CN101395183A - Anti-ephb4 antibodies and methods using same - Google Patents

Abstract

The invention provides anti-EphB4 antibodies, and compositions comprising and methods of using these antibodies.

Description

Anti-ephb 4 antibodies and using method thereof

Related application

The application requires the U.S. Provisional Application No.60/756 that submitted on January 5th, 2006 according to 35USC § 119, the U.S. Provisional Application No.60/760 that on January 20th, 889 and 2006 submitted to, and 892 right of priority is by with reference to its complete content income this paper.

Technical field

The present invention relates generally to biology field.In particular, the present invention pays close attention to anti-EphB4 antibody and uses thereof.

Background technology

The growth of vascular supply (vascular supply) is the basic demand of many physiologicals and pathologic process.Tissue in the active growth such as embryo and tumour require competent blood supply.They satisfy this needs by generating short angiogenesis factor, and described short angiogenesis factor is via being called the process promotion neovascularization that (angiogenesis) takes place blood vessel.The pipe formation of blood vessel is complicated but orderly biology incident relates to all perhaps many following steps: a) breed EC or differentiate EC from progenitor cell from existing endotheliocyte (EC); B) EC migration and joint are to form the rope spline structure; C) (tubulogenesis) has central lumen with formation blood vessel takes place in the dull back of blood vessel generating pipe; D) existing rope or blood vessel stretch out bud to form time angiogenic; E) (remodeling) and shaping (reshaping) take place further to reinvent in elementary plexus vasculosus; And f) endothelium pericyte (peri-endothelial cell) is raised and is surrounded interior leather hose, keep and adjusting function for blood vessel provides, this type of cell comprises the pericyte (pericyte) that is used for little capillary vessel, the myocardial cell who is used for the smooth muscle cell and the heart of great vessels.Hanahan，Science277：48-50(1997)；Hogan & Kolodziej，Nat.Rev.Genet.3：513-23(2002)；Lubarsky & Krasnow，Cell 112：19-28(2003)。

Establish now blood vessel fully and related to the pathogeny of various disease conditions.These comprise solid tumor and transfer, atherosclerosis, Terry's sign, vascular tumor, chronic inflammatory diseases, intraocular neovascular disorders such as proliferating retinopathy for example immunological rejection, rheumatoid arthritis and the psoriatic of diabetic retinopathy, age related macular degeneration (AMD), neovascular glaucoma, corneal transplant tissue and other tissue.Folkman etc., J.Biol.Chem.267:10931-34 (1992); Klagsbrun etc., Annu.Rev.Physiol.53:217-39 (1991); Garner A., " Vasculardiseases ", in Pathobiology of Ocular Disease.A Dynamic Approach, Garner A and Klintworth GK compile, and the 2nd edition, Marcel Dekker, NY, 1994, pp1625-1710.

In the situation of tumor growth, blood vessel take place (angiogenesis) for from hyperplasia to neoplastic conversion, and provide nutrition seemingly vital for growth of tumor and transfer.Folkman etc., Nature339:58 (1989).Neovascularization (neovascularization) allows tumour cell obtain growth vigor and the propagation autonomy of comparing with normal cell.Tumour starts from single abnormal cells usually since with the distance that can utilize capillary bed, this cell can only be bred the size to several cubic millimeters, and it can keep " dormancy " state and not further growth and propagation in long period of time.Some tumour cell turns to blood vessel generation phenotype to start endotheliocyte then, and described endothelial cell proliferation also becomes the new capillary vessel of maturation.The blood vessel of these new formation not only allows the primary tumor continued growth, and allows metastatic cancer cell propagate and build group (recolonization).Thereby, observed dependency between patient's survival of microvessel density in tumor biopsy and mammary cancer and several other tumours.Weidner etc., N.Engl.J.Med.324:1-6 (1991); Horak etc., Lancet340:1120-24 (1992); Macchiarini etc., Lancet340:145-46 (1992).Do not understand the accurate mechanism that the control blood vessel changes as yet fully, but think that the neovascularization of tumor mass is derived from the clean balance of numerous blood vessel generation stimulator and inhibition.Folkman，Nat.Med.1(1)：27-31(1995)。

The vascular development process is subjected to tight adjusting.Up to now, multiple molecule mostly is the secretion sex factor that is generated by peripheral cell, has demonstrated adjusting EC and has broken up, breeds, moves and be bonded into the rope spline structure.For example, vascular endothelial growth factor (VEGF) has been accredited as and has related to the key factor that stimulates blood vessel generation and induction of vascular permeability.Ferrara etc., Endocr.Rev.18:4-25 (1997).Even the allelic loss of single VEGF causes the discovery of embryonic death to point to the not replaceable effect that this factor is brought into play in the growth of vascular system and differentiation.In addition, VEGF has been shown as the crucial mediators of the neovascularization relevant with the intraocular illness with tumour.Ferrara etc., Endocr.Rev. sees above.VEGF mRNA crosses in people's tumour that majority is checked and expresses.Berkman etc., J.Clin.Invest.91:153-59 (1993); Brown etc., Human Pathol.26:86-91 (1995); Brown etc., Cancer Res.53:4727-35 (1993); Mattern etc., Brit.J.Cancer 73:931-34 (1996); Dvorak etc., Am.J.Pathol.146:1029-39 (1995).

Equally, in the eye liquid the active vascular proliferation among the concentration level of VEGF and diabetic and other ischemic dependency retinopathy patient have a height correlation.Aiello etc., N.Engl.J.Med.331:1480-87 (1994).In addition, studies have shown that the location of VEGF in AMD patient's choroid neovascularity film.Lopez etc., Invest.Ophthalmol.Vis.Sci.37:855-68 (1996).

Anti-VEGF neutrality antibody is contained growth (Kim etc., the Nature 362:841-44 (1993) of various human tumor cell line in nude mice; Warren etc., J.Clin.Invest.95:1789-97 (1995);

Deng, Cancer Res.56:4032-39 (1996); Melnyk etc., Cancer Res.56:921-24 (1996)), and in ischemic retinal illness model, also suppress intraocular blood vessel generation (Adamis etc., Arch.Ophthalmol.114:66-71 (1996)).Therefore, anti-VEGF monoclonal antibody or other VEGF function inhibitor are the material standed fors that is hopeful to be used for the treatment of tumour and multiple intraocular neovascularity illness.This antibody-like for example be recorded on January 14th, 1998 disclosed EP 817,648 and on October 15th, 1998 disclosed WO98/45331 and WO98/45332.A kind of VEGF antibody, rhuMAb-VEGF (bevacizumab) has obtained FDA approval and has united with chemotherapy regimen and be used for the treatment of metastatic colorectal cancer (CRC).And rhuMAb-VEGF is just accepted research in the clinical trial of the various cancer indications of many ongoing treatments.

EphB4 acceptor (" EphB4 " or " EphB4R ") is a member of eph receptor family, and this family constitutes tyrosine kinase receptor family (summary is seen Dodelet, Oncogene, 19:5614-5619,2000) maximum in the human genome.People eph receptor tyrosine kinase is divided into category-A and category-B according to sequence identity, and it has corresponding A type and the Type B part that is called ephrin (s).Signal conduction can take place with forward manner, and wherein receptor tyrosine kinase is subjected to the activation of part, perhaps takes place with reverse manner, wherein strides film ephrinB part and is subjected to activation with acceptor interaction.The eph receptor-ligand interaction connects with biological function widely, comprise that axon guidance, organizational boundary form, (vasculogenesis) and cell movement (Kullander etc. take place the dimension pipe, Nat.Rev.Mol.Cell.Biol., 3:475-486,2002; Cheng etc., Cytokine Growth Factor Rev., 13:75-85,2002; Coulthard etc., Int.J.Dev.Biol., 46:375-384,2002).EphB4 is in conjunction with the part such as ephrin-B1, ephrin-B2 and ephrin-B3.The EphB4 acceptor has extracellular region, and this extracellular region has the motif that is rich in halfcystine, extends across its aminoterminal part, then is two fibronectin II type motifs.Be characterized as the born of the same parents' internal area and the membrane-spanning domain in conservative kinases district in addition.

Obviously still need to have the medicament of the clinical attributes that is suitable for being developed to therapeutical agent most.Invention described herein has been satisfied these needs and other benefit is provided.

All reference by quoting herein with reference to complete income comprise patent application and publication.

Summary of the invention

The present invention's part is based on the evaluation of multiple EphB4 wedding agent (such as immune conjugate, antibody and fragment thereof).EphB4 is rendered as important and favourable treatment target, and the invention provides based on composition and method in conjunction with EphB4.As described herein, EphB4 wedding agent of the present invention provides important therapeutical agent and diagnostic reagent for use for the expression of target and EphB4-EphB4 part approach and/or active relevant pathological condition.Thereby, the invention provides with EphB4 in conjunction with relevant method, composition, test kit and goods.

The invention provides with EphB4 the antibody that combines (such as the specificity combination).

On the one hand, the invention provides isolating anti-EphB4 antibody, wherein the described antibody of total length IgG form with about 50pM or better binding affinity (binding affinity) specificity in conjunction with people EphB4.As establishing fully this area, part can use in the multiple assay method any to measure to the binding affinity of its acceptor, and represents with various quantitative value forms.Thereby in one embodiment, binding affinity reflects inherent binding affinity (for example having minimized avidity effect (avidity effect)) with the Kd value representation.Generally and preferably, no matter binding affinity is to carry out under acellular environment or the cell relevant environment for example in in-vitro measurements.In many assay methods known in the art any comprises that those are described herein, all can be used for obtaining the observed value of binding affinity, comprises for example Biacore, radioimmunoassay (RIA) and ELISA.

On the one hand, the invention provides ligand binding domain bonded isolated antibody with EphB4.In some embodiment, described isolated antibody with comprise the EphB4 ectodomain or form or combine by its polypeptide of forming basically by it.

On the one hand, the invention provides and the isolating anti-EphB4 antibody of EphB4 part competition EphB4 bonded.

On the one hand, the invention provides inhibition, reduction and/or the active isolating anti-EphB4 antibody of blocking-up EphB4.In some embodiment, the autophosphorylation of EphB4 is suppressed, reduces and/or blocks.

On the one hand, anti-EphB4 antibody of the present invention comprises:

(a) at least one, two, three, four or five be selected from down the group hypervariable region (HVR) sequences:

(i) HVR-L1, it comprises sequence A 1-A11, and wherein A1-A11 is RASQDVSTAVA (SEQ ID NO:9),

(ii) HVR-L2, it comprises sequence B 1-B7, and wherein B1-B7 is SASFLYS (SEQ IDNO:11),

(iii) HVR-L3, it comprises sequence C 1-C9, and wherein C1-C9 is QESTTTPPT (SEQID NO:15),

(iv) HVR-H1, it comprises sequence D 1-D10, and wherein D1-D10 is GFSISNYYLH (SEQ ID NO:2),

(v) HVR-H2, it comprises sequence E1-E18, wherein E1-E18 be GGIYLYGSSSEYADSVKG (SEQ ID NO:4) and

(vi) HVR-H3, it comprises sequence F1-F17, and wherein F1-F17 is ARGSGLRLGGLDYAMDY (SEQ ID NO:7); And

(b) HVR of at least one variation, the HVR sequence of wherein said variation comprises the modification of at least one residue of sequence shown in the SEQ ID NO:1-17.

On the one hand, the invention provides the antibody that comprises one, two, three, four, five or six HVR, wherein each HVR comprises the sequence that is selected from SEQ ID NO:1-17 or is formed or be made up of it basically by it, and wherein SEQ ID NO:9 or 10 is corresponding to HVR-L1, SEQ ID NO:11 or 12 is corresponding to HVR-L2, SEQ ID NO:13,14,15,16 or 17 is corresponding to HVR-L3, SEQ ID NO:1 or 2 is corresponding to HVR-H1, SEQ ID NO:3,4,5 or 6 corresponding to HVR-H2 and SEQ ID NO:7 or 8 corresponding to HVR-H3.

In one embodiment, antibody of the present invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, and wherein each comprises SEQ ID NO:9,11,13,1,3,7 successively.

In one embodiment, antibody of the present invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, and wherein each comprises SEQ ID NO:10,12,14,1,3,8 successively.

In one embodiment, antibody of the present invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, and wherein each comprises SEQ ID NO:9,11,15,2,4,7 successively.

In one embodiment, antibody of the present invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, and wherein each comprises SEQ ID NO:9,11,16,1,5,7 successively.

In one embodiment, antibody of the present invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, and wherein each comprises SEQ ID NO:9,11,17,1,6,7 successively.

The HVR of the variation in the antibody of the present invention can have the modification of one or more (such as two, three, four, five or more a plurality of) residue in the HVR.

In one embodiment, the HVR-L1 variant comprises 1-5 (1,2,3,4 or 5) and locates to substitute in the following column position of arbitrary combination: A6 (V or S), A7 (S or E), A8 (T or I), A9 (A or F) and A10 (V or L).

In one embodiment, the HVR-L2 variant comprises 1-2 (1 or 2) and locates to substitute in the following column position of arbitrary combination: B4 (F or N) and B6 (Y or E).

In one embodiment, the HVR-L3 variant comprises 1-7 (1,2,3,4,5,6 or 7) and locates to substitute in the following column position of arbitrary combination: C2 (Q, E or K), C3 (S or T), C4 (Y, N, T, E or A), C5 (T, A or Q), C6 (T, V or I), C8 (P, L or E) and C9 (T or S).

In one embodiment, the HVR-H1 variant comprises 1-3 (1,2 or 3) and locates to substitute in the following column position of arbitrary combination: D3 (T or S), D6 (G or N) and D9 (I or L).

In one embodiment, the HVR-H2 variant comprises 1-5 (1,2,3,4 or 5) and locates to substitute in the following column position of arbitrary combination: E5 (P or L), E7 (S or G), E8 (G or S), E10 (T, S or R) and E11 (D, E or G).

In one embodiment, it is alternative that the HVR-H3 variant comprises 1 place in descending column position: F3 (G or S).

Letter indication illustration in each back, position bracket substitutes (i.e. displacement) amino acid; It will be apparent for a person skilled in the art that and to use technology known in the art and/or described herein other amino acid of conventional assessment amino acid whose as an alternative suitability in background described herein.

On the one hand, the invention provides the antibody that comprises the HVR-H1 district, described HVR-H1 district comprises the sequence of SEQID NO:1 or 2.On the one hand, the invention provides the antibody that comprises the HVR-H2 district, described HVR-H2 district comprises SEQ ID NO:3,4,5 or 6 sequence.On the one hand, the invention provides the antibody that comprises the HVR-H3 district, described HVR-H3 district comprises the sequence of SEQ ID NO:7 or 8.In one embodiment, the invention provides the antibody that comprises the HVR-L1 district, described HVR-L1 district comprises the sequence of SEQID NO:9 or 10.In one embodiment, the invention provides the antibody that comprises the HVR-L2 district, described HVR-L2 district comprises the sequence of SEQ ID NO:11 or 12.In one embodiment, the invention provides the antibody that comprises the HVR-L3 district, described HVR-L3 district comprises SEQ ID NO:13,14,15,16 or 17 sequence.

On the one hand, the invention provides comprise at least one, the antibody of at least two or all three following sequences:

(i) HVR-H1 sequence, it comprises the sequence of SEQ ID NO:2;

(ii) HVR-H2 sequence, it comprises the sequence of SEQ ID NO:4;

(iii) HVR-H3 sequence, it comprises the sequence of SEQ ID NO:7.

On the one hand, the invention provides comprise at least one, the antibody of at least two or all three following sequences:

(i) HVR-L1 sequence, it comprises the sequence of SEQ ID NO:9;

(ii) HVR-L2 sequence, it comprises the sequence of SEQ ID NO:11;

(iii) HVR-L3 sequence, it comprises the sequence of SEQ ID NO:15.

As shown in Figure 1, the aminoacid sequence of SEQ ID NO:1-17 is numbered at each HVR (being H1, H2 or H3), and numbering is consistent with Kabat numbering system as described below.

On the one hand, the invention provides the antibody that comprises heavy chain HVR sequence shown in Figure 1.

On the one hand, the invention provides the antibody that comprises light chain HVR sequence shown in Figure 1.

Some embodiment of antibody of the present invention comprise hereinafter the antibody of humanization 4D5 shown in the SEQ ID NO:18 (huMAb4D5-8) (

, Genentech, Inc., South San Francisco, CA, USA) (also can be referring to U.S. Patent No. 6,407,213 and Lee et al., J.Mol.Biol. (2004), 340 (5): light chain variable territory 1073-93).

1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val AlaTrp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr SerGlyVal Pro Ser Arg Phe Ser Gly SerArgSer Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro ThrPhe Gly Gln Gly Thr Lys Val Glu Ile Lys 107 (SEQ ID NO:18) (the HVR residue indicates underscore)

In one embodiment, there is modification at the one or more places of sequence in the 30th, 66 or 91 (Asn, Arg and the His that indicates with runic/italic respectively hereinbefore), described huMAb4D5-8 light chain variable territory.In one embodiment, described huMAb4D5-8 sequence through modification comprises Ser at the 30th, comprises Gly at the 66th, and/or comprises Ser at the 91st.Thereby, in one embodiment, antibody of the present invention comprises the light chain variable territory, and it comprises hereinafter sequence shown in the SEQ ID NO:52: 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val AlaTrp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr SerGlyVal Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro ThrPhe Gly Gln Gly Thr Lys Val Glu Ile Lys 107 (SEQ ID NO:52) (the HVR residue indicates underscore)

Alternative residue with respect to huMAb4D5-8 indicates with runic/italic hereinbefore.

Antibody of the present invention can comprise any suitable frame variable domain sequence, if EphB4 obtain basically keeping in conjunction with activity.For example, in some embodiment, antibody of the present invention comprises people's subgroup III heavy chain framework consensus sequence.In an embodiment of these antibody, described framework consensus sequence comprises alternative at the 71st, 73 and/or 78.In some embodiment of these antibody, the 71st is A, and the 73rd is T, and/or the 78th is A.In one embodiment, these antibody comprise huMAb4D5-8 (

, Genentech, Inc., South San Francisco, CA, USA) (also can be referring to U.S. Patent No. 6,407,213 ﹠amp; 5,821,337 and Lee et al., J.Mol.Biol. (2004), 340 (5): heavy chain variable domain framework sequence 1073-93).In one embodiment, these antibody further comprise people κ I light chain framework consensus sequence.In one embodiment, these antibody comprise light chain HVR sequence (U.S. Patent No. 6,407,213 ﹠amp of huMAb4D5-8; 5,821,337).In one embodiment, these antibody comprise huMAb4D5-8 ( , Genentech, Inc., South San Francisco, CA, USA) (also can be referring to U.S. Patent No. 6,407,213 ﹠amp; 5,821,337 and Lee et al., J.Mol.Biol. (2004), 340 (5): light chain variable territory sequence 1073-93).

In one embodiment, antibody of the present invention comprises heavy chain variable domain, wherein said framework sequence comprises SEQ ID NO:19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36 and/or 37 sequence, and HVR H1, H2 and H3 sequence are respectively SEQ ID NO:9,11 and/or 15.In one embodiment, antibody of the present invention comprises the light chain variable territory, and wherein said framework sequence comprises SEQ ID NO:38,39,40 and/or 41 sequence, and HVR L1, L2 and L3 sequence are respectively SEQ ID NO:2,4 and/or 7.

In one embodiment, antibody of the present invention comprises heavy chain variable domain, and wherein said framework sequence comprises SEQ ID NO:46,47,48 and/or 49 sequence, and HVR H1, H2 and H3 sequence are respectively SEQ ID NOS:2,4 and/or 7.In one embodiment, antibody of the present invention comprises the light chain variable territory, and wherein said framework sequence comprises SEQ ID NO:42,43,44 and/or 45 sequence, and HVR L1, L2 and L3 sequence are respectively SEQ ID NOS:9,11 and/or 15.

In one embodiment, antibody of the present invention comprises heavy chain variable domain, and wherein said framework sequence comprises SEQ ID NOS:46,47,51 and 49 sequence, and HVR H1, H2 and H3 sequence are respectively SEQ ID NOS:2,4 and/or 7.In one embodiment, antibody of the present invention comprises the light chain variable territory, and wherein said framework sequence comprises SEQ ID NOS:42,43,50 and 45 sequence, and HVR L1, L2 and L3 sequence are respectively SEQ ID NOS:9,11 and/or 15.

In one embodiment, antibody of the present invention is affinity maturation, in order to obtain the target thing binding affinity of expectation.In one embodiment, the one or more places of the antibody of affinity maturation of the present invention in H28, H31, H34, H52a, H54, H55, H57, H58, H95, L29, L30, L31, L32, L33, L53, L55, L90, L91, L92, L93, L94, L96 or L97 amino acids comprise alternative.In one embodiment, the antibody of affinity maturation of the present invention comprises one or more following substituting: (a) in the heavy chain, and T28S, G31N, I34L, P52aL, S54G, G55S, T57S or R, D58E or G, G95S, perhaps, (b) in the light chain, V29S, S30E, T31I, A32F, V33L, F53N, Y55E, Q90E or K, S91T, Y92N, T, E or A, T93V or I, T94V or I, P96L or E, T97S.

In one embodiment, antibody of the present invention comprises heavy chain variable domain, and this heavy chain variable domain comprises the sequence of SEQ ID NO:53.In one embodiment, antibody of the present invention comprises the light chain variable territory, and this light chain variable territory comprises the sequence of SEQ ID NO:54.In one embodiment, antibody of the present invention comprises heavy chain variable domain and light chain variable territory, and heavy chain variable domain comprises the sequence of SEQ ID NO:53, and the light chain variable territory comprises the sequence of SEQ ID NO:54.

In one embodiment, antibody of the present invention comprises heavy chain variable domain, and this heavy chain variable domain comprises the sequence of SEQ ID NO:55.In one embodiment, antibody of the present invention comprises the light chain variable territory, and this light chain variable territory comprises the sequence of SEQ ID NO:56.In one embodiment, antibody of the present invention comprises heavy chain variable domain and light chain variable territory, and heavy chain variable domain comprises the sequence of SEQ ID NO:55, and the light chain variable territory comprises the sequence of SEQ ID NO:56.

In one embodiment, antibody of the present invention comprises heavy chain variable domain, and this heavy chain variable domain comprises the sequence of SEQ ID NO:57.In one embodiment, antibody of the present invention comprises the light chain variable territory, and this light chain variable territory comprises the sequence of SEQ ID NO:58.In one embodiment, antibody of the present invention comprises heavy chain variable domain and light chain variable territory, and heavy chain variable domain comprises the sequence of SEQ ID NO:57, and the light chain variable territory comprises the sequence of SEQ ID NO:58.

In one embodiment, antibody of the present invention comprises heavy chain variable domain, and this heavy chain variable domain comprises the sequence of SEQ ID NO:59.In one embodiment, antibody of the present invention comprises the light chain variable territory, and this light chain variable territory comprises the sequence of SEQ ID NO:60.In one embodiment, antibody of the present invention comprises heavy chain variable domain and light chain variable territory, and heavy chain variable domain comprises the sequence of SEQ ID NO:59, and the light chain variable territory comprises the sequence of SEQ ID NO:60.

In one embodiment, antibody of the present invention comprises heavy chain variable domain, and it comprises the sequence of SEQ ID NO:61.In one embodiment, antibody of the present invention comprises the light chain variable territory, and it comprises the sequence of SEQID NO:62.In one embodiment, antibody of the present invention comprises heavy chain variable domain and light chain variable territory, and heavy chain variable domain comprises the sequence of SEQ ID NO:61, and the light chain variable territory comprises the sequence of SEQ ID NO:62.

On the one hand, the invention provides the antibody that combines EphB4 with any above-mentioned antibody competition.On the one hand, the invention provides the antibody of going up identical epi-position with above-mentioned antibodies EphB4.

As known in the art and hereinafter more describe in detail, based on context with various definition known in the art (as hereinafter described), amino acid position/border of defining the antibody hypervariable region can change to some extent.Some position in the variable domain can be considered heterozygosis hypermutation position, because these positions can think in the hypervariable region according to a cover standard, but then thinks outside the hypervariable region according to the different standard of a cover.In the hypervariable region that extends (as hereinafter further definition), also can find one or more these positions.

In some embodiment, described antibody is monoclonal antibody.In some embodiment, described antibody is polyclonal antibody.In some embodiment, described antibody is selected from antibody, humanized antibody and people's antibody of chimeric antibody, affinity maturation.In some embodiment, described antibody is antibody fragment.In some embodiment, described antibody is Fab, Fab ', Fab '-SH, F (ab ') ₂, or scFv.

In one embodiment, described antibody is chimeric antibody, for example will migrate to the antibody of allogenic non-human sequence, human sequence or humanization sequence (for example framework and/or constant domain sequence) from the antigen binding sequence of inhuman donor.In one embodiment, described inhuman donor is a mouse.In one embodiment, the antigen binding sequence is a synthetic, for example obtains by mutagenesis (for example phage display screening etc.).In one embodiment, chimeric antibody of the present invention has V district of mouse and people's C district.In one embodiment, light chain V district of described mouse and people's κ light chain merges.In one embodiment, merge in heavy chain V district of described mouse and people's IgG1C district.

Humanized antibody of the present invention comprises those have amino acid replacement in FR antibody and have the variant of the affinity maturation of change in the CDR that transplants.Alternative amino acid among CDR or the FR is not limited to those and is present in amino acid in donor or the receptor antibody.In other embodiments, antibody of the present invention further comprises the change that causes effector functions to improve (comprising CDC and/or ADCC and B cell killing increased functionality) in the amino-acid residue place in the Fc district.Other antibody of the present invention comprises that those have the antibody of the specific change that improves stability.In other embodiments, the amino-acid residue place of antibody of the present invention in the Fc district comprises and causes the be reduced change of (for example CDC and/or ADCC function reduce and/or the B cell killing reduces) of effector functions.In some embodiment, antibody of the present invention be characterized as with NK cell (NK) cell on people's complement factor C1q and/or people Fc acceptor combine reduction (such as in conjunction with the disappearance).In some embodiment, antibody of the present invention be characterized as with people Fc γ RI, Fc γ RIIA and/or Fc γ RIIIA combine reduction (such as in conjunction with the disappearance).In some embodiment, antibody of the present invention is IgG class (for example IgG1 or IgG4), and comprises at least one sudden change (numbering is according to the EU index) in E233, L234, G236, D265, D270, N297, E318, K320, K322, A327, A330, P331 and/or P329.In some embodiment, described antibody comprises sudden change L234A/L235A or D265A/N297A.

On the one hand, the invention provides anti-EphB4 polypeptide, any antigen binding sequence that it comprises herein to be provided, wherein said anti-EphB4 polypeptide combines with the EphB4 specificity.

Antibody of the present invention combines (such as the specificity combination) with EphB4, and, in some embodiment, one or more aspects of adjustable EphB4 correlation effect include but not limited to the EphB4 activation, the conduction of EphB4 downstream molecules signal, the EphB4 ligand activation, the conduction of EphB4 part downstream molecules signal, to part (ephrin-B1 for example, ephrin-B2, and/or ephrin-B3) destroys with the EphB4 bonded, EphB4 phosphorylation and/or EphB4 multimerization, and/or EphB4 part phosphorylation, and/or to the destruction of relevant EphB4 of any biology and/or EphB4 part biological pathway, inhibition to the blood vessel generation, and/or to tumour, cell proliferative disorders or treatment for cancer and/or prevention, and/or to expressing with EphB4 and/or active (expressing and/or active the rising) relevant treatment of conditions or prevention such as EphB4.In some embodiment, antibody of the present invention combines with the EphB4 specificity.In some embodiment, described antibody combines with EphB4 ectodomain (ECD) specificity.In some embodiment, described antibody with form by the EphB4 ectodomain or combine by its polypeptid specificity of forming basically.In some embodiment, described antibody combines with the EphB4 specificity with about 50pM or stronger KD.In some embodiment, antibody of the present invention is in vivo and/or in external reduction, inhibition and/or blocking-up EphB4 activity.In some embodiment, described antibody reduces, suppresses and/or blocking-up EphB4 autophosphorylation.In some embodiment, the combining of described antibody competition and EphB4 part (reduce and/or blocking-up EphB4 part combines with EphB4).

On the one hand, the invention provides antibody of the present invention and be used for purposes such as the medicine of the treatment of conditions of cancer, tumour and/or cell proliferative disorders and/or preventative processing in preparation.In some embodiment, described illness is neuropathy or neurodegenerative disease.

On the one hand, the invention provides antibody of the present invention is used for suppressing the medicine that blood vessel takes place in preparation purposes.

On the one hand, the invention provides the composition that comprises one or more antibody of the present invention and carrier.In one embodiment, described carrier is that pharmacopedics is acceptable.

On the one hand, the invention provides the nucleic acid that code book is invented anti-EphB4 antibody.

On the one hand, the invention provides the carrier that comprises nucleic acid of the present invention.

On the one hand, the invention provides the composition that comprises one or more nucleic acid of the present invention and carrier.In one embodiment, described carrier is that pharmacopedics is acceptable.

On the one hand, the invention provides the host cell that comprises nucleic acid of the present invention or carrier.Carrier can be an any kind, and recombinant vectors for example is such as expression vector.Can use in the multiple host cell any.In one embodiment, host cell is a prokaryotic cell prokaryocyte, for example intestinal bacteria.In one embodiment, host cell is an eukaryotic cell, and mammalian cell for example is such as Chinese hamster ovary (CHO) cell.

On the one hand, the invention provides the method for preparing antibody of the present invention.For example, the invention provides the anti-EphB4 antibody of preparation (as defined herein, comprise total length and fragment thereof) or the method for immune conjugate, described method is included in the recombinant vectors of the present invention of expressing encoding said antibody (or its fragment) in the proper host cell, and reclaims described antibody.

On the one hand, the invention provides the goods that comprise container and be contained in the composition in this container, wherein said composition comprises one or more anti-EphB4 antibody of the present invention.In one embodiment, described composition comprises nucleic acid of the present invention.In one embodiment, the composition that comprises antibody further comprises carrier, and it is that pharmacopedics is acceptable in some embodiment.In one embodiment, goods of the present invention further comprise the specification sheets (such as the specification sheets about any method described herein) about the experimenter being used composition (for example antibody).

On the one hand, the invention provides the test kit that comprises first container and second container, in described first container composition that comprises the anti-EphB4 antibody of one or more the present invention is housed, in described second container buffer reagent is housed.In one embodiment, described buffer reagent is that pharmacopedics is acceptable.In one embodiment, the composition that comprises antibody further comprises carrier, and it is that pharmacopedics is acceptable in some embodiment.In one embodiment, test kit further comprises about the specification sheets to experimenter's applying said compositions (for example described antibody).

On the one hand, the invention provides the anti-EphB4 antibody of the present invention and be used for purposes such as the medicine of the treatment of conditions of cancer, tumour and/or cell proliferative disorders and/or preventative processing in preparation.In some embodiment, described illness is neuropathy or neurodegenerative disease.In some embodiment, described illness is with blood vessel relevant pathological condition to take place.

On the one hand, the invention provides nucleic acid of the present invention and be used for purposes such as the medicine of the treatment of conditions of cancer, tumour and/or cell proliferative disorders and/or preventative processing in preparation.In some embodiment, described illness is neuropathy or neurodegenerative disease.In some embodiment, described illness is with blood vessel relevant pathological condition to take place.

On the one hand, the invention provides carrier of the present invention and be used for purposes such as the medicine of the treatment of conditions of cancer, tumour and/or cell proliferative disorders and/or preventative processing in preparation.In some embodiment, described illness is neuropathy or neurodegenerative disease (neurodegenerative disorders) (neurodegenerative disease).In some embodiment, described illness is with blood vessel relevant pathological condition to take place.

On the one hand, the invention provides host cell of the present invention and be used for purposes such as the medicine of the treatment of conditions of cancer, tumour and/or cell proliferative disorders and/or preventative processing in preparation.In some embodiment, described illness is neuropathy or neurodegenerative disease.In some embodiment, described illness is with blood vessel relevant pathological condition to take place.

On the one hand, the invention provides goods of the present invention and be used for purposes such as the medicine of the treatment of conditions of cancer, tumour and/or cell proliferative disorders and/or preventative processing in preparation.In some embodiment, described illness is neuropathy or neurodegenerative disease.In some embodiment, described illness is with blood vessel relevant pathological condition to take place.

On the one hand, the invention provides test kit of the present invention and be used for purposes such as the medicine of the treatment of conditions of cancer, tumour and/or cell proliferative disorders and/or preventative processing in preparation.In some embodiment, described illness is neuropathy or neurodegenerative disease.In some embodiment, described illness is with blood vessel relevant pathological condition to take place.

The invention provides the method or the composition that can be used for regulation and control and EphB4 expression and/or active (such as expressing and/or active rising or reduction or undesired expression and/or activity) disease states associated.

On the one hand, the invention provides the method that is used for the treatment of or prevents EphB4 expression and/or active raise relevant tumour, cancer and/or cell proliferative disorders, described method comprises the anti-EphB4 antibody to experimenter's administrations significant quantity of this type of treatment of needs.

On the one hand, the invention provides the method that is used for killer cell (such as cancer or tumour cell), described method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.

On the one hand, the invention provides the method that is used to reduce, suppress, block or stop tumour or growth of cancers, described method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.

On the one hand, the invention provides the method that is used for the treatment of or prevents neuropathy or neurodegenerative disease or reparation injured nerve cell, described method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.

On the one hand, the invention provides and be used to promote neuronal development, breed, keep or the regenerated method that described method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.

On the one hand, the invention provides the method that is used to suppress blood vessel generation (angiogenesis), described method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.

On the one hand, the invention provides the method that is used for the treatment of the pathological condition relevant with the blood vessel generation, described method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.In some embodiment, the described pathological condition relevant with the blood vessel generation is tumour, cancer and/or cell proliferative disorders.In some embodiment, the described pathological condition relevant with the blood vessel generation is the intraocular neovascular disorders.

Method of the present invention can be used for influencing any suitable pathological state.Describe exemplary illness herein, comprised the cancer that is selected from down group: small cell lung cancer, neuroblastoma, melanoma, mammary cancer, cancer of the stomach, colorectal carcinoma (CRC), hepatocellular carcinoma.

In one embodiment, the cell of institute's target is a cancer cells in the inventive method.For example, cancer cells can be selected from breast cancer cell, colorectal cancer cell, lung carcinoma cell, papillary carcinoma cell, colon cancer cell, pancreatic cancer cell, ovarian cancer cell, cervical cancer cell, central nervous system cancer cells, osteogenic sarcoma cell, kidney cancer cell, hepatocellular carcinoma cells, transitional cell bladder carcinoma cell line, stomach cancer cell, head and neck squamous cancer cell, melanoma cells, leukemia cell and adenoma of colon cell.In one embodiment, the cell of institute's target is the cell of hyper-proliferative (hyperproliferative) and/or hyperplasia (hyperplastic) in the inventive method.In one embodiment, the cell of institute's target is dysplastic (dysplastic) cell in the inventive method.In another embodiment, the cell of institute's target is the cell that shifts in the inventive method.

Method of the present invention also can further comprise other treatment step.For example, in one embodiment, described method further comprises wherein the step that target cell and/or tissue (for example cancer cells) is exposed to radiotreatment or chemotherapeutics.

On the one hand, the invention provides another therapeutical agent of the anti-EphB4 antibody that comprises co-administered significant quantity and significant quantity (such as the method for antiangiogenic agent (anti-angiogenesis agent).For example, anti-EphB4 antibody is united with carcinostatic agent or antiangiogenic agent and is used for the treatment of various superfluous natural disposition or non-superfluous natural disposition illness.In one embodiment, described superfluous natural disposition (neoplastic) or non-superfluous natural disposition (non-neoplastic) illness are with blood vessel relevant pathological condition to take place.In some embodiment, another therapeutical agent is antiangiogenic agent, antineoplastic agent and/or chemotherapeutics.

Anti-EphB4 antibody can be with effectively another therapeutical agent be continuous or co-administered to those purposes, or in same composition or as the composition that separates.Using and can carrying out simultaneously of anti-EphB4 antibody and another therapeutical agent (for example carcinostatic agent, antiangiogenic agent) for example as single composition, perhaps as two or more different compositions, used the identical or different path of using.Perhaps/in addition, use and to carry out with any sequence.Perhaps/in addition, described step can be used as the order of any order and carries out carrying out (the steps can be performed as acombination of both sequentially and simultaneously, in any order) with the combination of carrying out the two simultaneously.In certain embodiments, can have between the using of two or more compositions scope from several minutes to a couple of days, to several weeks, to the interval of several months.For example, can use carcinostatic agent earlier, then use anti-EphB4 antibody.Yet, also imagined and used or used earlier anti-EphB4 antibody simultaneously.Thereby, on the one hand, the invention provides and comprise and use anti-EphB4 antibody, then use the method for antiangiogenic agent (such as VEGF antibody, such as rhuMAb-VEGF).In certain embodiments, between the using of two or more compositions if having time on the scope from several minutes to a couple of days, to several weeks, to the interval of several months.

In some aspects, the invention provides the method that anti-EphB4 antibody by using significant quantity and/or angiogenesis inhibitor and one or more chemotherapeutics are treated illness (such as tumour, cancer and/or cell proliferative disorders).Multiple chemotherapeutics can be used for combinational therapeutic methods of the present invention.This paper provides the exemplary and non-limiting tabulation of contemplated chemotherapeutics in " definition " part.Using of anti-EphB4 antibody and linking agent can be carried out simultaneously, for example as single composition, perhaps as two or more different compositions, uses the identical or different path of using.Perhaps/in addition, use and to carry out with any sequence.Perhaps/in addition, the combination of carrying out and carry out simultaneously the two of the described step order that can be used as any order is carried out.In certain embodiments, can go up if having time between the using of two or more compositions from several minutes to a couple of days, to several weeks, to the interval of several months.For example, can use chemotherapeutics earlier, then use anti-EphB4 antibody.Yet, also imagined and used or used earlier anti-EphB4 antibody simultaneously.Thereby, on the one hand, the invention provides and comprise and use anti-EphB4 antibody, then use the method for chemotherapeutics.In certain embodiments, can go up if having time between the using of two or more compositions from several minutes to a couple of days, to several weeks, to the interval of several months.

On the one hand, the invention provides and be used for strengthening the method for antiangiogenic agent in the experimenter's who suffers from the pathological condition relevant effect with the blood vessel generation, described method comprises anti-EphB4 antibody and the antiangiogenic agent to the co-administered significant quantity of experimenter, strengthens the inhibition activity of described antiangiogenic agent thus.

On the other hand, the invention provides the method that is used to detect EphB4, described method is included in and detects the anti-EphB4 antibody complex of EphB4-in the sample.Term " detection " comprises when being used for this paper qualitatively and/or quantitative detection (measurement level), has or do not have the reference contrast.

On the other hand, the invention provides to be used to diagnose and express with EphB4 and/or the method for active relevant illness, described method is included in from suffering from or suspecting the anti-EphB4 antibody complex of detection EphB4-in the patient's who suffers from described illness the biological sample.In some embodiment, it is the expression of rising or unusual expression that described EphB4 expresses.In some embodiment, described illness is tumour, cancer and/or cell proliferative disorders.

On the other hand, the invention provides any anti-EphB4 antibody described herein, wherein said anti-EphB4 antibody comprises detectable.

On the other hand, the invention provides the mixture of any anti-EphB4 antibody described herein and EphB4.In some embodiment, described mixture be in vivo or external.In some embodiment, described mixture comprises cancer cells.In some embodiment, described anti-EphB4 antibody is detectable label.

The accompanying drawing summary

Fig. 1: the heavy chain of anti-EphB4 antibody and light chain HVR ring sequence.The figure illustrates heavy chain HVR sequence, i.e. H1, H2 and H3, and light chain HVR sequence, i.e. L1, L2 and L3.Sequence numbering is as follows: (HVR-H1 is SEQ ID NO:1 to clone 30.35; HVR-H2 is SEQ ID NO:3; HVR-H3 is SEQ ID NO:7; HVR-L1 is SEQ ID NO:9; HVR-L2 is SEQ ID NO:11; HVR-L3 is SEQ ID NO:13); (HVR-H1 is SEQ ID NO:1 to clone 30.35.1D2; HVR-H2 is SEQ ID NO:3; HVR-H3 is SEQ ID NO:8; HVR-L1 is SEQ ID NO:10; HVR-L2 is SEQ ID NO:12; HVR-L3 is SEQ ID NO:14); (HVR-H1 is SEQ ID NO:2 to clone 30.35.2D8; HVR-H2 is SEQ ID NO:4; HVR-H3 is SEQ ID NO:7; HVR-L1 is SEQ ID NO:9; HVR-L2 is SEQ ID NO:11; HVR-L3 is SEQ IDNO:15); (HVR-H1 is SEQ ID NO:1 to clone 30.35.2D12; HVR-H2 is SEQ ID NO:5; HVR-H3 is SEQ ID NO:7; HVR-L1 is SEQ ID NO:9; HVR-L2 is SEQ ID NO:11; HVR-L3 is SEQ ID NO:16); (HVR-H1 is SEQ ID NO:1 and clone 30.35.2D13; HVR-H2 is SEQ ID NO:6; HVR-H3 is SEQ ID NO:7; HVR-L1 is SEQ ID NO:9; HVR-L2 is SEQ ID NO:11; HVR-L3 is SEQ ID NO:17).

The aminoacid sequence position is numbered according to following Kabat numbering system.

Fig. 2 A and 2B and Fig. 3 have described to be used to implement the total framework sequence of illustrative receptors people of the present invention, and sequence identifier is as follows:

Weight chain variable (VH) has framework (Fig. 2 A and 2B)

The total framework of people VH subgroup I deducts Kabat CDR (SEQ ID NO:19)

The total framework of people VH subgroup I deducts the hypervariable region (SEQ ID NO:20-22) of extension

The total framework of people VH subgroup II deducts Kabat CDR (SEQ ID NO:23)

The total framework of people VH subgroup II deducts the hypervariable region (SEQ ID NO:24-26) of extension

The total framework of people's subgroup hypotype III deducts Kabat CDR (SEQ ID NO:27)

The total framework of people VH subgroup III deducts the hypervariable region (SEQ ID NO:28-30) of extension

People VH acceptor framework deducts Kabat CDR (SEQ ID NO:31)

People VH acceptor framework deducts the hypervariable region (SEQ ID NO:32-33) of extension

People VH acceptor 2 frameworks deduct Kabat CDR (SEQ ID NO:34)

People VH acceptor 2 frameworks deduct the hypervariable region (SEQ ID NO:35-37) of extension

Light chain variable (VL) has framework (Fig. 3)

People VL κ subgroup I has framework (SEQ ID NO:38)

People VL κ subgroup II has framework (SEQ ID NO:39)

People VL κ subgroup III has framework (SEQ ID NO:40)

People VL κ subgroup IV has framework (SEQ ID NO:41)

Fig. 4 has described the framework region sequence of huMAb4D5-8 light chain and heavy chain.The numerical value indication of subscript/runic is according to the amino acid position of Kabat.

Fig. 5 has described huMAb4D5-8 light chain and heavy chain Modify/variationThe framework region sequence.The numerical value indication of subscript/runic is according to the amino acid position of Kabat.

Fig. 6 has described variable region of heavy chain and the variable region of light chain of antibody cloning 30.35,30.35.1D2,30.35.2D8,30.35.2D12 and 20.25.2D13.

Fig. 7: anti-EphB4 monoclonal antibody is blocked the conduction of EphB4 receptor signal in based on the assay method of cell.

Detailed Description Of The Invention

The invention provides and can be used for for example treating or prevention is expressed with EphB4 and/or the anti-EphB4 antibody of the morbid state that active (such as the expression that raises and/or active or undesired expression and/or activity) is relevant. In some embodiment, antibody of the present invention is used for the treatment of tumour, cancer and/or cell proliferative disorders.

On the other hand, anti-EphB4 antibody of the present invention can be used as the reagent that detects and/or separate EphB4, such as detect EphB4 in various tissues and cell type.

The method for preparing anti-EphB4 antibody, the polynucleotides that reach the anti-EphB4 antibody of coding have been the present invention further provides.

Current techique

The technology of describing herein or mentioning and rules have generally obtained fully understanding of those skilled in the art, and usually utilize conventional method to be adopted, method such as the extensive use of putting down in writing in the following document for example: Sambrook et al., Molecular Cloning:A Laboratory Manual 3rd.edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M.Ausubel, et al. eds., (2003)); Book series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2:A PRACTICAL APPROACH (M.J.MacPherson, B.D.Hames and G.R. Taylor eds. (1995)); Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL; And ANIMAL CELL CULTURE (R.I.Freshney, ed. (1987)).

Definition

" separation " antibody refers to identify and from a kind of composition of its natural surroundings separately and/or the antibody that reclaims. The contaminative composition of its natural surroundings refers to disturb the diagnosis of this antibody or the material of therapeutical uses, can comprise the solute of enzyme, hormone and other oroteins character or nonprotein character. In preferred embodiments, with antibody purification to (1) mensuration according to the Lowry method, antibody weight surpasses 95%, most preferably weight surpasses 99%, (2) be enough to by using the rotary-cup type sequenator to obtain the N-end of at least 15 residues or the degree of internal amino acid sequence, or (3) reach homogeneity according to the SDS-PAGE under reproducibility or the irreducibility condition and use Coomassie blue or preferred silver dyeing. Since at least a composition of antibody natural surroundings can not exist, the antibody that separates so comprises the original position antibody in the recombinant cell. Yet the antibody of separation will prepare by at least one purification step usually.

" separation " nucleic acid molecules refer to identify and with the natural origin of antibody nucleic acid in the common nucleic acid molecules that separates of at least a contaminative nucleic acid molecules of associated. The nucleic acid molecule differ that separates is in form or background when occurring in nature is found it. The nucleic acid molecules that separates the therefore nucleic acid molecules when being present in the n cell is had any different. Yet the nucleic acid molecules of separation comprises the nucleic acid molecules that comprises in the cell of common this antibody of expression, for example when the chromosome mapping of described nucleic acid molecules in described cell is different from its chromosome mapping in n cell.

Term " according to the variable domain residue numbering of Kabat " or " according to the amino acid position numbering of Kabat " and variant thereof refer to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, the antibody editor among the MD. (1991) is used for the numbering system of heavy chain variable domain or light chain variable territory (variable domain). Use this numbering system, actual linear amino acid sequence can comprise less or other amino acid, corresponding to shortening or the insertion of variable domain FR or CDR. For example, heavy chain variable domain can comprise the insertion residue (such as being residue 82a, 82b and 82c etc. according to Kabat) after single amino acid behind the H2 residue 52 inserts (be residue 52a according to Kabat) and heavy chain FR residue 82. The Kabat residue numbering of given antibody can be by determining antibody sequence and " standard " Kabat numbered sequence contrast homologous region.

Phrase " basically similar " or " substantially the same " when being used for this paper, represent two numerical value (common one relate to antibody of the present invention and another relate to reference to/antibody relatively) between sufficiently high similarity degree so that those skilled in the art will think that in the measured biological characteristics background of described numerical value (for example Kd value) difference between two numerical value has very little or do not have biology and/or significance,statistical. As with reference to/the function of this numerical value of antibody relatively, the difference between described two numerical value is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10%.

" binding affinity " is often referred between the single binding site of molecule (for example antibody) and its binding partners (for example antigen) all intensity of noncovalent interaction summations. Except as otherwise noted, when being used for this paper, " binding affinity " refers to reflect in conjunction with the interactional inherent binding affinity of 1:1 between the right member (for example antibody and antigen). Molecule X explains the common available dissociation constant of the affinity of its gametophyte Y (Kd). Affinity can be measured by the common method that this area is known, comprises described herein. Low-affinity antibody is conjugated antigen and be tending towards dissociating easily lentamente usually, and the common conjugated antigen and be tending towards keeping the combination of longer time faster of high-affinity antibody. The several different methods of measuring binding affinity is known in this area, and wherein any all can be used for purpose of the present invention. Concrete exemplary has hereinafter been described.

In one embodiment, that the radio-labelled antigen binding assay (RIA) that purpose antibody and antigen thereof by the described use of following determination method Fab pattern carry out is measured according to " Kd " of the present invention or " Kd value ": by under the condition of the titration series that has unlabelled antigen, with Cmin¹²⁵Then I labelled antigen balance Fab measures Fab to the solution binding affinity (Chen, et al., J Mol Biol 293:865-881 (1999)) of antigen with the antigen that the flat board of anti-Fab antibody sandwich catches combination. In order to determine condition determination, spend the night with the coated microtiter plate (Dynex) of anti-Fab antibody (Cappel Labs) with the seizure of 5 μ g/ml in the 50mM sodium carbonate (pH9.6), use subsequently 2% among the PBS (w/v) bovine serum albumin in room temperature (about 23 ℃) sealing 2-5 hour. In non-absorption dull and stereotyped (Nunc#269620), with 100pM or 26pM[¹²⁵I]-antigen mixes (for example with Presta et al., VEGF antibody among the Cancer Res. 57:4593-4599 (1997), the assessment of Fab-12 is consistent) with the purpose Fab of serial dilution. Then with purpose Fab incubated overnight; But, (for example 65 hours) reach balance with assurance to be incubated the sustainable longer time. After this, mixture is transferred to the seizure plate to carry out room temperature insulation (for example 1 hour). Then remove solution, and wash plate 8 times with the PBS that contains 0.1%Tween-20. After the dull and stereotyped drying, add 150 μ l/ hole scintillation solution (MicroScint-20; Packard), then upper to plate count 10 minutes in Topcount gamma counter (Packard). Select each Fab to provide to be less than or equal to 20% concentration of maximum combined to be used for the competitive binding assay method. According to another embodiment, Kd or Kd value are to use BIAcore by surperficial plasmon resonance determination method^TM-2000 or BIAcore^TM-3000 (BIAcore, Inc., Piscataway, NJ) use immobilized antigen CM5 chip to measure in about 10 response units (RU) in 25 ℃. In brief, specification according to the supplier activates carboxy methylation dextran biologic sensor chip (CM5, BIAcore Inc.) with hydrochloric acid N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS). With antigen diluent to 5 μ g/ml (about 0.2 μ M), then be injected into the coupling protein matter of about 10 response units of acquisition (RU) with 10mM sodium acetate pH4.8 with 5 μ l/ minutes flow velocity. After injecting antigen, inject the 1M monoethanolamine with sealing unreacted group. In order to carry out kinetic measurement, be infused in the Fab (0.78nM to 500nM) of twice serial dilution among the PBS (PBST) that contains 0.05%Tween-20 in 25 ℃ of flow velocitys with about 25 μ l/ minutes. Use simply one to one Lang Gemiuer (Langmuir) combination model (BIAcore Evaluation Software version3.2) by while match combination and the sensing figure calculations incorporated speed (k that dissociates_on) and the speed (k that dissociates_off). Equilibrium dissociation constant (Kd) is with ratio k_off/k _onCalculate. Referring to for example Chen, Y., et al., J Mol Biol 293:865-881 (1999). If according to surperficial plasmon resonance determination method above, association rate surpasses 10⁶M ^-1S ^-1Association rate can be measured with the fluorescent quenching technology so, namely according to using the measurement of stirring cuvette (stir red cuvette) in spectrometer such as the spectrophotometer that has been equipped with cut-off device (a stop-flow equipped spectrophometer) (Aviv Instruments) or the 8000 serial SLM-Aminco spectrophotometers (ThermoSpectronic), under the condition that has the cumulative antigen of concentration, measure PBS, the anti-antigen-antibody of the 20nM among the pH7.2 (Fab form) (excites=295nm 25 ℃ fluorescent emission intensity; Emission=340nm, 16nm band is logical) rising or reduction.

According to " association rate " of the present invention (on-rate, rate of association, association rate) or " k_on" also can use BIAcore by identical surperficial plasmon resonance technique mentioned above^TM-2000 or BIAcore^TM-3000 (BIAcore, Inc., Piscataway, NJ) use immobilized antigen CM5 chip to measure in about 10 response units (RU) in 25 ℃. In brief, specification according to the supplier activates carboxy methylation dextran biologic sensor chip (CM5, BIAcore Inc.) with hydrochloric acid N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS). With antigen diluent to 5 μ g/ml (about 0.2 μ M), then be injected into the coupling protein matter of about 10 response units of acquisition (RU) with 10mM sodium acetate pH4.8 with 5 μ l/ minutes flow velocity. After injecting antigen, inject the 1M monoethanolamine with sealing unreacted group. In order to carry out kinetic measurement, be infused in the Fab (0.78nM to 500nM) of twice serial dilution among the PBS (PBST) that contains 0.05% Tween-20 in 25 ℃ of flow velocitys with about 25 μ l/ minutes. Use simply one to one Lang Gemiuer (Langmuir) combination model (BIAcore Evaluation Software version 3.2) by while match combination and the sensing figure calculations incorporated speed (k that dissociates_on) and the speed (k that dissociates_off). Equilibrium dissociation constant (Kd) is with ratio k_off/k _onCalculate. Referring to for example Chen, Y., et al., J Mol Biol 293:865-881 (1999). Yet if according to surperficial plasmon resonance determination method above, association rate surpasses 10⁶M ^-1S ^-1Association rate is preferably measured with the fluorescent quenching technology so, namely according to using the measurement of stirring cuvette in spectrometer such as the spectrophotometer that has been equipped with cut-off device (Aviv Instruments) or the 8000 serial SLM-Aminco spectrophotometers (ThermoSpectronic), under the condition that has the cumulative antigen of concentration, measure PBS, the anti-antigen-antibody of the 20nM among the pH7.2 (Fab form) (excites=295nm 25 ℃ fluorescent emission intensity; Emission=340nm, 16nm band is logical) rising or reduction.

Term " carrier " means to transport the nucleic acid molecules of connected other nucleic acid when being used for this paper. One class carrier is " plasmid ", refers to wherein can connect the circular double stranded DNA ring of other DNA section. Another kind of carrier is phage vector. Another kind of carrier is viral vectors, wherein other DNA section can be connected in the viral genome. Some carrier can be in the host cell that it imports self-replicating (bacteria carrier and the additive type mammal carrier that for example have the bacterium origin of replication). Other carrier (for example non-add type mammal carrier) can be incorporated in the genome of host cell after importing host cell, thus along with host genome copies together. In addition, some carrier can instruct the gene expression that is operatively connected with it. Examples of such carriers is referred to herein as " recombinant expression carrier " (or referred to as " recombinant vector "). Usually, useful expression vector usually is the plasmid form in recombinant DNA technology. In this manual, " plasmid " and " carrier " is used interchangeably, because plasmid is the most common form of carrier.

" polynucleotides " or " nucleic acid " are used interchangeably in this article, refer to the nucleotide polymer of any length, comprise DNA and RNA. Nucleotides can be deoxyribonucleotide, ribonucleotide, nucleotides or base and/or its analog through modifying, or can or mix any substrate of polymer by synthetic reaction by DNA or RNA polymerase. Polynucleotides can comprise through the nucleotides of modifying, such as methylated nucleotide and analog thereof. If any, can before or after the assembling polymer, carry out the modification of nucleotide structure. Nucleotide sequence can be interrupted by the non-nucleotide component. Polynucleotides can further be modified after synthetic, such as by with the label coupling. The modification of other type for example comprises " cap ", one or more naturally occurring nucleotides are substituted with analog, modify between nucleotides such as connecting (such as methyl-phosphonate, phosphotriester, phosphoramidate (phosphoamidate), carbamate etc.) and the modification with electrically charged connection (such as thiophosphate, phosphorodithioate etc.) such as having neutral, contain the module of dangling (pendant moiety) such as such as the modification of protein (such as nuclease, toxin, antibody, signal peptide, polylysine etc.), the modification with intercalator (such as acridine, psoralen etc.), the modification that contains chelating agent (such as metal, radioactive metal, boron, oxidisability metal etc.), the modification that contains alkylating agent, the modification with modified connection (such as α end group isomery nucleic acid (anomeric nucleic acid) etc.) and the polynucleotides of unmodified form. In addition; usually any hydroxyl that is present in the carbohydrate can be replaced with for example phosphonic acids (phosphonate) group, phosphoric acid (phosphate) group; protect with the standard blocking group; or activation is connected with other of other nucleotides with preparation, perhaps can be coupled to solid and semi-solid holder. But 5 ' and 3 ' terminal OH phosphorylation or add the replacement of cap group module with amine or 1-20 carbon atom organic. Other hydroxyl also can be derivatized to the standard blocking group. Polynucleotides also can contain the ribose generally known this area or the analog form of deoxyribose carbohydrate, for example comprise 2 '-oxygen-methyl, 2 '-oxygen-pi-allyl, 2 '-fluoro-or 2 '-nitrogen-ribose repeatedly, carba sugars, α-end group isomerized sugar, epimerism sugar such as arabinose, wood sugar or lyxose, pyranose, furanose, sedoheptulose, without ring analogues and dealkalize yl nucleosides analog such as methylribonucleotide. The available alternative linking group is replaced one or more di-phosphate esters and is connected. These alternative linking groups include but not limited to following embodiment, and wherein phosphate is with P (O) S (" thioester " (thioate)), P (S) S (" two thioesters " (dithioate)), (O) NR₂(" carboxylic acid amide esters " (amidate)), P (O) R, P (O) OR ', CO or CH₂(" dimethoxym ethane " (formacetal)) substitutes, wherein R or R ' are independent separately is H or replacement or unsubstituted alkyl (1-20 C), optional ether (O-) connection, aryl, thiazolinyl, cycloalkyl, cycloalkenyl group or the aralkyl (araldyl) of containing. Be not all connections in the polynucleotides all must be identical. All polynucleotides that aforementioned description is applicable to mention herein comprise RNA and DNA.

" oligonucleotides " refers generally to short polynucleotides when being used for this paper, generally be strand, generally synthesizes, and length generally but be not must be less than about 200 nucleotides. Term " oligonucleotides " is not mutually exclusive with " polynucleotides ". Above about the description equality of polynucleotides and be applicable to oligonucleotides fully.

" percentage (%) amino acid sequence identity " about peptide or peptide sequence is defined as the contrast sequence and introduces where necessary breach with after obtaining largest percentage sequence homogeneity, and not with any conservative substituting when being considered as sequence homogeneity a part of, the percentage of the amino acid residue identical with amino acid residue in particular peptide or the peptide sequence in the candidate sequence. For the contrast of the measuring percentage amino acid sequence identity purpose various ways in can the art technology scope carries out, for example use the available computer software of the public, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine for the suitable parameter of measuring contrast, comprise institute's comparative sequences total length is obtained the required any algorithm of maximum contrast. Yet, for the purposes of the present invention, % amino acid sequence identity value be use sequence relatively computer program ALIGN-2 obtain, the complete source code of ALIGN-2 program wherein hereinafter is provided in the Table A. ALIGN-2 sequence comparison computer program is write by Genentech company, hereinafter source code shown in the Table A is submitted to (the US Copyright Office of U.S. Copyright Bureau together with user's literary composition file, Washington D.C., 20559), and with U.S. copyright registration TXU510087 register. The public can pass through Genentech company (South San Francisco, California) and obtain the ALIGN-2 program. The ALIGN2 program should be compiled in UNIX operating system, uses on the preferred digital UNIX V4.0D. The all sequences comparative parameter is by ALIGN-2 program setting and constant.

Adopting ALIGN-2 to come in the situation of comparing amino acid sequence, given amino acid sequence A with respect to (to), with (with) or for the % amino acid sequence identity of (against) given amino acid sequence B (perhaps can be expressed as have or comprise with respect to, with or for the given amino acid sequence A of a certain % amino acid sequence identity of given amino acid sequence B) following calculating:

Mark X/Y takes advantage of 100

Wherein X is to be the total number of atnino acid of identical match by sequence contrast program ALIGN-2 scoring in the A of this program and B contrast, and wherein Y is amino acid residue sum among the B. Can understand, if the length of the length of amino acid sequence A and amino acid sequence B is unequal, then A will be not equal to B with respect to the % amino acid sequence identity of A with respect to the % amino acid sequence identity of B.

Unless specify in addition, all % amino acid sequence identity values used herein all are described according to the preceding paragraph, use the ALIGN-2 computer program to obtain.

Term " EphB4 " (interchangeable being called " EphB4R "), when being used for this paper, unless expressly stated otherwise, or context be otherwise noted, refer to any (no matter being natural or synthetic) natural or variation EphB4 polypeptide. Naturally occurring brachymemma or secreted form (for example ectodomain sequence), naturally occurring variant form (for example alternative splicing form) and naturally occurring allelic variant clearly contained in term " native sequences ". Term " wild type EphB4 " refers generally to comprise the polypeptide of the amino acid sequence of the natural EphB4 of existence albumen. Term " wild type EphB4 sequence " refers generally to the amino acid sequence that finds in the natural EphB4 of existence.

Term " antibody " and " immunoglobulin (Ig) " are with broad sense Alternate, comprise monoclonal antibody (for example total length or complete monoclonal antibody), polyclonal antibody, multivalent antibody, multi-specificity antibody (bispecific antibody for example, as long as they show the BA of expectation), but also can comprise some antibody fragment (as more describing in detail) herein. Antibody can be the people, humanized and/or affinity maturation.

Term " variable " refers to that some part difference between antibody sequence in the variable domain is extensive and be used for every kind of specific antibodies to combination and the specific truth of its specific antigen. Yet variability is not the whole variable domain that is uniformly distributed in antibody. It concentrates on three sections that are called complementary determining region (CDR) or hypervariable region in light chain and the heavy chain variable domain. In the variable domain more the part of high conservative be called framework (FR). Each self-contained four FR district of the variable domain of natural heavy chain and light chain, they take β-folded piece conformation mostly, connect by three CDR that form loop connecting and form β-folded piece structure part in some situation. CDR in every chain is by keeping together that the FR district approaches very much, and facilitate the formation of the antigen binding site of antibody (referring to Kabat et al. with the CDR of another chain, Sequences of Proteins of Immunological Interest, the 5th edition, National Institutes of Health, Bethesda, MD. (1991)). Constant domain is not participated in the combination of antibody and antigen directly, but shows multiple effector functions, such as the participation of antibody in the cell of antibody dependent cellular.

Produce two identical Fabs with papain digestion antibody, be called " Fab " fragment, have separately an antigen binding site, and remaining " Fc " fragment, its title has reflected that it is easy to the ability of crystallization. Pepsin produces a F (ab ')₂Fragment, it has two antigen binding sites and still can crosslinked antigen.

" Fv " is the minimum antibody fragment that comprises complete antigen recognizing and binding site. In double-stranded Fv kind, this district is comprised of tight a, heavy chain variable domain of non-covalent combination and the dimer in a light chain variable territory. In the scFv kind, a heavy chain variable domain can link to each other by flexible peptide linker covalency with a light chain variable territory, so that light chain and heavy chain are similarly combining in " dimer " structure with double-stranded Fv kind. Just in this structure, three CDR of each variable domain interact and at V_H-V _LDetermined an antigen binding site on the dimer surface. Six CDR give antibody jointly with antigen-binding specificity. Yet, even single variable domain (or only comprise three CDR of antigen-specific half Fv) also has the ability of identification and conjugated antigen, be that affinity is lower than complete binding site.

The Fab fragment also comprises the constant domain of light chain and first constant domain (CH1) of heavy chain. The difference of Fab ' fragment and Fab fragment is that the carboxyl terminal of heavy chain CH1 domain has increased the minority residue, comprises the one or more cysteines from antibody hinge region. Fab '-SH is the appellation of constant domain cysteine residues wherein being carried the Fab ' of free sulphur alcohol radical herein. F (ab ')₂Antibody fragment is to generate as the paired Fab ' fragment that hinge cysteine is arranged between Fab ' fragment at first. Also know other chemical coupling of antibody fragment.

According to the amino acid sequence of its constant domain, can be included into a kind of in two kinds of distinct types from " light chain " of the antibody (immunoglobulin (Ig)) of any invertebrate species, be called card handkerchief (κ) and lambda (λ).

According to the amino acid sequence of its heavy chain constant domain, immunoglobulin (Ig) can be included into different classes. Immunoglobulin (Ig) has five large class: IgA, IgD, IgE, IgG and IgM, and wherein some can be further divided into subclass (isotype), for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Heavy chain constant domain that will be corresponding with inhomogeneous immunoglobulin (Ig) is called respectively α, δ, ε, γ and μ. The subunit structure of inhomogeneous immunoglobulin (Ig) and three-dimensional structure are well-known.

" antibody fragment " only comprises the part of complete antibody, usually with it relevant at least one, preferred great majority or all functions when wherein said part preferably keeps this part and is present in the complete antibody. The example of antibody fragment comprises Fab, Fab ', F (ab ')₂, and Fv fragment; Double antibody; Linear antibody; The single-chain antibody molecule; With the multi-specificity antibody that is formed by antibody fragment. In one embodiment, antibody fragment comprises the antigen binding site of complete antibody, so keeps the ability of conjugated antigen. In another embodiment, antibody fragment, the antibody fragment that for example comprises the Fc district keeps when usually being present in the complete antibody with Fc district usually at least one relevant with it biological function, such as regulate and control FcRn combination, antibody half life, ADCC function and complement combination. In one embodiment, antibody fragment is Half-life in vivo and complete antibody similar univalent antibody basically. For example, such antibody fragment can comprise an antigen combination arm and itself and can give the Fc sequence of this fragment with the body internal stability and link to each other.

Term " hypervariable region ", " HVR " or " HV " refer to alterable height on the antibody variable domains sequence and/or form the zone of the ring that structure defines when being used for this paper. Usually, antibody comprises six hypervariable regions: three in VH (H1, H2, H3), three in VL (L1, L2, L3). Use and contain the narration of many hypervariable regions herein. Kabat complementary determining region (CDR) is take sequence variability as the basis, and be the most frequently used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia changes the position (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)) that refers to structure ring into. The AbM hypervariable region represents trading off between Kabat CDR and the Chothia structure ring, and obtains the use of the AbM antibody modeling software of Oxford Molecular. " contact " hypervariable region take to obtainable compound crystal construction analysis as the basis. Hereinafter recorded in these hypervariable regions the residue of each.

Ring Kabat AbM Chothia contact

---   ------------ ----------- -----------  ----------     \

L1     L24-L34     L24-L34     L26-L32     L30-L36

L2     L50-L56     L50-L56     L50-L52     L46-L55

L3     L89-L97     L89-L97     L91-L96     L89-L96

H1     H31-H35B    H26-H35B    H26-H32     H30-H35B

(Kabat numbering)

H1     H31-H35     H26-H35     H26-H32     H30-H35

(Chothia numbering)

H2     H50-H65     H50-H58     H53-H55     H47-H58

H3     H95-H102    H95-H102    H96-H101    H93-H101

The hypervariable region can comprise following " hypervariable region of extension ": 26-35 (H1), the 50-65 among the 24-36 among the VL or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) and the VH or 49-65 (H2) and 93-102,94-102 or 95-102 (H3). For in these definition each, the variable region residue is according to Kabat etc., the numbering that sees above.

" framework " or " FR " residue refers to those residues except hypervariable region as defined herein residue in the variable domain.

" humanization " form of inhuman (for example mouse) antibody refers to that bottom line comprises the chimeric antibody derived from the sequence of non-human immunoglobulin. Largely, humanized antibody refers to the immunoglobulin (Ig) that the hypervariable region residue in the human immunoglobulin(HIg) (receptor antibody) is replaced with the hypervariable region residue of inhuman species (donor antibody) such as mouse, rat, rabbit or non-human primate with expectation specificity, affinity and ability. In some situation, framework region (FR) residue of human immunoglobulin(HIg) is replaced with corresponding inhuman residue. In addition, humanized antibody can be included in the residue that does not find in receptor antibody or the donor antibody. Carrying out these modifications is in order further to improve the performance of antibody. Generally speaking, humanized antibody will comprise at least one, common two whole following variable domains basically, wherein all or basically all hypermutation rings corresponding to the hypermutation ring of non-human immunoglobulin, and all or basically all FR are FR of human immunoglobulin(HIg) sequence. Optional at least part of constant region for immunoglobulin (Fc), the normally constant region of human immunoglobulin(HIg) of also will comprising of humanized antibody. More details are referring to Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); And Presta, Curr.Op.Struct.Biol. 2:593-596 (1992). Also can be referring to following summary and the list of references of quoting thereof: Vaswani and Hamilton, Ann.Allergy, Asthma ﹠ Immunol.1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op.Biotech. 5:428-433 (1994).

In " chimeric " antibody (immunoglobulin (Ig)) part of heavy chain and/or light chain with derived from the identical or homology of the corresponding sequence of individually defined thing species or genus in the antibody of specific antibodies classification or subclass, and the remainder of chain with derived from another species or belong to the identical or homology of corresponding sequence in the antibody of another antibody isotype or subclass, and the fragment of this antibody-like, as long as they show the BA (U.S. Patent No. 4 of expectation, 816,567; Morrison et al., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984)). Humanized antibody used herein is a subset of chimeric antibody.

" scFv " or " scFv " antibody fragment comprises the V of antibody_HAnd V_LDomain, wherein these domains are present on the polypeptide chain. Generally speaking, the scFv polypeptide is at V_HWith V_LScFv also comprises peptide linker between the domain, so that can form the required structure of conjugated antigen. About the summary of scFv referring to Pluckthun, in " The Pharmacology of Monoclonal Antibodies " the 113rd volume that Rosenburg and Moore compiles, Springer-Verlag, New York, pp.269-315 (1994).

Antigen " refer to the predetermined antigens of the alternative combination of antibody. Target antigen can be polypeptide, carbohydrate, nucleic acid, lipid, haptens or other naturally occurring or synthetic compound. Preferably, target antigen is polypeptide.

Term " double antibody " refers to have the small-sized antibody fragment of two antigen binding sites, and this fragment is at same polypeptide chain (V_H-V _L) in comprise continuous heavy chain variable domain (V_H) and light chain variable territory (V_L). , force the complementary structure territory pairing of these domains and another chain, thereby produce two antigen binding sites so that can not match between two domains on the same chain by using too short joint. Double antibody is more complete is recorded in for example EP 404,097; WO 93/11161; Hollinger et al., Proc.Natl.Acad.Sci. USA 90:6444-6448 (1993).

The antibody that " people's antibody " refers to have the amino acid sequence corresponding with the amino acid sequence of the antibody that is generated by the people and/or uses any technology for generating people's antibody disclosed herein to generate. This definition clear-cut of people's antibody is got rid of and is comprised non-human antigen in conjunction with the humanized antibody of residue.

" affinity maturation " antibody refers to have in one or more CDR of antibody a place or many places change, cause this antibody that the affinity of antigen is compared the antibody that improves to some extent with the parental antibody that does not have these changes. The antibody of preferred affinity maturation will have nanomole or even the affinity to target antigen of picomole magnitude. The antibody of affinity maturation can generate by rules known in the art. Marks et al., Bio/Technology 10:779-783 (1992) have put down in writing the affinity maturation that is undertaken by VH and the reorganization of VL domain. Put down in writing the random mutagenesis of CDR and/or framework residue with Publication about Document: Barbas et al., Proc. Nat.Acad.Sci.USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J.Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol.154 (7): 3310-9 (1995); Hawkins et al., J.Mol.Biol.226:889-896 (1992).

Antibody " effector functions " refers to the BA that those are attributable to antibody Fc district (native sequences Fc district or amino acid sequence variant Fc district) and change with antibody isotype. The example of antibody mediated effect device function comprises: C1q combination and CDC; The Fc receptors bind; The cytotoxicity (ADCC) of antibody dependent cellular mediation; Phagocytosis; Cell surface receptor (for example B-cell receptor) downward modulation; With the B cell activation.

" antibody dependent cellular mediation cytotoxicity " or " ADCC " refers to wherein be attached to secreting type Ig on the upper Fc acceptor (FcR) that exists of some cytotoxic cell (for example NK (NK) cell, neutrophil cell and macrophage) so that the target cell that these cytotoxic effect cells can specific binding carry antigen, the cytotoxicity form of killing subsequently target cell with cytotoxin. This antibody " arms " is cytotoxic cell (arm), and is the absolute requirement of this type of lethal effect. The main cell of mediation ADCC, the NK cell is only expressed Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII. Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991) the 464th page table 3 have summed up the FcR on the hematopoietic cell and have expressed. For the ADCC of purpose of appraisals molecule active, can carry out external ADCC determination method, such as U.S. Patent No. 5,500,362 or 5,821,337 or Presta U.S. Patent No. 6,737,056 in put down in writing. The effector cell who can be used for this type of determination method comprises PMNC (PBMC) and NK (NK) cell. Perhaps/in addition, the ADCC of purpose of appraisals molecule is active in vivo, for example in animal model, and such as Clynes et al., disclosed in PNAS (USA) 95:652-656 (1998).

The leucocyte that " people effector cell " refers to express one or more FcR and exercise effector functions. Preferably, this cell is expressed at least Fc γ RIII and is exercised the ADCC effector functions. The HL's of mediation ADCC example comprises PMNC (PBMC), NK (NK) cell, monocyte, cytotoxic T cell and neutrophil cell, preferred PBMC and NK cell. The effector cell can separate from its natural origin, for example blood.

" Fc acceptor " or " FcR " describes the acceptor in binding antibody Fc district. Preferred FcR is native sequences people FcR. In addition, preferred FcR is the FcR (γ acceptor) in conjunction with IgG antibody, comprises the acceptor of Fc γ RI, Fc γ RII and Fc γ RIII subclass, comprises allelic variant and the alternative splicing form of these acceptors. Fc γ RII acceptor comprises Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" inhibition acceptor "), and they have similar amino acid sequence, and difference is mainly in its cytoplasmic structure territory. Activated receptor Fc γ RIIA comprises immunity receptor based on the activation motif (ITAM) of tyrosine in its cytoplasmic structure territory. Suppress acceptor Fc γ RIIB in its cytoplasmic structure territory, comprise immunity receptor based on the inhibition motif (ITIM) of tyrosine (referring to summary

, Annu.Rev.Immunol.15:203-234 (1997)). The summary of FcR is referring to Ravetch and Kinet, Annu.Rev.Immunol.9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); De Haas et al., J.Lab.Clin.Med.126:330-41 (1995). Other FcR contained in this article in term " FcR ", comprises what will identify those futures. This term also comprises neonate's acceptor, FcRn, it is responsible for that Maternal immunoglobulin G is transferred to fetus (Guyer et al., J.Immunol.117:587 (1976) and Kim etal., J.Immunol.24:249 (1994)) and regulates the dynamic equilibrium of immunoglobulin (Ig). WO 00/42072 (Presta) has put down in writing the antibody variants in conjunction with raising or reduction to FcR. In this content of clearly taking in this patent publications as a reference. Also can be referring to Shieldsetal., J.Biol.Chem. 9 (2): 6591-6604 (2001).

Measurement is known (referring to for example Ghetie 1997, Hinton 2004) to the method for the combination of FcRn. Can measure Binding in vivo and the serum half-life of people FcRn high-affinity Binding peptide and people FcRn, for example at the transgenic mice of expressing people FcRn or in the human cell line of transfection, perhaps in the primate of having used the Fc variant polypeptide.

" CDC " or " CDC " when referring to have complement to the dissolving of target cell. The startup of CCP is initial in conjunction with the antibody (suitable subclass) that it closes the combination of associated antigen institute by complement system the first component (Clq). Start in order to assess complement, can carry out the CDC determination method, for example such as Gazzano-Santoro et al., put down in writing among the J.Immunol.Methods 202:163 (1996).

Polypeptide variants with Clq binding ability of the Fc region amino acid sequence of change and raising or reduction is recorded in U.S. Patent No. 6,194,551 B1 and WO 99/51642. In this content of clearly taking in those patent publications as a reference. Also can be referring to Idusogie et al., J.Immunol.164:4178-4184 (2000).

Contain the polypeptide that " Fc district polypeptide " refers to comprise the Fc district, such as antibody or immunoadhesin (definition sees below). The terminal lysine of the C-in Fc district (according to the residue 447 of EU numbering system) can be eliminated, for example in the process of purified polypeptide or the nucleic acid by the modified recombinant coded polypeptide. Therefore, the composition that comprises the polypeptide with Fc district according to the present invention can comprise polypeptide with K447, eliminated the polypeptide of all K447 or have mixture with the polypeptide that does not have the K447 residue.

" blocking-up " antibody or " Antagonism " antibody (antagonist antibody) refer to suppress or reduce the antibody of BA of the antigen of its combination. Preferred blocking antibody or antagonistic antibodies essence or suppress the BA of antigen fully.

" agonistic antibody (agonist antibody) " finger print when being used for this paper is intended the antibody of at least one functional activity of desired polypeptides.

With regard to this paper purpose, " acceptor people framework " is the framework that comprises the amino acid sequence of the VL of the total framework of derived from human immunoglobulin (Ig) framework or people or VH framework. " derived from " the acceptor people framework of the total framework of human immunoglobulin(HIg) framework or people can comprise identical with it amino acid sequence, perhaps can comprise the amino acid sequence that is pre-existing in and change. When the amino acid that is pre-existing in when existence changed, the preferred existence was no more than 5, and preferred 4 or still less, perhaps 3 or the amino acid that still less is pre-existing in change. When existing the amino acid that is pre-existing in to change among the VH, preferably those change three, two or position that only is arranged in 71H, 73H and 78H; For example, the amino acid residue that is positioned at those positions can be 71A, 73T and/or 78A. In one embodiment, VL acceptor people framework is identical with VL human immunoglobulin(HIg) Frame sequence or the total Frame sequence of people on sequence.

" people has framework " refers to the framework of modal amino acid residue in table human immunoglobulin(HIg) VL or the VH Frame sequence selected works. Usually, human immunoglobulin(HIg) VL or VH sequence selected works are from the variable region sequences hypotype. Usually, the sequence hypotype is the hypotype such as people such as Kabat. In one embodiment, for VL, hypotype is the hypotype κ I such as people such as Kabat. In one embodiment, for VH, hypotype is the hypotype III such as people such as Kabat.

" VH subgroup III has framework " comprises the consensus sequence that the amino acid sequence from the people's such as Kabat variable heavy chain hypotype III obtains. In one embodiment, the total framework amino acid sequence of VH hypotype III comprise following each sequence at least a portion or whole all: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:42)-H1-WVRQAPGKGL EWV (SEQ ID NO:43)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:44)-H3-WGQGTLVTVSS (SEQ ID NO:45).

" VL subgroup I has framework " comprises the consensus sequence that the amino acid sequence from the people's such as Kabat variable light chain K hypotype I obtains. In one embodiment, the total framework amino acid sequence of VL hypotype I comprises at least a portion of following each sequence or whole: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:46)-L1-WYQQKPGKAPKLL IY (SEQ ID NO:47)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:48)-L3-FGQGTKVEIK (SEQ ID NO:49).

" illness " or " disease " refers to that any meeting benefits from the illness of the treatment of substances/molecules of the present invention or method. This comprises chronic and acute disease or disease, comprises that those make mammal tend to the pathological condition of the illness of discussing. The non-limitative example of illness to be treated comprises pernicious and benign tumour herein; Cancer, blastoma and sarcoma.

Term " cell proliferative disorders " refers to the illness relevant with abnormal cell proliferation to a certain degree with " proliferative disorders ". In one embodiment, cell proliferative disorders refers to cancer.

" tumour " refers to all tumprigenicity Growth of Cells and propagation when being used for this paper, no matter be pernicious or optimum, and (pre-cancerous) and cancerous cells and tissue before all cancers. Term " cancer ", " carcinous ", " cell proliferative disorders ", " proliferative disorders " and " tumour " are not mutually exclusive when mentioning in this article.

Feature is generally the not modulated physiology illness of Growth of Cells/propagation in term " cancer " and " carcinous " sensing or the description mammal. The example of cancer includes but not limited to cancer, lymthoma, blastoma, sarcoma and leukaemia. The more specifically example of this type of cancer comprises squama cancer, peritoneal cancer, hepatocellular carcinoma, human primary gastrointestinal cancers, cancer of pancreas, spongioblastoma, cervical carcinoma, oophoroma, liver cancer (liver cancer), carcinoma of urinary bladder, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, carcinoma of endometrium or the cancer of the uterus, salivary-gland carcinoma, kidney, prostate cancer, carcinoma of vulva, thyroid cancer, liver cancer (hepatic carcinoma), cancer of the stomach, melanoma, and various types of head and the neck cancer of gland cancer, the lung of squamous cell carcinoma, ED-SCLC, non-small cell lung cancer, lung.

Imbalance occurs blood vessel can cause the many illnesss that can treat by the compositions and methods of the invention. These illnesss comprise nontumorous and tumorous illness. The tumprigenicity illness include but not limited to as described above those. The Non-cancerous illness includes but not limited to undesired or unusual hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, plaque psoriasis, sarcoidosis, atherosclerotic, atherosclerotic plaque, diabetic keratopathy and other proliferating retinopathy comprise retinopathy of prematurity, Terry's sign, age related macular degeneration, diabetic macular edema, the cornea neovascularization, the corneal graft neovascularization, corneal graft rejection, retina/choroid neovascularization, the neovascularization at canthus (rubescent), ocular neovascular disorders, reangiostenosis, arteriovenous malformation (AVM), meningoma, hemangioma, angiofibroma, thyroid gland hyperplasia (comprising Ge Leifusishi (Graves) disease), cornea and other tissue transplantation, chronic inflammation, pneumonia, ALI/ARDS, septicemia, primary pulmonary hypertension, malign lung hydrops (malignant pulmonary effusion), encephaledema (for example relevant with Acute Stroke/closed head injury/wound), the synovia inflammation, pannus among the RA forms, myositis ossificans, the hypertrophy bone forms, osteoarthritis (OA), RA, POD, endometriosis, sick (3rd spacing of fluid the diseases) (pancreatitis of the 3rd space fluid, compartment syndrome, burn, enteropathy), fibroma uteri (uterine fibroids), premature labor, chronic inflammation such as IBD (Crow engler (Crohn) disease and ulcerative colitis), the kidney allograft thing repels, inflammatory bowel disease, nephrotic syndrome, undesired or unusual tissue block growth (non-carcinous), bleeder's joint, hypertrophic scar, the inhibition of hair growth, Ao-Wei Er Shi (Osler-Weber) syndrome, the granuloma pyogenicum Terry's sign, chorionitis, trachoma, angiosynizesis, synovitis, dermatitis, pre-eclampsia, ascites, hydropericardium (such as relevant with pericarditis) and pleural effusion.

When being used for this paper, " treatment " or " processing " refer to attempt to change the clinical intervention of nature process of the individuality for the treatment of or cell, can be in order to prevent or in the process of clinicopathologia, to carry out. The desired effects for the treatment of comprises that any direct or indirect pathology consequence of prophylactic generation or recurrence, relief of symptoms, weakening disease, prevention shift, slow down the speed of PD, improve or the state that palliates a disease, and exempt or improve prognosis. In some embodiment, antibody of the present invention is for the generation that postpones disease or illness/development.

Term " neurodegenerative disease " and " neurodegenerative disorders " use with broad sense, comprise that its pathology relate to neuron sex change and/or handicapped all illnesss, include but not limited to peripheral nerve disease; The motor neuron illness is such as ALS (amylotrophic lateral sclerosis) (ALS, Lou GehrigShi sick), Bei Ershi (Bell) paralysis with relate to the various illness of Duchenne-Arandisease or paralysis; And other people's neurodegenerative disease, such as Alzheimer's, Parkinson's disease, epilepsy, multiple sclerosis, Heng Tingdunshi chorea, Down syndrome, merve deafness and plum Ni Ershi (Meniere) disease.

" peripheral nerve disease " refers to affect perineural neurodegenerative disorders, the most normal or the combination that shows as in motion, sensation, sensorimotor or the autonomic dysfunction. Peripheral nerve disease can be for example to obtain by heredity, can be derived from systemic disease, perhaps can be that for example antitumor agent or industry or environmental contaminants bring out toxic agent such as neurotoxicity medicine. The sex change that is characterized as peripheral sensory neuron of " esthesioneurosis on every side ", this can be idiopathic, can be (for example to use the treatment of chemotherapeutics as for example cytostatic therapy of diabetes (diabetic neuropathy), cancer, such as vincristine, cis-platinum, methotrexate (MTX), 3 '-repeatedly nitrogen-3 '-AZT or taxane, for example Taxol (paclitaxel) [

Bristol-Myers Squibb Oncology, Princeton, N.J.] and Taxotere (doxetaxel) [

Rorer, Antony, France]), the consequence of alcoholism, acquired immunodeficiency syndrome (AIDS) or genetic factor (genetic predisposition) occurs. The peripheral nerve disease that obtains by heredity for example comprises that the not plain Mu Shi of thunder (Refsum) is sick, restrains cured Bi Shi (Krabbe) disease, metachromatic leukodystrophy, Fa Bulishi (Fabry) disease, Dai-Suo Er Shi (Dejerine-Sottas) syndrome, abetalipoproteinemia and Xia Ke-Mali-Tu Si (Charcot-Marie-Tooth, CMT) disease (being also referred to as Proneal amyotrophia or hereditary motor and sensory neuropathy (HMSN)). The peripheral nerve disease of most of types slowly occurs/develops, course of disease several months or several years. In clinical practice, this type of neuropathy is called chronic. Sometimes peripheral nerve disease occurs fast/develops, and course of disease a couple of days, is called acute. Peripheral nerve disease is together impact sensation and kinesitherapy nerve usually, thereby causes mixed type sensation and motor neuropathy, but pure sensation and neuropathy pure motion also are known.

" individuality " refers to vertebrate, preferred mammal, more preferably people. Mammal includes, but not limited to livestock (such as ox), motion animal, pet (such as cat, dog and horse), primate, Mouse and rat.

For the purpose for the treatment of, " mammal " aim enters mammiferous any animal, comprises the people, domestic animal and livestock, and zoo, motion or pet animals, such as dog, horse, cat, ox etc. Preferably, mammal refers to the people.

" effective dose " refers at essential dosage and effectively realizes the treatment of expectation or the amount of preventive effect on the time.

" the treatment effective dose " of substances/molecules of the present invention, antagonist or activator can change according to cause the factors such as ability that expectation replys in individuality such as morbid state, age, sex and the body weight of individuality and this substances/molecules, activator or antagonist. The treatment effective dose refers to that also the treatment beneficial effect of this substances/molecules, activator or antagonist surpasses the amount of any poisonous or harmful consequence. " prevention effective dose " refers to the amount in essential dosage and the preventive effect that effective realization is expected on the time. Usually but not inevitable, because preventive dose is before seizure of disease or the early stage experimenter of being used for of disease, therefore prevent effective dose will be lower than the treatment effective dose.

Term " cytotoxic agent " refers to suppress when being used for this paper or prevents the function of cell and/or cause the material of cytoclasis. This term intention comprises: radio isotope, for example At²¹¹、I ¹³¹、I ¹²⁵、Y ⁹⁰、 Re ¹⁸⁶、Re ¹⁸⁸、Sm ¹⁵³、Bi ²¹²、P ³²Radio isotope with Lu; Chemotherapeutics, for example methotrexate (MTX) (methotrexate), adriamycin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine (vincristine), vincaleukoblastinum (vinblastine), Etoposide (etoposide)), Doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, Chlorambucil (chlorambucil), daunorubicin (daunorubicin) or other intercalator; Enzyme and fragment thereof are such as nucleolytic enzyme; Antibiotic; And toxin, such as the enzyme of little molecule toxin or bacterium, fungi, plant or animal origin toxin alive, comprise its fragment and/or variant; And the various antineoplastics or the anticarcinogen that hereinafter disclose. Hereinafter put down in writing other cytotoxic agent. Kill the destruction that the tumour efficacy-enhancing ingredient plays tumour cell.

" chemotherapeutics " refers to can be used for treating the chemical compound of cancer. The example of chemotherapeutics comprises alkylating agent class (alkylating agents), such as phosphinothioylidynetrisaziridine (thiotepa) and

Endoxan (cyclophosphamide); Alkyl sulfonate esters class (alkyl sulfonates) is such as busulfan (busulfan), Improsulfan (improsulfan) and croak pool Shu Fan (piposulfan); Aziridines (aziridines) is such as Benzodepa (benzodepa), card ripple quinone (carboquone), U.S. appropriate in sending (meturedepa) and uredepa (uredepa); Ethylenimine class (ethylenimines) and methylmelamine class (methylamelamines) comprise hemel (altretamine), triethylenemelamine (triethylenemelamine), APO (triethylenephosphoramide), TESPA (triethylenethiophosphoramide) and trimethylolmelamine (trimethylolomelamine); Annonaceousacetogenicompounds (acetogenins) (especially bullatacin (bullatacin) and bullatacin ketone (bullatacinone)); Delta-9-Tetrahydrocannabinol (tetrahydrocannabinol) (Dronabinol (dronabinol),

); β-lapachol (lapachone); Lapachol (lapachol); Colchicine class (colchicines); Betulic acid (betulinic acid); Camptothecine (camptothecin) (comprises synthetic analogues Hycamtin (topotecan)

CPT-11 (Irinotecan (irinotecan),

), acetyl camptothecine, scopoletin (scopoletin) and 9-aminocamptothecin); Bryostatin (bryostatin); Callystatin; CC-1065 (comprising its Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin (bizelesin) synthetic analogues); Podophyllotoxin (podophyllotoxin); Podophyllic acid (podophyllinic acid); Teniposide (teniposide); Hidden algae element class (cryptophycins) (particularly hidden algae element 1 and hidden algae element 8); Dolastatin (dolastatin); Duocarmycin (comprising synthetic analogues, KW-2189 and CB1-TM1); Eleutherobin (eleutherobin); Pancratistatin; Sarcodictyin; Sponge chalone (spongistatin); Nitrogen mustards (nitrogen mustards) is such as Chlorambucil (chlorambucil), Chlornaphazine (chlornaphazine), courage phosphamide (cholophosphamide), Estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), mustron (mechlorethamine oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide), uracil mastard (uracil mustard); Nitrosourea (nitrosoureas) is such as BCNU (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine) and Ranimustine (ranimustine); Antibiotics, such as Enediyne Antibiotic (enediyne) (Calicheamicin (calicheamicin) for example, especially Calicheamicin γ 1I and Calicheamicin ω I1 (referring to for example Agnew, Chem.Intl.Ed.Engl. 33:183-186 (1994)); Anthracycline antibiotic (dynemicin) comprises dynemicin A; Ai Sibo mycin (esperamicin); And Neocarzinostatin (neocarzinostatin) chromophore and related colour albumen Enediyne Antibiotic chromophore), aclacinomycin (aclacinomycin), D actinomycin D (actinomycin), anthramycin (anthramycin), azaserine (azaserine), bleomycin (bleomycin), act-C (cactinomycin), carabicin, carminomycin (carminomycin), cardinophyllin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), Detorubicin (detorubicin), 6-phenodiazine-5-oxygen-L-nor-leucine,Doxorubicin (doxorubicin) (comprises the morpholino Doxorubicin, cyano group morpholino Doxorubicin, 2-pyrroles is for Doxorubicin and deoxidation Doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycins) is such as mitomycin C, mycophenolic acid (mycophenolic acid), nogalamycin (nogalamycin), olivomycin (olivomycin), Peplomycin (peplomycin), potfiromycin, puromycin (puromycin), triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), streptonigrin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), Zinostatin (zinostatin), zorubicin (zorubicin); The antimetabolite class is such as methotrexate (MTX) (methotrexate) and 5 FU 5 fluorouracil (5-FU); Folacin is such as denopterin (denopterin), methotrexate (MTX) (methotrexate), pteropterin (pteropterin), Trimetrexate (trimetrexate); Purine analogue is such as fludarabine (fludarabine), Ismipur (mercaptopurine), ITG (thiamiprine), thioguanine (thioguanine); Pyrimidine analogue is such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (azauridine), Carmofur (carmofur), cytarabine (cytarabine), two BrdU (dideoxyuridine), FUDR (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); Androgens is such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), Mepitiostane (mepitiostane), Testolactone (testolactone); Anti-adrenal gland class is such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane); Folic acid supplement is such as folinic acid (folinic acid); Aceglatone (aceglatone); Aldophosphamide glucosides (aldophosphamide glycoside); Amino-laevulic acid (aminolevulinic acid); Eniluracil (eniluracil); Amsacrine (amsacrine); Bestrabucil; Bisantrene (bisantrene); Edatrexate (edatraxate); Defosfamide (defosfamide); Demecolcine (demecolcine); Diaziquone (diaziquone); Elfornithine; Elliptinium Acetate (elliptinium acetate); Epothilones (epothilone); Ethoglucid (etoglucid); Gallium nitrate; Hydroxyl urea (hydroxyurea); Lentinan (lentinan); Lonidamine (lonidamine); Maytansinoid class (maytansinoids) is such as maytansine (maytansine) and ansamitocin (ansamitocin); Mitoguazone (mitoguazone); Mitoxantrone (mitoxantrone); Croak does not reach alcohol (mopidamol); C-283 (nitracrine); Pentostatin (pentostatin); Phenamet (phenamet); THP (pirarubicin); Losoxantrone (losoxantrone); 2-ethyl hydrazides (ethylhydrazide); Procarbazine (procarbazine);

Polysaccharide compound (JHS Natural Products, Eugene, OR); Razoxane (razoxane); Rhizomycin (rhizoxin); Sizofiran (sizofiran); Spirogermanium (spirogermanium); Tenuazonic acid (tenuazonic acid); Triethyleneiminobenzoquinone (triaziquone); 2,2 ', 2 ＂-RA3; Trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verrucarin) A, roridin (roridin) A and the rhzomorph (anguidin) that crawls); Urethane (urethan); Eldisine (vindesine) (

); Dacarbazine (dacarbazine); Mannomustine (mannomustine); Dibromannitol (mitobronitol); Mitolactol (mitolactol); Croak pool bromine alkane (pipobroman); Gacytosine; Cytarabine (arabinoside) (" Ara-C "); Phosphinothioylidynetrisaziridine (thiotepa); Taxoid class (taxoids), for example

Taxol (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE^TMDo not contain cremophor (Cremophor), albumin transform nano particle formulation Taxol (American Pharmaceutical Partners, Schaumberg, Illinois) and

Taxotere (doxetaxel) (

-Poulenc Rorer, Antony, France); Chlorambucil (chlorambucil); Gemcitabine (gemcitabine)

6-thioguanine (thioguanine); Purinethol (mercaptopurine); Methotrexate (MTX) (methotrexate); Platinum analogs is such as cis-platinum (cisplatin) and carboplatin (carboplatin); Vincaleukoblastinum (vinblastine)

Platinum (platinum); Etoposide (etoposide) (VP-16); Ifosfamide (ifosfamide); Mitoxantrone (mitoxantrone); Vincristine (vincristine)

Oxaliplatin (oxaliplatin); Folinic acid (leucovorin); Vinorelbine (vinorelbine) NSC-279836 (novantrone); Edatrexate (edatrexate); Daunomycin (daunomycin); Aminopterin (aminopterin); Ibandronate (ibandronate); Topoisomerase enzyme inhibitor RFS2000; DFMO (DMFO); Retinoic acid-like class (retinoids) is such as retinoic acid (retinoic acid); Capecitabine (capecitabine)

The acceptable salt of the pharmacy of any above-mentioned substance, acid or derivative; And the combination of two or more above-mentioned substances, such as CHOP (abbreviation of endoxan, Doxorubicin, vincristine and prednisolone conjoint therapy) and FOLFOX (oxaliplatin (ELOXATIN^TM) abbreviation of therapeutic scheme of associating 5-FU and folinic acid).

This definition has also comprised adjusting, reduction, blocking-up or has suppressed to promote the antihormone agent of the hormone effect effect of cancer growth, and usually has been the form of system or whole body therapeutic. They self can be hormones. Example comprises anti-estrogens and SERM class (SERM), comprises that for example TAM (tamoxifen) (comprises

TAM),

Raloxifene (raloxifene), Droloxifene (droloxifene), 4-hydroxytamoxifen, Trioxifene (trioxifene), that Lip river former times fragrant (keoxifene), LY117018, Onapristone (onapristone) and

Toremifene (toremifene); The antiprogestin class; Adjust class (ERD) under the ERs; Work the medicament that suppresses or close the ovary effect, luteinizing hormone releasing hormone (LHRH) activator for example, such asWith

Leuprorelin acetate (leuprolide acetate), goserelin acetate (goserelin acetate), buserelin acetate (buserelin acetate) and Triptorelin (triptorelin); Anti-androgens is such as Drogenil (flutamide), Nilutamide (nilutamide) with than Ka Mite (bicalutamide); Other anti-androgens is such as Drogenil (flutamide), Nilutamide (nilutamide) with than Ka Mite (bicalutamide); And be suppressed in the adrenal gland aromatase inhibitor of the aromatase enzyme of regulating estrogen production, such as 4 (5)-imidazoles for example, aminoglutethimide (aminoglutethimide),

Megestrol acetate (megestrol acetate),

Exemestane (exemestane), formestane (formestane), Fadrozole (fadrozole),

Vorozole (vorozole),

Letrozole (letrozole) and

Anastrozole (anastrozole). In addition, this definition of chemotherapeutics comprises diphosphonates (bisphosphonates), such as clodronate (clodronate) (for example

Or

)、

Etidronate (etidronate), NE-58095,

Zoledronic acid/zoledronate (zoledronic acid/zoledronate),

Alendronate (alendronate),

Pamidronate (pamidronate),Tiludronate (tiludronate) orRisedronate (risedronate); And troxacitabine (troxacitabine) (DOX nucleosides cytimidine analog); The signal conduction that ASON, particularly those inhibition relate to adhesive cell propagation by way of in the ASON of gene expression, such as for example PKC-α, Raf, H-Ras and EGF-R ELISA (EGF-R); Vaccine, such as

Vaccine and gene therapy vaccine, for example

Vaccine,

Vaccine and

Vaccine;

Topoisomerase 1 inhibitor;RmRH; Lapatinib ditosylate (the dual EGFR-TK micromolecular inhibitor of ErbB-2 and EGFR is also referred to as GW572016); And the acceptable salt of pharmacy, acid or the derivative of any above-mentioned substance.

" growth inhibitor " refers to when being used for this paper external or suppress in vivo compound or the composition of cell (such as the cell of expressing EphB4) growth. Therefore, growth inhibitor can be the medicament that significantly reduces cell (such as the cell of the expressing EphB4) percentage that is in the S phase. The example of growth inhibitor comprises the cell cycle the advance medicament of (be in S phase beyond position) of blocking-up, such as inducing the medicament that G1 stagnates and the M phase stagnates. Classical M phase blocking agent comprises Changchun medicine class (vincas) (vincristine (vincristine) and vincaleukoblastinum (vinblastine)), taxanes (taxanes) and Topoisomerase II inhibitors such as Doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), Etoposide (etoposide) and bleomycin (bleomycin). Medicaments of those retardances G1 also overflow and enter the S phase and stagnate, for example DNA alkylating agent class such as TAM (tamoxifen), metacortandracin (prednisone), Dacarbazine (dacarbazine), chlormethine (mechlorethamine), cis-platinum (cisplatin), methotrexate (MTX) (methotrexate), 5 FU 5 fluorouracil (5-fluorouracil) and ara-C. More information can be referring to " The Molecular Basis of Cancer ", Mendelsohn and Israel compile, the 1st chapter is entitled as " Cell cycle regulation, oncogenes; and antieioplastic drugs ", the people such as Murakaini, WB Saunders, Philadelphia, 1995, especially the 13rd page. Taxanes (taxol (paclitaxel) and docetaxel (docetaxel)) is the anticarcinogen derived from yew tree. Derived from the docetaxel of European yew (

Rhone-Poulenc Rorer) be taxol (

Bristol-Myers Squibb) semi-synthetic analog. Taxol and docetaxel promote to be assembled into microtubule and by preventing that depolymerization from making microtubule stable, causes mitotic inhibition in the cell by the tubulin dimer.

" Doxorubicin (Doxorubicin) " is anthracycline antibiotic. The complete chemical name of Doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-three deoxidations-α-L-lysol-pyranohexose base) the oxygen base]-7; 8,9,10-tetrahydrochysene-6; 8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxyl group-5, the 12-naphthalenedione. (8S-cis)-and 10-[(3-amino-2, the oxy of 3,6-trideoxy-α-L-lyxo-hexapyranosyl)]-7,8,9,10-tetra hydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione

Term " anti-tumor compositions " refers to can be used for treating the composition of cancer, and it comprises at least a active therapeutic agent, for example " anticancer ". The example of therapeutic agent (anticancer is also referred to as " antitumor agent " in this article) includes but not limited to for example chemotherapeutics, growth inhibitor, cytotoxic agent, employed medicament in the radiotherapy, antiangiogenic agent, apoptosis agent, antitublin, toxin, with other medicament that is used for the treatment of cancer anti-VEGF neutrality antibody for example, the VEGF antagonist, anti-HER-2, anti-CD20, EGF-R ELISA (EGFR) antagonist (for example tyrosine kinase inhibitor), the HER1/EGFR inhibitor, erlotinib, cox 2 inhibitor (for example celecoxib (celecoxib)), interferon, cell factor, energy and ErbB2, ErbB3, ErbB4, or the antagonist (for example neutrality antibody) of one or more target thing combinations in the vegf receptor, the inhibitor of the receptor tyrosine kinase of platelet derived growth factor (PDGF) and/or stem cell factor (SCF) (for example imatinib mesylate (imatinib mesylate) (

Novartis)), TRAIL/Apo2, and other biologically active and organic chemistry agent etc.

Term " pro-drug " refers to that when being used for the application to compare the cytotoxicity of tumour cell less and can the enzymatic activation or change precursor or the derivative form of the active medicinal matter that has more active female medicine form into female medicine (parent drug). Referring to for example Wilman " Prodrugs in Cancer Chemotherapy ", Biochemical Society Transactions, 14, pp.375-382, " Prodrugs:A Chemical Approach to Targeted Drug Delivery " such as 615th Meeting Belfast (1986) and Stella, Directed Drug Delivery, Borchardt etc., compile pp.247-267, Humana Press (1985). Pro-drug of the present invention includes but not limited to phosphate-containing/ester prodrug, contains sulfo-phosphate/ester pro-drug, containing sulfate/ester prodrug, contain peptide pro-drug, the amino acid modified pro-drug of D-, glycosylation pro-drug, contain the beta-lactam pro-drug, contain the pro-drug of optional substituted benzene oxygen yl acetamide or contain optional substituted benzene acetamide pro-drug, can be converted into and have more activity and 5-flurocytosine and other 5-FUD pro-drug of the medicine of no cytotoxicity. The example of the cytotoxic drug of the prodrug form used for the present invention of can deriving includes but not limited to above-described those chemotherapeutics.

" antiangiogenic agent " or " angiogenesis inhibitor " refers to or directly or indirectly suppresses protein, recombinant protein, antibody or its conjugate or the fusion of (angiogenesis), Angiogenesis (vasculogenesis) or undesired vasopermeability occur blood vessel small molecular weight material, polynucleotides, polypeptide, separation. For example, antiangiogenic agent is antibody or other antagonist of blood vessel propellant defined above, for example the little molecule (for example PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinib malate), AMG706) of the antibody of the antibody of VEGF, vegf receptor, the conduction of blocking VEGF receptor signal. Antiangiogenic agent also comprises natural angiogenesis inhibitor, such as angiostatin (angiostatin), endostatin (endostatin) etc. Referring to for example Klagsbrun and D ' Amore Annu. Rev.Physiol.53:217-39 (1991); Streit and Detmar Oncogene22:3172-3179 (2003) (for example enumerating the table 3 of anti-angiogenic generation therapy in the chromoma); Ferrara ﹠ Alitalo Nature Medicine5 (12): 1359-1364 (1999); The Oncogene 22:6549-6556 (2003) (for example enumerating the table 2 of anti-angiogenic occurrence factor) such as Tonini; SatoInt.J.Clin.Oncol.8:200-206 (2003) (for example enumerating the table 1 of employed antiangiogenic agent in the clinical testing).

Composition and method of making the same of the present invention

The composition that comprises anti-EphB4 antibody is contained in the present invention, comprises Pharmaceutical composition; And comprise the polynucleotides of anti-EphB4 antibody coding sequence. When being used for this paper, composition comprises one or more in conjunction with the antibody of EphB4, and/or one or more comprise one or more polynucleotides in conjunction with the sequence of the antibody of EphB4 of coding. These compositions can further comprise suitable carrier, such as the acceptable excipient of pharmaceutics, comprise buffer, and they are well-known in the art.

The embodiment of antibody and the polynucleotides of separation is also contained in the present invention. The embodiment of basically pure antibody and polynucleotides is also contained in the present invention.

Anti-EphB4 antibody of the present invention is preferably monoclonal. Scope of the present invention also is provided by Fab, Fab ', Fab '-SH and the F (ab ') of the anti-EphB4 antibody that provides herein₂ These antibody fragments can create by conventional means, such as enzymatic digestion, perhaps can generate by recombinant technique. This type of antibody fragment can be chimeric or humanized. These fragments can be used for hereinafter listed diagnosis and therapeutic purposes.

Monoclonal antibody be by a group basically the antibody of homogeneity obtain, each antibody that namely consists of colony is identical, except having a sudden change with indivisible exist possible natural. Thus, modifier " monoclonal " indicates the feature of antibody, namely is not the mixture of different or polyclonal antibody.

Anti-EphB4 monoclonal antibody of the present invention can be used at first by Kohler et al., the hybridoma method of Nature 256:495 (1975) record prepares, perhaps can pass through recombinant DNA method (U.S. Patent No. 4,816,567) prepares.

In hybridoma method, immune mouse or other suitable host animal such as hamster, generate the lymphocyte that maybe can generate following antibody to cause, and described antibody is used for specific binding in the protein of immunity. The antibody of EphB4 generally by in animal repeatedly in subcutaneous (sc) or the peritonaeum (ip) inject EphB4 and adjuvant generates. EphB4 can prepare with method well-known in the art, and wherein some method has description in this article. For example, the recombinant production of EphB4 has description hereinafter. In one embodiment, animal is merged EphB4 derivative immunity to heavy chain immunoglobulin Fc part with comprising EphB4 ectodomain (ECD) and its. In a preferred embodiment, animal is used the EphB4-IgG1 fusion protein immunization. Animal usually for the immunogenic conjugate of EphB4 or derivative with monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT) carry out immunity, the solution intracutaneous injection is in a plurality of positions. After 2 weeks, animal is carried out booster immunization. After 7-14 days, to animal blood taking, and measure anti-EphB4 titre. Animal is carried out booster immunization, until titre reaches platform (plateaus).

Perhaps, can be at external immunological lymphocyte. Then, use suitable fusion agent such as polyethylene glycol that lymphocyte and myeloma cell are merged, to form hybridoma (Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103, Academic Press, 1986).

In suitable inoculation of medium and cultivation, described medium optimization contains parent myeloma cell's growth that inhibition do not merge or one or more materials of survival with the hybridoma of so preparation. For example, if parent myeloma cell lacks enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium that then is used for hybridoma typically will contain hypoxanthine, aminopterin-induced syndrome and thymidine (HAT culture medium), and these materials stop the growth of HGPRT deficient cells.

Preferred myeloma cell is that those efficiently merge, support the stable high level of selected antibody-producting cell to generate antibody and to the culture medium sensitivity such as the HAT culture medium. In these cells, preferred myeloma cell line is rat bone marrow tumour system, can restrain research institute's cell distributing center (Salk Institute Cell Distribution Center from Sol such as those, San Diego, California, USA) MOPC-21 that obtains and MPC-11 mouse tumor and can be from American type culture collection (American Type Culture Collection, Rockville, Maryland, USA) SP-2 that obtains or X63-Ag8-653 cell derive. Human myeloma and mouse-people's allos myeloma cell line has also been put down in writing for generating human monoclonal antibodies (Kozbor, J.Immunol.133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987).

The nutrient solution that can grow just therein to hybridoma is measured the generation for the monoclonal antibody of EphB4. Preferably, by immunoprecipitation or by external binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), measure the binding specificity of the monoclonal antibody that is generated by hybridoma.

The binding affinity of monoclonal antibody can be by for example Munson et al., and the Scatchard of Anal.Biochem. 107:220 (1980) analyzes to measure.

Evaluation obtain generating have the expectation specificity, avidity and/or active antibody hybridoma after, this clone can carry out subclone and cultivate (Goding by standard method by the limiting dilution rules, Monoclonal Antibodies:Principles and Practice, pp.59-103, AcademicPress, 1986).The substratum that is suitable for this purpose comprises for example D-MEM or RPMI-1640 substratum.In addition, hybridoma can carry out culturing in vivo as ascitic tumor in animal.

Can pass through routine immunization sphaeroprotein purifying rules,, subclone excretory monoclonal antibody and nutrient solution, ascites or serum suitably be separated such as for example albumin A-Sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

Anti-EphB4 antibody of the present invention can be cloned and creates by using the combinatorial libraries screening to have the active synthetic antibody of expectation.On principle, select the synthetic antibody clone by the screening phage library, described phage library contains shows the segmental phage of various antibody variable regions (Fv) of merging to bacteriophage coat protein.By at required antigenic this type of phage library of affinity chromatography elutriation.Expression can be adsorbed to antigen in conjunction with the segmental clone of required antigenic Fv, thus with the library in uncombined clone separate.Then with bonded clone wash-out from the antigen, and the further enrichment that can circulate by extra antigen absorption/wash-out.Any anti-EphB4 antibody of the present invention can followingly obtain, promptly design suitable antigen selection rules and select interested phage clone, then use Fv sequence and Kabat et al. from interested phage clone, Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda MD (1991), the anti-EphB4 antibody cloning of putting down in writing among the vols.1-3 of suitable constant region (Fc) sequence construct total length.

The antigen binding domain of antibody is formed by two about 110 amino acid whose variable (V) districts, respectively from heavy chain (VL) and light chain (VH), all presents three hypermutation rings or complementary determining region (CDR).Variable domain can functionally be illustrated on the phage, or as strand Fv (scFv) fragment (wherein VH links to each other by joint covalency short, flexibility with VL), perhaps as Fab fragment (wherein they merge and noncovalent interaction with constant domain separately), as Winter et al., Ann.Rev.Immunol., 12:433-455 (1994) is described.When being used for this paper, the phage clone of the phage clone of coding scFv and coding Fab is referred to as " Fv phage clone " or " Fv clone ".

The complete or collected works of VH and VL gene can pass through separately clone of polymerase chain reaction (PCR), and reorganization at random in phage library, can search for antigen then in conjunction with the clone, as Winter et al., Ann.Rev.Immunol., 12:433-455 (1994) is described.The library that the immunity of hanging oneself is originated can provide the antibody to the original high-affinity of immunity, need not to make up hybridoma.Perhaps, can clone non-immune complete or collected works, be used to nonself widely and self antigen provides single people's antibody sources, need not any immunity, as Griffiths et al., EMBO J, 12:725-734 (1993) is described.At last, non-immune library can also make up with synthesis mode, promptly clone the V gene of not resetting from stem cell, and use the PCR primer comprise stochastic sequence encode alterable height CDR3 district and be used in external realization rearrangement, as Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992) is described.

By merging, use filobactivirus to show antibody fragment with less important coat protein pIII.Antibody fragment can be shown as strand Fv fragment, wherein VH links to each other on same polypeptide chain by flexible polypeptide spacer with the VL structural domain, for example as Marks et al., J.Mol.Biol., 222:581-597 (1991) is described, perhaps be shown as the Fab fragment, wherein chain and pIII merge, another chain is secreted in the bacterial host cell pericentral siphon, in this assembling Fab-coat protein structure, it is illustrated on the phage surface by replacing some wild type coat proteins, for example as Hoogenboom et al., Nucl.Acids Res., 19:4133-4137 (1991) is described.

Generally speaking, obtain the nucleic acid of encoding antibody gene fragment from human or animal's immunocyte from results.Be partial to anti-EphB4 clone if wish the library, can give experimenter's immunity EphB4 producing antibody response so, and reclaim splenocyte and/or circulation B cell or other peripheral blood lymphocyte (PBL) and be used for library construction.In a preferred embodiment, the following human immunoglobulin gene fragment library that has obtained being partial to anti-EphB4 clone, promptly in carrying the functional human immunoglobulin gene array transgenic mice of (and lacking functional endogenous antibody generation system), produce anti-EphB4 antibody response, make the EphB4 immunity produce the B cell that generates at people's antibody of EphB4.Being created on of transgenic mice that generates people's antibody hereinafter has description.

The further enrichment of the reactive cell mass of can following acquisition anti-EphB4, promptly use suitable screening rules to separate to express the B cell of EphB4 specific membranes binding antibody, for example by the cellular segregation of carrying out or cell with the EphB4 affinity chromatography to the absorption of fluorescently-labeled EphB4 and follow-up fluorescence-activated cell sorting (FACS).

Perhaps, provide possible the better of antibody complete or collected works to represent, but also allowed and use EphB4 not have antigenic animal (people or inhuman) species to make up antibody library therein from the not splenocyte of immune donor and/or the use of B cell or other PBL.In order to make up the library of mixing external antibody gene, gather in the crops stem cell from the experimenter and do not reset the nucleic acid of antibody gene section so that coding to be provided.Can obtain interested immunocyte from multiple animal species (such as people, mouse, rat, Lagomorpha, luprine, dog, cat, pig, ox, horse and bird etc.).

Reclaim the nucleic acid and the amplification of encoding antibody variable gene segment (comprising VH and VL section) from interested cell.With regard to the VH and VL gene library that reset, can the required DNA of following acquisition, promptly from lymphocyte isolation of genomic DNA or mRNA, then use with 5 of VH that resets and VL gene ' and the primer of 3 ' terminal matching carry out polymerase chain reaction (PCR), as Orlandi et al., Proc.Natl.Acad.Sci. (USA), 86:3833-3837 (1989) is described, makes up diversity V gene complete or collected works thus for expressing.Can be from cDNA and genomic dna amplification V gene, reverse primer is positioned at 5 ' end of the exon of encoding mature V structural domain, and forward primer is based on J section inside, as Orlandi et al. (1989) and Ward et al., Nature, 341:544-546 (1989) is described.Yet, in order to increase from cDNA, reverse primer also can be based in the leading exon, as Jones et al., Biotechnol., 9:88-89 (1991) is described, forward primer is based in the constant region, as Sastry et al., Proc.Natl.Acad.Sci. (USA), 86:5728-5732 (1989) is described.In order to make complementary maximization, can mix degeneracy in the primer, as described in Orlandi et al. (1989) or Sastry et al. (1989).Preferably, following the library diversity is maximized, promptly use the PCR primer of each V gene family of target all obtainable VH and the VL that exists in the immunocyte nucleic acid samples that increase to reset, for example as Marks et al., J.Mol.Biol., 222:581-597 (1991) or Orum et al., Nucleic Acids Res., 21:4491-4498 (1993) is described.For the dna clone that will be increased in expression vector, can introduce rare restriction site as label at an end of PCR primer, as described in Orlandi et al. (1989), perhaps the primer with tape label carries out further pcr amplification, as Clackson et al., Nature, 352:624-628 (1991) is described.

The V gene complete or collected works of synthetic reorganization can derive from the V constant gene segment C external.Most of people VH constant gene segment Cs are cloned and are checked order (Tomlinson et al., J.Mol.Biol., 227:776-798 (1992)), and location (Matsuda et al., Nature Genet., 3:88-94 (1993)); These clones' section (comprise H1 and H2 ring all mainly construct) can be used for generating diversity VH gene complete or collected works, use the PCR primer of the multifarious H3 ring of encoding sequence and length, as Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992) is described.The VH complete or collected works also can followingly generate, and all sequences diversity concentrates on the long H3 ring of single length, as Barbas et al., and Proc.Natl.Acad.Sci.USA, 89:4457-4461 (1992) is described.People V κ and V λ section are cloned and are checked order (Williams and Winter, Eur.J.Immunol., 23:1456-1461 (1993)), and can be used for generating synthetic light chain complete or collected works.The antibody that coding is had considerable structure diversity based on the synthetic V gene complete or collected works of a series of VH and VL pleated sheet structure and L3 and H3 length.Behind the DNA of amplification coding V gene, according to Hoogenboom and Winter, J.Mol.Biol., the method for 227:381-388 (1992) can be the V constant gene segment C in external rearrangement kind.

The antibody fragment complete or collected works can followingly make up, promptly with several means with VH and VL gene gang.Can in different carriers, create each complete or collected works, and at the vitro recombination carrier, for example as Hogrefe et al., Gene, 128:119-126 (1993) is described, perhaps infects recombinant vectors, for example Waterhouse et al. external by combination, the loxP system of record among the Nucl.Acids Res., 21:2265-2266 (1993).The storage capacity that the interior recombination method of body utilizes the segmental double-stranded character of Fab to overcome and applies because of the intestinal bacteria transformation efficiency limits.Separately clone non-immune VH and VL complete or collected works, one is cloned into phagemid, and another is cloned into phage vector.Make each cell comprise a kind of various combination by the bacterium that contains phagemid with phage-infect and unite two libraries that storage capacity only is subjected to cell to have the restriction (about 10 of number then ¹²Individual clone).Two kinds of carriers are recombination signal in the inclusion body all, makes VH and VL gene recombination to single replicon, and is packaged into the phage virus grain altogether.These huge libraries provide a large amount of good avidity (Kd that has ^-1Be about 10 ^-8M) diversity antibody.

Perhaps, complete or collected works can be cloned into identical carrier successively, for example as Barbas et al., Proc.Natl.Acad.Sci.USA, 88:7978-7982 (1991) is described, perhaps is assembled together by PCR, clone then, as Clackson et al., Nature, 352:624-628 (1991) is described.The PCR assembling also can be used for VH is connected to form strand Fv (scFv) complete or collected works with the DNA of the flexible peptide spacer of coding with VL DNA.In another kind of technology, " cell in PCR assembling " is used for unite VH and VL gene by PCR in lymphocyte, clone then connect the complete or collected works of gene, as Embleton et al., Nucl.Acids Res., 20:3831-3837 (1992) is described.

The antibody that not immune library (natural or synthetic) generates can have medium avidity (Kd ^-1Be about 10 ⁶-10 ⁷M ^-1), but can also be following at in-vitro simulated affinity maturation, promptly make up and select secondary library once more, as Winter et al. (1994), it is described to see above.For example, at Hawkins et al., J.Mol.Biol., the method of 226:889-896 (1992) or Gram et al., Proc.Natl.Acad.Sci USA in the method for 89:3576-3580 (1992), uses the fallibility polysaccharase at external sudden change (the Leung etal. that introduces at random, Technique, 1:11-15 (1989)).In addition, can carry out affinity maturation, for example in selected indivedual Fv clones, use the primer that carries the stochastic sequence of crossing over CDR interested to carry out PCR and screen the more clone of high-affinity by the one or more CDR of random mutation.WO 9607754 (being disclosed on March 14th, 1996) has put down in writing and has been used in the complementary determining region induced mutation of light chain immunoglobulin to create the method in light chain gene library.Another kind of high efficiency method is VH or the VL structural domain that will select by phage display and derives from the not natural V of the existence domain variants combination of immune donor, and in the reorganization of number endless chain, screen more high-affinity, as Marks et al., Biotechnol., 10:779-783 (1992) is described.This technology allows that generation avidity is 10 ^-9The antibody of M scope and antibody fragment.

EphB4 nucleic acid and aminoacid sequence are known in the art.The nucleotide sequence of coding EphB4 can use the aminoacid sequence in required EphB4 zone to design.Perhaps, can use cDNA sequence (or its fragment), GenBank is numbered NM_004444's or is disclosed in U.S. Patent No. 5,635,177.The DNA of coding EphB4 can prepare by several different methods known in the art.These methods include but not limited to the al. by Engels et, Agnew.Chem.Int.Ed.Engl., any method of record among the 28:716-734 (1989), the chemosynthesis of carrying out such as three esters, phosphorous acid ester, phosphoramidate (phosphoramidite) and H-phosphonic acid ester method.In one embodiment, use the preferred codon of expression host cell institute in the design of the DNA of coding EphB4.Perhaps, the DNA of coding EphB4 can separate from genome or cDNA library.

After making up the DNA of coding EphB4, with dna molecular and expression vector, can be operatively connected such as the expression control sequenc in the plasmid, wherein said control sequence is subjected to the identification of this carrier transformed host cells.Generally speaking, plasmid vector comprises and duplicates and control sequence, and it is derived from the species compatible with host cell.Carrier carries replication site usually, and coding can provide the proteinic sequence of Phenotypic Selection in transformant.The carrier that is suitable for expressing in protokaryon and eukaryotic host cell is known in the art, and some further describes in this article.Can use most eukaryotes, such as yeast or derived from multicellular organisms such as mammiferous cell.

Optional is, the DNA of coding EphB4 can be operatively connected with the secretion leader sequence, cause expression product by secretory host cell in substratum.The example of secretion leader sequence comprises stII, ecotin, lamB, bleb GD, lpp, alkaline phosphatase, saccharase and α-factor.Be applicable to 36 the amino acid whose leader sequences (Abrahmsen et al., EMBO J., 4:3901 (1985)) that also have albumin A herein.

Host cell preferably transforms, and cultivates in conventional nutritional medium with expression of the present invention mentioned above or cloning vector transfection, and substratum can suitably be revised for the gene of evoked promoter, selection transformant or the required sequence of amplification coding.

Transfection refers to host cell picked-up expression vector, and no matter whether encoding sequence in fact expresses.Those of ordinary skills are up to many transfection methods, for example CaPO ₄Precipitation and electroporation.If there is any indication of this carrier-mediated transport in the host cell, think that then transfection is successful.The method that is used for transfection is well-known in the art, and some has description in this article.

Conversion refers to DNA is imported organism, makes DNA to duplicate, or as extra-chromosomal element, or pass through chromosomal integration.According to used host cell, use the standard technique that is suitable for described cell to transform.The method that is used to transform is well-known in the art, and some has description in this article.

The prokaryotic host cell that is used to generate EphB4 can be as Sambrook et al., and general described the cultivation sees above.

The mammalian host cell that is used for generating EphB4 can be cultivated at multiple substratum, and described substratum is well-known in the art, and some has description in this article.

The host cell that relates in this open book is contained the cell of vitro culture and the cell in the host animal.

The purifying of EphB4 can use art-recognized method to realize that this paper has described some of them.

The EphB4 of purifying can be attached to suitable matrix, such as sepharose 4B, acrylamide pearl, granulated glass sphere, Mierocrystalline cellulose, various acrylic copolymer, hydroxyl-metacrylate gel, polyacrylic acid and polymethyl acid copolymer, nylon, neutrality and ionophore, like that, the affinity chromatography that is used for the phage display clone is separated.EphB4 albumen can pass through Methods inEnzymology to adhering to of matrix, and the technology of record realizes among the vol.44 (1976).Protein ligands is attached to polysaccharide matrix, and for example agarose, dextran or cellulosic common technology relate to and use the halogen cyan activated carrier, subsequently the aliphatics of peptide part or primary aromatic amine are coupled to the matrix after the activation.

Perhaps, EphB4 can be used for wrapping the hole that is adsorbed plate, expresses being attached on the host cell of adsorption plate, or is used for cell sorting, perhaps be coupled to vitamin H and catch, perhaps be used for any other method that is used for elutriation phage display storehouse known in the art with pearl with the plain bag of strepto-affinity quilt.

Be suitable for to the condition of small part phage particle, making the phage library sample contact immobilized EphB4 in conjunction with sorbent material.Under the normal circumstances, select to comprise that the condition of pH, ionic strength, temperature or the like simulates physiological conditions.The phage that is bonded to solid phase is cleaned, and takes off with pickling then, for example as Barbas et al., Proc.Natl.Acad.Sci USA, 88:7978-7982 (1991) is described, perhaps takes off with alkali cleaning, for example as Marks et al., J.Mol.Biol., 222:581-597 (1991) is described, perhaps by EphB4 antigenic competition wash-out, for example with Clackson et al., Nature is in the similar rules of antigenic competition method of 352:624-628 (1991).Phage can enrichment 20-1 in single-wheel is selected, 000 times.In addition, the phage of enrichment can be cultivated in bacterial cultures, and carries out more wheels and select.

The efficient of selecting depends on many factors, comprises dissociated kinetics in the cleaning process, and whether a plurality of antibody fragments on the single phage can the while conjugated antigens.Antibody with the kinetics of comparatively fast dissociating (with weak binding affinity) can keep by the high antigen coated density that cleaning, the polyvalent phage of using the short period of time show, reach in the solid phase.High-density not only interacts by multivalence and has stablized phage, and helps the combination again of dissociated phage.Selection with antibody of the kinetics of dissociating more slowly (with strong binding affinity) can be showed (as Bass etal. by using cleaning for a long time and monovalent phages, Proteins, 8:309-314 (1990) and WO 92/09690 are described) and hang down antigen coated density (as Marks et al., Biotechnol., 10:779-783 (1992) is described) promote.

Might between the phage antibody that EphB4 is had different avidity, select, or even avidity is slightly variant.Yet the random mutagenesis (for example carrying out as some affinity maturation technology mentioned above) of selected antibody might produce many mutant, most conjugated antigens, and minority has higher avidity.By restriction EphB4, rare high-affinity phagocytosis physical efficiency competition is won.In order to keep the mutant of all higher affinity, can be with phage with excessive biotinylation EphB4 incubation, but the volumetric molar concentration of biotinylation EphB4 is lower than the target mole affinity costant of EphB4.Then with the paramagnetic beads of the plain bag of strepto-affinity quilt catch high-affinity in conjunction with phage.This type of " balance seizure " allowed according to binding affinity and selected antibody, and its susceptibility is allowed from excessive greatly low-affinity phage and isolated the mutant clone that avidity only exceeds 2 times.Can also operate the condition of cleaning the phage that is bonded to solid phase carries out based on dynamic (dynamical) differentiation of dissociating.

Anti-EphB4 clone can carry out activity and select.In one embodiment, the invention provides combining but do not block EphB4 part and the anti-EphB4 antibody of second protein (such as EphB1, EphB3, EphB4, EphB5 and/or EphB6) bonded between blocking-up EphB4 part (such as ephrin-B1, ephrin-B2 and/or ephrin-B3) and the EphB4.Fv clone corresponding to this type of anti-EphB4 antibody can followingly select: (1) separates anti-EphB4 clone from phage library as mentioned above, and optional by cultivate the isolating phage clone group that increases in suitable host bacterium; (2) select and think blocking-up respectively and do not block its active EphB4 and second protein; (3) make anti-EphB4 phage clone be adsorbed to immobilized EphB4; (4) use the second excessive protein to come the clone of any undesired, the identification EphB4 of wash-out in conjunction with determinant (it overlaps with the second combination of proteins determinant or be shared); And (5) are eluted in the clone of still adsorbing after the step (4).Optional is, has the clone of blocking characteristics of the blocking-up of expectation/and can not repeat selection rules described herein by one or many and come further enrichment.

Derive monoclonal antibody or phage display Fv clone's DNA of code book invention hybridoma is easy to use conventional rules to separate and order-checking (for example being designed to from the increase Oligonucleolide primers of interested heavy chain and light chain coding region of hybridoma or phage DNA template specificity by use).In case separate, DNA can be placed expression vector, then this expression vector transfection is not generated in the proteinic host cell of immunoglobulin (Ig) to script, such as Bacillus coli cells, ape and monkey COS cell, Chinese hamster ovary (CHO) cell or myeloma cell, in recombinant host cell, to obtain the synthetic of required monoclonal antibody.The recombinant expressed summary paper of DNA in bacterium about encoding antibody comprises Skerra et al., Curr.Opinion in Immunol., 5:256 (1993) and Pluckthun, Immunol.Revs, 130:151 (1992).

Code book invention Fv clone's DNA can the combined coding heavy chain and/or the known dna sequence (for example Shi Yi dna sequence dna can derive from Kabat et al., sees above) of constant region of light chain to form the clone of encode total length or part heavy chain and/or light chain.The constant region that will be appreciated that any isotype all can be used for this purpose, comprise IgG, IgM, IgA, IgD and IgE constant region, and this type of constant region can derive from anyone or animal species.Derived from the variable domain dna of a kind of animal (such as the people) species, the Fv clone of merging with the encoding sequence that forms " heterozygosis " total length heavy chain and/or light chain with the constant region DNA of another animal species is included in the definition of " chimeric " used herein and " heterozygosis " antibody then.In a preferred embodiment, the Fv of the variable DNA of derived from human clone and human constant region DNA merge to form encoding sequence complete people, total length or part heavy chain and/or light chain.

Can also modify the DNA of coding derived from the anti-EphB4 antibody of hybridoma of the present invention, for example by substituting, promptly the choose encoding sequence of heavy chain and constant region of light chain replaces homology mouse source sequence derived from hybridoma antibody (for example as Morrison et al., Proc.Natl.Acad.Sci.USA, the method among the 81:6851-6855 (1984)).Can further modify coding hybridoma or Fv and clone derive antibody or segmental DNA, engage the whole or part encoding sequence of immunoglobulin coding sequence and NIg polypeptide by covalency.Can prepare in this mode and have derive " chimeric " or " heterozygosis " antibody of binding specificity of antibody of Fv of the present invention clone or hybridoma clone.

Antibody fragment

Antibody fragment is contained in the present invention.In some cases, use antibody fragment to have superiority, rather than complete antibody.Segmental reduced size is allowed quick removing, and can cause being easier near solid tumor.

The multiple technologies that are used to generate antibody fragment have been developed.Traditionally, by the proteolytic digestion complete antibody derive these fragments (referring to for example Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1992); Brennan et al., Science 229:81 (1985)).Yet, can directly generate these fragments now by recombinant host cell.Fab, Fv and scFv antibody fragment all can so be allowed easily to generate these a large amount of fragments at expression in escherichia coli and by the intestinal bacteria secretion.Can be from phage antibody library discussed above the separation antibody fragment.Perhaps, can be directly reclaim Fab '-SH fragment and chemical coupling to form F (ab ') from intestinal bacteria ₂Fragment (Carter et al., Bio/Technology 10:163-167 (1992)).According to another kind of method, can directly separate F (ab ') from the recombinant host cell culture ₂Fragment.Comprise the Fab and the F (ab ') that remedy receptors bind epi-position residue, have the interior transformation period of body of prolongation ₂Fragment is recorded in U.S. Patent No. 5,869,046.Other technology that is used to generate antibody fragment will be conspicuous for skilled practitioner.In other embodiments, the antibody of selection is strand Fv fragment (scFv).Referring to WO 93/16185; U.S. Patent No. 5,571,894; And 5,587,458.Fv and sFv are the unique type that has complete binding site, lacks constant region; Reduce non-specific binding when so, they are suitable for using in vivo.Can make up the sFv fusion rotein to generate effector protein at the amino of sFv or the fusions of C-terminal.Referring to Antibody Engineering, Borrebaeck compiles, and is the same.Antibody fragment can also be " a linear antibody ", for example as U.S. Patent No. 5,641, is put down in writing in 870.This type of linear antibody fragment can be monospecific or dual specific.

Humanized antibody

Humanized antibody is contained in the present invention.The several different methods that is used for the humanization non-human antibody is known in this area.For example, humanized antibody can have one or more amino-acid residues of introducing from inhuman source.These inhuman amino-acid residues usually are called " input " residue, and they take from " input " variable region usually.Basically Winter and colleague's thereof the method for can following is carried out humanization (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988)), promptly use the corresponding sequence of inhuman hypervariable region sequence replacing people antibody.Therefore, this type of " humanization " antibody is chimeric antibody (United States Patent (USP) 4,816,567), and it is alternative with the corresponding sequence of inhuman species wherein significantly to be less than complete people variable region.In practice, humanized antibody is normally as servant's antibody, and wherein some hypervariable region residue and some possible FR residue substitute with the residue of rodents antibody class like the site.

Being used to prepare the people's light chain of humanized antibody and the selection of variable region of heavy chain is very important for reducing antigenicity.According to so-called " the suitableeest (best-fit) " method, the whole library of known person variable region sequences is screened with the variable region sequences of rodents antibody.Select people's framework (Sims et al., the J.Immunol.151:2296 (1993) as humanized antibody then with the immediate human sequence of rodents; Chothia et al., J.Mol.Biol.196:901 (1987)).Another kind method is used the consensus sequence deutero-specific frame by everyone antibody of specific light chain or heavy chain subclass (subgroup).Identical frames can be used for several different humanized antibodies (Carter et al., Proc.Natl.Acad.Sci.USA 89:4285 (1992); Presta et al., J.Immunol.151:2623 (1993)).

What is more important, antibody keep behind humanization antigenic high-affinity and other favourable biological characteristics.In order to reach this purpose, according to a kind of method, the process of analyzing parental array and each ways makes conceptual researches humanization product by the three-dimensional model that uses parental array and humanization sequence prepares humanized antibody.Usually can obtain the immunoglobulin (Ig) three-dimensional model, this is that those skilled in the art are familiar with.Also can obtain the computer program of the possible three-dimensional conformation structure of diagram and the selected candidate's immunoglobulin sequences of demonstration.By checking that these display images can analyze residue may act in candidate's immunoglobulin sequences performance function, promptly analyzing influence candidate immunoglobulin (Ig) is in conjunction with the residue of its antigenic ability.Like this, can from receptor sequence and list entries, select FR residue and combination, thereby the antibody feature that obtains expecting raises such as the avidity to target antigen.Generally speaking, the hypervariable region residue directly and the most substantially participates in influence to antigenic combination.

People's antibody

The anti-EphB4 antibody of people of the present invention can make up by uniting the Fv clone's variable domain sequence and the known people's constant domain sequence that are selected from people's charon phages displaying storehouse as mentioned above.Perhaps, can generate the anti-EphB4 antibody of human monoclonal of the present invention by hybridoma method.The human myeloma and the mouse-people's allos myeloma cell line that are used to generate human monoclonal antibodies are on the books, Kozbor J.Immunol. for example, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); And Boerner et al., J.Immunol., 147:86 (1991).

For example, might be created on the transgenic animal (for example mouse) that can after immunity, generate the complete complete or collected works of people's antibody under the situation that lacks endogenous immunoglobulin (Ig) generation now.For example, put down in writing the inhibition fully that the deletion of isozygotying of heavy chain of antibody joining region (JH) gene causes endogenous antibody to generate in chimeric and the germ line mutation mouse.Shifting a large amount of ethnic groups in this type of germ line mutation mouse is that immunoglobulin gene will cause attacking back generation people antibody at antigen.Referring to for example Jakobovits et al., Proc.Natl.Acad.Sci.USA90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann etal., Year in Immunol.7:33 (1993).

Gene reorganization also is used in external from inhuman (for example rodents) antibody people's antibody of deriving, and wherein people's antibody has avidity similar to initial non-human antibody and specificity.According to this method, it is also referred to as " the epi-position marking " (epitope imprinting), segmental heavy chain of non-human antibody or the variable region of light chain personnel selection V domain gene complete or collected works that obtain by display technique of bacteriophage replace as mentioned above, produce non-human chain-human chain scFv or Fab block polymer group.The selection of carrying out with antigen causes the separation of chimeric scFv of non-human chain/human chain or Fab, wherein human chain has been recovered antigen binding site eliminate corresponding non-human chain in one-level phage display clone after, be epi-position decision (marking, imprint) selection of human chain mating partner.When repeating this process, obtain people's antibody (, being disclosed on April 1st, 1993) referring to PCT WO 93/06213 with the non-human chain of replacement residue.Pass through CDR to transplant the non-human antibody's who carries out humanization different with traditional, this technology provides complete people's antibody, and they do not contain the FR or the CDR residue of inhuman origin.

Bi-specific antibody

Bi-specific antibody refer to at least two kinds not synantigen have the monoclonal antibody of binding specificity, preferred people's antibody or humanized antibody.In this case, one of binding specificity is at EphB4, and another of binding specificity is at any other antigen.Exemplary bi-specific antibody can be in conjunction with the proteinic two kinds of different epi-positions of EphB4.Bi-specific antibody also can be used for cytotoxic agent is positioned to express the cell of EphB4.These antibody have the EphB4 brachium conjunctivum and in conjunction with the arm of cytotoxic agent (for example Saponaria officinalis toxalbumin, anti-interferon-α, vinca alkaloids, ricin A chain, methotrexate or radio isotope haptens).Bi-specific antibody can be prepared into full length antibody or antibody fragment (F (ab ') for example ₂Bi-specific antibody).

The method that is used to make up bi-specific antibody is known in the art.Traditionally, the recombinant production of bi-specific antibody is based on the coexpression of two pairs of heavy chain immunoglobulin-light chains, and wherein two kinds of heavy chains have different specificity (Millstein and Cuello, Nature 305:537 (1983)).Because the random assignment of heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) generate the potential mixture of 10 kinds of different antibodies molecules, wherein have only a kind of correct dual specific structure that has.Usually quite trouble and product yield poorly the purifying of the correct molecule that is undertaken by the affinity chromatography step.Similarly rules are disclosed in WO 93/08829, are disclosed on May 13rd, 1993 and Traunecker et al., and EMBO is (1991) J.10:3655.

According to a kind of difference and preferred method, the antibody variable domains and the immunoglobulin (Ig) constant domain sequence that will have expectation binding specificity (antibody-antigen binding site) merge.Preferably, with comprise to small part hinge, C _H2 and C _HThe heavy chain immunoglobulin constant domain in 3 districts merges.Preferably at least a fusions, exist and comprise the first CH (C of light chain in conjunction with necessary site _H1).To encode the heavy chain immunoglobulin fusions and, when needed, the DNA of light chain immunoglobulin inserts expression vector separately, and cotransfection is in the appropriate host organism.The embodiment of optimum yield is provided when the three peptide species chain ratios that are used for making up do not wait, and this provides great handiness for adjusting the segmental mutual ratio of three peptide species.Yet, express when causing high yield with same ratio or when this ratio does not have special meaning at least two peptide species chains, the encoding sequence of two kinds or all three peptide species chains might be inserted an expression vector.

In a preferred embodiment of this method, bi-specific antibody is made of (second binding specificity is provided) heterozygosis heavy chain immunoglobulin that has first binding specificity on the arm and the heterozygosis heavy chain immunoglobulin-light chain on another arm.Because light chain immunoglobulin is the separating pathway of providing convenience of the existence in half bispecific molecule only, therefore find this unsymmetrical structure be convenient to will expectation the dual specific mixture make up with undesired immunoglobulin chain and separate.This method is disclosed in WO94/04690.About the further details that generate bi-specific antibody referring to for example Suresh et al., Methods in Enzymology 121:210 (1986).

According to another kind of method, can transform the interface between a pair of antibody molecule, with the per-cent maximization of the heterodimer that will reclaim from the reconstitution cell culture.Preferred interface comprises partial antibody constant domain C at least _H3 structural domains.In the method, one or more p1 amino acid side chains at first antibody molecule interface are replaced with larger side chain (for example tyrosine or tryptophane).By big amino acid side chain is replaced with less amino acid side chain (for example L-Ala or Threonine), on the interface of second antibody molecule, produce compensatory " cavity " with the same or similar size of bulky side chain.This provides the mechanism that improves heterodimer output than other undesired end product such as homodimer.

Bi-specific antibody comprises crosslinked or " allos coupling " antibody.For example, a kind of antibody in the allos conjugate can with affinity plain coupling, another kind of antibody and vitamin H coupling.For example, this antibody-like has been proposed to be used in the undesired cell of immune system cell target (U.S. Patent No. 4,676,980), and is used for the treatment of HIV infection (WO 91/00360, WO 92/00373 and EP 03089).Can use any cross-linking method easily to prepare allos coupling antibody.Suitable crosslinking agent is well-known in the art, is disclosed in U.S. Patent No. 4,676,980 together with many crosslinking technologicals.

Also put down in writing the technology that generates bi-specific antibody by antibody fragment in the document.For example, can use chemistry to connect and prepare bi-specific antibody.Brennan et al., Science 229:81 (1985) have put down in writing by proteolysis cutting complete antibody to generate F (ab ') ₂Segmental rules.These fragments are reduced under the situation that has two mercaptan complexing agent Sodium metaarsenites, with two mercaptan of stablizing vicinity and the formation that prevents intermolecular disulfide bond.Change the Fab ' fragment that produces into sulfo-nitrobenzoyl acid esters (TNB) derivative then.Then the reduction of one of Fab '-TNB derivative by mercaptoethylamine reverted to Fab '-mercaptan again, and mix, to form bi-specific antibody with the another kind of Fab '-TNB derivative of equimolar amount.The bi-specific antibody that produces can be used as the selectivity immobilized reagent of enzyme.

Up-to-date progress is convenient to directly reclaim Fab '-SH fragment from intestinal bacteria, but these fragment chemical couplings are to form bi-specific antibody.Shalaby et al., J.Exp.Med.175:217-225 (1992) have put down in writing the bi-specific antibody F (ab ') of full-length human ₂The generation of molecule.Separately secrete every kind of Fab ' fragment by intestinal bacteria, and carry out directed chemical coupling to form bi-specific antibody external.So the bi-specific antibody that forms can be in conjunction with crossing cell and the normal human T-cell who expresses the HER2 acceptor, and trigger the lytic activity of people's cytotoxic lymphocyte at HBT's target thing.

Also put down in writing from the reconstitution cell culture and directly generated and the multiple technologies of separating bispecific antibody fragment.For example, used leucine zipper to generate bi-specific antibody.Kostelny et al.，J.Immunol.148(5)：1547-1553(1992)。To be connected with the Fab ' part of two kinds of different antibodies by gene fusion from the proteic leucine zipper peptide of Fos and Jun.The antibody homodimer in hinge area reduction to form monomer, then again oxidation to form the antibody heterodimer.This method also can be used for generating the antibody homodimer.Hollinger et al., " double antibody " technology of Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993) record provides the replacement mechanism that makes up bispecific antibody fragment.This fragment comprises the heavy chain constant domain (V that links to each other by joint _H) and light chain constant domain (V _L), described joint is too short to make and can not match between two structural domains on same the chain.Therefore, force a V on the fragment _HAnd V _LComplementary V on structural domain and another fragment _LAnd V _HThe structural domain pairing forms two antigen binding sites thus.Also reported by using strand Fv (sFv) dimer to make up the another kind of strategy of bispecific antibody fragment.Referring to Gruber et al., J.Immunol.152:5368 (1994).

Imagined and had two antibody of tiring of surpassing.For example, can prepare three-specific antibody.Tutt et al.，J.Immunol.147：60(1991)。

Multivalent antibody

Multivalent antibody can be subjected to expressing the internalization (and/or alienation) of the cell of this antibody institute conjugated antigen faster than bivalent antibody.Antibody of the present invention can be recombinant expressed that generate, the multivalent antibody with three or more antigen binding sites (for example tetravalent antibody) that can be easy to the nucleic acid by the encoding antibody polypeptide chain (beyond the IgM classification).Multivalent antibody can comprise dimerization structural domain and three or more antigen binding site.Preferred dimerization structural domain comprises (or be made up of it) Fc district or hinge area.In this case, antibody will comprise the aminoterminal three or more antigen binding sites in Fc district and Fc district.Preferred herein multivalent antibody comprises (or be made up of it) three to about eight, but preferred four antigen binding sites.Multivalent antibody comprises at least one polypeptide chain (and preferred two polypeptide chains), and wherein said polypeptide chain comprises two or more variable domains.For example, polypeptide chain can comprise VD1-(X1) _n-VD2-(X2) _n-Fc, wherein VD1 is first variable domain, and VD2 is second variable domain, and Fc is a polypeptide chain in Fc district, X1 and X2 represented amino acid or polypeptide, and n is 0 or 1.For example, polypeptide chain can comprise: VH-CH1-flexible joint-VH-CH1-Fc district chain; Or VH-CH1-VH-CH1-Fc district chain.Multivalent antibody herein preferably further comprises at least two (and preferred four) light chain variable domain polypeptides.Multivalent antibody herein can comprise for example about two to about eight light chain variable domain polypeptides.The light chain variable domain polypeptide of this paper imagination comprises the light chain variable territory, and the optional CL structural domain that further comprises.

Antibody variants

In some embodiment, imagined the amino acid sequence modifications of antibody described herein.For example, may wish to improve binding affinity and/or other biological characteristics of antibody.The aminoacid sequence variant of antibody is to introduce antibody nucleic acid or synthesize preparation by peptide by suitable Nucleotide is changed.This type of modification comprises for example interior residue deletion and/or the insertion and/or alternative of antibody aminoacid sequence.Can carry out any deletion, insertion and alternative combinations to obtain final construction, if final construction has the feature of expectation.Can when the preparation sequence, amino acid change be introduced theme antibody aminoacid sequence.

Can be used for identifying in the antibody " alanine scanning mutagenesis " arranged, as Cunningham and Wells, described in the Science 244:1081-1085 (1989) as some residue of preferred mutagenesis position or the method in zone.Here, identify that a residue or one group of target residue are (as charged residue, such as arginine, aspartic acid, Histidine, Methionin and L-glutamic acid) and alternative with neutral or electronegative amino acid (most preferably L-Ala or Polyalanine), to influence amino acid and antigenic interaction.Then by or alternate site introduced more or other variant, weigh substituting the amino acid position of display function susceptibility.Thus, be predetermined although be used to introduce the site of variant amino acid sequence, yet the essence of sudden change itself needn't be predetermined.For example, in order to analyze the consequence of specifying the site sudden change, carry out L-Ala scanning or random mutagenesis, and expressed immunoglobulin (Ig) is screened the activity of expectation at target codon or zone.

Aminoacid sequence inserts the fusion comprise amino and/or C-terminal, and length range, and is inserted in the sequence of single or multiple amino-acid residues to the polypeptide that comprises up to a hundred or more residues by a residue.The terminal example that inserts comprises antibody with N end methionyl residue or the antibody that merges with the cytotoxicity polypeptide.Other of antibody molecule inserts variant and comprises that N or C end with antibody merge with enzyme (as being used for ADEPT) or the polypeptide of prolongation antibody serum transformation period.

That the glycosylation of polypeptide is typical or N-connects or the O-connection.N-connects and refers to that the carbohydrate module is attached to the side chain of asparagine residue.Tripeptide sequence l-asparagine-X-Serine and l-asparagine-X-Threonine (wherein X is any amino acid except that proline(Pro)) is the recognition sequence that carbohydrate module enzymatic is attached to the l-asparagine side chain.So, these two kinds of arbitrary existence of tripeptide sequence have produced the potential glycosylation site in the polypeptide.The glycosylation that O-connects refers to one of carbohydrate N-acetylgalactosamine, semi-lactosi or wood sugar are attached to hydroxy-amino-acid, and modal is Serine or Threonine, but also can use 5-oxyproline or 5-hydroxylysine.

Adding glycosylation site in antibody can make it comprise one or more above-mentioned tripeptide sequences and finish (being used for the glycosylation site that N-connects) easily by changing aminoacid sequence.Described change also can be by adding in the sequence of original antibody or substituting one or more Serines or threonine residues is carried out (being used for the glycosylation site that O-connects).

If antibody comprises the Fc district, then can change the carbohydrate that adheres on it.For example, (Presta, put down in writing in L.) has the ripe carbohydrate structure that lacks Fucose to be attached to the antibody in antibody Fc district to U.S. Patent application US 2003/0157108.Also can referring to US 2004/0093621 (Kyowa Hakko KogyoCo., Ltd.).Mentioned the antibody that five equilibrium N-acetyl-glucosamine (GlcNAc) arranged in WO 2003/011878 (people such as Jean-Mairet) and the United States Patent (USP) the 6th, 602, No. 684 (people such as Umana) in the carbohydrate that is attached to the antibody Fc district.Reported the antibody that at least one galactose residue is arranged among the WO 1997/30087 (people such as Patel) in the oligosaccharides that is attached to the antibody Fc district.About have antibody that the change carbohydrate is attached to its Fc district also can referring to WO 1998/58964 (Raju, S.) with WO 1999/22764 (Raju, S.).Also can be about having the glycosylated antigen binding molecules of improvement referring to US 2005/0123546 (Umana et al.).

Preferred glycosylation variant herein comprises the Fc district, and the carbohydrate structure that wherein is attached to the Fc district lacks Fucose.This type of variant has improved ADCC function.Optional is that the Fc district also comprises one or more amino acid replacements of further improvement ADCC, for example alternative (the Eu residue numbering) at Fc zone position 298,333 and/or 334 places.The example that relates to the publication of " taking off the Fucose type " or " Fucose shortage type " antibody comprises: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; Okazakiet al.J.Mol.Biol.336:1239-1249 (2004); Yamane-Ohnuki et al.Biotech.Bioeng.87:614 (2004).The example that the clone of fucosylated antibody is taken off in generation comprises Lec13 Chinese hamster ovary celI (the Ripka et al.Arch.Biochem.Biophys.249:533-545 (1986) of the fucosylated defective of protein; Application No. US 2003/0157108 A1, Presta, L; And WO 2004/056312 A1, Adams et al., especially embodiment 11) and knock out clone, such as α-1, the 6-fucose transferase gene, FUT8, the Chinese hamster ovary celI that knocks out (Yamane-Ohnuki et al.Biotech.Bioeng.87:614 (2004)).

Another kind of variant is the amino acid replacement variant.These variants have at least one amino-acid residue to substitute with different residues in antibody molecule.The most interesting site that substitutes mutagenesis comprises the hypervariable region, but has also imagined the FR change." preferred substituting " hurdle has shown conservative substituting in the table 1.Cause biologic activity to change if this type of substitutes, can be called the more substantial variations of " illustration substitutes " so in the importing table 1, or it is further described to see below the amino acid classification, and the screening product.

Table 1

Original residue	Illustration substitutes	Preferred substituting
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp，Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; Val; Met; Ala; Phe; Nor-leucine	Leu
			Leu(L)	Nor-leucine; Ile; Val; Met; Ala; Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr

Tyr(Y)	Trp；Phe；Thr；Ser	Phe
			Val(V)	Ile; Leu; Met; Phe; Ala; Nor-leucine	Leu

The substance of antagonist biological characteristics is modified to substitute significantly the difference on effect of keeping following aspect by selection and is realized: (a) structure of polypeptide main chain in the replacement area, for example (fold) sheet or helical conformation, (b) electric charge or the hydrophobicity of target site punishment, or (c) volume of side chain.

According to common side chain characteristic, the natural residue that exists can followingly divide into groups:

(1) hydrophobic: nor-leucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) tart: Asp, Glu;

(4) alkalescence: His, Lys, Arg;

(5) influence the residue of chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitute need be replaced another classification with the member of one of these classifications.

One class alternative variations relates to one or more hypervariable regions residue of alternative parental antibody (for example humanization or people's antibody).Usually, select to be used for the further gained variant of developing will have improvement with respect to the parental antibody that produces them biological characteristics.A kind of facilitated method that is used to generate this type of alternative variations relates to the affinity maturation that uses phage display.In brief, with several sites, hypervariable region (for example 6-7 site) sudden change, produce all possible amino acid replacement in each site.So the antibody that generates is illustrated on the filobactivirus particle, as with the fusions of the M13 gene III product of each particle internal packing.As disclosed herein the variant of phage display is screened its biologic activity (for example binding affinity) then.In order to identify the site, candidate hypervariable region that is used to modify, can carry out alanine scanning mutagenesis to identify antigen in conjunction with hypervariable region residue with significant contribution.Perhaps/in addition, analyze the crystalline structure of antigen-antibody complex to identify that the point of contact between antibody and the antigen may be useful.Described contact residues and contiguous residue are to carry out the alternate candidate locus according to the technology that this paper describes in detail.In case produce such variant, as described herein this group variant is screened, can be chosen in the antibody that has good characteristic in one or more related assays methods and be used for further exploitation.

The nucleic acid molecule of encoding antibody aminoacid sequence variant can prepare by the several different methods that this area is known.These methods include but not limited to separate (the situation of natural generation aminoacid sequence variant) from natural origin, perhaps carry out oligonucleotide mediated (or fixed point) mutagenesis, PCR mutagenesis and cassette mutagenesis by the antibody to the variant of preparation early or unmanifest pattern and prepare.

May wish in the Fc district of immunoglobulin polypeptides of the present invention to introduce a place or many places amino acid modified, generate the Fc region variants thus.The Fc region variants can be included in the people Fc region sequence (as human IgG1, IgG2, IgG3 or IgG4Fc district) that one or more amino acid positions (comprising hinge cysteine) comprise amino acid modified (as substituting).

According to the instruction of this description and this area, to have imagined in some embodiment, the corresponding antibody with wild-type of employed antibody is compared and can for example comprised a place or many places change in the Fc district in the inventive method.Compare with their wild type counterparts, these antibody will keep the needed identical characteristics of therapeutic efficiency basically.For example, think can in the Fc district, carry out causing Clq in conjunction with and/or CDC (CDC) change some change of (promptly or strengthen or weakening), for example described in the WO99/51642.Also can be referring to the Duncan and Winter that pays close attention to other example of Fc region variants, Nature 322:738-40 (1988); United States Patent (USP) 5,648,260; United States Patent (USP) 5,624,821; And WO94/29351.

WO00/42072 (Presta) and WO 2004/056312 (Lowman) have put down in writing the antibody variants in conjunction with raising or reduction to FcR.In this content of clearly taking in these patent publications as a reference.Also can be referring to Shields et al.J.Biol.Chem.9 (2): 6591-6604 (2001).Transformation period prolongs and to neonatal Fc receptor (FcRn) (it is responsible for parent IgG is transferred to fetus) (Guyer et al., J.Immunol.117:587 (1976) and Kim et al., J.Immunol.24:249 (1994)) the antibody in conjunction with improvement is recorded in US2005/0014934A1 (Hinton et al.).These antibody comprise and have a place or many places and improve Fc district and FcRn bonded alternate Fc.Have the Fc region amino acid sequence of change and the polypeptide variants of rising of Clq binding ability or reduction and be recorded in U.S. Patent No. 6,194,551B1, WO99/51642.In this content of clearly taking in these patent publications as a reference.Also can be referring to Idusogie et al.J.Immunol.164:4178-4184 (2000).

Antibody derivatives

Can further modify antibody of the present invention to comprise that this area is known and to be easy to the extra nonprotein character part that obtains.Preferably, the part that is suitable for the antibody derivatize is a water-soluble polymers.The limiting examples of water-soluble polymers includes but not limited to polyoxyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly--1,3-dioxolane, poly--1,3,6-trioxane, ethene/copolymer-maleic anhydride, polyamino acid (homopolymer or randomcopolymer) and dextran or poly-(n-V-Pyrol RC) polyoxyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylene polyvalent alcohol (as glycerine), polyvinyl alcohol and composition thereof.Because its stability in water, the polyoxyethylene glycol propionic aldehyde may have advantage aborning.Polymkeric substance can be any molecular weight, and can be ramose or unbranched.The polymkeric substance number that is attached on the antibody can change, and if adhered to and surpassed a polymkeric substance, they can be identical or different molecules so.Generally speaking, the number and/or the type of the polymkeric substance of derivatize be can be identified for, the concrete property of antibody or function included but not limited to wait to improve, whether antibody derivatives will be used to specify treatment under the condition etc. according to following consideration.

Screening has the antibody of desired characteristic

Can characterize their physico and biological function by the multiple assay method that this area is known to antibody of the present invention.In some embodiment, antibody has characterized with the next item down or multinomial: reduce or blocking-up EphB4 activation, reduce or the conduction of blocking-up EphB4 downstream molecules signal, reduce or blocking-up EphB4 ligand activation, reduce or the conduction of blocking-up EphB4 part downstream molecules signal, destroy or block ligand (ephrin-B1 for example, ephrin-B2, and/or ephrin-B3) with the combining of EphB4, EphB4 phosphorylation and/or EphB4 multimerization, and/or EphB4 part phosphorylation, and/or treat and/or prevent tumour, cell proliferative disorders or cancer, and/or treatment or prevention and EphB4 expression and/or active (expressing and/or active the rising) relevant illness such as EphB4.

Can include but not limited to the order-checking of N end, amino acid analysis, non-sex change size exclusion high pressure liquid chromatography (HPLC) (HPLC), mass spectrum, ion exchange chromatography and papain digestion by the antibody of the further purification Identification of a series of assay methods.

In certain embodiments of the invention, their biologic activity of antibody analysis to being generated herein.In some embodiment, to their antigen-binding activity of antibody test of the present invention.This area is known and antigen binding assay that can be used for this paper include but not limited to use such as technology such as Westem trace, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoprecipitation assay, fluorescence immunoassay and albumin A immunoassay any directly or the competitive binding assay method.Hereinafter in the embodiment part, provide exemplary antigen binding assay.

In another embodiment, the invention provides with 30.35,30.35.1D2 and/or the anti-EphB4 monoclonal antibody of 30.35.2D8 antibody competition EphB4 bonded.The antibody that this type of competitive antibody comprises the EphB4 epi-position discerned and antibody 30.35, the EphB4 epi-position that 30.35.1D2 and/or 30.35.2D8 discerned is identical or overlap.This type of competitive antibody can by the screening of antagonism EphB4 hybridoma supernatant liquor with pass through mark 30.35,30.35.1D2 and/or 30.35.2D8 antibody competition obtain combining of immobilization EphB4.With contain irrelevant antibody or do not contain detected bonded in the contrast binding mixture of antibody, compare through the amount of the antibody of mark, the hybridoma supernatant liquor that contains competitive antibody can reduce detected bonded in the test competition binding mixture, through the amount of the antibody of mark.Any competition binding assay described herein all is applicable to aforementioned rules.

On the other hand, the invention provides comprise 30.35, the anti-EphB4 monoclonal antibody of one or more (such as 2,3,4,5 and/or 6) HVR of 30.35.1D2 or 30.35.2D8 antibody.Comprise 30.35, the anti-EphB4 monoclonal antibody of one or more HVR of 30.35.1D2 and/or 30.35.2D8 can be as the structure that gets off, promptly as mentioned above with 30.35, one or more HVR of 30.35.1D2 and/or 30.35.2D8 are transplanted on the template antibody sequence, for example near the human antibody sequence of the corresponding mouse sequence of parental antibody or the consensus sequence of specific parental antibody light chain or everyone antibody of heavy chain subgroup, and in recombinant host cell, express gained chimeric light chain and/or weight chain variabl area sequence, there is or do not have the constant region sequence of following.

The anti-EphB4 antibody of the present invention with unique property described herein can followingly obtain, promptly by any antagonism of method easily EphB4 hybridoma colony screening desired characteristic.For example, block or do not block the anti-EphB4 monoclonal antibody of EphB4 part if desired in conjunction with EphB4, can in conjunction with competition assay, test candidate's antibody so, such as competitiveness in conjunction with ELISA, wherein plate hole wraps quilt with EphB4, the excessive antibody-solutions of purpose Eph part is taped against through on the plate that wraps quilt, and enzyme process detects bonded antibody, for example make bonded antibody contact coupling anti-Ig antibody or the anti-Ig antibody of biotinylation of HRP be arranged and manifest the HRP color reaction, for example by making the plate colour developing with strepto-affinity element-HRP and/or hydrogen peroxide and reading the plate instrument by spectrophotometry with ELISA and detect the HRP color reaction at 490nm.

Suppress if desired or the active anti-EphB4 antibody of activation EphB4, can in EphB4 phosphorylation assay method, test candidate's antibody so.This type of assay method is known in the art, describes such assay method of knurl in the embodiment part.

Cytostatic if desired anti-EphB4 antibody, so can be in measuring cytostatic external and/or body test candidate antibody in the assay method.This type of assay method is known in the art, and further description and illustration are arranged herein.

In one embodiment, the present invention has imagined and has had some but the improvement antibody of non-all effector functions, and this makes that its transformation period in the antibody body is important but some effector functions (such as complement and ADCC) is the material standed for that becomes expectation in unnecessary or deleterious many application.In certain embodiments, measure the Fc activity of the immunoglobulin (Ig) that generates to guarantee only to have kept desired characteristics.Can carry out external and/or cells in vivo toxicity test method to confirm CDC and/or the active reduction of ADCC/subdue.For example, can carry out Fc acceptor (FcR) binding assay to confirm antibody deficiency Fc γ R combination (therefore might lack the ADCC activity) but reservation FcRn binding ability.The main cell of mediation ADCC, the NK cell is only expressed Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991) the 464th page table 3 have summed up the FcR on the hematopoietic cell and have expressed.U.S. Patent No. 5,500 has been put down in writing the example of the active external test method of the ADCC that is used for the purpose of appraisals molecule in 362 or 5,821,337.The effector cell who can be used for this type of assay method comprises peripheral blood mononuclear cell (PBMC) and NK cell (NK) cell.Perhaps/in addition, the ADCC activity of purpose of appraisals molecule in vivo, for example in animal model, such as Clynes et al., disclosed in PNAS (USA) 95:652-656 (1998).Also can carry out the C1q binding assay to confirm that antibody can not be in conjunction with C1q and therefore lack the CDC activity.In order to assess complement activation, can carry out the CDC assay method, for example as Gazzano-Santoro et al., described in the J.Immunol.Methods 202:163 (1996).The method that also can use this area to know is carried out in FcRn combination and the body the removings/transformation period and is measured, and for example embodiment is described in partly.

Carrier, host cell and recombination method

For recombinant production antibody of the present invention, separate its nucleic acid of coding, and be inserted into replicable vector, be used for further clone (DNA cloning) or expression.Can use old process be easy to separate the DNA of encoding antibody and order-checking (as use can with the gene specific bonded oligonucleotide probe of encoding antibody heavy chain and light chain).Can utilize many carriers.The selection of carrier depends in part on the host cell that will use.Usually, preferred host cell is protokaryon or eucaryon (normally Mammals) origin.The constant region that will be appreciated that any isotype can be used for this purpose, comprises IgG, IgM, IgA, IgD and IgE constant region, and this type of constant region can obtain from anyone or animal species.

A. use prokaryotic host cell to generate antibody:

I. vector construction

Can use the standard recombinant technology to obtain the polynucleotide sequence of code book invention antibody polypeptides member.Can separate the polynucleotide sequence and the order-checking of expectation from antibody-producting cell such as hybridoma.Perhaps, can use Nucleotide synthesizer or round pcr synthetic polyribonucleotides.In case obtain, the sequence of coded polypeptide inserted in prokaryotic hosts, to be duplicated the also recombinant vectors of expressing heterologous polynucleotide.For the present invention, can use many carriers that this area is obtainable and know.The selection of appropriate carrier will depend primarily on the size of the nucleic acid that will insert carrier and the concrete host cell that will transform with carrier.According to its function (amplification or expressing heterologous polynucleotide, or the two furthermore) and with the consistency of its resident therein concrete host cell, every kind of carrier contains multiple member.Support element generally includes but is not limited to replication orgin, selected marker gene, promotor, ribosome bind site (RBS), signal sequence, heterologous nucleic acids insertion fragment and transcription termination sequence.

Generally speaking, the plasmid vector that uses with host cell comprise derived from the replicon and the control sequence of the compatible species of these hosts.Carrier carries replication site usually, and the flag sequence that Phenotypic Selection can be provided in transformant.For example, use plasmid pBR322 transformed into escherichia coli usually derived from species Escherichia coli.PBR322 comprises the gene of coding penbritin (Amp) and tsiklomitsin (Tet) resistance, and the means of light identification of transformed cell are provided thus.PBR322, its derivative or other microorganism plasmid or phage also can comprise or be modified and comprise and can be used to express the promotor of endogenous protein by the microorganism biological body.People such as Carter, United States Patent (USP) 5,648, in 237 write up be used to express the example of the pBR322 derivative of specific antibodies.

In addition, can the conversion carrier of the phage vector of replicon compatible with host microorganism and control sequence as these hosts will be comprised.For example, can use phage such as λ GEM.TM.-11 to make up and can be used for transforming the recombinant vectors of susceptible host cell such as intestinal bacteria LE392.

It is right that expression vector of the present invention can comprise two or more promotor-cistrons, their each polypeptide members of encoding.Promotor is the untranslated regulating and controlling sequence that is positioned at cistron upstream (5 '), the expression of its regulation and control cistron.Prokaryotic promoter is divided into two classes usually, induction type with composition.Inducible promoter refer to respond culture condition variation (as nutraceutical existence whether or temperature variation) and start the promotor that the elevated levels of the cistron that is subjected to its control is transcribed.

Be subjected to a large amount of promotors of multiple potential host cell identification as everyone knows.Insert carrier of the present invention by the promotor in the restriction enzyme digestion cutting-out source DNA and with isolating promoter sequence, the cistron DNA of the promotor of selecting and encode light chain or heavy chain can be able to be operatively connected thus.Natural promoter sequence and many allogeneic promoters all can be used for instructing the amplification and/or the expression of target gene.In some embodiment, use allogeneic promoter, because compare with natural target polypeptide promotor, they allow that usually the higher of expressed target gene transcribe and higher output yield.

The promotor that is applicable to prokaryotic hosts comprises PhoA promotor, beta-galactosidase enzymes and lactose promoter systems, tryptophane (trp) promoter systems and hybrid promoter such as tac or trc promotor.Yet it also is suitable that other promotor (such as other known bacterium or phage promoter) of function is arranged in bacterium.Their nucleotide sequence is delivered, the skilled work personnel can use joint that any required restriction site is provided or adapter that they and the cistron of coding target light chain and heavy chain can be operatively connected (Siebenlist et al., Cell 20:269 (1980)) thus.

In one aspect of the invention, each cistron in the recombinant vectors all comprises and instructs expressed polypeptide to wear the secretory signal sequence member of film transhipment.Generally speaking, signal sequence can be the member of carrier, and perhaps it can be a part of inserting the target polypeptid DNA of carrier.The signal sequence of selecting for the present invention should be the signal sequence that is subjected to host cell identification and processing (promptly being excised by signal peptidase).For nonrecognition and process the prokaryotic host cell of the natural signals sequence of heterologous polypeptide, the prokaryotic signal sequence of group substitutes: alkaline phosphatase, penicillinase, Ipp or heat-staple enterotoxin 1 I (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP with for example being selected from down with signal sequence.In one embodiment of the invention, the signal sequence that all uses in two of expression system cistrons is STII signal sequence or its variant.

On the other hand, can in the tenuigenin of host cell, take place, therefore need in each cistron, not have secretory signal sequence according to the generation of immunoglobulin (Ig) of the present invention.In that, light chain immunoglobulin and heavy chain are expressed in tenuigenin, are folded and assemble and formation functional immunity sphaeroprotein.Some host strain (as intestinal bacteria trxB-bacterial strain) provides and is beneficial to the tenuigenin condition that disulfide linkage forms, thereby allows the correct folding and assembling of expressed protein subunit.Proba and Pluckthun，Gene 159：203(1995))。

The prokaryotic host cell that is suitable for expressing antibody of the present invention comprises archeobacteria (Archaebacteria) and eubacterium (Eubacteria), such as Gram-negative or gram-positive organism.The example of useful bacterium comprises Escherichia (Escherichia) (as colon bacillus E.coli), bacillus (Bacillus) (as subtilis B.subtilis), enterobacter (Enterobacteria), Rhodopseudomonas (Pseudomonas) (as Pseudomonas aeruginosa P.aeruginosa) species, Salmonella typhimurium (Salmonella typhimurium), serratia marcescens (Serratia marcescans), Klebsiella (Klebsiella), proteus (Proteus), Shigella (Shigella), rhizobium (Rhizobium), Vitreoscilla (Vitreoscilla), or paracoccus (Paracoccus).In one embodiment, use gram-negative cells.In one embodiment, use Bacillus coli cells as host of the present invention.The example of coli strain comprises bacterial strain W3110 (Bachmann, Cellular and Molecular Biology, the 2nd volume, Washington, D.C., American Academy Of Microbiology, 1987, the 1190-1219 pages or leaves; ATCC preserving number 27,325) and derivative, comprise bacterial strain 33D3 (U.S. Patent number 5,639,635) with genotype W3110 Δ fhuA (Δ tonA) ptr3 lac Iq lacL8 Δ ompT Δ (nmpc-fepE) degP41 kanR.Other bacterial strain and derivative thereof also are suitable such as intestinal bacteria 294 (ATCC 31,446), intestinal bacteria B, intestinal bacteria λ 1776 (ATCC 31,537) and intestinal bacteria RV308 (ATCC 31,608).These examples are illustration and unrestricted.This area knows to be used to make up to have the method for specifying genotypic any above-mentioned bacterial derivation thing, referring to for example Bass et al., and Proteins 8:309-314 (1990).Usually must consider that the reproducibility of replicon in bacterial cell select the bacterium that suits.For example, when using well-known plasmid such as pBR322, pBR325, pACYC177 or pKN410 that replicon is provided, intestinal bacteria, serratia or Salmonellas species may be suitable for use as the host.Usually, host cell should be secreted the proteolytic ferment of minimum, and may wish to mix in cell cultures extra proteinase inhibitor.

Ii. antibody generates

With above-mentioned expression vector transformed host cell, and in the conventional nutritional medium of expecting the gene of sequence for evoked promoter, selection transformant or amplification coding and suitably changing, cultivate.

Transform and be about to DNA importing prokaryotic hosts, make DNA to duplicate, or as extra-chromosomal element or by the karyomit(e) composition.According to used host cell, use the standard technique that is suitable for these cells to transform.Adopt the calcium of calcium chloride to handle the bacterial cell that is generally used for having firm cell walls barrier.Another kind of method for transformation adopts polyoxyethylene glycol/DMSO.A kind of technology that also has of using is an electroporation.

That know in this area and be suitable for cultivating in the substratum of selected host cell and cultivate the prokaryotic cell prokaryocyte that is used to generate polypeptide of the present invention.The example of suitable culture medium comprises the LB substratum (Luria broth) that has added essential nutritional supplement.In some embodiment, substratum also contains the structure of with good grounds expression vector and the selective agent selected, allows the prokaryotic cell prokaryocyte growth that comprises expression vector with selectivity.For example, the substratum to the cell that is used for the culture expression ampicillin resistance gene adds penbritin.

Except carbon, nitrogen and inorganic phosphate source, also can contain any essential fill-in of proper concn, or add separately or as with the mixture of another kind of fill-in or substratum, such as compound nitrogen source.Optional is that substratum can contain one or more reductive agents that is selected from down group: gsh, halfcystine, cystamine, thioglycolate salt/ester, dithioerythritol and dithiothreitol (DTT).

Cultivate prokaryotic host cell in suitable temperature.For example, for cultivating intestinal bacteria, preferred temperature range is about 20 ℃ to about 39 ℃, more preferably from about 25 ℃ to about 37 ℃, even more preferably from about 30 ℃.Depend primarily on host organisms, the pH of substratum can be that scope is any pH of about 5 to about 9.For intestinal bacteria, pH preferably about 6.8 is to about 7.4, and more preferably from about 7.0.

If use inducible promoter in the expression vector of the present invention, express being suitable for activating under the condition of promotor induced protein so.In one aspect of the invention, use the PhoA promotor to control transcribing of polypeptide.Therefore, in order to induce, in phosphoric acid salt restriction substratum, cultivate through transformed host cells.Preferably, phosphoric acid salt restriction substratum is C.R.A.P substratum (referring to for example Simmons et al., J.Immunol.Methods 263:133-147 (2002)).According to the vector construct that is adopted, can adopt multiple other inductor, as road known in the art.

In one embodiment, expressed polypeptide of the present invention is secreted in the pericentral siphon of host cell and therefrom reclaims.Protein recovery is usually directed to destroy microorganisms, passes through such as means such as osmotic shock (osmoticshock), supersound process or cracking usually.In case cell is destroyed, can be by centrifugal or filtration clear cell debris or whole cell.Can be further purified protein by for example affine resin chromatography.Perhaps, protein may be transported in the nutrient solution and therefrom separate.Can be from the nutrient solution scavenger cell, and culture supernatants filtered and concentrate, be used to be further purified the protein that generates.Can use method such as the polyacrylamide gel electrophoresis of generally knowing (PAGE) further to separate and identify expressed protein with the Western engram analysis.

In one aspect of the invention, carry out antibody producing in a large number by fermenting process.Multiple extensive feed supplement-batch fermentation flow process can be used for producing recombinant protein.Large scale fermentation has at least 1000 liters capacity, preferred about 1,000 to 100,000 liter capacity.These fermentor tanks use agitator paddle to distribute oxygen and nutrient, especially glucose (preferred carbon source/energy).On a small scale fermentation is often referred at volume capacity and is no more than the fermentation of carrying out in about 100 liters fermentor tank, and scope can be about 1 to rise to about 100 liters.

During the fermentation, cell is being cultured to inducing of expectation density (, being in early stage stationary phase) back startup protein expression usually under conditions suitable at this stage cell as the about 180-220 of OD550.According to the vector construct that is adopted, can use multiple inductor, know as this area with above-described.Can be before inducing that cell cultures is the shorter time.Usually with the about 12-50 of cell induction hour, still can use longer or shorter induction time.

For output and the quality that improves polypeptide of the present invention, can revise multinomial fermentation condition.For example, for the correct assembling that improves secreted antibody polypeptides and folding, the additional carrier of expressing chaperone such as Dsb albumen (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (having the active a kind of peptidyl prolyl-cis of companion, trans-isomerase) that can overuse is come cotransformation host prokaryotic cell prokaryocyte.Proved that chaperone promotes the correct of heterologous protein that generates to fold and solubleness in bacterial host cell.Chen et al., J.Biol.Chem.274:19601-19605 (1999); People such as Georgiou, United States Patent (USP) 6,083,715; People such as Georgiou, United States Patent (USP) 6,027,888; Bothmann and Pluckthun, J.Biol.Chem.275:17100-17105 (2000); Ramm and Pluckthun, J.Biol.Chem.275:17106-17113 (2000); Arie et al., Mol.Microbiol.39:199-210 (2001)).

Minimum for the proteolysis of the expressed heterologous protein heterologous protein of proteolysis sensitivity (especially to) is reduced to, some host strain of proteolysis enzyme defect can be used for the present invention.For example, can modify the host cell bacterial strain, in the gene of the known bacteria protease of coding, carry out genetic mutation, such as proteolytic enzyme III, OmpT, DegP, Tsp, proteolytic enzyme I, proteolytic enzyme Mi, proteolytic enzyme V, proteolytic enzyme VI and combination thereof.Can obtain some e. coli protein enzyme defect bacterial strain, referring to for example Joly et al., (1998) see above; People such as Georgiou, United States Patent (USP) 5,264,365; People such as Georgiou, United States Patent (USP) 5,508,192; Hara et al., Microbial Drug Resistance 2:63-72 (1996).

In one embodiment, the coli strain that the plasmid of use proteolysis enzyme defect and one or more chaperones of process overexpression transforms in expression system of the present invention is as host cell.

Iii. antibody purification

The standard protein purification process that can adopt this area to know.Below flow process be the illustration of suitable purifying flow process: fractionation, ethanol sedimentation, reversed-phase HPLC, tripoli or Zeo-karb such as the chromatography on the DEAE, chromatofocusing, SDS-PAGE, the ammonium sulfate precipitation on the affine or ion exchange column of immunity and use for example gel-filtration of Sephadex G-75.

In one aspect, be used for the immunoaffinity purification of full length antibody product of the present invention with being fixed on albumin A on the solid phase.Albumin A is the 41kD cell wall protein from streptococcus aureus (Staphylococcus aureas), and it is with high-affinity binding antibody Fc district.Lindmark et al.，J.Immunol.Meth.62：1-13(1983))。Albumin A is fixed the pillar that solid phase on it preferably has glass or quartz surfaces, more preferably controllable bore diameter glass column or silicic acid post.In some applications, pillar attempts to prevent the non-special adhesion of pollutent with such as pack quilts such as glycerine.

As the first step of purifying, will be applied on the albumin A immobilization solid phase derived from the prepared product of cell culture as mentioned above, make purpose antibody specific combination albumin A.Clean solid phase then with the pollutent of removing with the non-specific combination of solid phase.Reclaim purpose antibody by wash-out from solid phase at last.

B. use eukaryotic host cell to generate antibody:

Support element generally include but be not limited to following one or more: signal sequence, replication orgin, one or more marker gene, enhancer element, promotor and transcription termination sequence.

(i) signal sequence member

The carrier that uses in eukaryotic host cell also can be held other polypeptide that comprises signal sequence or have special cleavage site at the N of purpose mature protein or polypeptide.Preferably be subjected to the allos signal sequence of host cell identification and processing (promptly being excised) by signal peptidase.In mammalian cell expression, can utilize mammalian signal sequence and viral secretory leading, for example hsv gD signal.

The DNA of these prosomas is connected in the reading frame of DNA of encoding antibody.

(ii) replication orgin

Usually, mammalian expression vector does not need the replication orgin member.For example, the SV40 starting point may only just be used because of comprising early promoter usually.

(iii) select gene component

Expression and cloning vector can comprise the selection gene, are also referred to as selection marker.Typically select the following protein of genes encoding: (a) give resistance, as penbritin, Xin Meisu, methotrexate or tsiklomitsin to microbiotic or other toxin; (b) supply corresponding auxotrophy; Or (c) provide the crucial nutrition that can not obtain from complex medium.

An example of selection scheme utilizes medicine to block the growth of host cell.Give the protein of drug resistance through those cells generations that heterologous gene successfully transforms, thereby survive selection scheme.The example that this type of dominance is selected uses medicine Xin Meisu, mycophenolic acid and Totomycin.

Another example that is suitable for the selection marker of mammalian cell is the selection marker that can identify the cell of the picked-up antibody nucleic acid of having the ability, such as the preferred primates metallothionein gene of DHFR, thymidine kinase, metallothionein(MT) I and II, adenosine deaminase, ornithine decarboxylase etc.

For example, at first, all transformants identify the cell of selecting gene transformation through DHFR in the substratum that contains methotrexate (Mtx, a kind of competitive antagonist of DHFR) by being cultivated.When adopting wild-type DHFR, suitable host cells is Chinese hamster ovary (CHO) clone (as ATCC CRL-9096) of the active defective of DHFR.

Perhaps, can by culturing cell in containing at the substratum of selective agent such as the aminoglycoside antibiotics of selection marker such as kantlex, Xin Meisu or G418 select encoded antibody, wild-type dhfr protein and another kind of selection marker such as aminoglycoside 3 '-dna sequence dna of phosphotransferase (APH) transforms or the host cell (the wild-type host who particularly comprises endogenous DHFR) of cotransformation.Referring to United States Patent (USP) 4,965,199.

(iv) promotor member

Expression and cloning vector comprise the promotor that is subjected to host organisms identification usually, and can be operatively connected with antibody polypeptides nucleic acid.Known eukaryotic promoter sequence.In fact, all eukaryotic genes all have the AT of being rich in district, and it is positioned at about 25 to 30 base places, initial upstream, site of transcribing.The another kind of sequence of 70 to 80 base place discoveries is CNCAAT districts in many gene transcription starting points upstream, and wherein N can be any Nucleotide.At 3 of most of eukaryotic genes ' end is the AATAAA sequence, and it may be the signal that adds polyadenylic acid (polyA) tail to 3 of encoding sequence ' end.In the suitable insertion carrier for expression of eukaryon of all these sequences.

In mammalian host cell by carrier transcribe that antibody polypeptides for example is subjected to obtaining from virus (such as polyomavirus, fowlpox virus, adenovirus (such as 2 type adenovirus), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40)) genome, from allos mammalian promoter (as actin promoter or immunoglobulin promoter), from the control of the promotor of heat-shocked promotor, if the compatible words of these promotors and host cell systems.

Obtain the early stage and late promoter of SV40 virus easily with the form of SV40 restriction fragment, this fragment also comprises SV40 virus replication starting point.Obtain the immediate early promoter of human cytomegalic inclusion disease virus easily with the form of HindIII E restriction fragment.United States Patent (USP) 4,419 discloses the system of bovine papilloma virus as carrier expressible dna in mammalian hosts that use in 446.United States Patent (USP) 4,601, put down in writing in 978 this system a kind of modification or, can use the Rous sarcoma virus long terminal repeat as promotor.

(v) enhancer element member

Usually improve higher eucaryotic cells transcribing to the DNA of code book invention antibody polypeptides by in carrier, inserting enhancer sequence.It is now know that from many enhancer sequence of mammalian genes (sphaeroprotein, elastoser, white protein, α-fetoprotein and Regular Insulin).Yet, use enhanser usually from eukaryotic cell virus.Example comprises enhanser (bp 100-270), the sub-enhanser of cytomegalovirus early promoter of SV40 replication orgin side in late period, the enhanser and the adenovirus enhanser of polyomavirus replication orgin side in late period.Also can be about the enhancing element that activates eukaryotic promoter referring to Yaniv, Nature 297:17-18 (1982).But the enhanser montage is positioned at 5 of antibody polypeptides encoding sequence ' or 3 ' position in carrier, but is preferably placed at 5 ' site of promotor.

(vi) Transcription Termination member

The expression vector that uses in eukaryotic host cell also comprises termination usually and transcribes and the necessary sequence of stable mRNA.This type of sequence can obtain from 5 of eucaryon or viral DNA or cDNA non-translational region ' end and 3 ' end once in a while usually.These zones are included in the non-translational region of mRNA of encoding antibody and are transcribed into the segmental Nucleotide section of polyadenylation.A kind of useful Transcription Termination member is Trobest polyadenylation district.Referring to WO94/11026 and wherein disclosed expression vector.

(the vii) selection of host cell and conversion

The host cell that is suitable for cloning or express the DNA in this paper carrier comprises higher eucaryotic cells described herein, comprises the vertebrate host cell.The breeding of vertebrate cells in cultivating (tissue culture) become old process.The example of useful mammalian host cell line has the (COS-7 of monkey kidney CV1 system that transforms through SV40, ATCC CRL 1651), human embryo kidney (HEK) system (293 cells or be 293 cells of suspension culture subclone, Graham et al., J.Gen.Virol.36:59 (1977)), baby hamster kidney cell (BHK, ATCC CCL 10), Chinese hamster ovary cell/-DHFR (CHO, Urlaub et al., Proc.Natl.Acad.Sci.USA77:4216 (1980)), mouse Sai Tuoli (Sertoli) cell (TM4, Mather, Biol.Reprod.23:243-251 (1980)), monkey-kidney cells (CV1, ATCC CCL 70), African green monkey kidney cell (VERO-76, ATCC CRL 1587), human cervical carcinoma cell (HELA, ATCC CCL2), Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL 34), ox mouse (buffalo rat) liver cell (BRL 3A, ATCC CRL 1442), human pneumonocyte (W138, ATCC CCL 75), human liver cell (Hep G2, HB 8065), (MMT 060562 for the mouse lacteal tumor, ATCC CCL 51), TRI cell (Mather et al., Annals N.Y.Acad.Sci.383:44-68 (1982)), MRC 5 cells, FS4 cell and human liver cell knurl (hepatoma) are (Hep G2).

In order to generate antibody,, and in the conventional nutritional medium of expecting the gene of sequence for evoked promoter, selection transformant or amplification coding and suitably changing, cultivate with expression mentioned above or cloning vector transformed host cell.

(the viii) cultivation of host cell

Can in multiple substratum, cultivate the host cell that is used to generate antibody of the present invention.Commercially available culture medium such as HamShi F10 (Sigma), minimum essential medium (MEM, Sigma), RPMI-1640 (Sigma) and DulbeccoShi revise the EagleShi substratum (DMEM Sigma) is suitable for cultivating host cell.In addition, can use any substratum of putting down in writing in the following document substratum: Ham et al., Meth.Enz.58:44 (1979) as host cell; Barnes et al., Anal.Biochem.102:255 (1980); United States Patent (USP) 4,767,704; 4,657,866; 4,927,762; 4,560,655; 5,122,469; WO90/03430; WO 87/00195; Or United States Patent (USP) reexamination 30,985.Any of these substratum as required hormone supplemented and/or other somatomedin (such as Regular Insulin, transferrin or Urogastron), salt (such as sodium-chlor, calcium, magnesium and phosphoric acid salt), buffer reagent (such as HEPES), Nucleotide (such as adenosine and thymidine), microbiotic (such as GENTAMYCIN ^TMMedicine), trace elements (being defined as the common mineral compound that exists with the final concentration of micro-molar range) and the glucose or the equivalent energy.Can also suitable concentration contain those skilled in the art will know that any other must fill-in.It is previous used that culture condition such as temperature, pH etc. are the host cell of expressing and selecting, and this is obvious for those of ordinary skill.

(ix) purifying antibody

When using recombinant technology, can in cell, generate antibody, perhaps direct secretion is in substratum.If in cell, generate antibody, so at first need to remove the particulate fragment by for example centrifugal or ultrafiltration, or host cell or crack fragment.If antibody-secreting is in substratum, at first commodity in use protein concentrates the concentrated supernatant liquor from these expression systems of filter (for example Amicon or Millipore Pellicon ultra filtration unit) so usually.Can in any above-mentioned steps, comprise proteinase inhibitor such as PMSF, and can comprise that microbiotic is to prevent the growth of external contaminant with the arrestin hydrolysis.

For example can use hydroxyapatite, gel electrophoresis, dialysis and affinity chromatography (preferred purification technique is an affinity chromatography) to come the antibody compositions of purifying from cell preparation.Albumin A depends on the kind and the isotype of any immunoglobulin Fc domain that exists in the antibody as the suitability of affinity ligand.Albumin A can be used for the antibody (Lindmark et al., J.Immunol.Meth.62:1-13 (1983)) of purifying based on people γ 1, γ 2 or γ 4 heavy chains.Protein G recommends to be used for all mouse isotypes and people γ 3 (Guss et al., EMBOJ.5:1567-1575 (1986)).What the accompanying matrix of affinity ligand was the most frequently used is agarose, but can use other matrix.The matrix of physically stable such as controllable bore diameter glass or poly-(vinylbenzene divinyl) benzene can obtain than agarose flow velocity and shorter process period faster.If antibody comprises the CH3 structural domain, then can use Bakerbond ABX ^TM(J.T.Baker, Phillipsburg NJ) carry out purifying to resin.According to antibody to be recycled, also can use other purified technology of protein such as the chromatography on the fractionation on the ion exchange column, ethanol sedimentation, reversed-phase HPLC, the tripoli, heparin SEPHAROSE ^TMOn chromatography, negatively charged ion or Zeo-karb (such as the poly aspartic acid post) on chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any preliminary purification step, the mixture that contains purpose antibody and pollutent can be hanged down the pH hydrophobic interaction chromatography, use the elution buffer of the about 2.5-4.5 of pH, preferably carry out at low salt concn (0-0.25M salt according to appointment).

Immune conjugate

The present invention also provides and has comprised coupling the immune conjugate of described herein any anti-EphB4 antibody of cytotoxic agent (interchangeable being called " antibody-drug conjugates " or " ADC "), described cytotoxic agent such as chemotherapeutics, medicine, growth inhibitor, toxin (as enzyme activity toxin or its fragment of bacterium, fungi, plant or animal origin) or radio isotope (promptly radiating conjugate) are arranged.

Antibody-drug conjugates is used for local purposes (Syrigos and Epenetos, the Anticancer Research 19:605-614 (1999) that poisons the cell agent or suppress cell reagent (promptly being used to kill or suppress the medicine of tumour cell) that deliver in cancer therapy; Niculescu-Duvaz and Springer, Adv.Drg.Del.Rev.26:151-172 (1997); United States Patent (USP) 4,975,278) the drug moiety target can be delivered to tumour, and the there carry out accumulating in the cell, and systemic application these may cause beyond the tumour cell of attempting to eliminate unacceptable without the link coupled pharmaceutical agent to Normocellular toxic level (Baldwin et al., Lancet 603-05 (on May 15th, 1986); Thorpe, " Antibody CarriersOf Cytotoxic Agents In Cancer Therapy:A Review ", in " Monoclonal Antibodies ｀ 84:Biological And Clinical Applications ", people such as A.Pinchera compile, the 475-506 page or leaf, 1985).Attempt to obtain maximum effect and minimum toxicity thus.Polyclonal antibody and monoclonal antibody all have report to can be used for these strategies (Rowland et al., Cancer Immunol.Immunother.21:183-87 (1986)).Employed medicine comprises daunomycin (daunomycin), Dx (doxorubicin), methotrexate (methotrexate) and vindesine (vindesine) (Rowland etal., 1986, see above) in these methods.Employed toxin comprises bacteriotoxin such as diphtheria toxin, plant poison such as ricin, small molecules toxin such as geldanamycin (geldanamycin) (Mandler et al., Jour.of the Nat.Cancer Inst.92 (19): 1573-1581 (2000) in antibody-toxin conjugated thing; Mandler et al., Bioorganic ﹠amp; Med.Chem.Letters 10:1025-1028 (2000); Mandleret al., Bioconjugate Chem.13:786-791 (2002)), maytansinoid class (EP1391213; Liu et al., Proc.Natl.Acad.Sci.USA 93:8618-8623 (1996)) and calicheamicin (Lode et al., Cancer Res.58:2928 (1998); Hinman et al., Cancer Res.53:3336-3342 (1993)).Toxin can be by comprising that tubulin combination, DNA combination or topoisomerase are suppressed at interior mechanism and bring into play the effect that it is poisoned cell and suppresses cell.Some cell toxicity medicament is tending towards inactivation or active the reduction with big antibody or protein acceptor ligand coupling the time.

ZEVALIN

(ibritumomab tiuxetan, Biogen/Idec) be by at the antigenic mouse IgG1 of the CD20 κ monoclonal antibody of on normal and malignant B surface, finding and the antibody-radio isotope conjugate (Wiseman et al., Eur.Jour.Nucl.Med.27 (7): the 766-77 (2000) that constitute by thiourea linker-bonded 111In of sequestrant institute or 90Y radio isotope; Wiseman et al., Blood 99 (12): 4336-42 (2002); Witzig et al., J.Clin.Oncol.20 (10): 2453-63 (2002); Witzig et al., J.Clin.Oncol.20 (15): 3262-69 (2002)).Although ZEVALIN has the activity at B cell Fei Hejiejinshi (Hodgkin) lymphoma (NHL), yet dispenser causes serious and long hemocytopenia in Most patients.MYLOTARG ^TM(gemtuzumabozogamicin, Wyeth Pharmaceuticals), the antibody-drug conjugates that promptly is connected with calicheamicin by people CD33 antibody and constitutes is used for through injection for curing acute myelogenous leukemia (Drugs of the Future 25 (7): 686 (2000) approval in 2000; United States Patent (USP) 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001).Cantuzumab mertansine (Immunogen Inc.), promptly be connected with maytansinoid drug moiety DM1 and the antibody-drug conjugates that constitutes through disulphide joint SPP by huC242 antibody, the II phase that is being used for the treatment of cancer such as colorectal carcinoma, carcinoma of the pancreas, cancer of the stomach and other cancer of expressing CanAg tests.MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), the antibody-drug conjugates that promptly is connected with maytansinoid drug moiety DM1 by anti-prostatic specific membrane antigen (PSMA) monoclonal antibody and constitutes is being used for the exploitation of the potential treatment of tumor of prostate.Synthetic analogues auristatin peptide, auristatin E (AE) and monomethyl auristatin (MMAE) and chimeric mAb cBR96 (special) and cAC10 (special) coupling (Doronina et al. with dolastatin (dolastatin) to the CD30 on the pernicious neoplastic hematologic disorder to the Lewis Y on the cancer, Nature Biotechnology 21 (7): 778-784 (2003)), and carrying out the therapeutic exploitation.

This paper (for example above) has described the chemotherapeutics that can be used for generating immune conjugate.Spendable enzyme activity toxin and fragment thereof comprise diphtheria toxin A chain, the non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa Pseudomonas aeruginosa), ricin (ricin) A chain, toxalbumin (abrin) A chain, capsule lotus root toxalbumin (modeccin) A chain, α-broom aspergillin (sarcin), tung oil tree (Aleutites fordii) toxalbumin, Dianthus caryophyllus L. (dianthin) toxalbumin, dyers' grapes (Phytolacaamericana) toxalbumin (PAPI, PAPII and PAP-S), balsam pear (Momordica charantia) inhibition, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) inhibition, white tree toxalbumin (gelonin), NSC-69529 (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (trichothecenes).Referring to disclosed WO 93/21232 on October 28th, 1993 for example.Multiple radionuclide can be used for generating radiation coupling antibody.Example comprises 212Bi, 131I, 131In, 90Y and 186Re.The conjugate of antibody and cytotoxic agent can use multiple bifunctional protein coupling agent to prepare; such as N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP); imino-sulfane (IT); imido-ester (all example hydrochloric acid hexanedioyl imido acid dimethyl esters); active ester class (such as suberic acid two succinimido esters); aldehydes (such as glutaraldehyde); double azido compound (such as two (right-the azido benzoyl base) hexanediamine); dual azepine derivatives (such as two (right-the diazobenzene formyl radical) hexanediamine); vulcabond is (such as toluene 2; the 6-vulcabond); with the dual-active fluorine cpd (such as 1; 5-two fluoro-2,4-dinitrobenzene) dual-function derivative.For example, can be as Vitetta et al., the ricin of preparation described in the Science 238:1098 (1987) immunotoxin.The 1-isothiocyanic acid phenmethyl of carbon-14 mark-3-methyl diethylene triaminepentaacetic acid(DTPA) (MX-DTPA) is the exemplary sequestrant that is used for radioactive nuleus thuja acid and antibody coupling.Referring to WO 94/11026.

This paper has also imagined antibody and one or more small molecules toxin such as calicheamicin (calicheamicin), maytansinoid class (maytansinoids), dolastatin class (dolostatins), aurostatins, trichothecin (trichothecene) and CC1065 and these toxin and has had the active segmental conjugate of toxin.

I. maytenin and maytansinoid class

In some embodiment, immune conjugate comprises coupling the antibody of the present invention of one or more maytansinoid molecules (total length or fragment).

The maytansinoid class is by suppressing the mitotic inhibitor that the tubulin multimerization plays a role.Maytenin obtains (United States Patent (USP) 3,896,111) from East Africa shrub tingia Caulis Mayteni (Maytenus serrata) separation at first.Find that subsequently certain micro-organisms also generates the maytansinoid class, such as maytansinol and C-3 maytansinol ester (United States Patent (USP) 4,151,042).For example following U.S. Patent Publication synthetic maytansinol and derivative and analogue: 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; And 4,371,533.

Maytansinoid class medicine module is attractive medicine module in antibody drug conjugates, because they: (i) be easy to relatively prepare by chemically modified, the derivatize of fermentation or tunning; (ii) be easy to the link coupled functional group derivatize that is suitable for by non-disulphide joint; (iii) stable in blood plasma; And (iv) effectively be at kinds of tumor cells.

For example following patent disclosure comprise immune conjugate and the preparation and the therepic use of maytansinoid class: United States Patent (USP) 5,208,020; 5,416,064; And European patent EP 0 425 235 B1, clearly its disclosure is collected herein by reference.Liu et al., Proc.Natl.Acad.Sci.USA 93:8618-8623 (1996) have put down in writing the immune conjugate that comprises the maytansinoid that is called DM1 that is connected with monoclonal antibody C242 at human colorectal cancer.Find that this conjugate has the height cytotoxicity at the colon cancer cell of cultivating, and show anti-tumor activity in the tumor growth assay method in vivo.Chari et al., Cancer Research 52:127-131 (1992) put down in writing maytansinoid wherein through the disulphide joint with combine CCL188 on antigenic murine antibody A7 or in conjunction with the another kind of mouse monoclonal antibody TA.1 link coupled immune conjugate of HER-2/neu oncogene.External be the cytotoxicity of having tested TA.1-maytansinoid conjugate on the SK-BR-3 at human breast cancer cell, each cell expressing 3 x 10 of this clone ⁵Individual HER-2 surface antigen.Drug conjugates has reached to a certain degree the cytotoxicity similar to free maytansinoid medicine, and this can improve by increasing each antibody molecule link coupled maytansinoid molecule number.A7-maytansinoid conjugate shows low systemic cytotoxicity in mouse.

Antibody-maytansinoid conjugate can prepare by the biologic activity that antibody is connected with the maytansinoid molecular chemistry and significantly do not weaken antibody or maytansinoid molecule.Referring to for example U.S. Patent No. 5,208,020, clearly its disclosure is collected herein by reference.Average 3-4 maytansinoid molecule of each antibody molecule coupling shows effect in the cytotoxicity that strengthens at target cell, and the function or the solubleness of antagonist do not have negative impact, although the toxin/antibody of an expectation even a molecule also will strengthen cytotoxicity than the use of naked antibody.The maytansinoid class is well known in the art, and can synthesize or separate from natural origin by known technology.For example United States Patent (USP) 5,208,020 and other patent mentioned above and non-patent deliver and disclose suitable maytansinoid class in the thing.The aromatic nucleus that preferred maytansinoid class is maytansinol and maytansinol molecule or other position maytansinol analogue through modifying is such as various maytansinol esters.

This area knows that many linking groups can be used for preparing antibody-maytansinoid conjugate, comprises for example United States Patent (USP) 5,208,020 or European patent 0 425 235 B1; Chari et al., CancerResearch 52:127-131 (1992); The U.S. Patent application No.10/960 that submitted on October 9th, 2004, disclosed in 602, clearly its disclosure is collected herein by reference.Antibody-maytansinoid class the conjugate that comprises joint member SMCC can be as the U.S. Patent application No.10/960 that submitted on October 8th, 2004, preparing disclosed in 602.Linking group comprises disulphide group, sulfide group, acid-unstable group, photo-labile group, the unstable group of peptase or the unstable group of esterase, as disclosed in the patent mentioned above, and preferred disulphide and sulfide group.Describe herein and illustration other linking group.

Can use multiple bifunctional protein coupling agent to prepare the conjugate of antibody and maytansinoid; such as N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP); succinimido-4-(N-maleimide amino methyl) hexanaphthene-1-carboxylicesters (SMCC); imino-sulfane (IT); imido-ester (all example hydrochloric acid hexanedioyl imido acid dimethyl esters); active ester class (such as suberic acid two succinimido esters); aldehydes (such as glutaraldehyde); double azido compound (such as two (right-the azido benzoyl base) hexanediamine); dual azepine derivatives (such as two (right-the diazobenzene formyl radical)-quadrols); vulcabond is (such as toluene 2; the 6-vulcabond); with the dual-active fluorine cpd (such as 1; 5-two fluoro-2,4-dinitrobenzene) dual-function derivative.Particularly preferred coupling agent comprises N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP) (Carlsson et al., Biochem.J.173:723-737 (1978)) and N-succinimido-4-(2-pyridylthio) valerate (SPP), provide disulfide linkage to connect thus.

According to the type that connects, joint can be attached to a plurality of positions of maytansinoid molecule.For example, can use conventional coupling technology to form ester bond by reaction with hydroxyl.Reaction can occur in C-3 position with hydroxyl, through C-14 position that methylol is modified, through the C-15 position of hydroxyl modified with have the C-20 position of hydroxyl.In a preferred embodiment, forming key in the C-3 position of maytansinol or maytansinol analogue connects.

Ii.Auristatin and dolastatin

In some embodiment, immune conjugate comprises and dolastatin class (dolastatins) or dolastatin peptide analogs and derivative, auristatin class link coupled antibody of the present invention (U.S. Patent No. 5,635,483; 5,780,588).Dolastatin class and auristatin class have demonstrated disturbs microtubule kinetics, GTP hydrolysis, and nuclear and cell fission (Woyke et al (2001) Antimicrob.Agents andChemother.45 (12): 3580-3584) and have that anticancer (US 5,663,149) and anti-mycotic activity (Pettitet al (1998) Antimicrob.Agents Chemother.42:2961-2965).Dolastatin or auristatin medicine module can be attached to antibody (WO 02/088172) via N (amino) end or C (carboxyl) end of peptide medicine module.

Exemplary auristatin embodiment comprises terminal monomethyl auristatin medicine module DE and the DF that connects of N-, be disclosed in " Monomethylvaline Compounds Capable of Conjugation toLigands ", US serial number No.10/983,340, submitted on November 5th, 2004, clearly be collected herein by reference its disclosure is complete.

Be typically, can prepare by between two or more amino acid and/or peptide fragment, forming peptide bond based on the medicine module of peptide.This type of peptide bond can prepare (referring to E. according to for example well-known liquid phase synthesizing method in chemistry of peptides field And K.L ü bke, The Peptides, volume1, pp76-136,1965, Academic Press).Auristatin/ dolastatin medicine module can prepare according to the method in the following document: US 5,635, and 483; US 5,780, and 588; Pettit et al (1989) J.Am.Chem.Soc.111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G.R., et al.Synthesis, 1996,719-725; Pettit et al (1996) J.Chem.Soc.PerkinTrans.1 5:859-863; And Doronina (2003) Nat Biotechnol21 (7): 778-784; " Monomethylvaline Compounds Capable of Conjugation to Ligands ", U.S. serial number No.10/983,340, submitted on November 5th, 2004, with its complete being collected herein by reference (disclosed and for example prepared joint and the method that is coupled to the monomethyl Xie Ansuan compound of joint such as MMAE and MMAF).

Iii. calicheamicin

In other said so counter taking, immune conjugate comprised the antibody of the present invention that coupling has one or more calicheamicin molecules.Calicheamicin microbiotic family can generate the double-stranded DNA fracture in inferior picomole concentration.About the preparation of calicheamicin family conjugate referring to United States Patent (USP) 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; 5,877,296 (all authorizing U.S. Cyanamid company).Available calicheamicin analog includes but not limited to γ 1I, α 2I, α 3I, N-ethanoyl-γ 1I, PSAG and θ I1 (Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998); And the above-mentioned United States Patent (USP) of authorizing U.S. Cyanamid company).The another kind of antitumor drug that can put together with antibody is QFA, and it is a kind of antifolic thing.Calicheamicin and QFA have action site in the born of the same parents, and are difficult for passing plasma membrane.Therefore, these reagent have strengthened their cytotoxic effect greatly via the cellular uptake of antibody-mediated internalization.

Iv. other cytotoxic agent

Can comprise BCNU, streptozocin (streptozoicin), vincristine(VCR) (vincristine), 5 FU 5 fluorouracil, United States Patent (USP) 5 with other antineoplastic agent of antibody coupling of the present invention, 053,394,5,770, the reagent family that is referred to as the LL-E33288 mixture of record, and Ai Sibo mycin class (esperamicins) (United States Patent (USP) 5 in 710,877,296).

Available enzyme activity toxin and fragment thereof comprise diphtheria toxin A chain, the non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa Pseudomonas aeruginosa), ricin (ricin) A chain, toxalbumin (abrin) A chain, capsule lotus root toxalbumin (modeccin) A chain, α-broom aspergillin (sarcin), tung oil tree (Aleutites fordii) toxalbumin, Dianthus caryophyllus L. (dianthin) toxalbumin, dyers' grapes (Phytolaca americana) toxalbumin (PAPI, PAPII and PAP-S), balsam pear (Momordicacharantia) inhibition, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) inhibition, white tree toxalbumin (gelonin), NSC-69529 (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (trichothecenes).Referring to the WO 93/21232 that for example announced on October 28th, 1993.

The present invention has also imagined antibody and has had the active compound of nucleolysis (as rnase or DNA endonuclease, such as deoxyribonuclease; The DNA enzyme) immune conjugate that forms between.

For the selective destruction tumour, antibody can comprise the height radioactive atom.Multiple radio isotope can be used for generating radiation coupling antibody.Example comprises At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³², Pb ²¹²Radio isotope with Lu.When conjugate is used to detect, can comprises radioactive atom and be used for scitiphotograph research, for example Tc ^99mOr I ¹²³, or comprise spin label be used for nucleus magnetic resonance (NMR) imaging (be also referred to as nuclear magnetic resonance, mri), such as iodo-123, iodine-131, indium-111, fluoro-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Can in a known way radioactivity or other marker be mixed conjugate.For example, but the biosynthesizing peptide, or by the synthetic peptide of chemical amino acid synthesis method, wherein use to relate to for example suitable amino acid precursor of fluoro-19 replacement hydrogen.Can adhere to marker by the cysteine residues in peptide, such as Tc99m or I123, Re186, Re188 and In111.Can adhere to Yttrium-90 through lysine residue.IODOGEN method (Frakeret al., Biochem.Biophys.Res.Commun.80:49-57 (1978)) can be used for mixing iodo-123." Monoclonal Antibodies in Immunoscintigraphy " (Chatal, CRC Press, 1989) write up other method.

Can use multiple bifunctional protein coupling agent to prepare the conjugate of antibody and cytotoxic agent; such as N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP); succinimido-4-(N-maleimide amino methyl) hexanaphthene-1-carboxylicesters (SMCC); imino-sulfane (IT); imido-ester (all example hydrochloric acid hexanedioyl imido acid dimethyl esters); active ester class (such as suberic acid two succinimido esters); aldehydes (such as glutaraldehyde); double azido compound (such as two (right-the azido benzoyl base) hexanediamine); dual azepine derivatives (such as two (right-the diazobenzene formyl radical)-quadrols); diisothio-cyanate is (such as toluene 2; the 6-vulcabond); with the dual-active fluorine cpd (such as 1; 5-two fluoro-2,4-dinitrobenzene) dual-function derivative.For example, can be as Vitetta et al., the ricin of preparation described in the Science 238:1098 (1987) immunotoxin.The 1-isothiocyanic acid phenmethyl of carbon-14 mark-3-methyl diethylene triaminepentaacetic acid(DTPA) (MX-DTPA) is the exemplary sequestrant that is used for radioactive nuleus thuja acid and antibody coupling.Referring to WO94/11026.Joint can be " can cut joint " of being convenient to discharge cell toxicity medicament in cell.For example, can use sour unstable joint, peptase responsive joint, photo-labile joint, dimethyl joint or contain disulphide joint (Chari et al., Cancer Research 52:127-131 (1992); United States Patent (USP) 5,208,020).

Compound of the present invention is clearly contained but is not limited to ADC with the preparation of following linking agent: commercialization is (as available from Pierce Biotechnology Inc., Rockford, IL, BMPS U.S.A.), EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (succinimido-(4-vinyl sulphone) benzoic ether).See 2003-2004 year application manual and products catalogue (Applications Handbookand Catalog) 467-498 page or leaf.

V. the preparation of antibody-drug conjugates

In antibody-drug conjugates of the present invention (ADC), antibody (Ab) is puted together through joint (L) and one or more drug moieties (D), for example each antibody coupling about 1 to about 20 drug moieties.Can adopt organic chemical reactions, condition and the reagent that those skilled in the art will know that to prepare the ADC of general formula I by several paths, comprise: the nucleophilic group of (1) antibody is through covalent linkage and divalence joint reagent react, form Ab-L, react with drug moiety D subsequently; (2) nucleophilic group of drug moiety forms D-L through covalent linkage and divalence joint reagent react, and the nucleophilic group with antibody reacts subsequently.The method for distinguishing that is used to prepare ADC has been described herein.

Ab-(L-D)p I

Joint can be made of one or more joint member.Exemplary joint member comprises 6-maleimide caproyl (＂ MC ＂); maleimide propionyl (＂ MP ＂); Xie Ansuan-citrulline (＂ val-cit ＂); L-Ala-phenylalanine (＂ ala-phe ＂); to amino carbobenzoxy-(Cbz) (＂ PAB ＂); 4-(2-pyridylthio) valeric acid N-succinimido ester (＂ SPP ＂); 4-(N-maleimide methyl) hexanaphthene-1 carboxylic acid N-succinimido ester (＂ SMCC '); (4-iodo-ethanoyl) benzaminic acid N-succinimido ester (＂ SIAB ＂).Other joint member is known in this area, and this paper has also described some.Also can be referring to " MonomethylvalineCompounds Capable of Conjugation to Ligands ", U.S. serial number No.10/983, on November 5th, 340,2004 submitted to, and its complete content is collected herein by reference.

In some embodiment, joint can comprise amino-acid residue.Exemplary amino acid joint member comprises dipeptides, tripeptides, tetrapeptide or pentapeptide.Exemplary dipeptides comprises: Xie Ansuan-citrulline (vc or val-cit), L-Ala-phenylalanine (af or ala-phe).Exemplary tripeptides comprises: glycine-Xie Ansuan-citrulline (gly-val-cit) and Gly-Gly-Gly (gly-gly-gly).The amino-acid residue that constitutes the amino acid joint member comprises those naturally occurring amino acid, and the amino acid analogue of accessory amino acid and non-natural existence, such as citrulline.The amino acid joint member can design at their the selectivity aspect of enzymatic cutting of certain enzyme (for example tumor correlated albumen enzyme, cathepsin B, C and D, or plasmin proteolytic enzyme) and optimize.

The nucleophilic group of antibody includes but not limited to: (i) N-terminal amino; (ii) side chain amino is as Methionin; (iii) side chain sulfydryl is as halfcystine; (iv) in the glycosylated antibodies sugar hydroxyl or amino.Amino, sulfydryl and hydroxyl are nucleophilic, can react with the electrophilic group on the shank and the formation covalent linkage, and joint reagent comprise: (i) active ester class, such as NHS ester, HOBt ester, haloformate and acid halide; (ii) alkyl and phenmethyl halogenide are such as Haloacetamide; (iii) aldehydes, ketone, carboxyl and maleimide base group.Some antibody has reducible interchain disulfide bond, i.e. the halfcystine bridge.Can handle by reductive agent such as DTT (dithiothreitol (DTT)) makes antibody have the reactive behavior of puting together with joint reagent.Each halfcystine bridge will form two reactive mercaptan nucleophiles in theory.Can cause amine to change mercaptan into via the reaction of Methionin and 2-imino-sulfane (TrautShi reagent), thereby extra nucleophilic group is introduced antibody.Can reactive thiol group be imported antibody (or its fragment) by importing one, two, three, four or more a plurality of cysteine residues (for example preparation comprises the mutant antibody of one or more non-natural cysteine amino).

Also can generate antibody-drug conjugates of the present invention by modified antibodies, promptly introduce can with the electrophilic part of nucleophilic substitution radical reaction on joint reagent or the medicine.The sugar of available for example periodate oxidation agent oxidation glycosylated antibodies, thus form can with the aldehydes or ketones group of the amine groups reaction of joint reagent or drug moiety.Gained imines Schiff base can form stable key, perhaps available for example hydroborate reagent reduction and form stable amine and connect.In one embodiment, the carbohydrate part of glycosylated antibodies can generate carbonyl (aldehyde and ketone) group with the reaction of galactose oxidase or sodium metaperiodate in protein, it can with the suitable radical reaction (Hermanson, Bioconjugate Techniques) on the medicine.In another embodiment, the protein that comprises N-terminal Serine or threonine residues can react with sodium metaperiodate, causes at first amino acid place generation aldehyde (Geoghegan ﹠amp; Stroh, Bioconjugate Chem.3:138-146 (1992); US5,362,852).This type of aldehyde can react with drug moiety or joint nucleophile.

Equally, nucleophilic group on the drug moiety includes but not limited to: amine, mercaptan, hydroxyl, hydrazides, oxime, hydrazine, thiosemicarbazone, hydrazinecarboxylate and aryl hydrazide group, they can react and the formation covalent linkage with the electrophilic group on the shank, and joint reagent comprises: (i) active ester class, such as NHS ester, HOBt ester, haloformate and acid halide; (ii) alkyl and phenmethyl halogenide are such as Haloacetamide; (iii) aldehydes, ketone, carboxyl and maleimide base group.

Perhaps, can synthesize by for example recombinant technology or peptide and prepare the fusion rotein that comprises antibody and cytotoxic agent.The length of DNA can comprise the zone of two parts of each own coding conjugate, or adjoins each other or by the zone of coding joint peptide separately, this joint peptide does not destroy the desired characteristic of conjugate.

In another embodiment, antibody and " acceptor " (such as Streptavidin) thereby coupling can be used for tumour target in advance, wherein to patient's administration of antibodies-acceptor conjugate, then use scavenging agent by removing unconjugated conjugate in the circulation, use then and cytotoxic agent (as the radioactive nuleus thuja acid) link coupled " part " (as the affinity element).

Medicinal proportional preparation

Antibody that can be by will having expectation purity and optional physiology can be accepted carrier, vehicle or stablizer and mix and prepare the treatment preparaton that comprises antibody of the present invention, with the form storage (Remington:The Science and Practice of Pharmacy 20thedition (2000)) of the aqueous solution, freeze-drying or other dry formulation.Acceptable carrier, vehicle or stablizer are nontoxic at dosage that is adopted and concentration to the recipient, and comprise buffer reagent, such as phosphoric acid salt, Citrate trianion and other organic acid; Antioxidant comprises xitix and methionine(Met); Sanitas is (such as octadecyl dimethyl benzyl ammonium chloride; Chlorination hexane diamine; Benzalkonium chloride, benzethonium chloride; Phenol, butanols or phenylcarbinol; Alkyl paraben is such as methyl p-hydroxybenzoate or propyl ester; Pyrocatechol; Resorcinol; Hexalin; The 3-amylalcohol; And meta-cresol); Lower molecular weight (being less than about 10 residues) polypeptide; Protein is such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is such as polyvinylpyrrolidone; Amino acid is such as glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrate comprise glucose, seminose or dextrin; Sequestrant is such as EDTA; Carbohydrate is such as sucrose, N.F,USP MANNITOL, trehalose or sorbyl alcohol; The salify counter ion is such as sodium; Metal composite (as the Zn-protein complex); And/or nonionogenic tenside, such as TWEEN ^TM, PLURONICS ^TMOr polyoxyethylene glycol (PEG).

Preparaton herein also can contain and surpass a kind of necessary active compound of concrete indication of treat, preferred activity complementation and do not have disadvantageous effect each other.Suitable is that this quasi-molecule is effectively to measure combination for predetermined purpose.

Activeconstituents also can wrap and for example be stated from by (for example being respectively Walocel MT 20.000PV or gelatin microcapsule and poly-(methyl methacrylate) microcapsule) in condensation technique or the microcapsule by the interfacial polymerization preparation, in gluey drug delivery system (for example liposome, white protein microsphere, microemulsion, nano particle and Nano capsule) or in macro emulsion.This type of technology is disclosed in for example Remington:The Scienceand Practice of Pharmacy 20th edition (2000).

The preparaton that is used for using in the body must be aseptic.This can be easy to by using aseptic membrane filtration to realize.

Can prepare and continue to discharge preparaton.The suitable example that continues the release preparaton comprises the solid hydrophobic polymkeric substance semipermeability matrix that contains immunoglobulin (Ig) of the present invention, and this matrix exists with the form of standardized product, as film or microcapsule.The example that continues release matrix comprises polyester, hydrogel (for example poly-(2-hydroxyethyl-methacrylic ester) or poly-(vinyl alcohol)), polylactide (United States Patent (USP) 3,773,919), the multipolymer of L-L-glutamic acid and γ-ethyl-L-glutamate, nondegradable ethane-acetic acid ethyenyl, degradable lactic acid-ethanol copolymer are such as LUPRON DEPOT ^TM(the Injectable microspheres body that constitutes by lactic acid-ethanol copolymer and leuprorelin acetate) and poly--D-(-)-3-hydroxybutyric acid.Though can continue to discharge molecule 1 more than 00 day such as polymkeric substance such as ethane-acetic acid ethyenyl and lactic acid-ethanols, the time of some hydrogel release protein is shorter.When encapsulated antibody was kept in vivo for a long time, they may sex change or gathering cause biologic activity loss and immunogenicity to change owing to be exposed to 37 ℃ wet environment.Can come stabilization strategy reasonable in design according to related mechanism.For example, if finding aggregation of multiple is to exchange and form intermolecular S-S key via mercaptan-disulphide, so can by modify sulfhydryl residue, by acidic solution freeze-drying, controlling moisture, the suitable additive of employing with develop specific polymer matrix composition and realize stablizing.

Purposes

That antibody of the present invention can be used for is for example external, ex vivo (ex vivo) and interior therapeutic method.

On the one hand, the invention provides treatment or prevention and express the method for rising and/or active raise relevant tumour, cancer and/or cell proliferative disorders with EphB4, this method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.

On the one hand, the invention provides the method for reduction, inhibition or prophylaxis of tumours or cancer growth, this method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.

Motion axon guidance among EphB4 and the developmental embryo and neural crest cell migration are connected.Thereby antibody of the present invention also can be used for treatment (comprising prevention) its pathology and relates to cell degradation/sex change or handicapped illness, such as treatment various (chronic) neurodegeneration (degeneration) illness and acute neural cell injury.This type of neurodegeneration (degeneration) illness includes but not limited to peripheral neuropathy; The motor neuron illness is such as amyotrophic lateral sclerosis (amylotrophic lateral sclerosis) (ALS, Lou GehrigShi disease), Bei Ershi (Bell) paralysis with relate to the various illness of Duchenne-Arandisease or paralysis; And other people's neurodegenerative disease, such as Alzheimer's, Parkinson's disease, epilepsy, multiple sclerosis, Heng Tingdunshi chorea (Huntington ' s chorea), mongolism (Down ' ssyndrome), neural heariing loss and plum Ni Ershi (Meniere) disease; And acute neural cell injury, for example by due to wound or the Spinal injury.

Antibody of the present invention also can be used for suppressing blood vessel to be taken place.In some embodiment, the position that blood vessel takes place is exactly tumour or cancer.

On the one hand, the invention provides and suppress the method that blood vessel takes place, this method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.

On the one hand, the invention provides the method for the treatment pathology illness relevant with the blood vessel generation, this method comprises that the experimenter to this type of treatment of needs uses the anti-EphB4 antibody of significant quantity.In some embodiment, the described pathology illness relevant with the blood vessel generation is tumour, cancer and/or cell proliferative disorders.In some embodiment, the described pathology illness relevant with the blood vessel generation is the intraocular neovascular disorders.

In addition, at least some antibody of the present invention can be in conjunction with the antigen from other species.Thereby, antibody of the present invention can be used in conjunction with the specific antigen activity, for example, in comprising antigenic cell culture, in the human experimenter or have antibody of the present invention with it in antigenic other Mammals of cross reaction (for example chimpanzee, baboon, marmoset, macaque and rhesus monkey, pig or mouse).In one embodiment, antibody of the present invention can be used for suppressing antigenic activity, makes antigenic activity be suppressed by antibody is contacted with antigen.Preferably, described antigen is the human protein molecule.

In one embodiment, antibody of the present invention can be used for the method in conjunction with the subject endoantigen of suffering from the illness relevant with antigen presentation rising and/or active rising, comprises that using antibody of the present invention to the experimenter makes the intravital antigen of experimenter obtain combination.Preferably, described antigen is that human protein molecule and described experimenter are people experimenters.Perhaps, the experimenter expresses the antigenic Mammals of antibody institute's bonded of the present invention.Or the experimenter can be the Mammals that has wherein imported antigen (for example by administration of antigens or by the antigen expressed transgenosis).Can antibody of the present invention be applied to people experimenter for therapeutic purpose.In addition, can antibody of the present invention be applied to for animal doctor's purpose and express the immunoglobulin (Ig) antigenic non-human mammal of cross reaction (for example primate, pig or mouse) with it, or human disease's animal model.About the latter, this type of animal model can be used for assessing the therapeutic efficiency (for example testing the dosage and the time-histories of dispenser) of antibody of the present invention.

Antibody of the present invention can be used for treating, inhibition, delay of progression, prevention/delay are recurred, improved or prevent expression and/or active relevant disease, illness or illness with one or more antigen molecules.

Exemplary illness comprises cancer knurl, lymphoma, blastoma, sarcoma and leukemia or lymph sample malignant tumour.The more specifically example of this type of cancer comprises squamous cell carcinoma (for example epithelium squamous cell carcinoma), lung cancer (comprises small cell lung cancer, nonsmall-cell lung cancer, the gland cancer of lung and the squamous cell carcinoma of lung), peritoneal cancer, hepatocellular carcinoma (hepatocellular cancer), cancer of the stomach (gastric or stomach cancer) (comprising gastrointestinal cancer), carcinoma of the pancreas, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, urethral carcinoma, hepatoma (hepatoma), mammary cancer, colorectal carcinoma, the rectum cancer, colorectal carcinoma, carcinoma of endometrium or uterus carcinoma, salivary-gland carcinoma, kidney (kidney or renal cancer), prostate cancer, carcinoma vulvae, thyroid carcinoma, liver cancer (hepatic carcinoma), anus cancer, penile cancer, melanoma, multiple myeloma and B cell lymphoma, brain, and head and neck cancer, and associated transitions.In some embodiment, cancer is selected from: small cell lung cancer, neuroblastoma (neuroblastomas), melanoma, mammary cancer, cancer of the stomach, colorectal carcinoma (CRC) and hepatocellular carcinoma.

In certain embodiments, will comprise coupling has the immune conjugate of the antibody of one or more cytotoxic agents to be applied to the patient.In some embodiment, immune conjugate and/or its institute's bonded antigen is by cell internalization, and the therapeutic efficiency that causes immune conjugate to kill and wound its institute bonded target cell improves.In one embodiment, the nucleic acid in cytotoxic agent target or the interference target cell.In one embodiment, cytotoxic agent target or interference microtubule polymerization.The example of this type of cytotoxic agent comprises any chemotherapeutics described herein (such as maytansinoid class, auristatin, dolastatin or calicheamicin), radio isotope or rnase or DNA endonuclease.

In treatment, antibody of the present invention can use separately, perhaps with other composition coupling.For example, antibody of the present invention can be used altogether with another kind of antibody, chemotherapeutics (comprising the chemotherapeutics mixture), other cytotoxic agent, antiangiogenic agent, cytokine and/or growth inhibitor.When antibody inhibiting tumor of the present invention is grown, may wish especially it is united other therapeutical agent that one or more suppress tumor growths.Perhaps/in addition, the patient can accept associating radiotherapy (therapy of for example outside beam irradiation or use radio-labeling medicament such as antibody).This type of conjoint therapy mentioned above comprise the associating dispenser (when two or more kit be contained in identical or the preparaton that separates in the time) and separately dispenser, in the later case, the using of antibody of the present invention can occur in before the using of adjuvant therapy and/or afterwards.

Conjoint therapy

As mentioned above, the invention provides conjoint therapy, wherein anti-EphB4 antibody is used with another therapy.For example, anti-EphB4 antibody and anticancer therapeutic agent or anti-neovascularization therapy are united use to treat various superfluous natural disposition or non-superfluous natural disposition illness.In one embodiment, described superfluous natural disposition or non-superfluous natural disposition illness be characterised in that with unusually or the blood vessel of not expecting relevant pathology illness takes place.Anti-EphB4 antibody can with to the effectively other medicament of those purposes in turn or co-administered, or in same composition or as the composition that separates.Perhaps/in addition, can use the multiple inhibitor of EphB4.

Using of anti-EphB4 antibody can be carried out simultaneously, for example as single composition or as two or more different compositions, uses the identical or different path of using.Perhaps/in addition, dispenser can be carried out in turn with arbitrary order.In certain embodiments, between the using of two or more compositions if having time scope be several minutes, timed interval of a couple of days, several weeks, several months.For example, can use carcinostatic agent earlier, then be the EphB4 inhibitor.Yet, also imagined and used or used earlier anti-EphB4 antibody simultaneously.

Depend on doctor or animal doctor's judgement with the significant quantity of the antibody combined therapeutical agent of using of anti-EphB4.Finishing dosage uses and adjusts to realize the maximum control to illness to be treated.This external dose depends on such as the type of therapeutical agent to be used and the factors such as particular patient of being treated.The suitable dose of carcinostatic agent is exactly currently used those, and can reduce owing to the combined action (working in coordination with) of carcinostatic agent and anti-EphB4 antibody.In certain embodiments, the combination of inhibitor strengthens the effect of single inhibitor.Term " enhancing " refers to that therapeutical agent dosage effect commonly used at it or approval is improved.

Be typically, anti-EphB4 antibody and carcinostatic agent are suitable for same or analogous disease with blocking-up or reduction pathology illness, the growth during such as tumor growth or cancer cells.In one embodiment, described carcinostatic agent is an antiangiogenic agent.

With the angiogenesis inhibitor therapy of related to cancer is to be devoted to suppress for supporting tumor growth that the nutrient strategy of cancer treatment that needed tumor vessel is grown is provided.Because blood vessel relates to the primary tumor growth and shifts the two, so the superfluous natural disposition growth that treatment not only can suppress tumour in the primary site takes place in blood vessel provided by the invention, and can prophylaxis of tumours in the transfer in Secondary cases site, thereby allow by other therapeutical agent and attack tumour.

This area has been identified and known many antiangiogenic agents, comprises those that this paper is listed, and is for example listed in the definition, also can be referring to for example Carmeliet and Jain, and Nature 407:249-257 (2000); Ferrara et al., Nature Reviews:Drug Discovery, 3:391-400 (2004); Sato Int.J.Clin.Oncol., 8:200-206 (2003).Also can be referring to U.S. Patent application US20030055006.In one embodiment, anti-EphB4 antibody and anti-VEGF neutrality antibody (or fragment) and/or other VEGF antagonist or vegf receptor antagonist (for example include but not limited to soluble VEGF-receptor (VEGFR-1 for example, VEGFR-2, VEGFR-3, neuropillins (NRP1 for example, NRP2)) fragment), can blocking VEGF or VEGFR fit, the anti-VEGFR antibody of neutrality, the low-molecular-weight depressor of VEGFR Tyrosylprotein kinase (RTK), the antisense strategy of VEGF, ribozyme at VEGF or vegf receptor, the antagonism variant of VEGF, and combinatorial association uses.Perhaps/in addition, optional is to use two or more angiogenesis inhibitors altogether to the patient outside VEGF antagonist and other medicament.In certain embodiments, can with anti-EphB4 antibody, VEGF antagonist and antiangiogenic agent co-administered one or more other therapeutical agent, for example carcinostatic agents.

Of the present invention aspect some, other therapeutical agent that can be used for the combination tumor therapy of anti-EphB4 antibody comprises other cancer therapy (treatment or its combination of for example operation, radiology treatment (for example relate to irradiation or use radioactive substance), chemotherapy, this paper is listed and this area is known carcinostatic agent).Perhaps/in addition, can be applied to the patient altogether in conjunction with the same antigen of this paper disclosure or different antigenic two or more antibody of two or more this paper disclosure.Sometimes, it may be useful also the patient being used one or more cytokines.

Chemotherapeutics

In some aspects, the invention provides blocking-up or reduce tumor growth or the method for growth of cancer cells, its by to susceptible in or diagnosis have the patient of cancer to use the EphB4 antagonist of significant quantity and/or angiogenesis inhibitor and one or more chemotherapeutics to realize.Multiple chemotherapeutics can be used for combinational therapeutic methods of the present invention.This paper provides the exemplary and non-limiting tabulation of contemplated chemotherapeutics in " definition ".

Will appreciate that as those of ordinary skills the optimal dose of chemotherapeutics is generally separately or unite other chemotherapeutics and change near using those dosage that adopt for a long time in the clinical treatment of this chemotherapeutics.According to the illness of being treated, dosage might change.The doctor who implements treatment can determine appropriate dosage for the experimenter is individual.

The recurrent tumor growth

The present invention also provides and has been used to suppress or the method and composition of prevention of recurrence tumor growth or recurrent growth of cancer cells.Recurrent tumor growth or recurrent growth of cancer cells are used for describing is accepting one or more current available therapies (cancer therapy for example, standard care scheme such as chemotherapy, radiotherapy, operation, hormonotherapy and/or biology therapy/immunotherapy, VEGF antibody therapy, particularly particular cancers) is not enough to treat this patient clinically or no longer obtains any beneficial effect or with the patient that one or more current available therapy for treating are crossed and make the situation of these other effective therapies of needs of patients by this therapy.When being used for this paper, this statement also refers to " do not have response/intractable (non-responsive/refractory) " patient's situation, for example describe the patient therapy is had response but suffer side effect, formation resistance, to therapy do not respond, unsatisfactory etc. to the response of therapy.In various embodiments, cancer is recurrent tumor growth or recurrent growth of cancer cells, wherein the cancer cells number does not have gratifying minimizing, perhaps increased, perhaps tumor size is not dwindled significantly, perhaps increased, perhaps the size of cancer cells or number fail further to dwindle or reduce.Determine whether cancer cells is that recurrent tumor growth or recurrent growth of cancer cells can carry out in vivo or external, measure any method of treatment by this area being used to of knowing, in such linguistic context, use the implication of art-recognized " recurrent " or " intractable " or " not having response " cancer cells validity.It is an example of recurrent tumor growth (relapse tumor growth) that antagonism VEGF treatment has the tumour of resistance.

The invention provides in the experimenter blocking-up or reduce the method for recurrent tumor growth or recurrent growth of cancer cells, it is realized with blocking-up or the recurrent tumor growth or the recurrent growth of cancer cells that reduce among the experimenter by using one or more anti-EphB4 antibody.In certain embodiments, can after cancer therapeutic agent, use described antagonist.In certain embodiments, use anti-EphB4 antibody simultaneously with cancer therapy.Perhaps/in addition, anti-EphB4 antibody therapy and another cancer therapy are alternately implemented, and this can carry out with any order.The method that one or more inhibiting antibodies come preventing cancer to show effect or recur of using is also contained in the present invention in the patient that the cancer tendency is arranged.In one embodiment, cancer therapy is to use the treatment of antiangiogenic agent, for example the VEGF antagonist.Listed those in those that antiangiogenic agent comprises that this area knows and this paper definition.In one embodiment, antiangiogenic agent be anti-VEGF neutrality antibody or fragment (for example humanized A4.6.1,

(Genentech, South San Francisco, CA), Y0317, M4, G6, B20,2C3 etc.).Referring to for example United States Patent (USP) 6,582,959,6,884,879,6,703,020; WO98/45332; WO96/30046; WO94/10202; EP 0666868B1; U.S. Patent application 20030206899,20030190317,20030203409,20050112126; Popkov et al., Journal of Immunological Methods288:149-164 (2004); And WO2005012359.Other medicament can be grown or the recurrent growth of cancer cells with VEGF antagonist and antibody combined the using with blocking-up or reduction recurrent tumor of anti-EphB4, for example sees that title is the part of " conjoint therapy " herein.

Antibody of the present invention (and auxiliary therapeutical agent) is used by any appropriate means, comprises in parenteral, subcutaneous, intraperitoneal, the lung and in the nose, and (if wishing topical therapeutic) used in the damage.The parenteral infusion comprises intramuscular, intravenously, intra-arterial, intraperitoneal or subcutaneous administration.In addition, pulse infusion antibody also is suitable, particularly the form that reduces gradually with antibody dosage.Can take medicine by any suitable path, for example by injection, such as intravenously or subcutaneous injection, this part depends on that dispenser is short-term or secular.

Can the mode consistent prepare with good medical practice, dosed administration and use antibody compositions of the present invention.The factor of considering in this content comprises the clinical condition of the concrete illness of being treated, the concrete Mammals of being treated, individual patients, the cause of illness, position, the method for dispenser, the schedule of dispenser and the other factors that the medical science practitioner knows of delivery medicament.Be not must but optional antibody is prepared with one or more medicaments that are used at present to prevent or treat the illness of discussing.The significant quantity of this type of other medicament depends on the type and the other factors discussed above of amount, illness or the treatment of the antibody of the present invention that exists in the preparaton.These are normally with used same dose above with use the path and use, or about 1-99% of used dosage so far.

For the prevention or the treatment of disease, the optimal dose of antibody of the present invention (when independent use or with other medicament such as chemotherapeutics coupling time) depends on that the severity of type, disease of type, the antibody of disease to be treated and the course of disease, administration of antibodies are used to prevent or the replying of therapeutic purpose, previous therapy, patient's clinical history and antagonist, and attending doctor's judgement.Antibody is disposable or suitable in a series of treatments be applied to the patient.According to the type and the severity of disease, about 1 μ g/kg is the initial candidate dosage that is applied to the patient to 15mg/kg (for example 0.1mg/kg-10mg/kg) antibody, no matter is for example to pass through the one or many separate administration, or pass through continuous infusion.Typical case every day dosage scope can for about 1 μ g/kg to 100mg/kg or more, depend on above-mentioned factor.For the repeat administration that continues a couple of days or longer time, according to illness, treatment is maintained to until the disease symptoms that expectation takes place and suppresses.The scope of the exemplary dosage of antibody is that about 0.05mg/kg is to about 10mg/kg.So, can use the potion or the multi-agent of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or its arbitrary combination) to the patient.Can intermittently use for described dose, for example weekly or per three weeks (once) (for example making the patient accept about 2 to about 20 doses, for example about 6 doses of antibody).Can use potion higher add supporting agent (loading dose), succeeded by potion or the lower dosage of multi-agent.A kind of exemplary dosage regimen comprises the supporting agent that adds of using the about 4mg/kg of potion, succeeded by the agent of keeping of about 2mg/kg antibody of potion weekly.Yet other dosages may also be useful.Can monitor the progress of this therapy easily by routine techniques and assay method.

Anti-EphB4 antibody of the present invention can be used for detecting the assay method (such as diagnosis or prognosis assay method) that the EphB4 in specific cells or the tissue expresses, and wherein said antibody is as described below to carry out mark and/or be fixed on the insoluble matrix.

On the other hand, the invention provides the method that is used to detect EphB4, this method comprises the anti-EphB4 antibody complex of the EphB4-in the test sample.Term " detection " comprises qualitative when being used for this paper and/or detection by quantitative (measurement level), has or do not have the reference contrast.

On the other hand, the invention provides that diagnosis is expressed with EphB4 and/or the method for active relevant illness, this method comprises from the anti-EphB4 antibody complex of biological sample detection EphB4-of suffering from or suspect the patient who suffers from described illness.In some embodiments, the EphB4 expression is expression or the expression of (not expecting) unusually that increases.In some embodiments, described illness is tumour, cancer and/or cell proliferative disorders.

On the other hand, the invention provides any anti-EphB4 antibody described herein, wherein said anti-EphB4 antibody comprises detectable marker.

On the other hand, the invention provides the mixture of any anti-EphB4 antibody described herein and EphB4.In some embodiments, mixture is intravital or external.In some embodiments, mixture comprises cancer cells.In some embodiments, anti-EphB4 antibody is detectable label.

The EphB4 that anti-EphB4 antibody can be used in a large amount of known detection assay methods any detects.For example, can followingly measure EphB4 to biological sample, promptly obtain sample from the expectation source, any EphB4 that sample is mixed with anti-EphB4 antibody to exist in permission antibody and the mixture forms antibody/EphB4 mixture, and detects any antibody/EphB4 mixture that exists in the mixture.Can prepare biological sample by the method that is suitable for specific sample known in the art uses for measuring.Select the method for biased sample and antibody and the method for detection antibody/EphB4 mixture according to the type of employed assay method.This type of assay method comprises immunohistochemistry, competitiveness and sandwich/sandwich assay method and steric inhibition assay method (steric inhibition assay).

The analytical procedure of EphB4 is all used one or more following reagent: through anti-EphB4 antibody, immobilized anti-EphB4 antibody and the space conjugate of the EphB4 analogue of mark, immobilized EphB4 analogue, process mark.Reagent through mark also is called " tracer agent ".

The marker that uses is any functional group (functionality) of detecting that does not disturb EphB4 and anti-EphB4 antibodies.Many markers become known for immunoassay, and example comprises the module that can directly detect, and such as fluorescence dye, chemoluminescence and radioactively labelled substance, and must react or module that derivatize could detect, such as enzyme.The example of this type of marker comprises:

The marker that uses is any functional group of detecting that does not disturb EphB4 and anti-EphB4 antibodies.Many markers become known for immunoassay, and example comprises the module that can directly detect, and such as fluorescence dye, chemoluminescence and radioactively labelled substance, and must react or module that derivatize could detect, such as enzyme.The example of this type of marker comprises radio isotope ³²P, ¹⁴C, ¹²⁵I, ³H and ¹³¹I, fluorophore such as rare earth inner complex or fluorescein and derivative thereof, rhodamine and derivative thereof, dansyl, Umbelliferone, luciferase, for example Lampyridea luciferase and bacteriofluorescein enzyme (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydro phthalazine diketone, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase enzymes, glucoamylase, N,O-Diacetylmuramidase, the carbohydrate oxydase is glucose oxidase for example, galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD), heterocycle oxydase such as uriKoxidase and XOD, coupling have uses enzyme that hydrogen peroxide comes oxidation dye precursors such as HRP, lactoperoxidase, or microperoxisome, vitamin H/affinity element, spin label, phage marker, stabilized radical, or the like.

The ordinary method that these markers is covalently bond to protein or polypeptide has been arranged.For example, coupling agent such as dialdehyde, carbodiimide, bismaleimides, two imido-ester, bis-diazotized-benzidine etc. can be used for to above-mentioned fluorescence, chemoluminescence and enzyme labelling thing on the antibody labeling.Referring to for example U.S. Patent No. 3,940,475 (fluorometry) and 3,645,090 (enzyme); Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J.Immunol.Methods, 40:219-230 (1981); And Nygren, J.Histochem.and Cytochem., 30:407-412 (1982).Preferred herein marker is an enzyme, such as horseradish peroxidase and alkaline phosphatase.Is a kind of standard operating procedure with antibody coupling for the those of ordinary skill of being familiar with immunoassay with this type of marker (comprising enzyme).Referring to for example O ' Sullivan et al., ＂ Methods for thePreparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay, ＂ is in Methods in Enzymology, ed.J.J.Langone and H.Van Vunakis, Vol.73 (Academic Press, New York, New York, 1981), pp.147-166.

Some assay method needs the immobilization of reagent.Immobilization can with resist EphB4 antibody with still in solution any EphB4 of free divide and open.For this reason, or by being adsorbed to water-fast matrix or surface (Bennich et al., U.S.3,720,760), anti-EphB4 antibody or EphB4 analogue are not dissolved by covalent coupling (for example utilizing glutaraldehyde cross-linking), or, anti-EphB4 antibody or EphB4 analogue are not dissolved for example by exempting to survey precipitation.

Can utilize immunohistochemistry and dyeing scheme to come protein expression in the sample for reference.The immunohistochemical staining of tissue slice has proved the reliable method that protein exists in a kind of assessment or the test sample.Immunohistochemistry (" IHC ") technology is utilized antibody to detect and is manifested the original position cell antigen, usually by adding lustre to or fluorescent method.For specimen preparation, can use tissue or cell sample from Mammals (being typically human patients).The example of sample includes but not limited to cancer cells, such as colorectal carcinoma, mammary cancer, prostate cancer, ovarian cancer, lung cancer, cancer of the stomach, carcinoma of the pancreas, lymphoma and leukaemia cancer cell.Can obtain sample by multiple rules known in the art, include but not limited to excision, suction or biopsy.Tissue can be fresh or refrigerated.In one embodiment, sample is fixing and is embedded in paraffin or the analogue.Can fix (promptly preserving) tissue sample by ordinary method.Those of ordinary skills will understand, and the selection of fixing agent is used for histological stain by sample or the purpose of other analysis decides.Those of ordinary skills also will understand, and fixedly duration depends on the size and the employed fixing agent of tissue sample.

IHC can implement with dyeing of other technology such as morphology and/or fluorescence in situ hybridization.Existing two kinds of IHC methods commonly used: direct and indirect measurement method.According to first kind of assay method, directly measure combining of antibody and target antigen (for example EphB4).This direct assay method is used the reagent through mark, and such as the first antibody of fluorescence labels or enzyme labelling, it does not need further antibody interaction just can manifest.In a kind of typical indirect measurement method, the link coupled first antibody is not bonded to antigen, and the second antibody through mark is bonded to first antibody then.If the second antibody coupling has the enzyme labelling thing, then add chromogenic substrate or fluorogenic substrate and manifest to provide antigenic.Because several second antibody can be reacted with the different epi-positions on the first antibody,, amplifies signal so taking place.

But be used for immunohistochemical first and/or second antibody carry out mark with detection module usually.Existing multiple marker, they can be divided into following type usually:

Outside specimen preparation rules discussed above, may be also need before the IHC, during or afterwards tissue slice is further processed.For example, can implement the epi-position repairing method, such as in citrate buffer, tissue sample being heated (referring to for example Leong et al.Appl.Immunohistochem.4 (3): 201 (1996)).

After optional sealing step, tissue slice is exposed to the enough time of first antibody under conditions suitable, make first antibody be bonded to the target protein antigen in the tissue sample.The suitable condition of realization this purpose can be determined by normal experiment.The combination degree of antibody and sample is measured by using any detectable discussed above.Preferably, marker is enzyme labelling thing (for example HRPO), its catalysis chromogenic substrate such as 3,3 '-chemical transformation of diaminobenzidine chromogen (chromogen).Preferably, the enzyme labelling thing is coupled to the antibody (for example first antibody be rabbit polyclonal antibody, second antibody be goat anti-rabbit antibodies) of specificity in conjunction with first antibody.

The sample that so prepares can be placed and covered.Carry out the slide glass assessment then, for example utilize microscope, and can adopt the conventional staining power standard of using in this area.The staining power standard can followingly be assessed:

Table 2

The dyeing pattern	Score
		In cell, do not observe dyeing.	0
In surpassing 10% cell, detect faint/just perceptible dyeing.	1+
		In surpassing 10% cell, observe weak to medium dyeing.	2+
In surpassing 10% cell, observe medium to strong dyeing.	3+

Typically, in the IHC assay method, must be divided into about 2+ or higher dyeing pattern is diagnostic and/or prognostic.In some embodiment, must be divided into about 1+ or higher dyeing pattern is diagnostic and/or prognostic.In other embodiments, must be divided into about 3 or higher dyeing pattern be diagnostic and/or prognostic.Should be appreciated that when using IHC to check and/or organizing from the cell of tumour or adenoma of colon, generally measure or assessment tumour cell and/or tissue (but not the matrix that may exist in the sample or surrounding tissue) in dyeing.

Other assay method is called competitiveness or sandwich/sandwich assay method, has clearly set up and be widely used in commercial diagnostic industry.

The ability that competitive assays depends on tracer agent EphB4 analogue and specimen EphB4 competes a limited number of anti-EphB4 antibody antigen-binding site.Usually before competition or after the competition anti-EphB4 antibody is not dissolved, the tracer agent and the EphB4 that will be bonded to anti-EphB4 antibody then open with unconjugated tracer agent and EphB4 branch.By toppling over (wherein make in conjunction with counterpart in advance and do not dissolve) or realizing this separation by centrifugal (wherein behind competitive reaction, making) in conjunction with the counterpart precipitation.The amount of the amount of EphB4 and bonded tracer agent is inversely proportional in the specimen, and the amount of described bonded tracer agent is measured by the amount of mark substance.Draw dosage-response curve of the EphB4 of known quantity, compare amount with the EphB4 that quantitatively determines to exist in the specimen with test result.When using enzyme as detectable, these assay methods are called the ELISA system.

Another kind of competitive assays is called " homogeneous phase " assay method (homogeneous assay), does not need to be separated.Herein, preparation is also used the conjugate of enzyme and EphB4, makes when anti-EphB4 antibodies EphB4 the existence change enzymic activity of anti-EphB4 antibody.In this case, EphB4 or its immunologic competence fragment are coupled to enzyme such as peroxidase with difunctional organic bridge.Select conjugate to use, make the combination of anti-EphB4 antibody suppress or strengthen the enzymic activity of marker with anti-EphB4 antibody.This method itself is called EMIT by broad practice.

Space conjugate (steric conjugate) is used for the sterically hindered method (sterichindrance method) of homogeneous assay method.Basically can not combine conjugate simultaneously with anti-EphB4 antibody by the covalently bound extremely little EphB4 fragment of lower molecular weight haptens being synthesized these conjugates, making at haptenic antibody.Under these assay method rules, the EphB4 that exists in the specimen will be in conjunction with anti-EphB4 antibody, thereby allow antihapten in conjunction with conjugate, causes the variation of conjugate haptens characteristic, for example variation of fluorescence when haptens is fluorophore.

Sandwich/sandwich assay method is particularly useful for the mensuration of EphB4 or anti-EphB4 antibody.In sandwich assay method (sequential sandwich assay) in turn, the anti-EphB4 antibody of immobilization is used to adsorb specimen EphB4, remove specimen by cleaning, the bonded EphB4 of institute is used to adsorb the second anti-EphB4 antibody through mark, then the tracer agent of institute's bonded material with remnants is separated.The amount of bonded tracer agent is directly proportional with specimen EphB4.In " synchronously " sandwich assay method (simultaneoussandwich assay), before adding the process anti-EphB4 of mark, do not separate specimen.Use anti-EphB4 monoclonal antibody to can be used for sample test EphB4 as a kind of antibody and the anti-EphB4 antibody of polyclone the assay method of sandwich in turn as another kind of antibody.

It above only is the exemplary detection assay method of EphB4.Now or after this other method of the anti-EphB4 TPPA of the use EphB4 of exploitation includes within the scope of the present invention, comprises bioassay method as herein described.

Goods

In another aspect of the present invention, provide to comprise the goods that can be used for treating, preventing and/or diagnose the material of disorder mentioned above.Goods comprise container and be attached on the described container or with the label or the package insert of its associated.Suitable containers comprises for example bottle, tubule, syringe etc.Container can be made from a variety of materials, such as glass or plastics.Himself is housed in the container or effectively treats, prevent and/or diagnose the composition of illness during other composition, and can have aseptic access port (for example container can be intravenous solution bag or the tubule with stopper that hypodermic needle can pierce through) in associating.At least a promoting agent in the composition is an antibody of the present invention.Label or package insert indication said composition are used for the treatment of the illness of selection, such as cancer.In addition, goods can comprise that (a) wherein is equipped with first container of composition, and wherein said composition comprises antibody of the present invention; (b) second container of composition is housed wherein, wherein said composition comprises other cytotoxic agent.Goods in this embodiment of the present invention can comprise that also indication first and second antibody compositions can be used for treating the package insert of specific illness such as cancer.Perhaps/in addition, goods also can comprise second (or 3rd) container, and the acceptable buffer reagent of pharmacopedics wherein is housed, such as injection bacteriostatic water (BWFI), phosphate buffered saline (PBS), woods Ge Shi (Ringer) solution and dextrose solution.It also can comprise other required material on commercial and the user's position, comprises other buffer reagent, thinner, filter, syringe needle and syringe.

Be the embodiment of the inventive method and composition below.Should be appreciated that according to general description provided above, can implement various other embodiments.

Embodiment

Embodiment 1: the generation of anti-EphB4 antibody

Anti-EphB4 antibody is to use phage display to generate, and wherein uses EphB4-His albumen to select phage.This area knows that several different methods can be used for generating the phage display storehouse, therefrom can obtain purpose antibody.A kind of method that generates purpose antibody is by as Lee et al., J.Mol.Biol. (2004), 340 (5): the described use phage antibody library of 1073-93.The clone who uses phage display to select uses phage E LISA to screen at EphB4-His albumen.Select unique clone to be used for further characterizing, to determine that can this phage antibody clone block EphB4-ephrinB2 and interact by phage competitive ELISA and blocking-up assay method.Clone 30.35 does well, thereby is selected for further analysis.In order to improve clone 30.35 avidity, in the background of YW30.35, generate the phage display storehouse, the selected HVR of each target carries out soft or rigid randomization (soft or hard randomization).Selected clone screens by phage E LISA, is expressed as Fab albumen then and uses Biacore to measure its avidity.Selected clone's reformatting becomes total length IgG, and uses Biacore to measure its avidity.The parent clone 30.35 and affinity maturation clone's sequence as shown in Figure 1.

Embodiment 2: the sign of anti-EphB4 antibody

In order to measure the binding affinity of anti-EphB4 monoclonal antibody, use BIAcore ^TM-3000 (Piscataway NJ) carries out surperficial plasmon resonance (SRP) and measures for BIAcore, Inc..In brief, according to supplier's specification sheets with hydrochloric acid N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy-succinamide (NHS) activate carboxymethylation dextran biologic sensor chip (CM5, BIAcoreInc.).To resist EphB4 antibody dilution to 5 μ g/ml with 10mM sodium acetate pH4.8, be injected into the coupling antibody of about 500 response units of acquisition (response unit RU) then with 5 μ l/ minutes flow velocity.Then, inject the 1M thanomin with sealing unreacted group.In order to carry out kinetic measurement, be infused in the people or the mouse EphB4-His molecule (0.7nM-500nM) of twice serial dilution among the PBS that contains 0.05% Tween-20 in 25 ℃ of flow velocitys with about 25 μ l/ minutes.Use Lang Gemiuer combination model (BIAcoreEvaluation Software version3.2) calculations incorporated speed (k simply one to one _On) and the speed (k that dissociates _Off).Equilibrium dissociation constant (Kd) is with ratio k _Off/ k _OnCalculate.This result of experiment is as shown in table 3." NA " expression is measured.

Table 3: anti-EphB4 antibody is to binding affinity and the kinetics of people and mouse EphB4

In different experiments, tested the cross reactivity of anti-EphB4 antibody cloning 30.35 at other EphB acceptor.In brief, the COS7 cell plasmid transient transfection of expressing total length people EphB1, people EphB2, people EphB4 or people EphB6.After the transfection 24 hours, cell carried out facs analysis to detect the combination of anti-EphB4 antibody, if any.Anti-EphB4 clone 30.35 not with people EphB1, people EphB2, people EphB3 or people EphB6 cross reaction.

Embodiment 3: anti-EphB4 antibody is blocked the conduction of EphB4 receptor signal in based on the assay method of cell

In order to prove the interactional ability of anti-EphB4 antibody blocking film in conjunction with EphrinB2 and EphB4, we have carried out the assay method based on cell, wherein present EphB4 and EphrinB2 by different cell types.The 3T3 cell of crossing expressing human EphrinB2 is used to stimulate expresses high-level EphB4 but the HUVEC cell of low-level EphrinB2, has tested anti-EphB4 antibody and has suppressed EphB4 activatory ability.

The 3T3 cell of crossing expressing human EphrinB2 is prepared as follows: people's total length EphrinB2 is cloned into pcDNA5/FRT carrier (Invitrogen), is used for subsequently with the handbook generation stable cell lines of 3T3.Flp cell (Invitrogen) according to manufacturers.

The 3T3 cell of crossing expressing human EphrinB2 is layered on and reaches 15 or 30 minutes on the HUVEC cell, has or do not exist anti-EphB4 antibody this moment.The following assessment of the activation of EphB4 acceptor, promptly immunoprecipitation EphB4 albumen uses anti-phosphoric acid-tyrosine antibody (antibody 4G10 then; Upstate) existence that detects the receptor tyrosine phosphorylation by the western trace whether.In brief, cell RIPA damping fluid cracking.Cell lysate adds anti-EphB4 antibody 35.2D8 by centrifugal clarification with 5 μ g/ samples.After 2 hours, immunocomplex uses the albumin A agarose electrophoresis in 4 ℃ of incubations.The EphB4 phosphorylation uses anti-phosphotyrosine antibody 4G10 (Upstate) to analyze with the concentration of 1 μ g/ml by the Western trace.

This result of experiment as shown in Figure 7.The 3T3 cell is layered on and causes significant EphB4 tyrosine phosphorylation (the 4th and 5 road) on the HUVEC cell.Preincubation in 30 minutes of HUVEC and anti-EphB4 antibody (clone 35.2D8,5 μ g/ml) are effectively eliminated the 3T3-EphrinB2 cell and are covered inductive EphB4 tyrosine phosphorylation (the 6th road).On the contrary, in HUVEC, do not cause EphB4 activation (the 2nd and 3 road), and untreated HUVEC cell is not showed the EphB4 activation with independent anti-EphB4 antibody treatment HUVEC cell.These results have established anti-EphB4 antibody block ligand-acceptor interaction in the environment of direct cell-cells contacting.

Although described above-mentioned aspect in more detail for the mode that the clear purpose of understanding has illustrated by way of example, specification sheets and embodiment should not be construed as the restriction invention scope.

Sequence table

＜110〉Genentech Inc (Genentech, Inc.)

＜120〉anti-ephb 4 antibodies and using method thereof

<130>P2300R1

<150>US 60/756,889

<151>2006-01-05

<150>US 60/760,892

<151>2006-01-20

<160>62

<210>1

<211>10

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-H1

<400>1

<210>2

<211>10

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-H1

<400>2

<210>3

<211>18

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-H2

<400>3

<210>4

<211>18

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-H2

<400>4

<210>5

<211>18

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-H2

<400>5

<210>6

<211>18

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-H2

<400>6

<210>7

<211>17

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-H3

<400>7

<210>8

<211>17

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-H3

<400>8

<210>9

<211>11

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-L1

<400>9

<210>10

<211>11

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-L1

<400>10

<210>11

<211>7

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-L2

<400>11

<210>12

<211>7

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-L2

<400>12

<210>13

<211>9

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-L3

<400>13

<210>14

<211>9

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-L3

<400>14

<210>15

<211>9

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-L3

<400>15

<210>16

<211>9

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-L3

<400>16

<210>17

<211>9

<212>PRT

＜213〉artificial sequence

<220>

<223>HVR-L3

<400>17

<210>18

<211>107

<212>PRT

＜213〉artificial sequence

<220>

<223>Light chain variable domain of huMab4D5-8

<400>18

<210>19

<211>87

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>19

<210>20

<211>81

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>20

<210>21

<211>80

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>21

<210>22

<211>79

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>22

<210>23

<211>87

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>23

<210>24

<211>81

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>24

<210>25

<211>80

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>25

<210>26

<211>79

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>26

<210>27

<211>87

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>27

<210>28

<211>81

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>28

<210>29

<211>80

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>29

<210>30

<211>79

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>30

<210>31

<211>87

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>31

<210>32

<211>81

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>32

<210>33

<211>80

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>33

<210>34

<211>87

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>34

<210>35

<211>81

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>35

<210>36

<211>80

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>36

<210>37

<211>79

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>37

<210>38

<211>80

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>38

<210>39

<211>80

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>39

<210>40

<211>80

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>40

<210>41

<211>80

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>41

<210>42

<211>23

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>42

<210>43

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>43

<210>44

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>44

<210>45

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>45

<210>46

<211>25

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>46

<210>47

<211>13

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>47

<210>48

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>48

<210>49

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>49

<210>50

<211>32

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>50

<210>51

<211>30

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>51

<210>52

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sequence is a synthetic

<400>52

<210>53

<211>128

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of heavy chain of Mab30.35

<400>53

<210>54

<211>108

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of light chain of Mab30.35

<400>54

<210>55

<211>128

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of heavy chain of Mab30.35.1D2

<400>55

<210>56

<211>108

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of light chain of Mab30.35.1D2

<400>56

<210>57

<211>128

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of heavy chain of Mab30.35.2D8

<400>57

<210>58

<211>108

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of light chain of Mab30.35.2D8

<400>58

<210>59

<211>128

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of heavy chain of Mab30.35.2D12

<400>59

<210>60

<211>108

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of light chain of Mab30.35.2D12

<400>60

<210>61

<211>128

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of heavy chain of Mab30.35.2D13

<400>61

<210>62

<211>108

<212>PRT

＜213〉artificial sequence

<220>

＜223〉variable region of light chain of Mab30.35.2D13

<400>62

Claims (46)

1. isolating anti-EphB4 antibody, it comprises:

(a) at least one, two, three, four or five be selected from down the group hypervariable region (HVR) sequences:

(i) HVR-L1, it comprises sequence A 1-A11, and wherein A1-A11 is RASQDVSTAVA (SEQ ID NO:9),

(ii) HVR-L2, it comprises sequence B 1-B7, and wherein B1-B7 is SASFLYS (SEQ IDNO:11),

(iii) HVR-L3, it comprises sequence C 1-C9, and wherein C1-C9 is QESTTTPPT (SEQID NO:15),

(iv) HVR-H1, it comprises sequence D 1-D10, and wherein D1-D10 is GFSISNYYLH (SEQ ID NO:2),

(v) HVR-H2, it comprises sequence E1-E18, wherein E1-E18 be GGIYLYGSSSEYADSVKG (SEQ ID NO:4) and

(vi) HVR-H3, it comprises sequence F1-F17, and wherein F1-F17 is ARGSGLRLGGLDYAMDY (SEQ ID NO:7); And

(b) HVR of at least one variation, the HVR sequence of wherein said variation comprises the modification of at least one residue of sequence shown in the SEQ ID NO:1-17.

2. the antibody of claim 1, wherein the HVR-L1 variant comprises 1-5 (1,2,3,4 or 5) and locates to substitute in the following column position of arbitrary combination: A6 (V or S), A7 (S or E), A8 (T or I), A9 (A or F) and A10 (V or L).

3. the antibody of claim 1, wherein the HVR-L2 variant comprises 1-2 (1 or 2) and locates to substitute in the following column position of arbitrary combination: B4 (F or N) and B6 (Y or E).

4. the antibody of claim 1, wherein the HVR-L3 variant comprises 1-7 (1,2,3,4,5,6 or 7) and locates to substitute in the following column position of arbitrary combination: C2 (Q, E or K), C3 (S or T), C4 (Y, N, T, E or A), C5 (T, A or Q), C6 (T, V or I), C8 (P, L or E) and C9 (T or S).

5. the antibody of claim 1, wherein the HVR-H1 variant comprises 1-3 (1,2 or 3) and locates to substitute in the following column position of arbitrary combination: D3 (T or S), D6 (G or N) and D9 (I or L).

6. the antibody of claim 1, wherein the HVR-H2 variant comprises 1-5 (1,2,3,4 or 5) and locates to substitute in the following column position of arbitrary combination: E5 (P or L), E7 (S or G), E8 (G or S), E10 (T, S or R) and E11 (D, E or G).

7. the antibody of claim 1, wherein the HVR-H3 variant comprises 1 place and substitutes in column position down: F3 (G or S).

8. isolating anti-EphB4 antibody, it comprises one, two, three, four, five or six HVR, wherein each HVR comprises the sequence that is selected from SEQ ID NO:1-17 or is formed or be made up of it basically by it, and wherein SEQ ID NO:9 or 10 is corresponding to HVR-L1, SEQ ID NO:11 or 12 is corresponding to HVR-L2, SEQ ID NO:13,14,15,16 or 17 is corresponding to HVR-L3, SEQ ID NO:1 or 2 is corresponding to HVR-H1, SEQ ID NO:3,4,5 or 6 corresponding to HVR-H2 and SEQ ID NO:7 or 8 corresponding to HVR-H3.

9. the antibody of claim 8, wherein said antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein HVR-L1 comprises SEQ ID NO:9, HVR-L2 comprises SEQ ID NO:11, HVR-L3 comprises SEQ ID NO:13, HVR-H1 comprises SEQ IDNO:1, and HVR-H2 comprises SEQ ID NO:3 and HVR-H3 comprises SEQ ID NO:7.

10. the antibody of claim 8, wherein said antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein HVR-L1 comprises SEQ ID NO:10, HVR-L2 comprises SEQ ID NO:12, HVR-L3 comprises SEQ ID NO:14, HVR-H1 comprises SEQ ID NO:1, and HVR-H2 comprises SEQ ID NO:3 and HVR-H3 comprises SEQ ID NO:8.

11. the antibody of claim 8, wherein said antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein HVR-L1 comprises SEQ ID NO:9, HVR-L2 comprises SEQ ID NO:11, HVR-L3 comprises SEQ ID NO:15, HVR-H1 comprises SEQ IDNO:2, and HVR-H2 comprises SEQ ID NO:4 and HVR-H3 comprises SEQ ID NO:7.

12. the antibody of claim 8, wherein said antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein HVR-L1 comprises SEQ ID NO:9, HVR-L2 comprises SEQ ID NO:11, HVR-L3 comprises SEQ ID NO:16, HVR-H1 comprises SEQ IDNO:1, and HVR-H2 comprises SEQ ID NO:5 and HVR-H3 and comprises SEQ ID NO:7 each comprises SEQ ID NO:9,11,16,1,5 and 7 successively.

13. the antibody of claim 8, wherein said antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein HVR-L1 comprises SEQ ID NO:9, HVR-L2 comprises SEQ ID NO:11, HVR-L3 comprises SEQ ID NO:17, HVR-H1 comprises SEQ IDNO:1, and HVR-H2 comprises SEQ ID NO:6 and HVR-H3 comprises SEQ ID NO:7.

14. each antibody of claim 1-13, wherein the part frame sequence is the total framework sequence of people at least.

15. the antibody of claim 1, wherein said modification are to substitute, insert or delete.

16. each antibody of claim 1-15, wherein said antibody comprise the total framework sequence of people κ subgroup.

17. each antibody of claim 1-15, wherein said antibody comprise the total framework sequence of heavy chain people subgroup III.

18. the antibody of claim 17, the wherein said antibody one or more positions in the 73rd, 73 or 78 comprise alternative.

19. the antibody of claim 18, wherein said substitute is one or more among R71A, N73T or the N78A.

20. polynucleotide, each antibody of its coding claim 1-19.

21. carrier, it comprises the polynucleotide of claim 20.

22. the carrier of claim 21, wherein said carrier is an expression vector.

23. host cell, it comprises the carrier of claim 21 or 22.

24. the host cell of claim 23, wherein said host cell is a protokaryon.

25. the host cell of claim 23, wherein said host cell is an eucaryon.

26. the host cell of claim 25, wherein said host cell is mammiferous.

27. prepare the method for anti-EphB4 antibody, described method comprises: (a) in proper host cell, express the carrier of claim 22, and (b) reclaim described antibody.

28. prepare the method for anti-EphB4 immune conjugate, described method comprises: (a) in proper host cell, express the carrier of claim 22, and (b) reclaim described antibody.

29. the method for claim 27 or 28, wherein said host cell is a protokaryon.

30. the method for claim 27 or 28, wherein said host cell is an eucaryon.

31. detect the method for EphB4, described method is included in and detects the anti-EphB4 antibody complex of EphB4-in the biological sample, the described anti-EphB4 antibody in the wherein said mixture is each anti-EphB4 antibody of claim 1-19.

32. the method for the illness that diagnosis is relevant with the EphB4 expression, described method is included in from suffering from or suspect and detect the anti-EphB4 antibody complex of EphB4-in the patient's who suffers from described illness the biological sample, and the described anti-EphB4 antibody in the wherein said mixture is each anti-EphB4 antibody of claim 1-19.

33. the method for claim 31 or 32, wherein said anti-EphB4 antibody is detectable label.

34. composition, it comprises each anti-EphB4 antibody of claim 1-19.

35. composition, it comprises each polynucleotide of claim 20-22.

36. the composition of claim 34 or 35, wherein said composition further comprises carrier.

37. suppress the method that blood vessel takes place, described method comprises that the experimenter to this type of treatment of needs uses each the anti-EphB4 antibody of claim 1-19 of significant quantity.

38. the method for claim 37, described method further comprises the antiangiogenic agent of described experimenter being used significant quantity.

39. the method for claim 38, wherein said antiangiogenic agent was used before or after using described anti-EphB4 antibody.

40. the method for claim 38, wherein said antiangiogenic agent and described anti-EphB4 antibody are used simultaneously.

41. each method of claim 38-40, wherein said antiangiogenic agent is the antagonist of vascular endothelial growth factor (VEGF).

42. the method for claim 41, wherein said VEGF antagonist is a VEGF antibody.

43. the method for claim 42, wherein said VEGF antibody is a rhuMAb-VEGF.

44. each method of claim 37-43, described method further comprises the chemotherapeutics of using significant quantity.

45. the method for treatment cancer, tumour and/or cell proliferative disorders, described method comprise that the experimenter to this type of treatment of needs uses each the anti-EphB4 antibody of claim 1-19 of significant quantity.

46. each anti-EphB4 antibody of claim 1-19 is in the purposes of preparation in the medicine, described medicine is used for treatment of conditions and/or preventative processing.

Images