method according to claim 1, wherein said antibody associates via peptide connexon and described toxin.

6. method according to claim 1, wherein said antibody associates via second antibody and described toxin.

7. separated antibody or its fragment that is bonded to EGFRvIII, described antibody further with the therapeutic agent coupling, wherein said therapeutic agent is the toxin that is selected from by AEFP, MMAE, AURISTATIN E, DM-1 and group that ZAP forms, and wherein said antibody comprises heavy chain amino acid sequence, and described heavy chain amino acid sequence comprises following complementary determining region (CDR):

(a) CDR1, it is formed by being selected from the sequence of forming group by the aminoacid sequence in following antibody CDR1 district:

(13.1.2 SEQ ID NO:138), 131 (SEQ ID NO:2), 170 (SEQ ID NO:4), 150 (SEQID 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), 342 (SEQ IDNO:16) and 333 (SEQ ID NO:17);

(b) CDR2, it is formed by being selected from the sequence of forming group by the aminoacid sequence in following antibody CDR2 district: 13.1.2 (SEQ ID NO:138), 131 (SEQ ID NO:2), 170 (SEQ ID NO:4), 150 (SEQID 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), 342 (SEQ IDNO:16) and 333 (SEQ ID NO:17); With

(c) CDR3, it is formed by being selected from the sequence of forming group by the aminoacid sequence in following antibody CDR3 district: 13.1.2 (SEQ ID NO:138), 131 (SEQ ID NO:2), 170 (SEQ ID NO:4), 150 (SEQID 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), 342 (SEQ IDNO:16) and 333 (SEQ ID NO:17).

8. an antibody or its fragment, it is bonded to EGFRvIII and it comprises heavy chain amino acid sequence, described heavy chain amino acid sequence is to be selected from by antibody 13.1.2 (SEQ ID NO:138), 131 (SEQ ID NO:2), 170 (SEQ IDNO: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 group that the heavy chain amino acid sequence of 342 (SEQ ID NO:16) and 333 (SEQ ID NO:17) is formed, described antibody further with the therapeutic agent coupling, wherein said therapeutic agent is to be selected from by DM-1, AEFP, MMAE, AURISTATIN E, toxin with group that ZAP forms.

9. segmental antibody according to claim 8, wherein said toxin associates via peptide connexon and described antibody.

10. segmental antibody according to claim 8, wherein said toxin associates via second antibody and described antibody.

11. an inhibition is expressed the method for relevant cell proliferation with EGFRvIII, it comprises with the antibody of effective dose or its fragment handles the cell of expressing EGFRvIII, wherein said antibody or its fragment are bonded to EGFRvIII, wherein said antibody be selected from by DM-1, AEFP, MMAE, the toxin of AURISTATIN E and group that ZAP forms associates, and wherein said antibody comprises heavy chain amino acid sequence, and described heavy chain amino acid sequence is to be selected from by antibody 13.1.2 (SEQ ID NO:138), 131 (SEQ ID NO:2), 170 (SEQ ID NO:4), 150 (SEQID 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 group that the heavy chain amino acid sequence of 342 (SEQ IDNO:16) and 333 (SEQ ID NO:17) is formed.

12. method according to claim 12, wherein said method is in vivo carried out.

13. method according to claim 12, wherein said method is carried out on mammal.

14. method according to claim 14, wherein said mammal is the people.

15. method according to claim 14, wherein said mammal suffers from the cancer that relates to epithelial cell proliferation.

16. method according to claim 16, wherein said cancer comprises pulmonary carcinoma, colon cancer, gastric cancer, renal carcinoma, carcinoma of prostate, breast carcinoma, glioblastoma multiforme or ovarian cancer.

17. the method for the cell proliferation of a cell that suppresses to express EGFRvIII, described method comprises with the antibody of effective dose or its fragment handles the cell of expressing EGFRvIII, wherein said antibody be selected from by DM-1, AEFP, MMAE, the toxin of AURISTATIN E and group that ZAP forms associates, and wherein said antibody has light-chain amino acid sequence, described light-chain amino acid sequence is to be selected from by being identified as SEQ ID NO:19,20,21,29,23,25,26,28,33,31 and 32 antibody 13.1.2,131,170,150,123,095,139,250,211,342, the group that 333 and 318 light-chain amino acid sequence is formed, wherein said separated polynucleotide molecule will be in conjunction with the peptide with the sequence that is identified as SEQ ID NO:56.

18. method according to claim 18, wherein said method is in vivo carried out.

19. method according to claim 18, wherein said method is carried out on mammal.

20. method according to claim 20, wherein said mammal is the people.

21. method according to claim 20, wherein said mammal suffers from the cancer that relates to epithelial cell proliferation.

22. method according to claim 22, wherein said cancer comprises pulmonary carcinoma, colon cancer, gastric cancer, renal carcinoma, carcinoma of prostate, breast carcinoma, glioblastoma multiforme or ovarian cancer.

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