CN101395183B - 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 submitted on January 5th, 2006 according to 35USC § 119, the U.S. Provisional Application No.60/760 that on January 20th, 889 and 2006 submits to, and 892 right of priority, by reference to its complete content is taken in to 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 active growth requires sufficient blood supply such as embryo and tumour.They meet this needs by generating short angiogenesis factor, and described short angiogenesis factor promotes neovascularization via the process that is called blood vessel generation (angiogenesis).The pipe formation of blood vessel is complicated but orderly biology event relates to all perhaps mainly with lower step: a) from existing endotheliocyte (EC), breed EC or differentiate EC from progenitor cell; B) EC moves and engages to form the rope spline structure; C) the dull rear generating pipe generation of blood vessel (tubulogenesis) has the blood vessel of central lumen with formation; D) existing rope or blood vessel stretch out bud to form time angiogenic; E) (remodeling) and shaping (reshaping) occur further to reinvent in elementary plexus vasculosus; And f) endothelium pericyte (peri-endothelial cell) is raised to surround interior leather hose, for blood vessel provides, maintain and adjusting function, this type of cell comprises pericyte (pericyte) for little capillary vessel, for the myocardial cell of 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 occur (angiogenesis) for from hyperplasia to neoplastic conversion, and provide nutrition seemingly vital for growth and the transfer of tumour.Folkman etc., Nature339:58 (1989).Growth vigor and propagation that neovascularization (neovascularization) allows tumour cell obtain and compares with normal cell are autonomous.Tumour starts from single abnormal cells usually, and due to the distance with utilizing 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.Then some tumour cell turns to blood vessel generation phenotype to start endotheliocyte, 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 the 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).Not yet fully understand and control the accurate mechanism that blood vessel changes, 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 subject to tight adjusting.Up to now, different kinds of molecules, mostly be the secretion sex factor generated by peripheral cell, demonstrated adjusting EC and broken up, breed, move 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).The allelic loss of even single VEGF causes the discovery of embryonic death to point to the effect that can not replace 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.Cross and express in people's tumour that VEGF mRNA checks at majority.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.Cancer73:931-34 (1996); Dvorak etc., Am.J.Pathol.146:1029-39 (1995).

Equally, in eye liquid the active vascular proliferation in 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 Nature362:841-44 (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 also suppress intraocular blood vessel generation (Adamis etc., Arch.Ophthalmol.114:66-71 (1996)) in ischemic retinal illness model.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 is recorded in disclosed EP817 on January 14th, 1998, on October 15th, 648 and 1998 disclosed WO98/45331 and WO98/45332.A kind of VEGF antibody, rhuMAb-VEGF (bevacizumab), obtained FDA and ratified to combine with chemotherapy regimen to 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 forms tyrosine kinase receptor family (summary is shown in Dodelet, Oncogene, 19:5614-5619,2000) maximum in 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 occur with forward manner, and wherein receptor tyrosine kinase is subject to the activation of part, or occurs with reverse manner, and wherein cross-film ephrinB part is subject to the 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. occur 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, is then two fibronectin II type motifs.Be characterized as in addition born of the same parents' internal area and the membrane-spanning domain in conservative kinases district.

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 met these needs and other benefit is provided.

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

Summary of the invention

The evaluation of the present invention's part based on 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 that to take in conjunction with EphB4 be basic composition and method.As described herein, the expression that EphB4 wedding agent of the present invention is target and EphB4-EphB4 part approach and/or active relevant pathological condition provide important therapeutical agent and diagnostic reagent for.Thereby, the invention provides with EphB4 in conjunction with relevant method, composition, test kit and goods.

The invention provides and be combined the antibody of (such as specific binding) with EphB4.

On the one hand, the invention provides the anti-EphB4 antibody of separation, wherein the described antibody of total length IgG form is with about 50pM or better binding affinity (binding affinity) specific binding people EphB4.As this area, establish fully, part can be measured with any in the many measure method the binding affinity of its acceptor, and means with various quantitative value forms.For example, thereby in one embodiment, binding affinity, with the Kd value representation, reflects inherent binding affinity (having minimized avidity effect (avidity effect)).Generally and preferably, binding affinity is measured in vitro, no matter be for example to carry out under acellular environment or cell relevant environment.Any in many assay methods known in the art, comprise 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 the antibody separated of being combined with the ligand binding domain of EphB4.In some embodiment, the antibody of described separation with comprise the EphB4 ectodomain consisting of or consisting essentially of polypeptide be combined.

On the one hand, the invention provides the anti-EphB4 antibody separated of being combined with EphB4 Ligand Competition EphB4.

On the one hand, the invention provides the anti-EphB4 antibody of the separation of inhibition, reduction and/or blocking-up EphB4 activity.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 hypervariable region (HVR) sequences that are selected from lower group:

(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, and wherein E1-E18 is 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 modification that the HVR sequence of wherein said variation comprises at least one residue of sequence shown in SEQ ID NO:1-17.

On the one hand, the invention provides and comprise one, two, three, four, the antibody of five or six HVR, wherein each HVR comprises the sequence that is selected from SEQ ID NO:1-17, or consisting of, or consisting essentially of, 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 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, with 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 antibody of the present invention can have the modification of one or more (such as two, three, four, five or more) residue in HVR.

In one embodiment, the HVR-L1 variant comprises 1-5 (1,2,3,4 or 5) and locates to substitute in the lower 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 lower 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 lower 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 lower 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 lower 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, the HVR-H3 variant comprises 1 place and substitutes in lower 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 can use other amino acid of the conventional assessment of technology known in the art and/or described herein amino acid whose suitability as an alternative in background described herein.

On the one hand, the invention provides the antibody that comprises the HVR-H1 district, the sequence that described HVR-H1 district comprises 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, the sequence that described HVR-H3 district comprises SEQ ID NO:7 or 8.In one embodiment, the invention provides the antibody that comprises the HVR-L1 district, the sequence that described HVR-L1 district comprises SEQID NO:9 or 10.In one embodiment, the invention provides the antibody that comprises the HVR-L2 district, the sequence that described HVR-L2 district comprises 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, the sequence that it comprises SEQ ID NO:2;

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

(iii) HVR-H3 sequence, the sequence that it comprises 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, the sequence that it comprises SEQ ID NO:9;

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

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

As shown in Figure 1, the aminoacid sequence of SEQ ID NO:1-17 is numbered for 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 the HVR of heavy chain shown in Fig. 1 sequence.

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

Some embodiment of antibody of the present invention comprise hereinafter the antibody of humanization 4D5 shown in 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).

1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys argAla 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 TyrSerglyVal Pro Ser Arg Phe Ser Gly Ser Arg Ser 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 Lys107 (SEQ ID NO:18) (the HVR residue indicates underscore)

In one embodiment, there is modification at the one or more places of described huMAb4D5-8 light chain variable territory sequence in the 30th, 66 or 91 (Asn, the Arg and the His that indicate with runic/italic respectively hereinbefore).In one embodiment, the 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, it comprises hereinafter sequence shown in SEQ ID NO:52:

1Asp 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 ValSer 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 Lys107 (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 framework variable domain sequence, if basically being retained in conjunction with activity of EphB4.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& 5,821,337 and Lee et al., J.Mol.Biol. (2004), 340 (5): heavy chain variable domain Frame sequence 1073-93).In one embodiment, these antibody further comprise people κ I light chain framework consensus sequence.In one embodiment, these antibody comprise huMAb4D5-8 light chain HVR sequence (U.S. Patent No. 6,407,213& 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 & 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 Frame 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 Frame 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 Frame 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 Frame 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 Frame 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 Frame 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, or, (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, the sequence that this heavy chain variable domain comprises SEQ ID NO:53.In one embodiment, antibody of the present invention comprises the light chain variable territory, the sequence that this light chain variable territory comprises SEQ ID NO:54.In one embodiment, antibody of the present invention comprises heavy chain variable domain and light chain variable territory, the sequence that heavy chain variable domain comprises SEQ ID NO:53, the sequence that the light chain variable territory comprises SEQ ID NO:54.

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

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

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

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

On the one hand, the invention provides and be combined the antibody of 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 (just as described below), amino acid position/border of defining the antibody hypervariable region can change to some extent.Some position in variable domain can be considered heterozygosis hypermutation position, because these positions can think in hypervariable region according to a set of standard, but thinks outside hypervariable region according to a set of different standard.Also can find one or more these positions in the hypervariable region extended (as hereinafter further definition).

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

Humanized antibody of the present invention comprises the antibody that those have amino acid replacement in FR and have the variant of the affinity maturation of change in the CDR transplanted.Alternative amino acid in CDR or FR is not limited to those and is present in the amino acid in donor or 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 in conjunction with reducing (such as in conjunction with disappearance).In some embodiment, be characterized as and the combination of people Fc γ RI, Fc γ RIIA and/or Fc γ RIIIA of antibody of the present invention reduce (such as in conjunction with 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 of providing herein is provided for it, wherein said anti-EphB4 polypeptide and EphB4 specific binding.

Antibody of the present invention is combined (such as specific binding) with EphB4, and, in some embodiment, can regulate and control one or more aspects of 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, for example, to part (ephrin-B1, ephrin-B2, the destruction of and/or ephrin-B3) being combined with EphB4, EphB4 phosphorylation and/or EphB4 multimerization, and/or EphB4 part phosphorylation, and/or to the destruction of the relevant EphB4 of any biology and/or EphB4 part biological pathway, the inhibition that blood vessel is occurred, and/or to tumour, treating and/or preventing of cell proliferative disorders or cancer, and/or to expressing to EphB4 and/or treatment or the prevention of the illness that active (expressing and/or active the rising such as EphB4) is relevant.In some embodiment, antibody of the present invention and EphB4 specific binding.In some embodiment, described antibody and EphB4 ectodomain (ECD) specific binding.In some embodiment, described antibody with by the EphB4 ectodomain, formed or consisting essentially of polypeptid specificity is combined.In some embodiment, described antibody is with about 50pM or stronger KD and EphB4 specific binding.In some embodiment, antibody of the present invention reduces in vivo and/or in vitro, suppresses and/or blocks the EphB4 activity.In some embodiment, described antibody reduces, suppresses and/or blocking-up EphB4 autophosphorylation.In some embodiment, the combination of described antibody competition and EphB4 part (reduce and/or block the EphB4 part and be combined with EphB4).

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

On the one hand, the invention provides the purposes of antibody of the present invention in the medicine for the preparation of suppressing the blood vessel generation.

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 any type, and recombinant vectors for example, such as expression vector.Can use any in multiple host cell.In one embodiment, host cell is prokaryotic cell prokaryocyte, for example intestinal bacteria.In one embodiment, host cell is eukaryotic cell, and mammalian cell for example, 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 appropriate host cells encoding said antibody (or its fragment), 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 for example, specification sheets (such as the specification sheets about any method described herein) about the experimenter being used to composition (antibody).

On the one hand, the invention provides the test kit that comprises the first container and second container, in described the first container, the 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 for example, specification sheets to experimenter's applying said compositions (described antibody).

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

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

On the one hand, the invention provides the purposes of carrier of the present invention in the medicine of the therapeutic of the illness for the preparation of such as cancer, tumour and/or cell proliferative disorders and/or preventative processing.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 occurs.

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

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

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

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

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 for the treatment of of needs.

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

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

On the one hand, the invention provides the method for promoting neuronal development, breed, maintain or regenerating, described method comprises that the experimenter to this type for the treatment of of needs uses the anti-EphB4 antibody of significant quantity.

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

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

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

In one embodiment, in the inventive method, the cell of institute's target is cancer cells.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, in the inventive method, the cell of institute's target is the cell of hyper-proliferative (hyperproliferative) and/or hyperplasia (hyperplastic).In one embodiment, in the inventive method, the cell of institute's target is dysplastic (dysplastic) cell.In another embodiment, in the inventive method, the cell of institute's target is the cell shifted.

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 combined 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 occurs.In some embodiment, another therapeutical agent is antiangiogenic agent, antineoplastic agent and/or chemotherapeutics.

Anti-EphB4 antibody can be with to those purposes, effectively another therapeutical agent be continuous or co-administered, or in same composition or as the composition separated.The using and can carry out simultaneously of anti-EphB4 antibody and another therapeutical agent (for example carcinostatic agent, antiangiogenic agent), for example, as single composition, or as two or more different composition, used the identical or different path of using.Perhaps/in addition, use and can 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, between the using of two or more compositions, can have scope from several minutes to a couple of days, to several weeks, to the interval of several months.For example, can first use carcinostatic agent, then use anti-EphB4 antibody.Yet, also imagined and used simultaneously or first used anti-EphB4 antibody.Thereby, on the one hand, the invention provides and comprise the method for using anti-EphB4 antibody, then using 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 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 list of contemplated chemotherapeutics in " definition " part.Using of anti-EphB4 antibody and linking agent can be carried out simultaneously, and for example, as single composition, or as two or more different composition, used the identical or different path of using.Perhaps/in addition, use and can carry out with any sequence.Perhaps/in addition, the order that described step can be used as any order is carried out and is carried out simultaneously the combination of the two and carries out.In certain embodiments, between the using of two or more compositions, can go up if having time from several minutes to a couple of days, to several weeks, to the interval of several months.For example, can first use chemotherapeutics, then use anti-EphB4 antibody.Yet, also imagined and used simultaneously or first used anti-EphB4 antibody.Thereby, on the one hand, the invention provides and comprise the method for using anti-EphB4 antibody, then using chemotherapeutics.In certain embodiments, between the using of two or more compositions, can go up if having time from several minutes to a couple of days, to several weeks, to the interval of several months.

On the one hand, the invention provides for strengthening antiangiogenic agent and suffering from the method for effect that the experimenter of relevant pathological condition occurs with blood vessel, described method comprises anti-EphB4 antibody and the antiangiogenic agent to the co-administered significant quantity of experimenter, strengthens thus the inhibition activity of described antiangiogenic agent.

On the other hand, the invention provides the method for detection of EphB4, described method is included in sample and detects the anti-EphB4 antibody complex of EphB4-.Term " detection " comprises qualitatively and/or quantitative detection (measurement level) having or contrast without reference for this paper the time.

On the other hand, the invention provides and express with EphB4 for diagnosis 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 biological sample.In some embodiment, it is the expression of rising or abnormal 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 in vitro.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); And clone 30.35.2D13, (HVR-H1 is SEQ ID NO:1; 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 for implementing the total Frame 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 VLK 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 the assay method based on 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 raise 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 detecting and/or separating the reagent of EphB4, such as in various tissues and cell type, detecting EphB4.

The method for preparing anti-EphB4 antibody, the polynucleotide 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 ordinary method to be adopted, method such as the widespread use of putting down in writing in 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.): PCR2:A PRACTICAL APPROACH (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)); Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY 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 reclaimed.The contaminative composition of its natural surroundings refers to disturb the diagnosis of this antibody or the material of therepic use, can comprise the solute of enzyme, hormone and other oroteins character or nonprotein character.In preferred embodiments, by 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 reductibility or irreducibility condition and use Coomassie blue or preferred silver dyeing.Since at least one composition of antibody natural surroundings can not exist, the antibody separated so comprises the original position antibody in reconstitution cell.Yet usually will prepare by least one purification step by the antibody of separation.

" separation " nucleic acid molecule refer to identify and with the natural origin of antibody nucleic acid at least one contaminative nucleic acid molecule of associated separates usually nucleic acid molecule.The nucleic acid molecule differ separated is in form or background when occurring in nature is found it.The nucleic acid molecule separated the therefore nucleic acid molecule when being present in n cell is had any different.For example, yet the nucleic acid molecule of separation comprises the nucleic acid molecule comprised in the cell of common this antibody of expression, when the chromosomal localization of described nucleic acid molecule in described cell is different from its chromosomal localization 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 in MD. (1991) is for the numbering system of heavy chain variable domain or light chain variable territory (variable domain).Use this numbering system, actual linear aminoacid 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 after H2 residue 52 inserts (being 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 " for this paper the time, mean 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 biological characteristics background for example, with described numerical value (Kd value) measured difference between two numerical value has very little or there is no biology and/or a significance,statistical.As with reference to/the function of this numerical value of antibody relatively, the difference between described two numerical value preferably is less than approximately 50%, preferably is less than approximately 40%, preferably is less than approximately 30%, preferably is less than approximately 20%, preferably is less than approximately 10%.

" binding affinity " for example is often referred to, for example, between the single binding site of molecule (antibody) and its binding partners (antigen) all intensity of noncovalent interaction summations.Except as otherwise noted, for this paper the time, " binding affinity " refers to that reflection for example, in conjunction with the interactional inherent binding affinity of 1:1 between right member (antibody and antigen).Molecule X explains the common available dissociation constant of the avidity of its mating partner Y (Kd).The common method that avidity can be known by this area is measured, and comprises described herein.Low-affinity antibody is conjugated antigen and be tending towards easily dissociating lentamente usually, and high-affinity antibody conjugated antigen and be tending towards keeping the combination of longer time faster usually.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, according to " Kd " of the present invention or " Kd value ", be that the radio-labelled antigen binding assay (RIA) that purpose antibody and antigen thereof by the described use of following assay method Fab pattern carry out is measured: under the condition by the titration series there being unlabelled antigen, with Cmin ¹²⁵i labelled antigen balance Fab, then with anti-Fab antibody, the antigen of coated flat board seizure combination is measured the solution binding affinity (Chen, et al., J Mol Biol293:865-881 (1999)) of Fab to antigen.In order to determine condition determination, catch with the coated microtiter plate (Dynex) of anti-Fab antibody (CappelLabs) and spend the night with 5 μ g/ml in 50mM sodium carbonate (pH9.6), use subsequently 2% in PBS (w/v) bovine serum albumin(BSA) in room temperature (approximately 23 ℃) sealing 2-5 hour.In non-absorption dull and stereotyped (Nunc#269620), by 100pM or 26pM[ ¹²⁵i]-antigen mixes (for example, with Presta et al., VEGF antibody in Cancer Res.57:4593-4599 (1997), the assessment of Fab-12 is consistent) with the purpose Fab of serial dilution.Then by 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 and for example catches plate, to carry out room temperature insulation (1 hour).Then remove solution, and wash plate 8 times with the PBS containing 0.1%Tween-20.After 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 for the competitive binding assay method.According to another embodiment, Kd or Kd value are to use BIAcore by surperficial plasmon resonance assay method ^tM-2000 or BIAcore ^tM-3000 (BIAcore, Inc., Piscataway, NJ) are used immobilized antigen CM5 chips to measure in about 10 response units (RU) in 25 ℃.In brief, hydrochloric acid N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimide (EDC) for specification sheets and N-hydroxy-succinamide (NHS) activation carboxymethylation dextran biologic sensor chip (CM5, BIAcoreInc.) according to the supplier., then with the flow velocity of 5 μ l/ minutes, be injected into and obtain the approximately coupling protein matter of 10 response units (RU) antigen diluent to 5 μ g/ml (approximately 0.2 μ M) with 10mM sodium acetate pH4.8.After injecting antigen, inject the 1M thanomin with sealing unreacted group.In order to carry out kinetic measurement, in 25 ℃ of flow velocitys with approximately 25 μ l/ minutes, be infused in the Fab (0.78nM to 500nM) of twice serial dilution in the PBS (PBST) containing 0.05%Tween-20.Use simply one to one Lang Gemiaoer (Langmuir) combination model (BIAcoreEvaluation Software version3.2) by while matching 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 Biol293:865-881 (1999).If, according to surperficial plasmon resonance assay method above, association rate surpasses 10 ⁶m ^-1s ^-1association rate can be measured by the fluorescent quenching technology so, 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 equippedspectrophometer) (Aviv Instruments) or 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 in pH7.2 (Fab form) (excites=295nm the fluorescent emission intensity of 25 ℃; 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 _onalso can use BIAcore by identical surperficial plasmon resonance technique mentioned above ^tM-2000 or BIAcore ^tM-3000 (BIAcore, Inc., Piscataway, NJ) are used immobilized antigen CM5 chips to measure in about 10 response units (RU) in 25 ℃.In brief, hydrochloric acid N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimide (EDC) for specification sheets and N-hydroxy-succinamide (NHS) activation carboxymethylation dextran biologic sensor chip (CM5, BIAcore Inc.) according to the supplier., then with the flow velocity of 5 μ l/ minutes, be injected into and obtain the approximately coupling protein matter of 10 response units (RU) antigen diluent to 5 μ g/ml (approximately 0.2 μ M) with 10mM sodium acetate pH4.8.After injecting antigen, inject the lM thanomin with sealing unreacted group.In order to carry out kinetic measurement, in 25 ℃ of flow velocitys with approximately 25 μ l/ minutes, be infused in the Fab (0.78nM to 500nM) of twice serial dilution in the PBS (PBST) containing 0.05%Tween-20.Use simply one to one Lang Gemiaoer (Langmuir) combination model (BIAcore Evaluation Software version3.2) by while matching 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 Biol293:865-881 (1999).Yet if, according to surperficial plasmon resonance assay method above, association rate surpasses 10 ⁶m ^-1s ^-1association rate is preferably measured by the fluorescent quenching technology so, 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 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 in pH7.2 (Fab form) (excites=295nm the fluorescent emission intensity of 25 ℃; Emission=340nm, 16nm band is logical) rising or reduction.

Term " carrier " means to transport the nucleic acid molecule of connected other nucleic acid for this paper the time.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 virus vector, wherein other DNA section can be connected in viral genome.Self-replicating in the host cell that some carrier can import at it (bacteria carrier and the additive type Mammals carrier that for example there is the bacterium replication orgin).For example, during other carrier (non-add type Mammals carrier) can be incorporated into the genome of host cell after importing host cell, thus along with host genome copies together.In addition, some carrier can instruct the genetic expression be operatively connected with it.Examples of such carriers is referred to herein as " recombinant expression vector " (or referred to as " recombinant vectors ").Usually, in recombinant DNA technology, useful expression vector is usually the plasmid form.In this manual, " plasmid " and " carrier " is used interchangeably, because plasmid is the most common form of carrier.

" polynucleotide " or " nucleic acid " are used interchangeably in this article, refer to the nucleotide polymer of any length, comprise DNA and RNA.Nucleotide can be deoxyribonucleotide, ribonucleotide, Nucleotide or base and/or its analogue through modifying, or can or mix any substrate of polymkeric substance by building-up reactions by DNA or RNA polymerase.Polynucleotide can comprise the Nucleotide through modifying, such as methylated nucleotide and analogue thereof.If any, to the modification of nucleotide structure, can before or after the assembling polymkeric substance, carry out.Nucleotide sequence can be interrupted by the non-nucleotide component.Polynucleotide can further be modified after synthetic, such as by with the marker coupling.The modification of other type for example comprises " cap ", one or more naturally occurring Nucleotide is substituted with analogue, modify between Nucleotide such as for example thering is neutral and connect (methyl-phosphonate for example, phosphotriester, phosphoramidate (phosphoamidate), carbamate etc.) and there is electrically charged connection (thiophosphatephosphorothioate for example, phosphorodithioate etc.) modification, contain the module of dangling (pendant moiety) such as for example protein (nuclease for example, toxin, antibody, signal peptide, polylysine etc.) modification, there is intercalator (acridine for example, psoralene etc.) modification, contain sequestrant (metal for example, radioactive metal, boron, oxidisability metal etc.) modification, the modification that contains alkylating agent, modification with modified connection (such as α end group isomery nucleic acid (anomericnucleic acid) etc.), and the polynucleotide of unmodified form.In addition; usually any hydroxyl be present in carbohydrate can be replaced with for example phosphonic acids (phosphonate) group, phosphoric acid (phosphate) group; with the standard blocking group, protect; or activation is connected with other of other Nucleotide with preparation, or can be coupled to solid and semi-solid upholder.But 5 ' with 3 ' end OH phosphorylation or by organic cap group module that adds of amine or 1-20 carbon atom, replace.Other hydroxyl also can be derivatized to the standard blocking group.Polynucleotide also can contain the ribose generally known this area or the analogue form of ribodesose carbohydrate, for example comprise 2 '-oxygen-methyl, 2 '-oxygen-allyl group, 2 '-fluoro-or 2 '-nitrogen-ribose repeatedly, carba sugars, α-end group isomerose, epimerization sugar such as pectinose, wood sugar or lyxose, pyranose, furanose, sedoheptulose, without ring analogues and dealkalize yl nucleosides analogue such as methylribonucleotide.The available alternative linking group is replaced one or more phosphodiesters and is connected.These alternative linking groups include but not limited to following embodiment, wherein P (O) S for phosphoric acid ester (" thioester " (thioate)), P (S) S (" dithio acid esters " (dithioate)), (O) NR ₂(" carboxylic acid amide esters " (amidate)), P (O) R, P (O) OR ', CO or CH ₂(" methylal〔Su〕 " (formacetal)) substitutes, wherein R or R ' are independent separately is H or replacement or unsubstituted alkyl (1-20 C), optionally contains ether (O-) connection, aryl, thiazolinyl, cycloalkyl, cycloalkenyl group or aralkyl (araldyl).Not all connections in polynucleotide must be all identical.Aforementioned description is applicable to all polynucleotide mentioned in this article, comprises RNA and DNA.

" oligonucleotide " refers generally to short polynucleotide for this paper the time, is generally strand, generally synthesizes, and length generally but be not to be less than approximately 200 Nucleotide.Term " oligonucleotide " is not mutually exclusive with " polynucleotide ".Above about the description equality of polynucleotide and be applicable to oligonucleotide fully.

" per-cent (%) 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 identity, and not by any conservative substituting while being considered as sequence identity a part of, the percentage of the amino-acid residue identical with amino-acid residue in particular peptide or peptide sequence in candidate sequence.Various ways for the contrast of measuring per-cent amino acid sequence identity purpose in can the art technology scope carries out, and for example uses 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 measuring the suitable parameter of contrast, comprise institute's comparative sequences total length is obtained to 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 Table A.ALIGN-2 sequence comparison computer program is write by Genentech company, hereinafter source code shown in Table A is submitted to (the US CopyrightOffice 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, on preferred digital UNIX V4.0D, uses.The all sequences comparative parameter is by ALIGN-2 program setting and constant.

Adopting during ALIGN-2 carrys out the situation of comparing amino acid sequence, given aminoacid sequence A with respect to (to), with (with) or for the % amino acid sequence identity of (against) given aminoacid sequence B (or can be expressed as have or comprise with respect to, with or for the given aminoacid sequence A of a certain % amino acid sequence identity of given aminoacid 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 the amino-acid residue sum in B.Can understand, if the length of the length of aminoacid sequence A and aminoacid sequence B is unequal, A will be not equal to the % amino acid sequence identity of B with respect to A with respect to the % amino acid sequence identity of B.

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

Term " EphB4 " (interchangeable being called " EphB4R "), for this paper the time, unless expressly stated otherwise, or context be otherwise noted, refer to (no matter being natural or synthetic) EphB4 polypeptide of any natural or variation.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 ".The polypeptide of the aminoacid sequence that term " wild-type EphB4 " refers generally to comprise the natural EphB4 of existence albumen.Term " wild-type EphB4 sequence " refers generally to the aminoacid sequence found 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 (bi-specific antibody for example, as long as they show the biologic activity of expectation), but also can comprise some antibody fragment (as more described in detail) herein.Antibody can be the people, humanized and/or affinity maturation.

Term " variable " refer to some part in variable domain between antibody sequence difference extensively and for every kind of specific antibodies combination and the specific truth to its specific antigen.Yet variability not is uniformly distributed in the whole variable domain of antibody.It concentrates in light chain and heavy chain variable domain three sections that are called complementary determining region (CDR) or hypervariable region.In variable domain more the part of high conservative be called framework (FR).Each self-contained Si Ge FR district of the variable domain of natural heavy chain and light chain, they take β-folded piece conformation mostly, by three CDR that form loop connecting and form β-folded piece structure part in some situation, connect.What the CDR in every chain approached by the FR district very much keeps together, and facilitate the formation of the antigen binding site of antibody (referring to Kabat et al. with together with the CDR of another chain, Sequences of Proteinsof 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, there is 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 recognition and binding site.In double-stranded Fv kind Zhong，Ci district, by tight a, heavy chain variable domain of non-covalent combination and the dimer in a light chain variable territory, formed.In the scFv kind, a heavy chain variable domain can be connected by flexible peptide linker covalency with a light chain variable territory, makes light chain and heavy chain similarly combine 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 comprising half Fv to three CDR of antigen-specific) also has the ability of identification and conjugated antigen, just avidity 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 C-terminal of heavy chain CH1 structural domain has increased the minority residue, comprises the one or more halfcystines from antibody hinge region.Fab '-SH is herein to appellation that wherein the constant domain cysteine residues carries the Fab ' of free sulphur alcohol radical.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 aminoacid 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 aminoacid 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, relevant at least one, preferably great majority or all functions with it usually when wherein said part preferably retains this part and is present in 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 formed by antibody fragment.In one embodiment, the antigen binding site that antibody fragment comprises complete antibody, so retain the ability of conjugated antigen.In another embodiment, antibody fragment, the antibody fragment that for example comprises the Fc district, retain while usually being present in complete antibody with the Fc district usually at least one relevant with it biological function, such as FcRn in conjunction with regulating and controlling, 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 brachium conjunctivum and itself and can give the Fc sequence of this fragment with the body internal stability and be connected.

Term " hypervariable region ", " HVR " or " HV " refer to alterable height on the antibody variable domains sequence and/or form the zone of the ring defined on structure for this paper the time.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) be take sequence variability as 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 Chothia structure ring, and obtains the use of the AbM antibody modeling software of Oxford Molecular." contact " hypervariable region is that take to the analysis of obtainable compound crystal structure is 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

Hypervariable region can comprise " hypervariable region of extension " as follows: 26-35 (H1), the 50-65 in the 24-36 in VL or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) and VH or 49-65 (H2) and 93-102,94-102 or 95-102 (H3).For each in these definition, 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 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 human normal immunoglobulin (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, avidity and ability.In some situation, the framework region of human normal immunoglobulin (FR) residue is replaced with corresponding inhuman residue.In addition, humanized antibody can be included in the residue do not found in receptor antibody or 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 normal immunoglobulin sequence.Humanized antibody optionally also will comprise at least part of constant region for immunoglobulin (Fc), the normally constant region of human normal immunoglobulin.More details are referring to Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature332:323-329 (1988); And Presta, Curr.Op.Struct.Biol.2:593-596 (1992).Also can be referring to following summary and the reference of quoting thereof: Vaswani andHamilton, Ann.Allergy, Asthma & Immunol.1:105-115 (1998); Harris, Biochem.Soc.Transactions23:1035-1038 (1995); Hurle and Gross, Curr.Op.Biotech.5:428-433 (1994).

The part of heavy chain and/or light chain and the identical or homology of the corresponding sequence in the antibody of specific antibodies classification or subclass derived from the Special Thing species or genus in " chimeric " antibody (immunoglobulin (Ig)), 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 biologic activity (U.S. Patent No. 4 of expectation, 816,567; Morrison et al., Proc.Natl.Acad.Sci.USA81:6851-6855 (1984)).Humanized antibody used herein is a subset of chimeric antibody.

VH and VL structural domain that " scFv " or " scFv " antibody fragment comprises antibody, wherein these structural domains are present on a polypeptide chain.Generally speaking, the scFv polypeptide also comprises peptide linker between VH and VL structural domain, makes scFv can form the required structure of conjugated antigen.About the summary of scFv referring to Pluckthun, in " The Pharmacology of MonoclonalAntibodies " the 113rd volume of compiling at Rosenburg and Moore, 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 connected heavy chain variable domain (V _h) and light chain variable territory (V _l).Can not match by using too short joint to make between two structural domains on the same chain, force the complementary structure territory pairing of these structural domains and another chain, thereby produce two antigen binding sites.Double antibody is more complete is recorded in for example EP404,097; WO93/11161; Hollinger et al., Proc.Natl.Acad.Sci.USA90:6444-6448 (1993).

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

" affinity maturation " antibody refers to have a place in one or more CDR of antibody or many places change, cause this antibody to compare with the parental antibody that there is no these changes the antibody improved to some extent to the avidity of antigen.The antibody of preferred affinity maturation will have nmole or the avidity to target antigen of picomole magnitude even.The antibody of affinity maturation can generate by rules known in the art.Marks et al., Bio/Technology10:779-783 (1992) has put down in writing the affinity maturation undertaken by VH and the reorganization of VL structural 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 biologic activity that those are attributable to antibody Fc district (native sequences Fc district or aminoacid 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; Phagolysis; Cell surface receptor (for example B-cell receptor) is lowered; With the B cell activation.

" antibody dependent cellular mediation cytotoxicity " or " ADCC " refers to wherein be attached to the target cell that for example, secretor type Ig on the upper Fc acceptor (FcR) existed of some cytotoxic cell (NK cell (NK) cell, neutrophilic granulocyte and scavenger cell) makes 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 that this type of lethal effect definitely requires.The main cell of mediation ADCC, the NK cell, only express Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Ravetchand Kinet, Annu.Rev.Immunol.9:457-92 (1991) the 464th page table 3 has been summed up the FcR on the hematopoietic cell and has been expressed.For the ADCC activity of purpose of appraisals molecule, can carry out external ADCC assay 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 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).

The white corpuscle that " people effector cell " refers to express one or more FcR and exercise effector functions.Preferably, this cell is at least expressed Fc γ RIII and is exercised the ADCC effector functions.The example of the human leukocyte of mediation ADCC comprises peripheral blood mononuclear cell (PBMC), NK cell (NK) cell, monocyte, cytotoxic T cell and neutrophilic granulocyte, preferably 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 aminoacid sequence, and difference is mainly in its cytoplasmic structure territory.Activated receptor Fc γ RIIA comprises the activation motif (ITAM) of immunity receptor based on tyrosine in its cytoplasmic structure territory.Suppress acceptor Fc γ RIIB and comprise the inhibition motif (ITIM) of immunity receptor based on tyrosine (referring to summary in its cytoplasmic structure territory

annu.Rev.Immunol.15:203-234 (1997)).The summary of FcR is referring to Ravetch and Kinet, Annu.Rev.Immunol.9:457-492 (1991); Capeletal., Immunomethods4:25-34 (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 newborn infant's acceptor, FcRn, it is responsible for the running balance that Maternal immunoglobulin G is transferred to fetus (Guyer et al., J.Immunol.117:587 (1976) and Kim et al., J.Immunol.24:249 (1994)) and regulates immunoglobulin (Ig).WO00/42072 (Presta) has put down in writing the antibody variants in conjunction with raising or reduction to FcR.The content of clearly taking in this patent publications at this as a reference.Also can be referring to Shields et al., J.Biol.Chem.9 (2): 6591-6604 (2001).

Measurement is known (referring to for example Ghetie1997, Hinton2004) 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, or in the primate of having used the Fc variant polypeptide.

" CDC " or " CDC " while referring to have complement to the dissolving of target cell.The startup of CCP is that the antibody (suitable subclass) that closes the combination of associated antigen institute in conjunction with it by complement system the first component (C1q) is initial.Start in order to assess complement, can carry out the CDC assay method, for example, as Gazzano-Santoro et al., put down in writing in J.Immunol.Methods202:163 (1996).

Polypeptide variants with C1q binding ability of the Fc region amino acid sequence of change and raising or reduction is recorded in U.S. Patent No. 6,194,551B1 and WO99/51642.The content of clearly taking in those patent publications at this as a reference.Also can be referring to Idusogie et al., J.Immunol.164:4178-4184 (2000).

Han“ Fc district polypeptide " refer to the polypeptide that comprises the Fc district, such as antibody or immunoadhesin (definition sees below).The C-end Methionin in Fc district (according to the residue 447 of EU numbering system) can be eliminated, for example, in the process of purified polypeptide or by the nucleic acid of 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 the mixture with the polypeptide that there is no the K447 residue.

" barrier " antibody or " Antagonism " antibody (antagonist antibody) refer to suppress or reduce the antibody of biologic activity of the antigen of its combination.Preferred blocking antibody or antagonistic antibodies essence or suppress the biologic activity of antigen fully.

" agonistic antibody (agonist antibody) " finger print for this paper the time 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 of the aminoacid sequence of the VL that comprises 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 normal immunoglobulin framework or people can comprise identical with it aminoacid sequence, or can comprise the aminoacid sequence be pre-existing in and change.When the amino acid be pre-existing in when existence changes, preferably exist and be no more than 5, preferably 4 or still less, or 3 or the amino acid that still less is pre-existing in change.While existing the amino acid be pre-existing in to change in VH, preferably those change three, two or a 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 normal immunoglobulin 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 normal immunoglobulin VL or VH Frame sequence selected works.Usually, human normal immunoglobulin VL or VH sequence selected works are from the variable region sequences hypotype.Usually, the sequence hypotype is the hypotype as people such as Kabat.In one embodiment, for VL, hypotype is the hypotype κ I as people such as Kabat.In one embodiment, for VH, hypotype is the hypotype III as people such as Kabat.

" VH subgroup III has framework " comprises the consensus sequence that the aminoacid sequence from the people's such as Kabat variable heavy chain hypotype III obtains.In one embodiment, at least a portion that the total framework amino acid sequence of VH hypotype III comprises following each sequence or whole whole:

EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO：42)-H1-WVRQAPGKGLEWV(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 aminoacid sequence from the people's such as Kabat variable light chain K hypotype I obtains.In one embodiment, at least a portion that the total framework amino acid sequence of VL hypotype I comprises following each sequence or whole:

DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO：46)-L1-WYQQKPGKAPKLLIY(SEQ ID NO：47)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQID 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 Mammals tend to the pathological condition of discussed illness.The non-limitative example of illness to be treated comprises pernicious and innocent 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 for this paper the time, no matter is pernicious or optimum, and all cancers before (pre-cancerous) and cancerous cells and tissue.Term " cancer ", " carcinous ", " cell proliferative disorders ", " proliferative disorders " and " tumour " are not mutually exclusive while mentioning in this article.

Term " cancer " and " carcinous " are pointed to or describe feature in Mammals and be generally the not modulated physiology illness of Growth of Cells/propagation.The example of cancer includes but not limited to cancer, lymphoma, blastoma, sarcoma and leukemia.The more specifically example of this type of cancer comprises squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer, the gland cancer of lung, the squama cancer of lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, carcinoma of the pancreas, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma (hepatoma), mammary cancer, colorectal carcinoma, colorectal carcinoma, carcinoma of endometrium or uterus carcinoma, salivary-gland carcinoma, kidney, prostate cancer, carcinoma vulvae, thyroid carcinoma, liver cancer (hepatic carcinoma), cancer of the stomach, melanoma, and various types of heads and neck cancer.

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 abnormal hypertrophy, sacroiliitis, rheumatoid arthritis (RA), psoriatic, plaque psoriasis, sarcoidosis, atherosclerosis, atherosclerotic plaque, diabetic 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, vascular restenosis, arteriovenous malformotion (AVM), meningioma, vascular tumor, hemangiofibroma, Tiroidina hyperplasia (comprising Ge Leifusishi (Graves) disease), cornea and other tissue transplantation, chronic inflammatory diseases, pneumonia, acute lung injury/ARDS, septicemia, primary pulmonary hypertension, malign lung hydrops (malignant pulmonary effusion), cerebral edema (for example relevant with Acute Stroke/closed head injury/wound), the synovia inflammation, pannus in RA forms, myositis ossificans, the hypertrophy bone forming, osteoarthritis (OA), refractory ascites, polycystic ovary disease, 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 inflammatory diseases such as IBD (Crow engler (Crohn) disease and ulcerative colitis), the kidney allograft thing repels, inflammatory bowel, nephrotic syndrome, undesired or abnormal tissue block growth (non-carcinous), bleeder's joint, hypertrophic cicatrix, the inhibition of hair growth, Ao-Wei Er Shi (Osler-Weber) syndrome, the botryomycosis hominis Terry's sign, scleroderma, trachoma, blood vessel adhesion, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as relevant with pericarditis) and hydrothorax.

For this paper the time, " treatment " or " processing " refer to attempt change the clinical intervention of nature process of the individuality for the treatment of or cell, can be in order to prevent or to carry out in the process of clinical pathology.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 progression of disease, improve or the state that palliates a disease, and exempt or improve prognosis.In some embodiment, antibody of the present invention is for postponing the generation of disease or illness/development.

Term " neurodegenerative disease " and " neurodegenerative disorders " are used with broad sense, comprise that its pathology relate to neuron sex change and/or handicapped all illnesss, include but not limited to peripheral neuropathy; The motor neuron illness, such as amyotrophic lateral sclerosis (amylotrophic lateral sclerosis) (ALS, LouGehrigShi 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, mongolism, nerve deafness and plum Ni Ershi (Meniere) disease.

" peripheral neuropathy " refers to affect perineural neurodegenerative disorders, the most often shows as one or combination in motion, sensation, sensorimotor or autonomic dysfunction.Peripheral neuropathy can be for example by heredity, to obtain, and can be derived from systemic disease, or can be that for example antineoplastic agent or industry or environmental pollutant 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(VCR), cis-platinum, methotrexate, 3 '-repeatedly nitrogen-3 '-deoxythymidine or Taxan, for example Taxol (paclitaxel)

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

-Poulenc Rorer, Antony, France]), the consequence of alcoholism, acquired immune deficiency syndrome (AIDS) (AIDS) or genetic factor (genetic predisposition) occurs.The peripheral neuropathy obtained by heredity comprises for example the not plain Mu Shi of thunder (Refsum) disease, the cured Bi Shi of gram (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) sick (also referred to as Proneal myatrophy or hereditary motor and sensory neuropathy (HMSN)).The peripheral neuropathy 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 neuropathy occurs fast/develops, and course of disease a couple of days, is called acute.Peripheral neuropathy is impact sensation and motorius together usually, thereby causes mixed type sensation and motor neuron, but pure sensation and neuropathy pure motion are also known.

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

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

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

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

Term " cytotoxic agent " refers to suppress or prevents the function of cell and/or cause the material of cytoclasis for this paper the time.This term intention comprises: radio isotope, for example At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², p ³²radio isotope with Lu; Chemotherapeutics, for example methotrexate (methotrexate), Zorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine(VCR) (vincristine), vinealeucoblastine(VLB) (vinblastine), Etoposide (etoposide)), Dx (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, Chlorambucil (chlorambucil), daunorubicin (daunorubicin) or other intercalator; Enzyme and fragment thereof, such as nucleolytic enzyme; Microbiotic; And toxin, such as the enzyme of small molecules toxin or bacterium, fungi, plant or animal origin toxin alive, comprise its fragment and/or variant; And the various antitumour drugs 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), such as busulfan (busulfan), Improsulfan (improsulfan) and croak pool Shu Fan (piposulfan), aziridines (aziridines), 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 (betulinicacid), 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), 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), such as BCNU (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine) and Ranimustine (ranimustine), antibiotics, for example, such as Enediyne Antibiotic (enediyne) (Calicheamicin (calicheamicin), especially Calicheamicin γ 1I and Calicheamicin ω I1 (referring to for example Agnew, Chem.Intl.Ed.Engl.33:183-186 (1994)), anthracycline antibiotic (dynemicin), comprise 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, such as methotrexate (MTX) (methotrexate) and 5 FU 5 fluorouracil (5-FU), folacin, such as denopterin (denopterin), methotrexate (MTX) (methotrexate), pteropterin (pteropterin), Trimetrexate (trimetrexate), purine analogue, such as fludarabine (fludarabine), Ismipur (mercaptopurine), ITG (thiamiprine), thioguanine (thioguanine), pyrimidine analogue, such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (azauridine), Carmofur (carmofur), cytarabine (cytarabine), two BrdU (dideoxyuridine), FUDR (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), androgens, such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), Mepitiostane (mepitiostane), Testolactone (testolactone), anti-adrenal gland class, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane), folic acid supplement, 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), 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 NaturalProducts, Eugene, OR), razoxane (razoxane), rhizomycin (rhizoxin), sizofiran (sizofiran), Spirogermanium (spirogermanium), tenuazonic acid (tenuazonic acid), triethyleneiminobenzoquinone (triaziquone), 2,2 ', 2 " RA3s, 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 ^TMcontaining cremophor (Cremophor), albumin transformation nano particle formulation Taxol (AmericanPharmaceutical Partners, Schaumberg, Illinois) and

Taxotere (doxetaxel) (

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

6-thioguanine (thioguanine), purinethol (mercaptopurine), methotrexate (MTX) (methotrexate), platinum analogs, 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), 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 has been usually the form of system or whole body therapeutic.They self can be hormones.Example comprises anti-estrogens and selective estrogen receptor modulators class (SERM), comprises that for example tamoxifen (tamoxifen) (comprises

tamoxifen),

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

toremifene (toremifene); The Mifepristone class; Adjust class (ERD) under estrogen receptor; Work the medicament that suppresses or close the ovary effect, r-hLH (LHRH) agonist for example, such as

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

magace (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),

pamldronate (pamidronate), tiludronate (tiludronate) or risedronate (risedronate); And troxacitabine (troxacitabine) (DOX nucleosides cytosine(Cyt) analogue); The signal conduction that antisense oligonucleotide, particularly those inhibition relate to adhesive cell propagation by way of in the antisense oligonucleotide of genetic 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 Tyrosylprotein kinase micromolecular inhibitor of ErbB-2 and EGFR, also referred to as GW572016); And the acceptable salt of pharmaceutics, acid or the derivative of any above-mentioned substance.

" growth inhibitor " refers to suppress in vitro or in vivo compound or the composition of cell (such as the cell of expressing EphB4) growth for this paper the time.Therefore, growth inhibitor can be the medicament that significantly reduces cell (such as the cell of the expressing EphB4) per-cent in the S phase.The example of growth inhibitor comprises the cell cycle the advance medicament of (position beyond the S phase) of blocking-up, such as inducing the medicament that G1 stagnates and the M phase stagnates.Classical M phase blocker comprises Changchun medicine class (vincas) (vincristine(VCR) (vincristine) and vinealeucoblastine(VLB) (vinblastine)), taxanes (taxanes) and Topoisomerase II inhibitors such as Dx (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 tamoxifen (tamoxifen), prednisone (prednisone), Dacarbazine (dacarbazine), chlormethine (mechlorethamine), cis-platinum (cisplatin), methotrexate (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, be entitled as " Cellcycle regulation, oncogenes; and antieioplastic drugs ", the people such as Murakaini, WBSaunders, 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 (

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

" Dx (Doxorubicin) " is anthracycline antibiotics.The complete chemical name of Dx is (8S-cis)-10-[(3-amino-2,3,6-tri-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)-10-[(3-amino-2，3，6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7，8，9，10-tetrahydro-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 one active therapeutic agent, for example " carcinostatic agent ".Therapeutical agent (carcinostatic agent, in this article also referred to as " antineoplastic agent ") example include but not limited to for example chemotherapeutics, growth inhibitor, cytotoxic agent, the medicament used in 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, rabbit, cytokine, energy and ErbB2, ErbB3, ErbB4, or the antagonist (for example neutrality antibody) of one or more target thing combinations in vegf receptor, the inhibitor of the receptor tyrosine kinase of Thr6 PDGF BB (PDGF) and/or STEM CELL FACTOR (SCF) (imatinib mesylate (imatinib mesylate) for example

novartis)), TRAIL/Apo2, and other biological activity and organic chemistry agent etc.

Term " prodrug " refers to that for the application the time 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).Prodrug of the present invention includes but not limited to phosphate-containing/ester prodrug, containing sulfo-phosphate/ester prodrug, containing sulfate/ester prodrug, containing the peptide prodrug, the amino acid modified prodrug of D-, the glycosylation prodrug, containing the beta-lactam prodrug, containing the prodrug of optional substituted benzene oxygen yl acetamide or containing the prodrug of optional substituted benzene ethanamide, can be converted into and have more activity and 5-flurocytosine and other 5-FUD prodrug of the medicine of no cytotoxicity.The example that can derive the cytotoxic drug of the prodrug form of using for the present invention 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 rotein of (angiogenesis), vasculogenesis (vasculogenesis) or undesired vascular permeability occur blood vessel small molecular weight material, polynucleotide, polypeptide, separation.For example, antiangiogenic agent is antibody or other antagonist of blood vessel propellant defined above, for example the small molecules (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 malignant melanoma); Ferrara& AlitaloNature Medicine5 (12): 1359-1364 (1999); The Oncogene22:6549-6556 such as Tonini (2003) (for example enumerating the table 2 of anti-angiogenic occurrence factor); SatoInt.J.Clin.Oncol.8:200-206 (2003) (for example enumerating the table 1 of the antiangiogenic agent used in clinical trial).

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 medicinal compositions; And the polynucleotide that comprise anti-EphB4 antibody coding sequence.For this paper the time, composition comprises one or more antibody in conjunction with EphB4, and/or one or more comprise one or more polynucleotide in conjunction with the sequence of the antibody of EphB4 of coding.These compositions can further comprise suitable carrier, such as the acceptable vehicle of pharmacopedics, comprise buffer reagent, and they are well-known in the art.

The embodiment of antibody and the polynucleotide of separation is also contained in the present invention.The embodiment of basically pure antibody and polynucleotide 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 provided herein ₂.These antibody fragments can create by conventional means, such as enzymatic digestion, or can generate by recombinant technology.This type of antibody fragment can be chimeric or humanized.These fragments can be used for hereinafter listed diagnosis and therapeutic purpose.

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

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

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

Perhaps, immunological lymphocyte in vitro.Then, use suitable fusogen such as polyoxyethylene glycol that lymphocyte and myeloma cell are merged, to form hybridoma (Goding, MonoclonalAntibodies:Principle sand Practice, pp.59-103, Academic Press, 1986).

The hybridoma of so preparation inoculate and cultivated in suitable medium, and described medium optimization contains one or more materials that parent myeloma cell that inhibition do not merge grows or survives.For example, if parent myeloma cell lacks enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the substratum for hybridoma typically will contain xanthoglobulin, aminopterin-induced syndrome and thymidine (HAT substratum), 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 substratum sensitivity such as the HAT substratum.In these cells, preferred myeloma cell line is rat bone marrow tumour system, can be from Sol gram institute cell distribution center (SalkInstitute Cell Distribution Center such as those, San Diego, California, USA) MOPC-21 obtained and MPC-11 mouse tumor and can be from American type culture collection (American Type CultureCollection, Rockville, Maryland, USA) SP-2 that obtains or X63-Ag8-653 cell derived.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 AntibodyProduction Techniques and Applications, pp.51-63, Marcel Dekker, Inc., NewYork, 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 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.

Obtain generating the hybridoma of the antibody with expectation specificity, avidity and/or activity in evaluation after, this clone can carry out subclone and be cultivated (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, such as for example albumin A-Sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibody of subclone secretion suitably be separated with nutrient solution, ascites or serum.

Anti-EphB4 antibody of the present invention can clone to create by with the combinatorial libraries screening, having the active synthetic antibody of expectation.On principle, select the synthetic antibody clone by the screening phage library, described phage library contains displaying and merges to the phage of various antibody variable regions (Fv) fragment of bacteriophage coat protein.By this type of phage library of affinity chromatography elutriation for required antigen.Expression can be adsorbed to antigen in conjunction with the clone of the Fv fragment of required antigen, thus with library in uncombined clone separate.Then combining clone wash-out from antigen, and the further enrichment that can circulate by extra antigen absorption/wash-out.Any anti-EphB4 antibody of the present invention can obtain as follows, design suitable antigen selection rules and select interested phage clone, then use Fv sequence and the Kabat et al. from interested phage clone, Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication91-3242, Bethesda MD (1991), the anti-EphB4 antibody cloning of suitable constant region (Fc) sequence construct total length of putting down in writing in vols.1-3.

The antigen binding domain of antibody by two approximately 110 amino acid whose variable (V) districts form, respectively from heavy chain (VL) and light chain (VH), all present three hypermutation rings or complementary determining region (CDR).Variable domain can functionally be illustrated on phage, or as scFv (scFv) fragment (wherein VH is connected by short, joint covalency 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.For this paper the time, 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 restructuring at random in phage library, then can search for antigen 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, without building hybridoma.Perhaps, can clone non-immune complete or collected works, be used to nonself and self antigen widely that single people's antibody sources is provided, without any immunity, as Griffiths et al., EMBO J, 12:725-734 (1993) is described.Finally, non-immune library can also build with synthesis mode, the V gene of not resetting from the stem cell clone, and with the PCR primer that comprises stochastic sequence encode alterable height the CDR3 district and be used for realizing in vitro resetting, as Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992) is described.

By merging with less important coat protein pIII, with filobactivirus, show antibody fragment.Antibody fragment can be shown as Single-Chain Fv Fragment of Murine, wherein VH is connected by flexible polypeptide spacer with the VL structural domain on same polypeptide chain, 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 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.If wish that library is partial to anti-EphB4 clone, can give so experimenter's immunity EphB4 to produce antibody response, and reclaim splenocyte and/or circulation B cell or other peripheral blood lymphocyte (PBL) for library construction.In a preferred embodiment, obtained as follows being partial to anti-EphB4 clone's human immunoglobulin gene fragment library, produce anti-EphB4 antibody response in the carrying function human immunoglobulin gene array transgenic mice of (and lacking functional endogenous antibody generation system), make the EphB4 immunity produce the B cell generated for people's antibody of EphB4.Being created on of transgenic mice that generates people's antibody hereinafter has description.

Can obtain as follows the further enrichment of the reactive cell mass of anti-EphB4, separate the B cell of expressing EphB4 specific membranes binding antibody by suitable screening rules, for example by the cellular segregation of being undertaken by the EphB4 affinity chromatography or cell to the absorption of fluorescently-labeled EphB4 and follow-up fluorescence-activated cell sorting (FACS).

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

Reclaim nucleic acid 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 obtain as follows required DNA, from separation of lymphocytes genomic dna or mRNA, then use with 5 of the VH reset 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, builds thus diversity V gene complete or collected works 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 be increased from cDNA, reverse primer also can be based in leading exon, as Jones et al., Biotechnol., 9:88-89 (1991) is described, forward primer is based in constant region, as Sastry et al., Proc.Natl.Acad.Sci. (USA), 86:5728-5732 (1989) is described.In order to make complementary the maximization, can mix degeneracy in primer, as described in Orlandi et al. (1989) or Sastry et al. (1989).Preferably, as follows the library diversity is maximized, with the PCR primer of each V gene family of target all obtainable VH and the VL existed that increase in the immunocyte nucleic acid samples, 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 by 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 with the primer of tape label, carry out further pcr amplification, as Clackson et al., Nature, 352:624-628 (1991) is described.

The V gene complete or collected works of synthetic restructuring can be derivative from the V constant gene segment C in vitro.Most people VH constant gene segment C is Cloning and sequencing (Tomlinson et al., J.Mol.Biol., 227:776-798 (1992)), and location (Matsuda et al., Nature Genet., 3:88-94 (1993)); These clones' section (all main structure that comprises H1 and H2 ring) 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 generate as follows, 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 VK and V λ section be Cloning and sequencing (Williams and Winter, Eur.J.Immunol., 23:1456-1461 (1993)), and can be used for generating synthetic light chain complete or collected works.Synthetic V gene complete or collected works based on a series of VH and VL pleated sheet structure and L3 and H3 length have coding the antibody of considerable structure diversity.After the DNA of amplification coding V gene, according to Hoogenboom and Winter, J.Mol.Biol., the method for 227:381-388 (1992), can reset germline V constant gene segment C in vitro.

The antibody fragment complete or collected works can build as follows, with several means by VH and VL gene gang.Can in different carriers, create each complete or collected works, and recombinant vectors in vitro, for example, as Hogrefe et al., Gene, 128:119-126 (1993) is described, or infects recombinant vectors by combination in vitro, for example Waterhouse et al., Nucl.Acids Res., the loxP system of record in 21:2265-2266 (1993).The storage capacity that the interior recombination method of body utilizes the double-stranded character of Fab fragment 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.Then combine two libraries by with phage-infect, containing the bacterium of phagemid, making each cell comprise a kind of various combination, storage capacity only is subject to cell to have the restriction (approximately 10 of number ¹²individual clone).Two kinds of carriers are recombination signal in occlusion body all, makes VH and VL gene recombination to single replicon, and is packaged into altogether the phage virus grain.These huge libraries provide a large amount of good avidity (Kd that has ^-1for approximately 10 ^-8m) diversity antibody.

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

The antibody of not immune library (natural or synthetic) generation can have medium avidity (Kd ^-1for approximately 10 ⁶-10 ⁷m ^-1), but can also simulate in vitro as follows affinity maturation, and build and again select secondary library, as Winter et al. (1994), see above described.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), used the fallibility polysaccharase to introduce at random in vitro sudden change (Leung etal., Technique, 1:11-15 (1989)).In addition, can carry out affinity maturation by the one or more CDR of random mutation, 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.WO9607754 (being disclosed on March 14th, 1996) has put down in writing for the complementary determining region induced mutation at 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 screen more high-affinity in several endless chain reorganization, 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 design with the aminoacid sequence in required EphB4 zone.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.Can prepare by several different methods known in the art by the DNA of coding EphB4.These methods include but not limited to the al. by Engels et, Agnew.Chem.Int.Ed.Engl., any method of record in 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 building the DNA of coding EphB4, by DNA molecular and expression vector, such as the expression control sequenc in plasmid, be operatively connected, wherein said control sequence is subject to the identification of the host cell of this carrier conversion.Generally speaking, plasmid vector comprises and copies and control sequence, and it is derived from the species compatible with host cell.Carrier carries replication site usually, and coding can provide the sequence of the protein 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, the DNA of coding EphB4 is operatively connected with the secretion leader sequence, causes expression product to be secreted in substratum by host cell.The example of secretion leader sequence comprises stII, ecotin, lamB, bleb GD, 1pp, 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.

Expression of the present invention mentioned above or cloning vector transfection for host cell, preferably transform, and cultivate in conventional nutritional medium, 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 for example, until many transfection methods, CaPO ₄precipitation and electroporation.If there is any indication of this carrier-mediated transport in host cell, think that transfection is successful.Method for transfection is well-known in the art, and some has description in this article.

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

Can be as Sambrook et al. for the prokaryotic host cell that generates EphB4, general described the cultivation sees above.

For the mammalian host cell that generates EphB4, can cultivate at multiple substratum, described substratum is well-known in the art, and some has description in this article.

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

The purifying of EphB4 can be realized by art-recognized method, this paper describes 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, for phage display clone's affinity chromatography, separate.EphB4 albumen can pass through Methods inEnzymology to adhering to of matrix, and in vol.44 (1976), the technology of record realizes.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 activation.

Perhaps, EphB4 can be used for the hole of coated adsorption plate, expresses being attached on the host cell of adsorption plate, or for cell sorting, perhaps be coupled to vitamin H and catch with the pearl coated with streptavidin, or for any other method for the elutriation phage display library known in the art.

Be suitable for, under the condition of at least part of phage particle in conjunction with sorbent material, making the phage library sample contact immobilized EphB4.Under normal circumstances, select to comprise that the condition of pH, ionic strength, temperature etc. simulates physiological conditions.The phage that is bonded to solid phase is cleaned, then de-with pickling, for example, as Barbas et al., Proc.Natl.Acad.Sci USA, 88:7978-7982 (1991) is described, or de-with alkali cleaning, for example, as Marks et al., J.Mol.Biol., 222:581-597 (1991) is described, or by EphB4 antigenic competition wash-out, for example with Clackson et al., Nature, 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 the more wheels selection.

The efficiency of selecting depends on many factors, comprises the kinetics of dissociating in cleaning process, and whether a plurality of antibody fragments on single phage can the while conjugated antigens.Antibody with very fast Dissociation (with weak binding avidity) can show by the cleaning with the short period of time, polyvalent phage, and solid phase in the antigen coated density of height retain.High-density not only interacts and has stablized phage by multivalence, and the combination again of the phage that is conducive to dissociate.Selection with antibody of slower Dissociation (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 WO92/09690 are described) and hang down antigen coated density (as Marks et al., Biotechnol., 10:779-783 (1992) is described) promote.

Likely between the phage antibody that EphB4 is there are to different avidity, selected, or even avidity is slightly discrepant.For example, yet the random mutagenesis of selected antibodies (affinity maturation technology as mentioned above as some is carried out) likely produces many mutant, most conjugated antigens, minority has higher avidity.By restriction EphB4, rare high-affinity phagocytosis physical efficiency competition is won.In order to retain the mutant of all higher affinity, can be by phage and incubation together with excessive biotinylation EphB4, but the volumetric molar concentration of biotinylation EphB4 is lower than the target mole affinity costant of EphB4.Then with streptavidin coated paramagnetic beads 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 greatly excessive 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 and carry out the differentiation based on Dissociation.

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

The derivative monoclonal antibody of code book invention hybridoma or phage display Fv clone's DNA 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).Once separate, DNA can be placed in to expression vector, then this expression vector is transfected into and does not originally generate in the host cell of immunoglobulin (Ig) protein, such as Bacillus coli cells, ape and monkey COS cell, Chinese hamster ovary (CHO) cell or myeloma cell, to obtain the synthetic of required monoclonal antibody in recombinant host cell.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 suitable DNA sequence dna can derive from Kabat et al., sees above) of constant region of light chain to form the clone of coding 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 then merging to form the encoding sequence of " 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.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, be that the encoding sequence of employment 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 in 81:6851-6855 (1984)).Can further modify the DNA of coding hybridoma or the derivative antibody of Fv clone or fragment, engage the encoding sequence all or in part of immunoglobulin coding sequence and NIg polypeptide by covalency.Can prepare in this mode " chimeric " or " heterozygosis " antibody of the binding specificity with Fv clone of the present invention or the derivative antibody of 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.The reduced size of fragment is allowed quick removing, and can cause being easier to approach solid tumor.

Developed for generating the multiple technologies of antibody fragment.Traditionally, by the proteolytic digestion complete antibody derive these fragments (referring to for example Morimoto et al., Journal of Biochemicaland Biophysical Methods24:107-117 (1992); Brennan et al., Science229:81 (1985)).Yet, can directly by recombinant host cell, generate these fragments now.Fab, Fv and scFv antibody fragment all can, at expression in escherichia coli and by the intestinal bacteria secretion, so be allowed and easily generate these a large amount of fragments.Can be from phage antibody library discussed above the separation antibody fragment.Perhaps, can be directly from intestinal bacteria, reclaim Fab '-SH fragment chemical coupling to form F (ab ') ₂fragment (Carter et al., Bio/Technology10:163-167 (1992)).According to another kind of method, can directly from the recombinant host cell culture, separate F (ab ') ₂fragment.Comprise the Fab and the F (ab ') that remedy the receptor binding domain residue, there is the Half-life in vivo of prolongation ₂fragment is recorded in U.S. Patent No. 5,869,046.For other technology of generating antibody fragment, for skilled practitioner, will be apparent.In other embodiments, the antibody of selection is Single-Chain Fv Fragment of Murine (scFv).Referring to WO93/16185; 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 build 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, the same.Antibody fragment can also be " linear antibody ", for example, as U.S. Patent No. 5,641, puts 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 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., Nature321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988)), 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), wherein significantly is less than complete people variable region and substitutes by the corresponding sequence of inhuman species.In practice, humanized antibody is normally as servant's antibody, and wherein the residue like site substitutes with the rodents antibody class for some hypervariable region residue and some possible FR residue.

For the preparation of people's light chain of humanized antibody and the selection of variable region of heavy chain, for reducing antigenicity, be very important.According to so-called " the suitableeest (best-fit) " method, with the variable region sequences of rodents antibody, the whole library of known person variable region sequences is screened.Then select people's framework (Sims et al., the J.Immunol.151:2296 (1993) as humanized antibody with the immediate human sequence of rodents; Chothia et al., J.Mol.Biol.196:901 (1987)).Another kind method is used the derivative specific frame of consensus sequence 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.USA89:4285 (1992); Presta et al., J.Immunol.151:2623 (1993)).

What is more important, antibody retains the high-affinity of antigen and other favourable biological characteristics after humanization.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 by parental array and humanization sequence prepares humanized antibody.But common adaptive immune sphaeroprotein 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 show that images can analyze residue may act in candidate's immunoglobulin sequences performance function, analyzing influence candidate immunoglobulin (Ig) is in conjunction with the residue of the ability of its antigen.Like this, can from receptor sequence and list entries, select FR residue combination, thereby obtain the antibody feature of expectation, raise such as the avidity to target antigen.Generally speaking, the hypervariable region residue directly and the most substantially participates in the combination of impact to antigen.

People's antibody

The anti-EphB4 antibody of people of the present invention can be selected from people's charon phages and shows that Fv clone's variable domain sequence in storehouse and known people's constant domain sequence build by combining as mentioned above.Perhaps, can become the anti-EphB4 antibody of human monoclonal of the present invention next life by hybridoma method.On the books for generating human myeloma and mouse-people's allos myeloma cell line of human monoclonal antibodies, 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, now likely be created in the situation that lacks endogenous immunoglobulin (Ig) generation and can generate afterwards in immunity the complete complete or collected works' of people's antibody transgenic animal (for example mouse).For example, put down in writing isozygotying of heavy chain of antibody joining region (JH) gene in chimeric and germ line mutation mouse and deleted the inhibition fully that causes endogenous antibody to generate.Shift a large amount of people's germline immunoglobulin genes and will cause generating people's antibody in this type of germ line mutation mouse after antigen is attacked.Referring to for example Jakobovits et al., Proc.Natl.Acad.Sci.USA90:2551 (1993); Jakobovits et al., Nature362:255-258 (1993); Bruggermann etal., Year in Immunol.7:33 (1993).

Gene shuffling also is used in external for example, from the derivative people's antibody of inhuman (rodents) antibody, and wherein people's antibody has the 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), the heavy chain of the non-human antibody's fragment obtained by display technique of bacteriophage as mentioned above or variable region of light chain employment V domain gene complete or collected works replace, and produce non-human chain-human chain scFv or Fab block polymer group.The selection of carrying out with antigen causes the separation of the 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 that epi-position determines (marking, imprint) selection of human chain mating partner.When repeating this process with the non-human chain of replacement residue, obtain people's antibody (referring to PCT WO93/06213, being disclosed on April 1st, 1993).From traditional to transplant the non-human antibody's who carries out humanization by CDR different, this technology provides complete people's antibody, and they are containing FR or the CDR residue of inhuman origin.

Bi-specific antibody

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

For the method that builds bi-specific antibody, be known in the art.Traditionally, the coexpression of the recombinant production of bi-specific antibody based on two pairs of heavy chain immunoglobulin-light chains, wherein two kinds of heavy chains have different specificity (Millstein and Cuello, Nature305:537 (1983)).Due to 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 only have a kind of correct dual specific structure that has.The purifying of the correct molecule usually undertaken by the affinity chromatography step quite bothers and product yields poorly.Similarly rules are disclosed in WO93/08829, 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, antibody variable domains and the immunoglobulin (Ig) constant domain sequence that will have expectation binding specificity (antibody-antigen binding site) merge.Preferably, with comprise at least part of hinge, C _h2 and C _hthe heavy chain immunoglobulin constant domain in 3rd district is merged.Preferably at least one 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 for building do not wait, and this provides great handiness for the mutual ratio of adjusting three peptide species fragments.Yet, at least two peptide species chains, with same ratio, express while causing high yield or when this ratio does not have special meaning, likely the encoding sequence of two kinds or all three peptide species chains is inserted to an expression vector.

In a preferred embodiment of the method, bi-specific antibody is by having the heterozygosis heavy chain immunoglobulin of the first binding specificity on an arm, and the heterozygosis heavy chain immunoglobulin-light chain on another arm forms (the second binding specificity is provided).Due to the light chain immunoglobulin separating pathway that only existence in half bispecific molecule is provided convenience, therefore find this unsymmetrical structure be convenient to will expectation the dual specific mixture combine and separate with undesired immunoglobulin chain.The 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 of the heterodimer that will reclaim from the recombinant cell culture thing, maximize.Preferred interface comprises at least part of antibody constant domain C _h3 structural domains.In the method, one or more p1 amino acid side chains at first antibody molecule interface for example, are replaced with larger side chain (tyrosine or tryptophane).Replace by large amino acid side chain being used for example, than p1 amino acid side chain (L-Ala or Threonine), produce compensatory " cavity " with the same or similar size of bulky side chain on the interface of second antibody molecule.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 element 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 (WO91/00360, WO92/00373 and EP03089).Can prepare allos coupling antibody with any cross-linking method easily.Suitable linking agent is well-known in the art, together with many crosslinking technologicals, is disclosed in U.S. Patent No. 4,676,980.

Also put down in writing the technology that is generated bi-specific antibody by antibody fragment in document.For example, can connect to prepare bi-specific antibody with chemistry.Brennan et al., Science229:81 (1985) has put down in writing by proteolysis and has cut complete antibody to generate F (ab ') ₂the rules of fragment.By these fragments in the situation that exist two mercaptan complexing agent Sodium metaarsenites to reduce, to stablize two contiguous mercaptan and to prevent the formation of intermolecular disulfide bond.Then change the Fab ' fragment produced into sulfo-nitrobenzoyl acid esters (TNB) derivative.Then by one of Fab '-TNB derivative, the reduction by mercaptoethylamine reverts to Fab '-mercaptan again, and mixes with the another kind of Fab ' of equimolar amount-TNB derivative, to form bi-specific antibody.The bi-specific antibody produced 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) has 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 in vitro directed chemical coupling to form bi-specific antibody.The bi-specific antibody so formed 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 for HBT's target thing.

Also put down in writing from the recombinant cell culture thing 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 by gene fusion, with the Fab ' part of two kinds of different antibodies, be connected from the leucine zipper peptide of Fos and Jun albumen.The antibody homodimer reduces to form monomer at hinge area, 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 builds bispecific antibody fragment.This fragment comprises the heavy chain constant domain (V connected by joint _h) and light chain constant domain (V _l), between too short two structural domains that make on the same chain of described joint, can not match.Therefore, force a V on fragment _hand V _lcomplementary V on structural domain and another fragment _land V _hthe structural domain pairing, form two antigen binding sites thus.Also reported by using scFv (sFv) dimer to build the another kind strategy of bispecific antibody fragment.Referring to Gruber et al., J.Immunol.152:5368 (1994).

Imagined and there are 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 subject to expressing than bivalent antibody the internalization (and/or alienation) of the cell of this antibody institute conjugated antigen faster.Antibody of the present invention can be recombinant expressed that generate, for example, the multivalent antibody with three or more antigen binding sites (tetravalent antibody) that can be easy to 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 consisting of) 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 multivalent antibody comprises (or consisting of) three to approximately eight herein, but preferred four antigen binding sites.Multivalent antibody comprises at least one polypeptide chain (and preferably 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 the first variable domain, VD2 is the second variable domain, a polypeptide chain in FcShi 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 preferably four) light chain variable domain polypeptides.Multivalent antibody herein for example can comprise approximately 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 optionally further comprises the CL structural domain.

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 change and introduce antibody nucleic acid or synthesize preparation by peptide by the Nucleotide by suitable.This type of modification comprises that for example the interior residue of antibody aminoacid sequence is deleted and/or inserts and/or substitutes.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 to theme antibody aminoacid sequence.

Can be used for identifying in antibody, as some residue of preferred mutagenesis position or the method in zone, " alanine scanning mutagenesis " arranged, as Cunningham and Wells, described in Science244:1081-1085 (1989).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 substitute with neutral or electronegative amino acid (most preferably L-Ala or Polyalanine), to affect the interaction of amino acid and antigen.Then pass through or alternate site is introduced to more or other variant, weighing substituting the amino acid position of display function susceptibility.Thus, although predetermine for the site of introducing variant amino acid sequence, yet the essence of sudden change itself needn't predetermine.For example, in order to analyze the consequence of specifying the site sudden change, at target codon or zone, carry out Alanine-scanning or random mutagenesis, and the activity that expressed immunoglobulin (Ig) screening is expected.

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 example that end inserts comprises the antibody with N end methionyl residue or the antibody merged with the cytotoxicity polypeptide.Other of antibody molecule inserts variant and comprises the N of antibody or C end and enzyme (as for ADEPT) or the fusion of 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 proline(Pro)) is carbohydrate module enzymatic to be attached to the recognition sequence of l-asparagine side chain.So, in polypeptide, these two kinds of arbitrary existence of tripeptide sequence have produced potential glycosylation site.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-OxoPro or 5-hydroxylysine.

Can make it comprise one or more above-mentioned tripeptide sequences and complete easily (glycosylation site connected for N-) by changing aminoacid sequence to adding glycosylation site in antibody.Described change also can be by adding in the sequence to original antibody or substituting one or more Serines or threonine residues is carried out (glycosylation site connected for O-).

If antibody comprises the Fc district, can change the carbohydrate adhered on it.For example, put down in writing the antibody that has the ripe carbohydrate structure that lacks Fucose to be attached to the antibody Fc district in U.S. Patent application US2003/0157108 (Presta, L.).Also can be referring to US2004/0093621 (Kyowa Hakko KogyoCo., Ltd.).Mentioned the antibody that decile N-acetyl-glucosamine (GlcNAc) arranged in the carbohydrate that is attached to the antibody Fc district in WO2003/011878 (people such as Jean-Mairet) and United States Patent (USP) the 6th, 602, No. 684 (people such as Umana).Reported the antibody that at least one galactose residue is arranged in the oligosaccharides that is attached to the antibody Fc district in WO1997/30087 (people such as Patel).Also can be referring to WO1998/58964 (Raju, S.) and WO1999/22764 (Raju, S.) about there being the change carbohydrate to be attached to the antibody in its Fc district.Also can be referring to US2005/0123546 (Umana et al.) about thering is the glycosylated antigen binding molecules of improvement.

Preferred glycosylation variants 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 Shi， 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 " de-Fucose type " or " Fucose shortage type " antibody comprises: US2003/0157108; WO2000/61739; WO2001/29246; US2003/0115614; US2002/0164328; US2004/0093621; US2004/0132140; US2004/0110704; US2004/0110282; US2004/0109865; WO2003/085119; WO2003/084570; WO2005/035586; WO2005/035778; WO2005/053742; Okazakiet al.J.Mol.Biol.336:1239-1249 (2004); Yamane-Ohnuki et al.Biotech.Bioeng.87:614 (2004).The example that generates the clone of de-fucosylated antibody comprises Lec13CHO cell (the Ripka et al.Arch.Biochem.Biophys.249:533-545 (1986) of the fucosylated defect of protein; Application No. US2003/0157108A1, Presta, L; And WO2004/056312A1, 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 knocked 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 hypervariable region, but has also imagined the FR change." preferably substitute " hurdle in table 1 and shown conservative substituting.If this type of substitutes, cause biologic activity to change, can in importing table 1, be called so the more substantial variations of " illustration substitutes ", or further described referring below to Amino Acid Classification, and the screening product.

Table 1

Original residue	Illustration substitutes	Preferably substitute
			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 and can be substituted significantly to realize to the difference on effect that maintains following aspect by selection: (a) structure of polypeptide main chain in the replacement area, for example (fold) sheet or helical conformation, (b) target site is punished sub electric charge or hydrophobicity, or (c) volume of side chain.

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

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

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

(3) acid: Asp, Glu;

(4) alkalescence: His, Lys, Arg;

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

(6) aromatic: Trp, Tyr, Phe.

Non-conservative alternative another classification of need to replacing 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 will there is the biological characteristics of improvement for the gained variant of further exploitation with respect to the parental antibody that produces them.Relate to for a kind of facilitated method that generates this type of alternative variations the affinity maturation that uses phage display.In brief, for example, by site, several hypervariable region (6-7 site) sudden change, in each site, produce all possible amino acid replacement.The antibody so generated is illustrated on the filobactivirus particle, as the fusions of the M13 gene III product with each particle internal packing.Then as disclosed herein the variant of phage display is screened to its biologic activity (for example binding affinity).In order to identify the site, candidate hypervariable region for modifying, can carry out alanine scanning mutagenesis to identify antigen in conjunction with the 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 antigen may be useful.Described contact residues and contiguous residue are to carry out alternative candidate locus according to technology detailed in this article.Once produce such variant, as described herein this group variant is screened, can be chosen in there is good characteristic in one or more related assays methods antibody for further exploitation.

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

May wish in the Fc district of immunoglobulin polypeptides of the present invention to introduce a place or many places amino acid modified, generate thus the Fc region variants.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 substituted).

According to the instruction of this description and this area, to have imagined in some embodiment, the antibody used in the inventive method antibody corresponding to wild-type is compared and can for example in the Fc district, comprised a place or many places change.With their wild type counterparts, compare, these antibody are the needed identical characteristics for the treatment of effect basically.For example, think in Ke Fc district carry out causing C1q in conjunction with and/or CDC (CDC) change some change of (or strengthen or weakening), for example, described in WO99/51642.Also can be referring to the Duncan and Winter that pays close attention to other example of Fc region variants, Nature322:738-40 (1988); United States Patent (USP) 5,648,260; United States Patent (USP) 5,624,821; And WO94/29351.

WO00/42072 (Presta) and WO2004/056312 (Lowman) have put down in writing the antibody variants in conjunction with raising or reduction to FcR.The content of clearly taking in these patent publications at this as a reference.Also can be referring to Shields et al.J.Biol.Chem.9 (2): 6591-6604 (2001).Increased Plasma Half-life and to neonatal Fc receptor (FcRn) (it is responsible for Maternal immunoglobulin G 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 the alternative Fc of being combined with FcRn in the Fc district.There is the Fc region amino acid sequence of change and the polypeptide variants of the rising of C1q binding ability or reduction and be recorded in U.S. Patent No. 6,194,551B1, WO99/51642.The content of clearly taking in these patent publications at this 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 obtained.Preferably, the part that is suitable for the antibody derivatize is 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-VP) polyoxyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylene polyvalent alcohol (as glycerine), polyvinyl alcohol and composition thereof.Stability due to it in water, the polyoxyethylene glycol propionic aldehyde may have advantage aborning.Polymkeric substance can be any molecular weight, and can be branch or unbranched.The polymkeric substance number be attached on antibody can change, and if adhered to and surpassed a polymkeric substance, they can be identical or different molecules so.Generally speaking, can be identified for number and/or the type of the polymkeric substance of derivatize according to following consideration, include but not limited to whether the concrete property of antibody to be improved or function, antibody derivatives will be used to specify treatment under condition etc.

Screening has the antibody of desired characteristic

The many measure method that can know by this area characterizes their physico/chemical properties and biological function to antibody of the present invention.In some embodiment, antibody has characterized following one or more: 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 combination 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 illness relevant with EphB4 expression and/or active (expressing and/or active the rising such as EphB4).

Can, by the antibody of the further purification Identification of a series of assay methods, 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.

In certain embodiments of the invention, their biologic activity of antibody analysis to generating herein.In some embodiment, to their antigen-binding activity of antibody test of the present invention.Antigen binding assay that this area is known and that can be used for this paper includes but not limited to use 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.Exemplary antigen binding assay hereinafter is provided in the embodiment part.

In another embodiment, the anti-EphB4 monoclonal antibody of the invention provides with 30.35,30.35.1D2 and/or 30.35.2D8 antibody competition EphB4 being combined.This type of competitive antibody comprises the identical or overlapping antibody of EphB4 epi-position that identified EphB4 epi-position is identified with antibody 30.35,30.35.1D2 and/or 30.35.2D8.This type of competitive antibody can by the screening of antagonism EphB4 hybridoma supernatant liquor with through mark 30.35,30.35.1D2 and/or 30.35.2D8 antibody competition to immobilization EphB4 in conjunction with obtaining.With the combination that contains irrelevant antibody or do not detect in binding mixture containing contrasting of antibody, through the amount of the antibody of mark, compare, the hybridoma supernatant liquor that contains competitive antibody can reduce the combination that detects in 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, as mentioned above by 30.35, one or more HVR of 30.35.1D2 and/or 30.35.2D8 are transplanted on the template antibody sequence, for example approach most 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 express gained chimeric light chain and/or weight chain variabl area sequence in recombinant host cell, have or without the constant region sequence of following.

The anti-EphB4 antibody of the present invention with unique property described herein can obtain as follows, by any antagonism of method easily EphB4 hybridoma colony screening desired characteristic.For example, if need to block or do not block the anti-EphB4 monoclonal antibody of EphB4 ligand binding EphB4, can in conjunction with competition assay, test candidate's antibody so, such as competitive binding ELISA, wherein plate hole is coated with EphB4, the excessive antibody-solutions of purpose Eph part is taped against through on coated plate, and enzyme process detects the antibody of combination, for example make the antibody contact coupling of combination 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 streptavidin-HRP and/or hydrogen peroxide and reading the plate instrument by spectrophotometry with ELISA and detect the HRP color reaction at 490nm.

If need to suppress or activate the anti-EphB4 antibody of EphB4 activity, 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.

If need cytostatic anti-EphB4 antibody, so can be in measuring cytostatic external and/or in vivoassay method test candidate antibody.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 it is important but some effector functions (such as complement and ADCC) is to become the material standed for of expectation in unnecessary or harmful many application at the antibody Half-life in vivo.In certain embodiments, measure the Fc activity of the immunoglobulin (Ig) that generates to guarantee only to have retained the characteristic of expectation.Can carry out external and/or in vivo cytotoxicity assay method with the reduction of confirming CDC and/or ADCC activity/subdue.For example, can carry out Fc acceptor (FcR) binding assay to confirm antibody deficiency Fc γ R in conjunction with (therefore likely lacking the ADCC activity) but to retain the FcRn binding ability.The main cell of mediation ADCC, the NK cell, only express 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 has been summed up the FcR on the hematopoietic cell and has been expressed.U.S. Patent No. 5,500, put down in writing the example for assessment of the external test method of the ADCC activity of molecules of interest 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 J.Immunol.Methods202:163 (1996).The method that also can use this area to know carries out removings/transformation period mensuration in FcRn combination and body, for example, described in the embodiment part.

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, for further clone's (DNA cloning) or expression.Can use old process to be easy to separate DNA the order-checking (as used the oligonucleotide probe that can be combined with the gene specific of encoding antibody heavy chain and light chain) of encoding antibody.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

But the Application standard recombinant technology obtains the polynucleotide sequence of code book invention antibody polypeptides member.Can separate from antibody-producting cell such as hybridoma polynucleotide sequence the order-checking of expectation.Perhaps, can use Nucleotide synthesizer or round pcr synthetic polyribonucleotides.Once obtain, the sequence of coded polypeptide inserted and can in prokaryotic hosts, be copied 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 nucleic acid that will insertion vector 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 its consistency of resident concrete host cell therein, 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 Insert Fragment and transcription termination sequence.

Generally speaking, the plasmid vector used together with host cell comprises derived from replicon and control sequence with 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, usually use the plasmid pBR322 derived from species Escherichia coli to transform intestinal bacteria.The gene that pBR322 comprises coding penbritin (Amp) and tsiklomitsin (Tet) resistance, provide the means of light identification of transformed cell thus.PBR322, its derivative or other microorganism plasmid or phage also can comprise or be modified and comprise can be by the microorganism biological body for expressing the promotor of endogenous protein.The people such as Carter, United States Patent (USP) 5,648, put down in writing the example of the pBR322 derivative for expressing specific antibodies in 237 in detail.

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

Expression vector of the present invention can comprise two or more promotor-cistrons pair, 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 subject to its control is transcribed.

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

The promotor that is applicable to prokaryotic hosts comprises that PhoA promotor, beta-galactosidase enzymes and Lac operon system, tryptophane (trp) promoter systems and hybrid promoter are such as tac or trc promotor.Yet it is also 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 or adapter that any required restriction site is provided that they and the cistron of coding target light chain and heavy chain are operatively connected to (Siebenlist et al., Cell20:269 (1980)) thus.

In one aspect of the invention, each cistron in recombinant vectors 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, or it can be the part of the target polypeptid DNA of insertion vector.The signal sequence of selecting for the present invention should be to be subject to the signal sequence that (being excised by signal peptidase) identified and processed to host cell.For nonrecognition and process the prokaryotic host cell of the natural signals sequence of heterologous polypeptide, signal sequence is substituted with being selected from for example prokaryotic signal sequence of lower group: alkaline phosphatase, penicillinase, Ipp or heat-staple enterotoxin 1 I (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP.In one embodiment of the invention, the signal sequence all used in two of expression system cistrons is STII signal sequence or its variant.

On the other hand, according to the generation of immunoglobulin (Ig) of the present invention, can in the tenuigenin of host cell, occur, therefore need in each cistron, not have secretory signal sequence.In that, light chain immunoglobulin and heavy chain are expressed, are folded and assemble and formation functional immunity sphaeroprotein in tenuigenin.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，Gene159：203(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; ATCC preserving number 27,325) and derivative, comprise the bacterial strain 33D3 (U.S. Patent number 5,639,635) with genotype W3110 Δ fhuA (Δ tonA) ptr3lac Iq lacL8 Δ ompT Δ (nmpc-fepE) degP41kanR.Other bacterial strain and derivative thereof are also suitable such as intestinal bacteria 294 (ATCC31,446), intestinal bacteria B, intestinal bacteria λ 1776 (ATCC31,537) and intestinal bacteria RV308 (ATCC31,608).These examples are illustration and unrestricted.This area knows for structure to have the method for specifying genotypic any above-mentioned bacterial derivation thing, referring to for example Bass et al., and Proteins8:309-314 (1990).Usually must consider that the reproducibility of replicon in bacterial cell select suitable bacterium.For example, when with well-known plasmid such as pBR322, pBR325, pACYC177 or pKN410, providing replicon, 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 extra proteinase inhibitor in cell cultures.

Ii. antibody generates

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

Transform and be about to DNA importing prokaryotic hosts, make DNA to be copied, or as extra-chromosomal element or by the karyomit(e) composition.According to host cell used, use the standard technique that is suitable for these cells to be transformed.Adopt the calcium of calcium chloride to process 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 electroporation.

That in this area, know and be suitable for cultivating in the substratum of selected host cell and cultivate for generating the prokaryotic cell prokaryocyte of 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, the selective agent that substratum is also selected containing the structure of with good grounds expression vector, allow the prokaryotic cell prokaryocyte growth that comprises expression vector with selectivity.For example, the substratum to the cell 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 the mixture with another kind of fill-in or substratum, such as compound nitrogen source.Optional, substratum can contain one or more reductive agents that is selected from lower 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 approximately 20 ℃ to approximately 39 ℃, more preferably from about 25 ℃ to approximately 37 ℃, and even more preferably from about 30 ℃.Depend primarily on host organisms, the pH of substratum can be that scope is approximately 5 to about any pH of 9.For intestinal bacteria, pH preferably approximately 6.8 to approximately 7.4, and more preferably from about 7.0.

If use inducible promoter in expression vector of the present invention, express being suitable for activating induced protein under the condition of promotor so.In one aspect of the invention, control transcribing of polypeptide by the PhoA promotor.Therefore, in order to induce, in phosphoric acid salt restriction substratum, cultivate the host cell through transforming.Preferably, phosphoric acid salt restriction substratum is C.R.A.P substratum (referring to for example Simmons et al., J.Immunol.Methods263:133-147 (2002)).According to adopted vector construct, 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 and reclaims.Protein recovery is usually directed to destroy microorganisms, usually by means such as osmotic shock (osmoticshock), supersound process or cracking.Once 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 nutrient solution and therefrom separate.Can be from the nutrient solution scavenger cell, and culture supernatants is filtered and concentrated, for being further purified generated protein.Can use method such as the polyacrylamide gel electrophoresis (PAGE) of generally knowing further to separate with the Western engram analysis and identify expressed protein.

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

During the fermentation, usually after cell being cultured under conditions suitable to expectation density (180-220 as about as OD550, at this phase cell in early stage stationary phase), start inducing of protein expression.According to adopted vector construct, can use multiple inductor, as this area, know with above-described.Can be before inducing by cell cultures shorter time.Usually by the about 12-50 hour of cell induction, 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 (a kind of peptidyl prolyl-cis with companion's activity, trans-isomerase) that can overuse is carried out cotransformation host prokaryotic cell prokaryocyte.Proved that chaperone promotes the correct of heterologous protein generated to fold and solubleness in bacterial host cell.Chen et al., J.Biol.Chem.274:19601-19605 (1999); The people such as Georgiou, United States Patent (USP) 6,083,715; The 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)).

For the proteolysis by the expressed heterologous protein heterologous protein of proteolysis sensitivity (especially to) be down to minimum, can be by some host strain of proteolysis enzyme defect for the present invention.For example, can modify the host cell bacterial strain, carry out genetic mutation in the gene of the known bacteria protease of coding, such as proteinase II I, 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; The people such as Georgiou, United States Patent (USP) 5,264,365; The people such as Georgiou, United States Patent (USP) 5,508,192; Hara et al., Microbial Drug Resistance2:63-72 (1996).

In one embodiment, use the proteolysis enzyme defect in expression system of the present invention and through the coli strain of the Plasmid Transformation of one or more chaperones of overexpression 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 precipitation, reversed-phase HPLC, tripoli or the Zeo-karb on the affine or ion exchange column of immunity is such as the chromatography on DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and use for example gel-filtration of Sephadex G-75.

In one aspect, albumin A on the solid phase immunoaffinity purification for full length antibody product of the present invention will be fixed on.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))。The solid phase that albumin A is fixed thereon preferably has the pillar of glass or quartz surfaces, more preferably controllable bore diameter glass column or silicic acid post.In some applications, pillar, with such as pack quilts such as glycerine, attempts to prevent the non-specific adhesion of pollutent.

As the first step of purifying, will be applied on albumin A immobilization solid phase derived from the prepared product of cell culture as mentioned above, make purpose antibody specific combination albumin A.Then clean the pollutent of solid phase with removing and solid phase non-specific binding.Finally by wash-out, from solid phase, reclaim purpose antibody.

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 used 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 subject to host cell and identify and process the allos signal sequence of (being excised by signal peptidase).In mammalian cell expression, can utilize mammalian signal sequence and viral secretory leading, for example the 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 comprising early promoter usually.

(iii) Select gene member

The cloning and expression carrier can comprise Select gene, also referred to as selection marker.The typical Select gene following protein of encoding: (a) give the resistance to microbiotic or other toxin, as penbritin, Liu Suanyan NEOMYCIN SULPHATE, methotrexate or tsiklomitsin; (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.Those Hemapoiesis that successfully transform through heterologous gene are given the protein of drug resistance, thereby survive selection scheme.The example of this type of dominant selection is used medicine Liu Suanyan NEOMYCIN SULPHATE, 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 by all transformants being cultivated in the substratum that contains methotrexate (Mtx, a kind of competitive antagonist of DHFR) identify the cell transformed through the DHFR Select gene.When adopting wild-type DHFR, suitable host cell is Chinese hamster ovary (CHO) clone (as ATCC CRL-9096) of the active defect of DHFR.

Perhaps, can by contain selective agent such as aminoglycoside antibiotics for selection marker as the substratum of kantlex, Liu Suanyan NEOMYCIN SULPHATE or G418 in culturing cell 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

The cloning and expression carrier comprises the promotor that is subject to host organisms identification usually, and is 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 approximately 25 to 30 base places, initial upstream, site of transcribing.In the another kind of sequence of being permitted to find at base place, 70 to 80 of polygenic transcriptional start point upstreams, be the CNCAAT district, wherein N can be any Nucleotide.At 3 of most of eukaryotic genes ' end, be the AATAAA sequence, it may be to add the signal of 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 subject 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 easily the early stage and late promoter of SV40 virus with the form of SV40 restriction fragment, this fragment also comprises SV40 virus replication starting point.Obtain easily the immediate early promoter of human cytomegalic inclusion disease virus with the form of HindIIIE restriction fragment.United States Patent (USP) 4,419, disclose the system of bovine papilloma virus as carrier expressible dna in mammalian hosts of using 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 by insert enhancer sequence in carrier, improve higher eucaryotic cells transcribing the DNA of code book invention antibody polypeptides.It is now know that from many enhancer sequence of mammalian genes (sphaeroprotein, elastoser, white protein, α-fetoprotein and Regular Insulin).Yet, usually use the enhanser from eukaryotic cell virus.Example comprises enhanser (bp100-270), the sub-enhanser of cytomegalovirus early promoter of SV40 replication orgin side in late period, enhanser and the adenovirus enhanser of polyomavirus replication orgin side in late period.Also can be referring to Yaniv about the enhancing element that activates eukaryotic promoter, Nature297:17-18 (1982).But the enhanser montage, in carrier, is positioned at 5 of antibody polypeptides encoding sequence ' or 3 ' position, but be preferably placed at 5 ' site of promotor.

(vi) Transcription Termination member

The expression vector used in eukaryotic host cell usually also comprises termination 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 district inclusions become the Nucleotide section of polyadenylation fragment at the non-translational region transcription of the mRNA of encoding antibody.A kind of useful Transcription Termination member is Trobest polyadenylation district.Reach wherein disclosed expression vector referring to WO94/11026.

(vii) selection of host cell and conversion

The host cell that is suitable for the DNA in clone or expression 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 monkey kidney CV1 system (COS-7, ATCC CRL1651) transformed through SV40, 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 CCL10), 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 CCL70), African green monkey kidney cell (VERO-76, ATCC CRL1587), human cervical carcinoma cell (HELA, ATCC CCL2), Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL34), ox mouse (buffalo rat) liver cell (BRL3A, ATCC CRL1442), human pneumonocyte (W138, ATCC CCL75), human liver cell (Hep G2, HB8065), mouse lacteal tumor (MMT060562, ATCC CCL51), TRI cell (Mather et al., Annals N.Y.Acad.Sci.383:44-68 (1982)), the MRC5 cell, FS4 cell and human liver cell knurl (hepatoma) are (Hep G2).

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

(viii) cultivation of host cell

Can in multiple substratum, cultivate for generating the host cell of 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 EagleShi substratum (DMEM, Sigma) and are suitable for cultivating host cell.In addition, can use in following document any substratum of putting down in writing substratum as host cell: Ham et al., Meth.Enz.58:44 (1979); 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; WO87/00195; Or United States Patent (USP) reexamination 30,985.Any 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 existed with the final concentration of micro-molar range) and 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 of antibody

When using recombinant technology, can in cell, generate antibody, or direct secretion is in substratum.If generate antibody in cell, 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 compression filter (for example Amicon or Millipore Pellicon ultra filtration unit) concentrates the supernatant liquor from these expression systems so usually.Can comprise that proteinase inhibitor is hydrolyzed with arrestin such as PMSF in any above-mentioned steps, and can comprise that microbiotic is to prevent the growth of external contaminant.

Can use the antibody compositions that for example hydroxyapatite, gel electrophoresis, dialysis and affinity chromatography (preferred purification technique is affinity chromatography) come purifying to prepare from cell.Albumin A depends on kind and the isotype of any immunoglobulin Fc domain existed in 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 is recommended 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, can use Bakerbond ABX ^tMresin (J.T.Baker, Phillipsburg, NJ) carries out purifying.According to antibody to be recycled, also can use chromatography, heparin SEPHAROSE on other oroteins purification technique such as the fractionation on ion exchange column, ethanol precipitation, reversed-phase HPLC, tripoli ^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 to the pH hydrophobic interaction chromatography, use the elution buffer of the about 2.5-4.5 of pH, preferably at low salt concn (0-0.25M salt according to appointment), carry out.

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 (radiating conjugate) are arranged.

Antibody-drug conjugates deliver to be poisoned the cell agent for part or is suppressed purposes (Syrigos and Epenetos, the Anticancer Research19:605-614 (1999) of cell reagent (for killing or the medicine of inhibition tumor cell) in cancer therapy; Niculescu-Duvaz and Springer, Adv.Drg.Del.Rev.26:151-172 (1997); United States Patent (USP) 4,975,278) can be by the drug moiety targeting Delivery to tumour, and carry out thin intracellular accumulation there, and these pharmaceutical agents without coupling of systemic application may cause unacceptable to Normocellular toxic level (Baldwin et al., Lancet603-05 (on May 15th, 1986) at the tumour cell of attempting to eliminate in addition; Thorpe, " Antibody CarriersOf Cytotoxic Agents In Cancer Therapy:A Review ", in " Monoclonal Antibodies ' 84:Biological And Clinical Applications ", the people such as A.Pinchera compile, the 475-506 page, 1985).Attempt thus to obtain maximum effect and minimum toxicity.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)).The medicine used in these methods comprises daunomycin (daunomycin), Dx (doxorubicin), methotrexate (methotrexate) and vindesine (vindesine) (Rowland etal., 1986, see above).The toxin used in antibody-toxin conjugated thing 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); Mandler et al., Bioorganic & Med.Chem.Letters10:1025-1028 (2000); Mandleret al., Bioconjugate Chem.13:786-791 (2002)), maytansinoid class (EP1391213; Liu et al., Proc.Natl.Acad.Sci.USA93:8618-8623 (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 binding, 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 activity decreased when the antibody with large or protein acceptor ligand coupling.

(ibritumomab tiuxetan, Biogen/Idec) be the antibody that formed by the mouse IgG1K monoclonal antibody of the CD20 antigen for finding and the 111In of being combined by thiourea linker-sequestrant or 90Y radio isotope-radio isotope conjugate (Wiseman et al., Eur.Jour.Nucl.Med.27 (7): 766-77 (2000) on normal and malignant B surface; Wiseman et al., Blood99 (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 for B cell non-Hodgkin's (Hodgkin) lymphoma (NHL), yet dispenser causes serious and long hemocytopenia in Most patients.MYLOTARG ^tM(gemtuzumabozogamicin, Wyeth Pharmaceuticals), the antibody-drug conjugates that is connected with calicheamicin by people CD33 antibody and forms, be used for through injection for curing acute myelogenous leukemia (Drugs ofthe Future25 (7): 686 (2000) approval in 2000; United States Patent (USP) 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001).Cantuzumab mertansine (Immunogen Inc.), the antibody-drug conjugates that is connected with maytansinoid drug moiety DM1 through disulphide joint SPP by huC242 antibody and forms, 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 is connected with maytansinoid drug moiety DM1 by anti-prostatic specific membrane antigen (PSMA) monoclonal antibody and forms, carrying out the exploitation for the potential treatment of tumor of prostate.By the synthetic analogues auristatin peptide of dolastatin (dolastatin), auristatin E (AE) and monomethyl auristatin (MMAE) and chimeric mAb cBR96 (special to the Lewis Y on cancer) and cAC10 (special to the CD30 on pernicious neoplastic hematologic disorder) coupling (Doronina et al., Nature Biotechnology21 (7): 778-784 (2003)), and carry 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 WO93/21232 on October 28th, 1993 for example.Multiple radionuclide can be used for generating radiation coupling antibody.Example comprises 212Bi, 131I, 131In, 90Y and 186Re.Can prepare with multiple bifunctional protein coupling agent by the conjugate of antibody and cytotoxic agent, 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 (p-azido benzoyl base) hexanediamine), dual azepine derivatives (such as two (p-diazobenzene formyl radical) hexanediamine), vulcabond is (such as toluene 2, the 6-vulcabond), with the double activated fluorine cpd (such as 1, 5-bis-fluoro-2, the 4-dinitrobenzene) dual-function derivative.For example, can be as Vitetta et al., Science238:1098 prepares the ricin immunotoxin described in (1987).The 1-isothiocyanic acid phenmethyl of carbon-14 mark-3-methyl diethylene triaminepentaacetic acid(DTPA) (MX-DTPA) is for the exemplary sequestrant by radioactive nuleus thuja acid and antibody coupling.Referring to WO94/11026.

This paper has also imagined the conjugate that 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 have the fragment of toxin activity.

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 the mitotic inhibitor played a role by suppressing the tubulin multimerization.Maytenin separates and obtains (United States Patent (USP) 3,896,111) from East Africa shrub tingia Caulis Mayteni (Maytenus serrata) 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 United States Patent (USP) discloses 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) relatively be easy to prepare by chemically modified, the derivatize of fermentation or tunning; (ii) be easy to being suitable for by functional group's derivatize of the coupling of non-disulphide joint; (iii) stable in blood plasma; And for kinds of tumor cells, be effectively (iv).

For example following patent discloses immune conjugate and preparation and the therepic use that comprises the maytansinoid class: United States Patent (USP) 5,208,020; 5,416,064; And European patent EP 0425235B1, clearly its disclosure is collected herein by reference.Liu et al., Proc.Natl.Acad.Sci.USA93:8618-8623 (1996) has put down in writing the immune conjugate that comprises the maytansinoid that is called DM1 be connected with monoclonal antibody C242 for human colorectal cancer.Find that this conjugate has the height cytotoxicity for the colon cancer cell of cultivating, and show anti-tumor activity in the tumor growth assay method in vivo.Chari et al., Cancer Research52:127-131 (1992) put down in writing maytansinoid wherein through the disulphide joint with in conjunction with CCL188 on antigen murine antibody A7 or in conjunction with the immune conjugate of the another kind of mouse monoclonal antibody TA.1 coupling of HER-2/neu oncogene.At human breast cancer cell, be the cytotoxicity of having tested TA.1-maytansinoid conjugate on SK-BR-3 in vitro, each cell expressing 3x10 of this clone ⁵individual HER-2 surface antigen.Drug conjugates has reached the cytotoxicity to a certain degree similar to free maytansinoid medicine, and this can improve by the maytansinoid molecule number that increases each antibody molecule coupling.A7-maytansinoid conjugate shows low systemic cytotoxicity in mouse.

Can prepare by biologic activity antibody is connected with the maytansinoid molecular chemistry and significantly do not weaken antibody or maytansinoid molecule by antibody-maytansinoid conjugate.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 strengthened for target cell, and function or the solubleness of antagonist do not have negative impact, although estimate that even toxin/the antibody of 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 in thing and disclose suitable maytansinoid class.The aromatic nucleus that preferred maytansinoid class is maytansinol and maytansinol molecule or other position maytansinol analogue through modifying, such as various maytansinol esters.

This area knows that many linking groups can be used for Dispersal risk-maytansinoid conjugate, comprises for example United States Patent (USP) 5,208,020 or European patent 0425235B1; Chari et al., CancerResearch52:127-131 (1992); The U.S. Patent application No.10/960 submitted on October 9th, 2004, disclosed in 602, clearly its disclosure is collected herein by reference.The antibody that comprises joint member SMCC-maytansinoid class conjugate can be as the U.S. Patent application No.10/960 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 patent mentioned above, and preferred disulphide and sulfide group.Describe herein and exemplified with other linking group.

Can carry out with multiple bifunctional protein coupling agent the conjugate of Dispersal risk 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 (p-azido benzoyl base) hexanediamine), dual azepine derivatives (such as two (p-diazobenzene formyl radical)-quadrols), vulcabond is (such as toluene 2, the 6-vulcabond), with the double activated fluorine cpd (such as 1, 5-bis-fluoro-2, the 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 thus disulfide linkage to connect.

According to the type connected, joint can be attached to a plurality of positions of maytansinoid molecule.For example, can with conventional coupling technology by with hydroxyl react to form ester bond.Reaction can occur in C-3 position with hydroxyl, the C-14 position of modifying through methylol, through the C-15 position of hydroxyl modified with there is 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 antibody of the present invention (U.S. Patent No. 5,635,483 with dolastatin class (dolastatins) or dolastatin peptide analogs and derivative, the coupling of auristatin class; 5,780,588).Dolastatin class and auristatin class have demonstrated disturbs microtubule dynamics, GTP hydrolysis, and core and cell fission (Woyke et al (2001) Antimicrob.Agents andChemother.45 (12): 3580-3584) and have an anticancer (US5,663,149) and anti-mycotic activity (Pettitet al (1998) Antimicrob.Agents Chemother.42:2961-2965).Dolastatin or auristatin medicine module can be attached to antibody (WO02/088172) via N (amino) end or C (carboxyl) end of peptide medicine module.

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

Be typically, can prepare by form peptide bond between two or more amino acid and/or peptide fragment by the medicine module based on peptide.Can prepare (referring to E. according to for example well-known liquid phase synthesizing method in chemistry of peptides field by this type of peptide bond

and K.L ü bke, The Peptides, volume1, pp76-136,1965, Academic Press).Can prepare according to the method with in Publication about Document by auristatin/ dolastatin medicine module: US5,635,483; US5,780,588; 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.15:859-863; And Doronina (2003) Nat Biotechnol21 (7): 778-784; " Monomethylvaline Compounds Capable of Conjugation to Ligands ", U.S. serial number No.10/983,340, submit on November 5th, 2004, by its complete being collected herein by reference (disclosed and for example prepared joint and the method that is coupled to the monomethyl α-amino-isovaleric acid compound of joint such as MMAE and MMAF).

Iii. calicheamicin

At other, say in counter taking, immune conjugate comprises 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 Research53:3336-3342 (1993); Lode et al., Cancer Research58:2925-2928 (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 born of the same parents, and are difficult for through 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 with other antineoplastic agent of antibody coupling of the present invention BCNU, streptozocin (streptozoicin), vincristine(VCR) (vincristine), 5 FU 5 fluorouracil, United States Patent (USP) 5,053,394,5,770, 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 (Aleutitesfordii) 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 WO93/21232 for example announced on October 28th, 1993.

The present invention has also imagined antibody and has had the compound of nucleolysis activity (as rnase or DNA endonuclease, such as deoxyribonuclease; The DNA enzyme) immune conjugate formed 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.By conjugate for detection of the time, can comprise radioactive atom and study for scitiphotograph, for example Tc ^99mor I ¹²³, or comprise spin label for nucleus magnetic resonance (NMR) imaging (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 to conjugate.For example, but the biosynthesizing peptide, or, by chemical amino acid synthesis method synthetic peptide, wherein use and 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) have put down in writing other method in detail.

Can carry out with multiple bifunctional protein coupling agent the conjugate of Dispersal risk 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 (p-azido benzoyl base) hexanediamine), dual azepine derivatives (such as two (p-diazobenzene formyl radical)-quadrols), diisothio-cyanate is (such as toluene 2, the 6-vulcabond), with the double activated fluorine cpd (such as 1, 5-bis-fluoro-2, the 4-dinitrobenzene) dual-function derivative.For example, can be as Vitetta et al., Science 238:1098 prepares the ricin immunotoxin described in (1987).The 1-isothiocyanic acid phenmethyl of carbon-14 mark-3-methyl diethylene triaminepentaacetic acid(DTPA) (MX-DTPA) is for the exemplary sequestrant by radioactive nuleus thuja acid and antibody coupling.Referring to WO94/11026.Joint can be " can cut joint " of being convenient to release cells cytotoxic drug in cell.For example, sour unstable joint, the responsive joint of peptase, photo-labile joint, dimethyl joint be can use or disulphide joint (Chari et al., Cancer Research52:127-131 (1992) contained; United States Patent (USP) 5,208,020).

Compound of the present invention is clearly contained but is not limited to the ADC prepared with following linking agent: commercialization is (as purchased from Pierce Biotechnology Inc., Rockford, IL, U.S.A.) BMPS, 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.

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 approximately 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, through covalent linkage and divalence joint reagent react, forms D-L, with the nucleophilic group of antibody, reacts subsequently.Method for distinguishing for the preparation of ADC has been described herein.

Ab-(L-D)pI

Joint can consist of one or more joint member.Exemplary joint member comprises 6-maleimide caproyl (" MC "), maleimide propionyl (" MP "), α-amino-isovaleric acid-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 '), (the iodo-ethanoyl of 4-) 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 submits 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: α-amino-isovaleric acid-citrulline (vc or val-cit), L-Ala-phenylalanine (af or ala-phe).Exemplary tripeptides comprises: glycine-α-amino-isovaleric acid-citrulline (gly-val-cit) and Gly-Gly-Gly (gly-gly-gly).The amino-acid residue that forms the amino acid joint member comprises those naturally occurring amino acid, and the amino acid analogue of less important amino acid and non-natural existence, such as citrulline.The amino acid joint member can for example, be designed and be optimized at their the selectivity aspect of enzymatic cutting of certain enzyme (tumor correlated albumen enzyme, cathepsin B, C and D, or fibrinolytic enzyme enzyme).

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

Also can become antibody-drug conjugates of the present invention next life by modified antibodies, introduce can with joint reagent or medicine on the electrophilic part of nucleophilic substitution radical reaction.The sugar of available for example periodate oxidation agent oxidation glycosylated antibodies, thus the aldehydes or ketones group that can react with the amine groups of joint reagent or drug moiety formed.Gained imines Schiff base can form stable key, or 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 reacting of galactose oxidase or sodium metaperiodate in protein, it can with medicine on suitable radical reaction (Hermanson, Bioconjugate Techniques).In another embodiment, the protein that comprises N-terminal Serine or threonine residues can react with sodium metaperiodate, causes generating at first amino acid place aldehyde (Geoghegan & 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 drug moiety includes but not limited to: amine, mercaptan, hydroxyl, hydrazides, oxime, hydrazine, thiosemicarbazone, hydrazinecarboxylate and aryl hydrazide group, they can react with the electrophilic group on shank and form covalent linkage, and joint reagent comprises: (i) active ester class, such as NHS ester, HOBt ester, haloformate and acid halide; (ii) alkyl and phenmethyl halogenide, such as Haloacetamide; (iii) aldehydes, ketone, carboxyl and maleimide base group.

Perhaps, can synthesize to prepare the fusion rotein that comprises antibody and cytotoxic agent by for example recombinant technology or peptide.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, can be by antibody and " acceptor " (such as Streptavidin) thus coupling for tumour target in advance, wherein to patient's administration of antibodies-acceptor conjugate, then use scavenging agent to remove unconjugated conjugate in circulation, then use " part " (as the affinity element) with cytotoxic agent (as the radioactive nuleus thuja acid) coupling.

Medicinal proportional preparation

Can accept carrier, vehicle or stablizer with optional physiology by the antibody that will there is expectation purity and mix to prepare the treatment preparaton that comprises antibody of the present invention, with the form of the aqueous solution, freeze-drying or other dry formulation, store (Remington:The Science and Practice ofPharmacy20thedition (2000)).Acceptable carrier, vehicle or stablizer are nontoxic at adopted dosage and concentration to the recipient, and comprise buffer reagent, such as phosphoric acid salt, Citrate trianion and other organic acid; Antioxidant, comprise 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, such as methyl p-hydroxybenzoate or propyl ester; Pyrocatechol; Resorcinol; Hexalin; The 3-amylalcohol; And meta-cresol); Lower molecular weight (being less than approximately 10 residues) polypeptide; Protein, such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, such as polyvinylpyrrolidone; Amino acid, such as glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrate, comprise glucose, seminose or dextrin; Sequestrant, such as EDTA; Carbohydrate, such as sucrose, N.F,USP MANNITOL, trehalose or sorbyl alcohol; The salify counter ion, 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 the necessary active compound of a kind of treat concrete indication, preferably active complementation and there is no each other disadvantageous effect.Suitable, this quasi-molecule is effectively to measure combination for predetermined purpose.

Activeconstituents also can wrap and for example for example be stated from, for example, by (being respectively Walocel MT 20.000PV or gelatin microcapsule and poly-(methyl methacrylate) microcapsule) in condensation technique or the microcapsule that prepare by interfacial polymerization, in gluey drug delivery system (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 Pharmacy20th edition (2000).

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

Can prepare the sustained release preparaton.The suitable example of sustained 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 of sustained 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 Pidolidone and γ-ethyl-Pidolidone ester, nondegradable ethane-acetic acid ethyenyl, degradable lactic acid-ethanol copolymer are such as LUPRON DEPOT ^tM(the Injectable microspheres body formed by lactic acid-ethanol copolymer and leuprorelin acetate) and poly--D-(-)-3-hydroxybutyrate.Although polymkeric substance such as ethane-acetic acid ethyenyl and lactic acid-ethanol can the sustained release molecule 1 more than 00 day, the time of some hydrogel release protein is shorter.When encapsulated antibody maintains in vivo for a long time, they may owing to being exposed to the wet environment of 37 ℃, sex change or gathering cause biologic activity loss and immunogenicity to change.Can carry out stabilization strategy reasonable in design according to related mechanism.For example, if discovery aggregation mechanism is to exchange and form intermolecular S-S key via mercaptan-disulphide, can and develops specific polymer matrix composition and realize stablizing by modifying sulfhydryl residue, by acidic solution freeze-drying, controlling moisture, the suitable additive of employing so.

Purposes

Antibody of the present invention can be used for 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, the method comprises that the experimenter to this type for the treatment of 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, the method comprises that the experimenter to this type for the treatment of of needs uses the anti-EphB4 antibody of significant quantity.

The motion axon guidance in EphB4 and 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, 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 Spinal injury.

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

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

On the one hand, the invention provides and treat the method that relevant pathology illness occurs with blood vessel, the method comprises that the experimenter to this type for the treatment of of needs uses the anti-EphB4 antibody of significant quantity.In some embodiment, described with blood vessel, relevant pathology illness to occur be tumour, cancer and/or cell proliferative disorders.In some embodiment, described with blood vessel, relevant pathology illness to occur be 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, for example, in comprising the cell culture of antigen, in the human experimenter or there is antibody of the present invention with it in other Mammals of the antigen of cross reaction (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, by making antibody, with antigen, contacts and makes antigenic activity be suppressed.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 antigen in subject obtain combination.Preferably, described antigen is that human protein molecule and described experimenter are people experimenters.Perhaps, the experimenter can be the Mammals of the antigen of expression antibody of the present invention institute combination.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 and express the immunoglobulin (Ig) non-human mammal of the antigen of cross reaction (for example primate, pig or mouse) with it for animal doctor's purpose, 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 dosage and the time-histories of dispenser) of antibody of the present invention.

Antibody of the present invention can be used for treatment, inhibition, delay of progression, prevention/delay recurrence, improve or expression and/or relevant disease, illness or the illness of activity of prevention and 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, the antigen of immune conjugate and/or its institute's combination is by cell internalization, and the therapeutic efficiency that causes immune conjugate to kill and wound the target cell of its institute's combination improves.In one embodiment, the nucleic acid in cytotoxic agent target or 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 be used alone, or 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 growth of the present invention, may wish especially it is combined to other therapeutical agent that one or more suppress tumor growth.Perhaps/in addition, the patient can accept associating radiotherapy (for example outside beam irradiates or use the therapy of radio-labeling medicament such as antibody).This type of conjoint therapy mentioned above comprises associating dispenser (when two or more kit are contained in identical or the preparaton that separates) and separately dispenser, in the later case, before or after the using and can occur in the using of adjuvant therapy of antibody of the present invention.

Conjoint therapy

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

Using of anti-EphB4 antibody can be carried out simultaneously, for example as single composition or as two or more different composition, use 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 first use carcinostatic agent, be then the EphB4 inhibitor.Yet, also imagined and used simultaneously or first used anti-EphB4 antibody.

Depend on doctor or animal doctor's judgement with the significant quantity of the antibody combined therapeutical agent of using of anti-EphB4.Complete dosage and use and adjust 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 for the treatment of.The appropriate dose of carcinostatic agent is exactly currently used those, and can reduce due 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 the dosage effect that therapeutical agent is commonly used or ratified at it is improved.

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

With the anti-angiogenic generation therapy of related to cancer, be 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 in the primary site tumour occurs 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 has been known many antiangiogenic agents, comprises those that this paper is listed, for example listed in the definition, also can be referring to for example Carmeliet and Jain, and Nature407:249-257 (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 for VEGF or vegf receptor, the Antagonism variant of VEGF, and combinatorial association is used.Perhaps/in addition, optional, can outside VEGF antagonist and other medicament, to the patient, use altogether two or more angiogenesis inhibitors.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 irradiate or use radioactive substance), chemotherapy, this paper is listed and this area is known carcinostatic agent).Perhaps/in addition, two or more antibody of the not synantigen that the same antigen disclosed in conjunction with this paper or two or more this paper disclose can be applied to the patient altogether.Sometimes, it may be useful also the patient being used to 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 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 list of contemplated chemotherapeutics in " definition ".

As those of ordinary skills, will appreciate that, the optimal dose of chemotherapeutics generally changes near those dosage that other chemotherapeutics adopts in using the clinical treatment of this chemotherapeutics for a long time alone or in combination.According to treated illness, dosage likely changes.The doctor who implements treatment can determine for the experimenter is individual suitable dosage.

The recurrent tumor growth

The present invention also provides the method and composition for inhibition or prevention of recurrence tumor growth or recurrent cancer Growth of Cells.Recurrent tumor growth or recurrent cancer Growth of Cells are being accepted one or more current available therapies (cancer therapy for example for describing, standard care scheme such as chemotherapy, radiotherapy, operation, hormonotherapy and/or biological therapy/immunotherapy, VEGF antibody therapy, particularly particular cancers) or the patient who crosses with one or more current available therapy for treating be not enough to clinically treat this patient or no longer by this therapy, obtain the situation that any beneficial effect makes these other effective therapies of needs of patients.For this paper the time, this statement also refers to " without response/intractable (non-responsive/refractory) " patient's situation, such as describe the patient therapy is had to 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 cancer Growth of Cells, wherein the cancer cells number does not have gratifying minimizing, perhaps increased, perhaps tumor size is not dwindled significantly, perhaps increased, or the size of cancer cells or number fail further dwindle or reduce.Determine whether cancer cells is that recurrent tumor growth or recurrent cancer Growth of Cells can carry out in vivo or in vitro, by this area, know for measuring treatment to any method of cancer cells validity, use the implication of art-recognized " recurrent " or " intractable " or " without response " in such linguistic context.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 cancer Growth of Cells, it is realized with blocking-up or the recurrent tumor growth or the recurrent cancer Growth of Cells that reduce in 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 with cancer therapy simultaneously.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 in the patient that the cancer tendency is arranged of using is also contained in the present invention.In one embodiment, cancer therapy is the treatment of using antiangiogenic agent, for example 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; EP0666868B1; U.S. Patent application 20030206899,20030190317,20030203409,20050112126; Popkov et al., Journal of Immunological Methods288:149-164 (2004); And WO2005012359.Other medicament can with the VEGF antagonist and anti-EphB4 is antibody combined is used to block or reduces recurrent tumor growth or recurrent cancer Growth of Cells, for example sees the part that title is " conjoint therapy " herein.

Antibody of the present invention (and auxiliary therapeutical agent) is used by any appropriate means, comprises in parenteral, subcutaneous, intraperitoneal, lung and in nose, and in damage, (if wishing topical therapeutic) used.The parenteral infusion comprises intramuscular, intravenously, intra-arterial, intraperitoneal or subcutaneous administration.In addition, pulse infusion antibody is also suitable, the form particularly reduced 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 long-term.

Can the mode consistent with good medical practice prepare, dosed administration and use antibody compositions of the present invention.The factor of considering in this content comprises the clinical condition of treated concrete illness, the concrete Mammals for the treatment of, 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 optionally by antibody with together with one or more medicaments of prevention or the treatment illness of discussing, prepare at present.The significant quantity of this type of other medicament depends on type and the other factors discussed above of amount, illness or the treatment of the antibody of the present invention existed in preparaton.These are normally with same dose used above with use path and use, or about 1-99% of dosage used so far.

For 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 for the replying of prevention or 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 type and the severity of disease, about 1 μ g/kg for example, is the initial candidate dosage that is applied to the patient to 15mg/kg (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.The scope of the every per daily dose of typical case 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 inhibition of expectation occurs.The scope of the exemplary dosage of antibody is that about 0.05mg/kg is to about 10mg/kg.So, can use 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 every 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 that maintains of about 2mg/kg antibody of potion weekly.Yet other dosages may be also useful.Can monitor easily the progress of this therapy 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 tissue expresses, and wherein said antibody is as described below to carry out mark and/or is fixed on insoluble matrix.

On the other hand, the invention provides the method for detection of EphB4, the method comprises the anti-EphB4 antibody complex of EphB4-detected in sample.Term " detection " comprises qualitative and/or detection by quantitative (measurement level) for this paper the time, has or contrasts without reference.

On the other hand, the invention provides the method for the diagnosis illness relevant with EphB4 expression and/or activity, the method comprises the anti-EphB4 antibody complex of biological sample detection EphB4-from 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) extremely increased.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 in body or external.In some embodiments, mixture comprises cancer cells.In some embodiments, anti-EphB4 antibody is detectable label.

Anti-EphB4 antibody can be used for any EphB4 in a large amount of known detection assay methods and detects.For example, can measure EphB4 to biological sample as follows, obtain sample from the expectation source, sample is mixed allow any EphB4 existed in antibody and mixture to form antibody/EphB4 mixture with anti-EphB4 antibody, and any antibody existed in the detection mixture/EphB4 mixture.Can prepare biological sample for measuring by the method that is suitable for specific sample known in the art.Select the method for biased sample and antibody and the method for detection antibody/EphB4 mixture according to the type of used 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 used 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 module that can direct-detection, 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 used is any functional group of detecting that does not disturb EphB4 and anti-EphB4 antibodies.Many markers become known for immunoassay, and example comprises module that can direct-detection, 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 is such as Rare Earth Chelate or fluorescein and derivative thereof, rhodamine and derivative thereof, dansyl, Umbelliferone, luciferase, for example Fluc 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), the heterocycle oxydase is such as uriKoxidase and XOD, coupling has with hydrogen peroxide and comes the enzyme of oxidation dye precursors such as HRP, lactoperoxidase, or microperoxisome, vitamin H/affinity element, spin label, the phage marker, stabilized radical, etc..

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 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 marker is enzyme herein, such as horseradish peroxidase and alkaline phosphatase.By this type of marker (comprising enzyme), with antibody coupling, for the those of ordinary skill of being familiar with immunoassay, be a kind of standard operating procedure.Referring to for example O ' Sullivan et al., " Methods for thePreparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay; " 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 will resist EphB4 antibody and any EphB4 still dissociated in solution to divide out.For this reason, or by being adsorbed to water-fast matrix or surface (Bennich et al., U.S.3,720,760), for example, by covalent coupling (utilizing glutaraldehyde cross-linking), before the assay method step, make anti-EphB4 antibody or EphB4 analogue not dissolve, or, for example by exempting to survey precipitation, after the assay method step, make anti-EphB4 antibody or EphB4 analogue not dissolve.

Can utilize immunohistochemistry and dyeing scheme to carry out the protein expression in sample for reference.The immunohistochemical staining of tissue slice has proved a kind of assessment or has detected the reliable method that in sample, protein exists.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 sample 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 freezing.In one embodiment, sample is fixing and is embedded in paraffin or analogue.Can fix by ordinary method (preserving) tissue sample.Those of ordinary skills will understand, and the selection of fixing agent is 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 of tissue sample and the fixing agent used.

IHC can implement together with the dyeing of other technology such as morphology and/or fluorescence in situ hybridization.Existing two kinds of IHC methods commonly used: direct and Indirect Determination.According to the first assay method, directly measure the combination of antibody and target antigen (for example EphB4).This direct measuring 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 Determination, the first antibody of coupling is not bonded to antigen, and then the second antibody through mark is bonded to first antibody.If the second antibody coupling has the enzyme labelling thing, add chromogenic substrate or fluorogenic substrate so that manifesting of antigen to be provided.Because several second antibody can be reacted from the different epi-positions on first antibody, so occurring, amplifies signal.

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

Outside sample preparation rules discussed above, may be also need to be before 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 first antibody enough time under conditions suitable, makes first antibody be bonded to the target protein antigen in tissue sample.Realize that the suitable condition of this purpose can determine by normal experiment.The combination degree of antibody and sample is by measuring by 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 is rabbit polyclonal antibody, and second antibody is goat anti-rabbit antibodies) of specific binding first antibody.

The sample so prepared can be placed and covered.Then carry out the slide glass assessment, for example utilize microscope, and can adopt the conventional staining power standard of using in this area.The staining power standard can be assessed as follows:

Table 2

the dyeing pattern	score
		do not observe dyeing in cell.	0
faint/just perceptible dyeing detected in surpassing 10% cell.	1+
		in surpassing 10% cell, observe weak to medium dyeing.	2+
in surpassing 10% cell, observe medium to strong dyeing.	3+

Typically, must be divided into about 2+ in the IHC assay method 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 approximately 3 or higher dyeing pattern be diagnostic and/or prognostic.Should be appreciated that when using the IHC inspection from the cell of tumour or adenoma of colon and/or organizing, generally measure or assess the dyeing in tumour cell and/or tissue (but not the matrix that may exist in sample or surrounding tissue).

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

Competitive assays depends on the ability of tracer agent EphB4 analogue and a limited number of anti-EphB4 antibody antigen-binding site of specimen EphB4 competition.Usually before competition or after competition, anti-EphB4 antibody is not dissolved, then divide out with unconjugated tracer agent and EphB4 in connection with tracer agent and EphB4 to anti-EphB4 antibody.By toppling over (wherein making in advance not dissolve in conjunction with counterpart) or realize this separation by centrifugal (wherein making in conjunction with the counterpart precipitation) after competitive reaction.In specimen, the amount of the tracer agent of the amount of EphB4 and combination is inversely proportional to, and the amount of the tracer agent of described combination is measured by the amount of mark substance.Draw dosage-response curve of the EphB4 of known quantity, with test result, compare quantitatively determine the amount of the EphB4 existed in specimen.When using enzyme as detectable, these assay methods are called the ELISA system.

Another kind of competitive assays, be called " homogeneous phase " assay method (homogeneous assay), do not need to be separated., prepare and use the conjugate of enzyme and EphB4 herein, make when anti-EphB4 antibodies EphB4, the existence of anti-EphB4 antibody changes enzymic activity.In this case, EphB4 or its immunologic competence fragment are coupled to enzyme such as peroxidase by difunctional organic bridge.Select conjugate to use together with anti-EphB4 antibody, make the enzymic activity in conjunction with inhibition or enhancing marker of anti-EphB4 antibody.This method itself, by broad practice, is called EMIT.

Space conjugate (steric conjugate) is for the sterically hindered method (sterichindrance method) of homogeneous assay method.By the covalently bound extremely little EphB4 fragment of lower molecular weight haptens is synthesized to these conjugates, making basically can not be with anti-EphB4 antibody simultaneously in conjunction with conjugate for haptenic antibody.Under these assay method rules, the EphB4 existed in specimen is in connection with anti-EphB4 antibody, thereby the permission antihapten causes the variation of conjugate haptens characteristic, for example variation of fluorescence when haptens is fluorophore in conjunction with conjugate.

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 for adsorbing specimen EphB4, remove specimen by cleaning, the EphB4 of institute's combination, for adsorbing the second anti-EphB4 antibody through mark, then separates the material of institute's combination and remaining tracer agent.In conjunction with the amount of tracer agent to specimen EphB4, be directly proportional.In " synchronously " sandwich assay method (simultaneoussandwich assay), before adding the process anti-EphB4 of mark, do not separate specimen.The assay method of sandwich in turn as another kind of antibody can be used for sample test EphB4 as a kind of antibody and Anti-TNF-α EphB4 antibody to use anti-EphB4 monoclonal antibody.

It is only above 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 and comprised the goods that can be used for the treatment, prevent and/or diagnose the material of disorder mentioned above.Goods comprise container and be attached on described container or with label or the package insert of its associated.Suitable container comprises such as bottle, tubule, syringe etc.Container can be made from a variety of materials, such as glass or plastics.Himself is housed in container or effectively treats, prevent and/or diagnose the composition of illness in associating during other composition, and can there is aseptic access port (for example container can be intravenous solution bag or the tubule with stopper that hypodermic needle can pierce through).At least one promoting agent in composition is 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 the first container of composition, and wherein said composition comprises antibody of the present invention; (b) second container of composition wherein is housed, wherein said composition comprises other cytotoxic agent.Goods in this embodiment of the present invention also can comprise that indication the first and second antibody compositions can be used for treating the package insert of specific illness 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 material required on business and user's position, comprises other buffer reagent, thinner, filter, syringe needle and syringe.

Below the embodiment of the inventive method and composition.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 with EphB4-His albumen, selects phage.This area knows that several different methods can be used for generating phage display library, 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-ELISA to be screened for EphB4-His albumen.Select unique clone for further characterizing by phage competitive ELISA and blocking-up assay method, to determine that can this phage antibody clone block EphB4-ephrinB2 and interact.Clone 30.35 does well, thereby is selected for further analysis.In order to improve clone 30.35 avidity, generate phage display library in the background of YW30.35, the selected HVR of each target carries out soft or rigid randomization (soft or hard randomization).Selected clone is screened by Phage-ELISA, then is expressed as Fab albumen 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 (BIAcore, Inc., Piscataway, NJ) carry out surperficial plasmon resonance (SRP) and measure.In brief, hydrochloric acid N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimide (EDC) for specification sheets and N-hydroxy-succinamide (NHS) activation carboxymethylation dextran biologic sensor chip (CM5, BIAcoreInc.) according to supplier.To resist EphB4 antibody dilution to 5 μ g/ml with 10mM sodium acetate pH4.8, and then with the flow velocity of 5 μ l/ minutes, be injected into and obtain the approximately coupling antibody of 500 response units (response unit RU).Then, inject the 1M thanomin with sealing unreacted group.In order to carry out kinetic measurement, people or the mouse EphB4-His molecule (0.7nM-500nM) of twice serial dilution in 25 ℃ of flow velocitys with approximately 25 μ l/ minutes are infused in containing the PBS of 0.05%Tween-20.Use Lang Gemiaoer 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.The result of this experiment is as shown in table 3." NA " means to be measured.

Table 3: binding affinity and the kinetics of anti-EphB4 antibody on human and mouse EphB4

In different experiments, tested the cross reactivity of anti-EphB4 antibody cloning 30.35 for other EphB acceptor.In brief, the plasmid transient transfection of expressing total length people EphB1, people EphB2, people EphB4 or people EphB6 for the COS7 cell.After 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 the assay method based on 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 by different cell types, present EphB4 and EphrinB2.Cross the 3T3 cell of expressing people EphrinB2 and express high-level EphB4 but the HUVEC cell of low-level EphrinB2 for stimulating, tested the ability of anti-EphB4 antibody suppression EphB4 activation.

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

Cross the 3T3 cell of expressing people EphrinB2 and be layered on the HUVEC cell and reach 15 or 30 minutes, now have or do not exist anti-EphB4 antibody.The activation of EphB4 acceptor is assessed as follows, and immunoprecipitation EphB4 albumen, then used anti-phosphoric acid-tyrosine antibody (antibody 4G10; Upstate) existence that detects the receptor tyrosine phosphorylation by the western trace whether.In brief, RIPA damping fluid cracking for cell.Cell lysate, by centrifugal clarification, adds anti-EphB4 antibody 35.2D8 with 5 μ g/ samples.In 4 ℃ of incubations, after 2 hours, immunocomplex is used the albumin A agarose electrophoresis.The EphB4 phosphorylation is used antiphosphotyrosine antibody 4G10 (Upstate) to be analyzed with the concentration of 1 μ g/ml by the Western trace.

The result of this experiment as shown in Figure 7.The 3T3 cell is layered on the HUVEC cell and causes significant EphB4 tyrosine phosphorylation (the 4th and 5 road).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 the EphB4 tyrosine phosphorylation (the 6th road) of inducing.On the contrary, with independent anti-EphB4 antibody treatment HUVEC cell, do not cause EphB4 activation (the 2nd and 3 road) in HUVEC, and untreated HUVEC cell is not showed the EphB4 activation.These results have been established anti-EphB4 antibody block ligand-acceptor interaction in the environment of direct cell-cells contacting.

Although described in more detail above-mentioned aspect 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.

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<212>PRT

<213 > artificial sequence

<220>

<223>HVR-H2

<400>5

Gly Gly Ile Tyr Leu Tyr Ser Gly Ser Arg Gly Tyr Ala Asp Ser

1 5 10 15

Val Lys Gly

<210>6

<211>18

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-H2

<400>6

Gly Gly Ile Tyr Leu Tyr Ser Gly Ser Thr Asp Tyr Ala Asp Ser

1 5 10 15

Val Lys Gly

<210>7

<211>17

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-H3

<400>7

Ala Arg Gly Ser Gly Leu Arg Leu Gly Gly Leu Asp Tyr Ala Met

1 5 10 15

Asp Tyr

<210>8

<211>17

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-H3

<400>8

Ala Arg Ser Ser Gly Leu Arg Leu Gly Gly Leu Asp Tyr Ala Met

1 5 10 15

Asp Tyr

<210>9

<211>11

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-L1

<400>9

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

5 10

<210>10

<211>11

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-L1

<400>10

Arg Ala Ser Gln Asp Ser Glu Ile Phe Leu Ala

5 10

<210>11

<211>7

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-L2

<400>11

Ser Ala Ser Phe Leu Tyr Ser

5

<210>12

<211>7

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-L2

<400>12

Ser Ala Ser Asn Leu Glu Ser

5

<210>13

<211>9

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-L3

<400>13

Gln Gln Ser Tyr Thr Thr Pro Pro Thr

5

<210>14

<211>9

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-L3

<400>14

Gln Gln Ser Asn Ala Val Pro Leu Thr

5

<210>15

<211>9

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-L3

<400>15

Gln Glu Ser Thr Thr Thr Pro Pro Thr

5

<210>16

<211>9

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-L3

<400>16

Gln Lys Ser Glu Thr Ile Pro Pro Ser

5

<210>17

<211>9

<212>PRT

<213 > artificial sequence

<220>

<223>HVR-L3

<400>17

Gln Gln Thr Ala Gln Thr Pro Glu Thr

5

<210>18

<211>107

<212>PRT

<213 > artificial sequence

<220>

<223>Light chain variable domain of huMab4D5-8

<400>18

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

1 5 10 15

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

20 25 30

Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser

50 55 60

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

65 70 75

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

80 85 90

His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys

<210>19

<211>87

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>19

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

1 5 10 15

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

20 25 30

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

35 40 45

Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu

50 55 60

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

65 70 75

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

80 85

<210>20

<211>81

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>20

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

1 5 10 15

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Trp Val Arg Gln Ala

20 25 30

Pro Gly Gln Gly Leu Glu Trp Met Arg Val Thr Ile Thr Ala Asp

35 40 45

Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Gly Gln Gly Thr

65 70 75

Leu Val Thr Val Ser Ser

80

<210>21

<211>80

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>21

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

1 5 10 15

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Trp Val Arg Gln Ala

20 25 30

Pro Gly Gln Gly Leu Glu Trp Met Arg Val Thr Ile Thr Ala Asp

35 40 45

Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Trp Gly Gln Gly Thr Leu

65 70 75

Val Thr Val Ser Ser

80

<210>22

<211>79

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>22

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

1 5 10 15

Ala Ser Val Lys Val Ser Cys Lys Ala Ser Trp Val Arg Gln Ala

20 25 30

Pro Gly Gln Gly Leu Glu Trp Met Arg Val Thr Ile Thr Ala Asp

35 40 45

Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr Leu Val

65 70 75

Thr Val Ser Ser

<210>23

<211>87

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

65 70 75

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

80 85

<210>24

<211>81

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>24

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

1 5 10 15

Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Trp Ile Arg Gln Pro

20 25 30

Pro Gly Lys Gly Leu Glu Trp Ile Arg Val Thr Ile Ser Val Asp

35 40 45

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

50 55 60

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

65 70 75

Leu Val Thr Val Ser Ser

80

<210>25

<211>80

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>25

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

1 5 10 15

Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Trp Ile Arg Gln Pro

20 25 30

Pro Gly Lys Gly Leu Glu Trp Ile Arg Val Thr Ile Ser Val Asp

35 40 45

Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Se r Val Thr Ala

50 55 60

Ala Asp Thr Ala Val Tyr Tyr Cys Ala Trp Gly Gln Gly Thr Leu

65 70 75

Val Thr Val Ser Ser

80

<210>26

<211>79

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>26

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

1 5 10 15

Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Trp Ile Arg Gln Pro

20 25 30

Pro Gly Lys Gly Leu Glu Trp Ile Arg Val Thr Ile Ser Val Asp

35 40 45

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

50 55 60

Ala Asp Thr Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr Leu Val

65 70 75

Thr Val Ser Ser

<210>27

<211>87

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75

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

80 85

<210>28

<211>81

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>28

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

1 5 10 15

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

20 25 30

Pro Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Arg Asp

35 40 45

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Gly Gln Gly Thr

65 70 75

Leu Val Thr Val Ser Ser

80

<210>29

<211>80

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>29

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

1 5 10 15

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

20 25 30

Pro Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Arg Asp

35 40 45

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Trp Gly Gln Gly Thr Leu

65 70 75

Val Thr Val Ser Ser

80

<210>30

<211>79

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>30

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

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Trp Val Arg Gln Al a

20 25 30

Pro Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Arg Asp

35 40 45

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr Leu Val

65 70 75

Thr Val Ser Ser

<210>31

<211>87

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>31

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

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75

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

80 85

<210>32

<211>81

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>32

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

1 5 10 15

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

20 25 30

Pro Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Ala Asp

35 40 45

Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gln Gly Thr

65 70 75

Leu Val Thr Val Ser Ser

80

<210>33

<211>80

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>33

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

1 5 10 15

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

20 25 30

Pro Gly Lys Gly Leu Glu Trp Val Arg Phe Thr IleSer Ala Asp

35 40 45

Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Ser Trp Gly Gln Gly Thr Leu

65 70 75

Val Thr Val Ser Ser

80

<210>34

<211>87

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>34

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

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75

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

80 85

<210>35

<211>81

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>35

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

1 5 10 15

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

20 25 30

Pro Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Ala Asp

35 40 45

Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Gly Gln Gly Thr

65 70 75

Leu Val Thr Val Ser Ser

80

<210>36

<211>80

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>36

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

1 5 10 15

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

20 25 30

Pro Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Ala Asp

35 40 45

Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Trp Gly Gln Gly Thr Leu

65 70 75

Val Thr Val Ser Ser

80

<210>37

<211>79

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>37

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

1 5 10 15

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

20 25 30

Pro Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Ala Asp

35 40 45

Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

50 55 60

Glu Asp Thr Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr Leu Val

65 70 75

Thr Val Ser Ser

<210>38

<211>80

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>38

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

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Trp Tyr Gln Gln Lys Pro Gly

20 25 30

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

35 40 45

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

50 55 60

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gly Gln Gly Thr

65 70 75

Lys Val Glu Ile Lys

80

<210>39

<211>80

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>39

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

1 5 10 15

Gly Glu Pro Ala Ser Ile Ser Cys Trp Tyr Leu Gln Lys Pro Gly

20 25 30

Gln Ser Pro Gln Leu Leu Ile Tyr Gly Val Pro Asp Arg Phe Ser

35 40 45

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

50 55 60

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gly Gln Gly Thr

65 70 75

Lys Val Glu Ile Lys

80

<210>40

<211>80

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>40

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

1 5 10 15

Gly Glu Arg Ala Thr Leu Ser Cys Trp Tyr Gln Gln Lys Pro Gly

20 25 30

Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ile Pro Asp Arg Phe Ser

35 40 45

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

50 55 60

Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gly Gln Gly Thr

65 70 75

Lys Val Glu Ile Lys

80

<210>41

<211>80

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>41

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

1 5 10 15

Gly Glu Arg Ala Thr Ile Asn Cys Trp Tyr Gln Gln Lys Pro Gly

20 25 30

Gln Pro Pro Lys Leu Leu Ile Tyr Gly Val Pro Asp Arg Phe Ser

35 40 45

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

50 55 60

Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Phe Gly Gln Gly Thr

65 70 75

Lys Val Glu Ile Lys

80

<210>42

<211>23

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>42

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

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys

20

<210>43

<211>15

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>43

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

1 5 10 15

<210>44

<211>32

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>44

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

1 5 10 15

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

20 25 30

Tyr Cys

<210>45

<211>10

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>45

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

5 10

<210>46

<211>25

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>46

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

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>47

<211>13

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>47

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

5 10

<210>48

<211>30

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>48

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

1 5 10 15

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

20 25 30

<210>49

<211>11

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>49

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

5 10

<210>50

<211>32

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>50

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

1 5 10 15

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

20 25 30

Tyr Cys

<210>51

<211>30

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>51

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

1 5 10 15

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

20 25 30

<210>52

<211>107

<212>PRT

<213 > artificial sequence

<220>

<223 > sequence is synthesized

<400>52

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

1 5 10 15

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

20 25 30

Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser

50 55 60

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

65 70 75

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

80 85 90

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys

<210>53

<211>128

<212>PRT

<213 > artificial sequence

<220>

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

<400>53

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

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Val Gly Gly Ile Tyr Pro Tyr Ser Gly Ser Thr Asp Tyr

50 55 60

Ala Asp Ser Val Lys Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe

65 70 75

Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met

80 85 90

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

95 100 105

Gly Ser Gly Leu Arg Leu Gly Gly Leu Asp Tyr Ala Met Asp Tyr

110 115 120

Trp Gly Gln Gly Thr Leu Val Thr

125

<210>54

<211>108

<212>PRT

<213 > artificial sequence

<220>

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

<400>54

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

1 5 10 15

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

20 25 30

Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser

50 55 60

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

65 70 75

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

80 85 90

Ser Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys Arg

<210>55

<211>128

<212>PRT

<213 > artificial sequence

<220>

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

<400>55

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

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Val Gly Gly Ile Tyr Pro Tyr Ser Gly Ser Thr Asp Tyr

50 55 60

Ala Asp Ser Val Lys Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe

65 70 75

Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met

80 85 90

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

95 100 105

Ser Ser Gly Leu Arg Leu Gly Gly Leu Asp Tyr Ala Met Asp Tyr

110 115 120

Trp Gly Gln Gly Thr Leu Val Thr

125

<210>56

<211>108

<212>PRT

<213 > artificial sequence

<220>

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

<400>56

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

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ser Glu

20 25 30

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

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

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

65 70 75

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

80 85 90

Ser Asn Ala Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys Arg

<210>57

<211>128

<212>PRT

<213 > artificial sequence

<220>

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

<400>57

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

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Ile Ser

20 25 30

Asn Tyr Tyr Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Gly Gly Ile Tyr Leu Tyr Gly Ser Ser Ser Glu Tyr

50 55 60

Ala Asp Ser Val Lys Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe

65 70 75

Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met

80 85 90

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

95 100 105

Gly Ser Gly Leu Arg Leu Gly Gly Leu Asp Tyr Ala Met Asp Tyr

110 115 120

Trp Gly Gln Gly Thr Leu Val Thr

125

<210>58

<211>108

<212>PRT

<213 > artificial sequence

<220>

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

<400>58

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

1 5 10 15

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

20 25 30

Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser

50 55 60

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

65 70 75

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

80 85 90

Ser Thr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys Arg

<210>59

<211>128

<212>PRT

<213 > artificial sequence

<220>

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

<400>59

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

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Val Gly Gly Ile Tyr Leu Tyr Ser Gly Ser Arg Gly Tyr

50 55 60

Ala Asp Ser Val Lys Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe

65 70 75

Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met

80 85 90

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

95 100 105

Gly Ser Gly Leu Arg Leu Gly Gly Leu Asp Tyr Ala Met Asp Tyr

110 115 120

Trp Gly Gln Gly Thr Leu Val Thr

125

<210>60

<211>108

<212>PRT

<213 > artificial sequence

<220>

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

<400>60

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

1 5 10 15

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

20 25 30

Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser

50 55 60

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

65 70 75

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

80 85 90

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

95 100 105

Ile Lys Arg

<210>61

<211>128

<212>PRT

<213 > artificial sequence

<220>

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

<400>61

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

1 5 10 15

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

20 25 30

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

35 40 45

Glu Trp Val Gly Gly Ile Tyr Leu Tyr Ser Gly Ser Thr Asp Tyr

50 55 60

Ala Asp Ser Val Lys Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe

65 70 75

Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met

80 85 90

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

95 100 105

Gly Ser Gly Leu Arg Leu Gly Gly Leu Asp Tyr Ala Met Asp Tyr

110 115 120

Trp Gly Gln Gly Thr Leu Val Thr

125

<210>62

<211>108

<212>PRT

<213 > artificial sequence

<220>

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

<400>62

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

1 5 10 15

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

20 25 30

Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser

50 55 60

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

65 70 75

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

80 85 90

Thr Ala Gln Thr Pro Glu Thr Phe Gly Gln Gly Thr Lys Val Glu

95 100 105

Ile Lys Arg

Claims (34)

1. the anti-EphB4 antibody of a separation, described antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein 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, and HVR-H2 is that SEQ ID NO:3 and HVR-H3 are SEQ ID NO:7.

2. the anti-EphB4 antibody of a separation, described antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein 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:1, and HVR-H2 is that SEQ ID NO:3 and HVR-H3 are SEQ ID NO:8.

3. the anti-EphB4 antibody of a separation, described antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein HVR-L1 is SEQ ID NO:9, HVR-L2 is SEQ ID NO:11, HVR-L3 is SEQ ID NO:15, HVR-H1 is SEQ ID NO:2, and HVR-H2 is that SEQ ID NO:4 and HVR-H3 are SEQ ID NO:7.

4. the anti-EphB4 antibody of a separation, described antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein 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 HVR-H2 is that SEQ ID NO:5 and HVR-H3 are SEQ ID NO:7.

5. the anti-EphB4 antibody of a separation, described antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein HVR-L1 is SEQ ID NO:9, HVR-L2 is SEQ ID NO:11, HVR-L3 is SEQ ID NO:17, HVR-H1 is SEQ ID NO:1, and HVR-H2 is that SEQ ID NO:6 and HVR-H3 are SEQ ID NO:7.

6. the antibody of claim 1-5 any one, wherein Frame sequence is the total Frame sequence of people at least partly.

7. the antibody of claim 1-6 any one, wherein said antibody comprises the total Frame sequence of people κ subgroup.

8. the antibody of claim 1-6 any one, wherein said antibody comprises the total Frame sequence of heavy chain people subgroup III.

9. the antibody of claim 8, the one or more positions of wherein said antibody in the 71st, 73 or 78 of total Frame sequences of heavy chain people subgroup III comprise alternative.

10. the antibody of claim 9, wherein said substituting is one or more in R71A, N73T or N78A.

11. polynucleotide, the antibody of its coding claim 1-10 any one.

12. carrier, the polynucleotide that it comprises claim 11.

13. the carrier of claim 12, wherein said carrier is expression vector.

14. host cell, the carrier that it comprises claim 12 or 13.

15. the host cell of claim 14, wherein said host cell is protokaryon.

16. the host cell of claim 14, wherein said host cell is eucaryon.

17. the host cell of claim 16, wherein said host cell is mammiferous.

18. prepare the method for anti-EphB4 antibody, described method comprises: (a) at the carrier of appropriate host cells claim 13, and (b) reclaim described antibody.

19. prepare the method for anti-EphB4 immune conjugate, described method comprises: (a) at the carrier of appropriate host cells claim 13, and (b) reclaim described antibody.

20. the method for claim 18 or 19, wherein said host cell is protokaryon.

21. the method for claim 18 or 19, wherein said host cell is eucaryon.

22. the purposes of the anti-EphB4 antibody of claim 1-10 any one in the goods for the preparation of detecting EphB4, described detection is included in biological sample and detects the anti-EphB4 antibody complex of EphB4-, and the described anti-EphB4 antibody in wherein said mixture is the anti-EphB4 antibody of claim 1-10 any one.

23. the purposes of claim 22, wherein said anti-EphB4 antibody is detectable label.

24. composition, the anti-EphB4 antibody that it comprises claim 1-10 any one.

25. composition, the polynucleotide that it comprises claim 11.

26. the composition of claim 24 or 25, wherein said composition further comprises carrier.

27. the purposes in the medicine that the anti-EphB4 antibody of claim 1-10 any one occurs at preparation inhibition blood vessel.

28. the purposes of claim 27, wherein said medicine is the medicine with the antiangiogenic agent coupling.

29. the purposes of claim 28, wherein said antiangiogenic agent was used before or after using described anti-EphB4 antibody.

30. the purposes of claim 28, wherein said antiangiogenic agent and described anti-EphB4 antibody are used simultaneously.

31. the purposes of claim 28-30 any one, wherein said antiangiogenic agent is the antagonist of vascular endothelial growth factor (VEGF).

32. the purposes of claim 31, wherein said VEGF antagonist is VEGF antibody.

33. the purposes of claim 32, wherein said VEGF antibody is rhuMAb-VEGF.

34. the purposes of claim 27-33 any one, wherein said medicine also with the chemotherapeutics coupling.

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