CN101309933A - Toxin conjugated eph receptor antibodies - Google Patents

Abstract

The present invention relates to compositions and methods for inducing cell death or stasis in cancer cells or other hyperproliferative cells using anti-EphA2 or anti-EphA4 antibodies conjugated to toxins.

Description

Toxin conjugated Eph receptor antibody

The right of priority of the U.S. Provisional Patent Application that the application requires to submit on September 7th, 2005 U.S. Provisional Patent Application is submitted to number on November 14th, 60/714,362 and 2005 number 60/735,966, this paper is included in each application by reference in.

Invention field

[001] the invention provides employing and come inducing cancer cell or the necrocytosis of other super hyperplasia sexual cells (hyperproliferative cell) or the composition and the method for stagnation with toxin conjugated resisting-EphA2 or anti--EphA4 antibody.

Background of invention

Cancer

[002] vegetation, or tumour are the vegetation matter that unusual not controlled cell growth causes, and it can be optimum or virulent.Innocent tumour usually remains in original position.Malignant tumour can be referred to as cancer.Term " virulent " thus the described tumour of ordinary representation can attack or destroy adjacent body structure and be diffused into distal site and cause death and (look back visible Robbins and Angell, 1976, Basic Pathology, second edition, (the W.B.Saunders Co. of Philadelphia WBS company, Philadelphia), 68-122 page or leaf).Cancer can appear at many positions of health, and according to its origin and behavior is different.Cancerous cells can be destroyed a part of body that they originate from, and other position diffusions to body also begin new growth there and cause more havoc then.

[003] there are every year 1200000 Americans to suffer from cancer.Cancer is the second largest cause of the death of the U.S., if continue development with this trend, expects cancer in 2010 and will be called the first cause of the death.Lung cancer and prostate cancer are the No.1 male cancer killers of the U.S..Lung cancer and mammary cancer are No.1 women's cancer killers of the U.S..In the U.S., what just have among per two male sex that a people will be at its life is diagnosed out cancer sometime.In the U.S., what just have among per three women that a people will be at its life is diagnosed out cancer sometime.Present methods of treatment is as surgical operation, chemotherapy and radiation, common invalid or severe side effect is arranged.

Shift

[004] the most threatening cancer form of life is betided usually when a group tumour cell obtains to ability that the far-end and the external source position of health are settled down.The cell of these transfers is survived by the limitation of breaking common restrictive cell and settling down in different tissue.For example, if typically the breast epithelial cell is transferred to lung then can't be grown or survive usually, but lung's transfer is the major cause that causes breast cancer incidence and mortality ratio.Nearest evidence hint, the appearance that metastatic cell is propagated in health are far early than the clinical manifestation of primary tumor.These small transitional cells can keep dormancy several months or several years after detecting or removing primary tumor.Therefore, understand better mechanism that metastatic cell can grow in the external source microenvironment and survive be designed for the therapy of resisting metastatic cancer for improvement and make early detection and the diagnosis that shifts the location most important.

The conduction of cancer cells signal

[005] cancer is a kind of abnormal signal transduction disease.The adherent dependence that unusual cell signaling has been broken cell growth and survival limits (Rhim etc., Critical Reviews in Oncogenesis8:305,1997; Patarca, Critical Reviews in Oncogenesis 7:343,1996; Malik etc., Biochimica et Biophysica Acta 1287:73,1996; Cance etc., Breast CancerRes Treat 35:105,1995).Tyrosine kinase activity is induced by ECM is adherent, in fact, and the expression of Tyrosylprotein kinase or function in malignant cell, raise usually (Rhim etc., Critical Reviews inOncogenesis 8:305,1997; Cance etc., Breast Cancer Res Treat 35:105,1995; Hunter, Cell 88:333,1997).Based on essential this fact of tyrosine kinase activity of malignant cell growth, Tyrosylprotein kinase has been called target spot (Levitzki etc., Science 267:1782,1995 of new therapy; Kondapaka etc., Molecular﹠amp; Cellular Endocrinology 117:53,1996; Fry etc., Current Opinion in BioTechnology 6:662,1995).Unfortunately, the obstacle relevant with selectively targeted tumour cell limited the application of these medicines usually.Specifically, tyrosine kinase activity is for the function and the survival most important usually (Levitzki etc., Science 267:1782,1995) of benign tissue.For minimizing indirect toxicity, key is to identify that target selectivity in tumour cell is crossed the Tyrosylprotein kinase of expression then.

The Eph family of receptor tyrosine kinase

[006] the Eph family of acceptor be family maximum in the receptor tyrosine kinase (RTK) (Gale etc., 1997, Cell Tissue Research 290 (2): 227-241 and Dodelet etc., 2000, Oncogene 19 (49): 5614-9).It is the important medium of regulating the cell-cell communication of cell adhesion, shape and mobility that Eph acceptor and their film are joined protein ligands in conjunction with liver.The many aspects of Eph RTK signal conduction incident control fetal development, especially neural growth (Kullander etc. is seen in review, 2002, and Nat.Rev.Mol.Cell Biol.3:473 and Mamling etc., 2002, Trends Biochem Sci 27:514-520.The acceptor of Eph subfamily has single kinase domain usually and comprises and is rich in Cys structural domain and 2 fibronectin III type multiple extracellular regions (seeing Figure 18).Can join the similarity of protein receptor ectodomain sequence and they according to liver joins albumen-A and liver to liver and joins the avidity of albumen-B part they are divided into two groups.Many members of Eph acceptor have been accredited as that cancer takes place and the vital signs of progress and/or instrumentality (referring to, for example, Thaker etc., 2004, Clin.Cancer Res.10:5145; Fox BP etc., 2004, Biochem.Biophys.Res.Commun.318:882; Nakada etc., 2004, Cancer Res.64:3179; Coffman etc., 2003, Cancer Res.63:7907; Also can look back and see Dodelet etc., 2000, Oncogene19:5614).For known and Eph acceptor related to cancer, the effect of EphA2 and EphA4 and expression pattern are fully characterized.

[007] EphA2 expresses in becoming the human epithelial cell, and level is lower and at cell-cell adhesion position enrichment (Zantek etc., 1999, Cell Growth﹠amp therein to find EphA2; Diff10:629; Lindberg etc., 1990, Mol﹠amp; Cell Biol 10:6316).Because EphA2 joins albumin A 1-A5 in conjunction with the liver that is anchored to cytolemma so this Subcellular Localization is important (EphNomenclature Committee, 1997, Cell 90:403; Gale etc., 1997, Cell﹠amp; Tissue Res 290:227).The main consequence of part bonded is the autophosphorylation (Lindberg etc., 1990, the same) of EphA2.Yet, be different from other receptor tyrosine kinases, EphA2 lack part in conjunction with or keep enzymic activity (Zantek etc., 1999, the same) during the Tyrosine O-phosphate inclusion.EphA2 joins albumen-A1 with liver and raise (Ogawa etc., 2000, Oncogene19:6043 in the transformant that comprises mammary gland, prostate gland, colon, lung, kidney, skin and many different tumours of oesophagus cancer; Zelinski etc., 2001, Cancer Res 61:2301; Walker-Daniels etc., 1999, Prostate41:275; Easty etc., 1995, Int J Cancer 60:129; Nemoto etc., 1997, Pathobiology 65:195; Hess etc., 2001, Cancer Res 61 (8): 3250-5).

[008] EphA4 expresses in brain, the heart, lung, muscle, kidney, placenta, pancreas (Fox etc., 1995, Oncogene 10:897) and melanophore (Easty etc., 1997, Int.J.Cancer 71:1061).EphA4 joins albumin A 1, A2, A3, A4, A5, B2 and B3 (Pasquale in conjunction with liver, 1997, Curr.Opin.In Cell Biology 9:608), also binding partner B61, AL 1/RAGS, LERK4, Htk-L and Elk-L3 (Martone etc., 1997, Brain Research 771:238).Part in conjunction with cause EphA4 the tyrosine residues autophosphorylation (Ellis etc., 1996, Oncogene12:1727).The EphA4 tyrosine phosphorylation forms the protein bound district contain Src homeodomain 2/3 (SH2/SH3), as cytoplasmic tyrosine kinase p59fyn (Ellis etc., the same; Cheng etc., Cytokine andGrowth Factor Reviews 13:75,2002).The cell adhesion that the activation of EphA4 causes depending on cadherin among the Africa xenopus embryo is lost (Winning etc., Differentiation 70:46,2002; Cheng etc., the same), this has pointed out the effect of EphA4 in tumor vessel takes place; Yet the effect of EphA4 in cancer progression is not clear.As if EphA4 raise (Kuang etc., Nucleic Acids Res.26:1116,1998 in mammary cancer, the esophageal carcinoma and carcinoma of the pancreas; Meric etc., Clinical Cancer Res.8:361,2002; Nemoto etc., Pathobiology 65:195,1997; Logsdon etc., Cancer Res.63:2649,2003), but downward modulation (Easty etc., the same) in melanoma tissue.

[009] EphB2 and EphB4 acceptor are also crossed in some tumor tissues and are expressed.The main EphB4 of discovery crosses in having high-level pernicious-2 wetting property ductal breast cancer and expresses (Berclaz etc., 1996, Biochem Biophys Res Commun 226:869), and EphB2 crosses expression (Kiynokawa etc. in most of tumor stomaches, 1994, Cancer Res 54:3645).Simultaneously, and the expression excessively in many tumor cell lines of these two kinds of acceptors (Berclaz etc., the same; Kiynokawa etc., the same; Bennett etc., 1995, PNAS USA92:1866).EphB2 and EphB4 raise (Liu etc., 2002, Cancer 94:934 in colon cancer tissue; Stephenson etc., 2001, BMC Mol Biol2:15).In addition, EphB2 and EphB4 also are important (Wang etc., 1998, Cell 93:741 for the blood vessel generation of embryo and possibility tumour; Gerety, 1999Mol Cell 4:403 such as S.S.).

Cancer therapy

[010] bottleneck of antimetastatic agents exploitation is the pilot system that is used for designing and estimating these medicines.The cell that most conventional cancer therapy target is being grown fast.Yet cancer cells is not necessarily grown comparatively fast, but it can be survived under the condition that normal cell can not be survived and grow and grow (Lawrence and Steeg, 1996, World J.Urol.14:124-130).These basic differences between normal cell and malignant cell behavior provide chance for the treatment target-seeking.Small metastatic tumour spreads this example at whole body and has emphasized to estimate potential chemotherapeutic under external source and three-dimensional microenvironment.Growth and the survival of many standard cancer drug experimental measurement tumour cells under typical cells culture condition (that is monolayer growth).Yet the cell behavior in the two dimension test can not be predicted the interior behavior of the body of tumour cell usually reliably.

[011] present, cancer therapy can comprise surgical operation, chemotherapy, hormonotherapy and/or radiotherapy with eliminate the intravital tumour cell of patient (referring to, for example, Stockdale, 1998, " cancer patients's management principle " (Principles of Cancer Patient Management), be selected from " the science U.S.: medical science " (Scientific American:Medicine) that Rubenstein and Federman compile, the 3rd volume, the 12nd chapter, IV part).All these methods are for the patient and the remarkable shortcoming of Yan Douyou.For example, surgical operation may can't be implemented or do not accepted by the patient owing to patient's health problem.In addition, surgical operation may not be removed tumor tissues fully.Radiotherapy is only just effective when tumor tissues is higher than healthy tissues for radiating susceptibility, and radiotherapy also can cause severe side effect.Hormonotherapy is used seldom separately, although and effectively also just be used for usually preventing after removing most of cancer cells with the other treatment method or the delay cancer return.

[012], many chemotherapeutic that are used for treating cancer is arranged now as for chemotherapy.Most of cancer chemotherapy method play a role by suppressing that DNA is synthetic (referring to, for example, Gilman etc., " Gu Deman and gill are graceful: the pharmacological basis of methods of treatment " (Goodman and Gilman ' s:ThePharmacological Basis of Therapeutics), the 8th edition (Pa Jiameng press (PergamonPress), New York, 1990)).Like this, chemotherapeutic itself is nonspecific.Except nearly all chemotherapeutic all poisonous, chemotherapy also can cause significant and dangerous usually side effect, comprise serious feel sick, bone marrow depression, immunosuppression etc. (referring to, for example, Stockdale, 1998, " cancer patients's management principle " (Principles of Cancer Patient Management) is selected from " Scientific Beauty compatriots: medical science " (Scientific American:Medicine) that Rubenstein and Federman compile, the 3rd volume, the 12nd chapter, the 10th part).In addition, even if use the combination of chemotherapeutic, many tumour cells have resistance to chemotherapeutic maybe can produce resistance.

[013] cancer therapy also comprises biotherapy or immunotherapy now.The limited amount of biotherapy/immunotherapy is although and have more specificity many still simultaneously target healthy cell and cancerous cells than chemotherapeutic.In addition, this therapy can produce the side effect such as fash or swelling, influenza-like symptom (comprising heating, cough or tired), digestive tube problem or anaphylaxis.

[014] therefore, significant need has other cancer treatment methods, especially needs the methods of treatment more special to the target cancer cells.The member of Eph receptor family is accredited as the tumour cell mark, and this makes them become strong treatment target spot.Therefore, but specificity target-seeking and the cancer treatment method that destroys the one or more members' of unconventionality expression Eph receptor family tumour cell will be the strong instruments of treatment and preventing cancer.

The antibody of treatment cancer

[015] antibody is the immunoglobulin (Ig) in conjunction with specific antigen.In the most of Mammalss that comprise human and mouse, antibody is made of paired polypeptide heavy chain and light chain.Each chain is made of two different zones, becomes variable region (Fv) and constant region (Fc).The antigen of this molecule is contained in conjunction with determinant and responsible for target antigen in light chain and heavy chain Fv zone.Thereby the Fc district has determined the type (or isotype) (for example IgG) of antibody and has been responsible for causing important biochemical event in conjunction with many natural proteins.

[016] the Fc district of antibody and the many ligand interactions that comprise Fc acceptor and other parts give a series of critical functions that are called effector function.An important family of IgG class Fc acceptor is Fc γ acceptor (Fc γ R).Communication between these receptor-mediated antibody and the immune system cell branch (Raghavan etc., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch etc., 2001, Annu Rev Immunol 19:275-290).In the mankind, this protein families comprises: Fc γ RI (CID64) comprises isotype Fc γ RIA, Fc γ RIB and Fc γ RIC; Fc γ RII (CD32) comprises isotype Fc γ RIIA, Fc γ RIIB and Fc γ RIIC; And Fc γ RIII (CID16), comprise isotype Fc γ RIIIA and Fc γ RIIB (Jefferis etc., 2002, Immunol Lett 82:57-65).These acceptors have mediation and Fc bonded ectodomain usually, stride born of the same parents' intracellular domain of some signals conduction incidents in film district and the possibility mediated cell.These different Fc γ R hypotypes are expressed (Ravetch etc. is seen in review, 1991, Annu Rev Immunol 9:457-492) on different cell types.For example, in the mankind, find that Fc γ RIIIB only expresses on neutrophilic leukocyte, express and find that Fc γ RIIIA kills and wounds on (NK) cell and subgroup of T cell in scavenger cell, monocyte, neutrality.

[017] formation of Fc/Fc γ R mixture makes the effector cell focus on antigen-binding site, this causes signal conduction incident and important follow-up immunization to be replied usually in cell, attacks as discharging inflammatory mediator, B cell-stimulating, endocytosis, phagolysis and cell toxicant.The mediated cell poison is the potential mechanism that antibody destroys target cell with the ability of engulfing effector function.The non-specific cell poison cell identification of expressing Fc γ R is incorporated into the antibody of target cell and causes the molten born of the same parents' of target cell cell-mediated reaction to be called cell-mediated cytotoxicity (the ADCC) (Raghavan etc. that depend on antibody subsequently, 1996, AnnuRev Cell Dev Biol 12:181-220; Ghetie etc., 2000, Annu Rev Immunol18:739-766; Ravetch etc., 2001, Annu Rev Immunol 19:275-290).Note, the main cell (primary cell) of mediation ADCC, the NK cell is only expressed Fc γ RIIIA, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII (Ravetch etc., 1991, the same).

[018] another kind of important Fc part is complement proteins C1q.The Fc mediation that is incorporated into C1q is called the process of the cytotoxicity (CDC) that depends on complement (Ward etc. is seen in review, 1995, TherImmunol 2:77-94).C1q can be in conjunction with 6 kinds of antibody, although just be enough to the activating complement cascade in conjunction with two kinds of IgG.Thereby C1q and C1r and C1s serine protease form the C1 mixture that mixture forms complement pathway.

[019] some key features of antibody include but not limited to: to the specificity of target, the transformation period that can mediate immunological effect mechanism and grow in serum, these make antibody and related immune globulin molecule become strong methods of treatment.Many monoclonal antibodies are in the development phase at present or have used the various illnesss that comprise cancer with treatment in treatments.The example of these antibody comprises: the Vitaxin of Mai Di Immunivest Corp. of Midi Miu Ni company (MedImmune)

, a kind of humanized beta 2 integrin alpha v β 3 antibody (for example, the open WO 2003/075957 of PCT); The Trastuzumab (Herceptin) of Genentech, Inc. (US) 460 Point San Bruno Blvd, South San Francisco, CA, 94080 (Genentech)

, the humanization that a kind of approval is used for the treatment of mammary cancer resists-Her2/neu antibody (for example, U.S.5,677,171); The CNTO 95 of Sai Tuke company (Centocor), a kind of human beta 2 integrin alpha v antibody (the open WO 02/12501 of PCT); The Rituxan of IDEC/ Genentech, Inc. (US) 460 Point San Bruno Blvd, South San Francisco, CA, 94080/Roche Holding Ag (IDEC/Genentech/Roche)

, a kind of approval is used for the treatment of the chimeric anti-CD 20 antibodies (for example, U.S.5,736,137) of non-Hodgkin lymphoma; And the Erbitux of immune clone company (ImClone)

, a kind of anti-EGFR-antibodies (for example, U.S.4,943,533).

[020] antibody is tumoricidal may mechanism have multiple, comprise by blocking required growth pathway and come antiproliferative, cause the intracellular signal conduction of apoptosis, the downward modulation that strengthens acceptor and/or reverse, ADCC, CDC and promote adaptive immunity reaction (Cragg etc., 1999, Curr OpinImmunol 11:541-547; Glennie etc., 2000, Immunol Today 21:403-410).Yet, although be widely used, in clinical use also not antagonist be optimized and the anticancer potential of many antibody is not the best.Therefore, significant need strengthens the ability that antibody destroys the target cancer cells.

Antibody-drug conjugates

[021] a kind of effective ways that strengthen the antitumor potential of antibody relate to cell toxicity medicament or toxin with can be linked to each other by the mAb of target cell internalization.These reagent are called antibody-drug conjugates (ADC) and immunotoxin.After being applied to the patient, ADC and immunotoxin are incorporated into target cell and the internalization that becomes by their antibody moiety, thereby make medicine or toxin can bring into play their effectiveness.Referring to, for example, U.S. Patent Application Publication No. US2005/0180972A1, US2005/0123536A1.Also can referring to, for example, Hamblett etc., Clin Canc Res, 10:7063-7070,1999/10/15, Law etc., Clin Canc Res, 10:7842-7851,2004/12/01, Francisco etc., Neoplasia, 102 (4): 1458-1465,2003/08/15, Russell etc., Clin Canc Res, 11:843-852,2005/01/15, Doronina etc., Nat Biotech, 21 (7): 778-784,2003/07, all documents are included this paper by reference in full in.

[022] quoting or discussing and to be interpreted as and admit that they are prior aries of the present invention reference in the literary composition.

Summary of the invention

[023] the invention provides the antibody drug conjugates (ADC) that a specific specificity is incorporated into the internalization of EphA2, wherein, described ADC is coupled to toxin.The present invention also provides a kind of ADC, and wherein, described ADC comprises toxin, self-sacrifice (self-immolative) transcribed spacer and joint.In one embodiment, described joint is the Val-Cit joint.In other embodiments, described toxin is anti--tubulin agent.In further embodiment, described toxin is Ao Ruisitating (auristatin), for example, and Ao Ruisitating E, Ao Ruisitating F, MMAE or MMAF.

[024] the present invention also provides the method for a kind of anticancer growth, and this method comprises and give the pharmaceutically composition of significant quantity of object that described composition comprises (a) ADC of the present invention; (b) pharmaceutically acceptable carrier.In one embodiment, described cancer cells is melanoma cancer cells, prostate cancer cell, lung carcinoma cell, breast cancer cell, colon cancer cell, kidney cancer cell, ovarian cancer cell or pancreatic cancer cell.

[025] the present invention also provides a kind of treatment method for cancer, and this method comprises and give the pharmaceutically composition of significant quantity of object that described composition comprises (a) ADC of the present invention; (b) pharmaceutically acceptable carrier.Again in another embodiment, described cancer is selected from melanoma, prostate cancer, lung cancer, mammary cancer, colorectal carcinoma, kidney, ovarian cancer and carcinoma of the pancreas.

Definition

[026] in the literary composition, term " agonist " refers to comprise any compound of the activity that can strengthen other molecules, activation or the function of protein, polypeptide, peptide, antibody, antibody fragment, macromole or small molecules (less than 10kD).EphA2 or EphA4 agonist can promote EphA2 or proteic phosphorylation of EphA4 and degraded.The EphA2 of identification EphA2 or EphA4 or EphA4 antibody can also suppress or the anticancer phenotype is not (for example, colony in the soft agar forms or forms the tubulose network in three-dimensional substrates film or extracellular matrix goods), but and be exposed to the EphA2 or the EphA4 epi-position of cancer cells, and can have or not have lower K with respect to non-cancer cells preferred combination or debond _OffSpeed.

[027] in the literary composition, term " immunologic opsonin is incorporated into the Eph acceptor " and similar terms refer to that specificity is incorporated at least a Eph acceptor or its segmental peptide, polypeptide, protein, fusion rotein and antibody or its fragment.Term " immunologic opsonin " can be replaced with term term " specificity " and use.Immunologic opsonin is incorporated at least a Eph acceptor or its segmental peptide, polypeptide, protein or antibody can be than peptide, polypeptide or the protein of low-affinity in conjunction with other, and described avidity is by for example immunoassay, BIAcore or other test determinations known in the art.Immunologic opsonin be incorporated at least a Eph acceptor or its segmental antibody or fragment can with the related antigen cross reaction.Preferably, immunologic opsonin is incorporated at least a Eph acceptor or its segmental antibody or fragment preferentially surpasses in conjunction with other antigen in conjunction with at least a Eph acceptor.Yet, in the definition of the antibody of " immunologic opsonin is incorporated into the Eph acceptor ", the present invention specifically comprises having multiple specific antibody ((Cao etc. is seen in review for example, two or more discontinuous antigens to be had specific antibody, 2003, Adv Drug Deliv Rev 55:171; Hudson etc., 2003, Nat Med 1:129)).For example, bi-specific antibody has two kinds of different binding specificities that merge.In the simplest this situation, bi-specific antibody will be in conjunction with two adjacent epi-positions on the single target antigen, such antibody not can with other antigens (as mentioned above) cross reaction.Perhaps, bi-specific antibody can be in conjunction with two kinds of synantigens not.Such antibody mediated immunity specificity is incorporated into two kinds of differing moleculars, but other uncorrelated molecules of debond.When mentioning one or more particular complex, the multi-specificity antibody of another kind of type can be discerned the shared subunit of many subunits mixture.In addition, specificity in conjunction with the antibody of Eph acceptor can with relevant Eph acceptor (or RTK) cross reaction.

[028] for example can or be proficient in other technologies known to those skilled in the art and identify that specificity is incorporated into Eph acceptor or its segmental antibody or fragment by immunity test, BIAcore.Employing such as (RIA) and enzyme linked immunosorbent assay experimental techniques such as (ELISA) record, and the avidity when specificity is incorporated into Eph acceptor or its segmental antibody or its fragment in conjunction with Eph acceptor or its fragment is higher than the avidity in conjunction with any cross-reactive antigen.The discussion of antibodies specific can referring to, for example, " basic immunology " (Fundamental Immunology) that Paul compiles, second edition, 1989, Lei Wen press (Raven Press), New York, 332-336 page or leaf.

[029] term " specificity is incorporated into antibody or its fragment of EphA2 or EphA4 " refers to that in the text specificity is incorporated into EphA2 or EphA4 polypeptide or EphA2 or EphA4 polypeptide fragment and non-specificity antibody or its fragment in conjunction with other non--EphA2 or non--EphA4 polypeptide.Preferably, specificity be incorporated into EphA2 or EphA4 polypeptide or its segmental antibody or fragment not with other antigen generation non-specific cross-reactions (for example, in suitable immunity test, non--EphA2 or non--EphA4 albumen such as BSA can't combine with its competition).For example, can or be proficient in other technologies known to those skilled in the art and identify that specificity is incorporated into the antibody or the fragment of EphA2 or EphA4 polypeptide by immunity test.Antibody of the present invention includes but not limited to: the Fvs (sdFv) that the antibody that synthetic antibody, monoclonal antibody, reorganization produce, intracellular antibody, multi-specificity antibody (comprising bi-specific antibody), people's antibody, humanized antibody, chimeric antibody, synthetic antibody, strand Fv (scFv) (comprising dual specific scFv), single-chain antibody Fab fragment, F (ab ') fragment, disulfide linkage connect, anti--idiotype (anti--Id) antibody and above-mentioned any epi-position-binding fragment.Specifically, antibody of the present invention comprises the immunocompetence part of immunoglobulin molecules and immunoglobulin molecules, promptly, contain the molecule that specificity is incorporated into EphA2 or the antigenic antigen binding site of EphA4 (for example, one or more complementary determining regions (CDR) of anti--EphA2 or anti--EphA4 antibody).Preferably, specificity is incorporated into the segmental exciting antibody of EphA2 or EphA4 polypeptide or its or its fragment preferred combination EphA2 or EphA4 and can be obviously exciting other molecules or activity.

[030] in the literary composition, term " cancer " refers to certain disease, its related cell has the potential that is transferred to distal site and shows the phenotypic character that is different from non-cancer cells, described phenotypic character for example refers to form colony in three-dimensional substrates thing (for example soft agar) or at three-dimensional substrates film or extracellular matrix goods, for example MATRIGEL ^TMMiddle tubulose network or the pseudostructure of forming.Non-cancer cells can not form colony in soft agar, can not form ball-like structure clearly in three-dimensional Ranvier's membrane or extracellular matrix goods.Cancer cells obtains the series of features sexual function in its growth course, though be to pass through different mechanisms.This function comprise evade apoptosis, growth signals self-sufficient, antagonism-growth signals is insensitive, the tissue intrusion/transfer ability, infinite copy potential and persistent vasculogenesis.Term " cancer cells " refers to comprise precancer and pernicious cancer cells.

[031] in the literary composition, term " cancer cell phenotype inhibition " refers to that compound prevents or reduces that cancer cells colony in the soft agar forms or at three-dimensional substrates film or extracellular matrix goods or detect the ability that the tubulose network forms in any other method that cancer cell phenotype reduces, described additive method for example detects the test (for example, the colony that reduces in the monolayer cell cultivation forms) that the cell proliferation contact inhibition increases.It also can cause colony to reduce or eliminate when compound added the soft agar that has formed the cancer cells colony when cancer cell phenotype is suppressed, and perhaps can reduce or eliminate the degree of the tubulose network in three-dimensional substrates film or the extracellular matrix goods.The EphA2 of anticancer phenotype or EphA4 antibody can also be exciting or not exciting EphA2 or EphA4 and can have or not have lower K _OffSpeed.

[032] in the literary composition, term " delivery vector " refers to can be used to curative or prophylactic agent are applied to object, especially people's material.Delivery vector can preferentially be delivered to treatment/prophylactic agent specific cell subtype.For example, by the inherent feature of delivery vector or by with carrier mutually the link coupled part, comprise within it part (perhaps with carrier-bound part, thereby make this part and this delivery vector maintain together, and then make this part be enough to the targeted delivery carrier) make the cell of some type of delivery vector target, described part, for example, by in conjunction with representing the cell subtype characteristic cell surface molecule of institute's target specificity in conjunction with specific cell subtype.Delivery vector also can improve transformation period in the body of the reagent that will send and/or the bioavailability of the reagent that will send.The non-limitative example of delivery vector is virus vector, virus-like particle, polycation carrier, peptide carrier, liposome and hybrid vector.In concrete embodiment, described delivery vector directly is not coupled to the part in conjunction with EphA2 and/or EphA4.In other embodiments, described delivery vector is not the antibody in conjunction with EphA2 and/or EphA4.

[033] in the literary composition, term " derivative " refers to contain in the context of protein drug (for example, protein, polypeptide, peptide and antibody) because of introducing the protein drug that amino-acid residue replaces, lacks and/or add the aminoacid sequence that changes.Term " derivative " refers in the text, such as but not limited to, the fragment, the specificity that comprise polypeptide, EphA2 or the EphA4 polypeptide of EphA2 or EphA4 amino acid sequence of polypeptide are incorporated into the antibody of EphA2 or EphA4 polypeptide or the antibody fragment that specificity is incorporated into EphA2 or EphA4 polypeptide, they change (that is sudden change) by introducing amino-acid residue replacement, disappearance or adding.In some embodiments, antibody derivatives or its fragment comprise one or more amino-acid residues and replace, lack or add in CDR.Compare with the non-antibody of deriving, antibody derivatives can have essentially identical, better or worse binding ability.In concrete embodiment, 1,2,3,4 or 5 residue of CDR is substituted, deletes or add (that is, being suddenlyd change).Term " derivative " also refers to modified protein drug in the text, promptly links to each other with this protein drug covalency by the molecule with certain type.Term " derivative " also refers in the text, such as but not limited to, through EphA2 or the EphA4 polypeptide of modification, the fragment of EphA2 or EphA4 polypeptide, specificity is incorporated into the antibody of EphA2 or EphA4 polypeptide, or specificity is incorporated into the antibody fragment of EphA2 or EphA4 polypeptide, and the molecule that is about to any kind is covalently bound to polypeptide.Such as but not limited to, can utilize the cutting of known protection/blocking group, proteolysis, be connected with cell ligand or other protein etc. and modify fragment, antibody or the antibody fragment of EphA2 or EphA4 polypeptide, EphA2 or EphA4 polypeptide by for example glycosylation, acetylize, pegization, phosphorylation, amidation, derivatize.Can adopt technology well known by persons skilled in the art to modify fragment, antibody or the antibody fragment of derivative, EphA2 or the EphA4 polypeptide of EphA2 or EphA4 polypeptide by chemically modified, described technology includes but not limited to that the metabolism of specificity chemical chop, acetylize, formylation, tunicamycin is synthetic etc.In addition, the derivative of protein drug can contain one or more atypia amino acid.For example, fragment, antibody or the antibody fragment of the derivative of EphA2 or EphA4 polypeptide, EphA2 or EphA4 polypeptide can contain one or more atypia amino acid.In one embodiment, polypeptide derivative has the similar or identical functions of fragment, antibody or antibody fragment with EphA2 described here or EphA4 polypeptide, EphA2 or EphA4 polypeptide.In other embodiments, compare with unaltered polypeptide, what fragment, antibody or the antibody fragment of the derivative of EphA2 or EphA4 polypeptide, EphA2 or EphA4 polypeptide had is active different.For example, deutero-antibody or its fragment can combine or more anti-proteolysis more firmly with its epi-position.

[034] term " epi-position " refers in the text in animal, preferably in Mammals, most preferably has the part of antigenicity or active EphA2 of immunogenicity or EphA4 polypeptide in the mouse or the mankind.Having the active epi-position of immunogenicity is the part that can cause the EphA2 or the EphA4 polypeptide of antibody response in animal.Having the active epi-position of antigenicity is by any method well known in the art, that for example record by immunity test and part antibodies specific bonded EphA2 or EphA4 polypeptide.It is immunogenic that antigenic epitopes needs not to be.

[035] in the literary composition, term " EphA2 " or " EphA4 " refer to by Eph NK (EphNomenclature Committee) (Eph NK, 1997, Cell, 90:403-404) any Eph receptor polypeptides of identifying and approving.In concrete embodiment, EphA2 or EphA4 receptor polypeptides or its fragment are from any species.In one embodiment, EphA2 or EphA4 receptor polypeptides or its fragment are from the people.The Nucleotide of Eph receptor polypeptides and/or aminoacid sequence can (for example, find in GenBank), perhaps can adopt and be proficient in clone known to those skilled in the art and sequencing technologies is measured this Nucleotide and/or aminoacid sequence at document or public database.For example, the GenBank accession number of the Nucleotide of people EphA2 and aminoacid sequence is respectively NM_004431.2 and NP_004422.2.The GenBank accession number of the Nucleotide of people EphA4 and aminoacid sequence is respectively NM_004438.3 and NP_004429.1.

[036] in the literary composition, have referred to term " liver is joined albumen " or " liver is joined protein ligands " or will by Eph NK (Eph NK, 1997, Cell 90:403-404) identifies and any liver of approval is joined protein ligands.Liver of the present invention is joined albumen and includes but not limited to: liver is joined albumin A 1, liver and joins albumin A 2, liver and join albumin A 3, liver and join albumin A 4, liver and join that albumin A 5, liver are joined protein B 1, liver joins protein B 2 and liver is joined protein B 3.In concrete embodiment, liver is joined protein polypeptide, and especially liver is joined albumin A 1, from any species.In other embodiments, liver is joined protein polypeptide, and especially liver is joined albumin A 1, from the people.Liver is joined the Nucleotide of protein polypeptide and/or aminoacid sequence and can (for example, find in GenBank), perhaps can adopt and be proficient in clone known to those skilled in the art and sequencing technologies is measured this Nucleotide and/or aminoacid sequence at document or public database.For example, the people liver is joined the Nucleotide of albumin A 1 variant 1 and the GenBank accession number of aminoacid sequence is respectively NM_004428.2 and NP_004419.2.The people liver is joined the Nucleotide of albumin A 1 variant 2 and the GenBank accession number of aminoacid sequence is respectively NM_182685.1 and NP_872626.1.

[037] when relating to polypeptide described herein, contains at least 5 contiguous amino acid residues in the aminoacid sequence of antibody that EphA2 or EphA4 polypeptide or specificity be incorporated into EphA2 or EphA4 polypeptide in peptide that term " fragment " is comprised or the amino acid sequence of polypeptide, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.Preferably, antibody fragment is epi-position-binding fragment.

[038] in the literary composition, term " humanized antibody " refers to the antibody of inhuman (for example, Muridae) form, and this antibody contains the chimeric antibody of the sequence that is derived from non-human immunoglobulin on a small quantity.The major part of humanized antibody is human normal immunoglobulin (receptor's antibody), the inhuman species of hypervariable region residue of receptor's antibody wherein, for example the hypervariable region residue with required specificity, avidity and binding ability of mouse, rat, rabbit or non-human primates antibody (donor antibody) substitutes.In some instances, the framework region of human normal immunoglobulin (FR) residue can be substituted by corresponding inhuman residue.In addition, humanized antibody can contain the residue that does not have in receptor's antibody or donor antibody.Carry out these modifications and can further optimize the antibody performance.Humanized antibody can contain at least one usually, basically whole (residue) of common two variable domains, wherein all or all basically hypervariable region are corresponding to non-human immunoglobulin, and all or all basically FR are the human normal immunoglobulin sequences.Humanized antibody also will be chosen at least a portion that comprises constant region for immunoglobulin (Fc) wantonly, this part normally specificity is incorporated into the part of the human normal immunoglobulin of EphA2 or EphA4 polypeptide, change (that is sudden change) by introducing amino-acid residue replacement, disappearance or adding.In some embodiments, humanized antibody is a derivative.This humanized antibody comprises amino-acid residue and replaces, lacks or add in one or more inhuman CDR.Compare with non-deutero-humanized antibody, the humanized antibody derivative can have essentially identical binding ability, better binding ability or worse binding ability.In concrete embodiment, 1,2,3,4 or 5 amino-acid residue of CDR is substituted, deletes or add (that is sudden change).The more details of relevant humanized antibody are seen european patent number EP 239,400, EP 592,106 and EP 519,596; International publication number WO 91/09967 and WO 93/17105; U.S. Patent number 5,225,539,5,530,101,5,565,332,5,585,089,5,766,886 and 6,407,213; And Padlan, 1991, MolecularImmunology 28 (4/5): 489-498; Studnicka etc., 1994, Protein Engineering7 (6): 805-814; Roguska etc., 1994, PNAS 91:969-973; Tan etc., 2002, J.Immunol.169:1119-25; Caldas etc., 2000, Protein Eng.13:353-60; Morea etc., 2000, Methods 20:267-79; Baca etc., 1997, J.Biol.Chem.272:10678-84; Roguska etc., 1996, Protein Eng.9:895-904; Couto etc., 1995, Cancer Res.55 (23Supp): 5973s-5977s; Couto etc., 1995, Cancer Res.55:1717-22; Sandhu, 1994, Gene 150:409-10; Pedersen etc., 1994, J. Mol.Biol.235:959-73; Jones etc., 1986, Nature 321:522-525; Reichmann etc., 1988, Nature 332:323-329; And Presta, 1992, Curr.Op.Struct.Biol.2:593-596.

[039] in the literary composition, term " hypervariable region " refers to be responsible for and antigen bonded amino-acid residue in the antibody.Hypervariable region comprises amino-acid residue (that is, the residue 24-34 (L1) of variable region of light chain, 50-56 (L2) and 89-97 (L3), the residue 31-35 (H1) of variable region of heavy chain, 50-65 (H2) and the 95-102 (H3) of " complementary determining region " or " CDR "; Kabat etc., " immunology protein of interest matter sequence " (Sequences of Proteins of Immunological Interest), the 5th edition, (the Public Health Service of publilc health service department of NIH, National Institutes of Health), Maryland State Bei Sesida (Bethesda, MD), 1991) and/or the residue of " hypermutation ring " (promptly, the residue 26-32 (L1) of variable region of light chain, 50-52 (L2) and 91-96 (L3), the residue 26-32 (H1) of variable region of heavy chain, 53-55 (H2) and 96-101 (H3); Chothia and Lesk, 1987, J.Mol.Biol., 196:, 901-917).The CDR residue of Eph099B-208.261 and Eph099B-233.152 is listed in table 2." framework region " or " FR " residue is the variable region residue except that hypervariable region residue defined herein.

[040] in the literary composition, term " combination " refers to use more than one therapy (for example, preventing and/or treating medicine).When using term " combination " to the order that prevents and/or treats medicine that is applied to the object of suffering from super hyperplasia sexual cell disease, especially cancer without limits.(for example treat agent for first kind, prophylactic agent or curative) can suffered from, just suffering from or easily suffering from super hyperplasia sexual cell disease, especially the object of cancer is used second kind and (is for example treated agent, prophylactic agent or curative) before (for example, 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 the week before), simultaneously, or (for example, 1 minute afterwards, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 week back) uses.Described treatment agent (for example, prophylactic agent or curative) is applied to object with a definite sequence and at certain time intervals, thus the benefit that treatment agent of the present invention can play a role and be better than other application processes to provide with other medicaments.Any extra treatment agent (for example, prophylactic agent or curative) can any order give with other extra therapy (for example, prophylactic agent or curative) together.

[041] in the literary composition, term " low tolerance " refers to that the patient is treated side effect and makes it surpass the state that the treatment benefit does not benefit and/or stops to treat because of undesirable action and/or harmful side effect from treatment.

[042] in the literary composition, term " control ", " control " and " control method " refer to that object obtains beneficial effect from using to treat in the agent (for example, prophylactic agent or curative), but do not cause curing this disease.In some embodiments, use one or more to object and treat next " control " disease of agent (for example, prophylactic agent or curative), thereby prevent this progression of disease or deterioration.

[043] in the literary composition, term " unresponsive/refractory " with one or more present available therapies (is for example described, cancer therapy), for example chemotherapy, radiotherapy, surgical operation, hormonotherapy and/or biotherapy/immunotherapy, when particularly the standard care scheme of particular cancers is treated the patient, described therapy is not enough to treat these patients clinically, for example treatment is kept insensitive, thereby makes them need other effective treatment.Though this phrase also can be described the patient therapy is responded, be subjected to side effect, recurrence, generation resistance etc.In various embodiments, " unresponsive/refractory " expression at least obviously some cancer cells is not killed or is failed to stop their cytodifferentiation.Utilize in the literary composition " refractory " in this area acceptable implication, by any method known in the art in vivo or the external test cancer cells whether be that " unresponsive/refractory " checks the effect of treatment to cancer cells.In various embodiments, the cancer cells number obviously reduces during treatment, or when increasing, cancer is " unresponsive/refractory ".

[044] in the literary composition, the effectiveness of curative improved when term " enhancing " referred to the dose application of conventional or approval.

[045] in the literary composition, term " prevention " and " preventing " refer to by using generation, recurrence or the diffusion that treatment agent of the present invention (for example, prophylactic agent or curative) wards off disease.

[046] in the literary composition, term " prophylactic agent " refers to can be used to prevent to cross with EphA2 or EphA4 any medicament of outbreak, recurrence or the diffusion of expressing relevant disease or illness and/or cell super proliferative disease, especially cancer.In concrete embodiment, any composition that term " prophylactic agent " refers to comprise treatment or prevents the following component of significant quantity: (a) be coupled to (or continuous) delivery vector in conjunction with the part of EphA2 and/or EphA4 by other modes; (b) one or more treatments or prevent the treatment or the prophylactic agent of described super proliferative disease; (c) pharmaceutically acceptable carrier.In some embodiments, term " prophylactic agent " refers to that the exciting antibody of EphA2 or EphA4, EphA2 or EphA4 cancer cell phenotype suppress the EphA2 of antibody, exposure or EphA4 epitope antibodies or with less than 3 * 10 ^-3s ^-1K _OffAntibody (for example, listed any antibody among Eph099B-102.147, Eph099B-208.261, Eph099B-210.248, Eph099B-233.152, EA44 or the table 2-4 or 6) in conjunction with EphA2 or EphA4.In concrete embodiment, the exciting antibody of EphA4 that is used for the present composition and method is EA44, this is a kind of anti--EphA4scFV antibody that is disclosed in U.S.'s non-provisional application sequence number 10/863,729 of submitting on June 7th, 2004, and this application is included this paper by reference in full in.The cell of expressing anti--EphA4scFv EA44 was preserved in American Type Culture Collection (post-office box 1549 on June 4th, 2004 according to being specified in of " Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure " (Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedures), the Manassas, Virginia 20108), its specified accession number is PTA-6044.In some other embodiment, term " prophylactic agent " refers to cancer chemotherapy, radiotherapy, hormonotherapy, biotherapy (for example, immunotherapy) and/or EphA2 of the present invention or EphA4 antibody.In other embodiments, more than one prophylactic agent is capable of being combined gives.

[047] in the literary composition, " prevention significant quantity " refers to treat agent (for example, prophylactic agent) consumption is enough to prevent the super proliferative disease of cell, outbreak, recurrence or the diffusion of preferred cancer.The prevention significant quantity also can refer to (for example treat agent, prophylactic agent) consumption is enough to prevent super proliferative disease, especially refer to outbreak, recurrence or the diffusion of cancer, include but not limited to super proliferative disease tendency those (objects), for example have in the heredity cancer tendency or before contacted carcinogenic those (objects).The prevention significant quantity can refer to that also the consumption for the treatment of agent (for example, prophylactic agent) can provide the prevention benefit for the super proliferative disease of prevention.In addition, with regard to prophylactic agent of the present invention, the prevention significant quantity refers to independent or can provide the prevention benefit with the consumption of the prophylactic agent of other medicaments combinations when the super proliferative disease of prevention.During with the coupling of the EphA2 of the present invention of certain consumption or EphA4 antibody, this term can comprise that can improve overall preventive effect maybe can strengthen another prevention for the treatment of agent (for example, prophylactic agent) and render a service or synergistic with it consumption.

[048] in the literary composition, " scheme " comprises administration time table and dosage regimen.

[049] in the literary composition, term " side effect " comprises the harmful and detrimentally affect of prophylactic agent or curative.Detrimentally affect usually is undesirable, but undesirable influence is not necessarily bad.The detrimentally affect of prophylactic agent or curative may be deleterious uncomfortable or dangerous.The side effect of chemotherapy includes but not limited to: gastrointestinal toxicity, such as but not limited to early stage and late period diarrhoea and flatulence, nauseating, vomiting, poor appetite, oligoleukocythemia, anaemia, neutrophil minimizing, weakness, abdominal cramps, heating, pain, weight loss, dehydration, alopecia, insomnia, expiratory dyspnea, dizzy, mucositis, xerostomia and kidney depletion and constipation, influence to N﹠M, temporary or permanent damage to kidney and bladder, influenza-like symptom, fluid retention and temporary or permanent sterile.The side effect of radiotherapy includes but not limited to: tired, dry and forfeiture appetite.The side effect of biotherapy/immunotherapy includes but not limited to: medicine-feeding part generation fash or swelling, influenza-like symptom is for example generated heat, is shivered and tired, digestive tube problem and anaphylaxis.The side effect of hormonotherapy includes but not limited to: feel sick, fertility Issue, depression, forfeiture appetite, eyes problem, headache and weight fluctuations.Patient known in the art can experience a lot of other undesirable actions usually.Many undesirable actions are described in " doctor's desk reference " (Physicians ' Desk Reference), (the 58th edition, 2004).

[050] in the literary composition, term " strand Fv " or " scFv " refer to comprise the antibody fragment of antibody VH and VL structural domain, and wherein these structural domains are present in the polypeptide chain.The Fv polypeptide generally also contains between VH and VL structural domain can make scFv form the peptide linker of antigen in conjunction with desired structure.The summary of scFv is seen Pluckthun, publish in " pharmacology of monoclonal antibody " (The Pharmacology of Monoclonal Antibodies) that Rosenburg and Moore compile, the 113rd volume, Springer Wei Ge press (Springer-Verlag), New York, the 269-315 page or leaf, 1994.In concrete embodiment, scFv comprises dual specific scFv and humanization scFv.

[051] in the literary composition, term " object " and " patient " are used interchangeably.

[052] in the literary composition, object is Mammals preferably, and as non-human primate (for example, milk cow, pig, horse, cat, dog, rat etc.) and primates (for example, monkey and people), optimum is chosen.

[053] in the literary composition, term " targeting moiety " or " bound fraction " refer to strengthen this reagent and transport to target tissue or cell subtype with identical characteristics when being connected in other reagent (as delivery vector or other compounds), thereby improve this reagent in target tissue or cell subtype or any part of partial concn on every side.For example, targeting moiety can be in conjunction with the molecule on part or all cells surface in target tissue or the cell subtype.In concrete embodiment, targeting moiety is in conjunction with EphA2 or EphA4.In other embodiments, targeting moiety is in conjunction with the EphA2 on the cancer cells or EphA4 (for example, not being incorporated into the EphA2 or the EphA4 of part) and EphA2 or EphA4 (for example, being incorporated into the EphA2 or the EphA4 of part) on the non-cancer cells of debond.

[054] in the literary composition, term " treatment ", " treatment " and " methods of treatment " refer to particularly eradicate, remove, improve or controlled primary, regionality or metastatic cancer tissue because of giving the symptom that disease or illness had been eradicated, alleviated or alleviated to one or more curatives.In some embodiments, this term refers to reduce as far as possible or delayed because of one or more treatment agent (for example, prophylactic agent or curative) of object of suffering from this disease the diffusion of cancer.

[055] in the literary composition, term " curative " refers to can be used to prevent, treat or control is crossed with EphA2 or EphA4 and expressed relevant disease or illness and/or the super proliferative disease of cell or illness, especially cancer.In concrete embodiment, any composition that term " curative " refers to comprise treatment or prevents the following component of significant quantity: (a) be coupled to (or continuous) delivery vector in conjunction with the part of EphA2 and/or EphA4 by other modes; (b) one or more treatments or prevent the treatment or the prophylactic agent of described super proliferative disease; (c) pharmaceutically acceptable carrier.In some embodiments, term " curative " refers to that the exciting antibody of EphA2 or EphA4, EphA2 or EphA4 cancer cell phenotype suppress the EphA2 of antibody, exposure or EphA4 epitope antibodies or with less than 3 * 10 ^-3s ^-1K _OffAntibody (for example, listed any antibody among Eph099B-102.147, Eph099B-208.261, Eph099B-210.248, Eph099B-233.152, EA44 or the table 2-4 or 6) in conjunction with EphA2 or EphA4.In some other embodiment, term " curative " refers to that cancer chemotherapeutic agent, radiotherapeutic agents, hormone are treated agent, agent and/or EphA2 of the present invention or EphA4 antibody are treated in biological treatment agent/immunity.In other embodiments, more than one curative is capable of being combined gives.

[056] in the literary composition, " treatment significant quantity " (for example refers to treat agent, curative) consumption is enough to treat or control is crossed expression relevant disease or illness and/or the super proliferative disease of cell with EphA2 or EphA4, preferably, this consumption is enough to destroy, change, control or remove primary, regionality or metastatic cancer tissue.The treatment significant quantity also can refer to treat agent, and (for example, curative) consumption is enough to postpone or reduce as far as possible the outbreak of super proliferative disease, for example, postpones or reduce as far as possible the diffusion of cancer.The treatment significant quantity can refer to that also the consumption for the treatment of agent (for example, curative) can provide the treatment benefit when treatment or control cancer.In addition, the present invention's treatment significant quantity for the treatment of agent (for example, curative) refers to independent or can provide the treatment benefit with other consumptions for the treatment of the curative of agent combination when treatment or the super proliferative disease of control.During with the EphA2 of the present invention of certain consumption or the coupling of EphA4 antibody, this term can comprise can improve overall result of treatment, alleviates or avoid undesirable reaction, maybe can strengthen another treatment for the treatment of agent (for example, curative) and render a service or synergistic with it consumption.

[057] in the literary composition, term " therapy " refers to can be used for preventing, treat, controlling or improve any scheme, method and/or the medicament of super proliferative disease.In some embodiments, term " therapy " refers to be proficient in those skilled in the art such as known biotherapy, supportive treatment and/or other therapies that can be used for treating, control, preventing or improve one or more symptoms of super proliferative disease or this disease of medical worker.

[058] should understand, alleged complementary determining region (CDR) residue of this paper numbering is Kabat etc. (1991, NIH publication 91-3242, technical intelligence service department (National TechnicalInformation Service) of country, the numbering of this Ping Feierde of Virginia (Springfield, VA)).Specifically, residue 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) are positioned at variable region of light chain, and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) are positioned at variable region of heavy chain.Notice widely different between antibody and the antibody (rely on and define the homology that can not show) with the Kabat consensus sequence.The high specific of framework residue inserts the numbering system that is used for the Fv district to often needing with " room " residue.Will be appreciated that the alleged CDR of this paper defines as (the same) such as Kabat.In addition, the identity of some single residue that is in any given Kabat site numbering may be between antibody chain and antibody chain because of species between or allelotrope divergence (allelic divergence) different.

[059] when having two or more, certain term is defined as that adopt this area and/or when accepting, except as otherwise noted, the term definition in the literary composition will comprise all these implications.A concrete example is, adopts term " CDR " to be described in the discontinuous antigen binding site of finding in the heavy chain of polypeptide and the variable region of light chain.This specific region is by Kabat etc. (1991, NIH publication 91-3242, technical intelligence service department of country, this Ping Feierde of Virginia) and Chothia etc. (1987, J.Mol.Biol.196:901-17) and MacCallum etc. (1996, J.Mol.Biol.262:732-45) describe, include them in this paper respectively by reference, this definition will comprise the overlapping or subclass of amino-acid residue when they are compared mutually.However, adopt any definition to represent that the CDR of antibody or its variant is included within the scope as the term that defines and use in the literary composition.The suitable amino-acid residue that comprises the CDR that each reference defines that quotes as mentioned is listed in the table below 1 to show comparison.The accurate residue number that comprises specific CDR will be according to the sequence of CDR with size and different.

[060] those are proficient in those skilled in the art and can determine which residue has comprised the specific CDR in the antibody variable region amino acid sequence according to routine.

Table 1:CDR defines

Kabat ¹ Chothia ² MacCallum ³

VH?CDR1 31-35 26-32 30-35

VH?CDR2 50-65 53-55 47-58

VH?CDR3 95-102 96-101 93-101

VL?CDR1 24-34 26-32 30-36

VL?CDR2 50-56 50-52 46-55

VL，CDR3 89-97 91-96 89-96

¹Nomenclature according to (the same) such as Kabat is numbered residue

²Nomenclature according to (the same) such as Chothia is numbered residue

³Nomenclature according to (the same) such as MacCallum is numbered residue

The accompanying drawing summary

[061] for setting forth the present invention, described some embodiment of the present invention in the drawings.Yet, the invention is not restricted to the accurate configuration and the means of embodiment described in the figure.

[062] Fig. 1.The light-chain amino acid sequence of various resisting-EphA2 and anti--EphA4 antibody.

[063] Fig. 2.The heavy chain amino acid sequence of various resisting-EphA2 and EphA4 antibody.

[064] Fig. 3.The variable chains aminoacid sequence of anti--EphA2 antibody G5 and 3F2.

[065] Fig. 4.The variable chains aminoacid sequence of anti--EphA2 antibody EA2,4H5 and 10D9.

[066] Fig. 5.Variable heavy chain (the V of anti--various avidity-mature forms of Eph antibody GEA44 _H) and light chain (V _L) aminoacid sequence.The aminoacid sequence of following heavy chain of antibody is listed in the first half of this figure: GEA44,1A4,1B10,1D11,1G11,2C9,3A12,3C6,6B7,6B4 and 11H1 (SEQ ID No is respectively 115,117,119,121,123,125,127,129,131,133 and 135).The aminoacid sequence of following light chain of antibody is listed in the Lower Half of this figure: GEA44,1A4,1B10,1D11,1G11,2C9,3A12,3C6,6B7,6B4 and 11H1 (SEQ ID No is respectively 116,117,120,122,124,126,128,130,132,134 and 136).Sequence in the frame is partly represented CDR (Kabat definition).

[067] Fig. 6.General (pan)-Eph antibody 10C12 (GEA10C12) variable heavy chain (V _H) and variable light chain (V _L) Nucleotide and aminoacid sequence.Variable region heavy chain Nucleotide and aminoacid sequence are listed in the first half (SEQ ID No is respectively 141 and 140) of this figure; Variable region light chain Nucleotide and aminoacid sequence are listed in the Lower Half (SEQ ID No is respectively 142 and 141) of this figure.

[068] Fig. 7 A-7B.Various phage-derived anti--EphA2 antibody variable heavy chain (V _H) and light chain (V _L) aminoacid sequence and comparison.Fig. 7 A:5A8,1C1,1D3,1F12,1H3,2B12 (SEQ ID No. is respectively 53,3,33,13,23 and 43) are listed in the aminoacid sequence of following heavy chain of antibody and comparison.Fig. 7 B:5A8,1C1,1D3,1F12,1H3,2B12 (SEQ ID No. is respectively 54,4,34,14,24 and 43) are listed in the aminoacid sequence of following light chain of antibody and comparison.Sequence in the frame is partly represented CDR (Kabat definition).

[069] Fig. 8.Nucleotide sequence and the amino acid variable region sequences (SEQ ID No.1-4) of anti--EphA2 antibody 1C1.

[070] Fig. 9.Nucleotide sequence and the amino acid variable region sequences (SEQ ID No.11-14) of anti--EphA2 antibody 1F12.

[071] Figure 10.Nucleotide sequence and the amino acid variable region sequences (SEQ ID No.21-24) of anti--EphA2 antibody 1H3.

[072] Figure 11.Nucleotide sequence and the amino acid variable region sequences (SEQ ID No.31-34) of anti--EphA2 antibody 1D3.

[073] Figure 12.Nucleotide sequence and the amino acid variable region sequences (SEQ ID No.41-44) of anti--EphA2 antibody 2B12.

[074] Figure 13.Nucleotide sequence and the amino acid variable region sequences (SEQ ID No.51-54) of anti--EphA2 antibody 5A8.

[075] Figure 14.The nucleotide sequence and the aminoacid sequence (SEQ ID No.111-114) of anti--EphA2 antibody 1C1,1F12,1H3,1D3,2B12 and 5A8 constant region (heavy chain or κ light chain).

[076] Figure 15 A-15B.By flow cytometry more various anti--EphA2 antibody combines with the cell surface of various clones.Figure 14 A has compared antibody 1C1,1F12,1H3 and the 3F2 combination on following clone: A549, Hey-A8, PC3, KC-231, Panc-02.03, SK Mel-28, ACHN, 498, D-145, HT-29, SKOV-3 and SW-480.Figure 14 B has compared antibody 1C1,1F12 and the combination of 3F2 on following clone: Balb/3T3, NIH/3T3, CT26, F98, RG2, YPEN.

[077] Figure 16.Several differences are anti--comparison of EphA2 antibody internalization.The internalization of relatively anti--EphA2 antibody B233, EA5 and B208 and contrast in MCF-10A clone.

[078] Figure 17.Several differences are anti--comparison of EphA2 antibody internalization.The relatively internalization of anti--EphA2 antibody B233, B208, EA2, G5,3F2,1C1, C2,3B2 and contrast in following clone: PC3, SK Mel-28, HuVec, MCF10A and CT26.

[079] Figure 18.Confirm the internalization of anti--EphA2 antibody G5 by immunofluorescence.PC3 cell personnel selection α-EphA2mAb (G5; Figure A and B) or R347 isotype contrast (figure C) mark.Then cell is cultivated 0 (not internalization: figure A and C) or (internalization: in 60 minutes figure B) so that invest the antibody internalization of cell surface under growth conditions.Then all cells fixed (4% formaldehyde), changed processing (0.5%Triton X-100) also with AlexaFluor 488-Ab dyeing thoroughly, add then to resist and fade film solid media and carry out the fluorescent microscope detection.Figure B confirmed G5 anti--internalization of EphA2 antibody.

[080] Figure 19 A-19C.Confirm anti--EphA2 antibody 1C1 (Figure 18 A), 1F12 (Figure 18 B) and 3F2 (Figure 18 C) internalization on the HuVec cell by immunofluorescence.

[081] Figure 20.Confirm anti--EphA2 antibody 1C1 and 1F12 internalization on Ct-26 and PC-3 cell by immunofluorescence.

[082] Figure 21.Confirm that in following clone anti--EphA2 antibody 1C1 and 1F12 can make EphA2 phosphorylation: CT26,4T1, F98, YPEN1, PC3 and ES2.

[083] Figure 22.Confirm that in the HuVec cell anti--EphA2 antibody 1C1,1F12 and 3F2 can make the EphA2 phosphorylation.

[084] Figure 23.This figure summed up various anti--characteristic of EphA2 antibody (1C1,1F12,1H3,1D3,2B12 and 5A8) (activation, internalization and organize cross reactivity (TCR)).

[085] Figure 24.This figure has summed up anti--EphA2 antibody 1C1 and the specificity of 1F12 to the acceptor Eph different Muridae members of family.1C1 confirms that the energy specificity is in conjunction with mouse EphA2 and 4.1F12 is proved can be in conjunction with mouse EphA2,3,4,5,6,7 and 8, and the while can also be in conjunction with mouse EphB1 and 2.

[086] Figure 25.The chemical structure that has shown monomethyl Ao Ruisitating E comprises transcribed spacer part and VC joint.

[087] Figure 26.The chemical structure that has shown monomethyl Ao Ruisitating F comprises transcribed spacer part and VC joint.

[088] Figure 27.The chemical structure that has shown monomethyl Ao Ruisitating E and F comprises transcribed spacer part and two different joints (Xie Ansuan-citrulline and maleimide caproyl-citrulline).

[089] Figure 28.The coupling of ADC.The figure illustrates the coupling of typical resisting-EphA2 antibody.Each antibody molecule is by 4 medicine joints of the average coupling of suitable peptide linker (Hamblett etc., Clinical Cancer Research 2004).

[090] Figure 29.The coupling of anti--EphA2 antibody and mcMMAF.This figure has summed up output (mg) and other characteristics (% polymerization and endotoxin concns) of mcMMAF link coupled 1C1,1F12 and 1H3 antibody.

[091] Figure 30.Contain the comparison that the different joints of anti--EphA2 antibody and drug regimen suppress the growth in vitro of several different carcinoma clones.Anti--EphA2 antibody the G5 that will be coupled to vcMMAF in SKMEL, PC-3 and MDA231 clone compares with the anti--EphA2 antibody 3F2 that is coupled to vcMMAE, vcMMAF or mcMMAF.The concentration range of test is 0.001-100 μ g/ml.The results are summarized among the different figure of 3 width of cloth.

[092] Figure 31.Compare with the contrast 1A7 antibody that is connected in MMAF, the growth in vitro that is connected in anti--EphA2 antibody EA5 of the MMAF that has the vc joint suppresses.Following clone: A549, MDA231 and A375 have been tested.The concentration range of test is 0.001-100 μ g/ml.The results are summarized among the different figure of 4 width of cloth.

[093] Figure 32 A-32C.Compare with free MMAE with the EA5 in the EA5 that is connected in the contrast 1A7 antibody of MMAF, no MMAF, the competition, the growth in vitro that is connected in anti--EphA2 antibody EA5 of the MMAF that has the vc joint suppresses.Following clone: MDA231 (Figure 32 A), A549 (Figure 32 B) and A375 (Figure 32 C) have been tested.The concentration range of test is 0.001-100 μ g/ml.

[094] Figure 33.Compare with free MMAE with the EA5 in the contrast 1A7 antibody that is connected in MMAF, the competition, the growth in vitro that is connected in anti--EphA2 antibody EA5 of the MMAF that has the vc joint suppresses.Following clone: HCT-116 and SW620 have been tested.The concentration range of test is 0.001-100 μ g/ml.The results are summarized among the different figure of 2 width of cloth.

[095] Figure 34.Compare with free MMAE with the contrast 1A7 antibody that is connected in MMAF, independent EA5, the EA5 in the competition, the anti--EphA2 antibody EA5 that is connected in the MMAF that has the vc joint is to MDA-231 cells in vitro growth-inhibiting.The concentration range of test is 0.001-100 μ g/ml.The results are summarized among the different figure of 2 width of cloth.

[096] Figure 35.Compare with free MMAE with the G5 in the contrast 1A7 antibody that is connected in MMAF, the competition, the anti--EphA2 antibody G5 that is connected in the MMAF that has the vc joint is to PC-3 cell and MDA-MB-468 cells in vitro growth-inhibiting.The concentration range of test is 0.001-100 μ g/ml.The results are summarized among the different figure of 4 width of cloth.

[097] Figure 36.Be connected in the anti--EphA2 antibody G5 of the MMAF that has the vc joint and EA5 to A498 cell, PC-3 cell and MDA-MB-468 cells in vitro growth-inhibiting.The concentration range of test is 0.001-100 μ g/ml.The results are summarized among the different figure of 3 width of cloth.

[098] Figure 37.Compare with independent G5 with the G5 in the contrast R3-47 control antibodies that is connected in MMAF, the competition, the anti--EphA2 antibody G5 that is connected in the MMAF that has the vc joint is to PC-3 cell, 231KC cell and T-231 cells in vitro growth-inhibiting.The concentration range of test is 0.001-100 μ g/ml.The results are summarized among the different figure of 4 width of cloth.

[099] Figure 38.Compare with free MMAE with the 3F2 in the competition, anti--EphA2 antibody G5vcMMAF, 3F2vcMMAF, 3F2vcMMAE are to normal HuVEC cells in vitro growth-inhibiting.The concentration range of test is 0.001-100 μ g/ml.The results are summarized among the different figure of 2 width of cloth.

[0100] Figure 39.Compare with free MMAE with the G5 in the contrast R3-47 antibody that is connected in MMAF, the competition, the anti--EphA2 antibody G5 that is connected in the MMAF that has the vc joint is to PC-3 cells in vitro growth-inhibiting.The concentration range of test is 0.001-100 μ g/ml.

[0101] Figure 40.Summary with the clone of the anti--EphA2 antibody G5 vitro test that is coupled to the MMAF that has the vc joint.

[0102] Figure 41.The average IC of the anti--EphA2 antibody G5 that measures from a different set of EphA2 positive cell line ₅₀Value (μ g/ml).This IC50 value is obtained by the growth in vitro inhibition test extrapolation of carrying out on clone.

[0103] Figure 42.From anti--EphA2 antibody 3F2vcMMAE of a different set of EphA2 positive cell line mensuration and the IC50 value of 3F2mcMMAF.This IC50 value is obtained by the growth in vitro inhibition test extrapolation of carrying out on clone.Sort from high to low according to the EphA2 expression level, the clone of test is as follows: HEY-A8, PANC.02.03, KC231, PC3, DU-145, ACHN, A498, A549, SKMEL28.

[0104] Figure 43 A-43B.From anti--EphA2 antibody 3F2mcMMAF, 1C1mcMMAF of a different set of EphA2 positive human cancerous cell line mensuration and the IC50 value of 1F12mcMMAF.This IC50 value is obtained by the growth in vitro inhibition test extrapolation of carrying out on clone.The clone of test is as follows: PC3, KC231, SKOV3 and HEY-A8.Figure 44 B has shown and has used acceptable IC in the body ₅₀Concentration.

[0105] Figure 44.Compare with free MMAE, be connected in anti--EphA2 antibody 1C1, the 1F12 of the MMAF that has the mf joint and 3F2 MCF10-A and HuVec cells in vitro growth-inhibiting.Also summed up EphA2 combining among another width of cloth figure at the EphA2 of HUVEC and MCF10-A cell surface expression and test antibody and surface expression.

[0106] Figure 45.Same antibody (comprising the corresponding antibodies that they are not connected) in anti--EphA2 antibody 1C1, the 1F12 that is connected in the MMAF that has the mf joint and 3F2 and the competition is to the growth inhibiting comparison of PC-3 cells in vitro.The concentration range of test is 0.001-10 μ g/cc.

[0107] Figure 46.Be connected in the MMAF that has the mf joint and be connected in anti--EphA2 antibody 1C1 of the MMAE that has the vc joint and 1F12 to KC-231 and PC3 cells in vitro growth-inhibiting.The coupling antibody that in this group experiment, has compared different batches.The concentration range of test is 0.001-100 μ g/cc.

[0108] Figure 47.1C1 and the cross species activity of 1F12 in EphA2+ clone of anti--EphA2ADC.In following cell, relatively be connected in anti--EphA2 antibody 1C1 and the 1F12 and the contrast R347 antibody that is connected in the MMAF that has the mc joint of the MMAF that has the mc joint: F98 (rat glioma), PC3 (human prostata cancer), CT26 (mouse junction cancer) and CYNO-MK.The concentration range of test is 0.001-10 μ g/cc.

[0109] Figure 48.In the PC-3 PC-3, relatively be coupled to anti--EphA2G5 antibody of the MMAF that has the vc joint and link coupled G5 not in the body, be coupled to the contrast IgG of MMAF and link coupled contrast IgG not.The dosage of antibody is 20 μ g and 200 μ g.

[0110] Figure 49.In the MDA-MB-231KC MCF-7, relatively be coupled to the MMAF that has the vc joint in the body and resist-EphA2G5 antibody and the contrast IgG that is coupled to MMAF.The dosage of antibody is 20 μ g, 50 μ g and 100 μ g.

[0111] Figure 50.In the PC3 PC-3, relatively be coupled to the MMAF that has the vc joint in the body or have anti--EphA2G5 antibody and the contrast IgG that is coupled to MMAF of MMAF of mc joint and coupling and link coupled contrast R347 not.The dosage of antibody is 20 μ g and 200 μ g.

[0112] Figure 51.In the PC-3 PC-3, relatively be coupled to anti--EphA23F2 antibody and the anti--EphA23F2 antibody that is coupled to the MMAF that has the mc joint and the contrast R347 and the PBS that are coupled to MMAE or MMAF of the MMAF that has the vc joint in the body.For the MMAE conjugate, antibody dosage is 3mg/kg (60 μ g), and for MMAF, antibody dosage is 10mg/kg (200 μ g).

[0113] Figure 52.In the PC-3 PC-3, relatively be coupled to anti--EphA2 antibody 1C1 and 1F12 of the MMAF that has the mc joint in the body and be coupled to contrast R347 and the PBS of MMAF.Antibody dosage is 3mg/kg (60 μ g) or 1mg/kg (20 μ g).

[0114] Figure 53 A-53C.In the PC-3 PC-3, relatively be coupled to the MMAF that has the vc joint in the body or be coupled to anti--EphA2 antibody 1C1 and 1F12 and PBS of the MMAF that has the mc joint.Antibody dosage is 6.0mg/kg (Figure 54 A), 3.0mg/kg (Figure 54 B) and 1.0mg/kg (Figure 54 C).

[0115] Figure 54.In the MDA-231KC MCF-7, relatively be coupled in the body MMAF that has the mc joint anti--EphA2 antibody 1C1 and 1F12 and PBS, be coupled to the contrast R347 of MMAF and not link coupled resist-EphA2 antibody 3F2-3M.Antibody dosage is 1mg/kg (20 μ g), 3mg/kg (60 μ g), 6mg/kg (120 μ g) or 10mg/kg (200 μ g).

[0116] Figure 55.In the MDA-231KC MCF-7, relatively be coupled to the MMAE that has the vc joint in the body or be coupled to anti--EphA2 antibody 1C1 and 1F12 and PBS of the MMAF that has the mc joint.Antibody dosage is 1mg/kg (20 μ g) or 6mg/kg (120 μ g).

[0117] Figure 56 A-56C.In the MDA-231KC MCF-7, relatively be coupled to the MMAE that has the vc joint in the body or be coupled to anti--EphA2 antibody 1C1 and 1F12 and PBS of the MMAF that has the mc joint.The dosage of antibody is 6.0mg/kg (Figure 57 A), 3.0mg/kg (Figure 57 B) and 1.0mg/kg (Figure 57 C).

[0118] Figure 57.The growth-inhibiting of free MMAE medicine.Detected some different mouse, rat, people and MC and tied up to external sensitivity, and summed up the IC50 (μ M) that obtains free MMAE.

[0119] Figure 58.The toxicity of the anti--EphA2ADC that measures with the body weight loss of Balb/c mouse.Tested in vivo be coupled to the MMAE that has the vc joint or be coupled to anti--EphA2 antibody 1C1 of the MMAF that has the mc joint and 1F12 to determine that dispenser is to the influence of mouse body weight than using contrast PBS.The dosage of vcMMAE antibody is 40mg/kg, 50mg/kg and 60mg/kg.The dosage of 1C1-mcMMAF antibody is 120mg/kg, 180mg/kg and 240mg/kg.The dosage of 1F12-mcMMAF antibody is 90mg/kg, 120mg/kg, 180mg/kg, 210mg/kg and 240mg/kg.

[0120] Figure 59.The treatment window (therapeutic window) of anti--EphA2ADC.This figure has summed up the potential treatment window of 1C1-mcMMAF, the 1C1-vcMMAE, 1F12-mcMMAF and the 1F12-vcMMAE that observe based on data in external and the body.

Detailed Description Of The Invention

[0121] receptor tyrosine kinase (RTK) is to import signal into cytoplasmic transmembrane molecule from born of the same parents' external environment. The Eph family of RTK is the subfamily of RTK maximum. This organizes to be rich in, and the zone of cysteine and two III type fibronectins in the ectodomain repeat is feature. The Eph acceptor is joined albumen by second family-liver of cell surface anchorin and is activated. The member that Eph EGFR-TK and liver are joined protein ligands mediates receptor-ligand binding signal conduction (Bruckner etc., 1997, Science 275:1640 afterwards; Holland etc., 1996, Nature 383:722). Known this two-way signaling conduction can affect the process that relates to cell interaction, forms (Boyd etc., 2001 Sci STKE RE20 such as cell adherence, cell migration and organizational boundary; Schmucher etc., 2001, Cell 105:701-4; Kullander etc., 2002Nat.Rev.Mol.Cell Biol.3:475). The Eph acceptor is associated with generation and the progress of cancer recently.

[0122] as cell surface molecule, the Eph acceptor is the target molecule of the easy contact of the directed therapy of antibody. For example, excessively expressing in the tumour cell of EphA2, the surface receptor increase of existence causes unsettled cell-cells contacting, and this can destroy Cycle Regulation and cause growth of tumour cell, propagation and invasion and attack. The exposed antibody of anti-Eph receptor family different members (for example EphA2) phosphorylation, internalization and the degraded by acceptor demonstrate agonist activity (referring to, for example, U.S. Patent number 6,927,203, U.S. Provisional Application number 60/717,209, U.S. Patent Application Publication No. US2006/0121042-A1, U.S. Patent Application No. 09/952,560,10/994,129,10/436,782,10/863,729 and 11/203,251, each patent is included this paper by reference in full in). In one embodiment, ADC of the present invention is the variant of the antibody of at least a Eph acceptor of specific binding. The Eph acceptor of ADC specific binding of the present invention includes but not limited to EphA1, EphA2, EphA3a, EphA3b, EphA4, EphA5a, EphA5b, EphA6, EphA7, EphA8, EphB1, EphB2a, EphB2b, EphB3, EphB4 and EphB6.

[0123] those skilled in the art will understand, Eph acceptor of the present invention be with known Eph acceptor have the certain degree homology molecule (referring to, for example, the same), thereby according to its amino acid sequence or it can be classified as Eph receptor family molecule. Adopt MegaAlign program (DNASTAR) to carry out the paired comparison (Thompson etc., 1994 Nucleic Acids Res 22:4673-80) of known people Eph acceptor with Clustal W algorithm. Result (Figure 18) shows that in Eph receptor family member, each protein has the similarity of height in a plurality of zones. Especially can estimate, be proficient in the antibody that those skilled in the art can produce the some zones of Eph acceptor, thereby allow described antibody that cross reaction takes place between the family member or have more the specificity of limitation, so that described antibody is only with family member of high-affinity specific binding. For identifying for generation of protein specific or in conjunction with the potential immunogenic peptide of the antibody of one or more Eph acceptors, can adopt Protean program (DNASTAR) to detect the antigenicity index of each protein with the Jameson-Wolf algorithm. Can identify the high zone of antigenicity index among all members of Eph receptor family and in the middle of one or more family members those zones of high conservative will be the fabulous candidate target that obtains more than one family members' of identification antibody. And adopt zone not too cautious may produce the special antibody to a kind of Eph receptor family member.

In [0124] one embodiment, the Eph acceptor that exists on the ADC preferred combination tumour cell of the present invention and not in conjunction with the Eph acceptor that exists on the non-tumor cell. In other embodiments, the ADC of the present invention normal structure that do not dye, described normal structure includes but not limited to: brain, lung, pancreas, liver, prostate, the heart, ovary, skin, kidney, intestines and stomach. Adopt immune labeled method well known in the art to identify easily combination and the specific staining method of antibody, these methods include but not limited to: immunohistochemistry and fluorescence activated cell scanning/sorting (FACS). Concrete method and strategy can be at " immunohistochemistry introduction " (Introduction to Immunocytochemistry) of Polak and Van Noorden, second edition (1997), (the Springer Verlag of New York Springer Wei Ge publishing house, N.Y.), " fluorescence probe and the specializes in chemistry material handbook (Handbook of Fluorescent Probes and Research Chemicals) of Haugland, the 9th edition (2004) and (the Molecular Probes of Eugene, Ore molecular probe company, Inc., Eugene, Oreg) find in the composition handbook published and the catalogue etc.

[0125] in other embodiments, ADC of the present invention is the variant of the antibody of specific binding EphA2 and/or EphA4, their derivative, analog and epi-position-binding fragment, such as but not limited to: this paper and PCT publication number WO 04/014292, WO 03/094859 and U.S. Patent Application Serial Number 10/863, those that disclose in 729, each document is in full included this paper in by reference, and list in table 2-4,6 or Fig. 1-59 in any antibody. In concrete embodiment, ADC of the present invention is the antibody of specific binding EphA2 and/or EphA4, it comprise 12G3H11 and/or 3F2 and/or 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8 and/or table 2-4 6 or Fig. 1-59 in all or part of variable region (for example, one or more CDR) of listed any antibody.

[0126] the present invention also comprises the application that at least a Eph acceptor is had the ADC of the present invention of high binding affinity. In concrete embodiment, association rate constant or the k of the ADC of the present invention of at least a Eph acceptor of specific binding_onSpeed ((Ab)+antigen (Ag)^k _on← Ab-Ag) be: at least 10⁵M ^-1s ^-1, at least 5 * 10⁵M ^-s ^-1, at least 10⁶M ^-1s ^-1, at least 5 * 10⁶M ^-1s ^-1, 107M at least^-1s ^-1, at least 5 * 10⁷M ^-1s ^-1, or at least 10⁸M ^-1s ^-1 In further concrete embodiment, association rate constant or the k of the ADC of the present invention of at least a Eph acceptor of specific binding_onSpeed ((Ab)+antigen (Ag)^k _on← Ab-Ag) be: at least about 10⁵M ^-1s ^-1, at least about 5 * 10⁵M ^-s ^-1, at least about 10⁶M ^-1s ^-1, at least about 5 * 10⁶M ^-1s ^-1, at least about 10⁷M ^-1s ^-1, at least about 5 * 10⁷M ^-1s ^-1, or at least about 10⁸M ^-1s ^-1 In other embodiments, the k of the ADC of at least a Eph acceptor of specific binding_onFor: at least 2 * 10⁵M ^-1s ^-1, at least 5 * 10⁵M ^-1s ^-1, at least 10⁶M ^-1s ^-1, at least 5 * 10⁶M ^-1s ^-1, at least 10⁷M ^-1s ^-1, at least 5 * 10⁷M ^-1s ^-1, or at least 10⁸M ^-1s ^-1 In further embodiment, the k of the ADC of at least a Eph acceptor of specific binding_onFor: at least about 2 * 10⁵M ^-1s ^-1, at least about 5 * 10⁵M ^-1s ^-1, at least about 10⁶M ^-1s ^-1, at least about 5 * 10⁶M ^-1s ^-1, at least about 10⁷M ^-1s ^-1, at least about 5 * 10⁷M ^-1s ^-1, or at least about 10⁸M ^-1s ^-1。

[0127] in other embodiments, specific binding is in the K of the ADC of the present invention of at least a Eph acceptor_offSpeed ((Ab)+antigen (Ag)^k _off← Ab-Ag) be: less than 10^-1s ^-1, less than 5 * 10^-1s ^-1, less than 10^-2s ^-1, less than 5 * 10^-2s ^-1, less than 10^-3s ^-1, less than 5 * 10^-3s ^-1, less than 10^-4s ^-1, less than 5 * 10^-4s ^-1, less than 10^-5s ^-1, less than 5 * 10^-5s ^-1, less than 10^-6s ^-1, less than 5 * 10^-6s ^-1, less than 10^-7s ^-1, less than 5 * 10^-7s ^-1, less than 10^-8s ^-1, less than 5 * 10^-8s ^-1, less than 10^-9s ^-1, less than 5 * 10^-9s ^-1, or less than 10^-10-1s ^-1 Again in other embodiments, specific binding is in the K of the ADC of the present invention of at least a Eph acceptor_offSpeed ((Ab)+antigen (Ag)^k _off← Ab-Ag) be: less than about 10^-1s ^-1, less than about 5 * 10^-1s ^-1, less than about 10^-2s ^-1, less than about 5 * 10^-2s ^-1, less than about 10^-3s ^-1, less than about 5 * 10^-3s ^-1, less than about 10^-4s ^-1, less than about 5 * 10^-4s ^-1, less than about 10^-5s ^-1, less than about 5 * 10^-5s ^-1, less than about 10^-6s ^-1, less than about 5 * 10^-6s ^-1, less than about 10^-7s ^-1, less than about 5 * 10^-7s ^-1, less than about 10^-8s ^-1, less than about 5 * 10^-8s ^-1, less than about 10^-9s ^-1, less than about 5 * 10^-9s ^-1, or less than about 10^-10-1s ^-1 In further embodiment, specific binding is in the K of the ADC of at least a Eph acceptor_offFor: less than 5 * 10^-4s ^-1, less than 10^-5s ^-1, less than 5 * 10^-5s ^-1, less than 10^-6s ^-1, less than 5 * 10^-6s ^-1, less than 10^-7s ^-1, less than 5 * 10^-7s ^-1, less than 10^-8s ^-1, less than 5 * 10^-8s ^-1, less than 10^-9s ^-1, less than 5 * 10^-9s ^-1, or less than 10^-10s ^-1 In other embodiments, specific binding is in the K of the ADC of at least a Eph acceptor_offFor: less than about 5 * 10^-4s ^-1, less than about 10^-5s ^-1, less than about 5 * 10^-5s ^-1, less than about 10^-6s ^-1, less than about 5 * 10^-6s ^-1, less than about 10^-7s ^-1, less than about 5 * 10^-7s ^-1, less than about 10^-8s ^-1, less than about 5 * 10^-8s ^-1, less than about 10^-9s ^-1, less than about 5 * 10^-9s ^-1, or less than about 10^-10s ^-1。

[0128] in other embodiments, specific binding is in affinity costant or the K of the ADC of the present invention of at least a Eph acceptor_a(k _on/k _off) be: at least 10²M ^-1, at least 5 * 10²M ^-1, at least 10³M ^-1, at least 5 * 10³M ^-1, at least 10⁴M ^-1, at least 5 * 10⁴M ^-1, at least 10⁵M ^-1, at least 5 * 10⁵M ^-1, at least 10⁶M ^-1, at least 5 * 10⁶M ^-1, at least 10⁷M ^-1, at least 5 * 10⁷M ^-1, at least 10⁸M ^-1, at least 5 * 10⁸M ^-1, at least 10⁹M ^-1, at least 5 * 10⁹M ^-1, at least 10¹⁰M ^-1, at least 5 * 10¹M ^-1, at least 10¹¹M ^-1, at least 5 * 10¹¹M ^-1, at least 10¹²M ^-1, at least 5 * 10¹²M, at least 10¹³M ^-1, at least 5 * 10¹³M ^-1, at least 10¹⁴M ^-1, at least 5 * 10¹⁴M ^-1, at least 10¹⁵M ^-1, or at least 5 * 10¹⁵M ^-1 In further embodiment, specific binding is in affinity costant or the K of the ADC of the present invention of at least a Eph acceptor_a(k _on/k _off) be: at least about 10²M ^-1, at least about 5 * 10²M ^-1, at least about 10³M ^-1, at least about 5 * 10³M ^-1, at least about 10⁴M ^-1, at least about 5 * 10⁴M ^-1, at least about 10⁵M ^-1, at least about 5 * 10⁵M ^-1, at least about 10⁶M ^-1, at least about 5 * 10⁶M ^-1, at least about 10⁷M ^-1, at least about 5 * 10⁷M ^-1, at least about 10⁸M ^-1, at least about 5 * 10⁸M ^-1, at least about 10⁹M ^-1, at least about 5 * 10⁹M ^-1, at least about 10¹⁰M ^-1, at least about 5 * 10¹M ^-1, at least about 10¹¹M ^-1, at least about 5 * 10¹¹M ^-1, at least about 10¹²M ^-1, at least about 5 * 10¹²M, at least about 10¹³M ^-1, at least about 5 * 10¹³M ^-1, at least about 10¹⁴M ^-1, at least about 5 * 10¹⁴M ^-1, at least about 10¹⁵M ^-1, or at least about 5 * 10¹⁵M ^-1。

[0129] more in another embodiment, specific binding is in dissociation constant or the K of the ADC of at least a Eph acceptor_d(k _off/k _on) be: less than 10^-2M is less than 5 * 10^-2M is less than 10^-3M is less than 5 * 10^-3M is less than 10^-4M is less than 5 * 10^-4M is less than 10^-5M is less than 5 * 10^-5M is less than 10^-6M is less than 5 * 10^-6M is less than 10^-7M is less than 5 * 10^-7M is less than 10^-8M is less than 5 * 10^-8M is less than 10^-9M is less than 5 * 10^-9M is less than 10^-10M is less than 5 * 10^-10M is less than 10^-11M is less than 5 * 10^-11M is less than 10^-12M is less than 5 * 10^-12M is less than 10^-13M is less than 5 * 10^-13M is less than 10^-14M is less than 5 * 10^-14M is less than 10^-15M, or less than 5 * 10^-15M. In further embodiment, specific binding is in dissociation constant or the K of the ADC of at least a Eph acceptor_d(k _off/k _on) be: less than about 10^-2M is less than about 5 * 10^-2M is less than about 10^-3M is less than about 5 * 10^-3M is less than about 10^-4M is less than about 5 * 10^-4M is less than about 10^-5M is less than about 5 * 10^-5M is less than about 10^-6M is less than about 5 * 10^-6M is less than about 10^-7M is less than about 5 * 10^-7M is less than about 10^-8M is less than about 5 * 10^-8M is less than about 10^-9M is less than about 5 * 10^-9M is less than about 10^-10M is less than about 5 * 10^-10M is less than about 10^-11M is less than about 5 * 10^-11M is less than about 10^-12M is less than about 5 * 10^-12M is less than about 10^-13M is less than about 5 * 10^-13M is less than about 10^-14M is less than about 5 * 10^-14M is less than about 10^-15M, or less than about 5 * 10^-15M。

[0130] as mentioned above, its antibody moiety comprises specific binding in the variable region of at least a Eph acceptor among the ADC that the present invention includes. The present invention also comprises specific binding in the ADC of at least a Eph acceptor, and with respect to comparable molecule, its ADCC and/or CDC are active different and one or more Fc parts (for example, Fc γ Rs, C1q) are had modified binding affinity. Referring to, for example, U.S. Patent Application Publication No. 2006/0039904A1. The present invention specifically comprises the ADC derived from anti--Eph receptor antibody or its fragment, described antibody or its fragment include but not limited to: Eph099B-102.147 (ATCC accession number PTA-4572), Eph099B-208.261 (ATCC accession number PTA-4573), Eph099B-210.248 (ATCC accession number PTA-4574), Eph099B-233.152 (ATCC accession number PTA-5194), (PCT publication number WO 03/094859, it includes this paper by reference in full in); EA2 (ATCC accession number PTA-4380), EA3, EA4, EA5 (ATCC accession number PTA-4381), (PCT publication number WO 04/014292, it includes this paper by reference in full in); LX-13 and scFv EA44 (ATCC accession number PTA-6044), (U.S. Patent Application Serial Number 10/863,729, it includes in full this paper by reference in), G2 and 12G3H11 with and analog, derivative or fragment. Especially estimate, ADC of the present invention can comprise 12G3H11 (seeing Table 2) and/or table 2-4 or 6 or Fig. 1-59 in all or part variable region (for example, one or more CDR) of listed any antibody.

In [0131] one embodiment, described ADC is the ADC of 12G3H11, and this is the exciting monoclonal antibody of a kind of humanization in conjunction with EphA2. The amino acid sequence of variable region of heavy chain and variable region of light chain is seen respectively SEQ ID NO:165 and SEQ ID NO:166 (seeing Fig. 1 and 2) in the text. In other embodiments, ADC of the present invention competes in conjunction with EphA2 in conjunction with identical epi-position or with 12G3H11 with 12G3H11. In another embodiment, specific binding is not the ADC of 12G3H11 in the ADC of the present invention of Eph acceptor.

In [0132] one embodiment, described ADC is the ADC of 3F2, and this is the exciting monoclonal antibody of a kind of humanization in conjunction with EphA2 (see U.S. Patent application 11/203,251, it includes this paper by reference in full in). The amino acid sequence of variable region of heavy chain and variable region of light chain is seen respectively SEQ ID NO:63 and SEQ ID NO:64 (Fig. 3) in the text. In other embodiments, ADC of the present invention competes in conjunction with EphA2 in conjunction with identical epi-position or with 3F2 with 3F2. In another embodiment, immunity-specific binding is not the ADC of 3F2 in the ADC of the present invention of Eph acceptor. In other embodiments, ADC of the present invention is not the ADC of 3F2.

[0133] in other embodiments, described ADC is the ADC of G5, and this is the exciting monoclonal antibody of a kind of humanization in conjunction with EphA2. The amino acid sequence of G5 heavy chain and variable region of light chain is seen respectively SEQ ID NO:103 and SEQ ID NO:104 (Fig. 1 and 2) in the text.

[0134] in other embodiments, described ADC is the ADC of anti--EphA2 antibody 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8. The amino acid sequence of these heavy chain of antibody and variable region of light chain is shown in Fig. 1-14 (SEQ ID NO.165 and 166,87 and 88,95 and 96,103 and 104,140 and 142,137 and 138,63 and 64,3 and 4,13 and 14,23 and 24,33 and 34,43 and 44 and 53 and 54).

In [0135] one embodiment, ADC of the present invention compares other Eph acceptors preferentially in conjunction with EphA2. In other embodiments, ADC of the present invention compares other Eph acceptors preferentially in conjunction with EphA4. Again in other embodiments, immune response takes place in ADC of the present invention and one or more Eph receptor complexes (for example, Eph acceptor-liver is joined the protein ligands compound). Again in other embodiments, more than one Eph acceptors of ADC specific binding of the present invention. The Eph receptor combination of the ADC institute combination of more than one Eph acceptors of specific binding can represent with following formula: EphA (x)+EphB (y); EphA (x)+EphA (x); EphB (y)+EphB (y); Wherein, (x) be 1,2,3,3a, 3b, 4,5,5a, 5b, 6,7 or 8, (y) be 1,2,2a, 2b, 3,4,5 or 6. In concrete embodiment, be combined with the ADC of more than one Eph acceptor generation specific immune responses, for example, EphA2+EphA4, or EphA2+EphA3, or EphA2+EphB4, or EphA4+EphA3, or EphA4+EphB4. Especially estimate that the ADC of more than one Eph acceptors of specific binding is bispecific antibodies. Can estimate that also the ADC of more than one Eph acceptors of specific binding is the antibody in conjunction with total epi-position between two or more Eph. Estimate that also the ADC of more than one Eph acceptors of specific binding is the antibody with one or more Eph acceptor cross reactions. In addition, ADC of the present invention is to more than one Eph acceptors (for example, EphA2 and EphA4)) have identical immunoreactivity, perhaps, described ADC can be better than other acceptors to a kind of immune response of Eph acceptor.

[0136] the present invention includes specific binding in the ADC of EphA2, the variable heavy chain that described antibody comprises (" VH ") domain has the amino acid sequence of the VH domain of 12G3H11, Eph099B-102.147, Eph099B-208.261 (" B208 "), Eph099B-210.248 (" B210 "), Eph099B-233.152 (" B233 "), EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8. The present invention also comprises specific binding in the ADC of EphA2, and the variable light chain that described antibody comprises (" VL ") domain has the amino acid sequence of the VL domain of 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8. The present invention also comprises specific binding in the ADC of EphA2, and described antibody comprises the VH domain described in the literary composition with the VL domain described in the literary composition or other VL domain combinations. The present invention also comprises specific binding in the ADC of EphA2, and described ADC comprises the VL domain described in the literary composition with the VH domain described in the literary composition or other VH domain combinations.

[0137] the present invention includes specific binding in the ADC of EphA4, the variable heavy chain that described antibody comprises (" VH ") domain has the amino acid sequence of the VH domain of LX-13 or scFv EA44. The present invention also comprises specific binding in the ADC of EphA4, and the variable light chain that described antibody comprises (" VL ") domain has the amino acid sequence of the VL domain of LX-13 or scFv EA44. The present invention also comprises specific binding in the ADC of EphA4, and described antibody comprises the VH domain described in the literary composition with the VL domain described in the literary composition or other VL domain combinations. The present invention also comprises specific binding in the ADC of EphA4, and described ADC comprises the VL domain described in the literary composition with the VH domain described in the literary composition or other VH domain combinations.

[0138] the present invention includes specific binding in the ADC of Eph acceptor, the VH CDR that described antibody comprises has the amino acid sequence of listed any VH CDR in following table 2 or 3. The present invention also comprises specific binding in the ADC of Eph acceptor, and the VL CDR that described antibody comprises has the amino acid sequence of listed any VL CDR in following table 2 or 3. The present invention also comprises specific binding in the ADC of Eph acceptor, and described ADC comprises one or more VH CDR listed in table 2 or 3 and one or more VL CDR. The present invention also comprises specific binding in the ADC of Eph acceptor, and described ADC comprises the some or all of any combination among the listed VHCDR and VLCDR in following table 2 or 3.

The CDR sequence of table 2:12G3H11 and 3F2

CDR	Sequence	SEQ ID NO：
			12G3H11 VH1	DYSM N	***
12G3H11 VH2	FIRNKANDYTTEYADSVKG	***
			12G3H11 VH3	YPRHHAMDS	***
12G3H11 VL1	RASQSISNNLH	***
			12G3H11 VL2	YAFQSIS	***
12G3H11 VL3	QQANSWPLT	***
			3F2 VH1	DYSMN	65
3F2 VH2	FIRNKANAYTTEYSASVKG	66
			3F2 VH3	YPRYHAMDS	67
3F2 VL1	RASQSISNNLH	68
			3F2 VL2	YGFQSIS	69
3F2 VL3	QQANSWPLT					70

The CDR sequence of table 3:1C1,1F12,1H3,1D3,2B12 and 5A8

CDR	Sequence	Seq ID No.
			1C1VH1	HYMMA	5
1C1VH2	RIGPSGGPTHYADSVKG
			6
1C1VH3	YDSGYDYVAVAGPAEYFQH
			7
1C1VL1	RASQSISTWLA
			8
1C1VL2	KASNLHT
			9
1C1VL3	QQYNSYSRT
			10
1F12VH1	RYQMM				15
		1F12VH2	SISPSGGVTLYADSVKG
	16
		1F12VH3	ELLGTVVVPVAWKMRGYFDY
	17
		1F12VL1	RASQSVSSNLA
	18
		1F12VL2	GASTRAST	19
1F12VL3	QQYNNWPPLT					20
		1H3VH1	MYAMR				25
1H3VH2	VIGPSGGWTPYADSVKG					26
		1H3VH3	DRGIYGMDV				27
1H3VL1	RASQGISSYLA
			28
1H3VL2	AASTLQS
			29
1H3VL3	LELNNYPFT				30
		1D3VH1	PYDML				35

1D3VH2	RIGSSGGYTKYADSVKG		36
				1D3VH3	ARSVVVSSDAFDI		37
1D3VL1	RASQGISKWLA
			38
1D3VL2	GASTLQS				39
		1D3VL3	QQYNDYPLT				40
2B12VH1	NYNMY			45
		2B12VH2	VIVPSGKTSYADSVKG			46
2B12VH3	SYGGGFDY
			47
2B12VL1	RASQDILTWLA
			48
2B12VL2	AASSLQS				49
		2B12VL3	QQAIRFPLT				50
5A8VH1	YYRMY				55
		5A8VH2	SIYSSGGPTYYADSVKG				56
5A8VH3	DMGTGFWSGWGLGSDY
			57
5A8VL1	RASQGISSWLA			58
		5A8VL2	AASSLQS
	59
		5A8VL3	QQANSFPLT		60

Table 4: representational resisting-the Eph receptor antibody

Antibody/hybridoma	EphR	The ATCC numbering	Preservation date	Number of patent application
					Eph099B-102.147	EphA2	PTA-4572	2002-8-7	WO 03/094859
Eph099B208.261	EphA2	PTA-4573	2002-8-7	WO 03/094859
					Eph099B-210.248	EphA2	PTA-4574	2002-8-7	WO 03/094859
Eph099B-233.152	EphA2	PTA-5194	2003-5-12	WO 03/094859
					EA2	EphA2	PTA-4380	2003-5-22	WO 04/014292
EA5	EphA2	PTA-4381	2003-5-22	WO 04/014292
					EA44	EphA4	PTA-6044	2004-6-4	10/863,729
3F2	EphA2									11/203,251

[0139] the present invention also comprises and the ADC of 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8,5A8LX-13 or scFv EA44 or its antigen-binding fragment competition in conjunction with the Eph acceptor. Competition experiments is to be proficient in well-known to those having ordinary skill in the artly, and it can be used to identify this antibody-like. In concrete embodiment, analyze and can record by the ORIGEN that knows, the antibody of the present invention of 1 μ g/ml can prevent the Eph receptors bind of 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8,5A8LX-13 or scFv EA44 and the biotin-mark of 75%, 80%, 85% or 90% ORIGEN TAG mark.

[0140] the present invention also provides and has comprised the ADC that is proficient in framework region known to those skilled in the art. In one embodiment, the segment area of antibody of the present invention or its fragment is the people's or humanized.

[0141] the present invention includes the ADC of the amino acid sequence that contains 12G3H11,3F2, Eph099B-102.147, Eph099B-208.261, Eph099B-210.248, Eph099B-233.152, EA2, EA3, EA4, EA5,3F2,1C1,1F12,1H3,1D3,2B12,5A8LX-13 or scFv EA44, described amino acid sequence also has sudden change except any other replacement or change (for example, Fc replaces) in framework region or variable region. In one embodiment, the sudden change in these antibody is kept or has been strengthened affinity and/or the affinity of antibody for the Eph acceptor of their specific bindings. Be proficient in standard technique known to those skilled in the art (for example, immunity test) and can be used to detect antibody to the affinity of specific antigen.

[0142] the present invention includes normally separate, the coding specific binding is in the application of the nucleic acid molecules of the antibody moiety of the ADC of Eph acceptor. In concrete embodiment, the nucleic acid molecule encoding specific binding that separates is in the ADC of Eph acceptor, and described ADC has the amino acid sequence of 12G3H11, the Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8,5A8LX-13 or the scFv EA44 that contain one or more Fc replacements. In other embodiments, the nucleic acid molecule encoding specific binding that separates is in the ADC of Eph acceptor, and the VH domain that described ADC comprises has the amino acid sequence of the VH domain of 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8,5A8LX-13 or scFv EA44. In other embodiments, the nucleic acid molecule encoding specific binding that separates is in the ADC of Eph acceptor, and the VL domain that described antibody comprises has the amino acid sequence of the VL domain of 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8,5A8LX-13 or scFv EA44.

[0143] the present invention includes the coding specific binding in the application of the nucleic acid molecules of the separation of the ADC of Eph acceptor, the VH CDR that described ADC comprises have listed any VH CDR in table 2 or 3 and/or be derived from table 4 or 6 in the amino acid sequence of VH CDR of listed any heavy chain of antibody. Specifically, the present invention includes the coding specific binding in the application of the nucleic acid molecules of the separation of the ADC of Eph acceptor, 1,2 or the more VH CDR that described antibody comprises have listed any VH CDR in table 2 or 3 and/or be derived from table 4 or 6 in the amino acid sequence of VH CDR of listed any heavy chain of antibody.

[0144] the present invention includes the coding specific binding in the application of the nucleic acid molecules of the separation of the ADC of Eph acceptor, the VL CDR that described ADC comprises has listed any VL CDR in table 2 or 3 and/or source derived from the amino acid sequence of the VL CDR of listed any light chain of antibody in table 4 or 6. Specifically, the present invention includes the coding specific binding in the application of the nucleic acid molecules of the separation of the ADC of Eph acceptor, 1,2 or the more VL CDR that described antibody comprises have listed any VL CDR in table 2 or 3 and/or be derived from table 4 or 6 in the amino acid sequence of VL CDR of listed any light chain of antibody.

[0145] the present invention includes specific binding in the application of the ADC of Eph acceptor, described ADC comprises specific binding as herein described in the derivative of the VH of Eph acceptor domain, VH CDR, VL domain or VL CDR. Being proficient in standard technique known to those skilled in the art can be used to (for example introduce sudden change in the nucleotide sequence of code book invention antibody, add, delete and/or replace), these technology comprise, for example, the mutagenesis of direct mutagenesis and PCR-mediation is generally used for producing 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor. In one embodiment, VH and/or VL CDR derivative comprise less than 25 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors with respect to original VH and/or VL CDR, less than 20 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, less than 15 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, less than 10 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, less than 5 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, less than 4 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, less than 3 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, or less than 2 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors. In other embodiments, VH and/or VL CDR derivative are at the nonessential amino acid residue of one or more predictions (namely, be not that the antibody specific binding is in the key amino acid residue of Eph acceptor) on carried out conservative amino acid replacement (for example, the same). Perhaps, for example can be on all or part of VH and/or VL CDR coded sequence introduce at random sudden change by saturation mutagenesis, and the biologically active that can screen the gained mutant keeps activated mutant to identify. The antibody that can express coding after the mutagenesis also can be identified the activity of antibody.

[0146] present invention resides in the ADC that has 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8,5A8LX-13 or scFv EA44 that one or more additional amino acid residues replace in variable light chain (VL) domain and/or variable heavy chain (VH) domain. The present invention also is included in the ADC of 12G3H11, the Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8,5A8LX-13 or the scFv EA44 that have one or more additional amino acid residues replacements among one or more VL CDR and/or the one or more VH CDR. For example, can detect in vitro and in vivo by at 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8, the VH domain of the ADC of 5A8LX-13 or scFv EA44, VH CDR, the antibody that introduce to replace among VL domain and/or the VL CDR and produce in conjunction with the ability of Eph acceptor (by, for example, immunity test includes but not limited to ELISA and BIAcore) or their mediations, prevention, treatment, control or improve the ability of cancer or one or more cancer symptoms.

[0147] the present invention also comprises specific binding in the ADC of at least a Eph acceptor or the application of its fragment, the variable heavy chain that described ADC comprises and/or the amino acid sequence of variable light chain and 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8, the variable heavy chain of 5A8LX-13 or scFv EA44 and/or the amino acid sequence of light chain have at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homogeny. The present invention also comprises specific binding in the ADC of at least a Eph acceptor or the application of its fragment, the variable heavy chain that described ADC comprises and/or the amino acid sequence of variable light chain and 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8, the variable heavy chain of 5A8LX-13 or scFv EA44 and/or the amino acid sequence of light chain have at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homogeny. The present invention comprises that also specific binding is in the ADC of at least a Eph acceptor or the application of its fragment, the amino acid sequence of one or more CDR that described antibody or antibody fragment comprise and 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8, the amino acid sequence of one or more CDR of 5A8LX-13 or scFv EA44 has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homogeny. The present invention comprises that also specific binding is in the ADC of at least a Eph acceptor or the application of its fragment, the amino acid sequence of one or more CDR that described antibody or antibody fragment comprise and 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8, the amino acid sequence of one or more CDR of 5A8LX-13 or scFv EA44 has at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homogeny. Can be by being proficient in any method known to those skilled in the art, comprise that BLAST protein retrieves to measure two seed amino acid sequence homogeny percentages.

[0148] the present invention also comprises specific binding in the ADC of at least a Eph acceptor or the application of its fragment, and described ADC is by nucleotide sequence coded with the nucleotide sequence hybridization of 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8,5A8LX-13 or scFv EA44 under stringent condition. In other embodiments, the present invention includes specific binding in the ADC of Eph acceptor or its fragment, one or more CDR that described ADC comprises are by nucleotide sequence coded with the nucleotide sequence hybridization of one or more CDR of 12G3H11, Eph099B-102.147, B208, B210, B233, EA2, EA3, EA4, EA5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8,5A8LX-13 or scFv EA44 under stringent condition. Stringent hybridization condition includes but not limited to: hybridize with the DNA that is combined on the filter membrane in 6 * sodium chloride/sodium citrate (SSC), wash one or many at about 50-65 ℃ with 0.2 * SSC/0.1%SDS then for about 45 ℃; The height stringent condition be for example about 45 ℃ in 6 * SSC with the DNA hybridization that is combined on the filter membrane, then about 60 ℃ with 0.1 * SSC/0.2% SDS washing one or many; Or any other stringent hybridization condition well known by persons skilled in the art (referring to, Ausubel for example, F.M. wait volume, 1989, " up-to-date molecular biology method " (Current Protocols in Molecular Biology), the first volume, (the Green Publishing Associates of Green's cooperation in publishing company, Inc.) and (the John Wiley﹠Sons of John Willie father and son publishing company, Inc.), New York, 6.3.1 page or leaf-6.3.6 page or leaf and 2.10.3 page or leaf).

[0149] the invention provides specific binding in the antibody drug conjugates of EphA2 polypeptide. The present invention also provides the antibody in conjunction with people EphA2 polypeptide, mouse EphA2 polypeptide and rat EphA2 polypeptide. In some embodiments, single antibody cloning can be in conjunction with the EphA2 polypeptide of people, Mouse and rat. In other embodiments, single antibody cloning is only in conjunction with people EphA2, or only in conjunction with mouse EphA2, or only in conjunction with rat EphA2. Again in other embodiments, single antibody cloning is in conjunction with the EphA2 of people and mouse, or in conjunction with the EphA2 of people and rat, or in conjunction with the EphA2 of rat and mouse.

[0150] specifically, the invention provides specific binding in following antibody or ADC:12G3H11 or its antigen-binding fragment of EphA2 polypeptide, Eph099B-102.147 or its Fab, B208 or its Fab, B210 or its Fab, B233 or its Fab, EA2 or its Fab, EA3 or its Fab, EA4 or its Fab, EA5 or its Fab, 10C12 or its Fab, 4H5 or its Fab, 10G9 or its Fab, 3F2 or its Fab, 5A8LX-13 or its Fab, scFv EA44 or its Fab, 1C1 or its antigen-binding fragment, 1F12 or its antigen-binding fragment, 1H3 or its antigen-binding fragment, 1D3 or its antigen-binding fragment, 2B 12 or its antigen-binding fragment, and 5A8 or its antigen-binding fragment. In one embodiment, specific binding is 1C1 or its antigen-binding fragment (for example, one or more CDR of 1C1) in the antibody of EphA2 polypeptide. In other embodiments, specific binding is 1F12 or its antigen-binding fragment (for example, one or more CDR of CDR1F12) in the antibody of EphA2 polypeptide. In further embodiment, specific binding is 1H3 or its antigen-binding fragment (for example, one or more CDR of 1H3) in the antibody of EphA2 polypeptide. In other embodiments, specific binding is 1D3 or its antigen-binding fragment (for example, one or more CDR of 1D3) in the antibody of EphA2 polypeptide. Again in another embodiment, specific binding is 2B12 or its antigen-binding fragment (for example, one or more CDR of 2B12) in the antibody of EphA2 polypeptide. In further embodiment, specific binding is 5A8 or its antigen-binding fragment (for example, one or more CDR of 5A8) in the antibody of EphA2 polypeptide.

[0151] the invention provides antibody or the ADC of specific binding EphA2 polypeptide, the VH domain that described antibody comprises has 12G3H11 (Fig. 1,2; SEQ ID NO.:165), B233 (Fig. 1,2; SEQ ID NO.:87), B208 (Fig. 1,2; SEQ ID NO.:95), B210 (Fig. 1,2), G5 (Fig. 1,2; SEQ ID NO.:103), 10C12 (Fig. 6; SEQ ID NO.:140), 4H5 (Fig. 4; SEQ ID NO.:138), 10G9 (Fig. 4), 3F2 (Fig. 3; SEQ ID NO.:63), 1C1 (Fig. 7 A and 8; SEQ ID NO.:3), 1F12 (Fig. 7 A and 9; SEQ ID NO.:13), 1H3 (Fig. 7 A and 10; SEQ ID NO.:23), 1D3 (Fig. 7 A and 11; SEQ ID NO.:33), 2B12 (Fig. 7 A and 12; SEQ ID NO.:43) or 5A8 (Fig. 7 A and 13; The amino acid sequence of VH domain SEQ ID NO.:53).

[0152] the invention provides specific binding in the antibody of EphA2 polypeptide, the VH CDR that described antibody comprises has the amino acid sequence of listed arbitrary VHCDR in following table 2 or 3. Specifically, the invention provides specific binding in the antibody of EphA2 polypeptide, described antibody comprises (perhaps, being made of it) 1,2,3,4,5 or more VH CDR with the amino acid sequence of listed any VH CDR in following table 2 or 3.

In [0153] one embodiment, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:5, the VH CDR1 of 15,25,35,45,55 or 65 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:6, the VH CDR2 of 16,26,36,46,56 or 66 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:7, the VH CDR3 of 17,27,37,47,57 or 67 amino acid sequence.

[0154] in other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:5, the VH CDR1 of 15,25,35,45,55 or 65 amino acid sequence and have the VH CDR2 of SEQ ID NO.:6,16,26,36,46,56 or 66 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:5, the VH CDR1 of 15,25,35,45,55 or 65 amino acid sequence and have the VH CDR3 of SEQ ID NO.:7,17,27,37,47,57 or 67 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:6, the VH CDR2 of 16,26,36,46,56 or 66 amino acid sequence and have the VH CDR3 of SEQ ID NO.:7,17,27,37,47,57 or 67 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:5, the VH CDR1 of 15,25,35,45,55 or 65 amino acid sequence, have the VH CDR2 of SEQ ID NO.:6,16,26,36,46,56 or 66 amino acid sequence and have the VH CDR3 of SEQ ID NO.:7,17,27,37,47,57 or 67 amino acid sequence.

[0155] the invention provides specific binding in the antibody of EphA2 polypeptide, the VL domain that described antibody comprises has 12G3H11 (Fig. 1,2; SEQ ID NO.:166), B233 (Fig. 1,2; SEQ ID NO.:88), B208 (Fig. 1,2; SEQ ID NO.:96), B210 (Fig. 1,2), G5 (Fig. 3; SEQ ID NO.:104), 10C12 (Fig. 6; SEQ ID NO.:142), 4H5 (Fig. 4; SEQ ID NO.:137), 10G9 (Fig. 4), 3F2 (Fig. 3; SEQ ID NO.:64), 1C1 (Fig. 7 B and 8; SEQ ID NO.:4), 1F12 (Fig. 7 B; SEQ ID NO.:14), 1H3 (Fig. 7 B and 10; SEQ ID NO.:24), 1D3 (Fig. 7 B and 11; SEQ ID NO.:34), 2B12 (Fig. 7 B and 12; SEQ ID NO.:44) or 5A8 (Fig. 7 B and 13; The amino acid sequence of VL domain SEQ ID NO.:54).

[0156] the present invention also provides the antibody of specific binding in the EphA2 polypeptide, and the VL CDR that described antibody comprises has the amino acid sequence of listed arbitrary VL CDR in following table 2 or 3. Specifically, the invention provides specific binding in the antibody of EphA2 polypeptide, described antibody comprises (perhaps, consisted of or substantially be made of it by it) 1,2,3 or more VL CDR with the amino acid sequence of listed any VL CDR in following table 2 or 3. In one embodiment, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:8, the VL CDR1 of 18,28,38,48,58 or 68 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:9, the VL CDR2 of 19,29,39,49,59 or 69 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:10, the VL CDR3 of 20,30,40,50,60 or 70 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:8, the VL CDR1 of 18,28,38,48,58 or 68 amino acid sequence and have the VL CDR2 of SEQ ID NO.:9,19,29,39,49,59 or 69 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:8, the VL CDR1 of 18,28,38,48,58 or 68 amino acid sequence and have the VL CDR3 of SEQ ID NO.:10,20,30,40,50,60 or 70 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:9, the VL CDR2 of 19,29,39,49,59 or 69 amino acid sequence and have the VL CDR3 of SEQ ID NO.:10,20,30,40,50,60 or 70 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:8, the VL CDR1 of 18,28,38,48,58 or 68 amino acid sequence, VL CDR2 with SEQ ID NO.:9, amino acid sequence of 19,29,39,49,59 or 69, with the VL CDR3 with SEQ ID NO.:10, amino acid sequence of 20,30,40,50,60 or 70, as the part of this antibody.

[0157] the invention provides specific binding in the antibody of EphA2 polypeptide, described antibody comprises the VH domain described here of being combined with VL domain described here or other known VL domains. The present invention also provides the antibody of specific binding in the EphA2 polypeptide, and described antibody comprises the VL domain described here of being combined with VH domain described here or other known VH domains.

[0158] the invention provides specific binding in the antibody of EphA2 polypeptide, described antibody comprises one or more VHCDR listed in table 2 or 3 and one or more VLCDR. Specifically, the invention provides specific binding in the antibody of EphA2 polypeptide, described antibody comprises (perhaps, consisted of or substantially be made of it by it): VH CDR1 and VL CDR1; VH CDR1 and VL CDR2; VH CDR1 and VL CDR3; VH CDR2 and VL CDR1; VH CDR2 and VL CDR2; VH CDR2 and VL CDR3; VH CDR3 and VH CDR1; VH CDR3 and VL CDR2; VH CDR3 and VL CDR3; VH1CDR1, VH CDR2 and VL CDR1; VH CDR1, VH CDR2 and VL CDR2; VH CDR1, VH CDR2 and VL CDR3; VH CDR2, VHCDR3 and VL CDR1, VH CDR2, VH CDR3 and VL CDR2; VH CDR2, VH CDR2 and VL CDR3; VH CDR1, VL CDR1 and VL CDR2; VH CDR1, VL CDR1 and VL CDR3; VH CDR2, VL CDR1 and VL CDR2; VH CDR2, VL CDR1 and VL CDR3; VH CDR3, VL CDR1 and VL CDR2; VH CDR3, VL CDR1 and VL CDR3; VH CDR1, VH CDR2, VH CDR3 and VL CDR1; VH CDR1, VH CDR2, VH CDR3 and VL CDR2; VH CDR1, VH CDR2, VH CDR3 and VL CDR3; VH CDR1, VH CDR2, VL CDR1 and VL CDR2; VH CDR1, VH CDR2, VL CDR1 and VL CDR3; VH CDR1, VH CDR3, VL CDR1 and VL CDR2; VH CDR1, VH CDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VL CDR1 and VL CDR2; VH CDR2, VH CDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VL CDR2 and VL CDR3; VH CDR1, VH CDR2, VH CDR3, VL CDR1 and VL CDR2; VH CDR1, VH CDR2, VH CDR3, VL CDR1 and VL CDR3; VH CDR1, VH CDR2, VL CDR1, VL CDR2 and VL CDR3; VH CDR1, VH CDR3, VL CDR1, VL CDR2 and VL CDR3; VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3; Perhaps go up any combination of listed VH CDR and VL CDR in table 2 or 3.

In [0159] one embodiment, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:5, the VH CDR1 of 15,25,35,45,55 or 65 amino acid sequence and have the VL CDR1 of SEQ ID NO.:8,18,28,38,48,58 or 68 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:5, the VH CDR1 of 15,25,35,45,55 or 65 amino acid sequence and have the VL CDR2 of SEQ ID NO.:9,19,29,39,49,59 or 69 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:5, the VH CDR1 of 15,25,35,45,55 or 65 amino acid sequence and have the VL CDR3 of SEQ ID NO.:10,20,30,40,50,60 or 70 amino acid sequence.

In [0160] one embodiment, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:6, the VH CDR2 of 16,26,36,46,56 or 66 amino acid sequence and have the VL CDR1 of SEQ ID NO.:8,18,28,38,48,58 or 68 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:6, the VH CDR2 of 16,26,36,46,56 or 66 amino acid sequence and have the VL CDR2 of SEQ ID NO.:9,19,29,39,49,59 or 69 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:6, the VH CDR2 of 16,26,36,46,56 or 66 amino acid sequence and have the VL CDR3 of SEQ ID NO.:10,20,30,40,50,60 or 70 amino acid sequence.

In [0161] one embodiment, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:7, the VH CDR3 of 17,27,37,47,57 or 67 amino acid sequence and have the VL CDR1 of SEQ ID NO.:8,18,28,38,48,58 or 68 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:7, the VH CDR3 of 17,27,37,47,57 or 67 amino acid sequence and have the VL CDR2 of SEQ ID NO.:9,19,29,39,49,59 or 69 amino acid sequence. In other embodiments, specific binding comprise in the antibody of EphA2 polypeptide have SEQ ID NO.:7,17,27,37,47,57 or 67 amino acid sequence VH CDR3 and have the VL CDR3 of SEQ ID NO.:10,20,30,40,50,60 or 70 amino acid sequence.

[0162] the invention provides specific binding in the antibody of EphA2 polypeptide, described antibody is by the nucleic acid sequence encoding of the nucleotide sequence with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 or its antigen-binding fragment. In concrete embodiment, the VH domain that specific binding comprises in the antibody of EphA2 polypeptide is by the nucleic acid sequence encoding of the nucleotide sequence of the VH domain with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8. In other embodiments, the VL domain that comprises in the antibody of EphA2 polypeptide of specific binding is by the nucleic acid sequence encoding of the nucleotide sequence of the VL domain with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8. In other embodiments, the VH domain that comprises in the antibody of EphA2 polypeptide of specific binding and VL domain are by the nucleic acid sequence encoding of the nucleotide sequence of the VH domain with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 and VL domain.

[0163] in other embodiments, the VH CDR that comprises in the antibody of EphA2 polypeptide of specific binding is by the nucleic acid sequence encoding of the nucleotide sequence of the VH CDR with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8. In other embodiments, the VL CDR that comprises in the antibody of EphA2 polypeptide of specific binding is by the nucleic acid sequence encoding of the nucleotide sequence of the VL CDR with 2G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8. In other embodiments, the VH CDR that comprises in the antibody of EphA2 polypeptide of specific binding and VL CDR are by the nucleic acid sequence encoding of the nucleotide sequence of the VH CDR with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 and VL CDR.

[0164] the invention provides a kind of normally isolating, coding specificity and be incorporated into the nucleic acid molecule of the antibody of the present invention of EphA2 polypeptide.Specifically, the invention provides the isolated nucleic acid molecule that a kind of specificity of encoding is incorporated into the antibody of EphA2 polypeptide, described antibody has the aminoacid sequence of 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 or its antigen-binding fragment.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody has the aminoacid sequence of 1C1.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody has the aminoacid sequence of 1F12.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody has the aminoacid sequence of 1H3.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody has the aminoacid sequence of 1D3.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody has the aminoacid sequence of 2B12.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody has the aminoacid sequence of 5A8.

[0165] the invention provides the isolated nucleic acid molecule that a kind of specificity of encoding is incorporated into the antibody of EphA2 polypeptide, described antibody comprises the VH structural domain that (perhaps, constituted or be made of it substantially by it) has the VH structural domain aminoacid sequence of 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VH structural domain of the aminoacid sequence of the VH structural domain with 1C1.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VH structural domain of the aminoacid sequence of the VH structural domain with 1F12.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VH structural domain of the aminoacid sequence of the VH structural domain with 1H3.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VH structural domain of the aminoacid sequence of the VH structural domain with 1D3.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VH structural domain of the aminoacid sequence of the VH structural domain with 2B12.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VH structural domain of the aminoacid sequence of the VH structural domain with 5A8.

[0166] the invention provides the isolated nucleic acid molecule that a kind of specificity of encoding is incorporated into the antibody of EphA2 polypeptide, described antibody comprises the VH CDR that (perhaps, constituted or be made of it substantially by it) has the aminoacid sequence of listed any VH CDR in last table 2 or 3.Specifically, the invention provides the isolated nucleic acid molecule that a kind of specificity of encoding is incorporated into the antibody of EphA2 polypeptide, described antibody comprises 1,2,3,4,5 or more a plurality of VH CDR with the aminoacid sequence of listed any VH CDR in last table 2 or 3.In one embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VH CDR1 of the aminoacid sequence with listed VH CDR1 in last table 2 or 3.In other embodiments, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VH CDR2 of the aminoacid sequence with listed VH CDR2 in last table 2 or 3.In other embodiments, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VH CDR3 of the aminoacid sequence with listed VH CDR3 in last table 2 or 3.

[0167] the invention provides the isolated nucleic acid molecule that a kind of specificity of encoding is incorporated into the antibody of EphA2 polypeptide, described antibody comprises the VL structural domain of aminoacid sequence that (perhaps, constituted or be made of it substantially by it) has the VL structural domain of 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VL structural domain of the aminoacid sequence of the VL structural domain with 1C1.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VL structural domain of the aminoacid sequence of the VL structural domain with 1F12.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VL structural domain of the aminoacid sequence of the VL structural domain with 1H3.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VL structural domain of the aminoacid sequence of the VL structural domain with 1D3.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VL structural domain of the aminoacid sequence of the VL structural domain with 2B12.In concrete embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VL structural domain of the aminoacid sequence of the VL structural domain with 5A8.

[0168] the present invention also provides a kind of specificity of encoding to be incorporated into the isolated nucleic acid molecule of the antibody of EphA2 polypeptide, described antibody comprises the VL CDR that (perhaps, constituted or be made of it substantially by it) has the aminoacid sequence of listed any VL CDR in last table 2 or 3.Specifically, the invention provides the isolated nucleic acid molecule that a kind of specificity of encoding is incorporated into the antibody of EphA2 polypeptide, described antibody comprises 1,2,3 or more a plurality of VL CDR with the aminoacid sequence of listed any VL CDR in last table 2 or 3.In one embodiment, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VL CDR1 of the aminoacid sequence with listed VH CDR1 in last table 2 or 3.In other embodiments, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VL CDR2 of the aminoacid sequence with listed VHCDR2 in last table 2 or 3.In other embodiments, isolated nucleic acid molecule coding specificity is incorporated into the antibody of EphA2 polypeptide, and described antibody comprises the VL CDR3 of the aminoacid sequence with listed VH CDR3 in last table 2 or 3.

[0169] the invention provides the nucleic acid molecule that the coding specificity is incorporated into the antibody of EphA2 polypeptide, described antibody comprises one or more VH CDR listed in table 2 or 3 and one or more VL CDR.Specifically, the invention provides the isolated nucleic acid molecule that a kind of specificity of encoding is incorporated into the antibody of EphA2 polypeptide, described antibody comprises (perhaps, constituted or be made of it substantially by it) VH CDR1 and VL CDR1; VH CDR1 and VL CDR2; VH CDR1 and VL CDR3; VH CDR2 and VL CDR1; VH CDR2 and VL CDR2; VH CDR2 and VL CDR3; VH CDR3 and VH CDR1; VH CDR3 and VL CDR2; VH CDR3 and VL CDR3; VH1 CDR1, VH CDR2 and VL CDR1; VH CDR1, VH CDR2 and VL CDR2; VH CDR1, VH CDR2 and VL CDR3; VH CDR2, VH CDR3 and VL CDR1, VH CDR2, VH CDR3 and VL CDR2; VH CDR2, VH CDR2 and VL CDR3; VH CDR1, VL CDR1 and VL CDR2; VH CDR1, VL CDR1 and VL CDR3; VH CDR2, VL CDR1 and VL CDR2; VH CDR2, VL CDR1 and VL CDR3; VH CDR3, VL CDR1 and VL CDR2; VH CDR3, VL CDR1 and VL CDR3; VH CDR1, VH CDR2, VH CDR3 and VL CDR1; VH CDR1, VH CDR2, VH CDR3 and VL CDR2; VH CDR1, VH CDR2, VH CDR3 and VL CDR3; VH CDR1, VH CDR2, VL CDR1 and VL CDR2; VH CDR1, VH CDR2, VL CDR1 and VL CDR3; VH CDR1, VH CDR3, VL CDR1 and VL CDR2; VH CDR1, VH CDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VL CDR1 and VL CDR2; VH CDR2, VH CDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VL CDR2 and VL CDR3; VH CDR1, VH CDR2, VH CDR3, VL CDR1 and VL CDR2; VH CDR1, VH CDR2, VH CDR3, VL CDR1 and VL CDR3; VH CDR1, VH CDR2, VL CDR1, VL CDR2 and VL CDR3; VH CDR1, VH CDR3, VL CDR1, VL CDR2 and VL CDR3; VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3; Or go up the arbitrary combination of listed VH CDR and VL CDR in the table 2 or 3.

[0170] below the further embodiment of the present invention, and number consecutively:

1. EphA2 antibody or ADC, its comprise have 12G3H11, variable heavy chain (VH) structural domain of the aminoacid sequence of the VH structural domain of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8, wherein, described antibodies specific is incorporated into the EphA2 polypeptide.

2. EphA2 antibody or ADC, its comprise have 12G3H11, variable light chain (VL) structural domain of the aminoacid sequence of the VL structural domain of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8, wherein, described antibodies specific is incorporated into the EphA2 polypeptide.

3. embodiment 1 described antibody or ADC, it also comprises the VL structural domain of the aminoacid sequence of the VL structural domain with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

4. EphA2 antibody or ADC, its comprise have 12G3H11, the complementary determining region (CDR) of the aminoacid sequence of the CDR of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8, wherein, described antibody or ADC specificity are incorporated into the EphA2 polypeptide.

5. embodiment 4 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VH CDR of the aminoacid sequence of the VH CDR of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

6. embodiment 4 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR of the aminoacid sequence of the VL CDR of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

7. embodiment 5 described antibody or ADC also comprise the VL CDR of the aminoacid sequence of the VL CDR with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

8. embodiment 5 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VH CDR1 of the aminoacid sequence of the VH CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

9. embodiment 5 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VH CDR2 of the aminoacid sequence of the VH CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

10. embodiment 5 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VH CDR3 of the aminoacid sequence of the VH CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

11. embodiment 8 described antibody or ADC, wherein, described antibody or ADC also comprise the VH CDR2 of the aminoacid sequence of the VH CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

12. embodiment 8 described antibody or ADC, wherein, described antibody or ADC also comprise the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

13. embodiment 9 described antibody or ADC, wherein, described antibody or ADC also comprise the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

14. embodiment 11 described antibody or ADC, wherein, described antibody or ADC also comprise the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

15. embodiment 7 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VH CDR1 of the aminoacid sequence of the VH of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

16. embodiment 7 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VH CDR2 of the aminoacid sequence of the VH CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

17. embodiment 7 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VH CDR3 of the aminoacid sequence of the VH CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

18. embodiment 15 described antibody or ADC, wherein, described antibody or ADC also comprise the VH CDR2 of the aminoacid sequence of the VH CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

19. embodiment 15 described antibody or ADC, wherein, described antibody or ADC also comprise the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

20. embodiment 16 described antibody or ADC, wherein, described antibody or ADC also comprise the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

21. embodiment 18 described antibody or ADC, wherein, described antibody or ADC also comprise the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

22. embodiment 6 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

23. embodiment 6 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

24. embodiment 6 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

25. embodiment 22 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

26. embodiment 22 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

27. embodiment 23 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

28. embodiment 25 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

29. embodiment 7 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

30. embodiment 7 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

31. embodiment 7 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

32. embodiment 29 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

33. embodiment 29 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

34. embodiment 30 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

35. embodiment 32 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

36. embodiment 15 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

37. embodiment 15 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

38. embodiment 15 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

39. embodiment 36 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

40. embodiment 36 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

41. embodiment 37 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

42. embodiment 39 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

43. embodiment 16 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

44. embodiment 16 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

45. embodiment 16 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

46. embodiment 43 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

47. embodiment 43 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

48. embodiment 44 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

49. embodiment 46 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

50. embodiment 17 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

51. embodiment 17 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

52. embodiment 17 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

53. embodiment 50 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

54. embodiment 50 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

55. embodiment 51 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

56. embodiment 53 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

57. embodiment 18 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

58. embodiment 18 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

59. embodiment 18 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

60. embodiment 57 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

61. embodiment 57 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

62. embodiment 58 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

63. embodiment 60 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

64. embodiment 19 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

65. embodiment 19 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

66. embodiment 19 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

67. embodiment 64 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

68. embodiment 64 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

69. embodiment 65 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

70. embodiment 67 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

71. embodiment 20 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

72. embodiment 20 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

73. embodiment 20 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

74. embodiment 71 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

75. embodiment 71 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

76. embodiment 72 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

77. embodiment 74 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

78. embodiment 21 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

79. embodiment 21 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

80. embodiment 21 described antibody or ADC, wherein, described antibody or ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

81. embodiment 78 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

82. embodiment 78 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

83. embodiment 79 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

84. embodiment 81 described antibody or ADC, wherein, described antibody or ADC also comprise the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

[0171] the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody comprises the derivative of VH structural domain, VH CDR, VL structural domain or VL CDR that specificity described in the literary composition is incorporated into the EphA2 polypeptide.Can adopt standard technique well known by persons skilled in the art will suddenly change (for example, disappearance, add and/or replace) introduce the nucleotide sequence of code book invention antibody, these technology for example comprise: the site-directed mutagenesis and the PCR-mediated mutagenesis that cause aminoacid replacement.Preferably, this derivative comprises with respect to initial molecule and is less than 25 aminoacid replacement, is less than 20 aminoacid replacement, is less than 15 aminoacid replacement, is less than 10 aminoacid replacement, is less than 5 aminoacid replacement, is less than 4 aminoacid replacement, is less than 3 aminoacid replacement or is less than 2 aminoacid replacement.In concrete embodiment, this derivative carries out conservative amino acid and replaces on the non-essential amino acid residue (that is, not being the key amino acid residue that antibodies specific is incorporated into the EphA2 polypeptide) of one or more predictions." conservative amino acid replacement " is the situation with the amino-acid residue substituted amino acid residue with the similar side chain of electric charge.This area defines the amino-acid residue family with the similar side chain of electric charge.These families comprise that the amino acid with basic side chain is (as Methionin, arginine, Histidine), amino acid with acid side-chain is (as aspartic acid, L-glutamic acid), amino acid with uncharged polar side chain is (as glycine, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), amino acid with non-polar sidechain is (as L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), amino acid with β-branched building block is (as Threonine, Xie Ansuan, Isoleucine) and the amino acid with aromatic side chains (as tyrosine, phenylalanine, tryptophane, Histidine).Perhaps, for example can be on all or part of encoding sequence introduce sudden change at random, and the biological activity that can screen the gained mutant remains with active mutant with evaluation by saturation mutagenesis.The antibody that can express coding after the mutagenesis also can be identified the activity of antibody.

[0172] the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody is included in the aminoacid sequence of 12G3H11, the B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or the 5A8 that have one or more amino-acid residues replacements in variable light chain (VL) structural domain and/or variable heavy chain (VH) structural domain.The antibody that the present invention also provides specificity to be incorporated into the EphA2 polypeptide, described antibody are included in the aminoacid sequence of 12G3H11, the B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or the 5A8 that have one or more amino-acid residues replacements among one or more VL CDR and/or the one or more VH CDR.The antibody that the present invention also provides specificity to be incorporated into the EphA2 polypeptide, described antibody are included in has 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 that one or more amino-acid residues replace or the aminoacid sequence of its VH and/or VL structural domain in one or more VH frameworks and/or the one or more VL framework.For example, can in external and/or body, detect the ability of passing through in the VH of 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 structural domain, VH CDR, VL structural domain, VL CDR and/or framework, to introduce ability or their inhibition that replaces the antibodies EphA2 polypeptide that produces or the ability that reduces the EphA2 receptor activation or their activation EphA2.

[0173] in concrete embodiment, the antibody that specificity is incorporated into the EphA2 polypeptide is included in stringent condition, the height stringent condition, or other stringent hybridization conditions are following and coding 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3, the nucleotide sequence of the nucleotide sequence hybridization of 2B12 or 5A8 or its antigen-binding fragment, described stringent condition for example, hybridize with the DNA that is incorporated into filter membrane in 6X sodium chloride/sodium citrate (SSC) at about 45 ℃, wash one or many at about 50-65 ℃ with 0.2X SSC/0.1%SDS then, described height stringent condition for example, about 45 ℃ in 6X SSC be incorporated into the nucleic acid hybridization of filter membrane, then about 68 ℃ with 0.1X SSC/0.2%SDS washing one or many, described other stringent hybridization conditions be proficient in known to those skilled in the art (referring to, for example, Ausubel, F.M. wait volume, 1989, " up-to-date molecular biology method ", the first roll, Green publishes cooperative venture and John Willie father and son publishing company, New York, 6.3.1 page or leaf-6.3.6 page or leaf and 2.10.3 page or leaf).

[0174] in other embodiments, the antibody that specificity is incorporated into the EphA2 polypeptide comprises the aminoacid sequence of VH structural domain or the aminoacid sequence of VL structural domain, and described aminoacid sequence by described in the text stringent condition down or be proficient under other stringent hybridization conditions known to those skilled in the art nucleotide sequence coded with the nucleotide sequence hybridization of the VH of coding 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 or VL structural domain.In other embodiments, the antibody that specificity is incorporated into the EphA2 polypeptide comprises the aminoacid sequence of VH structural domain and the aminoacid sequence of VL structural domain, and described aminoacid sequence by described in the text stringent condition down or be proficient under other stringent hybridization conditions known to those skilled in the art nucleotide sequence coded with the nucleotide sequence hybridization of the VH of coding 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B 12 or 5A8 and VL structural domain.In other embodiments, the antibody that specificity is incorporated into the EphA2 polypeptide comprises the aminoacid sequence of VH CDR or the aminoacid sequence of VL CDR, described aminoacid sequence by described in the text stringent condition down or under being proficient in other stringent hybridization conditions known to those skilled in the art with coding on nucleotide sequence hybridization nucleotide sequence coded of listed arbitrary VH CDR or VL CDR in the table 2 or 3.In other embodiments, the antibody that specificity is incorporated into the EphA2 polypeptide comprises the aminoacid sequence of VH CDR and the aminoacid sequence of VL CDR, described aminoacid sequence by described in the text stringent condition down or under being proficient in other stringent hybridization conditions known to those skilled in the art with coding on the nucleotide sequence hybridization of listed arbitrary VL CDR nucleotide sequence coded in listed arbitrary VHCDR and the last table 2 or 3 in the table 2 or 3.

[0175] in other embodiments, the invention provides the antibody that a specific specificity is incorporated into the EphA2 polypeptide, described antibody comprises the nucleotide sequence coded VH structural domain and/or the VL structural domain of hybridizing with the VH structural domain of 1C1 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 1 and 2) by under stringent condition.In other embodiments, the invention provides the antibody that a specific specificity is incorporated into the EphA2 polypeptide, described antibody comprises the nucleotide sequence coded VH structural domain and/or the VL structural domain of hybridizing with the VH structural domain of 1F12 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 11 and 12) by under stringent condition.In other embodiments, the invention provides the antibody that a specific specificity is incorporated into the EphA2 polypeptide, described antibody comprises the nucleotide sequence coded VH structural domain and/or the VL structural domain of hybridizing with the VH structural domain of 1H3 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 21 and 22) by under stringent condition.In other embodiments, the invention provides the antibody that a specific specificity is incorporated into the EphA2 polypeptide, described antibody comprises the nucleotide sequence coded VH structural domain and/or the VL structural domain of hybridizing with the VH structural domain of 1D3 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 31 and 32) by under stringent condition.In other embodiments, the invention provides the antibody that a specific specificity is incorporated into the EphA2 polypeptide, described antibody comprises the nucleotide sequence coded VH structural domain and/or the VL structural domain of hybridizing with the VH structural domain of 2B12 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 41 and 42) by under stringent condition.In other embodiments, the invention provides the antibody that a specific specificity is incorporated into the EphA2 polypeptide, described antibody comprises the nucleotide sequence coded VH structural domain and/or the VL structural domain of hybridizing with the VH structural domain of 5A8 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 51 and 52) by under stringent condition.

[0176] in other embodiments, the invention provides the antibody that a specific specificity is incorporated into the EphA2 polypeptide, described antibody comprises the nucleotide sequence coded VH CDR and/or the VL CDR of hybridizing with the VH CDR of 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 and/or the nucleotide sequence of VL CDR (Fig. 1-13) by under stringent condition.

[0177] in concrete embodiment, the aminoacid sequence that the antibody that specificity is incorporated into the EphA2 polypeptide comprises with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 or its antigen-binding fragment has at least 35%, preferred at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical aminoacid sequence.In other embodiments, the VH structural domain that the antibody that specificity is incorporated into the EphA2 polypeptide comprises with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 has at least 35%, the aminoacid sequence of preferred at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical VH structural domain.In other embodiments, the VL structural domain that the antibody that specificity is incorporated into the EphA2 polypeptide comprises with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 has at least 35%, the aminoacid sequence of preferred at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical VL structural domain.

[0178] in other embodiments, the antibody that specificity is incorporated into the EphA2 polypeptide comprise with last table 2 or 3 in listed any VL CDR have at least 35%, the aminoacid sequence of preferred at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical one or more VL CDR.In other embodiments, the antibody that specificity is incorporated into the EphA2 polypeptide comprise with last table 2 or 3 in listed arbitrary VL CDR have at least 35%, the aminoacid sequence of preferred at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical one or more VL CDR.

[0179] in other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody is by showing at least 65% with the nucleotides sequence of coding 1C1, and preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% is identical nucleotide sequence coded.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody is by showing at least 65% with the nucleotides sequence of coding 1F12, and preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% is identical nucleotide sequence coded.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody is by showing at least 65% with the nucleotides sequence of coding 1H3, and preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% is identical nucleotide sequence coded.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody is by showing at least 65% with the nucleotides sequence of coding 1D3, and preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% is identical nucleotide sequence coded.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody is by showing at least 65% with the nucleotides sequence of coding 2B12, and preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% is identical nucleotide sequence coded.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody is by showing at least 65% with the nucleotides sequence of coding 5A8, and preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% is identical nucleotide sequence coded.

[0180] in other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody comprises by having 65% with the VH structural domain of 1C1 and/or the nucleotide sequence of VL structural domain (SEQ IDNO is respectively 1 and 2) at least, preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical nucleotide sequence coded VH structural domain and/or VL structural domain.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody comprises by having 65% with the VH structural domain of 1F12 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 11 and 12) at least, preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical nucleotide sequence coded VH structural domain and/or VL structural domain.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody comprises by having 65% with the VH structural domain of 1H3 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 21 and 22) at least, preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical nucleotide sequence coded VH structural domain and/or VL structural domain.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody comprises by having 65% with the VH structural domain of 1D3 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 31 and 32) at least, preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical nucleotide sequence coded VH structural domain and/or VL structural domain.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody comprises by having 65% with the VH structural domain of 2B12 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 41 and 42) at least, preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical nucleotide sequence coded VH structural domain and/or VL structural domain.In other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody comprises by having 65% with the VH structural domain of 5A8 and/or the nucleotide sequence of VL structural domain (SEQ ID NO is respectively 51 and 52) at least, preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical nucleotide sequence coded VH structural domain and/or VL structural domain.

[0181] in other embodiments, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody comprises by having 65% with the VHCDR of 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 and/or the nucleotide sequence of VL CDR (Fig. 1-13) at least, preferred at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical nucleotide sequence coded VH CDR and/or VL CDR.

[0182] the present invention includes the antibody that combines the EphA2 polypeptide with the antibody competition described in the literary composition.Specifically, the present invention includes with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 or its antigen-binding fragment competition and combine the antibody of EphA2 polypeptide.In concrete embodiment, present invention resides in competition experiments described herein or the competition experiments well known in the art and can make 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 compare photograph with combining of EphA2 polypeptide, for example PBS reduces at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, perhaps 25-50%, 45-75%, or the antibody of 75-99%.In other embodiments, present invention resides in the ELISA competition experiments and can make 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 compare photograph with combining of EphA2 polypeptide, for example PBS reduces at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, perhaps 25-50%, 45-75%, or the antibody of 75-99%.

[0183] the ELISA competition experiments can be carried out in the following manner: be the reorganization EphA2 of 10 μ g/ml with the PBS compound concentration.In each hole of ELISA 98 hole titer plate, add 100 these solution of μ l and spend the night 4-8 ℃ of cultivation.Wash the ELISA flat board to remove excessive reorganization EphA2 with the PBS that is added with 0.1% tween.The final concentration that adds 100 μ, 1 usefulness PBS preparation is that 1% bovine serum albumin (BSA) is to seal nonspecific protein-protein interaction.Room temperature was placed after 1 hour, washing ELISA flat board.Prepare the unlabelled competition antibody of concentration range with confining liquid from 1 μ g/ml to 0.01 μ g/ml.Control wells only contains confining liquid or the control antibodies of concentration range from 1 μ g/ml to 0.01 μ g/ml.In the competition antibody diluent, add (for example, 1C1) with the fixedly final concentration of 1 μ g/ml with the test antibody of horseradish peroxidase-labeled.Triplicate 100 μ l test antibodies and the competition mixtures of antibodies of adding in the hole of ELISA flat board, flat board was cultivated 1 hour in room temperature.Flush away remains unconjugated antibody.In each hole, add 100 μ l horseradish peroxidase substrates to detect the bonded test antibody.Flat board was cultivated 30 minutes in room temperature, and read absorbancy with automatic flat bed reader.Calculate the mean value in triplicate hole.Compare with control wells, the absorbancy that records with the antibody of test antibody sufficient competition reduces.

[0184] in other embodiments, present invention resides in and to make antibody compare photograph in competition experiments described herein or the competition experiments well known in the art with combining of EphA2 polypeptide, for example PBS reduces at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, perhaps 25-50%, 45-75%, or the antibody of 75-99%, described antibody comprises (or be made of it) 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3, antigen-binding fragment of 2B12 or 5A8 (for example, but be not limited to the VH structural domain, VH CDR, VL structural domain or VL CDR).

[0185] in other embodiments, present invention resides in and to make antibody compare photograph in the ELISA competition experiments with combining of EphA2 polypeptide, for example PBS reduces at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, perhaps 25-50%, 45-75%, or the antibody of 75-99%, described antibody comprises (perhaps, constitute or constitute by it substantially by it) 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3, antigen-binding fragment of 2B12 or 5A8 (for example, but be not limited to the VH structural domain, VH CDR, VL structural domain or VLCDR).

[0186] the present invention includes polypeptide or the protein that the VH structural domain competition that contains (perhaps, constituted or be made of it substantially by it) and 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 combines the VH structural domain of EphA2 polypeptide.The present invention comprises that also the VL structural domain competition that contains (perhaps, constituted or be made of it substantially by it) and 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 combines the polypeptide or the protein of the VL structural domain of EphA2 polypeptide.

[0187] the present invention includes and contain polypeptide or the protein that (perhaps, constituted or be made of it substantially by it) and last table 2 or 3 listed VH CDR competitions combine the VH CDR of EphA2 polypeptide.The present invention also comprises and contains polypeptide or the protein that (perhaps, being made of it) and last table 2 or 3 listed VL CDR competitions combine the VL CDR of EphA2 polypeptide.

[0188] the specificity antibody that is incorporated into the EphA2 polypeptide comprises modified derivative, that is, form covalently bound thereby make the molecule of any kind be covalently bonded in antibody.For example; but be not limited thereto; antibody derivatives comprises modified antibody, for example by glycosylation, acetylize, pegization, phosphorylation, amidation, with known protecting group/blocking groups derive, proteolysis cuts, be connected to cell ligand or other protein, or the like.Can carry out any chemically modified by known technology, described technology includes but not limited to: the metabolism of specificity chemical chop, acetylize, formylation, tunicamycin is synthetic etc.In addition, described derivative can contain one or more atypia amino acid.

[0189] the present invention also provides the antibody that specificity is incorporated into the EphA2 polypeptide, and described antibody comprises is proficient in framework region known to those skilled in the art (for example, people or inhuman framework).Described framework region can be a framework region natural generation or total.Preferably, the fragment district of antibody of the present invention is people's (people's framework region tabulation can referring to, for example, Chothia etc., 1998, J.Mol.Biol.278:457-479, the document is included this paper by reference in full in).

[0190] the present invention includes the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody is included in have sudden change in the framework region aminoacid sequence of 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 of (for example, one or more aminoacid replacement).In some embodiments, the specificity antibody that is incorporated into the EphA2 polypeptide is included in the aminoacid sequence that has 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 that one or more amino-acid residues replace in the framework region of VH and/or VL structural domain.

[0191] the present invention comprises that also specificity is incorporated into the antibody of EphA2 polypeptide, described antibody is included in and has sudden change in variable region and the framework region aminoacid sequence of 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or the 5A8 of (for example, one or more amino-acid residues replace).

[0192] in some embodiments, antibody of the present invention does not comprise the antibody that some specificity is incorporated into the EphA2 polypeptide.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not 1C1.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not 1F12.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not 1H3.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not 1D3.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not 2B12.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not 5A8.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not 3F2.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not EA5.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not G5.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not EA2.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not B233.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not B208.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not 10C12.In one embodiment, antibody of the present invention is the EphA2 binding antibody, and prerequisite is that described EphA2 binding antibody is not B210.

[0193] in concrete embodiment, peptide that has antigenic epitopes and the polypeptide of antibodies EphA2 of the present invention, and described peptide that has an antigenic epitopes and polypeptide comprise following aminoacid sequence or are made of following aminoacid sequence: at least 4 of the EphA2 that finds in any species, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50 contiguous amino acid residues, and, preferably, about 30 contiguous amino acids of about 15-.The length that comprises the polypeptide of immunogenicity or antigenic epitopes is at least 8, at least 10, at least 15, at least 20, at least 25, at least 30 or at least 35 amino-acid residues.

[0194] having peptide, polypeptide and the fragment thereof of EphA2 epi-position can be by any ordinary method manufacturing.Referring to, for example, Houghten, R.A., 1985, " ordinary method of the quick a large amount of peptides of solid phase synthesis: the specificity of Ag-Ab on the single amino acids level " (General method for the rapidsolid-phase synthesis of large numbers of peptides:specificity ofantigen-antibody interaction at the level of individual amino acids), Proc.Natl.Acad.Sci.USA 82:5131-5135; The method of this " simultaneously a plurality of peptides synthetic (SMPS) " is further described in the U.S. Patent number 4,631,211 (1986) of Houghten etc.

[0195] the invention provides the one or more variable regions that comprise antibody described in the literary composition or peptide, polypeptide and/or the protein of hypervariable region.Preferably, comprise the one or more variable regions of antibody of the present invention or peptide, polypeptide or the protein of hypervariable region and also comprise the allogeneic amino acid sequence.In some embodiments, this allogeneic amino acid sequence comprises at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 75 contiguous amino acid residues, at least 100 contiguous amino acid residues or more a plurality of contiguous amino acid residue.This peptide, polypeptide and/or protein can be described as fusion rotein.

[0196] in embodiment, peptide, polypeptide or the proteinic length that comprises one or more variable regions of antibody of the present invention or hypervariable region is 10 amino-acid residues, 15 amino-acid residues, 20 amino-acid residues, 25 amino-acid residues, 30 amino-acid residues, 35 amino-acid residues, 40 amino-acid residues, 45 amino-acid residues, 50 amino-acid residues, 75 amino-acid residues, 100 amino-acid residues, 125 amino-acid residues, 150 amino-acid residues or amino acids residue more.In some embodiments, comprise the one or more variable regions of antibody of the present invention or peptide, polypeptide or the protein specific of hypervariable region and be incorporated into the EphA2 polypeptide.In other embodiments, peptide, polypeptide or the protein that comprises one or more variable regions of antibody of the present invention or hypervariable region not specificity in conjunction with the EphA2 polypeptide.

[0197] in concrete embodiment, peptide provided by the invention, polypeptide and/or protein comprise the VH structural domain and/or the VL structural domain (seeing the above table 2 and 3) of one of antibody described in the literary composition.In further embodiment, peptide provided by the invention, polypeptide and/or protein comprise one or more CDR of the aminoacid sequence with listed any CDR in last table 2 or 3.According to these embodiments, above-mentioned peptide, polypeptide or protein also can comprise the allogeneic amino acid sequence.

[0198] peptide, polypeptide or the protein that comprises one or more variable regions or hypervariable region can be used for, for example, make anti--idiotype antibody, and then can be used for prevention, treatment and/or alleviation and disease or one or more relevant symptoms of illness (for example, cancer, super propagation or hypoproliferation disease) again.Anti--the idiotype antibody that is produced also can be used for immunity test, and ELISA for example comprises with detection and to be used for producing this anti--contained variable region of peptide, polypeptide or protein of idiotype antibody or the antibody of hypervariable region.

[0199] the invention provides the antibody that the transformation period prolongs in the body specificity is incorporated into the EphA2 polypeptide.Specifically, the invention provides the antibody that specificity is incorporated into the EphA2 polypeptide, described antibody is at object, preferred mammal, most preferably the transformation period of philtrum is greater than 3 days, greater than 7 days, greater than 10 days, be preferably greater than 15 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months.

[0200] for (for example prolonging antibody, monoclonal antibody, single-chain antibody and Fab fragment) in vivo serum circulation, for example, can inert polymer molecule such as high molecular weight polyethylene glycol be connected to the antibody that has or do not have multifunction conjunction by N-or the C-epsilon-amino terminal or that on lysine residue, exist that polyoxyethylene glycol (PEG) locus specificity is coupled to antibody.Can adopt linear or branched chain polymer is derived to reduce biologic activity forfeiture as far as possible.Can monitor the coupling degree closely by SDS-PAGE and mass spectrum, thereby guarantee PEG molecule and the correct coupling of antibody.Can unreacted PEG and antibody-PEG conjugate be separated by size exclusion chromatography or ion exchange chromatography.Can adopt the method well-known to those having ordinary skill in the art of being proficient in, for example by immunity test as herein described detect PEG-deutero-antibody in conjunction with effect in activity and the body.

[0201] also can in IgG constant region or its FcRn binding fragment (preferred Fc or hinge-Fc structural domain fragment), introduce one or more amino acid modified (that is, replace, insert or deletion) to produce the antibody that the transformation period prolongs in the body.Referring to, for example, international publication number WO 98/23289; International publication number WO 97/34631; International publication number WO 02/060919; U.S. Patent Application Publication No. 2006/0039904A1 and U.S. Patent number 6,277,375, each document are included this paper by reference in full in.

[0202] in addition, can be with antibody and albumin coupling so that this antibody or antibody fragment are stable more in vivo or have the transformation period in the longer body.This technology is well known in the art, referring to, for example, international publication number WO 93/15199, WO 93/15200 and WO 01/77137; With european patent number EP 413,622, all patents are included this paper by reference in.

[0203] for ADC of the present invention is worked as required, in case selection to be coupled to the key of the antibody of toxin be this antibodies in the target cell surface (for example EphA2 or EphA4) promptly by this cell internalization.In case internalization, the link coupled toxin can discharge or keep combination, so that bring into play its toxic effect in cell.

[0204] antibody moiety of ADC of the present invention can include but not limited to: antibody, intracellular antibody, multi-specificity antibody, bi-specific antibody, people's antibody, humanized antibody, chimeric antibody, synthetic antibody, strand FvFc (scFvFc), strand Fv (scFv) and anti--idiotype that synthetic antibody, monoclonal antibody, the reorganization of few clonal antibody produce (resists-Id) antibody.Specifically, the antibody that is used for the inventive method comprises the immunocompetence part of immunoglobulin molecules and immunoglobulin molecules.Antibody of the present invention can be any kind (for example, IgG, IgE, IgM, IgD, IgA and IgY), kind (for example, IgG ₁, IgG ₂, IgG ₃, IgG ₄, IgA ₁And IgA ₂) or the immunoglobulin molecules of subclass.

[0205] antibody moiety of ADC of the present invention can be from any animal-origin, comprising bird and Mammals (for example, people, mouse, donkey, sheep, rabbit, goat, cavy, camel, horse or chicken).Preferably, described antibody is people or Humanized monoclonal antibodies.In the literary composition, " people " antibody comprises the antibody of the aminoacid sequence with human normal immunoglobulin, comprises that separation is from the human normal immunoglobulin storehouse or separate antibody from the mouse of expressing human gene antibody.

[0206] antibody has iso-electric point (pI) as all polypeptide, the pH when it is normally defined polypeptide and does not carry net charge.This area knows that proteinic solubleness is minimum usually when the pH of solution equals isoelectric point of protein (pI).Number that can be by changing ionizable residue in the antibody and position are regulated pI and are optimized solvability.For example, can handle the pI of polypeptide by suitable aminoacid replacement (for example, use charged amino acid, replace uncharged residue as Methionin) as L-Ala.Do not want to be subjected to the constraint of any particular theory, the antibody aminoacid replacement that causes described antibody pI to change can improve the solubleness and/or the stability of this antibody.Those skilled in the art will know which kind of aminoacid replacement is for the required pI of the most suitable acquisition of certain antibody specific.Can adopt the whole bag of tricks, include but not limited to: isoelectric focusing and various computerized algorithm (referring to, Bjellqvist etc. for example, 1993, Electrophoresis 14:1023) measures proteinic pI.In one embodiment, the pI of ADC of the present invention is higher than about 6.5, about 7.0, about 7.5, about 8.0, about 8.5 or about 9.0.In other embodiments, the pI of ADC of the present invention is higher than 6.5,7.0,7.5,8.0,8.5 or 9.0.In one embodiment, the replacement that causes the pI of ADC of the present invention to change will can significantly not reduce its binding affinity to the Eph acceptor.PI value defined used herein is the pI of main charged form.Can adopt the whole bag of tricks, include but not limited to: isoelectric focusing and various computerized algorithm (referring to, Bjellqvist etc. for example, 1993, Electrophoresis 14:1023) measures proteinic pI.

[0207] Tm of monoclonal antibody structural domain is the good index of antibody thermostability, has also shown its storage time.Tm is low to show the more/less stable of cohesion, and Tm higher show cohesion less/stability better.Therefore, the preferred higher antibody of Tm.In one embodiment, the Tm value of the Fab structural domain of ADC is higher than at least 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃, 105 ℃, 110 ℃, 115 ℃ or 120 ℃.In another embodiment, the Tm value of the Fab structural domain of ADC is higher than at least about 50 ℃, about 55 ℃, about 60 ℃, about 65 ℃, about 70 ℃, about 75 ℃, about 80 ℃, about 85 ℃, about 90 ℃, about 95 ℃, about 100 ℃, about 105 ℃, about 110 ℃, about 115 ℃ or about 120 ℃.Can adopt any standard method known in the art, for example differential scanning calorimetry detect protein domain (for example, the Fab structural domain) pyrolysis chain temperature (referring to, Vermeer etc. for example, 2000, Biophys.J., 78:394-404; Vermeer etc., 2000, Biophys.J., 79:2150-2154).

[0208] antibody moiety of ADC of the present invention can be monospecific, dual specific, tri-specific or polyspecific more.But the multi-specificity antibody specificity is incorporated into the different epi-positions of required target molecule, but perhaps specificity is incorporated into target molecule and allos epi-position, as heterologous polypeptide or solid support material.Referring to, for example, international publication number WO 94/04690; WO 93/17715; WO 92/08802; WO91/00360; With WO 92/05793; Tutt etc., 1991, J.Immunol.147:60-69; U.S. Patent number 4,474,893,4,714,681,4,925,648,5,573,920, and 5,601, and 819; With Kostelny etc., 1992, J.Immunol.148:1547; Each document is included this paper by reference in full in).In one embodiment, a kind of Eph acceptor is had a kind of binding specificity, and any other antigen (that is, another kind of Eph acceptor, liver are joined albumen, signal conduction or effector molecule) is had another kind of binding specificity.

[0209] multi-specificity antibody at least two kinds not synantigen have binding specificity.Although this molecule will only (be bi-specific antibody, BsAbs), have extra specific antibody such as three-specific antibody and be also included within the present invention in conjunction with two kinds of antigens usually.The example of BsAb includes but not limited to that those arm is at beta 2 integrin alpha _vβ ₃And another arm is at any other antigenic antibody.The method of making bi-specific antibody is known in the art.Traditionally, prepare the total length bi-specific antibody according to the coexpression of two pairs of heavy chain immunoglobulin-light chains, two chains wherein have different specificity (Millstein etc., 1983, Nature, 305:537-539, it includes this paper by reference in full in).Because the random assignment of heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) may produce the mixture of different antibodies molecule, wherein have only a kind of correct dual specific structure that has.The purifying of correct molecule (normally finishing by the affinity chromatography step) is trouble quite, and output is very low.Similarly method is disclosed in WO 93/08829, and Traunecker etc., 1991, EMBOJ., 10:3655-3659.More directly method is to produce two-two to resist, i.e. the tetravalence bi-specific antibody.Two-two anti-methods that produce are known in the artly (for example to see Lu etc., 2003, J Immunol method 279:219-32; Marvin etc., 2005, Acta Pharmacolical Sinica 26:649).

[0210], the antibody variable territory (antibody-antigen binding site) with required binding specificity is merged with immunoglobulin (Ig) constant domain sequence according to another kind of method.Preferably merge with the heavy chain immunoglobulin constant domain that comprises to small part hinge region, CH2 district and CH3 district.In one embodiment, exist at least a fusions and contain first CH (CH1) that combines essential site with light chain.The DNA of coding heavy chain immunoglobulin fusions and the light chain immunoglobulin that may need is inserted into different expression vectors, and cotransfection is gone into the appropriate host biology.When making up, adopt three peptide species chains of inequality proportion can provide in the embodiment of optimum yield, provide very big handiness for regulating the segmental mutual ratio of three peptide species like this.Yet, when expressing with equal proportion that at least two polypeptide chains can obtain high yield or when this ratio is not particularly important, also the encoding sequence of two or all three polypeptide chains can being inserted a kind of expression vector.

[0211] in an embodiment of this method, described bi-specific antibody is made of (second binding specificity is provided) the hybrid immunoglobulins heavy chain-light chain in the hybrid immunoglobulins heavy chain that has first binding specificity in the first arm (for example, Eph acceptor) and another arm.Find that this unsymmetrical structure helps required dual specific compound is separated with unwanted immunoglobulin chain composition, because only in half of this bispecific molecule, have light chain immunoglobulin and separation method easily is provided.This method is seen WO 94/04690 (including this paper by reference in full in).Other details that produces bi-specific antibody as seen, Suresh etc. for example, 1986, " Enzymology method " (Methodsin Enzymology), 121:210 (including this paper by reference in full in).According to the described other method of WO96/27011 (by reference in full include in this paper), can engineered a pair of antibody molecule to improve the per-cent that from the reconstitution cell culture, reclaims heterodimer (heterodimer) as far as possible.At least a portion of antibody constant region CH3 structural domain is contained at preferred interface.In this method, substitute one or more p1 amino acid side chains at first antibody molecule interface with larger side chain (for example, tyrosine or tryptophane).Substituting bulky side chain amino acid by the amino acid (for example, L-Ala or Threonine) with less side chain can produce on the interface of second antibody molecule and bulky side chain size same or analogous compensatory " hole ".This provides increase heterodimer productive rate to surpass the not mechanism of end product (for example homodimer (homodimer)).

[0212] in concrete embodiment, the used antibody of the inventive method is that dual specific T cell is convened antibody (bispecific T cell engager) (BiTE).It is the T cell can be redirected and the bi-specific antibody of antigen-specific elimination target that dual specific T cell is convened antibody (BiTE).The BiTE molecule its molecule one end contain can with the T cell antigen (for example, CD3) bonded antigen binding domains, (at the other end) contain can with the antigen bonded antigen binding domains on the target cell.In recent years, include this paper WO 99/54440 as a reference in and described BiTE.This publication has been described the novel strand multifunctional polypeptides that contains CD19 and CD3 antigen binding site (CD19 * CD3).This molecular source is from two kinds of antibody, and CD19 a kind of and on the B cell combines, and another kind of antibody combines with CD3 on the T cell.The variable region of these different antibodies connects by a peptide sequence, thereby produces a kind of molecule.This publication has also been described and has been utilized the snappiness joint to connect heavy chain (V _H) and light chain (V _LThereby) variable domains generation single chain bispecific antibody.

The part that can comprise [0213] in an embodiment of the invention, the BiTE molecule with polypeptide of interest (for example, Eph acceptor and/or liver are joined albumen) specificity bonded antibody or part.For example, can with the V of polypeptide of interest (for example, Eph acceptor and/or liver are joined albumen) specificity bonded antibody _HAnd/or V _L(for example, scFV) with anti--CD3 bound fraction, this meromixis of above-mentioned molecule for example, thus produce the BiTE molecule of target polypeptide of interest (for example, Eph acceptor and/or liver are joined albumen).Except (for example at polypeptide of interest, Eph acceptor and/or liver are joined albumen) the heavy chain and/or light chain variable structural domain of antibody outside, can with polypeptide of interest (for example, Eph acceptor and/or liver are joined albumen) other molecule of bonded can comprise the BiTE molecule, acceptor (for example, Eph acceptor and/or liver are joined albumen) for example.In another embodiment, the BiTE molecule can comprise can with other T cell antigen (except that CD3) bonded molecule.For example, a part of the present invention contained can with T cell antigen, for example CD2, CD4, CD8, CD11a, TCR and CD28 specificity bonded part and/or antibody.This inventory be not limit and just explanation can be used as the part of BiTE molecule with other molecule of T cell antigen specificity bonded.These molecules can comprise the VH and/or the VL part of antibody or native ligand (for example, LFA3, its native ligand is CD3).

[0214] bi-specific antibody comprises crosslinked or " different conjugate (heteroconjugate) " antibody.For example, a kind of antibody in this heterogeneous conjugate can with avidin coupling, another antibody and vitamin H coupling.Someone proposes, and for example this antibody can make the deleterious cell of immune system cell target (U.S. Patent number 4,676,980) and can be used for treating HIV and infect (WO 91/00360, WO 92/200373 and EP03089).Above-mentioned bibliography is included this paper by reference separately in full in.Can adopt conventional cross-linking method to prepare heterogeneous conjugate antibody.Well known suitable crosslinking agent and many crosslinking technologicals can be referring to U.S. Patent number 4,676,980.Above-mentioned bibliography is included this paper separately by reference in full in.

[0215] comprises having the above antibody that is mixed with at least one hinge modification of the present invention of two valencys.For example, can prepare three-specific antibody.Referring to, Tutt etc. for example, J.Immunol., 147:60,1991, include it in this paper by reference.

[0216] antibody moiety of ADC of the present invention comprises single domain antibody, comprises that camelization (camelized) single domain antibody (for example sees Muyldermans etc., 2001, TrendsBiochem.Sci.26:230; Nuttall etc., 2000, Cur.Pharm.Biotech.1:253; Reichmann and Muyldermans, 1999, J.Immunol.Meth.231:25; International publication number WO 94/04678 and WO 94/25591; U.S. Patent number 6,005,079; Include it in this paper by reference).

[0217] other special antibody of considering is " few clone " antibody.Term used herein " few clonal antibody " refers to the predetermined mixture of different monoclonal antibodies.Referring to, for example, the open WO95/20401 of PCT; U.S. Patent number 5,789,208 and 6,335,163, include them in this paper by reference.Preferably, few clonal antibody is made of the antibody predetermined mixture at one or more epi-positions that produces in a cell.More preferably, few clonal antibody contain can with a plurality of heavy chains of common light chain paired, thereby produce antibody (for example, the open WO 04/009618 of PCT includes it in this paper by reference) with polyspecific.When target molecule (for example, beta 2 integrin alpha of needs target _vβ ₃) on a plurality of epi-position the time, few clonal antibody is particularly useful.Those skilled in the art will know that antibody or the mixtures of antibodies that maybe can determine which kind of type are applicable to required purpose and needs.

In [0218] one embodiment, ADC of the present invention can or can be modified changing its glycosylation by chemically modified (for example, can connect one or more chemical parts on antibody), and then changes one or more functional performances of this antibody.

[0219] more in other embodiments, the glycosylation of ADC of the present invention is modified.The antibody (that is this antibody deficiency glycosylation) that for example, can prepare de-glycosylation (aglycosylate).Thereby can change glycosylation, for example improve the avidity of this antibody target antigen.This carbohydrate modification can be with, for example change of one or more glycosylation sites in the antibody sequence.For example, thus can make one or more aminoacid replacement and eliminate the glycosylation in this site with the glycosylation site of eliminating one or more variable regions framework.This de-glycosylation can improve this antibody to antigenic avidity.U.S. Patent number 5,714,350 and 6,350,861 have been described in further detail this method, and each document is included this paper by reference in full in.

[0220] additionally or alternatively, ADC can be made type of glycosylation and change, for example low fucosylation (hypofucosylated) antibody that reduces of fucosido residue content or contain the two cross-section structure enhanced antibody of GlcNAc.Proved that the glycosylation pattern of this change has improved the ADCC ability of antibody.This carbohydrate modification can with, for example in host cell, express the antibody that glycosylation mechanism changes.This area has been described the cell that glycosylation mechanism changes, expresses the antibody that recombinant antibodies of the present invention produces the glycosylation change thereby these cells can be used as host cell.Can referring to, Shields for example, R.L. etc., (2002), J.Biol.Chem., 277:26733-26740; Umana etc., (1999), Nat.Biotech., 17:176-1, and european patent number EP 1,176,195; The open WO 03/035835 of PCT; WO 99/54342, and each document is included this paper by reference in full in.

[0221] more in other embodiments, the glycosylation of ADC of the present invention is modified.The antibody (that is this antibody deficiency glycosylation) that for example, can prepare de-glycosylation (aglycosylate).Thereby can change glycosylation, for example improve the avidity of this antibody target antigen.This carbohydrate modification can be with, for example change of one or more glycosylation sites in the antibody sequence.For example, thus can make one or more aminoacid replacement and eliminate the glycosylation in this site with the glycosylation site of eliminating one or more variable regions framework.This de-glycosylation can improve this antibody to antigenic avidity.U.S. Patent number 5,714,350 and 6,350,861 have been described in further detail this method, and each document is included this paper by reference in full in.

[0222] additionally or alternatively, ADC can be made type of glycosylation and change, for example low fucosylation (hypofucosylated) antibody that reduces of fucosido residue content or contain the two cross-section structure enhanced antibody of GlcNAc.Proved that the glycosylation pattern of this change has improved the ADCC ability of antibody.This carbohydrate modification can with, for example in host cell, express the antibody that glycosylation mechanism changes.This area has been described the cell that glycosylation mechanism changes, expresses recombinant antibodies of the present invention and produces and have the glycosylated antibody of change thereby these cells can be used as host cell.Can referring to, Shields for example, R.L. etc., (2002), J.Biol.Chem., 277:26733-26740; Umana etc., (1999), Nat.Biotech., 17:176-1, and european patent number EP 1,176,195; The open WO 03/035835 of PCT; WO 99/54342, and each document is included this paper by reference in full in.

[0223] the present invention also comprises the active Fc variant antibody that improves of antibody dependent cellular mediated cell toxicity.The non-limitative example of this Fc variant antibody is disclosed in U.S. Patent application 11/203,253 (submissions on August 15th, 2005, and open as U.S. Patent Application Publication No. US2006/0039904A1) and 11/203,251 (submissions on August 15th, 2005), U.S. Provisional Patent Application 60/674,674 (submissions on April 26th, 2005) and 60/713,711 (submission on September 6th, 2005), each piece document is included this paper in as a reference in full.

Antibody coupling matter

[0224] the present invention includes utilize antibody or its fragment and the reorganization of allos reagent merge or chemical coupling (comprising covalency and non-covalent coupling) to produce fusion rotein simultaneously as target molecule and anti--EphA2 or anti--EphA4 reagent.Described allos reagent can be polypeptide (or its fragment, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acid whose polypeptide), nucleic acid, small molecules (less than 1000 dalton) or inorganic or organic compound.Merge and not necessarily leave no choice but directly (fusion), and can merge by joint sequence.The antibody that can adopt methods known in the art to use in vivo to be blended in or to be coupled to allos reagent is with the progress of detection, treatment, control or monitoring of diseases.Referring to, for example, international open WO 93/21232; EP 439,095; Naramura etc., 1994, Immunol.Lett.39:91-99; United States Patent (USP) 5,474,981; Gillies etc., 1992, PNAS 89:1428-1432; With Fell etc., 1991, J.Immunol.146:2446-2452 includes them in this paper by reference in full.In some embodiments, the disease that detect, treat, control or monitor was to express the malignant cancer of EphA2 or EphA4.The disease that detect in other embodiments,, treat, control or monitor is and expresses the relevant precancerous condition (pre-cancerouscondition) of cell of EphA2 or EphA4 excessively.In concrete embodiment, described precancerous condition is high level prostatic intraepithelial neoplasm sample pathology (PIN), mammary gland fibroadenoma, fibrocystic disease or compound nevi.

[0225] the present invention also comprises and contains the allos combination of agents thing that is blended in or is coupled to antibody fragment.For example, described heterologous polypeptide can be blended in or be coupled to Fab fragment, Fd fragment, Fv fragment, F (ab) ₂Fragment or its part.The method that polypeptide is blended in or is coupled to antibody moiety is known in the art.Referring to, for example, U.S. Patent number 5,336,603,5,622,929,5,359,046,5,349,053,5,447,851 and 5,112,946; EP 307,434; EP 367,166; International publication number WO 96/04388 and WO 91/06570; Ashkenazi etc., 1991, PNAS 88:10535-10539; Zheng etc., 1995, J.Immunol.154:5590-5600; With Vil etc., 1992, PNAS89:11337-11341 (described reference is included this paper by reference in full in).

[0226] can reorganize by gene, motif reorganization, exon reorganization and/or codon reorganization technology such as (being referred to as " DNA reorganization ") produce other fusion roteins, for example EA2-5, Eph099B-102.147, Eph099B-208.261, Eph099B-210.248, Eph099B-233.152, table 2 or 3 or Fig. 1-59 in listed any antibody or EA44 (or the EphA2/EphA4 epitope antibodies of the exciting antibody of any other EphA2/EphA4 or EphA2/EphA4 cancer cell phenotype inhibition antibody or exposure or with less than 3 * 10 ^-3s ^-1K _OffEphA2/EphA4 antibody in conjunction with EphA2 or EphA4) fusion rotein.DNA reorganization can be used to change antibody of the present invention or its segmental activity (for example, avidity is higher and dissociate speed lower antibody or its fragment).Generally can be referring to U.S. Patent number 5,605,793; 5,811,238; 5,830,721; 5,834,252; With 5,837,458, and Patten etc., 1997, Curr.Opinion Biotechnol.8:724-33; Harayama, 1998, TrendsBiotechnol.16:76; Hansson etc., 1999, J.Mol.Biol.287:265; With Lorenzo and Blasco, 1998, BioTechniques 24:308 (including these patents and publication in this paper separately in full by reference).Carry out random mutagenesis by fallibility PCR, random nucleotide insertion or other method earlier, reorganization can change antibody or its fragment again, perhaps Bian Ma antibody or its fragment.Can with the coding each several part can be with one or more components of one or more parts of the polynucleotide of EphA2 or EphA4 specificity bonded antibody or antibody fragment and one or more allos reagent, motif, section, partly, structural domain, fragment or the like reorganization (recombine).

In [0227] one embodiment, antibody of the present invention or its fragment or variant can be coupled to flag sequence (for example peptide) so that purifying.In some embodiments, described marker amino acid sequence is the peptide of six Histidines, the label that provides in the pQE carrier (proper (QIAGEN of root company for example, Inc.), main road, special Butterworth Eton is looked into No. 9259 in the California, 91311 (Eton Avenue, Chatsworth, CA, 91311)), wherein many by commercial acquisition.As Gentz etc., 1989, Proc.Natl.Acad.Sci.USA, described in the 86:821-824, six-Histidine is that the purifying of fusion rotein is provided convenience.Other peptide tag that can be used for purifying includes but not limited to: corresponding to hemagglutinin " HA " label (Wilson etc., Cell, 37:767, (1984)) and " flag " label derived from the influenza hemagglutinin protein epi-position.

[0228] in other embodiments, antibody of the present invention or its fragment or variant are coupled to diagnosis or detection reagent.The generation or the progress of cancer monitored or predicted to the part that this antibody can be used as the clinical detection process, for example measures the effect of specific therapy.In addition, this antibody can be used for monitoring or predicting the generation or the progress of the precancerous condition (for example, high level prostatic intraepithelial neoplasm sample pathology (PIN), mammary gland fibroadenoma, fibrocystic disease or compound nevi) relevant with the cell of expressing EphA2 or EphA4 excessively.In one embodiment, the EphA2 of exposure or EphA4 epitope antibodies are coupled to diagnosis or detection reagent.

[0229] antibody and detectable substance coupling can be realized this diagnosis and detection, described detectable substance includes but not limited to: various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase; Prothetic group is such as but not limited to Streptavidin/vitamin H and avidin/biotin; Fluorescent material is such as but not limited to Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine base amine fluorescein (dichlorotriazinylamine fluorescein), dansyl chloride or phycoerythrin; Luminescent material, such as but not limited to; The noclilucence material is such as but not limited to luciferase, luciferin and aequorin; Radioactive substance, such as but not limited to bismuth ( ²¹³Bi), carbon ( ¹⁴C), chromium ( ⁵¹Cr), cobalt ( ⁵⁷Co), fluorine ( ¹⁸F), gadolinium ( ¹⁵³Gd, ¹⁵⁹Gd), gallium ( ⁶⁸Ga, ⁶⁷Ga), germanium ( ⁶⁸Ge), holmium ( ¹⁶⁶Ho), indium ( ¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), iodine ( ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanum ( ¹⁴⁰La), lutetium ( ¹⁷⁷Lu), manganese ( ⁵⁴Mn), molybdenum ( ⁹⁹Mo), palladium ( ¹⁰³Pd), phosphorus ( ³²P), praseodymium ( ¹⁴²Pr), promethium ( ¹⁴⁹Pm), rhenium ( ¹⁸⁶Re, ¹⁸⁸Re), rhodium ( ¹⁰⁵Rh), ruthenium ( ⁹⁷Ru), samarium ( ¹⁵³Sm), scandium ( ⁴⁷Sc), selenium ( ⁷⁵Se), strontium ( ⁸⁵Sr), sulphur ( ³⁵S), technetium ( ⁹⁹Tc), thallium ( ²⁰¹Ti), tin ( ¹¹³Sn, ¹¹⁷Sn), tritium ( ³H), xenon ( ¹³³Xe), ytterbium ( ¹⁶⁹Yb, ¹⁷⁵Yb), yttrium ( ⁹⁰Y), zinc ( ⁶⁵Zn); The positron emitting metal and the on-radiation paramagnetic metal ion that are used for various positron emission tomographies.

[0230] in other embodiments, antibody of the present invention or its fragment or variant are coupled to curative such as cytotoxin (for example, cytostatics or cytocide), curative or radioactive metal ion (for example, α-gamma ray emission body).Cytotoxin or cytotoxic agent comprise the deleterious any reagent of pair cell.Its example comprises: taxol, cytochalasin B, Gramicidin D, the pyridine of bromination second, ipecamine, mitomycin, etoposide, teniposide (tenoposide), vincristine(VCR), vincaleucoblastine, colchicine, Zorubicin, daunorubicin, dihydroxyl anthracin diketone (dihydroxyanthracin dione), istizin, mithramycin, dactinomycin, 1-dehydrogenation testosterone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte, with tetracycline and their analogue or homologue.Curative includes but not limited to: metabolic antagonist (for example, methotrexate, Ismipur, 6-thioguanine, cytosine arabinoside, 5 FU 5 fluorouracil, dacarbazine); Alkylating agent (for example, mustargen, thioepa chlorambucil, melphalan, Carmustine (BCNU) and lomustine (CCNU); Endoxan, busulfan, mitobronitol, streptozotocin, ametycin and cis dichloro diamines platinum (II) (DDP) and cis-platinum); Anthracycline antibiotics (for example, daunorubicin (being called daunomycin in the past) and Zorubicin); Microbiotic (for example, dactinomycin (being called actinomycin in the past), bleomycin, mithramycin and anthramycin (AMC)); And antimitotic agent (for example, vincristine(VCR) and vincaleucoblastine).

In [0231] one embodiment, described cytotoxic agent is selected from enediyne (enediyne), lexitropsin, duocarmycin, Taxan, tetracycline, dolastatin, maytansinol (maytansinoid) and vinca alkaloids.In other embodiments, described cytotoxic agent is taxol, docetaxel, CC-1065, SN-38, Hycamtin, morpholino-Zorubicin, rhizomycin, cyano group morpholino-Zorubicin, dolastatin-10, Quinomycin A, combretastatin, calicheamycin (calicheamicin), maytenin, DM-1, Ao Ruisitating or other dolastatin derivatives, as Ao Ruisitating E or Ao Ruisitating F, AEB, AEVB, AEFP, MMAE (monomethyl Ao Ruisitating E), MMAF (monomethyl Ao Ruisitating F), soft coral alcohol or T-1384.The structrual description of MMAE and MMAF is in Figure 25-27.

[0232] more in other embodiments, the cytotoxic agent of ADC of the present invention is anti--tubulin agent.In more concrete embodiment, described cytotoxic agent is selected from: vinca alkaloids, podophyllotoxin, Taxan, baccatin derivative, beads algal rim peptide (cryptophysin), maytansinol, combretastatin and dolastatin.In more concrete embodiment, described cytotoxic agent is a vincristine(VCR), vincaleucoblastine, vindesine, vinorelbine, VP-16, camptothecine, taxol, docetaxel, epithilone A, epithilone B, but promise azoles, colchicine (coichicine), Colchiceinamidum (colcimid), Emcyt, Cemadotin, dish suberite lactone, maytenin, DM-1, Ao Ruisitating or other dolastatin derivatives are as Ao Ruisitating E or Ao Ruisitating F, AEB, AEVB, AEFP, MMAE (monomethyl Ao Ruisitating E), MMAF (monomethyl Ao Ruisitating F), soft coral alcohol or T-1384.

[0233] in concrete embodiment, the cytotoxic agent of ADC of the present invention is MMAE.In other embodiments, the cytotoxic agent of ADC of the present invention is AEFP.In further concrete embodiment, the cytotoxic agent of ADC of the present invention is MMAF.

[0234] the further example and their structure of toxin, transcribed spacer, joint, tract (stretcher) etc. can be at U.S. Patent Application Publication No. 2006/0074008A1,2005/0238649A1,2005/0123536A1,2005/0180972A1,2005/0113308A1,2004/0157782A1, U.S. Patent number 6,884,869B2, U.S. Patent number 5,635, find in 483, all these include this paper by reference in full in.

[0235] as discussed herein, be used for to comprise conventional chemotherapeutic, as Zorubicin, taxol, melphalan, vinca alkaloids, methotrexate, ametycin, etoposide etc. with ADC link coupled medicine of the present invention.In addition, also can adopt conditionality to stablize joint will link to each other with ADC to form effective immune conjugate such as potent agents such as CC-1065 analogue, calichiamicin, maytenin, dolastatin 10 analogue, rhizomycin and palytoxins.

[0236] in some embodiments, the medicine that ADC of the present invention comprised is stronger at least 40 times than Zorubicin to the effect of the cell of expression EphA2 or EphA4.This medicine includes but not limited to: the DNA minor groove binding comprises enediyne and lexitropsin, duocarmycin, Taxan (comprising taxol and docetaxel), tetracycline, vinca alkaloids, CC-1065, SN-38, Hycamtin, morpholino-Zorubicin, rhizomycin, cyano group morpholino-Zorubicin, Quinomycin A, combretastatin, T-1384, epithilone A and B, Emcyt, beads algal rim peptide, Cemadotin, maytansinol, dolastatin, for example, Ao Ruisitating E, dolastatin 10, MMAE, MMAF, dish suberite lactone, soft coral alcohol, and mitoxantrone.

[0237] in some concrete embodiment, ADC of the present invention comprises the enediyne part.In concrete embodiment, described enediyne partly is a calicheamycin.The enediyne compound is cutting double-stranded DNA by the diradical that produces through the Bergman cyclisation.

[0238] in some embodiment, described cytotoxic agent or cytostatics are Ao Ruisitating E or Ao Ruisitating F, or derivatives thereof.In further embodiment, described Ao Ruisitating E derivative is the ester that is formed by Ao Ruisitating E and ketone acid.For example, Ao Ruisitating E can be with reaction generates AEB and AEVB respectively to acetamido benzoate or benzoyl valeric acid.Other Ao Ruisitating derivatives comprise MMAE, MMAF and AEFP.

[0239] the same with dolastatin-10, synthetic and structure and the derivative thereof of Ao Ruisitating E also are known in this area, are described in U.S. Patent Application Publication No. 2003/0083263A1 and 2005/0009751A1; International patent application no PCT/US02/13435, U.S. Patent number 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; With 4,486,414, all these include this paper by reference in full in.

[0240] in concrete embodiment, described medicine is the DNA minor groove binding.The example of this compound and their the synthetic U.S. Patent number 6,130,237 that is disclosed in are included it in this paper by reference in full.In some embodiments, described medicine is the CBI compound.

[0241] In some embodiments of the present invention, ADC of the present invention comprises anti--tubulin agent.Described anti--the tubulin agent is the compound of the treatment cancer known of a class.The example of anti--tubulin agent includes but not limited to: Taxan (for example, Taxol.RTM. (taxol), docetaxel), and T67 (Tularik), Vinca, and Ao Ruisitating (for example, Ao Ruisitating E, AEB, AEVB, MMAE, MMAF, AEFP).The antitublin that such comprised also has: vinca alkaloids comprises vincristine(VCR) and vincaleucoblastine, vindesine and vinorelbine; Taxan such as taxol and docetaxel and baccatin derivative, epithilone A and B, but the promise azoles, Fluracil (luorouraci) and Colchiceinamidum, Emcyt, beads algal rim peptide, Cemadotin, maytansinol, combretastatin, dolastatin, dish suberite lactone and soft coral alcohol.

[0242] in concrete embodiment, described medicine is maytansinol (class resists-the tubulin agent).In more concrete embodiment, described medicine is a maytenin.Moreover, in concrete embodiment, described cytotoxic agent or cytostatics be DM-1 (Immunogen Inc. (and ImmunoGen, Inc.); Also can be) referring to Chari etc. 1992, Cancer Res 52:127-131.Maytenin is a kind of natural product, thereby it can suppress tubulin polymerization and causes mitotic division termination and necrocytosis.Therefore, as if the mechanism of action of maytenin is similar with vincristine(VCR) and vincaleucoblastine.Yet the vitro cytotoxicity of maytenin is than the about by force 200-1 of these vinca alkaloids, 000 times.In other embodiments, described medicine is AEFP.

[0243] in some embodiment of the present invention, described medicine is not to be longer than 50,100 or 200 amino acid whose polypeptide, for example toxin.In the specific embodiment of the present invention, described medicine is not a ricin.

[0244] in other embodiment of the present invention, ADC of the present invention does not comprise one or more cytotoxic agents or cytostatics, comprises the reagent of following non-mutual eliminating type: alkylating agent, anthracycline antibiotics, microbiotic, dihydrofolic acid antagonist, metabolic antagonist, antitublin, Ao Ruisitating, the chemotherapy sensitizing agent, DNA minor groove binding, dna replication dna inhibitor, duocarmycin, etoposide is fluoridized pyrimidine, lexitropsin, nitrosourea, cis-platinum, the purine metabolic antagonist, tetracycline, radiotherapy sensitizing agent, steroid, Taxan, topoisomerase enzyme inhibitor, vinca alkaloids, purine antagonist, and dihydrofolate reductase inhibitor.In more concrete embodiment, described efficient medicine be not following one or more: male sex hormone, anthramycin (AMC), asparaginase, U-18496, azathioprine, bleomycin, busulfan, buthionine sulphoximine, camptothecine, carboplatin, carmustine (BSNU), CC-1065, Chlorambucil, cis-platinum, Fluracil, endoxan, cytosine arabinoside, cytosine arabinoside (cytidine arabinoside), cytochalasin B, Dacarbazine, dactinomycin (being called actinomycin in the past), daunorubicin, decarbazine, docetaxel, Zorubicin, oestrogenic hormon, floxuridine, 5 FU 5 fluorouracil, Gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mustargen, melphalan, Ismipur, methotrexate, mithramycin, ametycin, mitoxantrone, nitroimidazole, taxol, Plicamycin, Procarbazine (procarbizine), streptozotocin, teniposide (tenoposide), 6-mercapto guanine, thiophene is for group, Hycamtin, vincaleucoblastine, vincristine(VCR), vinorelbine, VP-16, VM-26, azathioprine, mycophenlate mofetil, methotrexate, acycloguanosine, gangcyclovir, zidovudine, vidarabine, ribavirin (ribavarin), Zidovodine, cytosine arabinoside, Symmetrel, two deoxyuridines, iododeoxyuridine, Trisodium phosphonoformate hexahydrate (poscarnet), and trifluridine.

[0245] in some embodiments, described cytotoxic agent or cytostatics are dolastatins.In more concrete embodiment, described dolastatin is a Ao Ruisi Statins type.In the specific embodiment of the present invention, described cytotoxic agent or cytostatics are MMAE.In other embodiment of the present invention, described cytotoxic agent or cytostatics are AEFP.In other embodiment of the present invention, described cytotoxic agent or cytostatics are MMAF.

[0246] in other embodiments, antibody of the present invention or its fragment or variant are coupled to curative or the drug moiety that can modify the particular organisms reaction.Described curative or drug moiety can not be thought and only limit to typical chemotherapeutic.For example, described drug moiety can be to have required bioactive protein or polypeptide.This protein can comprise that for example, toxin is as toxalbumin, ricin A, Pseudomonas exotoxin, Toxins,exo-, cholera or diphtheria toxin; Protein, as tumour necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, Thr6 PDGF BB, tissue plasminogen activator, apoptosis material (as TNF-α, TNF-β), AIM I (referring to international publication number WO97/33899), AIM II (referring to international publication number WO 97/34911), Fas part (Takahashi etc., 1994, Int.Immunol., 6:1567-1574) and VEGI (referring to international publication number WO99/23105); Thrombotic medicine or angiogenesis inhibitor medicine, for example angiostatin or endostatin; Perhaps biological response modifier, as lymphokine (for example, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-4 (" IL-4 "), interleukin-6 (" IL-6 "), interleukin-7 (" IL-7 "), interleukin-9 (" IL-9 "), interleukin-15 (" IL-15 "), il-1 2 (" IL-12 "), rHuGM-CSF (" GM-CSF ") and granulocyte colony-stimulating factor (" G-CSF ")) or somatomedin (as, tethelin (" GH ")).

[0247] in other embodiments, antibody of the present invention or its fragment or variant are coupled to curative, as radio active material or be used for the macrocyclic chelants (example of radio active material sees above) of coupling isotopic ion.In some embodiments, described macrocyclic chelants be can through linkers link to each other with antibody 1,4,7,10-tetraazacyclododecanand-N, N ', N ", N "-tetraacethyl (DOTA).This linkers will further be discussed hereinafter, and they are normally known in the art and be described in Denardo etc., and 1998, Clin Cancer Res.4 (10): 2483-90; Peterson etc., 1999, Bioconjug.Chem.10 (4): 553; Zimmerman etc., 1999, Nucl.Med.Biol.26 (8): 943-50, each piece document is included this paper in as a reference in full.

[0248] in concrete embodiment, described coupling antibody is that preferred combination is exposed to the EphA2 on cancer cells rather than the non-cancer cells or the EphA2 or the EphA4 antibody (that is, the EphA2 of exposure or EphA4 epitope antibodies) of EphA4 epi-position.In other embodiments, described coupling antibody is not EA2 or EA4.In other embodiments, described coupling antibody is not EA44.

[0249] therapeutic partly being coupled to antibody is that technology is to know.Can each several part be coupled to antibody by any method known in the art, described method includes but not limited to: aldehyde/Schiff connecting key, sulfydryl connecting key, sour unstable connecting key, cis rhizome of Chinese monkshood connecting key, hydrazone connecting key, enzyme degradable connecting key (usually can be referring to Garnett, 2002, Adv.Drug Deliv.Rev.53:171-216).The other technologies that therapeutic partly are coupled to antibody are also known, can referring to, for example, Arnon etc., " monoclonal antibody that is used for the immune target of medicine in the cancer therapy " (Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy), publish in " monoclonal antibody and cancer therapy " (Monoclonal Antibodies And Cancer Therapy), volumes such as Reisfeld, 243-56 page or leaf ((the Alan R.Liss of A Lanlisi company, Inc.), 1985); Hellstrom etc., " be used for the antibody that medicine is sent " (Antibodies For Drug Delivery), publish in " controlled drug is sent " (Controlled Drug Delivery), (second edition), volumes such as Robinson, the 623-53 page or leaf (MD company (Marcel Dekker, Inc.), 1987); Thorpe, " the antibody vehicle of the cytotoxic drug in the cancer therapy: summary " (Antibody Carriers Of Cytotoxic Agents In CancerTherapy:A Review), publish in " monoclonal antibody ' 84: biology and clinical application " (Monoclonal Antibodies ' 84:Biological And Clinical Applications), volumes such as Pinchera, the 475-506 page or leaf, (1985); " therapeutic of radiolabelled antibody is used in the cancer therapy " (Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy), publish in " monoclonal antibody of cancer detection and treatment " (Monoclonal Antibodies For Cancer Detection And Therapy), volumes such as Baldwin, 303-16 page or leaf (academic press (Academic Press), 1985), and Thorpe etc., 1982, Immunol.Rev.62:119-58.The method that antibody is blended in or is coupled to polypeptide portion is known in the art.Referring to, for example, U.S. Patent number 5,336,603,5,622,929,5,359,046,5,349,053,5,447,851 and 5,112,946; EP 307,434; EP 367,166; International publication number WO 96/04388 and WO 91/06570; Ashkenazi etc., 1991, PNAS 88:10535-10539; Zheng etc., 1995, J.Immunol.154:5590-5600; With Vil etc., 1992, PNAS 89:11337-11341.The fusion of antibody and certain part not necessarily must directly be merged, and can merge by joint sequence.This linkers is that this area routine is known, is described in Denardo etc., 1998, and Clin Cancer Res.4:2483-90; Peterson etc., 1999, Bioconjug.Chem.10:553; Zimmerman etc., 1999, Nucl.Med.Biol.26:943-50; Garnett, 2002, Adv.DrugDeliv.Rev.53:171-216, each document include this paper by reference in full in.

[0250] can adopt two kinds of methods to reduce the extracellular activity of medicine at ADC of the present invention institute target as far as possible: first method is, can use antibody, thereby medicine (being included in the medicine that produces under the effect of prodrug saccharase) concentrates on the cell surface of activated lymphocytes in conjunction with cytolemma rather than soluble E phA2 or EphA4.The coupling mode that a kind of active more preferably method that reduces the medicine that is incorporated into antibody of the present invention as far as possible is a medicine can reduce pharmaceutical activity, unless they are hydrolyzed or cut off antibody.Can adopt this method medicine to be connected in the antibody that has joint, described joint to the cell surface environment of activated lymphocytes (for example, the protease activity that is present in the cell surface of activated lymphocyte) sensitivity, perhaps when described conjugate is activated the lymphocyte picked-up, to the internal medium sensitivity of the activated lymphocyte that conjugate ran into (for example, in endosome or lysosome environment, such as by pH susceptibility or proteolytic enzyme susceptibility).The example that can be used for joint of the present invention is disclosed in U.S. Patent Application Publication No. 2005/0123536A1,2005/0180972A1,2005/0113308A1,2004/0157782A1 and U.S. Patent number 6,884,869B2, all documents include this paper by reference in full in.

In [0251] one embodiment, described joint be the acid labile hydrazone that in lysosome, is hydrolyzed or hydrazides group (referring to, for example, U.S. Patent number 5,622,929).In other embodiments, can medicine be linked (Dubowchik and Walker, 1999, Pharm.Therapeutics 83:67-123 on the antibody by other acid labile joints such as cis-aconitic acid acid amides, ortho ester, acetal and ketal; Neville etc., 1989, Biol.Chem.264:14653-14661).This joint is relatively stable under neutral pH environment such as blood environment, and when being lower than pH 5 (approximately being lysosomal pH) instability.

[0252] in other embodiments, adopt the peptide transcribed spacer (spacer) that is excised by the intracellular protein enzyme that medicine is connected in antibody of the present invention.The target enzyme comprises cathepsin B and D and plasmin, thus known they can both in target cell, discharge active medicine (Dubowchik and Walker, 1999, Pharm.Therapeutics 83:67-123) by hydrolysis dipeptide medicament derivative.Adopt the benefit of intracellular protein hydrolysis drug release to be that medicine is very high by the serum stability of highly attenuated and conjugate when coupling.

[0253] more in other embodiments, described joint is propanedioic acid joint (Johnson etc., 1995.AntiCancer Res.15:1387-93), maleimide aminobenzyl (maleimidobenzoyl) joint (Lau etc., 1995, Bioorg-Med-Chem.3 (10): 1299-1304) or '-N-amide analogue (Lau etc., 1995, Bioorg-Med-Chem.3 (103:1305-12).

[0254] as mentioned above, normally drug coupling is prepared ADC in antibody by joint.Therefore, most of ADC of the present invention is anti-except comprising-EphA2 or EphA4 antibody and highly effective medicine and/or promote also to comprise joint the medicine of internalization.Any joint known in the art all can be used for ADC of the present invention, for example, difunctionality reagent (as dialdehyde or imido-ester) or ramose hydrazone joint (referring to, for example, U.S. Patent number 5,824,805 is included it in this paper by reference in full).

[0255] in some non-limiting embodiment of the present invention, under certain condition can be cut in drug moiety and the joint area between the antibody moiety of ADC, wherein, the excision of joint or release can discharge drug moiety from antibody moiety.Preferably, described joint easily cut or hydrolysis under the environment in born of the same parents.

In [0256] one embodiment, reach certain value or to surpass certain value then can be cut in drug moiety and the joint area between the antibody moiety of ADC if pH changes.In other embodiments of the present invention, around lysosome, for example described joint can be cut down near acidic conditions (that is, pH 5-5.5 or lower).In other embodiments, described joint is the peptide acyl joint that can be removed by peptase or proteolytic cleavage, and described enzyme includes but not limited to lysosomal protein enzyme, embrane-associated protein enzyme, intracellular protein enzyme or endosome proteolytic enzyme.Typically, described length of said joint has two amino acid at least, and more typically, its length has three amino acid at least.Preferably can be expressed the peptide acyl joint of the enzyme cutting that exists in the cancer (cell) of EphA2 or EphA4.For example, can use the peptide acyl joint (for example, the Gly-Phe-Leu-Gly joint) that can be organized proteolytic enzyme-B cutting, kethepsin-B is a kind of proteolytic enzyme that depends on mercaptan of expressing at the cancerous tissue camber.Other these type of joints are described in, and for example, U.S. Patent number 6,214,345 is included it in this paper by reference in full.

[0257] in the embodiment of the some other non-mutual eliminating of the present invention, can promote cell internalization with the anti--EphA2 of ADC of the present invention or the joint of EphA4 antibody and drug coupling.In some embodiments, joint-drug moiety of ADC can promote cell internalization.In some embodiments, select specific joint so that the structure of complete ADC can promote cell internalization.

[0258] in the specific embodiment of the present invention, Xie Ansuan-citrulline derivative is used as joint (val-cit joint).The synthesizing of Zorubicin that has the val-cit joint described (U.S. Patent number 6,214,345 of Dubowchik and Firestone is included it in this paper by reference in full) before.

[0259] in further concrete embodiment, described joint is maleimide caproyl-citrulline joint or maleimide caproyl-Xie Ansuan-citrulline joint.

[0260] in other embodiments, described joint is the phe-lys joint.

[0261] in other embodiments, described joint be the thioether joint (referring to, for example, authorize the U.S. Patent number 5,622,929 of Willner etc., include it in this paper in full by reference).

[0262] more in another embodiment, described joint be the hydrazone joint (referring to, for example, authorize the U.S. Patent number 5,122,368 of Greenfield etc. and authorize 5,824,805 of King etc., include them in this paper in full by reference).

[0263] again in some other concrete embodiment, described joint is the disulphide joint.Many disulphide joints known in the art, comprising but be not limited to the joint that available SATA (N-succinimido-S-acetyl thio acetic ester), SPDP (N-succinimido-3-(2-pyridyl dithio) propionic ester), SPDB (N-succinimido-3-(2-pyridyl dithio) butyric ester) and SMPT (N-succinimido-oxygen base carbonyl-Alpha-Methyl-α-(2-pyridyl-dithio) toluene) form.SPDB and SMPT (referring to, for example, Thorpe etc., 1987, Cancer Res., 47:5924-5931; Wawrzynczak etc., 1987, be selected from " immune conjugate: the antibody coupling matter in cancer radiation and the therapy " (Immunoconjugates:Antibody Conjugates in Radioimagery and Therapyof Cancer), C.W.Vogel compiles, Oxford University Press (Oxford U.Press), the 28-55 page or leaf; Also can include it in this paper in full by reference) referring to the U.S. Patent number 4,880,935 of Thorpe etc.

[0264] various terminal that can be used for the compositions and methods of the invention is described in U.S. Patent Application Publication No. US 2004/0018194A1, includes it in this paper in full by reference.

[0265] again in other embodiments of the present invention, (connector unit of anti--EphA2 or anti--EphA4ADC) connects cytotoxic agent or cytostatics (drug unit for anti--EphA2 or EphA4 antibody-joint-drug conjugates;-D) and anti--EphA2 or EphA4 antibody unit (A).In the literary composition, term is anti--and EphA2 or anti--EphA4ADC comprise anti--EphA2 or the anti--EphA4 antibody drug conjugates that has and have connector unit.In some embodiments, described connector unit has following general formula:

[0266]-T _a-W _w-Y _y-

[0267] in the formula:

[0268]-T-is the tract unit;

[0269] a is 0 or 1;

[0270] respectively-W-independently is the amino acid unit;

[0271] w independently is the integer of 2-12;

[0272]-Y-is the transcribed spacer unit; With

[0273] y is 0,1 or 2.

[0274] the tract unit (will resist when T-) existing-EphA2 or anti--EphA4 antibody unit is connected in the amino acid unit (W-).Can be present in natural on anti--EphA2 or anti--EphA4 antibody or include but not limited to: sulfydryl, amino, hydroxyl, different hydroxyl of carbohydrate, and carboxyl through chemically treated useful functional group.Preferred functional group is sulfydryl and amino.Sulfydryl can-EphA2 anti-by reducing or the intramolecular disulfide bond of anti--EphA4 antibody produce.Perhaps, sulfydryl can by generate with 2-imino-tetramethylene sulfide (iminothiolane) (Traut reagent) or other sulfydryls the agent reduction anti--amino of EphA2 or anti--EphA4 antibody Methionin part produces.In concrete embodiment, described anti--EphA2 or anti--EphA4 antibody is recombinant antibodies and carries one or more Methionins through engineered one-tenth.In other embodiments, described reorganization is anti--and EphA2 or anti--EphA4 antibody carries extra sulfydryl through engineered one-tenth, for example, extra halfcystine.

[0275] in some concrete embodiment, described tract unit forms key with anti--EphA2 or the unitary sulphur atom of anti--EphA4 antibody.The sulfydryl that described sulphur atom can be derived from anti--EphA2 of being reduced or anti--EphA4 antibody (A) (SH).At some in other the embodiment, the disulfide linkage of described tract unit between anti--unitary sulphur atom of CD30 antibody and the unitary sulphur atom of tract with resist-CD30 antibody unit (A) links to each other.

[0276] again in other embodiments, the reactive group of described tract contain can with the reactive site of the amino reaction of anti--EphA2 or anti--EphA4 antibody.Described amino can be arginine or lysine amino.Suitable amine reactive site includes but not limited to: activatory ester class, and as succinimide ester, 4-nitrophenyl ester, pentafluorophenyl group ester, acid anhydride, acid chloride, SULPHURYL CHLORIDE, isocyanic ester and lsothiocyanates.

[0277] again in another aspect of the present invention, the reactive functional groups of tract comprise can with the reactive site of the carbohydrate group reaction of the modification that exists on anti--EphA2 or anti--EphA4 antibody.In concrete embodiment, described anti--EphA2 or anti--EphA4 antibody by the enzyme glycosylation so that sugar moieties to be provided.Described sugar can be by such as reagent mild oxidation such as sodium periodates, and the carbonyl unit of the oxidized sugar of gained can with the tract condensation that contains such as functional groups such as hydrazides, oxime, reactive amine, hydrazine, thiosemicarbazone, carboxylic acid hydrazine and aryl hydrazides, as Kaneko, T. etc., Bioconiugate Chem1991,2,133-41 described those.

[0278] if there is the transcribed spacer unit, (W-) ((Y-), if there is no the transcribed spacer unit then is connected to the tract unit cytotoxic agent or cytostatics (drug unit T-) to be connected to the transcribed spacer unit with the tract unit in described amino acid unit; D).

[0279]-W _w-be dipeptides, tripeptides, tetrapeptide, penta peptide, six peptides, seven peptides, octapeptide, nonapeptide, decapeptide, 11 peptides or dodecapeptide unit.The amino acid unit of described connector unit can be included but not limited to cancer-related proteolytic enzyme the cutting of interior enzyme with discharge drug unit (D), drug unit after release in vivo by protonated so that cell toxicity medicament (D) to be provided.

[0280] in one embodiment, described amino acid unit is phenylalanine-Methionin dipeptides (phe-lys or a FK joint).In other embodiments, described amino acid unit is Xie Ansuan-citrulline dipeptides (val-cit or a VC joint).

[0281] described spacerarm unit (Y-) is connected to drug unit with the amino acid unit when existing.The spacerarm unit has two types usually: self-sacrifice type and non-self-sacrifice type.Some or all spacerarms unit in the non-self-sacrifice type spacerarm unit still combines with drug unit after enzyme downcuts the amino acid unit on anti--EphA2 or anti--EphA4 antibody-joint-drug conjugates or medicine-linker compounds.The unitary example of non-self-sacrifice type spacerarm includes but not limited to (glycine-glycine) spacerarm unit and glycine spacerarm unit.When contain glycine-glycine spacerarm unit or the unitary the present invention of glycine spacerarm anti--EphA2 or anti--when EphA4 antibody-joint-drug conjugates was cut by tumour cell dependency proteolytic enzyme, cancer cells dependency proteolytic enzyme or lymphocyte dependency proteolytic enzyme enzyme, glycine-glycine-drug moiety or glycine-drug moiety were from A-T-W _w-on cut down.Be to discharge medicine, hydrolysis reaction can take place independently in the target cell with the key between cutting glycine-drug unit.

[0282] other examples of self-sacrifice type spacerarm include but not limited to: with the phenolic compound of PAB group equivalence on electronics, as the 2-aminooimidazole-the 5-carbinol derivatives (for example, see Hay etc., Bioorg.Med.Chem.Lett., 1999,9,2237) and adjacent-or right-aminobenzyl acetal.Can adopt the spacerarm that is easy to cyclisation after the amido linkage hydrolysis, as replacing and unsubstituted 4-aminobutyric acid acid amides (Rodrigues etc., Chemistry, Biology, 1995,2,223), loop systems (Storm etc., J.Amer.Chem.Soc., 1972 that suitably replace, 94,5815) and 2-aminophenyl propionic acid acid amides (Amsberry etc., J.Org.Chem., 1990,55,5867).At the alpha-position of glycine the elimination that contains drug amine (Kingsbury etc., J.Med.Chem., 1984,27,1447) of replacement being arranged also is the example that can be used for the self-sacrifice type spacerarm strategy of antibody-joint of the present invention-drug conjugates.

[0283] in concrete embodiment, anti--EphA2 or the EphA4 antibody of ADC of the present invention are coupled to cytotoxic agent by joint, and wherein said joint is a peptide linker.In concrete embodiment, anti--EphA2 or the EphA4 antibody of ADC of the present invention are coupled to cytotoxic agent by joint, and wherein said joint is val-cit joint, phe-lys joint, hydrazone joint or disulphide joint.In some embodiments, anti--EphA2 or the EphA4 antibody of ADC of the present invention are coupled to cytotoxic agent by peptide linker.

[0284] in some embodiments; conjugate of the present invention is anti--EphA2-Xie Ansuan-citrulline-MMAE (anti--EphA2-val-citMMAE or anti--EphA2-vcMMAE); or anti--EphA2-Xie Ansuan-citrulline-MMAF; or anti--EphA2-maleimide glycyl-citrulline-MMAE; or anti--EphA2-maleimide glycyl-citrulline-MMAF, or anti--EphA2-Xie Ansuan-citrulline-AEFP (anti--EphA2-val-citAEFP or anti--EphA2-vcAEFP).In concrete embodiment, conjugate of the present invention is G5-Xie Ansuan-citrulline-MMAE (G5-val-citMMAE or G5-vcMMAE) or G5-Xie Ansuan-citrulline-AEFP (G5-val-citAEFP or G5-vcAEFP).

[0285] in further concrete embodiment, conjugate of the present invention is to be connected to-antibody that is selected from antibody described in Fig. 1 of the present invention-59 of Xie Ansuan-citrulline-MMAE, be connected to-Xie Ansuan-citrulline-MMAF, be connected to-maleimide glycyl-citrulline-MMAE, be connected to maleimide glycyl-citrulline-MMAF or be connected to-Xie Ansuan-citrulline-AEFP.

[0286] in some embodiments, conjugate of the present invention is anti--EphA4-Xie Ansuan-citrulline-MMAE (anti--EphA4-val-citMMAE or anti--EphA4-vcMMAE) or anti--EphA4-Xie Ansuan-citrulline-AEFP (anti--EphA4-val-citAEFP or anti--EphA4-vcAEFP).

[0287] in other embodiments, conjugate of the present invention is anti--EphA2-phenylalanine-Methionin-MMAE (anti--EphA2-phe-lysMMAE or anti--EphA2-fkMMAE) or anti--EphA2-phenylalanine-Methionin-AEFP (anti--EphA2-phe-lysAEFP or anti--EphA2-fkAEFP).

[0288] in concrete embodiment, conjugate of the present invention is G5-phenylalanine-Methionin-MMAE (G5-phe-lysMMAE or G5-fkMMAE) or G5-phenylalanine-Methionin-AEFP (G5-phe-lysAEFP or G5-fkAEFP).

[0289] in concrete embodiment, conjugate of the present invention be connected to-phenylalanine-Methionin-MMAE or phenylalanine-Methionin-MMAF or-antibody that is selected from antibody described in Fig. 1 of the present invention-59 of phenylalanine-Methionin-AEFP.

[0290] in other embodiments, conjugate of the present invention is anti--EphA4-phenylalanine-Methionin-MMAE (anti--EphA4-phe-lysMMAE or anti--EphA4-fkMMAE) or anti--EphA4-phenylalanine-Methionin-AEFP (anti--EphA4-phe-lysAEFP or anti--EphA4-fkAEFP).

[0291] therefore, in concrete embodiment, the invention provides the method for cancer of treatment target, described method comprise give the described treatment significant quantity of this object contain (a) G5-val-cit-MMAE; (b) pharmaceutical composition of pharmaceutically acceptable carrier.

[0292] in other embodiments, the invention provides the method for cancer of treatment target, described method comprise give the described treatment significant quantity of this object contain (a) G5-val-cit-AEFP; (b) pharmaceutical composition of pharmaceutically acceptable carrier.

[0293] in other embodiments, the invention provides the method for cancer of treatment target, described method comprise give the described treatment significant quantity of this object contain (a) G5-val-cit-MMAF; (b) pharmaceutical composition of pharmaceutically acceptable carrier.

[0294] in some embodiments, anti--EphA2 or the EphA4 antibody of ADC of the present invention are coupled to cytotoxic agent by joint, and wherein said joint can be cut less than 5.5 o'clock at pH.In concrete embodiment, described joint can be cut less than 5.0 o'clock at pH.

[0295] in some embodiments, anti--EphA2 or the EphA4 antibody of ADC of the present invention are coupled to cytotoxic agent by joint, and wherein said joint can be cut by proteolytic enzyme.In concrete embodiment, described proteolytic enzyme is the lysosomal protein enzyme.In other embodiment, described proteolytic enzyme is embrane-associated protein enzyme, intracellular protein enzyme or endosome proteolytic enzyme etc.

[0296] or, antibody can be coupled to second antibody forming as U.S. Patent number 4,676, the heterogeneous conjugate of the antibody described in 980, this patent full text is by reference included this paper in.

[0297] antibody also is attachable to solid support, and this immunity test or purifying for target antigen is particularly useful.This solid support includes but not limited to: glass, Mierocrystalline cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

In [0298] one embodiment, antibody of the present invention is in case in conjunction with promptly being entered cell by internalization, wherein internalization comparison according to the antibody height at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160% or at least about 170%, as described herein.

[0299] in other embodiments, antibody of the present invention is in case in conjunction with promptly being entered cell by internalization, internalization comparison wherein is according to the high 1-10% of antibody, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170%, as described herein.

[0300] in other embodiments, antibody of the present invention is in case in conjunction with promptly being entered cell by internalization, record by the internalization test of using the second saponin(e antibody to carry out, internalization comparison wherein is according to the high 1-10% of antibody, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170%.

[0301] in other embodiments, antibody of the present invention can activated receptor when combining with cell and internalization, and does not demonstrate the cross reactivity of organizing with human heart, and is as described herein.

[0302] in other embodiments, antibody of the present invention can activated receptor when combining with cell and internalization, and when the cross reactivity of organizing that does not demonstrate when using than low dosage with human heart, as described herein.

[0303] in other embodiments, antibody of the present invention can activated receptor when combining with cell but can internalization, and does not demonstrate the cross reactivity of organizing with human heart, and is as described herein.

[0304] in other embodiments, antibodies various kinds of cell type of the present invention, the rising comparison that records its mean fluorecence is shone the antibody height at least about 10%, at least about 100%, at least about 500%, at least about 1000%, at least about 1500%, at least about 2000%, at least about 2500%, at least about 3000%, at least about 3500%, at least about 4000%, at least about 4500%, at least about 5000%, at least about 5500%, at least about 6000%, at least about 6500%, at least about 7000%, at least about 7500%, at least about 8000%, at least about 8500%, at least about 9000%, at least about 9500% or at least about 10000%, as described herein.

[0305] in other embodiments, antibodies various human cell type of the present invention, comprising but be not limited to: A-549, Hey-A8, PC3, KC-231, Panc-02.03, SKMel.28, ACHN, 496, D-145, HT-29, SKOV-3, or SW-480, as described herein.

[0306] in other embodiments, antibodies specific of the present invention is in conjunction with mouse cell lines CT26, and is as described herein.

[0307] antibodies specific of the present invention in other embodiments is in conjunction with some rat cell types, and described type includes but not limited to F98, RG2 or YPEN, and is as described herein.

[0308] in other embodiments, antibodies specific of the present invention is F98 and YPEN in conjunction with mouse cell line CT26 and rat cell, and is as described herein.

[0309] in other embodiments, antibodies specific of the present invention is in conjunction with rat cell type F98 and YPEN, and described binding ratio is high at least about 2 times, about 5 times, about 10 times or about 100 times, as described herein with combining of rat cell type RG2.

[0310] in other embodiments, antibodies specific of the present invention is in conjunction with mouse cell type CT26, and described binding ratio is high at least about 2 times, about 5 times, about 10 times or about 100 times, as described herein with combining of mouse cell type Balb/3T3 or NIH3T3.

[0311] in other embodiments, antibody of the present invention can stimulate the EphA2 phosphorylation when being applied to the HuVec cell, as described herein.

[0312] in other embodiments, in the HuVec test cell line, antibody of the present invention stimulate the comparison of EphA2 phosphorylation according to the antibody height at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160% or at least about 170%, as described herein.

[0313] in other embodiments, antibody of the present invention in mouse cell lines internal stimulus EphA2 phosphorylation comparison according to the antibody height at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160% or at least about 170%, described mouse cell lines includes but not limited to CT26 and 4T1, and is as described herein.

[0314] in other embodiments, antibody of the present invention is F98 internal stimulus EphA2 phosphorylation comparison according to the antibody height at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160% or at least about 170% at rat cell, and is as described herein.

[0315] in other embodiments, antibody of the present invention in human cell line's internal stimulus EphA2 phosphorylation comparison according to the antibody height at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160% or at least about 170%, described human cell line includes but not limited to PC3 and ES2, and is as described herein.

[0316] in other embodiments, antibodies specific of the present invention is in conjunction with mouse Eph protein families, and described family includes but not limited to mEphA and mEphB.

[0317] in other embodiments, antibodies specific of the present invention is incorporated into the member of mouse Eph family, and described member includes but not limited to mEphA2 and mEphB2.

[0318] in other embodiments, the shown IC50 dosage that goes out of antibody of the present invention than external IC50 low at least about 1 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 100 times or at least about 500 times, as described herein.

[0319] in other embodiments, antibody of the present invention is low at least about 2 times, 5 times, 10 times or 100 times to the IC50 of the IC50 comparison KC231 clone of PC3 clone.

[0320] in other embodiments, compare with control antibodies, antibody of the present invention is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160% or at least about 170% to the inhibition of tumor growth in mouse heteroplastic transplantation model as herein described.

[0321] in other embodiments, compare with control antibodies, antibody of the present invention is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160% or at least about 170% to the promoter action of tumor regression in mouse heteroplastic transplantation model as herein described.

[0322] in other embodiments, compare with control antibodies, antibody of the present invention is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160% or at least about 170% to the inhibition of metastases in mouse heteroplastic transplantation model as herein described.

[0323] in other embodiments, compare with control antibodies, the inhibition that antibody of the present invention takes place tumor vessel in mouse heteroplastic transplantation model as herein described is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160% or at least about 170%.

[0324] in other embodiments, compare interior human cell line with including but not limited to SKMel.28, ACHN, 496, D-145, HT-29, SKOV-3 or SW-480, antibody of the present invention can be preferential in conjunction with including but not limited to A-549, Hey-A8, PC3, KC-231, Panc-02.03 interior human cell line at least about 2 times, 5 times, 10 times or 100 times, as described herein.

Make the method for antibody

[0325] can make antibody or its fragment with synthetic antibody by any method known in the art, specifically, by chemosynthesis or preferably pass through recombination and expression techniques.

[0326] can adopt various techniques known in the art, comprise and utilize hybridoma, recombinant chou and display technique of bacteriophage or their combination to prepare monoclonal antibody.For example, can adopt hybridoma technology to prepare monoclonal antibody, this technology comprise known in the art and following document is instructed those: Harlow etc., " antibody: laboratory manual " (Antibodies:A Laboratory Manual), (press of cold spring harbor laboratory, second edition, 1988); Hammerling etc., publish in " monoclonal antibody and T quadroma " (Monoclonal Antibodies and T-Cell Hybridomas), 563-681, (New York Yi Laisiweier (Elsevier, N.Y.), 1981) (described reference is included this paper in as a reference in full).Term used herein " monoclonal antibody " is not limited to the antibody by the hybridoma technology generation.Term " monoclonal antibody " refers to be derived from a clone's (comprising any eucaryon, protokaryon or phage clone) antibody, rather than refers to its production method.

[0327] the well known ordinary method of utilizing hybridoma technology preparation and screening specific antibody.In brief, available EphA2 or EphA4 (full length protein or its fragment, for example ectodomain or ligand binding domains) immune mouse is in case detect immune response, for example in mice serum, detect the specific antibody of EphA2 or EphA4, promptly collect mouse spleen and separating Morr. cell.By knowing technology splenocyte and any suitable myeloma cell (for example clone SP20 cell that can obtain from ATCC) are merged then.Select hybridoma, clone by limiting dilution assay.Then by secretion among the means known in the art checks hybridoma clone can with the cell of polypeptide bonded antibody of the present invention.Can clone immune mouse by positive hybridoma and produce the ascites that contains high-level antibody usually.

[0328] therefore, can produce monoclonal antibody by following steps: cultivation can be secreted the hybridoma of antibody of the present invention, and wherein said hybridoma preferably merges acquisition by the splenocyte and the myeloma cell that will separate personal EphA2 or EphA4 or its fragment mice immunized; Secrete in the hybridoma that the screening fusion obtains then and can clone with the hybridoma of EphA2 or EphA4 bonded antibody.

[0329] can be by the antibody fragment of any technology generation energy identification specificity EphA2 well known by persons skilled in the art or EphA4 epi-position.For example, available enzyme produces F (ab ') 2 fragments as papoid (producing the Fab fragment) or stomach trypsin), prepare Fab of the present invention and F (ab ') 2 fragments by proteolysis cutting immunoglobulin molecules.F (ab ') 2 fragments contain the CH1 structural domain of variable region, constant region of light chain and heavy chain.In addition, also can adopt various phage display method known in the art to produce antibody of the present invention.

[0330] in the phage display method, the function antibody structural domain is showed in the phage particle surface of carrying its coded polynucleotide sequence.Specifically, the dna sequence dna of amplification coding VH and VL structural domain from animal cDNA library (as people or the adenoid cDNA of mouse library).Utilize PCR will the encode DNA of VH and VL structural domain and the reorganization of scFV joint and be cloned into phagemid carrier (as pCANTAB 6 or pComb 3HSS).The carrier electroporation is entered intestinal bacteria (E.coli) and utilizes the helper phage ehec infection.Used phage is a filobactivirus often in these methods, comprises fd and M13, and often with VH and VL structural domain and phage gene III or gene VIII reorganization fusion.Can utilize antigen, as applying marking antigen, in conjunction with or be trapped in the antigen selection of solid surface or pearl or differentiate express phage in conjunction with the antigen binding domains of interested EphA2 epi-position.The example that can be used for producing the phage display method of antibody of the present invention comprises the method that discloses in the following document: Brinkman etc., 1995, and J.Immunol.Methods 182:41-50; Ames etc., 1995, J.Immunol.Methods 184:177; Kettleborough etc., 1994, Eur.J.Immunol.24:952-958; Persic etc., 1997, Gene 187:9; Burton etc., 1994, Advances in Immunology 57:191-280; International application no PCT/GB91/01134; International publication number WO 90/02809, WO 91/10737, and WO 92/01047, and WO 92/18619, WO93/11236, WO 95/15982, and WO 95/20401, and WO97/13844; And U.S. Patent number 5,698,426,5,223,409,5,403,484,5,580,717,5,427,908,5,750,753,5,821,047,5,571,698,5,427,908,5,516,637,5,780,225,5,658,727,5,733,743 and 5,969,108; Each document is included this paper by reference in full in.

[0331] can screen with EphA2 and combine, especially with the ectodomain bonded phage of EphA2 or EphA4.Also can be (for example to the activity of exciting EphA2 or EphA4, improve the phosphorylation of EphA2 or EphA4, reduce the level of EphA2 or EphA4) or cancer cell phenotype suppresses activity, and (for example, the colony that reduces in the soft agar forms or three-dimensional substrates film or extracellular matrix goods such as MATRIGEL ^TMIn the tubulose network form) or preferential in conjunction with cancer cells but not the ability of EphA2 that exposes on the non-cancer cells or EphA4 epi-position (for example, weak in conjunction with the EphA2 or the EphA4 that combine with the part that participates in cell-cells contacting, and EphA2 or EphA4 that good combination does not combine with the part that participates in cell-cells contacting) screen.

[0332] as described in the above-mentioned reference, after phage is selected, the antibody coding zone of separable phage also is used to produce whole antibody, comprise people's antibody or any required Fab, and, comprise mammalian cell, insect cell, vegetable cell, yeast and bacterium as expressing among following what required host in office.Also can use recombination method well known in the art to produce Fab, Fab ' and F (ab ') 2 segmental methods, as international publication number WO 92/22324; Mullinax etc., 1992, BioTechniques12:864; Sawai etc., 1995, AJRI34:26; With Better etc., 1988, the method that Science 240:1041 (it is for referencial use to include described document in this paper in full) is disclosed.

[0333], can utilize the PCR primer of the flanking sequence that contains VH or VL nucleotide sequence, restriction site and this restriction site of protection increase VH or VL sequence among the scFV clone for producing complete antibody.Adopt clone technology well known by persons skilled in the art, the VH structural domain of pcr amplification can be cloned into and express the VH constant region, for example in the carrier of people γ 4 constant regions, the VL structural domain of pcr amplification is cloned into expresses the VL constant region, for example in the carrier of people κ or λ constant region.Preferably, the carrier of expressing VH or VL structural domain comprises the cloning site and the selective marker of EF-1 α promotor, secretion signal, variable domains, constant domain, for example Xin Meisu.Also VH and VL structural domain can be cloned in the carrier of expressing required constant region.Adopt technology known in the art that heavy chain is changed carrier and light chain conversion carrier then and be transfected into clone jointly, can express full length antibody thereby produce, for example stable the or instantaneous clone of IgG.

[0334] for some application, be included in and use antibody and vitro detection test in the human body, preferably utilize people's antibody or chimeric antibody.For therapeutic treatment people patient, people's antibody or humanized antibody are desirable especially completely.Can be by prepared in various methods people's antibody known in the art, what comprise the antibody library that utilizes the derived from human immunoglobulin sequences above-mentionedly has a liking for the thalline methods of exhibiting.Also referring to U.S. Patent number 4,444,887 and 4,716,111; The open WO 98/46645 of PCT, WO 98/50433, WO98/24893, WO98/16654, WO 96/34096, WO 96/33735 and WO 91/10741; Each document is included this paper by reference in full in.

[0335] also can utilize the endogenous immunoglobulin (Ig) that to express function, but the transgenic mice of energy expressing human immunoglobulin gene produces people's antibody.For example, people's heavy chain and light chain immunoglobulin gene mixture can be introduced in the mouse embryo stem cell at random or through homologous recombination.Perhaps, except people's heavy chain and light chain gene, people variable region, constant region and diversity region (gene) can be introduced in the mouse embryo stem cell.Can introduce human immunoglobulin gene's seat by homologous recombination makes murine heavy chain and light chain immunoglobulin gene not have function respectively or simultaneously.Specifically, the deletion JH district of isozygotying has stoped the endogenous antibody generation.The embryonic stem cell that amplification is modified goes in the blastocyst its microinjection to produce the mosaic type mouse.Make the mating of mosaic type mouse to produce the offspring of isozygotying of energy expressing human antibody then.With the antigen of selecting, all or part of of polypeptide of the present invention this transgenic mice of immunity in a usual manner for example.Can adopt conventional hybridization knurl technology to obtain at this antigenic monoclonal antibody from the transgenic mice of immunity.The contained human normal immunoglobulin transgenosis of transgenic mice is reset during the B cytodifferentiation, experiences classification conversion and somatic mutation then.Therefore, adopt this technology can produce treatment and go up useful IgG, IgA, IgM and IgE antibody.The summary that produces this technology of people's antibody can be referring to Lonberg and Huszar, 1995, Int.Rev.Immunol., 13:65-93.The scheme that produces this technology of people's antibody and human monoclonal antibodies and produce this antibody describe in detail can referring to, for example international publication number WO98/24893, WO 96/34096, WO 96/33735; With U.S. Patent number 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318 and 5,939,598, each piece document is included this paper in as a reference in full.In addition, can employ for example antibody manufacturing company (Abgenix, the Inc. of California Freemont, Freemont, CA) and the wheat moral Simon Rex company of Princeton, New York (NJ) etc. company adopts above-mentioned similar technique to provide at selected antigenic people's antibody for Medarex, Princeton.

[0336] chimeric antibody is that an antibody different piece is derived from the molecule of different immunoglobulin molecules, as has the variable region that comes from the non-human antibody and the antibody of human normal immunoglobulin constant region.The method that produces chimeric antibody is known in the art.Referring to, for example, Morrison, 1985, Science229:1202; Oi etc., 1986, BioTechniques 4:214; Gillies etc., 1989, J.Immunol.Methods 125:191-202; And U.S. Patent number 6,311,415,5,807,715,4,816,567 and 4,816,397, it is for referencial use to include it in this paper in full.Comprise one or more CDR that come from inhuman species and can utilize well known few techniques to produce with the chimeric antibody that comes from the framework region of human normal immunoglobulin molecule, these technology comprise that for example, (EP 239,400 in the CDR-grafting; International publication number WO 91/09967; With U.S. Patent number 5,225,539,5,530,101 and 5,585,089), frosting (veneering) or reinvent (resurfacing) (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28 (4/5): 489-498; Studnicka etc., 1994, Proteinengineering 7:805; With Roguska etc., 1994, PNAS 91:969), chain reorganization (U.S. Patent number 5,565,332).In one embodiment, chimeric antibody specificity of the present invention comprises 1,2 or 3 VL CDR in conjunction with EphA2 and in people's framework region, described VL CDR has the aminoacid sequence of any VL CDR of EA2-5, Eph099B-102.147, Eph099B-208.261, Eph099B-210.248, Eph099B-233.152.

[0337] in other embodiments, chimeric antibody specificity of the present invention comprises 1,2 or 3 VL CDR in conjunction with EphA4 and in people's framework region, described VL CDR has the aminoacid sequence (of U.S.'s non-provisional application sequence number 10/863,729 that on June 7th, 2004 submitted to) of any VL CDR of EA44.In other embodiments, chimeric antibody specificity of the present invention comprises 1,2 or 3 VH CDR in conjunction with EphA2 and in people's framework region, described VH CDR has the aminoacid sequence of any VH CDR of EA2, EA5,12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

[0338] in other embodiments, chimeric antibody specificity of the present invention comprises 1,2 or 3 VH CDR in conjunction with EphA4 and in people's framework region, described VH CDR has the aminoacid sequence (of U.S.'s non-provisional application sequence number 10/863,729 that on June 7th, 2004 submitted to) of any VH CDR of EA44.In other embodiments, chimeric antibody specificity of the present invention comprises 1 in conjunction with EphA2 and in people's framework region, 2 or 3 VL CDR and 1,2 or 3 VH CDR, described VL CDR has EA2, EA5,12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3, the aminoacid sequence of any VL CDR of 2B12 or 5A8, described VH CDR has EA2, EA5,12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3, the aminoacid sequence of any VH CDR of 2B12 or 5A8.In other embodiments, chimeric antibody specificity of the present invention comprises 1,2 or 3 VL CDR and 1,2 or 3 VH CDR in conjunction with EphA4 and in people's framework region, described VL CDR has the aminoacid sequence of any VL CDR of EA44, and described VH CDR has the aminoacid sequence of any VH CDR of EA44.

[0339] in further embodiment, chimeric antibody specificity of the present invention comprises 3 VL CDR and 3 VH CDR in conjunction with EphA2 and in people's framework region, described VL CDR has EA2, EA5,12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3, the aminoacid sequence of any VL CDR of 2B12 or 5A8, described VH CDR has EA2, EA5,12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3, the aminoacid sequence of any VH CDR of 2B12 or 5A8.

[0340] the corresponding residue of the CDR donor antibody commonly used of the framework residue in the framework region replaces to change, and preferably raising or reduction antigen are in conjunction with (ability).Available method well known in the art identifies that these frameworks replace, for example by the interaction modeling of CDR and framework residue is identified for antigen in conjunction with important framework residue, with carry out the sequence comparison with evaluation be positioned at specific position unusual framework residue (referring to, for example, U.S. Patent number 5,585,089; With Riechmann etc., 1988, Nature332:323 includes it in this paper by reference).

[0341] humanized antibody be can with certain predetermined antigens bonded antibody or its variant or its fragment, its framework region has the aminoacid sequence of human normal immunoglobulin basically, its CDR has the aminoacid sequence of non-human immunoglobulin basically.Humanized antibody comprises all (at least one basically, common two) variable domains, wherein all or all CDR districts are corresponding to non-human immunoglobulin (promptly basically, donor antibody) those CDR, all or basically all framework regions are those framework regions of human normal immunoglobulin consensus sequence.Preferably, humanized antibody also comprises normally at least a portion of the constant region for immunoglobulin of human normal immunoglobulin (Fc).This antibody generally contains the light chain and the variable domains of heavy chain at least.This antibody also can contain CH1 district, hinge region, CH2 district, CH3 district and the CH4 district of heavy chain.Can comprise that IgM, IgG, IgD, IgA and IgE and any isotype (comprise IgG from the immunoglobulin (Ig) of any kind ₁, IgG ₂, IgG ₃And IgG ₄) the selection humanized antibody.Constant domain normally needs the active complement of humanized antibody showed cell toxicity in conjunction with constant domain, normally IgG ₁Class.When not needing this cellular cytoxicity activity, constant region can be IgG ₂Class.Humanized antibody can contain the sequence of broad variety or isotype, and those of ordinary skills know that selection particular constant structural domain is to optimize required effector function.The framework region of humanized antibody and CDR district need not and female antibody sequence, for example donor CDR is accurately corresponding, perhaps can come the total framework of mutagenesis, thereby make the CDR in this site or framework residue not correspond to total antibody or input antibody (import antibody) by replacement, insertion or the disappearance of at least one residue.Yet this sudden change is not widely.Usually have at least 75%, more commonly have 90%, most preferably greater than 95% humanized antibody residue those residues corresponding to female antibody framework district (FR) and CDR sequence.

[0342] can adopt various techniques known in the art to prepare humanized antibody, these technology include but not limited to: CDR-grafting (european patent number EP 239,400; International publication number WO91/09967; And U.S. Patent number 5,225,539,5,530,101 and 5,585,089), frosting or reinvent (european patent number EP 592,106 and EP 519,596; Padlan, 1991, MolecularImmunology 28 (4/5): 489-498; Studnicka etc., 1994, Protein Engineering7 (6): 805-814; With Roguska etc., 1994, PNAS 91:969-973), chain reorganization (U.S. Patent number 5,565,332) and the disclosed technology of following document: for example U.S. Patent number 6,407, and 213,5,766,886,5,585,089, international publication number WO 9317105, Tan etc., 2002, J.Immunol.169:1119-25, Caldas etc., 2000, Protein Eng.13:353-60, Morea etc., 2000, method 20:267-79, Baca etc., 1997, J.Biol.Chem.272:10678-84, Roguska etc., 1996, Protein Eng.9:895-904, Couto etc., 1995, Cancer Res.55 (23Supp): 5973s-5977s, Couto etc., 1995, CancerRes.55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen etc., 1994, J.Mol.Biol.235:959-73, Jones etc., 1986, Nature 321:522-525, Riechmann etc., 1988, Nature 332:323, and Presta, 1992, Curr.Op.Struct.Biol.2:593-596.The corresponding residue of the CDR donor antibody commonly used of the framework residue in the framework region replaces to change, and preferably improves antigen in conjunction with (ability).Can identify that these frameworks replace by method well known in the art, for example by the interaction modeling of CDR and framework residue being identified for antigen in conjunction with important framework residue with carry out the sequence comparison is positioned at specific position with evaluation unusual framework residue.(referring to, Queen etc. for example, U.S. Patent number 5,585,089; Riechmann etc., 1988, Nature, 332:323 includes it in this paper by reference).

[0343] in addition, and then the anti--idiotype antibody that can adopt technology well known to those skilled in the art to utilize antibody of the present invention to produce.(referring to, for example, Greenspan and Bona, 1989, FASEB is J.7:437-444; And Nissinoff, 1991, J.Immunol.147:2429-2438).The invention provides the method for using the polynucleotide that contain code book invention antibody or its segmental nucleotide sequence.

The polynucleotide of encoding antibody

[0344] method of the present invention also is included under for example above defined height strictness, medium strictness or the low stringent hybridization condition polynucleotide with the multi-nucleotide hybrid of code book invention antibody.

[0345] can obtain described polynucleotide and measure the nucleotide sequence of these polynucleotide by any method known in the art.Because the aminoacid sequence of known antibodies, therefore can adopt method well known in the art to measure the nucleotide sequence of these antibody of coding, that is, the assembling mode of the Nucleotide codon of known coded specific amino acids can produce code book invention antibody or its segmental nucleic acid.The polynucleotide that can assemble this encoding antibody from the oligonucleotide of chemosynthesis (for example, Kutmeier etc., 1994, BioTechniques, 17:242 is described), in brief, this method comprises the synthetic overlapping oligonucleotide that contains this polypeptid coding sequence each several part, annealing also connects those oligonucleotide, the oligonucleotide that adopts pcr amplification to connect then.

[0346] or, can produce the polynucleotide of encoding antibody from the nucleic acid in suitable source.If contain clone's non-availability of the nucleic acid of the specific antibodies of encoding, but the sequence of this polypeptide is known (for example, see Figure 19), but then chemosynthesis or by pcr amplification or by clone from suitable source (for example, antibody cDNA library, or by the cDNA library of its generation, or from the preferred poly A+RNA of isolating nucleic acid wherein, express any tissue or the cell of this antibody, as selecting to be used for expressing the hybridoma of antibody of the present invention, for example with PTA-4572, PTA-4573, PTA-4574, PTA-4380, PTA-4381 is preserved in the clone of ATCC) obtain the nucleic acid of coding immunoglobulin (Ig), described pcr amplification adopts and can hold the synthetic primer of hybridization with this sequence 3 ' and 5 ', and described clone adopts the specific oligonucleotide probe of the specific gene sequence that will identify, for example the cDNA in the cDNA library of encoding antibody clone.Can adopt any method well known in the art that the amplification of nucleic acid that PCR produces is cloned in the reproducible cloning vector then.

[0347] in case measured the nucleotide sequence of antibody, can adopt nucleotide sequence working method well known in the art to operate the nucleotide sequence of antibody to produce the different antibody of aminoacid sequence, for example produce aminoacid replacement, disappearance and/or insertion, described method is recombinant DNA technology for example, site-directed mutagenesis, PCR etc. (referring to, for example, the described technology of following document: Sambrook etc., 1990, " molecular cloning, laboratory manual " (Molecular Cloning, A LaboratoryManual), second edition, cold spring harbor laboratory, cold spring port, volume such as New York and Ausubel, 1989, " up-to-date molecular biology method ", John Willie father and son publishing company, New York, these documents are included this paper in as a reference in full).

[0348] in concrete embodiment, can adopt conventional recombinant DNA technology that one or more CDR are inserted framework region.Described framework region can be natural generation or the consensus sequence framework region, preferred people's framework region (tabulation of people's framework region can referring to, for example, Chothia etc., 1998, J.Mol.Biol.278:457-479).Be incorporated into the antibody of EphA2 or EphA4 by the polynucleotide optimized encoding energy specificity of group frame district and CDR generation.Preferably, as mentioned above, can in framework region, produce one or more aminoacid replacement, and preferably, described aminoacid replacement can improve antibody and its antigenic combination.In addition, thus can adopt these methods that the one or more variable region cysteine residues that participate in intrachain disulfide bond and form are made aminoacid replacement or disappearance produces the antibody that lacks one or more intrachain disulfide bonds.The present invention includes other change of the polynucleotide that those skilled in the art will know that.

Antibody recombinant expressed

[0349] the recombinant expressed expression vector that needs to make up the polynucleotide that contain this antibody of encoding of antibody of the present invention, its derivative, analogue or fragment (for example, the heavy chain of antibody of the present invention or light chain or its part or single-chain antibody of the present invention).In case having obtained code book invents the heavy chain of certain antibody molecule or certain antibody or light chain or its part and (preferably contains heavy chain or light chain variable structural domain, but polynucleotide not necessarily) can adopt technology well known in the art to produce the used carrier of antibody by the recombinant DNA technology preparation.Therefore, this paper has described the polynucleotide that contain the nucleotide sequence of encoding antibody by expression and has prepared method of protein.Sequence that can adopt method well known to those skilled in the art to make up to contain encoding antibody and the suitable expression vector of transcribing and translate control signal.These methods comprise, for example genetic recombination in extracorporeal recombinant DNA technology, synthetic technology and the body.

[0350] therefore, the invention provides heavy chain or light chain variable structural domain or its part or the heavy chain or the light chain CDR of the heavy chain that contains coding antibody of the present invention, antibody or light chain, antibody, and the replicable vector of the nucleotide sequence that links to each other with the promotor operability.This carrier can comprise that the nucleotide sequence of this antibody constant region of encoding is (referring to, the open WO 86/05807 of PCT for example; The open WO89/01036 of PCT; With U.S. Patent number 5,122,464), the variable region of this antibody can be cloned into heavy chain that this carrier comes The expressed, complete light chain or the two.

[0351] can expression vector be transported in the host cell by routine techniques, cultivate cells transfected to produce antibody of the present invention by routine techniques then.Therefore, the present invention includes and contain code book invention antibody or its fragment or its heavy chain or light chain or its part or single-chain antibody of the present invention, and the host cell of the polynucleotide that link to each other with the allogeneic promoter operability.In expressing some embodiment of double-stranded antibody, thus can be in host cell the immunoglobulin molecules of carrier The expressed of co expression encoding heavy chain and light chain, the following detailed description in detail.

[0352] can utilize various host expresses carrier systems express antibody of the present invention (referring to, for example, U.S. Patent number 5,807,715).This host expression system provides and has produced the vehicle that is purified behind the encoding sequence interested, and provides with suitable nucleotide coding sequence and transform or cell that can expressed in situ antibody of the present invention during transfection.These systems include but not limited to: microorganism, for example bacterium (as intestinal bacteria (E.coli), Bacillus subtillis (B.subtilis)) that transforms with the recombinant bacteria phage DNA that contains antibody coding sequence, plasmid DNA or cosmid DNA expression vector; Yeast (as pichia spp (Saccharomyces Pichia)) with the recombination microzyme expression vector conversion that contains antibody coding sequence; Insect cell system with the recombinant virus expression vector that contains antibody coding sequence (for example, baculovirus) infection; With the recombinant virus expression vector that contains antibody coding sequence (for example, cauliflower mosaic virus, CaMV; Tobacco mosaic virus (TMV) TMV) infects or with the recombinant plasmid expression vector that contains antibody coding sequence (for example, Ti-plasmids) plant transformed cell system; Or comprise contain be derived from genomic promotor of mammalian cell (as metallothionein promoter) or mammalian virus promotor (as gland virus stage starting; The mammal cell line system (as COS, CHO, BHK, 293, NS0,3T3, PerC6 cell) of recombinant expression construct thing vaccinia virus 7.5K promotor).Preferably utilize, more preferably use the eukaryotic cell recombinant antibodies of The expressed (particularly for) expressing recombinant antibody such as colibacillary bacterial cell.

[0353] for example, mammalian cell (as Chinese hamster ovary cell (CHO)) is effective expression system (Foecking etc., Gene, 1986, the 45:101 of antibody together with carrier (as the main middle early gene promoter sub-element of human cytomegalic inclusion disease virus); Cockett etc., BioTechnology, 1990,8:2).In concrete embodiment, the expression of certain antibody or its segmental nucleotide sequence of encoding is regulated by constitutive promoter, inducible promoter or organizing specific type promotor, described antibody capable specificity is incorporated into EphA2 or EphA4 and exciting EphA2 or EphA4, the anticancer phenotype preferentially is combined on the cancer cells but not at the EphA2 or epi-position on the EphA4 and/or the K that expose or increase selectively on the non-cancer cells _OffLess than 3 * 10 ^-3s ^-1

[0354] in bacterial system, be favourable according to the many expression vectors of the application choice of expressed antibody.For example, for producing the pharmaceutical composition of certain antibody, in the time will producing a large amount of this protein, required carrier should be able to instruct expresses the high-level fusion protein product that is easy to purifying.This carrier includes but not limited to: coli expression carrier pUR278 (Ruther etc., 1983, EMBO 12:1791), thus the antibody coding sequence in this carrier can connect into carrier separately and the lacZ coding region produces fusion rotein with frame; PIN carrier (Inouye and Inouye, 1985, Nucleic Acids Res.13:3101-3109; Van Heeke﹠amp; Schuster, 1989, J.Biol.Chem.24:5503-5509); Or the like.Also can utilize the pGEX carrier to express external polypeptide, for example contain the fusion rotein of glutathione S-transferase (GST).This fusion rotein generally is a solubility, by with the absorption of matrix gsh-agar beads with combine then in the presence of free glutathione the wash-out purifying from the cracked cell of being not difficult.The pGEX carrier design is cut the site for containing zymoplasm or factor Xa proteolytic enzyme, thereby can partly discharge clone's target gene product from GST.

[0355] in insect (cell) system, autographa california nuclear polyhedrosis virus (AcNPV) is as the carrier of expressing alien gene.This virus can be grown in greedy noctuid (Spodoptera frugiperda) cell in meadow.Antibody coding sequence can be cloned into the nonessential zone (for example luorourac gene) of this virus separately and place under the control of AcNPV promotor (for example luorourac promotor).

[0356] in mammalian host cell, can utilize many virus expression systems.In the situation of utilizing adenovirus as expression vector, interested antibody coding sequence and adenovirus can be transcribed/translated the control complex body, for example late promoter links to each other with three fens leader sequences.Can this mosaic type gene be inserted the adenoviral gene group by reorganization in external or the body then.Insert that virus genomic nonessential zone (for example, area E 1 or E3) can obtain to survive and can in the host who infects, express the recombinant virus (for example, referring to Logan and Shenk, 1984, PNAS 81:6355-6359) of antibody molecule interested.Also need the specificity start signal effectively to translate the antibody coding sequence that is inserted.These signals comprise the ATG initiator codon and adjoin sequence.In addition, initiator codon must be arranged in the frame of required encoding sequence to guarantee to translate whole insertion (sequence).These exogenous translation control signals and initiator codon can be various sources, natural and synthetic.Can improve the efficient of expressing by comprising suitable transcriptional enhancer element, transcription terminator etc.(referring to Bittner etc., 1987, Methods in Enzymol.153:516-544).

[0357] in addition, can select and to regulate the expression of insertion sequence or the host cell strain of modification and processed gene product with required ad hoc fashion.This modification of protein (for example, glycosylation) and processing (for example, cutting) may be most important to this proteinic function.Different host cells have characteristic and specific mechanism for the translation post-treatment and the modification of protein and gene product.Can select suitable clone or host system to guarantee the correct modification and the processing of expressed extraneous protein.For this purpose, can utilize and have primary transcript and suitably process the eukaryotic host cell of gene product glycosylation and phosphorylation cell mechanism.This mammalian host cell includes but not limited to: CHO, VERO, BHK, HeLa, COS, MDCK, 293,3T3, W138, BT483, Hs578T, HTB2, BT2O, NS1 and T47D, NS0 (endogenous does not produce the rat bone marrow tumour cell system of any immunoglobulin chain), CRL7O3O and HsS78Bst cell.

[0358] be that extended high rate amount ground produces recombinant protein, best stably express.For example, can be engineered can stably express antibody clone.Except utilization contains the expression vector of virus replication starting point, the available DNA transformed host cell that is subjected to suitable expression controlling elements (for example promotor, enhancer sequence, transcription terminator, polyadenylic acid site etc.) and can selects marking of control.After introducing foreign DNA, culturing engineering is transformed in enrichment medium cell 1-2 days.Change selective medium then over to.But the selective marker in the recombinant plasmid can be given selecting the resistance of (pressure), and the karyomit(e) and the growth that make cell plasmid stably can be integrated into them form the cell kitchen range, and then clone and amplification are clone.The preferred clone that adopts this method to come engineered expressing antibodies.This engineered clone be particularly useful for the screening and the assessment can with the direct or indirect interactional compound of antibody.

[0359] can adopt many selective systems, include but not limited to: herpes simplex virus thymidine kinase (Wigler etc., 1977, Cell 11:223), glutamine synthetase, xanthoglobulin-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1992, Proc.Natl.Acad.Sci.USA, 48:202) and adenine phosphoribosyl transferase (Lowy etc., 1980, Cell, 22:817) gene can be respectively applied in tk-, gs-, hgprt-or the aprt-cell.Also can utilize the basis of metabolic antagonist resistance: give dhfr (Wigler etc., 1980, PNAS, 77:357 to the methotrexate resistance as the following gene of selection; O ' Hare etc., 1981, PNAS, 78:1527); Give gpt to the mycophenolic acid resistance (Mulligan and Berg, 1981, PNAS, 78:2072); Give neo (Wu and Wu, 1991, Biotherapy, 3:87 to aminoglycoside G-418 resistance; Tolstoshev, 1993, Ann.Rev.Pharmacol.Toxicol., 32:573; Mulligan, 1993, Science, 260:926; Morgan and Anderson, 1993, Ann.Rev.Biochem., 62:191; May, 1993, TIB TECH, 11:155); With give hygro to hygromycin resistance (Santerre etc., 1984, Gene, 30:147).Can conventionally use the known ordinary method in recombinant DNA technology field and select required recombinant clone, this method is described in volumes such as Ausubel, " up-to-date molecular biology method " (Current Protocols in Molecular Biology), (the John Wiley﹠amp of John Willie father and son publishing company; Sons), New York (1993); Kriegler, " transgenosis and expression, laboratory manual " (Gene Transfer and Expression, A LaboratoryManual), Stockton press (Stockton Press), New York, (1990); Volumes such as Dracopoli, " human genome fresh approach " (Current Protocols in Human Genetics), the 12nd and 13 chapters, (the John Wiley﹠amp of John Willie father and son publishing company; Sons), New York, (1994); Colberre-Garapin etc., 1981, J.Mol.Biol., 150:1 includes them in this paper by reference in full.

[0360] (summary is seen Bebbington and Hentschel can to improve the expression level of antibody by carrier amplification, " for the gene of cloning by expression in Mammals utilizes carrier according to gene amplification in dna clone " (The use of vectors based on gene amplification for theexpression of cloned genes in mammalian cells in DNA cloning), the 3rd volume, (academic press (Academic Press), New York, 1987)).In the time of in certain is marked at the carrier system of expressing antibodies, can increasing, improve the copy number that the inhibitor level that exists in the host cell nutrient solution will increase this marker gene.Because the zone of being increased links to each other with antibody gene, antibody production also be increased (Crouse etc., 1983, Mol.Cell.Biol., 3:257).

[0361] available two kinds of common transfection host cells of expression vector of the present invention, the first vector encoded heavy chain polypeptides derived, the polypeptide of the second vector encoded derived light chain.But these two kinds of carriers can contain identical selective marker, thereby can express heavy chain polypeptide and light chain polypeptide with being equal to.Perhaps, the two a kind of carrier of utilizable energy coding (with expressing) heavy chain polypeptide and light chain polypeptide.In this case, light chain should be placed heavy chain before in order to avoid deleterious free heavy chain excessive (Proudfoot, 1986, Nature, 322:562; Kohler, 1980, PNAS, 77:2197).The encoding sequence of heavy chain and light chain can comprise cDNA or genomic dna.

[0362] in case produced antibody of the present invention by recombinant expressed, can come purifying by the known any method in immunoglobulin molecules purifying field, for example chromatography (as, ion-exchange, avidity are particularly utilized the avidity to this specific antigen behind A albumen and molecular size column chromatography), any other standard technique of centrifugal, difference solvability or protein purification.In addition, antibody of the present invention or its fragment and other allogeneic polypeptide sequence described herein or known in the art can be merged to promote purifying.

The preventing/treating method

[0363] the present invention includes in method treatment, prevention or the controlled member with crossing of EphA2 or EphA4 and express relevant disease or illness and/or the super proliferative disease of cell, especially cancer, method, described method comprise give significant quantity can targeted expression EphA2 or the cell of EphA4 and suppress EphA2 or the expression of EphA4 or function, and/or super hyperplasia sexual cell disease had treat or the composition of prevention effects.In one embodiment, method of the present invention comprises to object uses a kind of composition, and said composition comprises EphA2 or the EphA4 targeting moiety that links to each other with anti-super hyperplasia sexual cell treatment of diseases or prophylactic agent.In other embodiments, method of the present invention comprises to object uses a kind of composition that contains nucleic acid, and described nucleic acid comprises nucleotide sequence and the treatment of the anti-super proliferative disease of coding or the nucleotide sequence of prophylactic agent of coding EphA2 or EphA4 targeting moiety.In other embodiments, method of the present invention comprises to object uses a kind of composition, said composition comprises the nucleic acid of EphA2 or the EphA4 targeting moiety and the nucleotide sequence of treatment that contains the anti-super proliferative disease of coding or prophylactic agent, and wherein said targeting moiety directly links to each other with described nucleic acid or links to each other by the delivery vector that is delivered in the cell of expressing EphA2 or EphA4.In concrete embodiment, described EphA2 or EphA4 targeting moiety can also suppress expression or the activity of EphA2 or EphA4.

[0364] the present invention includes in treatment, prevention or the controlled member with crossing of EphA2 or EphA4 and express relevant disease or illness and/or the super proliferative disease of cell, preferred cancer, method, described method comprises that use can target EphA2 or EphA4 and/or suppress expression or active one or more ADC of EphA2 or EphA4, and wherein said ADC comprises that EphA2 or the exciting antibody of EphA4, EphA2 or EphA4 intracellular antibody or EphA2 or EphA4 cancer cell phenotype suppress the EphA2 of antibody or exposure or EphA4 epitope antibodies or with less than 3 * 10 ^-1s ^-1K _OffIn conjunction with EphA2 or the EphA4 antibody of EphA2 or EphA4, preferably the exciting antibody of one or more monoclonal EphA2 or EphA4, EphA2 or EphA4 intracellular antibody, BiTE molecule or EphA2 or EphA4 cancer cell phenotype suppress the EphA2 of antibody or exposure or EphA4 epitope antibodies or with less than 3 * 10 ^-1s ^-1K _OffEphA2 or EphA4 antibody in conjunction with EphA2 or EphA4.In concrete embodiment, the disease that treat, prevent or control is a malignant cancer.In other embodiments, the disease that treat, prevent or control is and expresses the relevant precancerous condition of cell of EphA2 or EphA4 excessively.In more concrete embodiment, described precancerous condition is high level prostatic intraepithelial neoplasm sample pathology (PIN), mammary gland fibroadenoma, fibrocystic disease or compound nevi.

In [0365] one embodiment, composition of the present invention can be used for the treatment of, crossing of prevention or control and EphA2 or EphA4 express relevant disease or one or more other treatment medicine combined administrations of illness, super proliferative disease and/or cancer.In some embodiments, the other treatment medicine that one or more compositions of the present invention and one or more are used for the treatment of cancer is applied to Mammals simultaneously, preferred people.Term " simultaneously " is not limited to use prophylactic agent or curative in the identical time, and be meant sequentially and in certain time interval, give object, thereby make the benefit that the present composition can play a role and be better than otherwise using to provide with other medicaments with the present composition and other pharmacy applications.For example, can use various prophylactic agents or curative successively with any order in the identical moment or in different time points; Yet,, should use them so that required treatment or preventive effect is provided in the very approaching time if not the while dispenser.Can adopt any suitable form and use various curatives respectively by any suitable way.In other embodiments, composition of the present invention before surgical operation, simultaneously or use afterwards.Preferably, described surgical operation can be removed local tumor fully or be reduced the size of tumour greatly.Surgical operation also can be used as preventative means or is used to ease the pain.

[0366] in further embodiment, the present composition comprises one or more EphA2 antibody that is made of following antibody: EA2, EA5,12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8 or table 23 or Fig. 1-59 in listed any antibody, wherein said antibody is as the medicament of EphA2-targeting moiety or anti-super hyperplasia sexual cell disease.In one embodiment, composition of the present invention comprise the antibody that constitutes by following antibody: EA2, EA5, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8 or table 23 or Fig. 1-59 in listed any by humanized antibody.In other embodiments, EA2 is provided, EA5, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8, the perhaps variant of table 1 or 2 listed any antibody, for example, especially the variant that in variable domains, has one or more aminoacid replacement, with EA2, EA5, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8, perhaps table 23 or Fig. 1-59 in listed any antibody compare, described variant has the activity of raising, binding ability or the like.

[0367] again in further embodiment, composition of the present invention comprises one or more EphA2 antibody (described in for example following document: U.S.'s non-provisional application sequence number 10/994,129 (submissions on November 19th, 2004), 10/436,782 (submission on May 12nd, 2003) and U.S. Provisional Application sequence numbers 60/583,184 (submissions on June 25th, 2004), 60/624,153 (submissions on November 2nd, 2004), 60/601,634 (submissions on August 16th, 2004), 60/608,852 (submissions on September 13rd, 2004), all documents are included this paper by reference in full in), wherein said antibody is as the medicament of EphA2 targeting moiety or anti-super hyperplasia sexual cell disease.

[0368] more in other embodiments, composition of the present invention comprises one or more EphA4 antibody that are made of EA44 and (is described in, for example, U.S.'s non-provisional application sequence number 10/863 that on June 7th, 2004 submitted to, 729), wherein said antibody is as the medicament of EphA4 targeting moiety or anti-super hyperplasia sexual cell disease.In further embodiment, composition of the present invention comprises the antibody that is made of humanization EA44.In other embodiments, provide the variant of EA44, for example, especially had the variant of one or more aminoacid replacement in variable domains, compared with EA44, described variant has activity, binding ability of raising or the like.

Patient group

[0369] the invention provides treatment, prevention and control and express relevant disease or illness and/or super hyperplasia sexual cell disease with crossing of EphA2 or EphA4, especially cancer, method, this method is by this object that needs treatment or one or more compositions of the present invention of prevention significant quantity are arranged.In other embodiments, composition of the present invention can with one or more other treatment medicine combined administrations.Described object is Mammals preferably, as non-human primate (for example, ox, pig, horse, cat, dog, rat etc.) and primates (for example, monkey is as cynomolgus monkey (cynomolgous monkey) and people).In other embodiments, described to liking the people.

The object lesson of the cancer of the method treatment that [0370] available the present invention comprised included but not limited to express the cancer of EphA2 or EphA4.In further embodiment, described cancer is the cancer in epithelial cell source.The example of this cancer has lung cancer, colorectal carcinoma, prostate cancer, mammary cancer and skin carcinoma.Other cancers comprise bladder cancer, carcinoma of the pancreas and renal cell carcinoma and melanoma.Other cancers are listed by way of example but not are only limited to hereinafter described those.In concrete embodiment, method of the present invention can be used for treating and/or preventing primary tumor and shifts.

[0371] method and composition of the present invention comprise to suffer from or estimate with cancered object/patient (for example, have the genetic predisposition of specific types of cancer, once contacted carcinogens or be in the alleviation of particular cancers) use one or more compositions of the present invention.In the literary composition, " cancer " refers to primary or metastatic cancer.Can carry out treating or treating to cancer before this patient.Method and composition of the present invention can be used as a line or two wires cancer therapy.The present invention comprises that also treatment accepting the patient of other cancer therapies, and method and composition of the present invention can be used on before these other cancer therapies any side effect occurs or do not tolerate.The present invention also comprises and uses one or more compositions of the present invention with treatment or alleviate the method for intractable patient's symptom.In some embodiments, the cancer of certain therapy refractory is meant that at least some integral parts of cancer cells are not killed or their cell fission does not stop.Thereby can by any method known in the art in vivo or external evaluation cancer cells whether be the validity of determining the cancer cells treatment of refractory, " refractory " definition of adopting this area to accept during evaluation.In various embodiments, cancer is a refractory when the number of cancer cells does not significantly reduce or increases to some extent.The present invention also comprises and uses that one or more EphA2 or EphA4 ADC (as EphA2 or EphA4-targeting moiety and/or anticarcinogen) take place with the cancer of preventing cancer-prone patient or the method for recurrence.Preferably, the antibody moiety of ADC be following one or more: EA2, EA5, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8 or table 2 or 3 or Fig. 1-59 in listed any antibody.In other embodiments, the exciting antibody of EphA4 that is used for ADC composition of the present invention and method is EA44.

[0372] in concrete embodiment, using composition of the present invention reduces resistance or the sensitivity that some hormone, radiation and chemotherapy medicine produce with inverse cancer cell, thereby make cancer cells again to one or more sensitivities in these reagent, just can use (or continuing to use) these reagent then with treatment or control cancer, comprise the prevention transfer.In concrete embodiment, the present composition is applied to the patient that cytokine IL-6 level raises, and known IL-6 level raises relevant to producing resistance such as different treatment plans such as chemotherapy and hormonotherapy with cancer cells.In other embodiments, composition of the present invention is applied to the reactivity reduction of tamoxifen treatment or the patient with breast cancer of refractory.In other embodiments, composition of the present invention is applied to the patient that cytokine IL-6 level raises, and known IL-6 level raises relevant to producing resistance such as different treatment plans such as chemotherapy and hormonotherapy with cancer cells.

[0373] in other embodiments, the invention provides treatment patient's method for cancer, this method with one or more compositions of the present invention and any other therapy combined administration in the patient who confirms no longer to adopt these therapies with other therapy refractories.Preferably, according to the present invention, can be with any antibody listed in EA2, EA5, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12,5A8, the table 2 or 3 or among the EA44 one or more as anti--EphA2 or anti--EphA4ADC.In some embodiments, the patient with method treatment of the present invention is the patient who treated with chemotherapy, radiotherapy, hormonotherapy or biotherapy/immunotherapy.Although comprise intractable patient among these patients and adopt the patient who has the cancer therapy treatment now but still suffer from cancer.In other embodiments, described patient uses one or more compositions of the present invention and is used for the preventing cancer recurrence through treating and not having disease activity.

[0374] in some embodiments, existing therapy is a chemotherapy.In concrete embodiment, existing therapy comprises uses chemotherapeutics (agent), comprising but be not limited to: methotrexate, taxol, purinethol, mercapto guanine, hydroxyurea, cytosine arabinoside, endoxan, ifosfamide, nitrosourea, cis-platinum, carboplatin, mitomycin, Dacarbazine, Procarbazine, etoposide, camptothecine (campathecin), bleomycin, Zorubicin, idarubicin, daunorubicin, dactinomycin, Plicamycin, mitoxantrone, asparaginase, vincaleucoblastine, vincristine(VCR), vinorelbine, taxol, docetaxel, or the like.Comprise the patient who treated with radiotherapy, hormonotherapy and/or biotherapy/immunotherapy among these patients.Also comprise the patient who accepted the cancer surgeries treatment among these patients.

[0375] or, the present invention comprises that also treatment accepting or accepting the patient's of radiotherapy method.Comprise the patient who is using or treating with chemotherapy, hormonotherapy and/or biotherapy/immunotherapy before among these patients.Also comprise the patient who accepted the cancer surgeries treatment among these patients.

[0376] in other embodiments, the present invention includes the method that the patient of hormonotherapy and/or biotherapy/immunotherapy was being accepted or accepting in treatment.Comprise the patient who uses or use before chemotherapy and/or radiotherapy in the treatment among these patients.Also comprise the patient who accepted the cancer surgeries treatment among these patients.

[0377] in addition, the present invention also provides the cancer treatment method as the alternative medicine of chemotherapy, radiotherapy, hormonotherapy and/or biotherapy/immunotherapy, above-mentioned therapy has confirmed maybe may confirm the toxicity of treatment target excessive, i.e. the side effect that is caused is unacceptable maybe can not be stood.Randomly, can use such as other cancer therapies treatments such as surgical operation, chemotherapy, radiotherapy, hormonotherapy or biotherapy with the object of the inventive method treatment, this depends on that finding which kind of therapy is unacceptable maybe can not stand.

[0378] in other embodiments, the present invention gives the patient with one or more present compositions and does not make up with the treatment cancer with any other cancer therapy, has proved that described patient is to these therapy refractories.In concrete embodiment, use one or more present compositions of patient of other cancer therapy refractories and do not take (other) cancer therapy.

[0379] in other embodiments, can use the present composition to patient to treat this disease and to reduce the possibility that develops into malignant cancer with precancerous condition relevant with the cell of expressing EphA2 or EphA4 excessively.In concrete embodiment, described precancerous condition is high level prostatic intraepithelial neoplasm sample pathology (PIN), mammary gland fibroadenoma, fibrocystic disease or compound nevi.

[0380] more in other embodiments, the invention provides treatment, prevent and control the super hyperplasia sexual cell disease of non-cancer, especially express the method for diseases associated with the mistake of EphA2 or EphA4, described disease includes but not limited to: asthma, chronic obstructive pulmonary disease (COPD), restenosis (unstriated muscle restenosis and/or endothelium restenosis), psoriatic, or the like.These methods comprise and above-mentioned treatment, prevention and the similar method of control method for cancer, for example, composition of the present invention and combination treatment can be applied to the patient of specific therapy refractory, or the like.

Cancer

[0381] cancer and the relative disease of available method and composition treatment of the present invention, prevention or control include but not limited to the cancer that epithelial cell is originated.The example of this cancer comprise following these: leukemia, such as but not limited to, acute leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia is as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia and myelodysplastic syndrome; Chronic leukemia, such as but not limited to, chronic myeloid (granulocytic) leukemia, lymphocytic leukemia, hairy cell; Polycythemia vera; Lymphoma, such as but not limited to, Hodgkin's disease, non-Hodgkin lymphoma; Multiple myeloma, such as but not limited to, SMM, nonsecreting type myelomatosis, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Macroglobulinemia Waldenstron; The MG that meaning is uncertain; Benign monoclonal gammopathy; Heavy chain disease; Bone and reticular tissue sarcoma, such as but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing sarcoma, pernicious giant cell tumor, the fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma, fibrosarcoma, Kaposi sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdosarcoma, synovial sarcoma; Cerebral tumor, such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-neurospongioma, acoustic nerve neurilemmoma, craniopharyngioma, medulloblastoma, meningioma, pinealoma, pineocytoma, primary brain lymphoma; Mammary cancer includes but not limited to, duct carcinoma, gland cancer, leaflet (minicell) cancer, intraductal carcinoma, marrow sample mammary cancer, mucus breast cancer, tubulose mammary cancer, nipple breast cancer, Paget's disease, inflammatory breast cancer; Adrenal carcinoma, such as but not limited to, pheochromocytoma and adrenocortical carcinoma; Thyroid carcinoma, such as but not limited to, corpora mammillaria or folliculus thyroid carcinoma, medullary thyroid carcinoma and undifferentiated type thyroid carcinoma; Carcinoma of the pancreas, such as but not limited to, insulinoma, gastrinoma, glucagonoma, VIPoma, Somatostatin secreting tumor and cancer or islet cells tumour; The pituitary gland cancer, such as but not limited to, hypercortisolism, prolactin secretion tumour, acromegaly, diabetes; Cancer eye, such as but not limited to, eye melanoma such as iris melanoma, melanoma of choroid, ciliary body melanoma, retinoblastoma; Carcinoma of vagina, as squamous cell carcinoma, gland cancer, malignant melanoma; External genital tumor, as squamous cell carcinoma, melanoma, gland cancer, rodent cancer, sarcoma, Paget's disease; Cervical cancer, such as but not limited to, squamous cell carcinoma and gland cancer; Uterus carcinoma, such as but not limited to, carcinoma of endometrium and sarcoma of uterus; Ovarian cancer, such as but not limited to, epithelial ovarian cancer, borderline tumor, gonioma, mesenchymoma; The esophageal carcinoma, such as but not limited to, squamous cell carcinoma, gland cancer, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmoma, verrucous carcinoma, and oat cell (minicell) cancer; Cancer of the stomach, such as but not limited to, gland cancer, gill fungus shape (polyp), ulcer, shallow diffusion, diffusivity diffusion, malignant lymphoma, liposarcoma, fibrosarcoma, sarcocarcinoma; Colorectal carcinoma; The rectum cancer; Liver cancer, such as but not limited to, hepatocellular carcinoma and hepatoblastoma; Carcinoma of gallbladder is as gland cancer; Cholangiocarcinoma, such as but not limited to, luoroura, nodositas cholangiocarcinoma and diffusivity cholangiocarcinoma; Lung cancer, as nonsmall-cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), gland cancer, large cell carcinoma and small cell lung cancer; Carcinoma of testis, such as but not limited to, gonioma, spermocytoma, undifferentiated carcinoma, classical (typical case) cancer, spermatocytic seminoma, nonseminoma, embryonal carcinoma cell, embryonal carcinoma, teratoma, choriocarcinoma (yolk sac tumor), prostate cancer, such as but not limited to, tumour in the prostatic epithelium, gland cancer, leiomyosarcoma, rhabdosarcoma; Penal cancers; Oral carcinoma, such as but not limited to, squamous cell carcinoma; The substrate cancer; The sialisterium cancer, such as but not limited to, mucoepidermoid adenocarcinoma, adenoid carcinoma; The pharynx cancer, such as but not limited to, squamous cell cancer, wart; Skin carcinoma, such as but not limited to, rodent cancer, squamous cell carcinoma and melanoma, superficial spreading melanoma, NM, lentigo, malignant melanoma, acral lentiginous melanoma; Kidney, such as but not limited to, renal cell carcinoma, gland cancer, grawitz's tumors of kidney, fibrosarcoma, transitional cell carcinoma (renal plevis and/or uterus); Wilms' tumor; Bladder cancer, such as but not limited to, transitional cell carcinoma, squamous cell cancer, gland cancer, sarcocarcinoma.In addition, cancer comprises myxosarcoma, osteosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial cancer, cystadenocarcinoma, bronchogenic carcinoma, syringocarcinoma, sebaceous carcinoma, papillary carcinoma and papillary carcinoma (can be referring to Fishman etc. for looking back these diseases, 1985, Medicine, second edition, Philadelphia JBL, Inc. (J.B.Lippincott Co), Philadelphia) and Murphy etc., 1997, " the decision-making of knowing the inside story: cancer diagnosis, treatment and rehabilitation pandect " (Informed Decisions:The Complete Book of Cancer Diagnosis, Treatment, and Recovery), Viking Penguin, U.S. penguin books company (Penguin Books U.S.A., Inc.)).

[0382] therefore, method and composition of the present invention also can be used for treatment or prevents various cancers or other paraplasm diseases, described paraplasm disease include, but is not limited to following these: cancer comprises bladder cancer, mammary cancer, colorectal carcinoma, kidney, liver cancer, lung cancer, ovarian cancer, carcinoma of the pancreas, cancer of the stomach, cervical cancer, thyroid carcinoma and skin carcinoma; Comprise squamous cell carcinoma; Lymphatic system hematopoiesis tumour comprises white corpuscle, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B cell lymphoma, t cell lymphoma, Burkitt lymphoma; Myeloid lineage hematopoiesis tumour comprises acute and chronic granulocytic leukemia and promyelocytic leukemia; The tumour in mesenchymal cell source comprises fibrosarcoma and rhabdosarcoma; Other tumours comprise melanoma, spermocytoma, teratocarcinoma, neuroblastoma and glioma; The tumour of maincenter and peripheral nervous system comprises astrocytoma, neuroblastoma, glioma, and schwannoma; The tumour in mesenchymal cell source comprises fibrosarcoma, rhabdosarcoma, and osteosarcoma; With other tumours, comprise melanoma, xeroderma pitmentosum, keratoacanthoma, spermocytoma, thyroid follcular carcinoma and teratocarcinoma.Also comprise with the cancer due to the inventive method and the distortion of combination treatment apoptosis.This cancer can include but not limited to follicular lymphoma, has the cancer of p53 sudden change, the hormone dependent tumor of mammary gland, prostate gland and ovary, and precancerous lesion such as familial adenomatous polyposis and myelodysplastic syndrome.In concrete embodiment, treatment or prevention be pernicious or paraplasm variation (as metaplasia and heteroplasia) in skin, lung, colon, mammary gland, prostate gland, bladder, kidney, pancreas, ovary or the uterus, perhaps super proliferative disease.In some embodiment, the treatment or the prevention be sarcoma, melanoma or white corpuscle.

[0383] in some embodiments, described cancer is virulent and crosses expression EphA2 or EphA4.In other embodiments, the disease that treat is and expresses the relevant precancerous condition of cell of EphA2 or EphA4 excessively.In concrete embodiment, described precancerous condition is high level prostatic intraepithelial neoplasm sample pathology (PIN), mammary gland fibroadenoma, fibrocystic disease or compound nevi.

[0384] in other embodiments, method and composition of the present invention is used for the treatment of and/or Breast Cancer Prevention, colorectal carcinoma, ovarian cancer, lung cancer and prostate cancer and melanoma, illustrates below but is not limited thereto.

The treatment of mammary cancer

[0385] in concrete embodiment, uses one or more compositions of the present invention of significant quantity to the patient with breast cancer.In one embodiment, the invention provides the method for a kind of prevention, treatment or control mammary cancer, this method comprises and gives the patient: (a) of the present invention anti--EphA2 or anti--EphA4ADC and (b) pharmaceutically acceptable carrier.In other embodiments, composition of the present invention can be used with one or more other agent combination that are used for the mammary cancer therapy of significant quantity.The medicament that is used for the mammary cancer therapy includes but not limited to: Zorubicin, epirubicin, the combination of adriamycin and cyclophosphamide (AC), the combination of endoxan, Zorubicin and 5 FU 5 fluorouracil (CAF), the combination of endoxan, epirubicin and 5 FU 5 fluorouracil (CEF), Trastuzumab, tamoxifen, the composition of tamoxifen and the agent of cell toxicant chemotherapeutics, Taxan (as docetaxel and taxol).In further embodiment, composition of the present invention can comprise Taxan and add the standard adriamycin and cyclophosphamide, or is used in combination with them, with the assisting therapy tubercle positive, local mammary cancer.

[0386] in concrete embodiment, use composition of the present invention to treat described disease and to reduce the possibility that they develop into malignant breast carcinomas for the patient who suffers from preceding fibroadenoma of mammary cancer or fiber cystic disease.In other embodiments, to treatment, especially hormonotherapy more specifically is that the responseless patient of tamoxifen therapy uses composition of the present invention to treat cancer and/or to make patient no longer refractory or generation reaction.

The treatment of colorectal carcinoma

[0387] in concrete embodiment, use one or more compositions of the present invention of significant quantity for the colorectal carcinoma patient.In other embodiments, composition of the present invention comprise significant quantity one or more be used for the colorectal carcinoma therapy other reagent or with its coupling, described other reagent include but not limited to: the combination of 5-FU and folinic acid, the combination of 5-FU and LEVAMISOLE HCL, the combination (IFL) of irinotecan (CPT-11) or irinotecan, 5-FU and folinic acid.

Treatment of prostate cancer

[0388] in concrete embodiment, uses one or more compositions of the present invention of significant quantity to patients with prostate cancer.In other embodiments, composition of the present invention comprise significant quantity one or more be used for the prostate cancer therapy other reagent or with its coupling, described other reagent include but not limited to: external beam radiotherapy, a matter is implanted radio isotope (that is I, ¹²⁵Palladium, iridium), Leuprolide or other LHRH agonists, non-steroid class androgen antagonist (flutamide, Nilutamide, bicalutamide), steroid class androgen antagonist (cyproterone acetate), the combination of Leuprolide and flutamide, oestrogenic hormon such as DES, Chlortrianisoestrol, Ethinylestradiol, link coupled oestrogenic hormon U.S.P., the DES-bisphosphate, radio isotope such as strontium-89, the composition of external beam radiotherapy and strontium-89, two wires hormonotherapy such as aminoglutethimide, hydrocortisone, inactive flutamide, progesterone and KETOKONAZOL, low dosage Bo Nisong, or report and can make symptom produce subjective other chemotherapy regimens that improve and reduce the PSA level, comprising docetaxel, taxol, Emcyt/docetaxel, Emcyt/etoposide, Emcyt/vincaleucoblastine, and Emcyt/taxol.

[0389] in concrete embodiment, use composition of the present invention to treat described disease and illness and to reduce the possibility that they develop into the malignant prostate cancer for high-level prostatic intraepithelial neoplasm sample pathology (PIN) patient.

Melanomatous treatment

[0390] in concrete embodiment, uses one or more compositions of the present invention of significant quantity to melanoma patients.In other embodiments, composition of the present invention comprise significant quantity one or more be used for the melanoma cancer therapy other reagent or with its coupling, described other reagent include but not limited to: Dacarbazine (DTIC), nitrosourea such as carmustine (BCNU) and lomustine (CCNU), have the active reagent of appropriate single agents and comprise vinca alkaloids, platinic compound and Taxan, Dartmouth (Dartmouth) scheme (cis-platinum, BCNU and DTIC), interferon alpha (IFN-A), and interleukin-2 (IL-2).In concrete embodiment, one or more of significant quantity exciting monoclonal antibody of the present invention can (ILP) add melphalan (L-PAM) with stripped intensification limbs perfusions (isolated hyperthermic limbperfusion), add or do not add tumor necrosis factor-alpha (TNF-α), be applied to together have that brain shifts, bone shifts and the patient of compression of spinal cord so that and radiotherapy realize that together alleviating of symptom and tumour to a certain extent dwindle.

[0391] in concrete embodiment, use composition of the present invention to treat this disease and to reduce the possibility that it develops into malignant melanoma for the preceding compound nevi patient of cancer.

The treatment of ovarian cancer

[0392] in concrete embodiment, uses one or more compositions of the present invention of significant quantity to ovarian cancer patients.In other embodiments, composition of the present invention comprise significant quantity one or more be used for the ovarian cancer therapy other reagent or with its coupling, described other reagent include but not limited to: intraperitoneal radiotherapy such as P ³²Therapy, belly and the full radiotherapy of basin bone, cis-platinum, the composition of taxol (taxol) or docetaxel (Taxotere) and cis-platinum or carboplatin, the composition of endoxan and cis-platinum, the composition of endoxan and carboplatin, the composition of 5-FU and folinic acid, etoposide, liposomal doxorubicin, gemcitabine or Hycamtin.Also comprise one or more present compositions of significant quantity and taxol combined administration in the patient who suffers from the platinum refractory disease.Comprise and treat the patient who suffers from intractable ovarian cancer, treatment comprises uses following medicament: use ifosfamide for platinum refractory disease patient, after the failure of cis-platinum assembled scheme, use altretamine (HMM) conduct and remedy chemotherapy, and use tamoxifen to the detectable patient of cytoplasmic oestrogen-receptor level on the tumour.

The treatment of lung cancer

[0393] in concrete embodiment, use one or more compositions of the present invention of significant quantity for the small cell lung cancer patient.In other embodiments, composition of the present invention comprise significant quantity one or more be used for the lung cancer therapy other reagent or with its coupling, described other reagent include but not limited to: chest radiotherapy, cis-platinum, vincristine(VCR), Zorubicin and etoposide (single using or coupling), the combination of endoxan, Zorubicin, vincristine(VCR)/etoposide and cis-platinum (CAV/EP), adopt the local mitigation symptoms of segmental bronchus inner laser therapy, segmental bronchus inner support, and/or brachytherapy.

[0394] in some embodiment, one or more that give nonsmall-cell lung cancer patient combined administration significant quantity are used for other reagent of lung cancer therapy and one or more present compositions of significant quantity, described other reagent include but not limited to: palliative radiation therapy, the combination of cis-platinum, vincaleucoblastine and mitomycin, the combination of cis-platinum and vinorelbine, taxol, docetaxel or gemcitabine, carboplatin and taxol, the interstital brachytherapy,IBT that is used for damage in the segmental bronchus or the combination of stereotaxic radiosurgery.

Other prophylactic agent/curatives

[0395] in some embodiment, the invention provides the method for a kind of prevention, treatment or the super hyperplasia sexual cell disease of control, this method comprises to the patient to be used: (a) of the present invention anti--EphA2 or anti--EphA4ADC and (b) pharmaceutically acceptable carrier.In some embodiments, the invention provides the method for a kind of prevention, treatment or the super hyperplasia sexual cell disease of control, this method comprises one or more other therapies of combined administration and one or more present compositions, described other therapies for example, but be not limited to chemotherapy, radiotherapy, hormonotherapy, biotherapy/immunotherapy and/or surgical operation.

[0396] can be used for prophylactic agent/curative of the present invention includes but not limited to: the protein molecule includes but not limited to peptide, polypeptide, protein (protein that comprises posttranslational modification), antibody or the like; Or the inorganic or organic compound of small molecules (less than 1000 dalton); Or nucleic acid molecule, include but not limited to two strands or single stranded DNA or two strands or single stranded RNA, and the triple helical nucleic acid molecule.Prevention/remedies can be derived from any known organism (including but not limited to: animal, plant, bacterium, fungi and protobiont or virus) or derived from the synthetic molecules storehouse.

[0397] in concrete embodiment, can be used for prophylactic agent/curative of the present invention is kinase inhibitor, and described kinases is such as but not limited to AB L, ACK, AFK, AKT (for example, AKT-1, AKT-2 and AKT-3), ALK, AMP-PK, ATM, Aurora1, Aurora2, bARK1, bArk2, BLK, BMX, BTK, CAK, CaM kinases, CDC2, CDK, CK, COT, CTD, DNA-PK, EGF-R, ErbB-1, ErbB-2, ErbB-3, ErbB-4, ERK (for example, ERK1, ERK2, ERK3, ERK4, ERK5, ERK6, ERK7), ERT-PK, FAK, FGR (for example, FGF1R, FGF2R), FLT is (for example, FLT-1, FLT-2, FLT-3, FLT-4), FRK, FYN, GSK (for example, GSK1, GSK2, GSK3-α, GSK3-β, GSK4, GSK5), G-protein linked receptor kinases (GRK), HCK, HER2, HKII, JAK (for example, JAK1, JAK2, JAK3, JAK4), JNK (for example, JNK1, JNK2, JNK3), KDR, KIT, the IGF-1 acceptor, IKK-1, IKK-2, INSR (insulin receptor), IRAK1, IRAK2, IRK, ITK, LCK, LOK, LYN, MAPK, MAPKAPK-1, MAPKAPK-2, MEK, MET, MFPK, MHCK, MLCK, MLK3, NEU, NIK, pdgf receptor α, pdgf receptor β, PHK, PI-3 kinases, PKA, PKB, PKC, PKG, PRK1, PYK2, the p38 kinases, p135tyk2, p34cdc2, p42cdc2, p42mapk, p44mpk, RAF, RET, RIP, RIP-2, RK, RON, the RS kinases, SRC, SYK, S6K, TAK1, TEC, TIE1, TIE2, TRKA, TXK, TYK2, UL13, VEGFR1, VEGFR2, YES, YRK, ZAP-70, and these kinase whose all hypotypes (are for example seen Hardie and Hanks (1995) " protein kinase factor handbook (The Protein Kinase FactsBook), I and II, the San Diego, CA academic press (Academic Press, SanDiego, Calif.)).In further embodiment, one or more can be used for prophylactic agent/curative of the present invention be the Eph receptor kinase inhibitor (for example, EphA2, EphA4).In concrete embodiment, one or more can be used for the inhibitor that prophylactic agent/curative of the present invention is EphA2 or EphA4.

[0398] in other embodiments, one or more can be used for prophylactic agent/curative of the present invention is angiogenesis inhibitor, such as but not limited to: Angiotensin (Profibrinolysin fragment); Anti-angiogenic formation Antithrombin III; Blood vessel enzyme (Angiozyme); ABT-627; Bay 12-9566; Benefin; RhuMAb-VEGF; BMS-275291; Cartilage deutero-inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagen XVIII fragment); CH-296; Gro-β; Halofuginone; Heparinase; Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin (hCG); IM-862; Interferon alpha/β/γ; Interferon, rabbit inducible protein (IP-10); Il-1 2; Kringle 5 (Profibrinolysin fragment); Marimastat; Inhibitors of metalloproteinase (TIMP); The 2-methoxyestradiol; MMI 270 (CGS 27023A); MoAbIMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placental ribonuclease inhibitor; Inhibitors of plasminogen activator inhibitor; PF4 (PF4); The prinomastat; Prolactin antagonist 16kD fragment; Proliferin associated protein (PRP); PTK 787/ZK 222594; Retinoid; Solimastat; Shark amine; SS 3304; SU 5416; SU6668; SU11248; Tetrahydrocortisol-S; Tetrathiomolybdate; Thalidomide; Thrombospondin-1 (TSP-1); TNP-470; Transforming growth factor-beta (TGF-β); Vasculostatin; Angiostatin (calreticulin fragment); ZD6126; ZD6474; Farnesyl transferase inhibitor (FTI); And diphosphonate.

[0399] in other embodiments, one or more can be used for prophylactic agent/curative of the present invention is anticarcinogen, for example, but is not limited to: U 42126; Aclarubicin; The hydrochloric acid acodazole; Acronine; U 73975; RIL-2; Altretamine; Ambomycin; The acetate ametantrone; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperline; A word used for translation is pricked cytidine; Ah's TEPA; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene hydrochloride; Two methylsulfonic acid Bisnafides; U 77779; Bleomycin sulfate; Brequinar sodium, Bropirimine; Busulfan; Sanarnycin; Clausterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin hydrochloride; U 80244; Cedefingol; Chlorambucil, Cirolemycin; Cis-platinum; Carat Qu Bin; The methylsulfonic acid crisnatol; Endoxan; Cytosine arabinoside; Dacarbazine, dactinomycin, daunorubicin hydrochloride; Decarbazine; Decitabine; U 78938; Dezaguanine; The methylsulfonic acid Dezaguanine; Diaziquone; Docetaxel; Zorubicin; Doxorubicin hydrochloride; Droloxifene; K-21060E; Dromostanolone propionate; Duazomycin; Edatrexate; Vaniqa; Elsamitrucin; Enloplatin; En Pumei; Epoxy third pyridine; Epirubicin hydrochloride; R 55104; Esorubicin hydrochloride; Estramustine phosphate sodium; Estramustine phosphate sodium; Etanidazole; Etoposide; The phosphoric acid etoposide; Etoprine; CGS-16949A; Fazarabine; Fenretinide; Floxuridine; Fludarabine phosphate; Fluracil; Flurocitabine; Fosquidone; Phosphotrienin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Thio ALP; Interleukin-22 (comprise recombinant interleukin 2, or rIL2); Intederon Alpha-2a; Interferon Alpha-2b; Interferon alfa-n1; Alferon N; Interferon beta-Ia; Interferon-gamma-Ib; Yi Pu platinum; U 101440E; Lanreotide acetate; Letrozole; Leuprorelin acetate; Liarozole hydrochloride; Lometrexol sodium; Lomustine; Losoxantrone hydrochloride; Aetinex; Maytenin; Mustine hydrochlcride; Magace; Melengestrol acetate; Melphalan; Menogaril; Purinethol; Methotrexate; Methotrexate sodium; U-197; Meturedepa; Mitindomide; Mi Tekaxin; Mitochromine; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nitrosourea; But promise azoles; U-15167; Ormaplatin; Oxisuran; Taxol; Asparaginase; Peliomycin; Neostigmine bromide; Perfosfamide; Pipobroman; A-20968; The hydrochloric acid piroxantrone; Plicamycin; Plomestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Tetracycline; Puromycin hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; The hydrochloric acid Safingol; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan sodium; Tegafur; Teloxandrone hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; ITG; Tioguanine; Plug is for group; Tiazofurine; Win-59075; FC-1157a; Trestolone; The phosphoric acid triciribine; Trimetrexate; Trimetrexate; Triptorelin; Tubulozole hydrochloride; Uracil mustard; Uredepa; Vapreotide; Visudyne; Vinblastine Sulfate; Vincristine sulphate; Vindesine; Vindesine sulfate; The sulfuric acid vinepidine; The sulfuric acid vinglycinate; Vinleurosine sulfate; Vinorelbine tartrate; Vinrosidine sulfate; The sulfuric acid vinzolidine; Vorozole; Zeniplatin; Zinostatin; Zorubicin hydrochloride.Preferred other anticarcinogens are 5 FU 5 fluorouracil and folinic acid.

[0400] in more concrete embodiment, the present invention also comprises and uses one or more compositions of the present invention so that the above-mentioned mammary cancer of preferred therapeutic, ovarian cancer, melanoma, prostate cancer, colorectal carcinoma and lung cancer, described composition comprise one or more therapeutical agents or with its coupling, described therapeutical agent is such as but not limited to anticarcinogen as shown in table 5.

Table 5

[0401] the present invention also comprises the combined administration present composition and radiotherapy, comprises using x ray, gamma-rays and other radioactive sources with destruction of cancer cells.In some embodiments, radiotherapy gives as external beam radiation or teletherapy, wherein radiates from the far-end radioactive source.In other embodiments, radiotherapy gives as inner therapy or brachytherapy, and wherein radioactive source places the interior place near cancer cells or lump of body.

[0402] dosage of novel remedies for cancer known in the art and they, route of administration and suggestion usage have been described in such as in " pharmacist's desk reference " (Physician ' s Desk Reference) documents such as (the 58th editions, 2004).

Preparation

[0403] can utilize one or more physiologically acceptable carriers or vehicle to prepare pharmaceutical compositions for use of the present invention in a usual manner.Therefore, the present composition and their physiologically acceptable salt and solvate can be mixed with through sucking or be blown into (by oral cavity or nose) or oral, parenteral or mucous membrane (for example, containing clothes, vagina, rectum, hypogloeeis) administration.In other embodiments, adopt part or general parenteral admin.

[0404] for oral administration, described pharmaceutical composition can be taked, tablet or the capsule form of for example utilizing pharmaceutically acceptable vehicle to prepare by conventional methods, described vehicle for example has: tamanori (as, W-Gum, polyvinylpyrrolidone or the Vltra tears of gelatinization in advance (pregelatinised)); Weighting agent (as, lactose, Microcrystalline Cellulose or secondary calcium phosphate); Lubricant (as, Magnesium Stearate, talcum powder or silicon-dioxide); Disintegrating agent (as, yam starch or Explotab); Or wetting agent (as, Sodium Lauryl Sulphate BP/USP).Can be by method coated tablet well known in the art.The liquid preparation of orally give can be taked following form, for example solution, syrup or suspension, and perhaps the form that they can dryed product exists, and water or other suitable vehicle re-use after making up.Can utilize pharmaceutically acceptable additive to prepare this liquid preparation by conventional methods, described additive for example has: suspension agent (as, sorbitol syrups, derivatived cellulose or hydrogenant edible fat); Emulsifying agent (as, Yelkin TTS or gum arabic); Non-aqueous vehicle (as, Prunus amygdalus oil, grease, ethanol or fractionated vegetables oil); Sanitas (as, methyl p-hydroxybenzoate or propyl ester or Sorbic Acid).These preparations also can suitably contain buffering salt, seasonings, pigment and sweeting agent.

[0405] can suitably prepare the preparation of oral administration and the controlled release of active ingredient thing.

[0406] for containing the clothes administration, described composition can be taked the tablet or the lozenge form of usual manner preparation.

[0407] for inhalation, can be contained in the aerosol spray form in the compression wrap or in the atomizer, utilize suitable propelling agent, for example Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas come routine to send used prophylactic agent of the present invention or curative.In the situation of pressurized aerosol, can determine dose unit by the valve that the transmissibility metered amount is provided.Can be formulated as and contain compound and suitable powder base, for example powdered mixture of lactose or starch being used for for example gelatine capsule of sucker or insufflator and medicine box.

[0408] prophylactic agent or curative can be formulated as by injection, for example be used for parenteral admin by injecting or infusing continuously.The preparation that is used to inject can be a unit dosage, for example is contained in to contain in the ampoule or multi-dose container that has added sanitas.Described composition can be taked suspension, solution or the emulsion form such as oiliness or the preparation of water-based vehicle, and can contain formulated (formulatoryagent), for example suspension agent, stablizer and/or dispersion agent.Perhaps, activeconstituents can be a powder type, and with suitable vehicle, use for example aseptic apirogen water preparation back.

[0409] prophylactic agent or curative also can be mixed with rectal compositions, and for example suppository or enema,retention (retention enemas) for example can contain, as conventional suppository bases such as theobroma oil or other glyceryl ester.

[0410], also prophylactic agent or curative can be formulated as prolonged action preparation except other above-mentioned preparation.Can give this prolonged action preparation by implanting (for example, subcutaneous or intramuscular) or intramuscular injection.Therefore, for example available suitable polymerization or hydrophobic material (as, the emulsion of acceptable oil preparation) or ion exchange resin preparation prophylactic agent or curative, or be formulated as and be difficult for molten derivative, as be formulated as and be difficult for molten salt.

[0411] the present invention also provides and is packaged in sealed vessel, for example indicates ampoule or the prophylactic agent in the pouch or the curative of content.In one embodiment, prophylactic agent or curative provide to be contained in the aseptic freeze-dried powder in the sealed vessel or not have aqueous concentrate, and available, for example water or salt solution give object after being reconstructed into suitable concn.

[0412] in other embodiment of the present invention, the preparation and the administration of various chemotherapy known in the art, biology/immunotherapy and hormonotherapy medicine, at " doctor's desk reference " (Physicians ' DeskReference), the 56th edition, description is often arranged in (2002).For example, in some embodiment of the present invention, curative of the present invention can and provide by the described form preparation of table 5.

[0413] in other embodiments of the present invention, can be with the radiotherapy medicine, for example radio isotope is to be contained in liquid or the beverage orally give in the capsule.Also the radio isotope preparation can be configured to the intravenous injection agent.Skilled oncologist can determine preferred preparation and route of administration.

[0414] in some embodiments, for the intravenous injection agent, composition of the present invention can be mixed with 1mg/ml, 5mg/ml, 10mg/ml and 25mg/ml, for subcutaneous administration and intramuscularly repeatedly, can be mixed with 5mg/ml, 10mg/ml and 80mg/ml.

[0415] if desired, these compositions can be contained in and comprise in one or more unit dosage that contain activeconstituents packing or the packaging device.For example, described packing can comprise metal or plastic foil, for example Blister Package.In packing or the packaging device administration working instructions can be housed.

Dosage and frequency

[0416] can treat the effective level of agent (for example, prophylactic agent or curative) or composition by the present invention that the standard clinical method is measured prevention, treatment, control and/or alleviated super proliferative disease or its one or more symptoms.Administration frequency and dosage also will change with each patient's particular case, this depends on severity, route of administration and patient's age, body weight, the reaction of the concrete treatment agent that given (for example, concrete one or more treatments or prophylactic agent), illness, disease or situation and medication history in the past.For example, can give animal model with described composition, animal model for example described herein or well known by persons skilled in the art is measured the prevention of the present invention that can effectively prevent, treat, control and/or alleviate super proliferative disease or its one or more symptoms or the dosage of curative or composition.In addition, can choose the employing in vitro tests wantonly and help identify the optimal dose scope.Those skilled in the art can be by considering these factors and following, and for example the dosage recommended of reference report and " doctor's desk reference " (Physicians ' DeskReference) (the 58th edition, 2004) is selected suitable scheme.

[0417] in various embodiment, (for example treat agent, prophylactic agent or curative) administration frequency as follows: at interval less than 1 hour, about 1 hour at interval, about 1 hour to about 2 hours at interval, about 2 hours to about 3 hours at interval, about 3 hours to about 4 hours at interval, about 4 hours to about 5 hours at interval, about 5 hours to about 6 hours at interval, about 6 hours to about 7 hours at interval, about 7 hours to about 8 hours at interval, about 8 hours to about 9 hours at interval, about 9 hours to about 10 hours at interval, about 10 hours to about 11 hours at interval, about 11 hours to about 12 hours at interval, be no more than 24 hours at interval, perhaps be no more than 48 hours at interval.In some embodiments, when going to a doctor, the homogeneous patient uses two or more components.

[0418] the term treatment effectively effectively comprises dosage provided herein and frequency with prevention.Described dosage and frequency also will change with each patient's particular case usually, and this depends on the seriousness of the concrete treatment that given or prophylactic agent, cancer and type, route of administration and patient's age, body weight, reaction and medication history in the past.Suitable scheme can make a choice by dosage of recommending in the dosage considering to report in this factor and following (for example) document and " the doctor's desk reference " (Physicians ' Desk Reference) (the 58th edition, 2004) by being proficient in those skilled in the art.

[0419] micromolecular exemplary dosage comprise every kilogram of object or example weight be microgram or milligram level consumption small molecules (for example, about 1 mg/kg-Yue 500 mg/kg, about 100 mg/kg-Yue 5 mg/kg, or about 1 microgram/kilogram-Yue 50 microgram/kilograms).

[0420] for the antibody that the present invention includes, protein, polypeptide, peptide and fusion rotein, the dosage that gives the patient is normally counted 0.0001-100mg/kg with weight in patients.Preferably, in weight in patients, the dosage that gives the patient is 0.0001-20mg/kg, 0.0001-10mg/kg, 0.0001-5mg/kg, 0.0001-2mg/kg, 0.0001-1mg/kg, 0.0001-0.75mg/kg, 0.0001-0.5mg/kg, 0.0001-0.25mg/kg, 0.0001-0.15mg/kg, 0.0001-0.10mg/kg, 0.001-0.5mg/kg, 0.01-0.25mg/kg or 0.01-0.10mg/kg.Owing to can produce immunne response to external polypeptide, people's antibody is longer than the antibody of other species usually in the intravital transformation period of people.Therefore, people's antibody often can be taked than low dosage and lower administration frequency.In addition, thus can reduce antibody of the present invention or its segmental dosage and frequency by picked-up and the tissue infiltration that for example lipid modification improve antibody.

[0421] in concrete embodiment, in weight in patients, be prevention, treatment, the control and/or the super proliferative disease of reduction of patient or its one or more symptoms and the dosage of the ADC of the present invention that gives is 150 μ g/kg or following, 125 μ g/kg or following, 100 μ g/kg or following, 95 μ g/kg or following, 90 μ g/kg or following, 85 μ g/kg or following, 80 μ g/kg or following, 75 μ g/kg or following, 70 μ g/kg or following, 65 μ g/kg or following, 60 μ g/kg or following, 55 μ g/kg or following, 50 μ g/kg or following, 45 μ g/kg or following, 40 μ g/kg or following, 35 μ g/kg or following, 30 μ g/kg or following, 25 μ g/kg or following, 20 μ g/kg or following, 15 μ g/kg or following, 10 μ g/kg or following, 5 μ g/kg or following, 2.5 μ g/kg or following, 2 μ g/kg or following, 1.5 μ g/kg or following, 1 μ g/kg or following, 0.5 μ g/kg or following or 0.5 μ g/kg or following.In other embodiments, the dosage of the ADC of the present invention that gives for super proliferative disease or its one or more symptoms of prevention, treatment, control and/or reduction of patient is following unitary dose: 0.1-20mg, 0.1-15mg, 0.1-12mg, 0.1-10mg, 0.1-8mg, 0.1-7mg, 0.1-5mg, 0.1-2.5mg, 0.25-20mg, 0.25-15mg, 0.25-12mg, 0.25-10mg, 0.25-8mg, 0.25-7m g, 0.25-5mg, 0.5-2.5mg, 1-20mg, 1-15mg, 1-12mg, 1-10mg, 1-8mg, 1-7mg, 1-5mg or 1-2.5mg.

[0422] in other embodiments, give object one or more the present invention a or many parts of significant quantities and (for example treat the agent method, treatment or prophylactic agent), the serum titer that this significant quantity can make the present invention treat agent (for example, treatment or prophylactic agent) reaches at least 0.1 μ g/ml, at least 0.5 μ g/ml, at least 1 μ g/ml, at least 2 μ g/ml, at least 5 μ g/ml, at least 6 μ g/ml, at least 10 μ g/ml, at least 15 μ g/ml, at least 20 μ g/ml, at least 25 μ g/ml, at least 50 μ g/ml, at least 100 μ g/ml, at least 125 μ g/ml, at least 150 μ g/ml, at least 175 μ g/ml, at least 200 μ g/ml, at least 225 μ g/ml, at least 250 μ g/ml, at least 275 μ g/ml, at least 300 μ g/ml, at least 325 μ g/ml, at least 350 μ g/ml, at least 375 μ g/ml or at least 400 μ g/ml.Again in other embodiments, give one or more ADC of the present invention of object significant quantity, thereby the serum titer that makes ADC of the present invention reaches at least 0.1 μ g/ml, at least 0.5 μ g/ml, at least 1 μ g/ml, at least 2 μ g/ml, at least 5 μ g/ml, at least 6 μ g/ml, at least 10 μ g/ml, at least 15 μ g/ml, at least 20 μ g/ml, at least 25 μ g/ml, at least 50 μ g/ml, at least 100 μ g/ml, at least 125 μ g/ml, at least 150 μ g/ml, at least 175 μ g/ml, at least 200 μ g/ml, at least 225 μ g/ml, at least 250 μ g/ml, at least 275 μ g/ml, at least 300 μ g/ml, at least 325 μ g/ml, at least 350 μ g/ml, at least 375 μ g/ml or at least 400 μ g/ml, one or more ADC of the present invention that give significant quantity subsequently can make serum titer maintain at least 0.1 μ g/ml, at least 0.5 μ g/ml, at least 1 μ g/ml, at least 2 μ g/ml, at least 5 μ g/ml, at least 6 μ g/ml, at least 10 μ g/ml, at least 15 μ g/ml, at least 20 μ g/ml, at least 25 μ g/ml, at least 50 μ g/ml, at least 100 μ g/ml, at least 125 μ g/ml, at least 150 μ g/ml, at least 175 μ g/ml, at least 200 μ g/ml, at least 225 μ g/ml, at least 250 μ g/ml, at least 275 μ g/ml, at least 300 μ g/ml, at least 325 μ g/ml, at least 350 μ g/ml, at least 375 μ g/ml or at least 400 μ g/ml.According to these embodiments, can give object 1,2,3,4,5,6,7,8,9,10,11,12 or more times subsequent dose.

[0423] in concrete embodiment, the invention provides prevention, treatment, control and/or the super proliferative disease of reduction of patient or the method for its one or more symptoms, described method comprises that one or more the present invention that the following dosage of the object of these needs is arranged treat agent (for example, treatment or prophylactic agent), agent or composition are treated in combination: at least 10 μ g, preferred at least 15 μ g, at least 20 μ g, at least 25 μ g, at least 30 μ g, at least 35 μ g, at least 40 μ g, at least 45 μ g, at least 50 μ g, at least 55 μ g, at least 60 μ g, at least 65 μ g, at least 70 μ g, at least 75 μ g, at least 80 μ g, at least 85 μ g, at least 90 μ g, at least 95 μ g, at least 100 μ g, at least 105 μ g, at least 110 μ g, at least 115 μ g or at least 120 μ g.In other embodiments, the invention provides prevention, treatment, control and/or the super proliferative disease of reduction of patient or the method for its one or more symptoms, described method comprises per 3 days 1 time, preferred per 4 days 1 time, per 5 days 1 time, per 6 days 1 time, per 7 days 1 time, per 8 days 1 time, per 10 days 1 time, per 2 weeks 1 time, per 3 weeks have one or more ADC of the present invention of this following dosage of object that needs for 1 time or 1 time every month, agent or composition are treated in combination: at least 10 μ g, preferred at least 15 μ g, at least 20 μ g, at least 25 μ g, at least 30 μ g, at least 35 μ g, at least 40 μ g, at least 45 μ g, at least 50 μ g, at least 55 μ g, at least 60 μ g, at least 65 μ g, at least 70 μ g, at least 75 μ g, at least 80 μ g, at least 85 μ g, at least 90 μ g, at least 95 μ g, at least 100 μ g, at least 105 μ g, at least 110 μ g, at least 115 μ g or at least 120 μ g.

[0424] the invention provides the super proliferative disease of prevention, treatment, control and/or reduction of patient or the method for its one or more symptoms, described method comprises: the object that (a) needs a or the prevention of many doses or one or more ADC of the present invention, combination treatment agent or the composition of treatment significant quantity; After (b) monitoring gives the described treatment agent (for example, treatment or prophylactic agent) of doses number, blood plasma level/concentration of the ADC that gives described in the described object.In addition, preferably agent is treated in the prevention of 1,2,3,4,5,6,7,8,9,10,11 or 12 doses or one or more ADC of the present invention, composition or the combination of treatment significant quantity to described doses number.

[0425] in concrete embodiment, the invention provides prevention, treatment, the method of control and/or the super proliferative disease of alleviation or its one or more symptoms, described method comprises: object at least 10 μ g dosage (the preferred at least 15 μ g that these needs (a) are arranged, at least 20 μ g, at least 25 μ g, at least 30 μ g, at least 35 μ g, at least 40 μ g, at least 45 μ g, at least 50 μ g, at least 55 μ g, at least 60 μ g, at least 65 μ g, at least 70 μ g, at least 75 μ g, at least 80 μ g, at least 85 μ g, at least 90 μ g, at least 95 μ g or at least 100 μ g) one or more the present invention treat agent (for example, treatment or prophylactic agent); (b) when the blood plasma level of the ADC that is given in the described object be lower than 0.1 μ g/ml, preferably be lower than 0.25 μ g/ml, when being lower than 0.5 μ g/ml, being lower than 0.75 μ g/ml or being lower than 1 μ g/ml, give described object a or many parts of subsequent dose.In other embodiments, the invention provides prevention, treatment, control and/or alleviate super proliferative disease or the method for its one or more symptoms, described method comprises: the object portion of these needs or many parts at least 10 μ g dosage (preferred at least 15 μ g (a) are arranged, at least 20 μ g, at least 25 μ g, at least 30 μ g, at least 35 μ g, at least 40 μ g, at least 45 μ g, at least 50 μ g, at least 55 μ g, at least 60 μ g, at least 65 μ g, at least 70 μ g, at least 75 μ g, at least 80 μ g, at least 85 μ g, at least 90 μ g, at least 95 μ g or at least 100 μ g) one or more ADC of the present invention; After (b) monitoring gives the doses number, the blood plasma level of the ADC that gives in the described object; (c) when the blood plasma level of the ADC that gives in the described object be lower than 0.1 μ g/ml, preferably be lower than 0.25 μ g/ml, when being lower than 0.5 μ g/ml, being lower than 0.75 μ g/ml or being lower than 1 μ g/ml, give the ADC of the present invention of described object subsequent dose.Preferably, one or more ADC of the present invention of the described doses number significant quantity that is 1,2,3,4,5,6,7,8,9,10,11 or 12 doses.

[0426] according to the inventive method, can be co-administered being used for or being used for prevention, treatment, control at present and/or alleviating the treatment agent (for example, prophylactic agent or curative) of super proliferative disease or its one or more symptoms and one or more ADC treat, prevent, control and/or alleviate super proliferative disease or its one or more symptoms except that ADC of the present invention.Preferably, the dosage of used prophylactic agent of combination therapy of the present invention or curative is lower than and is used for or is used for prevention, treatment, control at present and/or alleviates those dosage of super proliferative disease or its one or more symptoms.The recommended dose that is used for the medicine of prevention, treatment, control and/or the super proliferative disease of alleviation or its one or more symptoms at present can obtain by any document from this area, described document includes but not limited to: volumes such as Hardman, 2001, " the therapeutic pharmacological basis " of Goodman and Gilman (Pharmacological Basis OfBasis Of Therapeutics), the 10th edition, McGee mountain, New York (McGraw-Hill, New York); " doctor's desk reference " (Physician ' s Desk Reference) (PDR), and the 58th edition, 2004, New Jersey Meng Teweier medical economics company limited (Medical Economics Co., Inc., Montvale, NJ); These two parts of documents are included this paper in as a reference in full.

[0427] in various embodiment, these treat agent (for example, prophylactic agent or curative) at interval less than 5 minutes, at interval less than 30 minutes, 1 hour at interval, about 1 hour at interval, about 1-is about 2 hours at interval, about 2 hours-Yue 3 hours at interval, about 3 hours-Yue 4 hours at interval, about 4 hours-Yue 5 hours at interval, about 5 hours-Yue 6 hours at interval, about 6 hours-Yue 7 hours at interval, about 7 hours-Yue 8 hours at interval, about 8 hours-Yue 9 hours at interval, about 9 hours-Yue 10 hours at interval, about 10 hours-Yue 11 hours at interval, about 11 hours-Yue 12 hours at interval, about 12 hours-18 hours at interval, 18 hours-24 hours at interval, 24 hours-36 hours at interval, 36 hours-48 hours at interval, 48 hours-52 hours at interval, 52 hours-60 hours at interval, 60 hours-72 hours at interval, 72 hours-84 hours at interval, interval 84 hours-96 hours or interval gave in 96 hours-120 hours.In other embodiments, when going to a doctor, use two or more and treat agent with a patient.

[0428] in some embodiments, one or more ADC of the present invention that give capable of circulation treat agent (for example, prophylactic agent or curative) with one or more other.The circulation therapy is included in and (for example gives the first treatment agent certain period, first prophylactic agent or curative), phase gives second and (for example treats agent at a time then, second prophylactic agent or curative), randomly, the phase gives the 3rd treatment agent (for example, prophylactic agent or curative) or the like at a time then, repeat this and give in turn, promptly carry out this and circulate and reduce one of convection potential agent and produce tolerance, avoid or reduce the side effect for the treatment of one of agent and/or improve the effectiveness for the treatment of agent.

[0395] in some embodiments, can give identical ADC of the present invention repeatedly, administration can be at interval at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or at least 6 months.In other embodiments, can give repeatedly except that ADC of the present invention same treatment agent (for example, prophylactic agent or curative), administration can be at interval at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or at least 6 months.

Embodiment

[0429] refers now to following examples and describe the present invention.Giving these embodiment only is to be illustrative purposes, and the present invention should not be construed as and is confined to these embodiment by any way, and be understood to include Yin Benwen explanation and conspicuous any and all change form.

Embodiment 1

The generation of various antibody construction things and expression

[0431] six kinds of Humanized monoclonal antibodies (G5,10D3,12G3,1E11,4C10,4B11) and a kind of people/little mouse chimeric antibody (EA5) of the anti-common antigen EphA2 of generation.All these antibody are expressed not good in mammalian cell.Replace to determine on these positions, having of the influence of one or more preferred amino acids residues at these antibody the 40th, 60 and/or 61 one or more heavy chains of generation separately output.Six kinds of humanized antibodies contain L-Ala on the H40 position, the H60 of these antibody and H61 position are replaced by L-Ala and aspartic acid respectively.The chimeric antibody EA5 of anti-same antigen does not contain any preferred amino acid at H40, H60 or H61 position.Produce two independent heavy chains of EA5, one contains replacement at 60 and 61, and another contains replacement at H40, H60 and H61 position.The concrete amino-acid residue (seeing Figure 1B) of modified heavy chain is as described below.In all situations, cause 40,60 and 61 replacements can significantly improve output (seeing Table 6) with one or more preferred heavy chain residues.What is interesting is that when EA5 did not contain any preferred amino acids, heavy chain A60/D61 combination itself can significantly improve productive rate.

Material and method

[0432] The generation of antigen-specific antibodies, sign and clone: the universal method of generation, screening, clone and expressing antibodies is well known by persons skilled in the art.Referring to, for example, " up-to-date molecular biology method ", volumes such as F.M.Ausubel, John Willie father and son publishing company (strange Chester, England, 1998); " molecular cloning laboratory manual " (Molecular Cloning:A LaboratoryManual), the third edition, volumes such as J.Sambrook, press of cold spring harbor laboratory (Cold SpringHarbor Laboratory Press) (cold spring port, New York, 2001); " antibody laboratory manual " (Antibodies:A Laboratory Manual), E.Harlow and D.Lane compile, press of cold spring harbor laboratory (cold spring port, New York, 1988); " antibody use laboratory manual " (Using Antibodies:A Laboratory Manual), E.Harlow and D.Lane compile, and press of cold spring harbor laboratory (cold spring port, New York, 1999) includes them in this paper by reference in full.

[0433] The generation that heavy chain replaces: with the variable region of heavy chain of the variable region of light chain of antibody cloning G5,10D3,12G3,1E11,4C10,4B11 and EA5 and antibody cloning G5,10D3,12G3,1E11,4C10,4B11 and EA5 be cloned into respectively mainly early stage immediately (hCMVie) enhanser of coding human cytomegalic inclusion disease virus, promotor and 5 '-mammalian expression vector (Boshart etc. of non-translational region, 1985, Cell 41:521-30).In this system, people γ 1 chain with people κ chain secrete (Johnson etc., 1997, J.Infect.Dis.176:1215-24).(Quick Change Multi Mutagenesis Kit, Stratagene CA) introduce all heavy chains according to the explanation of manufacturers by site-directed mutagenesis and replace the quick change multiple spot mutagenesis kit of employing California Si Jiate genome company.Specifically, adopt following primer in clone G5,10D3,12G3,1E11,4C10 and 4B11, to introduce S60A/A61D:

5 '-ACACAACAGAGTACGCTGACTCTGTGAAGGGTAGAGTCACCATT-3 '; Heavy chain antibody clone G5/M, 10D3/M, 12G3/M, 1E11/M, 4C10/M and 4B11/M have so been produced; Adopt following primer that N60A/Q61D is introduced EA5:

5 '-GTTACAATGGTGTTACTAGCTACGCCGACAAGTTCAAGGGCAAGGCCAC-3 ' and

5 '-GTGGCCTTGCCCTTGAACTTGTCGGCGTAGCTAGTAACACCATTGTAAC-3 ' has produced EA5/M ' thus; And adopt following primer that S40A/N60A/Q61D is introduced EA5:

5’-CTACATGCACTGGGTCAAGCAGGCCCATGGAAAGAGCCTTGAG-3’、

5’-CTCAAGGCTCTTTCCATGGGCCTGCTTGACCCAGTGCATGTAG-3’、

5 '-GTTACAATGGTGTTACTAGCTACGCCGACAAGTTCAAGGGCAAGGCCAC-3 ' and

5 '-GTGGCCTTGCCCTTGAACTTGTCGGCGTAGCTAGTAACACCATTGTAAC-3 ' has produced EA5/M thus.Notice that light chain keeps not changing (Figure 1A).Adopt ABI 3100 sequenators checking sequence.Adopt then fat transfection amine and standard method in the 6 hole plates of 35mm with various antibody construction thing transient transfection people's tire kidneys (HEK) 293 cells.After transfection, gather in the crops supernatant liquor (being called the 1st time and the 2nd cutting) 72 and 144 hours the time at twice.Detect excretory soluble human IgG1 (seeing below) according to output with combining of original antigen then.

[0434] Express the measurement of output: the expression output of measuring antibody cloning G5, G5/M, 10D3,10D3/M, 12G3,12G3/M, 1E11,1E11/M, 4C10,4C10/M, 4B11 and 4B11/Mut by ELISA.Adopting Anti-Human IgG ELISA to detect with 3 days is the interval antibody production of the transfection supernatant liquor (seeing above) of collection at twice.In brief, 96 hole Biocoat flat boards (San Jose, California BD biotechnology (the BD Biosciences of company of the anti-human IgG of goat will be coated with, San Jose, CA)) each hole and sample (supernatant liquor) or standard substance (human IgG, 0.5-100ng/ml) cultivate together, cultivate together with the anti-human IgG antibody's of goat horseradish peroxidase thing then.With 3,3 ', 5,5 '-tetramethyl benzidine detects peroxidase activity and uses 0.2M H ₂SO ₄Termination reaction.Read dull and stereotyped at 450nm.The results are summarized in table 6.

Table 6: the output of heavy chain modified antibodies improves ^a

^aHEK 293 cells are with various antibody construction thing transient transfections.

^bH1=is cutting (after the transfection 72 hours) for the first time.

^cH2=after-crop thing (after the transfection 144 hours).

^dIncrease multiple=various cuttings (H1, H2) result that the mean yield of unmodified antibody is divided by in mean yield of " Mut " antibody of middle heavy chain modification and the various cutting.

Embodiment 2

The solid phase elutriation is to identify clone 1C1

[0435] use the EphA2-Fc of 20 μ g/ml of 0.1M carbonate buffer solution (pH 9.6, Sigma company (Sigma)) preparation to be coated with immune test tube, 4 ℃ of cultivations are spent the night.Phage library (Fab310, Dyax Corp (Dyax)) precipitates with the 20%PEG (fluorine Lu Ka company (Fluka)) of 1/5 volume and is resuspended in PBS (pH 7.4).Then with 2% milk sealing phage library, with non--EphA2 bonded monoclonal antibody cancellation selected (to remove the Fc binding substances).After sealing and cancellation are selected, phage library is transferred to the immune test tube that scribbles EphaA2, this immunity test tube seals with 2% milk.Wash immune test tube 10-20 time with PBST (PBS+0.1% tween) after 2 hours, wash 10-20 time to remove unconjugated phage with PBS then.With 1ml 100mM triethylamine (Sigma company) bonded phage and add 0.5ml 1M Tris-HCl (pH 7.5, hero company (Invitrogen)) and neutralize under the wash-out from the immune test tube.Then 1 volume wash-out and neutral phage are mixed with 5 volume logarithmic phase TG1 cells (Nuo Fogen company (Novagen)) and 4 volume 2YT (Plutarch Novartis Co.,Ltd (Teknova)).Sample is cultivated 30 minutes (water-bath) in 37 ℃.Be resuspended in 2YT with the centrifugal sample of 4000g and with throw out then.Cell is coated on the 2YT agar plate (Plutarch Novartis Co.,Ltd) that contains Pyocianil and 2% glucose.Flat board spends the night in 30 ℃ of cultivations.Collect colony in second day and use helper phage (hero company) to infect.The cell that infects in the 2YT that contains Pyocianil (hero company) and kantlex (Sigma company) in 30 ℃ of overnight incubation to produce the height phage of tiring.From incubated overnight liquid, precipitate phage, carry out second according to said process then and take turns elutriation.Second takes turns elutriation is resisted-EphA2 antibody cloning 1C1.

The generation of anti--EphA2 antibody: 1F12,1H3,1D3,2B12 and 5A8

[0436] adopt display technique of bacteriophage to identify the Fab that is incorporated into EphA2.The phage library fab310 of Dyax Corp is used for the lysogenic bacteriophage elutriation.Phage library is selected (to remove the Fc binding substances) with the sealing of 2% milk and with non--EphA2 in conjunction with the monoclonal antibody cancellation.The nail (unit of length) pearl (dynabead) (wearing nail (unit of length) biotechnology company (Dynal Biotech)) of wearing of streptavidin coating is sealed with 1% milk.The nail (unit of length) pearl (hero company) of wearing that makes sealing and the selected phage of cancellation contact the biotinylated EphA2 of 2.9 μ g and EphA2-phage mixture is closed catches.With the phage of 1ml100mM triethylamine (Sigma company) elution of bound and add among the 0.5ml 1M Tris-HCl and elutriant.For infecting, the TG1 (Nuo Fogen company) and the 4 volume 2YT (Plutarch Novartis Co.,Ltd) that the phage elutriant and 5 volumes of 1 volume are in logarithmic phase mix.This mixture was cultivated 30 minutes in 37 ℃ water-bath.Infect the back and be resuspended in 2YT with centrifugal 5 minutes of 4000g and with throw out.The TG-1 cell is coated on the 2YT flat board (Plutarch Novartis Co.,Ltd) that contains 50ug/ml Pyocianil and 2% glucose upward and with flat board to spend the night in 30 ℃ of cultivations.Collect bacterial colony in second day and use helper phage (hero company) to infect.The cell that makes infection in the 2YT substratum that contains Pyocianil (hero company) and kantlex (Sigma company) grow overnight to produce the height phage of tiring.From incubated overnight liquid, concentrate phage by the PEG precipitation.The PEG precipitation adopts the PEG/NaCl solution (PEG of fluorine Lu Ka company) of nutrient solution 1/5 volume to carry out.Post precipitation is resuspended in the phage throw out 1ml PBS (pH 7.4, hero company) and is used for the next round elutriation.Carry out the two-wheeled elutriation again, this moment, the concentration of biotinylated EphA2 was reduced to 2.0 μ g.Anti--EphA2 antibody 1F12,1D3,1H3 and 2B12 take turns elutriation from second, and anti--EphA2 antibody 5A8 is from the third round elutriation.

The transient expression of anti--EphA2 antibody

[0437] is the IgG antibody of The expressed, adopts standard molecular biological technique that the variable region of heavy chain of antibody and light chain is cloned into the mammalian cell IgG expression vector pABOE that contains IgG1/ or IgG1/ antibody constant region.Heavy chain and light chain expression box are all under their CMVie promotor control separately.According to the method (hero company) of manufacturers the antibody gene transient transfection is gone into HEK 293F with the 293fectin transfection reagent.Collect 3 times in 9 days, make culture supernatant pass through A albumen post (GE health care company (GE health care)) afterwards and come protein purification.With the antibody of 50mM citrate buffer (pH 3.2) elution of bound, dialyse with PBS then.Analyze all proteins by the SDS-polyacrylamide gel electrophoresis, adopt BCA test kit (Pierre Si company (PIERCE)) to carry out quantitative ELISA to determine antibody concentration.

Embodiment 3

The cell surface combination of α-EphA2 antibody

[0438] 4 ℃ in 96 hole v base plates, with 1 μ g the one Ab (1C1,1F12,1H3 and 3F2a-EphA2Ab and R347 isotype contrast) with 2 * 10 in the 150 μ l FACS damping fluids (PBS+2% foetal calf serum+L-glutaminate) ⁵ Cell dyeing 30 minutes.Use 2 * cold PBS washed cell then and at 4 ℃ with the 2nd Ab (the anti-human IgG of phycoerythrin (Phycoerythin) link coupled goat, biogenic company (Biosource)) dyeing 30 minutes.FACS flow cytometry (FACS Calibur flow cytometer) with BD biotechnology company carries out fluorometric analysis.This result of experiment is summarized in Figure 14 A and 14B, and these results confirm that these antibody each have the ability in conjunction with the people who expresses on the tumour cell, mouse and rat EphA2.

Embodiment 4

The internalization of α-EphA2 antibody

[0439] will resist-EphA2 antibody (B233, B208 and EA5) and identification anti--monoclonal antibody (mAb) of second saporin (toxin) mark of EphA2 antibody cultivates and introduces the monolayer cell (MCF-10A) based on tumour cell altogether, cultivated 72-96 hour.Purpose is to measure necrocytosis, and necrocytosis shows that mAb mixture (conjugate of anti--EphA2 mAb and the 2nd mAb-saporin) is by internalization.This test is as the mAb of preliminary screening with the selection internalization.Referring to Kohls etc., Biotechniques, 2000-1; 28 (1): 162-5.This result of experiment is summarized in Figure 15 and 16 of this paper.

Embodiment 5

The fluorescent imaging of α-EphA2 antibody internalization

[0440] make cell (PC3, HUVEC or CT26) in 37 ℃/5%CO ₂Growth is 24-48 hour on the slide glass of Nunc Tek II8-chamber, and its concentration is the suitable growth medium 2.5-5.0 of the per 400 μ l in each chamber * 10 ⁴Individual cell.4 ℃ is an Ab (G5,1C1,1F12 or 3F2 anti--EphA2Ab and the contrast of R347 isotype) mark attached cell 30-45 minute of 50 μ g/ml with concentration.Use 2 * PBS washed cell then, then with growth medium covering cell and at 37 ℃/5%CO ₂Cultivate 0 minute, 20 minutes (Figure 18 A-C and 19) or 60 minutes (Figure 17) so that the one Ab internalization of cell surface bonded.

[0441] after the internalization, cell fixation (4% paraformaldehyde), Tonghua are handled (0.5%Triton X-100), and with the anti-human IgG Ab of the 2nd AlexaFluor 488 goats (biogenic company) mark, washed with 2 * cold PBS between each step.Cover cell with containing the VECTASHIELD sealing substratum (carrier laboratory company (Vector Labs)) of DAPI and cover glass at last, detect with fluorescence confocal microscope then and take pictures.Antibody internalization result of experiment is shown in the fluorescence microscopy picture of this paper Figure 16,17,18A, 18B, 18C and 19.EphA2 and exciting EphA2 antibodies and by the rapid internalization of these clones.

Embodiment 6

The EphA2 receptor activation

[0442] make cell in 37 ℃/5%CO ₂Grow overnight in 6 hole tissue culture plate, its concentration are the suitable growth mediums 0.5 * 10 of the every 3ml in each hole ⁶Individual cell.Remove the exhausted substratum in second day and be replaced with the fresh culture that contains 10 μ g1C1 or 1F12 α-EphA2Ab or the contrast of R347 isotype.With cell at 37 ℃/5%CO ₂Following cultivation is to activate the EphA2 acceptor.Use the 1%TritonX-100 lysis buffer cracking of phosphoric acid enzyme inhibitors mixture 1 and 2 (Sigma company) and adequate proteins enzyme inhibitors mixture tablet (Roche Holding Ag (Roche)) after the activation with 1 * cold PBS washed cell and on ice, additive adds according to the recommendation of manufacturers.Mix at 4 ℃ with lysate and spend the night being coupled to the D7 α-EphA2mAb of the anti-mouse IgG of rabbit and 50 μ l A albumen sepharose 4Bs in advance so that the protein immunoprecipitation.

[0443] differentiate the protein cleavage product according to the method for manufacturers with 10%Bis-Tris NuPAGE Western gel and by electrophoretic transfer to nitrocellulose filter (hero company).With trace and 1 μ g/ml first (mouse α Tyrosine O-phosphate IgG2bk, clone 4-G10, A Pusite company (Upstate)) and the second (peroxidase-link coupled goat anti-mouse IgG, Jackson's immune Research company (Jackson Immuno Research)) Ab cultivates together so that identify activatory (phosphorylation) protein band with Super Signal ECL test kit (Pierre Si company (Pierce)), and cultivates (develop) with the Hyperfilm of the husky biotech company of liking to be beautiful (Amersham Biosciences).Also can be referring to Coffman etc., Cancer Res.63:7907-7912,2003.Figure 20 that the results are summarized in this paper and 21 of EphA2 receptor activation test, result have confirmed that these antibody have the ability of EphA2 on activation various people, mouse and the rat tumor clone separately.

Embodiment 7

Eph acceptor cross reactivity ELISA test

[0444] for confirming that whether anti--EphA2 antibody 1C 1 and 1F12 show that with any Muridae member of acceptor Eph family any combination arranged, and have carried out following test.With PBS (pH 7.2) will resist-EphA antibody is diluted in 8 holes with 1: 2 since the concentration of 5 μ l/ml.With 50 μ l dilution antibody coating EIA/RIA ELISA flat board (Costar numbering 3690), 4 ℃ of cultivations are spent the night.Used El in second day _x405 automatic dull and stereotyped washer washings are dull and stereotyped, and washing procedure is the interval to vibrate 3 seconds for carrying out 5 sub-distribution/suction washing step with 1X PBST (1X PSB, 0.1% polysorbas20) therebetween.On a stacker towel, flat board is patted dry, sealed 1 hour with 240 μ l sealing damping fluid (2%BSA w/v is with 1X PBST preparation) under the room temperature.With Eph receptor biological elementization, the ratio of vitamin H/Eph acceptor molecule is 8 to the sulphur-NHS-vitamin H reagent (EZ-link sulfo-NHS-BiotinReagent) (Pierce numbering 21335) that connects with enzyme.Biotinylated Eph acceptor is with 50mM Tris-HCl (Invitrogen numbers 15506-017) cancellation and be diluted to 1 μ g/ml with the sealing damping fluid.Flat board is used El again _x405 automatic dull and stereotyped washer washings also pat dry.Adding the biotinylated Eph acceptor of 50 μ l dilution and 37 ℃ in each hole cultivated 1 hour.With flat board washing and dry, add the neutral avidin (neutravidin) of 50 μ l-HRP 1:12500 (Pierce numbering 31002) then as mentioned above.Cultivate after 1 hour flat board washing, Rotate 180 ° also washing once more for 37 ℃.Flat board patted dry and add 50 μ l SureBlueTMB peroxidase (KPL number 52-00-03) in each hole, cultivation (develop) is 5-10 minute then.With 50 μ l 0.2M H ₂SO ₄Termination reaction xx also reads the ELISA signal at 450nm.This result of experiment is summarized in Figure 23, and the result confirms that 1C1 can be in conjunction with mouse EphA2 and mouse EphA4, and 1F12 can be in conjunction with mouse EphA2,3,4,5,6,7,8 and mouse EphB1 and 2.

Embodiment 8

The growth in vitro inhibition test

[0445] The coupling of antibody

With Xie Ansuan-citrulline (vc) or maleimide caproyl-citrulline (mc) joint EphA2 is coupled to monomethyl Ao Ruisitating E (MMAE) or monomethyl Ao Ruisitating F (MMAF).According to the scheme (Doronina etc., the BioConjug Chem.2006 that describe before; Doronina etc., NatBiotech 2003) (Seattle Genetics Inc.) carries out antibody coupling in Seattle heredity company limited.

The growth in vitro inhibition test

[0446] make cell in 37 ℃/5%CO ₂Grow overnight in the 96 hole flat boards (FalconBD) that tissue culture is handled, cultivating concentration is to contain 2.0-3.0 * 10 in every hole 150 μ l growth mediums (RPMI 1640+10% foetal calf serum) ³Individual cell.Removed the exhausted substratum in second day and every hole is replaced with 120 μ l fresh cultures.It is dull and stereotyped and the various diluents of 30 μ l are transferred in the cell to prepare different drug dilution liquid.

[0447] with flat board at 37 ℃/5%CO ₂Cultivated again 3-4 days and with cell tire-total photogenic cell viability measures test kit (CellTiter-GloLuminescent Cell Viability Assay kit) (handkerchief agate coffee company (Promega)) results.Read the luminous cell survival that detects with Wallac Victor II flat bed reader.

[0448] in different growth in vitro inhibition tests, detects following cell: PC3, SKMEL-28, A549, MDA-MB-231,231KC, A375, HCT-116, SW620, MDA-MB-468, MDA-MB-435, T231, HUVEC, H460, M21, SKOV-3, HeyA8, Panc.02.03, DU145, ACHN, OVCAR-3, HT29, MCF10-A, F98 and CYNO-MK.In different growth in vitro inhibition tests, detect following antibody: G5vcMMAF, 3F2vcMMAE, 3F2vcMMAF, 3F2mcMMAF, EA5vcMMAF, 1A7MMAF, R347vcMMAF, R347mcMMAF, 1C1mcMMAF, 1F12mcMMAF, 1C1vcMMAE and 1F12vcMMAE.Figure 30 that the results are summarized in this paper-47 of the numerous different growth in vitro inhibition tests of being carried out, this result have confirmed that various EphA2 conjugates have the ability that specificity suppresses the tumor cell line growth of expression EphA2.

Embodiment 9

Detect effect in the body of anti--EphA2 ADC G5 with various cancer models

[0449] (human relations (Harlan, Somerville, NJ)) female mice subcutaneous injection 5 * 10 is breathed out in the Somerville, New Jersey to give the 4-6 athymia nu/nu in age in week ⁶Individual tumour cell.Shown in the accompanying drawing legend, the mean sizes of tumour reaches 100-150mm ³The back was given intraperitoneal cavity injection PBS or antibody drug conjugates in per 4 days, injected 5 doses altogether.Each treatment group is made of 10-12 mouse.

[0450] when pharmacological agent begins with regular (1-2 time weekly) measurement gross tumor volume of slide calliper rule.Figure 49 that the results are summarized in this paper-51 of these researchs.These results confirm, the G5 that is coupled to the MMAF that has the vc joint suppresses with dose-dependently mode specificity to grow in the body of PC3 and MDA-MB231 tumour.

Embodiment 10

In the prostate cancer model, detect effect in the body of anti--EphA2ADC 3F2

[0450] gives the 4-6 athymia nu/nu in age in week (human relations are breathed out in the Somerville, New Jersey) female mice subcutaneous injection 5 * 10 ⁶Individual tumour cell.Shown in the accompanying drawing legend, the mean sizes of tumour reaches 100-150mm ³The back was given intraperitoneal cavity injection PBS or antibody drug conjugates in per 4 days, injected 5 doses altogether.Each treatment group is made of 10-12 mouse.

[0451] when pharmacological agent begins with regular (1-2 time weekly) measurement gross tumor volume of slide calliper rule.Figure 52 that the results are summarized in this paper of this research.These results confirm, but the 3F2 specificity that is coupled to the MMAE that has the vc joint or has a MMAF of mc joint suppresses growth in the body of PC3 tumour.

Embodiment 11

In various cancer models, detect effect in the body of anti--EphA2ADC 1C1 and 1F12

[0452] gives the 4-6 athymia nu/nu in age in week (human relations are breathed out in the Somerville, New Jersey) female mice subcutaneous injection 5 * 10 ⁶Individual tumour cell.As shown in drawings, the mean sizes of tumour reaches 100-150mm ³The back was given intraperitoneal cavity injection PBS or antibody drug conjugates in per 4 days, injected 5 doses altogether.Dosage range is that 1mg/kg (20 μ g) arrives 10mg/kg (200 μ g), as described in respectively scheming as this paper (seeing the description of this paper Figure 53-56C).Each treatment group is made of 10-12 mouse.

[0453] when pharmacological agent begins with regular (1-2 time weekly) measurement gross tumor volume of slide calliper rule.Figure 53-the 56C that the results are summarized in this paper of these researchs.These results confirm, are coupled in the 1C1 of the MMAF that has the mc joint and 1F12 suppresses PC3 and MDA-MB231 tumour with dose-dependently mode specificity the body to grow, and well-tolerated.

Embodiment 12

Toxicity in vivo research

[0454] give the 4-6 female Balb/c mouse in age in week (human relations are breathed out in the Somerville, New Jersey) through tail vein injection (single bolus) PBS or antibody drug conjugates (be coupled to the MMAF that has the vc joint or be coupled to 1C1 and the 1F12 of the MMAF that has the mc joint), dosage level is as follows: vcMMAE antibody is 40mg/kg, 50mg/kg and 60mg/kg; 1C1-mcMMAF antibody is 120mg/kg, 180mg/kg and 240mg/kg; And 1F12-mcMMAF antibody is 90mg/kg, 120mg/kg, 180mg/kg, 210mg/kg and 240mg/kg.14 days daily observed value and measured body weight value behind the record drug administration.Each treatment group is made of 3-4 mouse.Any confirmation has the animal of symptom (bow-backed, breathe impaired, vigor and reduce, lose weight greater than 20%, or the like) to pass through CO ₂Suffocate and implement hommization euthanasia.Figure 58 that the results are summarized in this paper of these toxicity in vivo research has shown the relative tolerance of various antibody drug conjugates because with lose weight relevant.

[0455] though described the specific embodiment of the present invention for purposes of illustration, those skilled in the art should know and can make many changes and not break away from the described the present invention of the claims of enclosing all details.

[0456] this specification sheets mention all deliver thing, patent or patent application and all in full include this specification sheets in as a reference, just looking like every part, to deliver thing, patent or patent application special separately and show that separately to include this paper in full such as a reference.

Sequence table

＜110〉Med Muniz Co., Ltd (MedImmune and Seattle Genetics)

M.S. golden strange (Kinch, Michael S)

C. the Bach (Bachy, Christine)

D. safe this (Tice, David)

H. Wu (Wu, Herren)

High longevity (Gao, Changsou)

P.D. cent (Senter, Peter)

＜120〉toxin conjugated Eph receptor antibody

<130>EP120PCT

<150>60/735,966

<151>2005-11-14

<160>166

＜170〉PatentIn version 3 .3

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gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?cagagtcacc 60

atcacttgcc?gggccagtca?gagtattagt?acttggttgg?cctggtatca?gcagaaacca 120

gggaaagccc?ctaaactcct?gatctataag?gcatctaatt?tacatacggg?ggtcccatct 180

aggttcagcg?gcagtggatc?tggaacagaa?ttcagtctca?ccatcagcgg?cctgcagcct 240

gatgattttg?caacctatta?ttgccaacaa?tataatagtt?attctcggac?gttcggccaa 300

gggaccaagg?tggaaatcaa?a 321

<210>3

<211>128

<212>PRT

＜213〉artificial

<220>

＜223〉humanized antibody variable region

<400>3

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?His?Tyr

20 25 30

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

35 40 45

Ser?Arg?Ile?Gly?Pro?Ser?Gly?Gly?Pro?Thr?His?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?Tyr?Asp?Ser?Gly?Tyr?Asp?Tyr?Val?Ala?Val?Ala?Gly?Pro?Ala

100 105 110

Glu?Tyr?Phe?Gln?His?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120 125

<210>4

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉humanized antibody VL district

<400>4

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?Ser?Ile?Ser?Thr?Trp

20 25 30

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

35 40 45

Tyr?Lys?Ala?Ser?Asn?Leu?His?Thr?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

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

65 70 75 80

Asp?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Ser?Tyr?Ser?Arg

85 90 95

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

100 105

<210>5

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>5

His?Tyr?Met?Met?Ala

1 5

<210>6

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>6

Arg?Ile?Gly?Pro?Ser?Gly?Gly?Pro?Thr?His?Tyr?Ala?Asp?Ser?Val?Lys

1 5 10 15

Gly

<210>7

<211>19

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>7

Tyr?Asp?Ser?Gly?Tyr?Asp?Tyr?Val?Ala?Val?Ala?Gly?Pro?Ala?Glu?Tyr

1 5 10 15

Phe?Gln?His

<210>8

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>8

Arg?Ala?Ser?Gln?Ser?Ile?Ser?Thr?Trp?Leu?Ala

1 5 10

<210>9

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>9

Lys?Ala?Ser?Asn?Leu?His?Thr

1 5

<210>10

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>10

Gln?Gln?Tyr?Asn?Ser?Tyr?Ser?Arg?Thr

1 5

<210>11

<211>387

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>11

gaagttcaat?tgttagagtc?tggtggcggt?cttgttcagc?ctggtggttc?tttacgtctt 60

tcttgcgctg?cttccggatt?cactttctct?cgttaccaga?tgatgtgggt?tcgccaagct 120

cctggtaaag?gtttggagtg?ggtttcttct?atctctcctt?ctggtggcgt?tactctttat 180

gctgactccg?ttaaaggtcg?cttcactatc?tctagagaca?actctaagaa?tactctctac 240

ttgcagatga?acagcttaag?ggctgaggac?acagccgtgt?attactgtac?gagagaactt 300

ttgggtactg?tagtagtacc?agttgcatgg?aaaatgcgtg?gctactttga?ctactggggc 360

cagctcaccc?tggtcaccgt?ctcaagc 387

<210>12

<211>324

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>12

gacatccaga?tgacccagtc?tccaggcacc?ctgtctgtgt?ctccagggga?aagagccacc 60

ctctcctgca?gggccagtca?gagtgttagc?agcaacttag?cctggtacca?gcagaaacct 120

ggccaggctc?ccaggctcct?catctatggt?gcatccacca?gggccactgg?tatcccagcc 180

aggttcagtg?gcagtgggtc?tgggacagag?ttcactctca?ccatcagcag?catgcagtct 240

gaagattttg?cagtttatta?ctgtcagcag?tataataact?ggcccccgct?cactttcggc 300

ggagggacca?aggtggagat?caaa 324

<210>13

<211>129

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>13

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?Arg?Tyr

20 25 30

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

35 40 45

Ser?Ser?Ile?Ser?Pro?Ser?Gly?Gly?Val?Thr?Leu?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

Thr?Arg?Glu?Leu?Leu?Gly?Thr?Val?Val?Val?Pro?Val?Ala?Trp?Lys?Met

100 105 110

Arg?Gly?Tyr?Phe?Asp?Tyr?Trp?Gly?Gln?Leu?Thr?Leu?Val?Thr?Val?Ser

115 120 125

Ser

<210>14

<211>108

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>14

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

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Met?Gln?Ser

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Asn?Trp?Pro?Pro

85 90 95

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

100 105

<210>15

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>15

Arg?Tyr?Gln?Met?Met

1 5

<210>16

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>16

Ser?Ile?Ser?Pro?Ser?Gly?Gly?Val?Thr?Leu?Tyr?Ala?Asp?Ser?Val?Lys

1 5 10 15

Gly

<210>17

<211>20

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>17

Glu?Leu?Leu?Gly?Thr?Val?Val?Val?Pro?Val?Ala?Trp?Lys?Met?Arg?Gly

1 5 10 15

Tyr?Phe?Asp?Tyr

20

<210>18

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>18

Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala

1 5 10

<210>19

<211>8

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>19

Gly?Ala?Ser?Thr?Arg?Ala?Ser?Thr

1 5

<210>20

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>20

Gln?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Leu?Thr

1 5 10

<210>21

<211>354

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>21

gaagttcaat?tgttagagtc?tggtggcggt?cttgttcagc?ctggtggttc?tttacgtctt 60

tcttgcgctg?cttccggatt?cactttctct?atgtacgcta?tgcgttgggt?tcgccaagct 120

cctggtaaag?gtttggagtg?ggtttctgtt?atcggtcctt?ctggtggctg?gactccttat 180

gctgactccg?ttaaaggtcg?cttcactatc?tctagagaca?actctaagaa?tactctctac 240

ttgcagatga?acagcttaag?ggctgaggac?acggccgtgt?attactgtgc?gagagatcgg 300

ggcatttacg?gtatggacgt?ctggggccaa?gggaccacgg?tcaccgtctc?aagc 354

<210>22

<211>321

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>22

gacatccaga?tgacccagtc?tccatccttc?ctgtctgcat?ctgtgggaga?cagagtcacc 60

atcacttgcc?gggccagtca?gggcattagt?agttatttag?cctggtatca?gcaaaaacca 120

gggaaagccc?ctaagctcct?gatctatgct?gcatccactt?tgcaaagtgg?ggtcccatca 180

aggttcagcg?gcagtggatc?tgggacagaa?ttcactctca?caatcagcag?cctgcagcct 240

gaagattttg?caacttatta?ctgtctagaa?cttaataatt?accctttcac?tttcggcctt 300

gggaccaaag?tgcatatcaa?a 321

<210>23

<211>118

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>23

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?Met?Tyr

20 25 30

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

35 40 45

Ser?Val?Ile?Gly?Pro?Ser?Gly?Gly?Trp?Thr?Pro?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?Asp?Arg?Gly?Ile?Tyr?Gly?Met?Asp?Val?Trp?Gly?Gln?Gly?Thr

100 105 110

Thr?Val?Thr?Val?Ser?Ser

115

<210>24

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>24

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr?Ala?Ala?Ser?Thr?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?Glu?Leu?Asn?Asn?Tyr?Pro?Phe

85 90 95

Thr?Phe?Gly?Leu?Gly?Thr?Lys?Val?His?Ile?Lys

100 105

<210>25

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>25

Met?Tyr?Ala?Met?Arg

1 5

<210>26

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>26

Val?Ile?Gly?Pro?Ser?Gly?Gly?Trp?Thr?Pro?Tyr?Ala?Asp?Ser?Val?Lys

1 5 10 15

Gly

<210>27

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>27

Asp?Arg?Gly?Ile?Tyr?Gly?Met?Asp?Val

1 5

<210>28

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>28

Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Tyr?Leu?Ala

1 5 10

<210>29

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>29

Ala?Ala?Ser?Thr?Leu?Gln?Ser

1 5

<210>30

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>30

Leu?Glu?Leu?Asn?Asn?Tyr?Pro?Phe?Thr

1 5

<210>31

<211>366

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>31

gaagttcaat?tgttagagtc?tggtggcggt?cttgttcagc?ctggtggttc?tttacgtctt 60

tcttgcgctg?cttccggatt?cactttctct?ccttacgata?tgctttgggt?tcgccaagct 120

cctggtaaag?gtttggagtg?ggtttctcgt?atcggttctt?ctggtggcta?tactaagtat 180

gctgactccg?ttaaaggtcg?cttcactatc?tctagagaca?actctaagaa?tactctctac 240

ttgcagatga?acagcttaag?ggctgaggac?acggccgtgt?attactgtgc?gagagcccgc 300

agcgtagtgg?ttagctctga?tgcttttgat?atctggggcc?aagggacaat?ggtcaccgtc 360

tcaagc 366

<210>32

<211>321

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>32

gacatccaga?tgacccagtc?tccatcttct?gtgtctgcat?ctgtaggaga?cagagtcacc 60

atcacttgtc?gggcgagtca?gggtattagt?aagtggttag?cctggtatca?gcagaaacca 120

gggaaagccc?ctaagctcct?gatctttggt?gcatccactt?tgcaaagtgg?ggtcccatca 180

aagttcagcg?gcagtaaatc?tgggacagat?ttcactctca?ccatcagcag?cctgcagcct 240

gaagattctg?caacttatta?ctgccaacaa?tataatgatt?acccgctcac?tttcggcgga 300

gggaccaagg?tggagattaa?a 321

<210>33

<211>122

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>33

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?Pro?Tyr

20 25 30

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

35 40 45

Ser?Arg?Ile?Gly?Ser?Ser?Gly?Gly?Tyr?Thr?Lys?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?Ala?Arg?Ser?Val?Val?Val?Ser?Ser?Asp?Ala?Phe?Asp?Ile?Trp

100 105 110

Gly?Gln?Gly?Thr?Met?Val?Thr?Val?Ser?Ser

115 120

<210>34

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>34

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Ser?Lys?Trp

20 25 30

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

35 40 45

Phe?Gly?Ala?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Lys?Phe?Ser?Gly

50 55 60

Ser?Lys?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>35

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>35

Pro?Tyr?Asp?Met?Leu

1 5

<210>36

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>36

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

1 5 10 15

Gly

<210>37

<211>13

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>37

Ala?Arg?Ser?Val?Val?Val?Ser?Ser?Asp?Ala?Phe?Asp?Ile

1 5 10

<210>38

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>38

Arg?Ala?Ser?Gln?Gly?Ile?Ser?Lys?Trp?Leu?Ala

1 5 10

<210>39

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>39

Gly?Ala?Ser?Thr?Leu?Gln?Ser

1 5

<210>40

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>40

Gln?Gln?Tyr?Asn?Asp?Tyr?Pro?Leu?Thr

1 5

<210>41

<211>351

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>41

gaagttcaat?tgttagagtc?tggtggcggt?cttgttcagc?ctggtggttc?tttacgtctt 60

tcttgcgctg?cttccggatt?cactttctct?aattacaata?tgtattgggt?tcgccaagct 120

cctggtaaag?gtttggagtg?ggtttctgtt?atcgttcctt?ctggtggcaa?gacttcttat 180

gctgactccg?ttaaaggtcg?cttcactatc?tctagagaca?actctaagaa?tactctctac 240

ttgcagatga?acagcttaag?ggctgaggac?acggccgtgt?attactgtgc?gagatcgtac 300

ggagggggat?ttgactactg?gggccagggc?accctggtca?ccgtctcaag?c 351

<210>42

<211>321

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>42

gacatccaga?tgacccagtc?tccatcttcc?gtgtctgcat?ctgttggaga?caaagtcacc 60

atcacttgtc?gggcgagtca?ggatattctc?acctggttag?cctggtatca?gtggaaacca 120

gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca 180

aggttcagcg?gcagtggatc?tgggacagat?ttcactctca?tcatcgacac?cctgcagcct 240

gaggattttg?caacttacta?ctgtcaacag?gctatccgtt?tcccgctcac?tttcggcgga 300

gggaccaagg?tggagatcaa?g 321

<210>43

<211>117

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>43

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?Asn?Tyr

20 25 30

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

35 40 45

Ser?Val?Ile?Val?Pro?Ser?Gly?Gly?Lys?Thr?Ser?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?Ser?Tyr?Gly?Gly?Gly?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu

100 105 110

Val?Thr?Val?Ser?Ser

115

<210>44

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>44

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

1 5 10 15

Asp?Lys?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Leu?Thr?Trp

20 25 30

Leu?Ala?Trp?Tyr?Gln?Trp?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?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?Asp?Phe?Thr?Leu?Ile?Ile?Asp?Thr?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ala?Ile?Arg?Phe?Pro?Leu

85 90 95

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

100 105

<210>45

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>45

Asn?Tyr?Asn?Met?Tyr

1 5

<210>46

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>46

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

1 5 10 15

<210>47

<211>8

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>47

Ser?Tyr?Gly?Gly?Gly?Phe?Asp?Tyr

1 5

<210>48

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>48

Arg?Ala?Ser?Gln?Asp?Ile?Leu?Thr?Trp?Leu?Ala

1 5 10

<210>49

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>49

Ala?Ala?Ser?Ser?Leu?Gln?Ser

1 5

<210>50

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>50

Gln?Gln?Ala?Ile?Arg?Phe?Pro?Leu?Thr

1 5

<210>51

<211>375

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>51

gaagttcaat?tgttagagtc?tggtggcggt?cttgttcagc?ctggtggttc?tttacgtctt 60

tcttgcgctg?cttccggatt?cactttctct?tattaccgta?tgtattgggt?tcgccaagct 120

cctggtaaag?gtttggagtg?ggtttcttct?atctattctt?ctggtggccc?tacttattat 180

gctgactccg?ttaaaggtcg?cttcactatc?tctagagaca?actctaagaa?tactctctac 240

ttgcagatga?acagcttaag?ggctgaggac?acggccgtgt?attactgtgc?gaaagatatg 300

ggtaccggtt?tttggagtgg?ttggggccta?ggctctgact?actggggcca?gggaaccctg 360

gtcaccgtct?caagc 375

<210>52

<211>321

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>52

gacatccaga?tgacccagtc?tccatcttcc?gtgtctgcat?ctgtaggaga?cagagtcacc 60

atcacttgtc?gggcgagtca?gggtattagc?agctggttag?cctggtatca?gcagaaacca 120

gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca 180

aggttcagcg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?cctgcagcct 240

gaagattttg?caacttacta?ttgtcaacag?gctaacagtt?tccctctcac?tttcggcgga 300

gggaccaagg?tggagatcaa?a 321

<210>53

<211>125

<212>PRT

＜213〉artificial

<220>

＜223〉antibody variable region

<400>53

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?Tyr?Tyr

20 25 30

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

35 40 45

Ser?Ser?Ile?Tyr?Ser?Ser?Gly?Gly?Pro?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?Asp?Met?Gly?Thr?Gly?Phe?Trp?Ser?Gly?Trp?Gly?Leu?Gly?Ser

100 105 110

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

115 120 125

<210>54

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉antibody variable region

<400>54

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

1 5 10 15

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

20 25 30

Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?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?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>55

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>55

Tyr?Tyr?Arg?Met?Tyr

1 5

<210>56

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>56

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

1 5 10 15

Gly

<210>57

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>57

Asp?Met?Gly?Thr?Gly?Phe?Trp?Ser?Gly?Trp?Gly?Leu?Gly?Ser?Asp?Tyr

1 5 10 15

<210>58

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>58

Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Trp?Leu?Ala

1 5 10

<210>59

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>59

Ala?Ala?Ser?Ser?Leu?Gln?Ser

1 5

<210>60

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>60

Gln?Gln?Ala?Asn?Ser?Phe?Pro?Leu?Thr

1 5

<210>61

<211>361

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>61

gaggtgcagc?tggtggagtc?tgggggaggt?gtggtacggc?ctggggggtc?cctgagactc 60

tcctgtgcag?cctctgggtt?caccgtcagt?gattactcca?tgaactgggt?ccgccaggct 120

ccagggaagg?gcctggagtg?gattgggttt?attagaaaca?aagctaatgc?ctacacaaca 180

gagtacagtg?catctgtgaa?gggtagattc?accatctcaa?gagatgattc?aaaaaacacg 240

ctgtatctgc?aaatgaacag?cctgaaaacc?gaggacacag?ccgtgtatta?ctgtaccaca 300

taccctaggt?atcatgctat?ggactcctgg?ggccagggca?ccatggtcac?cgtctcctca 360

g 361

<210>62

<211>321

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>62

gccatccagt?tgactcagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?cagagtcacc 60

atcacttgca?gggccagcca?aagtattagc?aacaacctac?actggtacct?gcagaagcca 120

gggcagtctc?cacagctcct?gatctattat?ggcttccagt?ccatctctgg?ggtcccatca 180

aggttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240

gaagattttg?caacttacta?ctgtcaacag?gccaacagct?ggccgctcac?gttcggcgga 300

gggaccaagc?tggagatcaa?a 321

<210>63

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉antibody variable heavy chain

<400>63

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Phe?Ile?Arg?Asn?Lys?Ala?Asn?Ala?Tyr?Thr?Thr?Glu?Tyr?Ser?Ala

50 55 60

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

65 70 75 80

Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Lys?Thr?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Thr?Thr?Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Met?Val?Thr?Val?Ser?Ser

115 120

<210>64

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉antibody variable region

<400>64

Ala?Ile?Gln?Leu?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?Ser?Ile?Ser?Asn?Asn

20 25 30

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

35 40 45

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

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>65

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>65

Asp?Tyr?Ser?Met?Asn

1 5

<210>66

<211>19

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>66

Phe?Ile?Arg?Asn?Lys?Ala?Asn?Ala?Tyr?Thr?Thr?Glu?Tyr?Ser?Ala?Ser

1 5 10 15

Val?Lys?Gly

<210>67

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>67

Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser

1 5

<210>68

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>68

Arg?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Asn?Leu?His

1 5 10

<210>69

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>69

Tyr?Gly?Phe?Gln?Ser?Ile?Ser

1 5

<210>70

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>70

Gln?Gln?Ala?Asn?Ser?Trp?Pro?Leu?Thr

1 5

<210>71

<211>115

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>71

Asp?Val?Lys?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Lys?Pro?Gly?Gly

1 5 10 15

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

20 25 30

Thr?Met?Ser?Trp?Val?Arg?Gln?Thr?Pro?Glu?Lys?Arg?Leu?Glu?Trp?Val

35 40 45

Ala?Thr?Ile?Ser?Ser?Gly?Gly?Thr?Tyr?Thr?Tyr?Tyr?Pro?Asp?Ser?Val

50 55 60

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

65 70 75 80

Leu?Gln?Met?Ser?Ser?Leu?Lys?Ser?Glu?Asp?Thr?Ala?Met?Tyr?Tyr?Cys

85 90 95

Thr?Arg?Glu?Ala?Ile?Phe?Thr?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr

100 105 110

Val?Ser?Ala

115

<210>72

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉antibody variable region

<400>72

Asp?Ile?Lys?Met?Thr?Gln?Ser?Pro?Ser?Ser?Met?Tyr?Ala?Ser?Leu?Gly

1 5 10 15

Glu?Arg?Val?Thr?Ile?Thr?Cys?Lys?Ala?Ser?Gln?Asp?Ile?Asn?Asn?Tyr

20 25 30

Leu?Ser?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Lys?Ser?Pro?Lys?Thr?Leu?Ile

35 40 45

Tyr?Arg?Ala?Asn?Arg?Leu?Val?Asp?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Gln?Asp?Tyr?Ser?Leu?Thr?Ile?Ser?Ser?Leu?Glu?Tyr

65 70 75 80

Glu?Asp?Met?Gly?Ile?Tyr?Tyr?Cys?Leu?Lys?Tyr?Asp?Glu?Phe?Pro?Tyr

85 90 95

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

100 105

<210>73

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>73

Ser?Tyr?Thr?Met?Ser

1 5

<210>74

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>74

Thr?Ile?Ser?Ser?Gly?Gly?Thr?Tyr?Thr?Tyr?Tyr?Pro?Asp?Ser?Val?Lys

1 5 10 15

Gly

<210>75

<211>6

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>75

Glu?Ala?Ile?Phe?Thr?Tyr

1 5

<210>76

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>76

Lys?Ala?Ser?Gln?Asp?Ile?Asn?Asn?Tyr?Leu?Ser

1 5 10

<210>77

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>77

Arg?Ala?Asn?Arg?Leu?Val?Asp

1 5

<210>78

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>78

Leu?Lys?Tyr?Asp?Glu?Phe?Pro?Tyr?Thr

1 5

<210>79

<211>115

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>79

Glu?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Glu?Leu?Val?Lys?Thr?Gly?Ala

1 5 10 15

Ser?Val?Lys?Ile?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Ser?Phe?Thr?Gly?Tyr

20 25 30

Tyr?Met?His?Trp?Val?Lys?Gln?Ser?His?Gly?Lys?Ser?Leu?Glu?Trp?Ile

35 40 45

Gly?Tyr?Ile?Ser?Cys?Tyr?Asn?Gly?Val?Thr?Ser?Tyr?Asn?Gln?Lys?Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala?Arg?Ser?His?Ala?Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Ser?Val?Thr

100 105 110

Val?Ser?Ser

115

<210>80

<211>112

<212>PRT

＜213〉artificial

<220>

＜223〉antibody variable region

<400>80

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

1 5 10 15

Gln?Pro?Ala?Ser?Ile?Ser?Cys?Lys?Ser?Ser?Gln?Ser?Leu?Leu?Tyr?Ser

20 25 30

Asn?Gly?Lys?Thr?Tyr?Leu?Asn?Trp?Leu?Leu?Gln?Arg?Pro?Gly?Gln?Ser

35 40 45

Pro?Lys?Arg?Leu?Ile?Tyr?Leu?Val?Ser?Lys?Leu?Asp?Ser?Gly?Val?Pro

50 55 60

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

65 70 75 80

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

85 90 95

Ser?His?Phe?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys

100 105 110

<210>81

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>81

Gly?Tyr?Tyr?Met?His

1 5

<210>82

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>82

Tyr?Ile?Ser?Cys?Tyr?Asn?Gly?Val?Thr?Ser?Tyr?Asn?Gln?Lys?Phe?Lys

1 5 10 15

Gly

<210>83

<211>6

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>83

Ser?His?Ala?Met?Asp?Tyr

1 5

<210>84

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>84

Lys?Ser?Ser?Gln?Ser?Leu?Leu?Tyr?Ser?Asn?Gly?Lys?Thr?Tyr?Leu?Asn

1 5 10 15

<210>85

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>85

Leu?Val?Ser?Lys?Leu?Asp?Ser

1 5

<210>86

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>86

Val?Gln?Gly?Ser?His?Phe?Pro?Trp?Thr

1 5

<210>87

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>87

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu?Tyr?Leu?Gln?Met?Asn?Ala?Leu?Arg?Ala?Glu?Asp?Ser?Ala?Thr?Tyr

85 90 95

Tyr?Cys?Val?Arg?Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

115 120

<210>88

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉antibody variable region

<400>88

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

1 5 10 15

Asp?Ser?Val?Asn?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Asn

20 25 30

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

35 40 45

Lys?Tyr?Val?Phe?Gln?Ser?Ile?Ser?Gly?Ile?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Ser?Ile?Asn?Ser?Val?Glu?Thr

65 70 75 80

Glu?Asp?Phe?Gly?Met?Tyr?Phe?Cys?Gln?Gln?Ser?Asn?Ser?Trp?Pro?Leu

85 90 95

Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Leu?Lys

100 105

<210>89

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>89

Asp?Tyr?Ser?Met?Asn

1 5

<210>90

<211>19

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>90

Phe?Ile?Arg?Asn?Lys?Ala?Asn?Asp?Tyr?Thr?Thr?Glu?Tyr?Ser?Ala?Ser

1 5 10 15

Val?Lys?Gly

<210>91

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>91

Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser

1 5

<210>92

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>92

Arg?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Asn?Leu?His

1 5 10

<210>93

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>93

Tyr?Val?Phe?Gln?Ser?Ile?Ser

1 5

<210>94

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>94

Gln?Gln?Ser?Asn?Ser?Trp?Pro?Leu?Thr

1 5

<210>95

<211>118

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>95

Gln?Val?Gln?Leu?Gln?Gln?Pro?Gly?Ala?Glu?Leu?Val?Lys?Pro?Gly?Ala

1 5 10 15

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

20 25 30

Trp?Met?His?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35 40 45

Gly?Met?Ile?His?Pro?Asn?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala?Arg?Gly?Gly?Asn?Met?Val?Gly?Gly?Gly?Tyr?Trp?Gly?Gln?Gly?Thr

100 105 110

Thr?Leu?Thr?Val?Ser?Ser

115

<210>96

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉antibody variable region

<400>96

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

1 5 10 15

Glu?Lys?Val?Thr?Met?Thr?Cys?Ser?Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met

20 25 30

Tyr?Trp?Tyr?Gln?Gln?Lys?Pro?Arg?Ser?Ser?Pro?Lys?Pro?Trp?Ile?Tyr

35 40 45

Leu?Thr?Thr?Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser

50 55 60

Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Ser?Met?Glu?Ala?Glu

65 70 75 80

Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Ser?Asn?Pro?Phe?Thr

85 90 95

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

100 105

<210>97

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>97

Ser?Tyr?Trp?Met?His

1 5

<210>98

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>98

Met?Ile?His?Pro?Asn?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Glu?Lys?Phe?Lys

1 5 10 15

Ser

<210>99

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>99

Gly?Gly?Asn?Met?Val?Gly?Gly?Gly?Tyr

1 5

<210>100

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>100

Ser?Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr

1 5 10

<210>101

<211>7

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>101

Leu?Thr?Thr?Asn?Leu?Ala?Ser

1 5

<210>102

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>102

Gln?Gln?Trp?Ser?Ser?Asn?Pro?Phe?Thr

1 5

<210>103

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>103

Gln?Met?Gln?Leu?Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr

1 5 10 15

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

20 25 30

Ser?Met?Asn?Trp?Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Ile

35 40 45

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

50 55 60

Ser?Val?Lys?Gly?Arg?Val?Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr

65 70 75 80

Ala?Tyr?Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

115 120

<210>104

<211>108

<212>PRT

＜213〉artificial

<220>

＜223〉antibody variable region

<400>104

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?Ser?Ile?Ser?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Val?Phe?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>105

<211>5

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>105

Asp?Tyr?Ser?Met?Asn

1 5

<210>106

<211>19

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>106

Phe?Ile?Arg?Asn?Lys?Ala?Asn?Asp?Tyr?Thr?Thr?Glu?Tyr?Ser?Ala?Ser

1 5 10 15

Val?Lys?Gly

<210>107

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>107

Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser

1 5

<210>108

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>108

Arg?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Asn?Leu?His

1 5 10

<210>109

<211>7

<212>PRT

＜213〉artificial

<220>

<223>Antibcdy?CDR

<400>109

Tyr?Val?Phe?Gln?Ser?Ile?Ser

1 5

<210>110

<211>9

<212>PRT

＜213〉artificial

<220>

＜223〉antibody CDR

<400>110

Gln?Gln?Ser?Asn?Ser?Trp?Pro?Leu?Thr

1 5

<210>111

<211>990

<212>DNA

＜213〉artificial

<220>

＜223〉antibody constant region heavy chain

<400>111

gcgtcgacca?agggcccatc?cgtcttcccc?ctggcaccct?cctccaagag?cacctctggg 60

ggcacagcgg?ccctgggctg?cctggtcaag?gactacttcc?ccgaaccggt?gacggtgtcc 120

tggaactcag?gcgctctgac?cagcggcgtg?cacaccttcc?cggctgtcct?acagtcctca 180

ggactctact?ccctcagcag?cgtggtgacc?gtgccctcca?gcagcttggg?cacccagacc 240

tacatctgca?acgtgaatca?caagcccagc?aacaccaagg?tggacaagag?agttgagccc 300

aaatcttgtg?acaaaactca?cacatgccca?ccgtgcccag?cacctgaact?cctgggggga 360

ccgtcagtct?tcctcttccc?cccaaaaccc?aaggacaccc?tcatgatctc?ccggacccct 420

gaggtcacat?gcgtggtggt?ggacgtgagc?cacgaagacc?ctgaggtcaa?gttcaactgg 480

tacgtggacg?gcgtggaggt?gcataatgcc?aagacaaagc?cgcgggagga?gcagtacaac 540

agcacgtacc?gtgtggtcag?cgtcctcacc?gtcctgcacc?aggactggct?gaatggcaag 600

gagtacaagt?gcaaggtctc?caacaaagcc?ctcccagccc?ccatcgagaa?aaccatctcc 660

aaagccaaag?ggcagccccg?agaaccacag?gtctacaccc?tgcccccatc?ccgggaggag 720

atgaccaaga?accaggtcag?cctgacctgc?ctggtcaaag?gcttctatcc?cagcgacatc 780

gccgtggagt?gggagagcaa?tgggcagccg?gagaacaact?acaagaccac?gcctcccgtg 840

ctggactccg?acggctcctt?cttcctctat?agcaagctca?ccgtggacaa?gagcaggtgg 900

cagcagggga?acgtcttctc?atgctccgtg?atgcatgagg?ctctgcacaa?ccactacacg 960

cagaagagct?taagcctgtc?tccgggtaaa 990

<210>112

<211>321

<212>DNA

＜213〉artificial

<220>

＜223〉antibody constant region light chain

<400>112

cgaactgtgg?ctgcaccatc?tgtcttcatc?ttcccgccat?ctgatgagca?gttgaaatct 60

ggaactgcct?ctgttgtgtg?cctgctgaat?aacttctatc?ccagagaggc?caaagtacag 120

tggaaggtgg?ataacgccct?ccaatcgggt?aactcccagg?agagtgtcac?agagcaggac 180

agcaaggaca?gcacctacag?cctcagcagc?accctgacgc?tgagcaaagc?agactacgag 240

aaacacaaag?tctacgcctg?cgaagtcacc?catcagggcc?tgagctcgcc?cgtcacaaag 300

agcttcaaca?ggggagagtg?t 321

<210>113

<211>330

<212>PRT

＜213〉artificial

<220>

＜223〉antibody constant region heavy chain

<400>113

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

1 5 10 15

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

20 25 30

Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser

35 40 45

Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser

50 55 60

Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr

65 70 75 80

Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys

85 90 95

Arg?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys

100 105 110

Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro

115 120 125

Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys

130 135 140

Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp

145 150 155 160

Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu

165 170 175

Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu

180 185 190

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

195 200 205

Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly

210 215 220

Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Glu?Glu

225 230 235 240

Met?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr

245 250 255

Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn

260 265 270

Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe

275 280 285

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

290 295 300

Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr

305 310 315 320

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

325 330

<210>114

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉antibody constant region light chain

<400>114

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser

50 55 60

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

65 70 75 80

Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser

85 90 95

Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys

100 105

<210>115

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>115

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?Ile?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

Thr?Gly?Arg?Phe?Val?Phe?Ser?Leu?Asp?Thr?Sar?Val?Ser?Thr?Ala?Tyr

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Thr?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>116

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>116

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Ser?Ser?Trp?Thr

85 90 95

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

100 105

<210>117

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>117

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Phe?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>118

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>118

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Pro?Ser?Trp?Thr

85 90 95

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

100 105

<210>119

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>119

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?Ile?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Phe?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>120

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>120

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Trp?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Pro?Ser?Trp?Thr

85 90 95

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

100 105

<210>121

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>121

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?Ile?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Thr?Thr?Val?Tyr?Gly?Asp?Asn?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>122

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>122

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Pro?Ser?Trp?Thr

85 90 95

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

100 105

<210>123

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>123

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?lle?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Phe?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>124

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>124

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Arg?Pro?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Pro?Ser?Trp?Thr

85 90 95

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

100 105

<210>125

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>125

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?Ile?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Phe?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>126

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>126

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Arg?Pro?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Pro?Ser?Trp?Thr

85 90 95

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

100 105

<210>127

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>127

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?Ile?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Phe?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>128

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>128

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Trp?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Pro?Ser?Trp?Thr

85 90 95

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

100 105

<210>129

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>129

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?Ile?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Leu?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>130

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>130

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Ser?Ser?Trp?Thr

85 90 95

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

100 105

<210>131

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>131

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?Ile?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Phe?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>132

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>132

Glu?lle?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Pro?Ser?Trp?Thr

85 90 95

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

100 105

<210>133

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>133

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?Ile?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Leu?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>134

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>134

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Leu?Ser?Thr?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Pro?Ser?Trp?Thr

85 90 95

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

100 105

<210>135

<211>121

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>135

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly?Trp?Ile?Asn?Thr?Asn?Thr?Gly?Asn?Pro?Thr?Tyr?Ala?Gln?Gly?Phe

50 55 60

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

65 70 75 80

Leu?Gln?Ile?Ser?Ser?Leu?Lys?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Val?Arg?Leu?Thr?Val?Tyr?Gly?Asp?Gly?Met?Asp?Val?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>136

<211>106

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>136

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Ala

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Val?Glu?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Ser?Ser?Trp?Thr

85 90 95

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

100 105

<210>137

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>137

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Lys?Ala?Ser?Gln?Asp?Ile?Asn?Asn?Tyr

20 25 30

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

35 40 45

Tyr?Arg?Ala?Asn?Arg?Leu?Val?Asp?Gly?Val?Pro?Asp?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Tyr?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Asn?Asn?Ile?Glu?Ser

65 70 75 80

Glu?Asp?Ala?Ala?Tyr?Tyr?Phe?Cys?Leu?Lys?Tyr?Asp?Val?Phe?Pro?Tyr

85 90 95

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

100 105

<210>138

<211>115

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>138

Gln?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

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

35 40 45

Gly?Thr?Ile?Ser?Ser?Gly?Gly?Thr?Tyr?Thr?Tyr?Tyr?Pro?Asp?Ser?Val

50 55 60

Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Asn?Ser?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?Glu?Ala?Ile?Phe?Thr?Tyr?Trp?Gly?Arg?Gly?Thr?Leu?Val?Thr

100 105 110

Val?Ser?Ser

115

<210>139

<211>381

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>139

caggtgcagc?tgcaggagtc?gggcccagga?ctggtgaagc?cttcggggac?cctgtccctc 60

acctgcgctg?tctctggtgg?ctccatcagc?agtagtaact?ggtggagttg?ggtccgccag 120

cccccaggga?aggggctgga?gtggattggg?gaaatctatc?atagtgggag?caccaactac 180

aacccgtccc?tcaagagtcg?agtcaccata?tcagtagaca?agtccaagaa?ccagttctcc 240

ctgaagctga?gctctgtgac?cgccgcggac?acggccgtgt?attactgtgc?gagggggggt 300

atagcagcag?ctggttactg?gggcttgggg?tacaactggt?tcgacccctg?gggccaggga 360

accctggtca?ccgtctcctc?a 381

<210>140

<211>127

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>140

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

1 5 10 15

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

20 25 30

Asn?Trp?Trp?Ser?Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp

35 40 45

Ile?Gly?Glu?Ile?Tyr?His?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro?Ser?Leu

50 55 60

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

65 70 75 80

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

85 90 95

Ala?Arg?Gly?Gly?Ile?Ala?Ala?Ala?Gly?Tyr?Trp?Gly?Leu?Gly?Tyr?Asn

100 105 110

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

115 120 125

<210>141

<211>336

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construction

<400>141

cagtctgtgt?tgacgcagcc?gccctcagtg?tctggggccc?cagggcagag?ggtcaccatc 60

tcctgcactg?ggagcagctc?caacatcggg?gcaggttatg?atgtacactg?gtaccagcag 120

cttccaggaa?cagcccccaa?actcctcatc?tatggtaaca?gcaatcggcc?ctcaggggtc 180

cctgaccgat?tctctggctc?caagtctggc?acctcagcct?ccctggccat?cactgggctc 240

caggctgagg?atgaggctga?ttattactgc?cagtcctatg?acaacagcct?gagtggttcg 300

gtggttttcg?gcggagggac?caagctgacc?gtccta 336

<210>142

<211>112

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>142

Gln?Ser?Val?Leu?Thr?Gln?Pro?Pro?Ser?Val?Ser?Gly?Ala?Pro?Gly?Gln

1 5 10 15

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

20 25 30

Tyr?Asp?Val?His?Trp?Tyr?Gln?Gln?Leu?Pro?Gly?Thr?Ala?Pro?Lys?Leu

35 40 45

Leu?Ile?Tyr?Gly?Asn?Ser?Asn?Arg?Pro?Ser?Gly?Val?Pro?Asp?Arg?Phe

50 55 60

Ser?Gly?Ser?Lys?Ser?Gly?Thr?Ser?Ala?Ser?Leu?Ala?Ile?Thr?Gly?Leu

65 70 75 80

Gln?Ala?Glu?Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Gln?Ser?Tyr?Asp?Asn?Ser

85 90 95

Leu?Ser?Gly?Ser?Val?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu

100 105 110

<210>143

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>143

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Lys?Ala?Ser?Gln?Asp?Ile?Asn?Asn?Tyr

20 25 30

Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ale?Pro?Arg?Leu?Leu?Ile

35 40 45

Tyr?Arg?Ala?Asn?Arg?Leu?Val?Asp?Gly?Val?Pro?Asp?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Tyr?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Asn?Asn?Ile?Glu?Ser

65 70 75 80

Glu?Asp?Ala?Ala?Tyr?Tyr?Phe?Cys?Leu?Lys?Tyr?Asp?Glu?Phe?Pro?Tyr

85 90 95

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

100 105

<210>144

<211>115

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>144

Gln?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

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

35 40 45

Gly?Thr?Ile?Ser?Ser?Gly?Gly?Thr?Tyr?Thr?Tyr?Tyr?Pro?Asp?Ser?Val

50 55 60

Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Asn?Ser?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?Glu?Ala?Ile?Phe?Thr?Tyr?Trp?Gly?Arg?Gly?Thr?Leu?Val?Thr

100 105 110

Val?Ser?Ser

115

<210>145

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>145

Asp?Ile?Gln?Leu?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Leu?Ser?Pro?Gly

1 5 10 15

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

20 25 30

Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Asp?Gln?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Arg?Ala?Asn?Arg?Leu?Val?Asp?Gly?Val?Pro?Asp?Arg?Phe?Ser?Gly

50 55 60

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

65 70 75 80

Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Leu?Lys?Tyr?Asp?Glu?Phe?Pro?Tyr

85 90 95

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

100 105

<210>146

<211>115

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>146

Gln?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

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

35 40 45

Gly?Thr?Ile?Ser?Ser?Gly?Gly?Thr?Tyr?Thr?Tyr?Tyr?Pro?Asp?Ser?Val

50 55 60

Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Asn?Ser?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?Glu?Ala?Ile?Phe?Thr?Tyr?Trp?Gly?Arg?Gly?Thr?Leu?Val?Thr

100 105 110

Val?Ser?Ser

115

<210>147

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>147

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?Ser?Ile?Ser?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Val?Phe?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?lle?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>148

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>148

Gln?Met?Gln?Leu?Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr

1 5 10 15

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

20 25 30

Ser?Met?Asn?Trp?Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Ile

35 40 45

Gly?Phe?Ile?Arg?Asn?Lys?Ala?Asn?Asp?Tyr?Thr?Thr?Glu?Tyr?Ala?Asp

50 55 60

Ser?Val?Lys?Gly?Arg?Val?Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr

65 70 75 80

Ala?Tyr?Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

115 120

<210>149

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>149

Gln?Met?Gln?Leu?Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr

1 5 10 15

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

20 25 30

Ser?Met?Asn?Trp?Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Ile

35 40 45

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

50 55 60

Ser?Val?Lys?Gly?Arg?Val?Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr

65 70 75 80

Ala?Tyr?Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

115 120

<210>150

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>150

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

1 5 10 15

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

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Val?Phe?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>151

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>151

Gln?Met?Gln?Leu?Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr

1 5 10 15

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

20 25 30

Ser?Met?Asn?Trp?Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Leu

35 40 45

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

50 55 60

Ser?Val?Lys?Gly?Arg?Val?Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr

65 70 75 80

Ala?Tyr?Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

115 120

<210>152

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>152

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

1 5 10 15

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

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Val?Phe?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>153

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>153

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Lys?Thr?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Thr?Thr?Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Met?Val?Thr?Val?Ser?Ser

115 120

<210>154

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>154

Ala?Ile?Gln?Leu?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?Ser?Ile?Ser?Asn?Asn

20 25 30

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

35 40 45

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

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>155

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>155

Glu?Val?Gln?Leu?Val?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?Asp?Tyr

20 25 30

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

35 40 45

Ala?Phe?Ile?Arg?Asn?Lys?Ala?Asn?Asp?Tyr?Thr?Thr?Glu?Tyr?Ser?Ala

50 55 60

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

65 70 75 80

Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Met?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>156

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>156

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Ser?Leu?Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Asp?Ile?Lys

100 105

<210>157

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>157

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Lys?Thr?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Thr?Thr?Tyr?Pro?Arg?Tyr?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>158

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>158

Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Ser?Leu?Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Asp?Ile?Lys

100 105

<210>159

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>159

Gln?Met?Gln?Leu?Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr

1 5 10 15

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

20 25 30

Ser?Met?Thr?Trp?Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Leu

35 40 45

Gly?Phe?Ile?Arg?Asn?Lys?Ala?Asn?Ala?Tyr?Thr?Thr?Glu?Tyr?Ser?Ala

50 55 60

Ser?Val?Lys?Gly?Arg?Val?Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr

65 70 75 80

Ala?Tyr?Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Tyr?Pro?Arg?His?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

115 120

<210>160

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>160

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?Ser?Ile?Ser?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Ala?Phe?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>161

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>161

Gln?Met?Gln?Leu?Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr

1 5 10 15

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

20 25 30

Ser?Met?Asn?Trp?Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Ile

35 40 45

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

50 55 60

Ser?Val?Lys?Gly?Arg?Val?Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr

65 70 75 80

Ala?Tyr?Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Tyr?Pro?Arg?His?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

115 120

<210>162

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>162

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?Ser?Ile?Ser?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Ala?Phe?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>163

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>163

Gln?Met?Gln?Leu?Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr

1 5 10 15

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

20 25 30

Ser?Met?Thr?Trp?Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Leu

35 40 45

Gly?Phe?Ile?Arg?Asn?Lys?Ala?Asn?Ala?Tyr?Thr?Thr?Glu?Tyr?Ala?Asp

50 55 60

Ser?Val?Lys?Gly?Arg?Val?Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr

65 70 75 80

Ala?Tyr?Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Tyr?Pro?Arg?His?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

115 120

<210>164

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>164

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?Ser?Ile?Ser?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Ala?Phe?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

<210>165

<211>120

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>165

Gln?Met?Gln?Leu?Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr

1 5 10 15

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

20 25 30

Ser?Met?Asn?Trp?Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Ile

35 40 45

Gly?Phe?Ile?Arg?Asn?Lys?Ala?Asn?Asp?Tyr?Thr?Thr?Glu?Tyr?Ala?Asp

50 55 60

Ser?Val?Lys?Gly?Arg?Val?Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr

65 70 75 80

Ala?Tyr?Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Tyr?Pro?Arg?His?His?Ala?Met?Asp?Ser?Trp?Gly?Gln

100 105 110

Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

115 120

<210>166

<211>107

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construction

<400>166

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?Ser?Ile?Ser?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Ala?Phe?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

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

85 90 95

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

100 105

Claims (108)

1. a specific specificity is incorporated into the internalization antibody drug conjugates (ADC) of EphA2, it is characterized in that described medicine is a toxin.

2. ADC as claimed in claim is characterized in that, described ADC at least specificity in conjunction with people EphA2, mouse EphA2 and rat EphA2.

3. ADC as claimed in claim 1 or 2 is characterized in that described ADC comprises transcribed spacer and joint.

4. ADC as claimed in claim 3 is characterized in that, described joint is maleimide caproyl-citrulline joint.

5. ADC as claimed in claim 3 is characterized in that, described joint is Xie Ansuan-citrulline joint.

6. ADC as claimed in claim 1 is characterized in that, described toxin is anti--tubulin agent.

7. ADC as claimed in claim 6 is characterized in that, described resisting-the tubulin agent is Ao Ruisitating.

8. ADC as claimed in claim 7 is characterized in that, described Ao Ruisitating is Ao Ruisitating E or Ao Ruisitating F.

9. ADC as claimed in claim 8 is characterized in that, described Ao Ruisitating is monomethyl Ao Ruisitating F (MMAF).

10. ADC as claimed in claim 8 is characterized in that, described Ao Ruisitating is monomethyl Ao Ruisitating E (MMAE).

11. ADC as claimed in claim 1, its comprise have 12G3H11, variable heavy chain (VH) structural domain of the aminoacid sequence of the VH structural domain of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8, wherein, described ADC specificity is incorporated into the EphA2 polypeptide.

12. ADC as claimed in claim 1, its comprise have 12G3H11, variable light chain (VL) structural domain of the aminoacid sequence of the VL structural domain of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8, wherein, described ADC specificity is incorporated into the EphA2 polypeptide.

13. ADC as claimed in claim 11, it also comprises the VL structural domain of the aminoacid sequence of the VL structural domain with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

14. ADC as claimed in claim 1, its comprise have 12G3H11, the complementary determining region (CDR) of the aminoacid sequence of the CDR of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8, wherein, described ADC specificity is incorporated into the EphA2 polypeptide.

15. ADC as claimed in claim 14, it is characterized in that, described ADC comprise have 12G3H11, the VH CDR of the aminoacid sequence of the VH CDR of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

16. ADC as claimed in claim 14, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR of the aminoacid sequence of the VL CDR of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

17. ADC as claimed in claim 15, it also comprises the VL CDR of the aminoacid sequence of the VL CDR with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

18. ADC as claimed in claim 15, it is characterized in that, described ADC comprise have 12G3H11, the VH CDR1 of the aminoacid sequence of the VH CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

19. ADC as claimed in claim 15, it is characterized in that, described ADC comprise have 12G3H11, the VH CDR2 of the aminoacid sequence of the VH CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

20. ADC as claimed in claim 15, it is characterized in that, described ADC comprise have 12G3H11, the VH CDR3 of the aminoacid sequence of the VH CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

21. ADC as claimed in claim 18, it is characterized in that described ADC also comprises the VH CDR2 of the aminoacid sequence of the VH CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

22. ADC as claimed in claim 18, it is characterized in that described ADC also comprises the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

23. ADC as claimed in claim 19, it is characterized in that described ADC also comprises the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

24. ADC as claimed in claim 21, it is characterized in that described ADC also comprises the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

25. ADC as claimed in claim 17, it is characterized in that, described ADC comprise have 12G3H11, the VH CDR1 of the aminoacid sequence of the VH of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B 12 or 5A8.

26. ADC as claimed in claim 17, it is characterized in that, described ADC comprise have 12G3H11, the VH CDR2 of the aminoacid sequence of the VH CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B 12 or 5A8.

27. ADC as claimed in claim 17, it is characterized in that, described ADC comprise have 12G3H11, the VH CDR3 of the aminoacid sequence of the VH CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

28. ADC as claimed in claim 25, it is characterized in that described ADC also comprises the VH CDR2 of the aminoacid sequence of the VH CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

29. ADC as claimed in claim 25, it is characterized in that described ADC also comprises the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

30. ADC as claimed in claim 26, it is characterized in that described ADC also comprises the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

31. ADC as claimed in claim 28, it is characterized in that described ADC also comprises the VH CDR3 of the aminoacid sequence of the VH CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

32. ADC as claimed in claim 16, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

33. ADC as claimed in claim 16, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B 12 or 5A8.

34. ADC as claimed in claim 16, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

35. ADC as claimed in claim 32, it is characterized in that described ADC also comprises the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

36. ADC as claimed in claim 32, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

37. ADC as claimed in claim 33, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

38. ADC as claimed in claim 35, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

39. ADC as claimed in claim 17, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

40. ADC as claimed in claim 17, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

41. ADC as claimed in claim 17, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

42. ADC as claimed in claim 39, it is characterized in that described ADC also comprises the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

43. ADC as claimed in claim 39, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

44. ADC as claimed in claim 40, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

45. ADC as claimed in claim 42, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

46. ADC as claimed in claim 45, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

47. ADC as claimed in claim 25, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

48. ADC as claimed in claim 25, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

49. ADC as claimed in claim 46, it is characterized in that described ADC also comprises the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

50. ADC as claimed in claim 46, it is characterized in that described ADC also comprises the VLCDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

51. ADC as claimed in claim 47, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

52. ADC as claimed in claim 49, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

53. ADC as claimed in claim 26, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B 12 or 5A8.

54. ADC as claimed in claim 26, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

55. ADC as claimed in claim 26, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B 12 or 5A8.

56. ADC as claimed in claim 53, it is characterized in that described ADC also comprises the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

57. ADC as claimed in claim 53, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

58. ADC as claimed in claim 54, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

59. ADC as claimed in claim 56, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

60. ADC as claimed in claim 27, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

61. ADC as claimed in claim 27, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B 12 or 5A8.

62. ADC as claimed in claim 27, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

63. ADC as claimed in claim 60, it is characterized in that described ADC also comprises the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

64. ADC as claimed in claim 60, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

65. ADC as claimed in claim 61, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

66. as the described ADC of claim 63, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

67. ADC as claimed in claim 28, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

68. ADC as claimed in claim 28, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

69. ADC as claimed in claim 28, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

70. as the described ADC of claim 67, it is characterized in that described ADC also comprises the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

71. as the described ADC of claim 67, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

72. as the described ADC of claim 68, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

73. as the described ADC of claim 70, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

74. ADC as claimed in claim 29, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

75. ADC as claimed in claim 29, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

76. ADC as claimed in claim 29, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

77. as the described ADC of claim 74, it is characterized in that described ADC also comprises the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

78. as the described ADC of claim 74, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

79. as the described ADC of claim 75, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

80. as the described ADC of claim 77, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

81. ADC as claimed in claim 30, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

82. ADC as claimed in claim 30, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B 12 or 5A8.

83. ADC as claimed in claim 30, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

84. as the described ADC of claim 81, it is characterized in that described ADC also comprises the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

85. as the described ADC of claim 81, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

86. as the described ADC of claim 82, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

87. as the described ADC of claim 84, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

88. ADC as claimed in claim 31, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR1 of the aminoacid sequence of the VL CDR1 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

89. ADC as claimed in claim 31, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR2 of the aminoacid sequence of the VL CDR2 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

90. ADC as claimed in claim 31, it is characterized in that, described ADC comprise have 12G3H11, the VL CDR3 of the aminoacid sequence of the VL CDR3 of B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

91. as the described ADC of claim 88, it is characterized in that described ADC also comprises the VL CDR2 of the aminoacid sequence of the VL CDR2 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

92. as the described ADC of claim 88, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

93. as the described ADC of claim 89, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

94. as the described ADC of claim 91, it is characterized in that described ADC also comprises the VL CDR3 of the aminoacid sequence of the VL CDR3 with 12G3H11, B233, B208, B210, G5,10C12,4H5,10G9,3F2,1C1,1F12,1H3,1D3,2B12 or 5A8.

95. an isolating nucleic acid, it comprises coding as the weight chain variable structural domain of people, humanization or the chimeric form of ADC or the nucleotide sequence of light chain variable structural domain as described in each among the claim 1-94.

96. a carrier, it comprises as the described nucleic acid of claim 95.

97. a host cell, it comprises as the described carrier of claim 96.

98. a treatment has this patient's who needs method for cancer, described method comprise to described patient's administering therapeutic significant quantity as each described ADC among the claim 1-94.

99., it is characterized in that described dispenser makes the EphA2 phosphorylation level in the cancer cells increase with respect to the EphA2 phosphorylation level in the untreated cancer cells as the described method of claim 98.

100., it is characterized in that described cancer is the cancer in epithelial cell source as the described method of claim 99.

101., it is characterized in that described cancer comprises the cell of comparing expression EphA2 with the non-cancer cells with described cancer cell tissue type as the described method of claim 100.

102., it is characterized in that described cancer is cancer or the renal cell carcinoma or the melanoma of skin, lung, colon, mammary gland, prostate gland, bladder, kidney or pancreas as the described method of claim 100.

103., it is characterized in that described cancer is a metastatic cancer as the described method of claim 98.

104. as the described method of claim 98, described method comprises uses other anti-cancer therapies that are not EphA2 antibody.

105., it is characterized in that described other cancer therapies are selected from chemotherapy, biotherapy, immunotherapy, radiotherapy, hormonotherapy or surgical operation as the described method of claim 104.

106. a pharmaceutical composition, its comprise the treatment significant quantity as each described ADC and pharmaceutically acceptable carrier among the claim 1-94.

107. as the described pharmaceutical composition of claim 106, it comprises the anticarcinogen that is not EphA2 antibody.

108., it is characterized in that described anticarcinogen is chemotherapeutic, radiotherapy medicine, hormonotherapy medicine, biotherapy medicine or immunotherapy medicine as the described pharmaceutical composition of claim 107.

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