CN104650229A - Anti-KDR antibodies and methods of use - Google Patents

Abstract

The present invention provides anti-KDR monoclonal antibodies and related compositions, which may be used in any of a variety of therapeutic methods for the treatment of a variety of cancers, rheumatoid arthritis, diabetic retinopathy and other diseases associated with aberrant VEGF or KDR expression and/or activity.

Description

Anti-KDR antibody and using method

The divisional application that the application is application number is 201210063667.7, the applying date is on 03 12nd, 2012, denomination of invention is the Chinese invention patent application of " Anti-KDR antibody and using method ", this application requires that the applying date is on November 02nd, 2011, application number is the right of priority of the U.S. Provisional Application of 61/554,758.

Sequence table

The application has the sequence table of paper mold and computer-reader form, and it has accurately reacted correlated series as herein described.

Technical field

The present invention relates generally to anti-VEGF acceptor 2 (VEGFR2; Be also called containing kinase insert domain receptor or KDR) antibody, composition and using method thereof.These antibody can be used for such as treating and suppress in the method for various illness, described illness comprise age-relevant macular degeneration (AMD), diabetes and ischemic retinopathy, rheumatoid arthritis, psoriatic and various tumor disease (comprising renal cell carcinoma, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, ovarian cancer, melanoma and recurrent glioblastoma multiforme, leukemia and solid tumor).

Background technology

VEGFR2/KDR is primary vasculogenesis acceptor, and in conjunction with VEGF hypotype A, C, D and E, and splitting effect, angiogenic action and perviousness-enhancing (perviousness-enhancing) for the short silk of endothelial cell differentiation and VEGF, to act on VEGFR2/KDR be important.Anti-KDR antibody can prevent all known VEGF hypotypes to be combined with VEGFR2/KDR and commencing signal transmission.In addition, because the molecule of the more VEGF of tumors secrete, and the number of acceptor still relative constancy, even if under the existence of very high-caliber VEGF hypotype, receptor targeted increase suppresses the possibility of signal completely.

Vasculogenesis

The vasculogenesis formation of neovascularity (from the existing vascular system) is the strict event controlled, and normal physiological (such as fetal development, follicular development, wound restore and the pathologic conditions of such as tumor growth and progress (1,2)) is played an important role.

The formation of neovascularity is depended in the growth of primary tumor and metabolism.When not having neovascularization, tumour is understood necrosis or apoptosis and/or can not be grown more than 2 – 3mm ³size (3).Tumor-blood-vessel growth comprises multiple process, the tissue infiltration from preexist blood vessel comprising vascular remodeling, propagation, migration and triggered by specific angiogenesis factor, described angiogenesis factor is produced by tumour cell and surrounding substrate (1 – 4).

VEGF and vegf receptor

Identify that several somatomedin is the conditioning agent (5) of possible angiogenic growth.In these factors, vascular endothelial growth factor (VEGF) and acceptor thereof have shown and have played an important role for tumor-blood-vessel growth (6 – 9).

VEGF be have vasculogenesis favourable by force, 34 – 42kDa heparin of the equal dimerization of mitogenetic and Vascular permeability-enhanced activity (10,11)-in conjunction with glycoprotein.VEGF regulates and generates in the fetal development of adult life (12,13) and the process medium vessels of vasculogenesis.VEGF family member comprises VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E.Combined and mediates reactive by vegf receptor (VEGFR) VEGF.There are 3 kinds of VEGFR, comprise VEGFR1 (Flt-1), VEGFR2 (Flk-1/KDR) and VEGFR3 (14-16).

The physiological significance of VEGF and vegf receptor in vascularization has clearly been confirmed in gene knockout experiment (17-18).VEGFR1 Tyrosylprotein kinase has for the conservative motif required for kinase activity.But, be low (37,38) to the level of the phosphorylation of the VEGFR1 of VEGF-A response.The non-better definition of function of VEGFR-1, it can act on virtual/Decoy receptors (dummy/decoy receptor) with chelating VEGF from VEGFR-2 combination and adjustment VEGFR-2 signal.Show the lymphatic vessel generation that VEGFR-3 can mediate response VEGF-C and VEGF-D.VEGFR2/KDR is primary vasculogenesis acceptor, and in conjunction with VEGF hypotype A, C, D and E, and for endothelial cell differentiation and mitotic division, it to occur be important.

The structure of KDR and biology

VEGFR is receptor tyrosine kinase, and belongs to as the identical family of PDGF with the acceptor of fibroblast growth factor (FGF).VEGFR2/KDR is at ectodomain, membrane spaning domain and is divided into by kinases insertion the 200kDa glycoprotein be made up of the ring of individual immunoglobulin-like in two intracellular tyrosine kinase structural domains.Second and the 3rd the ring of immunoglobulin-like be height-avidity ligand-binding domain for VEGF, but first and the 4th the ring of immunoglobulin-like regulate ligand binding and Receptor dimerization respectively.Compared with the Kd of the 25pM for VEGFR1, VEGF uses the Kd of 75-250pM in conjunction with KDR.

KDR mainly expresses the cell surface at vascular endothelial cell.Also on the cell surface of hematopoietic cell, vascular smooth muscle cell (VSMC) and some malignant cells, find KDR.

KDR is principal recipient in the vasculogenesis and hemopoietic of development, and be VEGF mitogenetic, vasculogenesis with the main amboceptor of perviousness-enhancement.VEGFR2 ^–/–knock-out mice is presented at the embryonic death rate (41) that E8.5 – has defective blood-island formation and vasculogenesis for 9.5 times.On physiology, the combination of VEGF and KDR causes vascular remodeling, propagation, migration, invasion and attack and survival.Be combined once with VEGF, KDR Receptor dimerization, this transduction causing the activation of kinase domain and KDR receptor signal to transmit.Similar other receptor tyrosine kinases many, cause the main Intracellular signals pipeline of vasculogenesis to comprise MAPK and PI3 kinase activator.

The KDR of Antybody therapy is used for as molecule target

VEGFR2/VEGF axle is main path in tumor-blood-vessel growth.Multiple research shows: the process LAN of VEGF and KDR is strongly relevant to Infiltration and metastasis in human malignant lesion (6).Vegf receptor to occur in many human solid tumors (comprising bladder (21), breast (22,23), rectum (24,25), stomach and intestine (26), neurospongioma (12,27), kidney (28), melanoma (29) and neuroblastoma (30)) medium vessels and generate relevant.

In tumor-blood-vessel growth, the vital role of KDR directly confirms under study for action, and wherein the expression of dominant-negative KDR acceptor causes the growth-inhibiting of endothelial cell mitogen generation and the underlying nerve glioma tumor reduced in athymic mouse (31).The KDR inhibitor of small molecule tyrosine kinase inhibitors (TKI) and KDR-specific antibody is comprised to confirm this effect with soluble VEGF-receptor and use during other researchs use.

Except it is for except the effect of tumor-blood-vessel growth, some tumour cells of such as leukemia cell also find KDR, and by stimulating the autocrine loop KDR of leukemia growth (42,43) directly to occur by mediate tumor.

The suppression of KDR signal transmission can reduce vasculogenesis and postpone tumor growth (35,36).It is little-molecule tyrosine kinase inhibitor (TKI) that the great majority of target KDR are treated at present.TKI disturbs the combination of ATP or other substrates and Tyrosylprotein kinase, and destroys kinase catalytic activity.All TKI (similar Sutent) developed so far are reversibly combined with the ATP combining site of KDR kinase domain.

In various types of tumour (12,19), VEGF expresses at a high level, and the capillary vessel of recently growing is collected at VEGF-generation tumour cell (12) around.Strong upregulation vegf expression under hypoxic condition (those conditions relevant to the tumour (20) grown fast).By in the antibody of such as Avastin (33,34) with VEGF being the treatment of clinical approval, thus other vasculogenesis diseases of suppression cancer or such as AMD.But, the opposing that VEGF is blocked even is had been found that when associating chemotherapy gives.This opposing can be relevant with the expression of the increase of other angiogenic factors to the vascular system reinvented.With regard to itself, this area reservation demand is for the composition of the improvement for suppressing cancer and the other diseases relevant to vasculogenesis and method.

Although the vasculogenesis that TKI and Anti-KDR antibody can suppress KDR-to mediate, the advantage of antibody approach is more than TKI.Relative to TKI, Anti-KDR antibody is more special KDR target agent (that is, it can not suppress other vegf receptor).Because its high specific, Anti-KDR antibody can limit and/or avoid the effect of missing the target (off-target effect) that caused by lower specificity T KI (44) and toxicity.

In various types of tumour (12,19), VEGF expresses at a high level, and the capillary vessel of recently growing is collected at VEGF-generation tumour cell (12) around.Strong upregulation vegf expression under hypoxic condition (those conditions relevant to the tumour (20) grown fast).By in the antibody of such as Avastin (33,34) with VEGF being the treatment of clinical approval, thus other vasculogenesis diseases of suppression cancer or such as AMD.But, the resistance that VEGF is blocked even is had been found that when associating chemotherapy gives.This resistance can be relevant with the expression of the increase of other angiogenic factors to the vascular system reinvented.

Relative to the Avastin of only one of binding partner (VEGF-A), the combination of all known VEGF and the VEGFR2/KDR of expection Anti-KDR antibody prevention.Compare and only blocking VEGF-A, this can have the more far-reaching restraining effect for tumor-blood-vessel growth.It is possible that use the treatment of Anti-KDR antibody can be effective when Avastin resistance.Target KDR summarizes in Table 1 more than the potential advantages of VEGF.

The Antybody therapy of table 1. target KDR is more than the advantage of VEGF

Therefore, the antibody of target KDR and blocking-up KDR signal transmission can have higher specificity and target suppression more completely, therefore can have and apply widely in solid tumor and liquid tumor, and have the potential overcoming Avastin resistance.

Anti-KDR therapeutic antibodies in exploitation Ramucirumab (IMC-1121B)

The Ramucirumab developed by ImClone Systems/Eli Lilly is complete people (fully human) IgG1mAb in conjunction with people KDR (KD ≈ 50pM), and Ramucirumab blocking VEGF combines, thus inhibiting angiogenesis.Because Ramucirumab can not with mouse KDR cross reaction, substitute anti-mouse KDR antibody (DC-101) so produce, and anti-mouse KDR antibody (DC-101) will be substituted for POC preclinical study.

In the I clinical trial phase of patient with terminal cancer, Ramucirumab better tolerates all dosage regimens.The dose-limiting toxicity that mechanism is relevant is that hypertension and dvt are formed.The II phase testing data of nearest report (39) as monotherapy after KDR tyrosine kinase inhibitor in the patient with metastatic renal cell cancer.To there is PD or 8mg/kg Ramucirumab be used to patient's biweekly intravenous injection (IV) of Xarelto (sorafenib), Sutent or both intolerances.Within every six weeks, carry out tumor evaluation.Registration total 40 patients, and treat 39.19 patients's (49%) have the stable disease continuing to be greater than 5 months; Preliminary meta progresson free survival is 6 months.The more II phase of combine dacarbazine in melanoma, combine mitoxantrone/prednisone in the patient with prostate cancer, combining Carboplatin/taxol and combine oxaliplatin/folinic acid/5 FU 5 fluorouracil in the patient with NSCLC in the patient with colorectal carcinoma tests well afoot.

Study in assessment Ramucirumab 3 III phases in the patient with cancer of the stomach, gastroesophageal junction gland cancer and hepatocellular carcinoma recently.In transitivity adenocarcinoma of stomach, in the second metastatic colorectal cancer being and in the second nonsmall-cell lung cancer being, use or do not use the 3 phases research well afoot that three of the Ramucirumab of taxol other.

33C3

The 33C3 developed by AstraZeneca uses XenoMouse ^tMcomplete people's Anti-KDR antibody that technology produces.33C3, in conjunction with the Ig structural domain 4-7 of KDR, does not therefore have impact to the combination of VEGF-A and KDR.It is not competed at the interactional antibody of ligand binding site.33C3 has the high affinity (KD<1nM) for KDR, and suppresses the phosphorylation that the VEGF-A of KDR induces.In vitro, the potent suppression of 33C3 pipe range and tapping point in 2D vasculogenesis is analyzed number and in 3D analyzes endothelium pipe formed.In vivo, 33C3 is the inhibitor generating very effective vasculogenesis in analysis and human skin chimeric model (40) at people's interior cutaneous vessel.33C3 is the early clinic last stage of exploitation now.Owing to lacking the cross reactivity to mouse KDR, not yet test 33C3 in tumor model in vivo.

TTAC-0001

The TTAC-0001 (the complete people's Anti-KDR antibody produced by phage display) developed by PharmAbcine is preclinical phase now.Effect of the potent anti-vasculogenesis of the various cancer mouse model (45) of antibody display antagonism.

Summary of the invention

One side of the present disclosure provides antibody in conjunction with the separation of KDR or its antigen-binding fragment, comprises: the variable region of heavy chain in the VHCDR2 district that (i) comprises VHCDR1 district that SEQ ID NO:3 or 11 represents, SEQ ID NO:4 or 12 represents and the VHCDR3 district that SEQ ID NO:5 represents; And the variable region of light chain in the VLCDR2 district that (ii) comprises VLCDR1 district that SEQ ID NO:6 represents, SEQ ID NO:7 represents and the VLCDR3 district that SEQ ID NO:8 represents; Or the variant of described antibody or its antigen-binding fragment, comprise heavy chain and variable region of light chain, except replacing up to 8 seed amino acids in described CDR district, described heavy chain is identical with variable region of light chain with the described heavy chain of (ii) with (i) with variable region of light chain.In one embodiment, the antibody be separated as disclosed herein or its antigen-binding fragment comprise variable region of heavy chain, and described variable region of heavy chain comprises the aminoacid sequence that SEQ ID NO:1 represents.In another embodiment, the antibody be separated as disclosed herein, or its antigen-binding fragment comprises variable region of light chain, and described variable region of light chain comprises the aminoacid sequence that SEQ ID NO:2 represents.In one embodiment, Anti-KDR antibody disclosed herein in conjunction with people KDR, and with mouse or monkey KDR cross reaction.

Another aspect of the present disclosure provides antibody in conjunction with the separation of KDR or its antigen-binding fragment, and the antibody of described separation or its antigen-binding fragment comprise: (i) comprises the variable region of heavy chain of VHCDR1, VHCDR2 and VHCDR3 of antibody as shown in figure 11; And (ii) comprises the variable region of light chain of the correspondence of VLCDR1, VLCDR2 and VLCDR3 of antibody as shown in figure 11; Or the variant of described antibody or its antigen-binding fragment comprise heavy chain and variable region of light chain, except replacing up to 8 seed amino acids in described CDR district, described heavy chain is identical with variable region of light chain with the described heavy chain of (ii) with (i) with variable region of light chain.In one embodiment, the antibody of separation or its antigen-binding fragment comprise variable region of heavy chain, and described variable region of heavy chain comprises any one of the aminoacid sequence that SEQ ID NO:17-42 represents.In another embodiment, the antibody of separation or its antigen-binding fragment comprise variable region of light chain, and described variable region of light chain comprises any one of the aminoacid sequence that SEQ ID NO:43-68 represents.

Another aspect of the present disclosure provides antibody in conjunction with the separation of KDR or its antigen-binding fragment, antibody or its antigen-binding fragment of the described separation in conjunction with KDR comprise variable region of heavy chain, and described variable region of heavy chain comprises the aminoacid sequence that SEQID NO:1 represents.In one embodiment, the antibody be separated comprises variable region of heavy chain, described variable region of heavy chain comprises the aminoacid sequence that SEQ ID NO:1 represents, and the antibody be separated comprises variable region of light chain, described variable region of light chain comprises the aminoacid sequence represented with SEQ ID NO:2 and has at least 90% conforming aminoacid sequence.In one embodiment, this antibody comprises variable region of light chain, and described variable region of light chain comprises the aminoacid sequence that SEQ IDNO:2 represents.

Another aspect of the present disclosure provides antibody in conjunction with the separation of KDR or its antigen-binding fragment, antibody or its antigen-binding fragment of the described separation in conjunction with KDR comprise variable region of light chain, and described variable region of light chain comprises the aminoacid sequence that SEQID NO:2 represents.In one embodiment, variable region of light chain is comprised in conjunction with the antibody of the separation of KDR or its antigen-binding fragment, described variable region of light chain comprises the aminoacid sequence that SEQ ID NO:2 represents, and comprising variable region of heavy chain in conjunction with the antibody of the separation of KDR or its antigen-binding fragment, described variable region of heavy chain comprises the aminoacid sequence represented with SEQ ID NO:1 and has at least 90% conforming aminoacid sequence.

In some embodiment of the present disclosure, Anti-KDR antibody as herein described is humanized.Schematic VH and VL district as described herein in conjunction with the humanized antibody of KDR represents in SEQ ID NO:9 and 10.

In another embodiment of Anti-KDR antibody as herein described, antibody can comprise single-chain antibody, ScFv, the univalent antibody of shortage hinge area, miniantibody, Fab, Fab ' fragment, F (ab ') ₂fragment or whole antibody.

In certain embodiments, the antibody of separation as herein described comprises human IgG constant domain.In this, IgG constant domain can comprise IgG1CH1 structural domain, such as, as IgG1CH1 domain amino acid sequence that SEQ ID NO:16 represents.In certain embodiments, antibody as herein described comprises IgG constant domain, and described IgG constant domain comprises IgG1Fc district.

Another aspect of the present disclosure provides competes and the antibody be separated of the combination of people KDR or its antigen-binding fragment with Anti-KDR antibody as described herein.

Another aspect of the present disclosure provides with high affinity (such as 5.3x10 ^-11the avidity of the KD of M or lower) in conjunction with the antibody of the separation of KDR or its antigen-binding fragment.In this, the avidity of Anti-KDR antibody as disclosed herein can be about 3.5,4,4.5,5, or 5.5X 10 ^-11m.In certain embodiments, the antibody of separation of the present disclosure and mouse KDR or non-human primates KDR cross reaction.

Another aspect of the present disclosure provides antibody or its antigen-binding fragment of separation, the combination of the antibody be wherein separated or its antigen-binding fragment blocking VEGF and KDR; Suppress KDR signal transmission; Inhibition of endothelial cell proliferation; Tumor suppression vasculogenesis; Inhibition tumor cell grows; Or any one of aforementioned function or multinomial combination.In one embodiment, the antibody of separation or the combination of its antigen-binding fragment blocking VEGF and KDR; Suppress KDR signal transmission; Inhibition of endothelial cell proliferation; Tumor suppression vasculogenesis; Grow with inhibition tumor cell.

In one aspect of the invention, the antibody of separation or the direct Tumor suppression growth of its antigen-binding fragment.

The disclosure also provides the polynucleotide of the separation of Anti-KDR antibody as described herein of encoding and comprises the expression vector of this polynucleotide be separated and comprise the host cell be separated of this carrier.

The composition of the antibody that another aspect of the present disclosure providing package is separated containing physiologically acceptable vehicle and pharmaceutical effective amount as described herein or its antigen-binding fragment.

Another aspect of the present disclosure is provided for treating the method had to the patient of the cancer that VEGF or KDR of distortion expresses or activity is relevant, comprise the composition using antibody that comprise physiologically acceptable vehicle and pharmaceutical effective amount as described herein be separated or its antigen-binding fragment to patient, thus treatment and the cancer that VEGF or KDR of distortion expresses or activity is relevant.In this, antibody as herein described can be used for Therapeutic cancer, includes but not limited to: angiosarcoma, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma, recurrent glioblastoma multiforme and leukemia.

Another aspect of the present invention is provided for treating the method for the patient suffering from inflammatory diseases, comprises the composition of the significant quantity using any one or more pharmaceutically comprising antibody disclosed herein to patient, thus treatment suffers from the patient of inflammatory diseases.

Another aspect of the present disclosure is provided for treating the method for the patient with rheumatoid arthritis, comprise the composition using antibody that comprise physiologically acceptable vehicle and pharmaceutical effective amount as described herein be separated or its antigen-binding fragment to patient, thus treatment has the patient of rheumatoid arthritis.

Another aspect of the present disclosure is provided for treating the method with psoriatic patient, comprise the composition using antibody that comprise physiologically acceptable vehicle and pharmaceutical effective amount as described herein be separated or its antigen-binding fragment to patient, thus treatment has psoriatic patient.

Further aspect of the present disclosure is provided for treating the method for the patient of the disease suffering from angiogenesis-mediated, comprise the composition of the significant quantity using any one or more pharmaceutically comprising antibody disclosed herein to patient, thus treatment suffers from the patient of the disease of angiogenesis-mediated.In this, in certain embodiments, patient suffers from age relevant macular degeneration.

Another aspect of the present disclosure provides the antibody of the separation in conjunction with KDR described herein or its antigen-binding fragment or composition as herein described in the purposes of the medicine of the cancer for the preparation for the treatment of patient.

Another aspect of the present disclosure provides the antibody of the separation in conjunction with KDR described herein or its antigen-binding fragment or composition as herein described in the purposes to the medicine of the cancer that VEGF or KDR of distortion expresses or activity is relevant that has for the preparation for the treatment of patient.Preferably described cancer is selected from: angiosarcoma, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma, recurrent glioblastoma multiforme and leukemia.

Another aspect of the present disclosure provides the antibody of the separation in conjunction with KDR described herein or its antigen-binding fragment or composition as herein described in the purposes of the medicine of the inflammatory diseases for the preparation for the treatment of patient.

Another aspect of the present disclosure provides the antibody of the separation in conjunction with KDR described herein or its antigen-binding fragment or composition as herein described to treat the purposes of the medicine of the rheumatoid arthritis of patient in preparation.

Another aspect of the present disclosure provides the antibody of the separation in conjunction with KDR described herein or its antigen-binding fragment or composition as herein described to treat the purposes of the psoriatic medicine of patient in preparation.

Another aspect of the present disclosure provides the antibody of the separation in conjunction with KDR described herein or its antigen-binding fragment or composition as herein described purposes in the medicine of disease preparing the vasculogenesis-mediation for the treatment of patient.Preferably, wherein said patient suffer from age-relevant macular degeneration.

Accompanying drawing is sketched

Fig. 1: the graphic representation showing the selection result of the potent antibody of major part for the combination blocking KDR and VEGF.

Fig. 2: to the screening of the antibody of the phosphorylation of suppression KDR.(A) in the suppression of the middle KDR phosphorylation of enzyme linked immunosorbent assay (ELISA assay); (B) suppression of KDR phosphorylation in Western blot.Ab9530 is the contrast KDR neutralizing antibody from Abcam (Cambridge, MA, USA).

Fig. 3: APX004 selective binding people and mouse KDR, but not in conjunction with other people VEGFR family protein or VEGF.

Fig. 4: APX004 with the combination of the IC50 of 2.6nM potent blocking-up KDR and VEGF.

Fig. 5: APX004 suppresses the KDR phosphorylation of being induced by VEGF.

Fig. 6: APX004 suppresses HUVEC propagation with dose-dependent manner,

Fig. 7: to the termination (41 days) of In vivo study, compared with Avastin (69% suppresses), APX004 show more potent Anti-tumor activity (77% suppresses).

Fig. 8: APX004 illustrates that remarkable (p<0.01) Anti-tumor is active in H460 tumor model under 2.5mg/kg.

Fig. 9: APX004 significantly suppresses A375 tumor growth under 3mg/kg.Volume=(wide) ²x long/2.Symbol and rod represent mean value+standard deviation.

Figure 10: APX004 significantly suppresses HT29 tumor growth under 5mg/kg and 10mg/kg.Gross tumor volume is calculated: volume=(wide) according to equation ²x long/2.Symbol and rod represent mean value+standard deviation.

Figure 11 is the comparison in VH and the VL district of the Anti-KDR antibody that embodiment 1 is identified.Figure 11 A1 shows the amino acid/11-100 in VH district.Figure 11 A2 shows the remaining amino acid of VH.Figure 11 B1 shows the comparison of the amino acid/11-70 in VL district.Figure 11 B2 shows the remaining amino acid whose comparison in VL district.As being summarized in following chapters and sections " BRIEF DESCRIPTION OF THE SEQUENCES " and table 3, provide the SEQ ID No being presented at VH district in comparison of SEQ ID NO:1 and 17-42; The SEQ ID No being presented at VL district in comparison of SEQ ID NO:2 and 43-68 is provided.CDR is underscore part.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the aminoacid sequence in the VH district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:2 is the aminoacid sequence in the VL district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:3 is the aminoacid sequence in the VHCDR1 district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:4 is the aminoacid sequence in the VHCDR2 district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:5 is the aminoacid sequence in the VHCDR3 district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:6 is the aminoacid sequence in the VLCDR1 district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:7 is the aminoacid sequence in the VLCDR2 district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:8 is the aminoacid sequence in the VLCDR3 district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:9 is the aminoacid sequence of the humanized sequence in the VH district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:10 is the aminoacid sequence of the humanized sequence in the VL district of clone 36 rabbit Anti-KDR antibody.

SEQ ID NO:11 is the aminoacid sequence that humanization clones the VHCDR1 district of 36 Anti-KDR antibodies (APX004).

SEQ ID NO:12 is the aminoacid sequence that humanization clones the VHCDR2 district of 36 Anti-KDR antibodies (APX004).

SEQ ID NO:13 is the polynucleotide sequence in encoding human CK district.

SEQ ID NO:14 is the aminoacid sequence of people CK.

SEQ ID NO:15 is the polynucleotide sequence in encoding human IgG1CH1 district.

SEQ ID NO:16 is the aminoacid sequence in human IgG1 CH1 district.

SEQ ID NO:17-42 is the aminoacid sequence of the VH as summarized rabbit Anti-KDR antibody clone in table 3.

SEQ ID NO:43-68 is the aminoacid sequence of the VL as summarized rabbit Anti-KDR antibody clone in table 3.

SEQ ID NO:69-95 is if display is in fig. 11 for the aminoacid sequence of the VHCDR1 of rabbit Anti-KDR antibody clone.

SEQ ID NO:96-122 is if display is in fig. 11 for the aminoacid sequence of the VHCDR2 of rabbit Anti-KDR antibody clone.

SEQ ID NO:123-149 is if display is in fig. 11 for the aminoacid sequence of the VHCDR3 of rabbit Anti-KDR antibody clone.

SEQ ID NO:150-176 is if display is in fig. 11 for the aminoacid sequence of the VLCDR1 of rabbit Anti-KDR antibody clone.

SEQ ID NO:177-203 is if display is in fig. 11 for the aminoacid sequence of the VLCDR2 of rabbit Anti-KDR antibody clone.

SEQ ID NO:204-230 is if display is in fig. 11 for the aminoacid sequence of the VLCDR3 of rabbit Anti-KDR antibody clone.

Detailed Description Of The Invention

Anti-KDR antibody as herein described has high binding affinity (53pM) and selectivity for KDR.The interaction of antibody blocking KDR and VEGF as herein described, and the KDR phosphorylation of blocking VEGF-induction and endothelial cell proliferation.In certain embodiments, antibody as herein described and mouse KDR cross reaction, make do not need under alternative antibody (surrogate antibody) can test antibody in animal model in vivo.Antibody as herein described confirms the potent activities of Tumor suppression growth in human tumor xenograft model.Because intersect-and reactive, according to genotoxic potential in PK, PD, biomarker development, clinical front mouse model even before it test in non-human primates and people, can evaluation and characterization Anti-KDR antibody as herein described completely in body.

The disclosure relates to antibody and the antigen-binding fragment thereof of specific binding KDR, and what especially have special epitope specificity and functional property is antibody.One embodiment of the invention contain Humanized antibody specific and fragment thereof, and described Humanized antibody specific and fragment thereof can in conjunction with KDR; Block the combination of KDR and VEGF; With the downstream cellular signal transmission and the biological action that suppress VEGF induction.In more particular embodiment of the present invention, antibody as herein described is with about 5.3X 10 ^-11the avidity of M combines in conjunction with KDR and blocking-up KDR and VEGF specifically.

In further embodiment, the direct Tumor suppression growth of antibody as herein described.In this, some tumour expressing K DR, and the VEGF-KDR approach of autocrine loop can be used as with growth.Therefore, in certain embodiments, can be comprised by antibody-mediated Anti-tumor effect as herein described: (i) anti-vasculogenesis and/or (ii) directly Tumor suppression growth.

Embodiment of the present invention relate to Anti-KDR antibody or its antigen-binding fragment purposes for the diagnosis of the disease relevant to VEGF or its expression distorted and illness, assessment and treatment.Express to VEGF or KDR and/or active relevant illness treatment or in preventing use inscribe and state antibody, described illness includes but not limited to: rheumatoid arthritis, diabetes, and ischemic retinopathy, age-relevant macular degeneration, psoriatic, with the renal glomerulus relevant to proteinuria is loose, (angiosarcoma is comprised with various tumor disease, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma, with recurrent glioblastoma multiforme, leukemia, and solid tumor), and other diseases.

Contrary unless otherwise indicated, operation of the present invention adopts the ordinary method of virusology, immunology, microbiology, molecular biology and recombinant DNA technology in art technology, in order to the many ordinary methods of following description are described.Fully explain these technology in the literature.See, such as, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3 ^rded., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning:A Laboratory Manual (third edition, 2001); Maniatiset al.Molecular Cloning:A Laboratory Manual (1982); DNA Cloning:A PracticalApproach, vol.I & II (D.Glover, ed.); Oligonucleotide Synthesis (N.Gait, ed., 1984); Nucleic Acid Hybridization (B.Hames & S.Higgins, eds., 1985); Transcription andTranslation (B.Hames & S.Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar reference.

Except being clearly otherwise noted in non-textual, as in this specification sheets and appended claim use, singulative " a ", " an " and " the " comprise plural form.

Run through this specification sheets, except non-textual separately has needs, word " should be comprised (comprise) " or such as " comprise (comprises) " or the version of " making to comprise (comprising) " is interpreted as the inclusion of the group of described element or entirety or element or entirety, but not get rid of any other element or the group of entirety or element or entirety.

Unless expressly stated otherwise, in this specification the change that each embodiment is in addition necessary is applied to every other embodiment.

Standard technique can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transform (such as, electroporation, lipofection).According to the specification sheets of manufacturer or complete as routine in the art or as described hereinly carry out enzymatic reaction and purification technique.According to ordinary method well-known in the art and as various usually to run through that this specification sheets quotes and discuss usually can carry out these and relevant technology and operation more specifically described in reference.Unless provided concrete definition, the relevant nomenclature that uses and molecular biology as herein described, analytical chemistry, synthetic organic chemistry and medical and pharmaceutical chemical laboratory procedure and technology be all this area well-known and conventional use those.Standard technique can be used for recombinant technology; Molecular biosciences; Microorganism; Chemosynthesis; Chemical analysis; Medicine preparation, formula and send; With the treatment of patient.

Embodiment of the present invention relate to the antibody in conjunction with KDR.Especially, antibody as herein described with unexpectedly high avidity specifically in conjunction with KDR; The combination of blocking VEGF and KDR; Blocking VEGF is active; With there is the treatment function being used for the treatment of the disease relevant to the expression of distortion or the activity of VEGF.Antibody as herein described also has the favourable character of the ability of the biological action (such as, the phosphorylation of KDR, vasculogenesis, endothelial cell proliferation and other VEGF/KDR-well known by persons skilled in the art mediate effect) such as suppressing various VEGF/KDR-to mediate.Antibody as herein described also can have the effect for KDR receptor internalisation.

SEQ ID NO:1-12 and 17-230 represents the sequence of schematic antibody or antigen-binding fragment or its complementary determining region (CDR).

As well known in the art, antibody be arranged in the variable region epi-position recognition site of immunoglobulin molecules by least one can the immunoglobulin molecules of specific binding target (such as carbohydrate, polynucleotide, lipid, polypeptide etc.).As used herein, complete polyclone or monoclonal antibody not only contained in this term, also contains its fragment (such as dAb, Fab, Fab', F (ab') ₂, Fv), strand (ScFv), its synthesis variant, naturally occurring variant, comprise the fusion rotein of antibody moiety that any other with required specificity, humanized antibody, chimeric antibody and immunoglobulin molecules (comprising required specific antigen-combining site or fragment (epi-position recognition site)) modifies the antigen-binding fragment of configuration." disome " that built by gene fusion, multivalence or polyspecific fragment (WO94/13804; P.Holligeret al., Proc.Natl.Acad.Sci.USA 906444-6448,1993) be also the special shape of the antibody contained herein.The miniantibody comprising the scFv be connected with CH3 structural domain is also included within herein (S.Hu et al., CancerRes., 56,3055-3061,1996).Such as, see Ward, E.S.et al., Nature 341,544-546 (1989); Bird et al., Science, 242,423-426,1988; Huston et al., PNAS USA, 85,5879-5883,1988); PCT/US92/09965; WO94/13804; P.Holliger et al., Proc.Natl.Acad.Sci.USA 906444-6448,1993; Y.Reiter et al., Nature Biotech, 14,1239-1245,1996; S.Hu et al., Cancer Res., 56,3055-3061,1996.

As used herein term " antigen-binding fragment " refers to the polypeptide fragment of at least one CDR of heavy chain immunoglobulin containing combining target antigen (especially for KDR) and/or light chain.In this, the antigen-binding fragment of antibody described herein can comprise 1,2,3,4,5 or all 6 CDR of VH and the VL sequence shown in this article from the antibody in conjunction with KDR.Antigen-the binding fragment of KDR-specific antibody as herein described can in conjunction with KDR.In certain embodiments, antigen-binding fragment or comprise the antibody prevention of antigen-binding fragment or suppress the combination of VEGF and KDR and follow-up signal transmission event.In certain embodiments, antigen-binding fragment combines specifically and/or suppresses or the biological activity of mediator KDR.

Term " antigen " refers to the part of molecule or the molecule that can be combined by the selective binding agent of such as antibody, and described molecule or molecular moiety can also be used for can the antibody of epi-position of conjugated antigen to produce in animal.Antigen can have one or more epi-position.

Term " epi-position " comprises can any determinant of specific binding immunoglobulin (Ig) or φt cell receptor, preferred polypeptide determinant.Epi-position is the region of the antigen by antibodies.In certain embodiments, Epitopic determinants comprises the chemically reactive surface group of molecule, such as amino acid, sugared side chain, phosphoryl or alkylsulfonyl, and in certain embodiments, can have specific three dimensional constitutional features and/or specific charge characteristics.In certain embodiments.When antibody during preferential its target antigen of identification, then can say its specific binding antigen in albumen and/or macromolecular compounding mixture.When equilibrium dissociation constant≤10 ^-7or 10 ^-8during M, then can say antibodies specific conjugated antigen.In some embodiments, equilibrium dissociation constant can be≤10 ^-9m or≤10 ^-10m.

In certain embodiments, antibody as described herein and antigen-binding fragment thereof comprise heavy chain and light chain CDR collection (set), between framework region (FR) collection laying respectively at heavy chain and light chain, FR collection provides the supporting function of CDR and defines CDR spatial relation each other.Term as used herein " CDR collection " refers to the hypervariable region in heavy chain or light chain V district.From the N-end of heavy chain or light chain, these sections are called after " CDR1 ", " CDR2 " respectively, and " CDR3 ".Therefore, antigen-combining site comprises six CDR, comprises the CDR district in heavy chain and light chain V district separately.Polypeptide containing single CDR (such as CDRl, CDR2 or CDR3) in this article refers to " molecular recognition unit ".The crystallographic analysis of a large amount of antigen-antibody complexes is verified: the amino-acid residue of CDR defines with the antigen of combination and contacts widely, and wherein contacting maximum is the CDR3 of heavy chain.Therefore, molecular recognition unit is the specificity of primary responsibility antigen-combining site.

As used herein term " FR collection " refers to that the CDR in heavy chain or light chain V district concentrates four aminoacid sequences forming CDR both sides.Some FR residue can with the antigen contact combined; But FR primary responsibility V district is folded to form antigen-combining site, particularly directly adjoin the FR residue of CDR.In FR, some amino-acid residue and some constitutional features are high conservatives.In this, all V region sequences contain the inside two sulphur ring of about 90 amino-acid residues.When V district is folded into combination-position, CDR demonstrates the outstanding cyclic group sequence forming antigen-binding surface.It has been generally acknowledged that what affect that CDR ring is folded into the shape of some " specification " structure is the conserved structure district of FR instead of accurate cdr amino acid sequence.And some FR residue known take part in the non covalent contact between structural domain, and described non covalent contact stabilizes the interaction between heavy chain of antibody and light chain.

With reference to Kabat, E.A.et al., Sequences of Proteins of Immunological Interest. the 4th edition .US Department of Health and Human Services.1987 and renewal (now can obtain (immuno.bme.nwu.edu) on the internet) thereof, can measure structure and the position in immunoglobulin variable domain territory.

" monoclonal antibody " refers to homogeneous antibody population, and wherein monoclonal antibody is made up of the amino acid (naturally occurring or non-natural exists) of the selective binding participating in antigen.Monoclonal antibody is high special, is directed to anti-single epi-position.Intact monoclonal antibodies and complete-long monoclonal antibody not only contained in term " monoclonal antibody ", also contains its fragment (such as Fab, Fab', F (ab') ₂, Fv), strand (ScFv), its variant, fusion rotein containing antigen-binding portion, Humanized monoclonal antibodies, chimeric monoclonal antibodies and comprise configuration of required specific antigen-binding fragment (epi-position recognition site) and any other modification in conjunction with the immunoglobulin molecules of the ability of epi-position.This invention is not used in the restriction source of antibody or it is by the mode (such as selected by hybridoma, phage, recombinant expressed, transgenic animal etc.) produced.This term is whole immunoglobulin (Ig) and above-mentioned fragment etc. under being included in the definition of " antibody ".

Proteolytic enzyme papain preferably divides IgG molecule, thus produces some fragments, and wherein two fragments (F (ab) fragment) are respectively containing the covalency heterodimer comprising intact antigen binding site.Stomach en-can divide IgG molecule, thus provides several fragments, comprises the F (ab') containing two antigen-binding sites ₂fragment.Can produce by preferred proteolytic ferment division IgM the Fv fragment used according to certain embodiments of the present invention, under a few cases, divide IgG or IgA immunoglobulin molecules.But, use recombinant technology well known in the art to obtain Fv fragment more common.Fv fragment comprises the non-covalent VH::VL heterodimer containing antigen-binding site, and described antigen-binding site largely remains antigen recognition and the binding ability of natural antibody mole-cules.Inbar et al. (1972) Proc.Nat.Acad.Sci.USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; And Ehrlich et al. (1980) Biochem 19:4091-4096.

In certain embodiments, scFv or scFV antibody is contained.Such as, according to about have expectation specific selective receptor the application theory use standard molecular biological technique can prepare κ body (III et al., Prot.Eng.10:949-57 (1997); Miniantibody (Martin et al., EMBO J 13:5305-9 (1994); Disome (Holliger et al., PNAS 90:6444-8 (1993) or Janusins (Traunecker et al., EMBO J 10:3655-59 (1991) and Traunecker et al., Int.J.Cancer Suppl.7:51-52 (1992).In other embodiment another, the dual specific or chimeric antibody of containing the part that book is opened can be prepared.Such as, chimeric antibody can comprise CDR from different antibodies and framework region, can produce specific binding KDR (by a binding domains) and the bi-specific antibody in conjunction with the second molecule (by the second binding domains) simultaneously.These antibody can be prepared by recombinant molecule biotechnology, maybe these antibody physics can be conjugated in together.

ScFv (sFv) polypeptide with from comprising V _h-and V _lthe V of the gene fusion expression of-encoding gene _h:: V _lheterodimer is covalently bound, described V _h-and V _l-encoding gene is connected by peptide-encoding linker.Huston et al.(1988)Proc.Nat.Acad.Sci.USA 85(16):5879-5883。Described large metering method with identify for from antibody V district by natural polymerization-but chemically separated-light and heavy polypeptide chain is converted into the chemical structure of sFv molecule, described sFv molecule finally can be folded into the three-dimensional structure of the structure being similar to antigen-combining site.Such as, see the U.S. Patent No. 5,091,513 and 5,132,405 of Huston et al; With the U.S. Patent No. 4,946,778 of Ladner et al.

In certain embodiments, KDR binding antibody as described herein is the form with disome.Disome is the polymer of polypeptide, each polypeptide comprises the first structural domain, described first structural domain comprises the land of light chain immunoglobulin and the second structural domain, described second structural domain comprises the land of heavy chain immunoglobulin, two structural domains are connected (such as passing through peptide linker), but can not associate each other to form antigen-binding site: form antigen-binding site (WO94/13804) by the first structural domain of a peptide species in polymer with the association of the second structural domain of another polypeptide in polymer.

The dAb fragment of antibody is made up of (Ward, E.S.et al., Nature 341,544-546 (1989)) VH structural domain.

Use bi-specific antibody place, it can be conventional bi-specific antibody, described bi-specific antibody (Holliger can be prepared in every way, P. with Winter G.Current Opinion Biotechnol.4,446-449 (1993)), such as chemical preparation or from hybridoma preparation can be maybe any one of above-mentioned bispecific antibody fragment.Only use variable region can build disome without Fc region and scFv, this may reduce the effect that anti-idiotype reacts.

Relative to complete bi-specific antibody, dual specific disome is also useful especially, because they can easily build and express in intestinal bacteria (E.coli.).Use phage display (W094/13804) easily can screen the disome (such as, with other polypeptide many, antibody fragment) with suitable binding specificity from library.Such as, if disome arm can keep the specificity of constant orientation antigen X, so can prepare the library of wherein another arm change and select to have the antibody of appropriate specificity.Available projection enters hole (knobs-into-hole) engineering to prepare bispecific whole antibodies (J.B.B.Ridgeway et al., Protein Eng., 9,616-621,1996).

In certain embodiments, passable form antibody as herein described is provided. that the IgG4 antibody of the hinge area with removal is (see GenMab Utrecht, The Netherlands; Such as, also see US20090226421).The antibody technique of patent produces the less antibody formation (antibody format) that stable, that have expectation longer (compared with antibody formation little at present) treats window.IgG4 antibody is identified as inertia, therefore its can not with immune system response.Complete human IgG 4 antibody can being modified, by eliminating hinge area correctability complete human IgG 4 antibody of antibody, thus obtaining the half-molecule fragment relative to the complete IgG4 (GenMab, Utrecht) of correspondence with different stability matter.IgG4 molecule is halved and is only retained in a upper region, described in can in conjunction with pass associated antigen (such as, disease target), therefore only covalent attachment position on target cell.For some cancer cell surfaces antigen, the combination of this unit price can not irritation cancer cell with growth, as the antibody using and there is the divalence of identical antigen-specific can be seen, therefore technology can give the therapeutic choice for using conventional antibody may be difficult to the cancer of some types for the treatment of.When some forms of Therapeutic cancer, small size can be large benefit, this effect allowing the better distribution of the molecule exceeding larger solid tumor and potentiality to increase.

In certain embodiments, antibody of the present disclosure can take the form of nanometer body.To be encoded nanometer body by term single gene, and effectively result from nearly all protokaryon and eucaryon host, such as intestinal bacteria (such as, see U.S. Patent No. 6,765,087), mould (such as Aspergillus (Aspergillus) or Trichoderma (Trichoderma)) and yeast (such as yeast belong, Kluyvermyces, Chinese Sen Shi yeast belong (Hansenula) or Pichia (Pichia)) are (such as, see U.S. Patent No. 6,838,254).Production process can be measured, and has produced the nanometer body of many-kilogram quantities.Can by nanometer body formula for having the ready-to-use solution of long quality guaranteed period.

Height-flux based on the automatization of B-cell is selected, and nanometer cloning process (such as, see WO 06/079372) is the patented method of the nanometer body of target for generation of anti-expectation.

In certain embodiments, Anti-KDR antibody as disclosed herein or its antigen-binding fragment are humanized.This refers to the chimeric molecule usually using recombinant technology to prepare, and described chimeric molecule has by the antigen-combining site derived from non-human kind immunoglobulin like protein and the remaining immunoglobulin structure based on the structure of human normal immunoglobulin and/or the molecule of sequence.Antigen-combining site can comprise the variable domains completely merged in constant domain or the CDR only comprised on grafting framework region appropriate in variable domains.Epi-position combining site for wild-type or can modify epi-position combining site by one or more aminoacid replacement.This eliminates as immunogenic constant region in individual human, but still may immunne response foreign variable region (LoBuglio, A.F.et al., (1989) Proc NatlAcad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann etal., Nature (1988) 332:323-327).Humanized exemplary process for Anti-KDR antibody disclosed herein is included in United States Patent (USP) no.7,462, the method described in 697.According to certain embodiments of the present invention, schematic humanized antibody comprises SEQ ID NO:9,10,19 and 20 humanized sequence provided.

Other method not only pays close attention to the constant region providing people-derivative, also modifies constant region to transform them as person form as far as possible.Three complementarity-determining region (CDR) are contained in the variable region of known heavy chain and light chain, when replying described epi-position, described complementarity-determining region can change, and determined binding ability by the described complementarity-determining region that four framework region (FR) sides connect, described in given kind, framework region is guarded relatively, and estimates the skeleton (scaffolding) that described framework region provides CDR.When preparing non-human antibody about defined epitope, by the CDR grafting being derived from non-human antibody (reshape) " can be transformed " or " humanization " in variable region by FR again being present in people's antibody to be modified.By Sato, K., et al., (1993) Cancer Res 53:851-856.Riechmann, L., et al., (1988) Nature332:323-327; Verhoeyen, M., et al., (1988) Science 239:1534-1536; Kettleborough, C.A., et al., (1991) Protein Engineering 4:773-3783; Maeda, H., et al., (1991) HumanAntibodies Hybridoma 2:124-134; Gorman, S.D., et al., (1991) Proc Natl Acad Sci USA88:4181-4185; Tempest, P.R., et al., (1991) Bio/Technology 9:266-271; Co, M.S., et al., (1991) Proc Natl Acad Sci USA 88:2869-2873; Carter, P., et al., (1992) Proc Natl AcadSci USA 89:4285-4289; And Co, M.S.et al., (1992) J Immunol 148:1149-1154, report the application of the method for various antibody.In some embodiments, humanized antibody retains all CDR sequences (such as, containing the humanized murine's body from all six CDR of murine antibody).In other embodiments, humanized antibody has one or more CDR (, two, three, four, five, six), according to original antibodies, this can change, and described humanized antibody can be called one or more CDR " derivative " the one or more CDR from original antibodies.

In certain embodiments, antibody of the present disclosure can be chimeric antibody.In this, chimeric antibody is made up of the antigen-binding fragment of Anti-KDR antibody, and described Anti-KDR antibody is operably connected or otherwise is the heterology Fc part merging different antibodies.In certain embodiments, heterology Fc structural domain has people source.In other embodiments, heterology Fc structural domain from different Ig classes (from parental antibody), can comprise IgA (comprising subclass IgA1 and IgA2), IgD, IgE, IgG (comprising subclass IgG1, IgG2, IgG3 and IgG4) and IgM.In further embodiment, heterology Fc structural domain can be made up of CH2 and the CH3 structural domain from one or more different Ig classes.As implied above about humanized antibody, the anti-KDR antigen-binding fragment of chimeric antibody can comprise the CDR of only one or more antibody as herein described (such as, 1,2,3,4,5 or 6 CDR of antibody described herein), maybe can comprise whole variable domains (VL, VH or both).

In certain embodiments, KDR-binding antibody comprises the CDR of one or more antibody as herein described.In this, in some cases, show: the transfer that the only VHCDR3 of antibody can be carried out, but also retain the specific binding (Barbas et al., PNAS (1995) 92:2529-2533) of expectation.Also see McLane et al., PNAS (1995) 92:5214-5218, Barbas et al., J.Am.Chem.Soc. (1994) 116:2161-2162.

Marks et al (Bio/Technology, 1992, the method in the storehouse (repertoire) of Dispersal risk variable domains 10:779-783) is described, wherein the total primer of the 5' end in orientation or adjacent variable domains region is for combining the total primer of the 3rd framework region to people VH gene, thus provides the storehouse of the VH variable domains of disappearance CDR3.Marks et al further describes and how to be merged by the CDR3 of this storehouse and specific antibody.Use similar technology, can reorganize the derivative sequence of the CDR3-of (shuffle) current described antibody (using the storehouse of VH or the VL structural domain lacking CDR3), and complete VH or the VI structural domain of reorganization provides antibody in conjunction with KDR or its antigen-binding fragment in conjunction with homology VL or VH district.Then storehouse can be illustrated in suitable host system, such as, in the phage display system of W092/01047, its suitable antibody or antigen-binding fragment can be selected.Storehouse can by least about 10 ⁴single member and more than some orders of magnitude (such as, about 10 ⁶to 10 ⁸or 10 ¹⁰, or more) member composition.Stemmer (Nature, 1994,370:389-391) also discloses similar reorganization or the technology of combination, and Stemmer describes about the technology of β-lactamase gene, but observes the generation that the method can be used for antibody.

Alternatively use the random sudden change of VH and/or the VL gene of one or more selection to produce novel VH or VL district (the derivative sequence of one or more CDR-of invention embodiment shown in this article is carried in described novel VH or VL district) further, thus produce the sudden change in whole variable domains.Gram et al (1992, Proc.Natl.Acad.Sci., USA, 89:3576-3580) describes this technology, and it uses fallibility PCR (error-prone PCR).Spendable another kind of method carries out rite-directed mutagenesis to the CDR district of VH or VL gene.Barbas et al., (1994, Proc.Natl.Acad.Sci., USA, 91:3809-3813) and Schier et al (1996, J.Mol.Biol.263:551-567) disclose this technology.

In certain embodiments, the specificity VH of antibody as herein described and/or VL can be used for the library of screening complementary variable domains, thus qualification has the antibody of the character (such as the avidity that KDR increases) of expectation.These methods are described in such as Portolano et al., J.Immunol. (1993) 150:880-887; Clarkson et al., Nature (1991) 352:624-628.

Other method can be used with mixing and coupling CDR, thus qualification have the antibody of the binding activities (such as in conjunction with KDR) of expectation.Such as: Klimka et al., British Journal of Cancer (2000) 83:252-260 describes the screening method in the people VH library using mouse VL and have CDR3 and FR4 retained from mouse VH.After acquisition antibody, VH is screened in anti-human VL library, thus obtains the antibody of conjugated antigen.Beiboer et al., J.Mol.Biol. (2000) 296:833-849 describes the screening method using whole mouse heavy chain and people's light chain libraries.After acquisition antibody, the people VH library of a VL and tool mouse CDR3 with a grain of salt merges.Acquisition can the antibody of conjugated antigen.Raderet al., PNAS (1998) 95:8910-8915 describes the method for similar above-mentioned Beiboer et al.

These above-mentioned technology itself are known in the art.But according to several embodiments of the present invention as herein described, utilize ordinary method in this area, those skilled in the art can use these technology to obtain antibody or its antigen-binding fragment.

Be the method for obtaining the antibody antigen binding domains for KDR antigen-specific disclosed in going back herein, described method comprises one or more amino acid whose interpolations in the aminoacid sequence at VH structural domain described herein, disappearance, replacement or inserts the VH structural domain being provided as the amino acid sequence variation of VH structural domain; Optionally provide one or more VL structural domain in conjunction with VH structural domain; And test VH structural domain or one or more VH/VL combine, thus identify specific binding members or for KDR special with the antibody antigen binding domains optionally with one or more desirable propertieses.VL structural domain can have substantially aminoacid sequence as described herein.Can adopt similar method, the sequence variants of wherein one or more VL structural domains disclosed herein combines one or more VH structural domain.

" specific binding " or " preferably combine " (commutative use herein) antibody or polypeptide epi-position be the term that this area fully understands, and the method measuring this species specificity or preferred combination is also well-known in the art.Compared with selectable cell or material, if molecular reaction or associate frequently, faster, there is the time length more of a specified duration and/or with specific cells or material, there is larger avidity, molecule is considered to show " specific binding " or " preferred combine ".Compared with other materials of antibodies, if antibodies has larger avidity, avidity, is more prone to and/or has the longer time length, antibody " specific binding " or " preferably combining " target.Such as, specificity or preferably in conjunction with the antibody that the antibody of KDR epi-position is in conjunction with a kind of KDR epi-position, compared with other KDR epi-positions of antibodies or non-KDR epi-position, described combination has higher avidity, avidity, is more prone to and/or has the longer time length.It is also understood that such as by reading this definition, specificity or preferably can or can not specificity or preferably in conjunction with the second target in conjunction with the antibody (or part or epi-position) of the first target.With regard to itself, " specific binding " or " preferred combine " is not necessary (although it can comprise) exclusive combination.Usually, but be not necessary, the combination of reference is meant to preferred combination.

Immunity combines the non-covalent interaction typically referring to type, described type appears between immunoglobulin molecules and antigen, special for described antigen immune sphaeroprotein, such as by illustrating but nonrestrictive mode, due to electrostatic, ion, hydrophilic and/or hydrophobic magnetism or repulsive force, steric hindrance power (steric force), hydrogen bond, Van der Waals force (van der Waals forces) and other interact.According to interactional dissociation constant (K _d) intensity or the avidity of immune binding interactions, wherein less K can be represented _drepresent higher avidity.Use method well-known in the art can the Immunological binding properties of polypeptide of quantitative choosing.This method needs the speed measuring antigen-combining site/antigenic compound formation and dissociation, and wherein these speed depend on the concentration of complex partners, interactional avidity and affect the geometric parameter at two direction medium-rates comparably.Therefore, concentration and actual speed rate by calculating association and dissociation can measure " association rate constant " (K _on) and " dissociation rate constant " (K _off).K _off/ K _onratio can offset all parameters irrelevant with avidity, therefore equal dissociation constant K _d.Usually, see Davies et al. (1990) Annual Rev.Biochem.59:439-473.

In certain embodiments, Anti-KDR antibody as herein described has about 100, and 150,155,160,170,175,180,185,190,191,192,193,194,195,196,197, the avidity of 198 or 199 picomole, and in some embodiments, even higher avidity can be had for KDR antibody.

About the term " immunocompetence " of epi-position or " remaining immunocompetence " refer under different conditions (such as, making epi-position after reduction and Denaturing) antibody (such as, Anti-KDR antibody) in conjunction with epi-position ability.

According to the antibody of some preferred embodiment of the application or its antigen-binding fragment can for and the one of any antibody competition as herein described in conjunction with KDR, it is (i) specific binding antigen, and (ii) comprises VH and/or VL structural domain disclosed herein or comprise VH CDR3 disclosed herein or these variant arbitrarily.Such as use ELISA and/or by specific reporter molecule being tagged to a kind of antibody (under the existence of other unlabelled antibody, described antibody can be detected), can competition easily between In vivo analysis antibody, thus the specific antibody of the epi-position in conjunction with identical epi-position or overlap can be identified.Therefore, provide specific antibody or its antigen-binding fragment herein, described antibody or its antigen-binding fragment comprise and people's antibody antigen-combining site of antibody competition as herein described in conjunction with KDR.

In this, as used herein, term " competition ", " suppressing to combine " and " blocking-up combination " are (such as, the inhibition/blocking of the inhibition/blocking relating to the combination of VEGF and KDR or the combination relating to Anti-KDR antibody and KDR) use convertibly, and contain part and complete inhibition/blocking.VEGF combines and the inhibition/blocking of KDR preferably reduces or change the normal level or type that transmit in conjunction with the cell signal occurred during KDR as VEGF under not having to suppress or block.Suppress and block also to be intended to comprise when contacting Anti-KDR antibody as disclosed herein in the combination of VEGF and KDR compared to any measurable minimizing of part not contacting Anti-KDR antibody, such as, the blocking-up of VEGF and KDR decreases at least about 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

Compared with variable region, the constant region of immunoglobulin (Ig) shows less sequence polymorphism, and is responsible in conjunction with a large amount of native protein, thus causes important biochemical reaction.In people, there are five kinds of different types of antibody, comprise IgA (it comprises subclass IgA1 and IgA2), IgD, IgE, IgG (it comprises subclass IgG1, IgG2, IgG3 and IgG4) and IgM.Notable feature between these antibody types is their constant region, although delicate difference may be present in V district.

The Fc district of antibody and a large amount of Fc acceptor and ligand interaction, this gives a series of important functional capabilities, is called effector functions.Ig domain C H2 and CH3 is comprised for IgG, Fc district and introduces the N-end hinge of CH2.Important family for the Fc acceptor of IgG class is Fc γ acceptor (Fc γ Rs).Arm (cellular arm) (Raghavan et al., 1996, the Annu Rev CellDev Biol 12:181-220 of the communication between these receptor-mediated antibody and immune cell; Ravetch et al., 2001, Annu Rev Immunol 19:275-290).In people, this protein family comprises Fc γ RI (CD64) (comprising hypotype Fc γ RIa, Fc γ RIb and Fc γ RIc); Fc γ RII (CD32) (comprising hypotype Fc γ RIIa (comprising allotype H131 and R131), Fc γ RIIb (comprising Fc γ RIIb-1 and Fc γ RIIb-2) and Fc γ RIIc); With Fc γ RIII (CD16) (comprising hypotype Fc γ RIIIa (comprising allotype V158 and F158) and Fc γ RIIIb (comprising allotype Fc γ RIIIb-NA1 and Fc γ RIIIb-NA2)) (Jefferis et al., 2002, Immunol Lett 82:57-65).These acceptors typically have mediation in conjunction with the extracellular domain of Fc, membrane spaning domain and the intracellular domain that can mediate some signal transmission events in cell.These expression of receptor, in various immunocyte, comprise monocyte, scavenger cell, neutrophilic leukocyte, dendritic cell, eosinocyte, mastocyte, thrombocyte, B cell, large granular lymphocyte, Langerhans cell, NK cell (NK) cell and T cell.These effector cells are absorbed the position to the antigen combined by the formation of Fc/Fc γ R mixture, this typically causes transmitting event and the response of important follow-up immunization at Intracellular signals, release, B cell activations, the endocytosis of such as inflammatory mediator, engulfs and cytotoxin attack.

Mediates cytotoxic be potential mechanism with the ability of cytophagous effector functions, by the cell of this machine-processed antibody disrupts target.Cell-mediated reaction (cell of wherein expressing the non-specific cell toxin of Fc γ Rs identifies the antibody of combination and finally causes the molten born of the same parents of target cell on target cell) is referred to as cytotoxicity (ADCC) (the Raghavan et al. of antibody dependent cellular-mediation, 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu RevImmunol 19:275-290).Cell-mediated reaction (cell recognition wherein expressing the non-specific cell toxin of Fc γ Rs goes out the antibody that combines on target cell and finally causes engulfing of target cell) is referred to as engulfing (ADCP) of antibody dependent cellular-mediation.All Fc γ Rs on Fc in conjunction with identical region, under the N-terminal of Cg2 (CH2) structural domain and mentioned hinge.This interaction is abundant characterization (Sondermann et al. structurally, 2001, J MolBiol 309:737-749), and solved some structures (pdb poll code 1E4K) (the Sondermann et al. of people Fc of the extracellular structural domain in conjunction with people Fc γ RIIIb, 2000, Nature 406:267-273) (pdb poll code 1IIS and 1IIX) (Radaev et al., 2001, J Biol Chem 276:16469-16477).

For Fc γ Rs, different I gG subclass has different avidities, and the typical combination of IgG1 and IgG3 and acceptor is almost better than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65).On IgG Fc, all Fc γ Rs are in conjunction with identical region, but have different avidity: for IgG1, and high affinity binding agent (binder) Fc γ RI has 10 ^-8m ^-1kd, but low affinity receptor Fc γ RII and Fc γ RIII is combined in 10 usually respectively ^-6with 10 ^-5under.Fc γ RIIIa and the ectodomain of Fc γ RIIIb are 96% consistent, but Fc γ RIIIb does not have Intracellular signals transferring structure territory.And, but Fc γ RI, Fc γ RIIa/c and Fc γ RIIIa are the positive regulators of the activation of immunocomplex-triggering, it is characterized in that there is intracellular domain, described intracellular domain has immunity receptor TYR-Ji and activates motif (ITAM), Fc γ RIIb has immunity receptor TYR-Ji and suppresses motif (ITIM), and therefore it suppresses.Therefore, the former is called as activated receptor, and Fc γ RIIb is called as suppression acceptor.Acceptor is also at expression pattern and different on expression level on different immunocyte.The another level of complexity is the existence of a large amount of Fc γ R polymorphism in people's protein groups.The polymorphism relevant especially with clinical meaning is V158/F158Fc γ RIIIa.Compared with F158 allotype, human IgG1 with larger avidity in conjunction with V158 allotype.Be presented at difference in avidity and infer its to ADCC and/or ADCP act as anti-CD 20 antibodies Rituximab (rituximab) ( the registered trademark of IDEC Pharmaceuticals Corporation) the important determinative of effect.There is the allotypic patient of V158 and reply Rituximab treatment preferably; But, there is the allotypic patient of lower avidity F158 and reply poor (Cartron et al., 2002, Blood 99:754-758).The people of about 10-20% is that V158/V158 isozygotys; 45% is V158/F158 heterozygosis; And the people of 35-45% is that F158/F158 isozygotys (Lehrnbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758).Therefore, the people of 80-90% is the respondent (responder) of difference, and that is that they have at least one allelotrope of F158Fc γ RIIIa.

Fc district is also included within the activation of complement series connection.In classical complement pathway, C1 is by the Fc fragment of its C1q subunit in conjunction with IgG or IgM, and this has formed the mixture with antigen.In certain embodiments of the invention, the modification in Fc district comprises the modification of the ability of change (increase or reduce) KDR-specific antibody as described herein, thus activating complement system (such as, see United States Patent (USP) 7,740,847).In order to assess complement activation, CDC (CDC) can be carried out and analyze (such as, see Gazzano-Santoro et al., J.Immunol.Methods, 202:163 (1996)).

Therefore, in certain embodiments, the invention provides the Anti-KDR antibody in the Fc district with modification, the Fc district of described modification has the functional property of change, and that such as reduce or that strengthen CDC, ADCC or ADCP are active; Or the binding affinity of enhancing for specificity Fc γ R; Or the plasma half-life increased.Other the Fc districts modified contained herein describe, such as, and the United States Patent (USP) 7,317,091 delivered; 7,657,380; 7,662,925; 6,538,124; 6,528,624; 7,297,775; 7,364,731; Disclosed US Patent No. 2009092599; US20080131435; US20080138344; With disclosed international application WO2006/105338; WO2004/063351; WO2006/088494; In WO2007/024249.

Therefore, in certain embodiments, the antibody variable territory of the binding specificity with expectation is made to merge immunoglobulin constant domains sequence.In certain embodiments, merge and use Ig heavy chain constant domain, described Ig heavy chain constant domain comprises hinge, C at least partially _h2 and C _h3rd district.Preferably there is the first weight-chain constant region (C combining necessary position containing the light chain be present at least one fusion _h1).If the DNA merged by encode immunoglobulin heavy and the light chain immunoglobulin needed insert independent expression vector, and by its common-transfection in suitable host cell.When the unequal ratio of three polypeptide chains used in the structure provides the optimum yields of the bi-specific antibody of expectation, in the common property regulating three polypeptide fragments in embodiment, this provides greater flexibility.But, when causing high yield with the expression of equal ratio at least two polypeptide chains, or when ratio is for when expecting that the productive rate of chain combination does not have material impact, may insert for two or all three polypeptide chain encoding sequences in single expression vector.

Also antibody of the present invention (with antigen-binding fragment and variant thereof) can be modified to comprise epitope tag or mark, such as, in purifying or diagnostic use.There are many linking groups known in the art for the preparation of antibody conjugates, comprise and be such as disclosed in U.S. Patent No. 5,208,020 or European patent 0 425 235 B1, with those of Chari et al., CancerResearch 52:127-131 (1992).Linking group comprise as disulfide group disclosed in above-mentioned patent, thioether group, acid instability group, to photo-labile group, the unstable group of peptase or the unstable group of esterase, preferred disulphide and thioether group.

In the embodiment that another is contained, KDR-specific antibody as described herein can be made to put together or may be operably coupled to another therapeutic compound, be called conjugate herein.Conjugate can be cytotoxic agent, chemotherapeutics, cytokine, the agent of anti-vasculogenesis, tyrosine kinase inhibitor, toxin, radio isotope or other treatment promoting agent.The agent of chemotherapeutics, cytokine, anti-vasculogenesis, tyrosine kinase inhibitor and other treatment agent describe as above, and all these aforementioned therapies agent can find to be used as antibody conjugates.

In alternately embodiment, antibody and toxin (include but not limited to the enzyme activity toxin of small molecule toxins and bacterium, fungi, plant or animal-origin, comprise its fragment and/or variant) is made to put together or be operably connected.Small molecule toxins includes but not limited to saporin (saporin) (Kuroda K, et al., The Prostate 70:1286-1294 (2010); Lip, WL.et al., 2007Molecular Pharmaceutics 4:241-251; Quadros EV., et al., 2010Mol Cancer Ther; 9 (11); 3033 – 40; Polito L., et al.2009British Journal ofHaematology, 147,710 – 718), calicheamicin (calicheamicin), maytenin (maytansine) (U.S. Patent No. 5,208,020), trichothecene toxin (trichothene) and CC1065.Toxin includes but not limited to: RNase, gelonin (gelonin), alkynes diene, ricin, abrin, diphtheria toxin, Toxins,exo-, cholera, gelonin, Pseudomonas exotoxin (PE40), blunt Shigellae, clostridium perfringens toxoid and Pokeweed antiviral protein.

In one embodiment, antibody of the present disclosure or its antigen-binding fragment is made to be conjugated to ansamitocin molecule.Ansamitocin is the mitotic inhibitor by suppressing tubulin polymerization to act on.First ansamitocin is separated from East Africa shrub tingia Caulis Mayteni (Maytenus serrata) (U.S. Patent No. 3,896,111).Finally, find that some bacterium also produces ansamitocin, such as maytansinol and C-3 maytansinol ester (U.S. Patent No. 4,151,042).The maytansinol of synthesis and derivative and analogue open, such as, in U.S. Patent No. 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; With 4,371,533.Immunoconjugates containing ansamitocin and their therepic use are disclosed in such as U.S. Patent No. 5,208,020,5,416,064 and European patent EP 0 425 235 B1.Liu et al., Proc.Natl.Acad.Sci.USA 93:8618-8623 (1996) describe and comprise the immunoconjugates of the ansamitocin of called after DM1, described DM1 connects monoclonal antibody C242, by monoclonal antibody C242 orientation antagonism human colorectal cancer.The rectum cancer cell cultivated is found that conjugate be high cytotoxic, and in vivo in tumor growth analyses conjugate show anti-tumor activity.

Under the biological activity significantly not reducing antibody or ansamitocin molecule, carry out Dispersal risk-ansamitocin conjugate by chemistry connection antibody and ansamitocin molecule.Under the function not having negative influence antibody or solvability, mean value/the antibody molecule of 3-4 the ansamitocin molecule puted together has been presented at effect in the cytotoxicity of the enhancing of target cell, although in the naked antibody of use (naked antibody) period, even expect that the toxin/antibody of a part strengthens cytotoxicity.Ansamitocin is well-known in the art, and can be synthesized by known technology or be separated from plant origin.Suitable ansamitocin is disclosed in such as U.S. Patent No. 5,208,020 and in open with reference to other hereinbefore patents and non-patent.Preferred ansamitocin is other positions of maytansinol and the maytansinol analogue modified in aromatic nucleus or maytansinol molecule, such as various maytansinol ester.

Another target conjugate comprises the antibody being conjugated to one or more calicheamicin molecules.The calicheamicin family of antibody can produce the DNA break of double-strand under Asia-picomole (sub-picomolar) concentration.Also analog (Hinman et al., 1993, the Cancer Research 53:3336-3342 of spendable calicheamicin; Lode et al., 1998, Cancer Research 58:2925-2928) (U.S. Patent No. 5,714,586; U.S. Patent No. 5,712,374; U.S. Patent No. 5,264,586; U.S. Patent No. 5,773,001).Dolastatin (Dolastatin) 10 analogue of such as auristatin E (AE) and monomethylauristatin E (MMAE) can find conjugate (the Doronina et al. being used as disclosed antibody or its variant recently, 2003, Nat Biotechnol 21 (7): 778-84; Francisco et al., 2003Blood 102 (4): 1458-65).Useful enzyme activity toxin includes but not limited to: diphtheria A chain, the nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa (Pseudomonasaeruginosa)), ricin A chain, abrin A chain, modeccin A chain, α-sarcina, Aleurites fordii proteins, oleanolic acid albumen, pokeroot (Phytolaca Americana) albumen (PAPI, PAPII, and PAP-S), balsam pear (momordica charantia) inhibitor, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin, mitogellin, restrictocin (restrictocin), phenomycin, enomycin, with single-ended spore mould enol (tricothecene).Such as, see PCT WO 93/21232.The disclosure contains the embodiment wherein forming conjugate or fusion between KDR-specific antibody as described herein and the compound with nucleolytic activity further, such as the DNA endonuclease of rnase or such as deoxyribonuclease (DNase).

In alternately embodiment, by this paper-disclosed antibody conjugate or the radio isotope that is operably connected, thus radioactivity conjugate can be formed.Various radio isotope can be obtained for the preparation of radioactivity conjugate antibody.Example includes but not limited to ⁹⁰y, ¹²³i, ¹²⁵i, ¹³¹i, ¹⁸⁶re, ¹⁸⁸re, ²¹¹at and ²¹²bi.

At some in other embodiment, antibody conjugate as herein described can be made to treatment part, such as cytotoxin (such as, cytostatics or cytocide), therapeutical agent or radioelement (alpha radiation source, γ-source of radiation etc.).Cytotoxin or cytotoxic agent comprise any medicament harmful to cell.Example comprises taxol/taxol (paclitaxol), Cytochalasin B, Gramicidin D, ethidium bromide, ipecamine, mitomycin, Etoposide, teniposide, vincristine(VCR), vincaleucoblastine, colchicine, Zorubicin, daunorubicin, dihydroxyl anthracin diketone (dihydroxy anthracin dione), mitoxantrone, mithramycin, dactinomycin, 1-boldenone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte, with tetracycline and analogue thereof or homologue.A kind of preferred Exemplary cytotoxins is saporin (from Advanced Targeting Systems, San Diego, CA can obtain).Therapeutical agent includes but not limited to: metabolic antagonist (such as, methotrexate, Ismipur, 6-Tioguanine, cytosine arabinoside, 5 FU 5 fluorouracil decarbazine), alkylating agent (such as, mustargen, thioepa chlorambucil, melphalan, carmustine (BSNU), with lomustine (CCNU), cyclothosphamide, busulfan, mitobronitol, streptozotocin, ametycin, with cisplatin (II) (DDP) Platinol), anthracycline (such as daunorubicin (being daunomycin originally) and Zorubicin), microbiotic (such as gengshengmeisu (being actinomycin originally), bleomycin, mithramycin and anthramycin (AMC) and anti-mitogenic agent (such as vincristine(VCR) and vincaleucoblastine).

Such as, and in certain embodiments, KDR-specific antibody (comprising its function fragment provided herein, antigen-binding fragment) can put together treatment part, such as, for puting together radio active material or the macrocyclic chelants of radiometal ion.In certain embodiments, macrocyclic chelants is DOTA (DOTA), and it is attached antibody by linkers.This linkers is well known in the art, and is described in Denardo et al., 1998, Clin Cancer Res.4:2483-90; Peterson et al., 1999, Bioconjug.Chem.10:553; With Zimmerman et al., 1999, Nucl.Med.Biol.26:943-50.

In yet another embodiment, antibody can put together the pre-determined bit of " acceptor " (such as streptavidin) for tumour, wherein antagonist-receptor conjugate is applied to patient, then using scavenging agent from the recycle system, remove unconjugated conjugate, then using " part " (such as the avidin) for puting together cytotoxic agent (such as radioactive nucleotides).In alternately embodiment, antibody conjugate or the enzyme that is operably connected are to apply the prodrug therapy (ADEPT) of antibody dependent enzyme mediation.ADEPT can be used by following manner: make antibody conjugate or may be operably coupled to the enzyme activating prodrug, this enzyme makes prodrug (such as peptidyl chemotherapeutic agent, see PCT WO 81/01145) be converted into active anti-cancer drug.For example, see PCT WO 88/07378 and U.S. Patent No. 4,975,278.Enzyme component for the immunoconjugates of ADEPT comprises any enzyme that can act on prodrug by this way, and which is converted into active cytotoxins form more for making it.Enzyme for these methods and related embodiment includes but not limited to: for being the alkaline phosphatase of free drug by the pro-drug conversion containing phosphoric acid ester; For being the aryl sulphatase of free drug by the pro-drug conversion containing sulfuric ester; For the phonetic heavy stone used as an anchor of non-toxic 5-fluorine born of the same parents being converted into the phonetic heavy stone used as an anchor desaminase of born of the same parents of cancer therapy drug 5 FU 5 fluorouracil; For being the proteolytic enzyme of free drug by the pro-drug conversion containing peptide, such as Serratia Proteases, thermolysin, subtilisin, carboxypeptidase and kethepsin (such as cathepsin B and L); For transforming the D-alanyl carboxypeptidase of the prodrug containing D-aminoacid replacement base; Such as, for glycosylated prodrugs being converted into the carbohydrate-lyase of free drug ,-tilactase and neuraminidase; For the β-lactamase being free drug by the drug Transformation of use-lactam derivative; And such as, for the nitrogen place at their amine being used respectively the penicillin amidase of drug Transformation for free drug of nitrophenoxyacetyl or phenyl acetyl derivatize, Penicillin-V-Amidase or Penicillin-G-amidases.Or have the antibody of enzymic activity, in the art also referred to as " abzyme ", can be used for pro-drug conversion is free active medicine (for example, see Massey, 1987, Nature 328:457-458).Antibody-antibody enzyme conjugate can be prepared and is delivered to tumor cell group for by abzyme.

Immunoconjugates can use the agent of various bi-functional albumen coupling to prepare, such as N-succinimido-3-(2-pyridyl dimercapto) propionic ester (SPDP), succinimido-4-(N-maleimidomehyl) hexanaphthene-1-carboxylicesters, iminothiolane (IT), the bi-functional derivative of imido-ester (such as oneself two imido disalt dimethyl phthalates), active ester (such as two succinimidyl suberate), aldehydes (such as glutaraldehyde), two-azido cpd (such as two (p-azidobenzoyl) hexanediamine), two-diazo compound derivative (such as two-(p-diazoniumbenzoyl)-quadrol), vulcabond (such as toluene 2, 6-vulcabond) and two-active fluorine compounds (such as 1, 5-bis-fluoro-2, 4-dinitrobenzene).Specific coupling agent comprises N-succinimido-3-(2-pyridyl dimercapto) propionic ester (SPDP) (Carlsson et al., Biochem.J.173:723-737 [1978]) and is connected to improve disulphide with N-succinimido-4-(2-pyridyl sulfydryl) valerate (SPP).Joint can be " joint of cleavable " of the component release promoting one or more cleavables.Such as, acid-unstable joint (Cancer Research 52:127-131 (1992) can be used; U.S. Patent No. 5,208,020).

Also contain other modifications of antibody of the present invention (and polypeptide) herein.Such as, antibody can be connected to the one in various non-protein polymer, such as the multipolymer of polyoxyethylene glycol, polypropylene glycol, polyoxyalkylene or polyoxyethylene glycol and polypropylene glycol.Antibody can also be encapsulated in micro-capsule, and it is such as by condensation technique in colloidal drug delivery systems (such as liposome, albumin microsphere, micro emulsion, nano particle and nanocapsule) or large breast (macroemulsion) or interfacial polymerization (being such as respectively Walocel MT 20.000PV or gelatin microcapsules and poly-(methyl methacrylate) micro-capsule).These technology are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

As used herein, " vehicle " to be included under the dosage of use and concentration the cell exposed or Mammals is atoxic pharmaceutically acceptable vehicle, vehicle or stablizer.Usual physiologically acceptable vehicle is water-based pH buffered soln.Physiologically acceptable vehicle comprises buffering salt, such as phosphoric acid salt, Citrate trianion and other organic acids; Antioxidant, comprises xitix; Lower molecular weight (being less than about 10 residues) polypeptide; Albumen is serum albumin, gelatin or immunoglobulin (Ig) such as; Hydrophilic macromolecule is polyvinylpyrrolidone such as; Amino acid is glycine such as, glutamine, l-asparagine, arginine or Methionin; Monose, disaccharides and other carbohydrates (comprising glucose, seminose or dextrin); Sequestrant is EDTA such as; Sugar alcohol such as N.F,USP MANNITOL or Sorbitol Powder; Salify gegenion such as sodium; And/or nonionic surface active agent such as polysorbate20 (TWEEN ^tM) polyoxyethylene glycol (PEG) and poloxamer (PLURONICS ^tM) etc.

The various methods that the desired function performance of Anti-KDR antibody can use those of skill in the art known are evaluated: avidity/combination measures (such as surface plasma resonance, competitive inhibition measure); Cytotoxic assay, cell viability measure, in response to VEGF cell proliferation or differentiation assays (such as phosphorylation assay), use external or In vivo model anticancer and/or tumor growth.Other mensuration can test the activity of the response of antibody blocking as herein described normal VEGF/KDR-mediation, such as but not limited to the phosphorylation of KDR, vasculogenesis and endothelial cell proliferation.Antibody as herein described also can be tested with the effect for KDR receptor internalisation, in vitro and in vivo effect etc.In further embodiment, antibody can use suitable animal model build-in test herein.Antibody as herein described can the ability of external or their Tumor suppression of body build-in test growth.The scheme of the good foundation that these mensuration can use those of skill in the art known is carried out (for example, see Current Protocols in Molecular Biology (Greene Publ.Assoc.Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Edited by:John E.Coligan, Ada M.Kruisbeek, David H.Margulies, Ethan M.Shevach, WarrenStrober 2001John Wiley & Sons, NY, NY); Or commercial reagent box.

In certain embodiments, the present invention also provides the nucleic acid of the separation of coding antibody as described herein or its antigen-binding fragment, the nucleic acid of such as encode CDR or VH as described herein or VL structural domain.Nucleic acid comprises DNA and RNA.These and relevant embodiment can comprise the polynucleotide of coding in conjunction with the antibody of KDR as described herein.As used herein, term " polynucleotide be separated " meaning is the polynucleotide of genome, cDNA or synthetic source or some its combinations, by its source, the polynucleotide (1) be separated are not associated with all or a part of of the polynucleotide of the polynucleotide that wherein natural discovery is separated, (2) polynucleotide are connected to, described polynucleotide are natural not to be connected, or (3) under natural not as larger sequence a part and produce.

Term " is operably connected " and refers to that the component of this term application is in such relation, and it makes them implement the function of their inherences under suitable conditions.Such as, " being operably connected ", to the transcriptional control sequence of albumen coded sequence and its colligation, makes the expression of albumen coded sequence realize under the condition compatible with the transcriptional activity of control sequence.

As used herein, term " control sequence " refers to polynucleotide sequence, and it can affect the expression of their colligations or the encoding sequence that is operably connected, processing or intracellular targeting.The character of these control sequences can be depending on host microorganism.In certain embodiments, promotor, ribosome binding site and transcription termination sequence can be comprised for procaryotic transcriptional control sequence.In other specific embodiments, can promotor be comprised for Eukaryotic transcriptional control sequence, comprise the one or more recognition sites for transcription factor, transcription enhancer sequences, transcription termination sequence and polyadenylation se-quence.In certain embodiments, " control sequence " can comprise leader sequence and/or merge spouse's sequence.

The term " polynucleotide " related to herein refers to the nucleic acid polymer of strand or double-strand.In certain embodiments, the Nucleotide comprising polynucleotide can be the modified forms of ribonucleotide or deoxyribonucleotide or the arbitrary type of Nucleotide.Described modification comprises base modification such as bromouridine, ribose and modifies such as Arabinoside and 2', 3'-dideoxy ribose and inner nucleotide connect modifies such as thiophosphatephosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoramidate.Term " polynucleotide " is particularly including the DNA of strand and double chain form.

Term " naturally occurring Nucleotide " comprises deoxyribonucleotide and ribonucleotide.Term " Nucleotide modified " comprises the Nucleotide of glycosyl that is that have modification or that replace etc.Term " oligonucleotide connection " comprises oligonucleotide and connects, such as thiophosphatephosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoramidate etc.For example, see LaPlanche et al., 1986, Nucl.Acids Res., 14:9081; Stec et al., 1984, J.Am.Chem.Soc., 106:6077; Stein etal., 1988, Nucl.Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES:A PRACTICALAPPROACH, pp.87-108 (F.Eckstein, Ed.), Oxford University Press, Oxford England; Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, for any object, it is openly incorporated to herein by way of reference.Oligonucleotide can comprise detectable mark can detect oligonucleotide or its hybridization.

Term " carrier " is for referring to any molecule (such as nucleic acid, plasmid or virus) for coded message being transferred to host cell.Term " expression vector " refers to such carrier, and it is suitable for transformed host cell, and contains the nucleotide sequence of the expression instructing and/or control the heterologous nucleic acid sequence inserted.Expression includes but not limited to processing, such as, transcribe, translate and RNA montage (if there is intron).

As understood by a person skilled in the art, polynucleotide can comprise genome sequence, outer-genome and the sequence of plasmid-coding and the constant gene segment C (its expression maybe can be suitable for expressing protein, polypeptide, peptide etc.) of less through engineering approaches.These sections can be natural separation, or by those of skill in the art's synthetic modification.

Those skilled in the art it is also recognized that, polynucleotide can be strand (coding or antisense) or double-strand, and can be DNA (genome, cDNA or synthesis) or RNA molecule.RNA molecule can comprise: HnRNA molecule, and it contains intron and corresponds to DNA molecular with single to folk prescription formula; With mRNA molecule, it is not containing intron.In addition coding or non-coding sequence can but to be nonessentially present in according in polynucleotide of the present disclosure, and polynucleotide can but other molecules of nonessential connection and/or support materials.Polynucleotide can comprise native sequences maybe can comprise the encode variant of this sequence or the sequence of derivative.

Therefore, according to these and relevant embodiment, the disclosure also provides the polynucleotide of Anti-KDR antibody as herein described of encoding.In certain embodiments, providing package is containing the component of some and the whole polynucleotide in the polynucleotide sequence of coding antibody as described herein and this polynucleotide.

In the embodiment that other are relevant, polynucleotide variant can have consistence substantially with the polynucleotide sequence of coding Anti-KDR antibody as herein described.Such as, method as herein described (such as described below, to use the BLAST of canonical parameter to analyze) is used, polynucleotide can be polynucleotide, comprise, compared to reference polynucleotide sequence (sequence of antibody as herein described of such as encoding), there is at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, the sequence identity of 97%, 98% or 99% or higher.Those skilled in the art will recognize that, by considering codon degeneration, amino acid similarities, reading frame location etc., the adjustment that these numerical value can be suitable is to determine by the consistence of the correspondence of two nucleotide sequence coded albumen.

Typically, polynucleotide variant will replace containing one or more, will increase, lack and/or insert, preferably make the antibody relative to the polynucleotide sequence coding of being set forth by this paper specificity, the binding affinity of the antibody of being encoded by variant polynucleotides does not weaken in fact.

At some in other relevant embodiments, polynucleotide passage can comprise or substantially be made up of following: the continuous sequence continuity of all lengths, it is equal to or is complementary to the sequence of antibody as described herein of encoding.Such as, provide polynucleotide, comprise or be substantially made up of following: at least about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,45,50,55,60,65,70,75,80,85,90,95,100,110,120,130,140,150,200,300,400,500 or 1000 or more continuous nucleotide sequences, its encode antibody disclosed herein or its antigen-binding fragment and all intermediate lengths between them.Within a context, easy understand " intermediate length " refers to any length quoted between numerical value, such as 50,51,52,53 etc.; 100,101,102,103 etc.; 150,151,152,153 etc.; Comprise all integers between 200-500; 500-1,000 etc.Polynucleotide sequence described herein can at one end or two ends extended by other Nucleotide undiscovered in native sequences.This other sequence can be made up of following: at 1,2,3,4,5,6,7 of the either end place of polynucleotide of antibody as herein described or the two ends place of the polynucleotide of antibody as herein described of encoding of encoding, 8,9,10,11,12,13,14,15,16,17,18,19 or 20 Nucleotide.

In another embodiment, provide polynucleotide, it can hybridize coding antibody provided herein or the polynucleotide sequence of its antigen-binding fragment or its fragment or its complementary sequence under moderate to high stringency.Hybridization technique is that biology field is known.In order to describe, be included in 5X SSC, pre-wash in the solution of 0.5%SDS, 1.0mM EDTA (pH 8.0) for the suitable moderate stringency testing polynucleotide provided herein and other multi-nucleotide hybrids; At 50 DEG C-60 DEG C, hybridized overnight under 5X SSC; Then at 65 DEG C, use the 2X containing 0.1%SDS respectively, 0.5X and 0.2X SSC washes twice 20 minutes.It will be understood by those skilled in the art that the Stringency of hybridization can easily be handled, such as, by temperature that the salts contg and/or carry out changing hybridization solution is hybridized.Such as, in another embodiment, the strict hybridization conditions of suitable height comprise above-mentioned those, except hybridization temperature is increased to such as 60 DEG C-65 DEG C or 65 DEG C-70 DEG C.

In certain embodiments, above-mentioned polynucleotide, such as polynucleotide variant, fragment and hybridization sequences, in conjunction with the encoding antibody of KDR or its antigen-binding fragment.In other embodiments, the polynucleotide of these encoding antibodies or antigen-binding fragment or its CDR in conjunction with KDR at least about 50%, at least about 70%, and in certain embodiments, at least about 90% and concrete antibody sequence of setting forth herein.In further embodiment, the polynucleotide of these encoding antibodies or antigen-binding fragment or its CDR with the avidity larger than the antibody of setting forth herein in conjunction with KDR, such as, with at least about 105%, 106%, 107%, 108%, 109% or 110% and herein concrete antibody sequence of setting forth combine quantitatively.

As described elsewhere herein, representative polypeptide (such as, variant KDR-specific antibody provided herein, such as there is the antibody protein of antigen-binding fragment provided herein) the determination of three-dimensional structure can be determined by ordinary method, making one or more aminoacid replacement, increase, disappearance or insert the natural or alpha-non-natural amino acid selected can in fact modeling, with in order to determine whether that structural variant derivative like this retains the space filling performance of disclosed species at present.The known various computer program of those of skill in the art determines aminoacid replacement (or suitable polynucleotide of encoding amino acid sequence) suitable in antibody, makes such as keep avidity or realize better avidity.

Polynucleotide as herein described or its fragment, the no matter length of encoding sequence itself, other DNA sequence dnas can be combined, such as promotor, polyadenylation signal, in addition restriction endonuclease sites, multiple clone site, other coding sections etc., make the change that their total length can be larger.Therefore contain, can use the nucleic acid fragment of almost any length, wherein the easiness that is preferably produced of total length and the purposes in expection recombinant DNA scheme limit.Such as, contain total length about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, the schematic polynucleotide section of about 50 base pairs length etc. (comprising all intermediate lengths) is useful.

When many nucleotide sequences, comparison maximal phase of working as described below is seasonable, if the nucleotide sequence in two kinds of sequences is identical, then two kinds of sequences it is said it is " identical ".Comparison between two kinds of sequences is carried out typically via following manner: relative to comparison window comparative sequences, to identify the regional area similar with comparative sequences.As used herein, " comparison window " refers to the section at least about 20,30 to about 75,40 to about 50 continuous positions, and wherein after the best comparison of two kinds of sequences, sequence can compare the reference sequence of the continuous position of equal amts.

In order to compare, the best comparison of sequence can use the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), utilize default parameter to carry out.This program realizes several comparison charts described in following reference: Dayhoff, M.O. (1978) A model of evolutionary change inproteins – Matrices for detecting distant relationships.In Dayhoff, M.O. (ed.) Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation, WashingtonDC Vol.5, Suppl.3, pp.345-358; Hein J., Unified Approach to Alignment and Phylogenes, pp.626-645 (1990); Methods in Enzymology vol.183, Academic Press, Inc., San Diego, CA; Higgins, D.G.and Sharp, P.M., CABIOS 5:151-153 (1989); Myers, E.W.and MullerW., CABIOS 4:11-17 (1988); Robinson, E.D., Comb.Theor 11:105 (1971); Santou, N.Nes, M., Mol.Biol.Evol.4:406-425 (1987); Sneath, P.H.A.and Sokal, R.R., NumericalTaxonomy – the Principles and Practice of Numerical Taxonomy, Freeman Press, SanFrancisco, CA (1973); Wilbur, W.J.and Lipman, D.J., Proc.Natl.Acad., Sci.USA80:726-730 (1983).

Or, in order to compare, the best comparison of sequence can be undertaken by following manner: Smith and Waterman, the local identity algorithm of Add.APL.Math 2:482 (1981), Needleman and Wunsch, the consistence alignment algorithm of J.Mol.Biol.48:443 (1970), Pearson and Lipman, the inquiry of the similar approach of Proc.Natl.Acad.Sci.USA 85:2444 (1988), computer-implemented (the Wisconsin Genetics SoftwarePackage of these algorithms, Genetics Computer Group (GCG), 575Science Dr., Madison, GAP in WI, BESTFIT, BLAST, FASTA and TFASTA) or by checking.

The preferred example being suitable for a kind of algorithm determining sequence identity and sequence similarities per-cent is BLAST and BLAST 2.0 algorithm, it is described in Altschul et al. respectively, Nucl.Acids Res.25:3389-3402 (1977) and Altschul et al., J.Mol.Biol.215:403-410 (1990).BLAST and BLAST 2.0 such as can use parameter as herein described to determine the percentage of sequence identity between two or more polynucleotide.The software carrying out BLAST analysis obtains by National Center for Biotechnology Information is public.In an illustrative example, for nucleotide sequence, accumulation score (can reward mark for a pair coupling residue by operation parameter M; >0 always) and N (for mismatch residue punishment mark; <0 always) calculate.As the maximum value decline quantity X that accumulative alignment score reaches from it; When cumulative score to drop to owing to accumulating one or more negative point of residue alignments zero or following time; When reaching the end of one of two sequences; Stop the extension of all directions hit word.BLAST algorithm parameter W, T and X determine sensitivity and the speed of this algorithm.The default value word that BLASTN program (for nucleotide sequence) adopts long (W) is 11, expected value (E) is 10, BLOSUM 62 rating matrix is (see Henikoff and Henikoff, Proc.Natl.Acad.Sci.USA 89:10915 (1989)) comparison, B is 50, and expected value (E) is 10, M=5, N=4, and compare two chains.

In certain embodiments, for the best comparison of two sequences, the mensuration of " percentage of sequence identity " should pass through the comparison window of at least 20 positions of two sequences of more best comparison, and the polynucleotide in this comparison window or peptide sequence part can contain 20% or less than the interpolation of, usual 5-15% or 10-12% or disappearance (i.e. space) compared with reference sequence (containing adding or lack).Calculating this per-cent by measuring the identical position number of nucleic acid base, producing the number of matched position, by the total number of positions (i.e. window size) of this matched position number divided by reference sequence, then being multiplied by 100, drawing percentage of sequence identity.

It is appreciated that due to the degeneration of genes encoding, there is the nucleotide sequence of Multi-encoding antibody as described herein in those skilled in the art.Some in these polynucleotide have and encode in conjunction with the minimum sequence identity of the nucleotide sequence of the natural or initial polynucleotide sequence of the antibody of KDR.But the disclosure contains the polynucleotide changed due to the difference of Codon Usage clearly.In certain embodiments, specificity contains the codon optimized sequence expressed for Mammals.

Therefore, in another embodiment of the present invention, mutation scheme such as rite-directed mutagenesis may be used for variant and/or the derivative of preparing antibody as herein described.By the program, the specificity in peptide sequence is modified and can be undertaken by forming the sudden change of their potential polynucleotide.These technology provide simple scheme with preparation and cycle tests variant, such as, are incorporated to one or more above-mentioned considerations by one or more nucleotide sequence is changed introducing polynucleotide.

Rite-directed mutagenesis produces mutant by utilizing specific oligonucleotides sequence, this mutant code has the DNA sequence dna of required sudden change, the contiguous nucleotides of enough numbers provides the primer sequence of enough sizes and sequence complexity, to form the stable duplex across disappearance both sides, joining region.The sudden change in selected polynucleotide sequence is utilized to improve, change, reduce, modify or change the performance of these polynucleotide itself and/or change the performance of its coded polypeptide, activity, composition, stability or primary sequence.

In certain embodiments, the sudden change of the polynucleotide sequence of coding antibody disclosed herein or its antigen-binding fragment is contained in the present invention, such as, to change one or more performances of the polypeptide of coding, the avidity in the Fc district of the binding affinity of antibody or its antigen-binding fragment or the function in specific Fc district or specific Fc γ R.Site-directed mutagenesis technique is well known in the art, and is widely used in the variant producing polypeptide and polynucleotide.Such as, rite-directed mutagenesis is generally used for the specificity portion changing DNA molecular.In these embodiments, use the primer typically comprising about 14 to about 25 Nucleotide or such length, on the both sides of sequence intersection point, about 5 to about 10 residues change.

Those skilled in the art are it is appreciated that site-directed mutagenesis technique often adopts the phage vector of strand and double chain form.Typical carriers for rite-directed mutagenesis comprises carrier such as M13 phage.These phages are easy commercially available acquisitions, and its purposes is generally those skilled in the art and knows.Step target gene being transferred to phage from plasmid is cancelled in the directed sudden change also conventional Double stranded plasmids that adopts.

Usually, the first step of carrying out above-mentioned rite-directed mutagenesis obtains single-stranded vector or makes two of double-stranded vector chains dissociate separately (DNA sequence dna containing the required peptide of coding in its sequence).Preparation carries the Oligonucleolide primers (by synthesis method) of required sudden change complex sequences.Then make this primer and single-stranded vector annealed combination, through archaeal dna polymerase such as E.coli polysaccharase I Klenow fragment effect, complete the synthesis containing sudden change chain.Therefore, an initial non-mutated sequence of chain encoding in the heteroduplex body of formation, Article 2 chain contains required sudden change.Then with the suitable cell of this heteroduplex body vector as Bacillus coli cells, and to select containing the clone carrying mutant nucleotide sequence recombinant vectors.

By a kind of method that the sequence variants of the selected peptide coding DNA section of fixed-point mutation method preparation provides generation to come in handy sequence, but do not mean that the sequence variants and the DNA sequences encoding thereof that are limited to and obtain peptide by these methods.The recombinant vectors of such as available mutations agent peptide sequence as required in azanol process coding is to obtain sequence variants.Detail about these methods and scheme is shown in Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; With Maniatis et al., the instruction of 1982, be in various object, it is incorporated to by way of reference.

As used herein, term " mutation process of oligonucleotide orientation " refers to that template dependant process and carrier mediated propagation raise causing its starting point concentration of concentration ratio of specific nucleic acid molecules, or detectable signal such as the concentration of amplified material raises.As used herein, term " mutation process of oligonucleotide orientation " refers to that the primer molecule of template dependant extends process.Term template dependant molecule refers to the nucleic acid synthesis of RNA or DNA molecular, and the nucleic acid chains sequence of wherein new synthesis is matched rule by the complementary base known and determined (for example, see, Watson, 1987).Typically, carrier mediated method relates to and imports in DNA or RNA carrier by nucleic acid fragment, this carrier of clonal expansion, reclaims the nucleic acid fragment of amplification.The example of these methods is by U.S. Patent No. 4, and 237,224 provide, and it is all incorporated to herein by way of reference specifically.

In the other scheme preparing polypeptide variants, U.S. Patent No. 5 can be used, 837, the recursive sequence restructuring described in 458.In this scenario, the iterative loop carrying out recombinating and screening or select has the independent polynucleotide variant of the binding affinity such as strengthened with " evolution ".Some embodiment also provides plasmid, carrier, transcribe or the construct of expression cassette form, and it comprises at least one polynucleotide as described herein.

In many embodiments, the nucleic acid that coding topic states monoclonal antibody directly introduces host cell, and by cell incubation under the condition being enough to the antibody expression of inducing coding.The antibody of the disclosure uses standard technique well known to those skilled in the art to combine polypeptide provided herein and prepared by nucleotide sequence.Peptide sequence can be used for determining encoding the suitable nucleotide sequence of specific antibodies disclosed herein.According to standard method well known to those skilled in the art, nucleotide sequence can optimize specific cryptosystem " preference " reflecting various expression system.

The embodiment relevant according to some, provides recombinant host cell, and it comprises one or more constructs as described herein; To encode the nucleic acid of any antibody, CDR, VH or VL structural domain or its antigen-binding fragment; With the method for the product of preparation coding, the method comprises the nucleic acid of being encoded and expresses.Can realize expediently expressing by cultivating the recombinant host cell containing nucleic acid under suitable conditions.After expression preparation, antibody or its antigen-binding fragment can use any suitable technology separation and/or purifying, then use as required.

Antibody provided herein or its antigen-binding fragment and coding nucleic acid molecule and carrier can such as be separated and/or purifying from their natural surroundingses with pure or homogeneous form substantially, or in the case of nucleic acids not containing or be substantially free of except coding has nucleic acid or the source gene of the sequence of the polypeptide of desired function.Nucleic acid can comprise DNA or RNA and can all or part ofly be synthesis.Comprise the DNA molecular with particular sequence when relating to the nucleotide sequence of setting forth herein, and comprise the RNA molecule with particular sequence, wherein U replaces T, unless needed explanation in addition.

The system of polypeptide cloning and expressing in various different hosts cell is known.Suitable host cell comprises bacterium, mammalian cell, yeast and rhabdovirus system.Mammal cell line for expressing heterologous polypeptide is that this area is available, comprises Chinese hamster ovary cell, HeLa cell, baby hamster kidney cell, NSO mouse black-in tumor cell and its other multiple.Usually preferred host bacterium is E.coli.

Antibody and the expression of antigen-binding fragment in prokaryotic cell prokaryocyte such as E.coli are well known in the art.In order to summarize, for example, see Pluckthun, A.Bio/Technology 9:545-551 (1991).Those skilled in the art also can obtain the selection as the preparation of antibody or its antigen-binding fragment of eukaryotic expression in the medium, see summarizing in recent years, and such as Ref, M.E. (1993) Curr.Opinion Biotech.4:573-576; Trill J.J.et al. (1995) Curr.Opinion Biotech 6:553-560.

Suitable carrier can be selected or be built, and containing suitable adjustment sequence, comprises promoter sequence, terminator sequence, polyadenylation se-quence, enhancer sequence, marker gene and other sequences (if needs).Carrier can be plasmid, virus such as phage or phagemid (if needs).In order to further details, for example, see MolecularCloning:a Laboratory Manual:2nd edition, Sambrook et al., 1989, Cold Spring HarborLaboratory Press.For handling multiple known technology and the scheme of nucleic acid, such as in nucleic acid construct preparation, sudden change, order-checking, DNA introduced cell and genetic expression and analysis of protein be described in detail in Molecular Biology, Second Edition, Ausubel et al.eds., John Wiley & Sons, in 1992 in current scheme or in upgrading subsequently.

The cell of the nucleotide sequence maybe can introducing one or more antibody described herein of coding wherein introduced in term " host cell " for representing, its further expression maybe can express the target gene of selection, the gene of any antibody described herein of such as encoding.This term comprises the offspring of parental cell, and no matter whether this offspring is same as initial parents on morphology or in gene formation, as long as the gene selected exists.Therefore, the method comprising and these nucleic acid is introduced host cell is also contained.This is quoted to use and anyly obtains technology.For eukaryotic cell, suitable technology can comprise calcium phosphate transfection, DEAE-dextran, electroporation, and using transfection and the transduction of the liposome-mediation of retrovirus or other virus (such as cowpox), is baculovirus for insect cell.For bacterial cell, suitable technology can comprise calcium chloride transformation, the transfection of electroporation and use phage.Be cause or allow expression of nucleic acid after introducing, such as, under for the condition of genetic expression, cultivate host cell.In one embodiment, nucleic acid is introduced in the genome (such as karyomit(e)) of host cell.According to standard method, promote to introduce by the sequence comprising promotion and genome recombination.

In certain embodiments, the present invention also provides such method, and it is included in expression system and uses above-mentioned construct to express specific polypeptide, such as KDR-specific antibody as described herein.Gene is transferred to another kind from a kind of bacterium for representing by term " transduction ", usually passes through phage." transduction " also refers to and obtains and shift eukaryotic cell sequence by retrovirus.Term " transfection " for represent outside or exogenous DNA by the picked-up of cell, and when exogenous DNA is incorporated in film cell quilt " transfection ".Multiple rotaring dyeing technology is well known in the art, and is disclosed herein.Such as, see Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring HarborLaboratories; Davis et al., 1986, BASIC METHODS 1N MOLECULAR BIOLOGY, Elsevier; With Chu et al., 1981, Gene 13:197.These technology can be used for one or more exogenous DNA moieties to introduce in suitable host cell.

As used herein, term " conversion " is the change of the genetic characteristics of phalangeal cell, and when it is modified with during containing new DNA, cell transforms.Such as, if it is by from its native state genetic modification, cell is converted.After transfection or transduction, transfering DNA can be incorporated in the karyomit(e) of cell by physics and recombinate with the DNA of its cell, or can remain episomal element temporarily and can not be replicated, or can be used as plasmid and copy independently.When the partial replication of DNA by cell, cell is considered to stably be transformed.When materials for binding biological (as nucleic acid molecule, polypeptide, host cell etc.) uses, term " naturally occurring " or " natural " refers in natural middle discovery with not by material that people handles.Similarly, as used herein, " non-natural exists " or " non-natural " refer to material not in natural middle discovery or by people's structural modification or synthesis.

Term " polypeptide ", " albumen " and " peptide " and " glycoprotein " use interchangeably, and refer to the amino acid polymer not limiting any length-specific.Term does not get rid of modification, the increase of such as myristylation, sulfation, glycosylation, phosphorylation and signal sequence or disappearance.Term " polypeptide " or " albumen " refer to one or more amino acid chain, wherein each chain comprises the amino acid connected by covalent peptide bonds, and wherein said polypeptide or albumen can comprise non-covalent and/or connect multiple chain by covalent peptide bonds, it has native protein sequence, namely albumen by natural existence and specifically non-recombinant cell or genetically engineered or reconstitution cell produce, and comprise the aminoacid sequence with native protein molecule or disappearance, increase and/or replace the amino acid whose molecule of one or more native sequences.Term " polypeptide " and " albumen " specifically comprise the antibody in conjunction with KDR of the present disclosure, or lack, increase and/or replace the amino acid whose sequence of one or more Anti-KDR antibody.Therefore, " polypeptide " or " albumen " can comprise one (being called " monomer ") or multiple (being called " polymer ") amino acid chain.

The term that relates to herein " albumen be separated " refer to topic state albumen (1) containing at least some other typically at the albumen of natural middle discovery, (2) other albumen from identical sources are substantially free of, such as from the albumen of same species, (3) by the cell expressing from different plant species, (4) from its be associated in essence at least about 50% polynucleotide, lipid, be separated in carbohydrate or other materials, (5) unconnected (covalently or non-covalently interacting) a part of albumen (" albumen be separated " is associated in essence), (6) polypeptide that (covalently or non-covalently interacting) does not associate in essence is operationally associated, or (7) are natural can not produce.The albumen of this separation can by genomic dna, and cDNA, mRNA or other RNA encode, and it can be synthetic source or any combination.In certain embodiments, the albumen of separation is not contained in the albumen or polypeptide or other pollutents that find in its natural surroundings substantially, and interference uses by it (treat, diagnose, prevent, study or other).

Term " polypeptide fragment " refers to it can is monomer or high molecular polypeptide, and its amino terminals lacks, carboxy-terminal deletion, and/or inside lacks or replaces polypeptide that is naturally occurring or restructuring preparation.In certain embodiments, polypeptide fragment can comprise the amino acid chain of at least 5 to about 500 amino acid lengths.In certain embodiments, it is to be appreciated that fragment at least 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,55,60,65,70,75,80,85,90,95,100,110,150,200,250,300,350,400 or 450 amino acid lengths.Specific useful polypeptide fragment comprises functional domain, comprises antigen-binding domain or the fragment of antibody.When Anti-KDR antibody, useful fragment includes but not limited to: the CDR3 district of CDR district, particularly heavy chain or light chain; The variable region of heavy chain or light chain; Part antibody chain or only its variable region (comprising two CDR) etc.

Polypeptide can comprise signal (or leader) sequence in the N-terminal of albumen, and it is translated ground altogether or translates directed transfer protein afterwards.Polypeptide can also frame endomixis or put together joint or other sequences to be easy to synthesis, purifying or to differentiate polypeptide (such as poly-His), or strengthens the combination of polypeptide and solid carrier.

Peptide linker/spacer sequence also can be used for by being enough to guarantee that the distance that each polypeptide is folded into its secondary and/or three grades of results (if needs) is separated multiple polypeptides component.This peptide linker sequence can use standard technique well known in the art to introduce in fusion polypeptide.

Some peptide spacer sequence can such as be selected based on following: (1) adopts the ability of the conformation extended flexibly; (2) adopt can with the functional epitope on the first and second polypeptide their ability of interactional secondary structure; And/or (3) lack the hydrophobicity or charged residues that can react with polypeptide functional epi-position.

In an illustrative embodiments, peptide spacer sequence contains such as Gly, Asn and Ser residue.Other contiguous neutral amino acidss such as Thr and Ala also can be included in spacer sequence.

Other aminoacid sequences that usefully can be used as spacer comprise Maratea et al., Gene 40:3946 (1985); Murphy et al., Proc.Natl.Acad.Sci.USA 83:82588262 (1986); U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751, disclosed in 180 those.

Other schematic spacers can comprise such as Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (Chaudhary et al., 1990, and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp (Bird et al. Proc.Natl.Acad.Sci.U.S.A.87:1066-1070), 1988, Science 242:423-426).

In some embodiments, when the first and second polypeptide have non-essential N-terminal amino acid region (can be used for separation function structural domain and prevent steric influence), spacer sequence is not needed.Two kinds of encoding sequences can directly merge and without any spacer or use flexible poly-joint (being such as made up of the pentamer Gly-Gly-Gly-Gly-Ser of repetition 1 to 3 time).This spacer is used for building single-chain antibody (scFv) by following manner: be inserted in (Bird et al., 1988, Science 242:423-426 between VH and VL; Huston et al., 1988, Proc.Natl.Acad.Sci.U.S.A.85:5979-5883).

In certain embodiments, peptide spacer designed to be able to and carry out correct interaction between two β-sheet of the variable region forming single-chain antibody.

In some illustrative embodiments, peptide spacer is the amino acid of 1 to 5 amino acid, 5 to 10 amino acid, 5 to 25 amino acid, 5 to 50 amino acid, 10 to 25 amino acid, 10 to 50 amino acid, 10 to 100 amino acid or any interference ranges.

In other illustrative embodiments, peptide spacer comprises about 1, and 5,10,15,20,25,30,35,40,45,50 or longer amino acid whose length.

Contain the amino acid sequence modifications of antibody as herein described.Such as, binding affinity and/or the other biological performance of improving antibody can be expected.Such as, the amino acid sequence variation of antibody is prepared by following manner: the polynucleotide change of suitable Nucleotide being introduced encoding antibody or its A chain, or peptide symthesis.This modification comprise such as in the aminoacid sequence of antibody disappearance and/or insert and/or replace residue.Any combination can carrying out lacking, insert and replacing is to realize final antibody, and condition is that final construct has desired characteristic (such as combining with the high affinity of KDR).Amino acid change can also change the rear translation process of antibody, such as, change quantity or the position of glycosylation site.For polypeptide of the present invention above-mentioned any change and modify can be included in antibody of the present invention.

The disclosure provides the variant of antibody disclosed herein.In certain embodiments, these variant antibodies or antigen-binding fragment or its CDR in conjunction with KDR at least about 50%, at least about 70%, and in certain embodiments, at least about 90% and concrete antibody sequence of setting forth herein.In further embodiment, these variant antibodies or antigen-binding fragment or its CDR with the avidity larger than the antibody of setting forth herein in conjunction with KDR, such as with at least about 105%, 106%, 107%, 108%, 109% or 110% and herein concrete antibody sequence of setting forth combine quantitatively.

In certain embodiments, topic is stated antibody and can be had: a) variable region of heavy chain of aminoacid sequence and Anti-KDR antibody as herein described has at least 80% consistence, at least 95% consistence, at least 90%, at least 95% or at least 98% or 99% conforming variable region of heavy chain; With the variable region of light chain of b) aminoacid sequence and Anti-KDR antibody as herein described has at least 80% consistence, at least 85%, at least 90%, at least 95% or at least 98% or 99% conforming variable region of light chain.The aminoacid sequence of schematic heavy chain and light chain region is set forth in SEQ ID NO:1,2,9 and 10.

In certain embodiments, antibody can comprise: a) variable region of heavy chain comprises: the CDR1 district that i. aminoacid sequence is identical with the heavy chain CDR1 district of the antibody of selection as herein described; Ii. the CDR2 district that aminoacid sequence is identical with the heavy chain CDR2 district of the antibody of selection; The CDR3 district identical with the heavy chain CDR3 district of the antibody of selection with iii. aminoacid sequence; And b) light variable domains, comprise: the CDR1 district that i. aminoacid sequence is identical with the light chain CDR1 district of the antibody of selection; Ii. the CDR2 district that aminoacid sequence is identical with the light chain CDR2 district of the antibody of selection; The CDR3 district identical with the light chain CDR3 district of the antibody of selection with iii. aminoacid sequence; Wherein antibodies specific combines the target (such as KDR) selected.In further embodiment, antibody or its antigen-binding fragment are variants, antibody, and wherein variant comprises except at the most 8 in the CDR district in VH and VL district, 9,10,11,12, the heavy chain that 13,14,15 or more aminoacid replacement are identical with the antibody of selection and light chain.In this, can be 1,2 in the CDR district of the antibody selected, 3,4,5,6,7,8, or 9,10,11,12,13,14,15 more aminoacid replacement in certain embodiments.Can carry out in the CDR in VH and/or VL district replacing (for example, see Muller, 1998, Structure 6:1153-1167).

Representative polypeptide (such as, variant KDR-specific antibody provided herein, the antibody protein with antigen-binding fragment provided herein) the determination of three-dimensional structure can be determined by ordinary method, making one or more aminoacid replacement, increase, disappearance or insert the natural or alpha-non-natural amino acid selected can in fact modeling, with in order to determine whether that structural variant derivative like this retains the space filling performance of disclosed species at present.For example, see Donate etal., 1994Prot.Sci.3:2378; Bradley et al., Science 309:1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc.Nat.Acad.Sci.USA103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Raman et al.Science 327:1014-1018 (2010).Can be used for the example of some other and non-limiting computerized algorithms of these and related embodiment, such as the appropriate design of KDR-specific antibody antigen-binding domain provided herein, comprise: VMD, it uses 3-D image and build script to show, build and analyze Molecular Visualization program compared with mcroorganism analysis system (see the website ks.uiuc.edu/Research/vmd/ of Theoretical and Computational BiophysicsGroup, University of Illinois at Urbana-Champagne.Other computer programs multiple are known in the art, and can obtain for those of skill in the art, and its permission determines atomic size from the space-filling model (van der Waals radius) of energy-minimized conformations; GRID, it seeks the high affinity region determining different chemical group, thus strengthens combination; Monte Carlo retrieves, its computational mathematics comparison; And CHARMM (Brooks et al. (1983) J.Comput.Chem.4:187-217) and AMBER (Weiner et al (1981) J.Comput.Chem.106:765), it evaluates field of force computation and analysis (also see Eisenfield et al. (1991) Am.J.Physiol.261:C376-386; Lybrand (1991) J.Pharm.Belg.46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ.Health Perspect.61:185-190; With Kini et al. (1991) J.Biomol.Struct.Dyn.9:475-488).Various suitable computational computer program is also commercially available, such as from (Munich, Germany).

In another embodiment of the present invention, Anti-KDR antibody and humanization version thereof are derived from rabbit monoclonal antibodies, and use especially technology produces.These antibody are favourable, because they require that minmal sequence is modified, thus promote to keep functional performance after humanization technologies's (for example, see U.S. Patent No. 7,462, the 697) humanization using sudden change family guiding (MLG).Therefore, the exemplary process preparing Anti-KDR antibody of the present disclosure comprises rabbit monoclonal antibodies technology, such as, be described in United States Patent (USP) 5,675,063 and 7,429,487.In this, in certain embodiments, Anti-KDR antibody of the present disclosure produces in rabbit.In certain embodiments, the rabbit-source immortality B-lymphocyte of rabbit splenocyte can be merged for generation of hybrid cell (its Dispersal risk).Immortality B-lymphocyte can not express endogenous immunoglobulin heavy chain with detecting, and in certain embodiments can containing the heavy chain immunoglobulin-encoding gene changed.

composition and using method

Disclosure providing package containing the composition of KDR-specific antibody or its antigen-binding fragment and in various Curing circumstance the using of this composition.

Pure form or using of the KDR-specific antibody as herein described in suitable pharmaceutical composition can be implemented by the mode of any acceptance of serving the pharmacy application of similar application.Pharmaceutical composition or can contain the composition of antibody and suitable physiologically acceptable vehicle, thinner or vehicle and prepare by associating antibody, and the preparation of solid, semisolid, liquid or gas form can be formulated as, such as tablet, capsule, powder agent, granule, ointment, solution, suppository, injection, inhalation, gelifying agent, microballoon and aerosol.In addition, can there is (but nonessential) in composition in other drug activeconstituents (comprising other carcinostatic agents herein described in other places) and/or suitable vehicle (such as salt, buffer reagent and stablizer).Can realize using by multiple different approaches, under comprising oral, parenteral, nasal cavity, intravenously, corium, subcutaneous or local.Preferred method of application depends on the character of illness that is to be treated or that prevent.The amount of the development and/or transfer that reduce, suppress, prevent or postpone cancer is after application considered to effective.

In certain embodiments, applied amount is enough to cause tumour to be changed in quality, and indicates by the quantitative statistics of knurl of living reducing significantly (such as tumor quality reduces at least 50%) or changing (such as statistically reducing significantly) scan size.In other embodiments, applied amount is enough to cause, with the clinical relevant minimizing of the conditions of patients that VEGF or KDR expresses and/or activity is relevant, including but not limited to any one in various tumor disease, inflammatory diseases and angiogenesis-associated diseases.Therefore, one or more antibody as herein described of pharmaceutical effective amount are applied the clinical relevant minimizing causing conditions of patients, include but not limited to rheumatoid arthritis, diabetes and ischemic retinopathy, age-relevant macular degeneration, psoriatic and the renal glomerulus relevant to proteinuria hypertrophy, and various tumor disease, comprise angiosarcoma, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma and recurrent glioblastoma multiforme, leukemia and solid tumor.These clinical related symptoms are that skilled practitioners is known, and depend on disease to be treated and change.

The exact dosage desired for the treatment of and time length are the functions of disease for the treatment of, and can use known testing scheme or empirically be determined from its extrapolation by the composition tested in model system known in the art.The clinical trial controlled can also be carried out.Along with the seriousness of illness to be slowed down, dosage also can change.Pharmaceutical composition is formulated and uses to play the upper effective effect for the treatment of usually, makes less desirable minimize side effects simultaneously.Composition can be used once, or can be divided into repeatedly and more low dose ofly to use in break time.For any particular subject, according to individual need, particular dosage regimen can regulate along with the time.

Composition containing KDR-specific antibody can be used alone or in combination in the treatment of other known cancers, such as radiotherapy, amic therapy method, transplanting, immunotherapy, hormonotherapy, photodynamic therapy etc.Composition can also combined with antibiotic be used.

Therefore, the typical route of administration of these and relevant pharmaceutical composition include but not limited to oral, locally, transdermal, suction, parenteral, in sublingual, cheek, rectum, vagina and nose.As used herein, term parenteral comprises subcutaneous injection, intravenously, intramuscular, breastbone inner injection or infusion techniques.The activeconstituents bioavailable when composition being applied to patient making wherein to contain is formulated according to the pharmaceutical composition of certain embodiments of the present invention.The composition being applied to experimenter or patient can be adopted the form of one or more dose units, wherein such as tablet can be single dosage unit, and the container of the KDR-specific antibody of aerosol form described herein can keep multiple doses unit.The practical methods preparing these formulations is that those skilled in the art are known or obvious; For example, see Remington:The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacyand Science, 2000).Under any circumstance, composition to be administered contains the antibody of the present disclosure of pharmaceutical effective amount to come therapeutic goal disease or illness according to instruction herein.

Pharmaceutical composition can be the form of solid or liquid.In one embodiment, vehicle is particle, makes composition be such as tablet or powder type.Vehicle can be liquid, and wherein composition is such as oral oil, injectable liquids or aerosol, and it can be used for such as sucking using.When expecting Orally administered, pharmaceutical composition is preferably solid or liquid form, and wherein semisolid, semiliquid, suspendible and gel form are included in consideration form herein as solid or liquid.

As oral solids composition, pharmaceutical composition can be configured to powder, particle, compressing tablet, pill, capsule, Chewing gum, thin slice etc.This solids composition typically will contain one or more inert diluents or edible vehicle.In addition, can exist following in one or more: tackiness agent, such as carboxymethyl cellulose, ethyl cellulose, Microcrystalline Cellulose, tragacanth gum or gelatin; Excipients is as starch, lactose or dextrin; Disintegrating agent is Lalgine, sodium alginate, Primogel, W-Gum etc. such as; Lubricant such as Magnesium Stearate or Sterotex; Glidant is colloidal silica such as; Sweetening agents is as sucrose or asccharin; Flavoring agent is as peppermint, wintergreen oil or tangerine taste seasonings; And tinting material.When pharmaceutical composition is capsule (such as gelatine capsule) form, except the material of the above-mentioned type, it can contain liquid vehicle such as polyoxyethylene glycol or oil.

Pharmaceutical composition can be liquid form, such as elixir, syrup, solution, emulsion or suspensoid.As two examples, liquid may be used for oral or injected delivery.When expecting oral, except this compound, preferred composition contain in sweeting agent, sanitas, dyestuff/tinting material and odorant one or more.Expecting in the composition that injection is used, can comprised one or more in tensio-active agent, sanitas, wetting agent, dispersion agent, suspensoid, buffer reagent, stablizer and isotonic agent.

If they are the forms such as solution, suspension, composition of liquid medicine can comprise in further auxiliary one or more: sterile diluent, such as water for injection, the preferred physiological saline of salt brine solution, Ringer ' s solution, isotonic sodium chloride, fixed oil such as synthesize list or two glyceryl ester (can be used as solvent or suspension medium), polyoxyethylene glycol, glycerine, propylene glycol or other solvents; Antiseptic-germicide such as benzyl alcohol or nipagin; Antioxidant such as xitix or sodium pyrosulfate; Sequestrant is ethylenediamine tetraacetic acid (EDTA) such as; Buffer reagent such as acetate, Citrate trianion or phosphoric acid salt and for regulating the medicament such as sodium-chlor or dextrose of toxicity.Parenteral administration can be encapsulated in ampulla, disposable syringe or the multiple dose vials be made up of glass or plastics.Physiological saline is preferred auxiliary agent.Injectable pharmaceutical composition is preferably sterilizing.

Expect that parenteral or oral composition of liquid medicine should contain the KDR-specific antibody of amount disclosed herein, make to obtain suitable dosage.Typically, this amount is at least 0.01% of antibody in composition.When expecting oral, this amount can change into 0.1 of composition weight to about 70%.Some combination of oral medication is containing the antibody of 4% to about 75% of having an appointment.In certain embodiments, according to pharmaceutical composition of the present invention and preparation be prepared as make dilution before parenteral dosage units contain 0.01 to 10 % by weight antibody.

Pharmaceutical composition can be expected to be useful in topical application, and vehicle can comprise solution, emulsion, ointment or gel matrix suitably under these circumstances.Described matrix such as can comprise following in one or more: Vaseline, lanolin, polyoxyethylene glycol, beeswax, mineral oil, thinner such as water and alcohol and emulsifying agent and stablizer.Thickening material can be present in pharmaceutical composition for topical application.If be expected to be useful in transdermal administration, described composition can comprise transdermal patch or iontophoresis device.Pharmaceutical composition can be expected to be useful in rectal administration, and it is the form of such as suppository, incites somebody to action melting in the rectum and discharges medicine.Composition for rectal administration can containing oleaginous base as suitable non-irritating excipient.These matrix include but not limited to lanolin, theobroma oil and polyoxyethylene glycol.

Pharmaceutical composition can comprise multiple material, and it modifies the physical form of solid or liquid dosage unit.Such as, composition can comprise the material forming coated shells around activeconstituents.Form the material inertia typically of coated shells, and can be selected from such as granulated sugar, shellac and other enteric coating reagent.Or activeconstituents can be encapsulated in gelatine capsule.The pharmaceutical composition of solid or liquid form can comprise the medicament in conjunction with antibody of the present invention, thus the sending of ancillary compound.The suitable medicament that can play a role in this volume comprises other mono-clonals or polyclonal antibody, one or more albumen or liposome.Pharmaceutical composition can be made up of dose unit substantially, and described dose unit can be used as aerosol.Term aerosol is for representing the various systems from colloidal state character to the system be made up of pressurized package.Send and can be undertaken by the suitable pump system of liquefaction or gas under pressure or dispensing activeconstituents.Aerosol can be sent with delivering active ingredients in single-phase, two-phase or three-phase system.Aerosol send the container, promotor, valve, sub-container etc. that comprise needs, it can form test kit together.Those skilled in the art can determine preferred aerosol without the need to carrying out test.

Prepared by the method that pharmaceutical composition can be known by pharmaceutical field.Such as, expect to be prepared by following manner by injecting the pharmaceutical composition used: combined packet forms solution containing the composition of KDR-specific antibody as described herein and optional one or more salt, buffer reagent and/or stablizer and Sterile dilution water.Tensio-active agent can be added to promote to form homogeneous solution or suspension.Tensio-active agent is such compound, and itself and antibody compositions noncovalently interact with enhancing antibody stripping or homogeneous suspendible in aqueous delivery system.

Composition can to treat significant quantity to use, and it will depend on many factors to change, and comprises the activity of the chemical compound specifically bind (such as KDR-specific antibody) of use; The metabolic stability of compound effects and length; The age of patient, body weight, probably health, sex and diet; Method of application and time; Excretion rate; Medication combined; The seriousness of specific illness or illness; Therapy is carried out with carrying stating.Usually, the effectively every per daily dose for the treatment of is that (70kg Mammals) about 0.001mg/kg (i.e. 0.07mg) is to about 100mg/kg (i.e. 7.0g); Preferred treatment effective dose is that (70kg Mammals) about 0.01mg/kg (i.e. 0.7mg) is to about 50mg/kg (i.e. 3.5g); Preferred treatment effective dose is that (70kg Mammals) about 1mg/kg (i.e. 70mg) is to about 25mg/kg (i.e. 1.75g).In certain embodiments, those of skill in the art will recognize, can preferably use with milli gram/m (i.e. mg/m ²) dosage that represents.Such as, in order to mg/kg dosage being expressed as equivalent mg/m in any given material ²dosage, makes dosage be multiplied by the suitable km factor.In adult, 100mg/kg is equal to 100mg/kg x 37kg/m ²=3700mg/m ².For example, see FDAguidelines for Industry and Reviewers; Also see Freireich, EJ, et al.Quantitativecomparison of toxicity of anticancer agents in mouse, rat, dog, monkey and man.CancerChemother Rep.1966; 50 (4): 219-244.

The composition comprising KDR-specific antibody of the present disclosure also can while the using of one or more other treatment agent, before or after use.This combination therapy can comprise using of single pharmaceutical dosage formulation, goes containing the compounds of this invention and one or more other active agents, and comprises the using of composition of antibody of the present invention and the active medicine all in himself drug alone dosage particles.Such as, antibody as described herein and other active agents can be combined single oral dose composition (such as tablet or capsule) and are applied to patient, or with each medicament that independent oral dosage formulation is used.Similarly, antibody as described herein and other active agents can be combined and in single parenteral dose composition, are applied to patient, such as, at salt brine solution or the acceptable solution of other physiology, or with each medicament that independent parenteral dose preparation is used.If use individually dosed preparation, the composition comprising antibody and one or more other active agents can be used in (i.e. while) substantially simultaneously or in the independent poor time (namely successively with any order); Combination therapy is interpreted as and comprises all these schemes.

Therefore, in certain embodiments, also contain one or more other treatment agent of associating and use Anti-KDR antibody composition of the present disclosure.This therapeutical agent can be accepted as standard care in the art for specified disease as described herein, the disease of such as inflammatory diseases (such as rheumatoid arthritis or other inflammatory diseasess), various tumor disease and vasculogenesis-mediation (such as but not limited to age-relevant macular degeneration) in any one.The exemplary treatment agent contained comprises cytokine, somatomedin, steroid, NSAID, DMARD, antiphlogistic, chemotherapeutics, radiotherapy dose or other active agents and auxiliary agent.

In certain embodiments, Anti-KDR antibody disclosed herein can be combined any quantity chemotherapeutics and used.The example of chemotherapeutics comprises: alkylating agent, and such as thiophene is for group and endoxan (CYTOXAN ^tM), alkyl sulfonic ester, such as busulfan, improsulfan and piposulfan, ethylenimine, such as benzodopa, carboquone, meturedopa and uredopa, ethyleneimine and trimeric cyanamide comprise altretamine, Tretamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylammonium trimeric cyanamide, mustargen is Chlorambucil such as, Chlornaphazine, ring Glyciphosphoramide, Emcyt, ifosfamide, mustargen, Mechlorethaminoxide Hydrochloride, melphalan, Novoembichin, phenesterin(e), PM, trofosfamide, uracil mustard, nitrosoureas is carmustine such as, chlorozotocin, Fotemustine, lomustine, Nidran, MCNU, microbiotic is aclacinomysins such as, actinomycin, anthramycin, azaserine, bleomycin, sanarnycin, calicheamicin, carabicin, carminomycin, carzinophillin, chromomycins, gengshengmeisu, daunorubicin, detorubicin, 6-diaza-5-oxn-l-norieucin, Zorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, U-15167, Olivomycine, peplomycin, potfiromycin, tetracycline, triferricdoxorubicin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, anti-metabolites is as methotrexate and 5 FU 5 fluorouracil (5-FU), folacin is N10,9-dimethylfolic acid such as, methotrexate, Pteropterin, Trimetrexate, purine analogue is fludarabine such as, Ismipur, ITG, Tioguanine, pyrimidine analogue is cyclotidine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, di-deoxyuridine, doxifluridine, BH-AC, floxuridine, 5-FU such as, male sex hormone is U-22550, Drostanolone propionic salt, Epitiostanol, mepitiostane, testolactone such as, such as Rumi spy, mitotane, Qu Luosi are smooth for anti-suprarenin, folic acid supplement is folic acid such as, aceglatone, aldophosphamide glucosides, aminolevulinic acid, amsacrine, bestrabucil, Bisantrene, edatrexate, defofamine, Omaine, diaziquone, eflornithine, acetic acid elliptinium, Etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethyl hydrazides, hydrazine, PSK.RTM, tetrahydroform, sizofiran, Spirogermanium, tenuazonic acid, triaziquone, 2,2', 2 "-RA3s, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, Gacytosine, Arabinoside (" Ara-C "), endoxan, thiophene is for group, Taxan, such as taxol ( bristol-Myers SquibbOncology, Princeton, N.J.) and DTX ( rhne-Poulenc Rorer, Antony, France), Chlorambucil, gemcitabine, 6-Tioguanine, purinethol, methotrexate, platinum analogs such as Platinol and Carboplatin, vincaleucoblastine, platinum, Etoposide (VP-16), ifosfamide, ametycin, mitoxantrone, vincristine(VCR), Vinorelbine, navelbine, Novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase enzyme inhibitor RFS 2000, α-difluorometylornithine (DMFO), retinoic acid derivatives is Targretin such as ^tM(bexarotene), Panretin ^tM(alitretinoin), ONTAK ^tM(denileukin diftitox), Ai Sibo mycin, capecitabine, with above-mentioned any one pharmacy acceptable salt, acid or derivative.This definition also comprise for regulate or inhibitory hormone to the antihormone agent of the effect of tumour, as estrogen antagonist, comprise such as tamoxifen, raloxifene, aromatase enzyme and suppress 4 (5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); And androgen antagonist, as flutamide, Nilutamide, bicalutamide, Leuprolide and goserelin; With above-mentioned any one pharmacy acceptable salt, acid or derivative.

Multiple other treatment agent can with Anti-KDR antibody conbined usage as herein described.In one embodiment, antibody is used together with antiphlogistic.Antiphlogistic or medicine include but not limited to that steroid and glucocorticosteroid (comprise Betamethasone Valerate, budesonide, dexamethasone, hydrocortisone acetate, hydrogen hydroxyl corticoid, hydrogen hydroxyl corticoid, methyl Bo Nisonglong, Bo Nisonglong, prednisone, Tr), non-steroidal anti-inflammatory drug thing (NSAIDS) comprises acetylsalicylic acid, Ibuprofen BP/EP, Naproxen Base, methotrexate, sulfasalazine, leflunomide, anti-TNF medicine, endoxan and mycophenolate.

The composition comprising KDR-specific antibody described herein can be applied to the individuality suffering from disease as described herein, include but not limited to express with VEGF or KDR and/or active relevant illness, include but not limited to rheumatoid arthritis, diabetes and ischemic retinopathy, age-relevant macular degeneration, psoriatic and the renal glomerulus relevant to proteinuria hypertrophy, and various tumor disease comprises angiosarcoma, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma, with recurrent glioblastoma multiforme, leukemia and solid tumor.For the treatment for human disease in body, antibody as herein described is introduced in pharmaceutical composition usually before administration.Pharmaceutical composition comprises one or more antibody as herein described and the physiologically acceptable vehicle of combining herein described in elsewhere or vehicle.In order to pharmaceutical compositions, one or more compound any pharmaceutical vehicles well known by persons skilled in the art of significant quantity or vehicle, to be suitable for specific method of application.Pharmaceutical vehicles can be liquid, semiliquid or solid.For in parenteral, skin, the solution of subcutaneous or topical application or suspension can comprise such as sterile diluent (such as water), physiological saline, fixed oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetics; Antiseptic-germicide is (benzyl alcohol or nipagin) such as; Antioxidant (such as xitix and sodium pyrosulfate) and sequestrant (such as ethylenediamine tetraacetic acid (EDTA) (EDTA)); Buffer reagent (such as acetate, Citrate trianion and phosphoric acid salt).If intravenously is used, suitable vehicle comprises the salt solution (PBS) of physiological saline or phosphate buffered and contains the solution of thickening material and solubilizing agent (such as glucose, polyoxyethylene glycol, polypropylene glycol and composition thereof).

The composition comprising KDR-specific antibody as described herein can use vehicle to prepare, and described vehicle protection antibody is avoided eliminating fast from body, such as time delivery formulations or dressing.These vehicles comprise controlled release preparation, such as, such as but not limited to transplant and microencapsulated delivery system and biodegradable, biocompatibility macromolecule, ethylene vinyl acetate, polyanhydride, polyglycolic acid, poe, poly(lactic acid) and other materials well known by persons skilled in the art.

The methods for the treatment of used in conjunction with the antibody of KDR is provided herein.In one embodiment, antibody of the present invention has the patient relating to the disease that inappropriate KDR expresses in using, in context of the present disclosure, refer to that the KDR comprising distortion expresses or the disease that characterizes of activity and illness, because due to albumen amount or there is mutain or both changes (such as statistically significance increases or reduces).Because any reason may be excessive, include but not limited to molecular level process LAN, action site extend or accumulation performance or relative to normal detectable activity, KDR increased activity (such as with statistically significance mode).The excessive of this KDR can be measured relative to the activity of normal expression, performance or KDR intracellular signaling event, and described measurement can play a significant role in the exploitation of antibody as herein described and/or setting.

Especially, antibody of the present invention be used for the treatment of express with KDR relevant various.Such as, one embodiment of the invention provide the KDR-specific antibody disclosed herein by using pharmaceutical effective amount to cancer patients to carry out the method for Therapeutic cancer, include but not limited to express with VEGF or KDR and/or active relevant illness, include but not limited to various tumor disease, comprise angiosarcoma, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma, with recurrent glioblastoma multiforme, leukemia and solid tumor.After application, be considered to effective with statistically significance mode (namely relative to suitable contrast well known by persons skilled in the art) suppression, prevention or the delay development of cancer and/or the amount of transfer.

Embodiment provides KDR-specific antibody disclosed herein by using from pharmaceutical effective amount to cancer patients (such as in addition, after application, with statistically significance mode (namely relative to suitable contrast well known by persons skilled in the art) suppression, prevention or postpone cancer metastasis) come preventing cancer transfer method, include but not limited to cancer, include but not limited to various tumor disease, comprise angiosarcoma, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma and recurrent glioblastoma multiforme, leukemia and solid tumor.

Embodiment provides the KDR-specific antibody disclosed herein by using pharmaceutical effective amount to cancer patients to carry out the method for preventing cancer in addition, include but not limited to cancer, include but not limited to various tumor disease, comprise angiosarcoma, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma, and recurrent glioblastoma multiforme, leukemia and solid tumor.

Embodiment provides the KDR-specific antibody disclosed herein using pharmaceutical effective amount to one or more the patient suffered from these diseases to treat in addition, suppress the progress of cancer or the method for prevention, include but not limited to various tumor disease, such as angiosarcoma, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma, with recurrent glioblastoma multiforme, leukemia and solid tumor, and express and/or active other relevant illness with VEGF or KDR, include but not limited to rheumatoid arthritis, diabetes and ischemic retinopathy, age-relevant macular degeneration, psoriatic and the renal glomerulus relevant to proteinuria hypertrophy.

In further embodiment, antibody of the present invention is used for the treatment of various inflammatory diseases.Such as, one embodiment of the invention provide a kind of method for the treatment of inflammatory diseases, include but not limited to express with VEGF or KDR and/or active relevant inflammation, include but not limited to rheumatoid arthritis, diabetes, gout, cold pyrrole quinoline associated period syndrome, chronic obstructive pulmonary disease and various cardiovascular diseases such as arteriosclerosis and vasculitis.Antibody of the present invention is used for the treatment of inflammatory syndrome, it is characterized in that the attack of the aseptic inflammation in joint, serositis, fever and skin lesion.Inflammatory diseases includes but not limited to Crohn's disease, colitis, dermatitis, psoriatic, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematosus, ephritis, Parkinson's disease, ulcerative colitis, multiple sclerosis (MS), Alzheimer, sacroiliitis, rheumatoid arthritis, asthma and various cardiovascular diseases such as arteriosclerosis and vasculitis.In one embodiment, the disclosure provides the composition disclosed herein by using pharmaceutical effective amount to the patient needed to treat, reduce the method for seriousness or preventing inflammation or inflammatory diseases.

In another embodiment, Anti-KDR antibody of the present invention is for determining the structure of the antigen combined, and such as conformational epitope, then such as by chemistry modelization and SAR method, this structure can be used for developing the compound or simulate with this structure.

Other embodiment parts various of the present invention relate to the diagnostic use of the existence of the cell or tissue detecting expressing K DR.Therefore, the disclosure provides the method detecting KDR in sample, such as, detect the cell or tissue of expressing K DR.These methods can the application of various known detection mode, include but not limited to immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), in conjunction with whole mount in situ hybridization (WISH), fluorescent DNA in situ hybridization (FISH), flow cytometry, enzyme immunoassay (EIA) and enzyme-linked immunoassay (ELISA).

ISH is a kind of hybrid method, the complementary DNA of its applying marking or RNA chain (i.e. elementary bonding agent) to locate specific DNA or RNA sequence (original position) in the part or section of cell or tissue, if or organize enough little, all tissues (combining overall).Those skilled in the art will recognize that this is different from immunohistochemistry, it uses antibody as promotion bonding agent positioning protein in organizing segments.DNA ISH is used for genomic dna to determine chromosomal structure.Fluorescent DNA ISH (FISH) can such as medical diagnosis to evaluate karyomit(e) integrity.RNA ISH (hybridization histochemistry) is at organizing segments or in conjunction with whole in-vivo measurement and location mRNAs and other transcriptions.

In multiple embodiments, the detectable mark that antibody conjugate as herein described can directly or indirectly detect.In this, antibody " conjugate " refers to the Anti-KDR antibody of covalently bound detectable mark.In the present invention, DNA probe, rna probe, monoclonal antibody, its antigen-binding fragment and antibody derivatives thereof the antibody of fragment antibody or epitope tag (such as strand is variable-) can whole covalently bound detectable marks.In " direct-detection ", only use a kind of detectable antibody, i.e. elementary detectable antibody.Therefore, but direct-detection refers to that the antibody self puting together detectable mark detects, and without the need to increasing second antibody (secondary antibodies).

" detectable mark " is molecule or the material that can produce detectable (such as naked eyes, electronics or other modes detect) signal, the existence marked in described signal designation sample and/or concentration.When conjugation of antibodies, detectable mark can be used for the target of location and/or quantitative specific antibody orientation.Thus, the existence that hits of sample and/or concentration can detect by detecting the signal that produced by detectable mark.Detectable mark can directly or indirectly detect, and put together different specific antibody several different detectable marks can conbined usage to detect one or more targets.

The example of the detectable mark that can directly or indirectly detect comprises fluorescence dye and radioactive substance and metallic particles.On the contrary, indirect detection needs to apply one or more other antibody, i.e. secondary antibodies after application primary antibody.Therefore, described detection is carried out in the combination by detecting secondary antibodies or bonding agent and elementary detectable antibody.The example of the elementary detectable bonding agent or antibody adding secondary bonding agent or antibody is needed to comprise the detectable bonding agent of enzyme and the detectable bonding agent of haptens or antibody.

In some embodiments, detectable mark puts together the nucleic acid polymer (such as in ISH, WISH or FISH method) comprising the first bonding agent.In other embodiments, detectable mark puts together the antibody (such as in IHC method) comprising the first bonding agent.

The example can puting together the detectable mark of the antibody used in method of the present disclosure comprises fluorescent mark, enzyme labelling, radio isotope, chemiluminescent labeling, electrochemiluminescence mark, biological fluorescent labelling, polymer, polymeric particles, metallic particles, haptens and dyestuff.

Fluorescently-labeled example comprises 5-(with 6)-Fluoresceincarboxylic acid, 5-or 6-Fluoresceincarboxylic acid, 6-(fluorescein)-5-(with 6)-carboxyamido caproic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, with dyestuff (such as Cy2, Cy3 and Cy5), the tonka bean camphor optionally replaced comprises AMCA, PerCP, phycobiliprotein comprises R-PE (RPE) and allophycoerythrin (APC), texas Red, Princeton is red, green fluorescent protein (GFP) and analogue thereof, with the conjugate of R-PE or allophycoerythrin, inorganic fluorescent marks the particle of such as based semiconductor material, such as coated CdSe nano microcrystalline.

The example of polymeric particles mark comprises particulate or the latex particle of polystyrene, PMMA or silicon-dioxide, and it can chimeric fluorescent dyestuff or the macromolecule micelle containing dyestuff, enzyme or substrate or capsule.

The example of metallic particles mark comprises the gold grain of gold grain and coating, and it can lead to argentation to transform.Haptenic example comprises DNP, fluorescein isothiocyanate (FITC), vitamin H and digoxigenin.The example of enzyme labelling comprises horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), beta-galactosidase enzymes (GAL), G-6-P ester desaturase, β-N-acetylglucosaminidase, beta-Glucuronidase, saccharase, XOD, Photinus pyralis LUC and glucose oxidase (GO).Example for the normally used substrate of horseradish peroxidase comprises 3, 3'-diaminobenzidine (DAB), the diaminobenzidine that nickel strengthens, AEC (AEC), benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), Indophane indigo plant (IB), tetramethyl benzidine (TMB), the chloro-1-naphthols (CN) of 4-, naphthyl alcohol pyronin (α-NP), o-dianisidine (OD), the chloro-3-indolyl phosphate (BCIP) of the bromo-4-of 5-, nitroblue tetrazolium (NBT) diformazan (NBT), 2-(p-iodine substituted phenyl)-3-p-oil of mirbane-l-5-tetraphenylphosphonium chloride tetrazolium (INT), tetranitro tetrazole indigo plant (TNBT), the bromo-4-of 5-chloro-3-indoxyl base-β-D-galactoside/ferroferricyanide (BCIG/FF).

Example for the normally used substrate of alkaline phosphatase comprises naphthols-AS-B 1-phosphoric acid ester/fast red TR (NABP/FR), naphthols-AS-MX-phosphoric acid ester/fast red TR (NAMP/FR), naphthols-AS-B1-phosphoric acid ester/-fast red TR (NABP/FR), naphthols-AS-MX-phosphoric acid ester/fast red TR (NAMP/FR), naphthols-AS-B1-phosphoric acid ester/new magenta (NABP/NF), bromine chloro-indole based phosphates/nitroblue tetrazolium(NBT) (BCIP/NBT), the chloro-3-indyl of the bromo-4-of 5--b--d-galactopyranoside (BCIG).

The example of luminescence mark comprises luminol, isoluminol, acridinium ester, 1,2-dioxetane and pyrido pyridazine.The example of electrochemiluminescence mark comprises ruthenium derivative.Radiolabeled example comprises the radio isotope of iodine, cobalt, selenium, tritium, carbon, sulphur and phosphorus.

Detectable mark can connect any other molecule of antibody as herein described or specific binding target organism mark, such as antibody, nucleic acid probe or polymer.And, one of skill in the art will appreciate that detectable mark also can put together second and/or the 3rd and/or the 4th and/or the 5th bonding agent or antibody etc.And, one of skill in the art will appreciate that each other bonding agent or antibody for identifying target organism mark can be used as amplification of signal step.Biomarker can use such as opticmicroscope, fluorescent microscope, electron microscope (detectable material be such as dyestuff, colloid gold particle, luminescence reagent) to carry out naked eyes and detect.The detectable material of naked eyes in conjunction with biomarker can also use spectrophotometer to detect.If detectable material is radio isotope, detection can be undertaken by radioautography naked eyes, or uses the non-naked eyes of scintillometer to carry out.For example, see Larsson, 1988, Immunocytochemistry:Theory and Practice, (CRC Press, Boca Raton, Fla.); Methods inMolecular Biology, vol.801998, John D.Pound (ed.) (Humana Press, Totowa, N.J.).

The present invention also provides the test kit of the cell or tissue detecting KDR or expressing K DR in the sample to which, and wherein test kit contains: at least one antibody, polypeptide, polynucleotide, carrier or host cell as described herein.In certain embodiments, test kit can contain buffer reagent, enzyme, mark, substrate, pearl or be attached other surfaces etc. of antibody of the present invention; And working instructions.

Embodiment

Embodiment 1

The preparation of Anti-KDR antibody and humanization

The generation of Anti-KDR antibody

By New Zealand white rabbit restructuring KDR-rabbit Fc fusion protein immunization.The rabbit with the highest serum titre of specific binding people KDR is selected for hybridoma and produces.230 kinds of hybridomas are accredited as in conjunction with solubility KDR-Fc positive altogether.At these in 230, by using KDR-transfectant 293 cell, 100 kinds of clones also find in conjunction with cell surface KDR positive.Two positive hybridoma is selected for further functional characterization.

the functional screening of hybridoma

Block the screening of the functional antibodies of the combination of KDR and VEGF: evaluate the ability in conjunction with the combination of suppression KDR and the VEGF of the two positive Anti-KDR antibody (100 clones) of solubility KDR and cell surface KDR.In 100 kinds of anti-KDR clones, 41 kinds of discoveries show consistence and recombinant expressed, and purifying is used for characterizing further.Suppress the effect of the best 10 kinds of Anti-KDR antibodies of the combination of KDR and VEGF to be summarized in table 2 below, and be shown in Fig. 1.

Table 2: the IC50 of Anti-KDR antibody

Antibody	IC50(nM)
		15	17.48
23	21.37
		24	19.83
27	5.95
		36	6.25
43	7.54
		71	9.94
81	4.56
		83	9.77
92	32.94

Suppress the antibody screening of KDR phosphorylation: measure based on ligand receptor combination and the best 10 kinds of Anti-KDR antibodies of selection use HUVEC cells to test further in cell based KDR phosphorylation assay.Use Anti-KDR antibody at 37 DEG C, to process 1 hour with 5 μ g/ml (relative saturation dosage) in the HUVEC cell cultivated, add the people VEGF (R & D System) of 20ng/ml (optimal stimulus dosage) afterwards.The cell lysate of results is carried out quantification by protein concentration.The phosphorylation of KDR uses fluor enzyme linked immunosorbent assay to determine, this test uses rabbit anti-KDR YK-5 (the functional anti-KDR clone of non-) as can not antibody and the anti-phosphorylated tyrosine of mouse, and P-Tyr-100 (cell signal Transfer Technology) is as detecting antibody.The best 6 kinds of clones of KDR phosphorylation are suppressed to be shown in Fig. 2.In best 6 kinds of clones, clone 36 shows the most high inhibition (Fig. 2 A) of the KDR phosphorylation that VEGF-stimulates.The fluor ELISA result of clone 36 uses anti-fluor-KDRY996 polyclonal antibody (Epitomics, Burlingame, CA) to be confirmed further (Fig. 2 B) by Western blot.

The screening of cross species Anti-KDR antibody: in order to evaluate the effect blocking KDR therapy in zooscopy in vivo, wherein KDR only expresses on the endotheliocyte of host, best 6 kinds of Anti-KDR antibodies are screened for the cross reactivity with mouse KDR.Antibody #27, #36, #43, #68, #83 find and people and mouse KDR have cross reactivity (data are not shown).Table 3 is summarized in the three groups of rabbit antibody identified in screening below: 1) block KDR phosphorylation and also with the Anti-Human KDR antibody of mouse KDR cross reaction, comprise clone 4,6,14,27,30,36,68,69,81,83,91 and 95; 2) block KDR phosphorylation but the Anti-Human KDR antibody of discord mouse KDR cross reaction, comprise clone 5,13,15,17,23,24,25,43,71,77,92 and 93; 3) and mouse KDR cross reaction but do not block the Anti-Human KDR antibody of KDR phosphorylation, clone 40,42 and 50 is comprised.

Table 3: anti-KDR rabbit antibody general introduction

Figure 11 illustrates the comparison in VH and the VL district of Anti-KDR antibody.CDR marks underscore.VHCDR1 aminoacid sequence is provided in SEQ ID NO:69-95; VHCDR2 aminoacid sequence is provided in SEQ ID NO:96-122; VHCDR3 aminoacid sequence is provided in SEQ ID NO:123-149; VLCDR1 aminoacid sequence is provided in SEQ IDNO:150-176; VLCDR2 aminoacid sequence is provided in SEQ ID NO:177-203; VLCDR3 aminoacid sequence is provided in SEQ ID NO:204-230.

Based on the effect of the anti-phosphorylation of people KDR and the cross reactivity with mouse KDR, clone 36 is selected as first candidate's Anti-KDR antibody.

Restructuring Anti-KDR antibody clone 36

Pcr amplification is passed through from the DNA fragmentation of the L chain of the rabbit igg of clone 36 and the variable region (VH) of H chain.The fragment of L chain is cloned in pTT5 carrier at Hind III and Not I site, and VH fragment is cloned in the constant region of the H chain set up in pTT5 carrier at Hind III and Kpn I site.For each hybridoma, three kinds of DNA clones of L or H chain are sequenced, and the plasmid with consensus sequence is identified and for recombinant expressed.In order to expressing recombinant antibody.The plasmid co-transfection of L and H chain is in 293-6E cell (National Research Council Canada).Gather in the crops supernatant after 5 days, and use enzyme linked immunosorbent assay to come quantitatively to measure front IgG concentration with measurement function.

humanization design

Sudden change connects (MLG) humanization technologies guided and is used for making leading clone 36 humanization.First, heavy chain (VH) and light chain (VK) variable region sequences of cloning 36 divide (blast) for people germline VH and VK database.Immediate human germ line sequences is accredited as humanization template.Secondly, the rabbit residue in framework region relates to CDR contact potentially, or in chain, contact is identified based on the knowledge from people and rabbit antibody.Be considered to for the non-key residue of the structure-activity of antibody based on from humanized rabbit antibody before knowledge and identify.

The light chain of humanization 36 (APX004) and heavy chain framework and human germ line sequences have the consistence of 93%.Except the humanization of framework, MLG method allows our further humanization and human germ line sequences to have CDR1 and CDR2 of the heavy chain of 47% to 58% homology.APX004 and KDR is found to be similar to its parent's rabbit monoclonal antibodies 36 (see Fig. 4) in conjunction with effect.The clone humanization VH of 36 and the aminoacid sequence in VL district are set forth in SEQ ID NO:9 and 10 respectively.The aminoacid sequence of VHCDR1 and VHCDR2 of humanization clone 36 is set forth in SEQ ID NO:11 and 12.The aminoacid sequence that humanization clones the VHCDR3 of 36 Anti-KDR antibodies (APX004) is identical with the parent VHCDR3 set forth in SEQ ID NO:5.Humanization clones the VLCDR1 of 36 Anti-KDR antibodies (APX004), and the aminoacid sequence of VLCDR2 with VLCDR3 is identical with the rabbit parent VLCDR sequence set forth in SEQ ID NO:6-8.

the expression of humanization clone

The DNA of humanization VK and VH of coding clone R-36 is by MCLab (South San Francisco, CA, USA) synthesis.DNA fragmentation comprises the Kozak sequence that signal peptide and 5 ' is held.In order to the humanization version of cloning by expression 36, humanization VK fragment is cloned at Hind III and Nhe I in the people CK built in pTT5 carrier.Humanization VH is cloned in the human IgG1 CH1 built in pTT5 carrier at Hind III and BsiW I site.The DNA of people CK and aminoacid sequence (being respectively SEQ ID NO:13 and 14) and IgG1CH1 (being respectively SEQ ID NO:15 and 16) are selected for constant region.The humanization version of clone 36 is at 293-6E cells, also quantitative by UV280 by protein A column purification after dialysing for PBS damping fluid.

Embodiment 2

APX004 in conjunction with selectivity

APX004 is the humanization IgG1 antibody for KDR (VEGFR2).Its high affinity (Kd=5.3x10 ^-11m) combine and specific binding people KDR.APX004 and monkey and mouse KDR cross reaction.APX004 blocks the combination of KDR and VEGF, and suppresses KDR phosphorylation, thus causes suppressing KDR downstream signal transmission and biological function, such as endothelial cell proliferation and vasculogenesis.

APX004 evaluates by ELISA is used for VEGFR family protein in conjunction with selectivity.The rabbit Fc-fusion rotein of people KDR, the mouse KDR of 1 μ g/ml, human VEGFR-3 1, human VEGFR-3 3, human OX 40 L and people VEGF is coated on elisa plate altogether, and the APX004 of then with 1 μ g/ml is hatched.In conjunction with the APX004 IgG that uses goat anti-human HRP-to put together detect.

As shown in Figure 3, APX004 selective binding people and mouse KDR, but not in conjunction with other people VEGFR family protein or VEGF.Therefore, APX004 can effect and PK research in test body in mouse tumor model.Because monkey KDR and people KDR has the sequence identity (only having conserved amino acid difference in non-ligand binding domains) of 99.9% in extracellular domain, therefore APX004 should be able to identify monkey KDR.

Embodiment 3

APX004 blocks the combination of KDR and VEGF

ELISA-base KDR-VEGF combines to measure and is developed and blocks the APX004 of KDR in conjunction with VEGF for evaluating.2 μ g/ml VEGF are coated on elisa plate altogether.APX004, parent rabbit antibody R-36 or IgG1 Isotype control antibodies preincubate together with 5 μ g/ml recombinant human KDR, transfers to the elisa plate that VEGF-A is coated afterwards.KDR in conjunction with fixing VEGF is detected by the anti-KDR monoclonal antibody of mouse, then adds the goat anti mouse IgG (Fisher Scientific/Pierce Biotechnology, Rockford, IL) puting together alkaline phosphatase.By elisa plate and p-nitrophenyl phosphate Substrate development, and be recorded in the absorbancy at 405nm place.All experiments carry out three times.

As shown in Figure 4, APX004 blocks the combination of KDR and VEGF effectively with IC502.6nM.The humanization of R-36 does not affect it and suppresses KDR in conjunction with the effect of VEGF.

Embodiment 3

APX004 suppresses KDR phosphorylation

In order to evaluate the restraining effect of the KDR phosphorylation that APX004 induces for VEGF-, by the HUVEC cell preincubate 1h of the antibody of various concentration and cultivation, increase the people VEGF (R & D System) of 5nM afterwards.KDR phosphorylation is detected by Western blot.Cell lysate from the HUVEC cell of the process of the albumen containing equivalent is resolved on 4 – 20%SDS – PAGE gels, and by protein delivery to pvdf membrane (Millipore, Billerica, MA).By stain successively with anti-fluor-KDR antibody, all KDR antibody and anti-alpha-tubulin antibody (Epitomics) detection.Primary antibody divests by washing in glycine buffer before the other primary antibody pre-detection of use.After the film of pollution and suitable horseradish peroxidase-put together secondary antibodies (FisherScientific/Pierce Biotechnology) being hatched, then using ECL reagent development (GE HealthcareBio-Sciences), signal specific is visual in X-ray film.As shown in Figure 5, APX004 suppresses the KDR phosphorylation of VEGF induction.

Embodiment 4

APX004 suppresses HUVEC propagation

In order to measure the inhibition of the HUVEC propagation that APX004 induces for VEGF-, by the APX004 of various concentration with in 96-orifice plate 4,000 cells/well adds HUVEC substratum, and hatches 1 hour, adds the VEGF (ultimate density) of 15ng/ml afterwards.HUVEC cell hatches 72 hours further at 37 DEG C, afterwards by 10% add each hole.After hatching at 24 hours, HUVEC cell viability reads fluorescence intensity to measure under the excitation of 530nm and the transmitting at 590nm place by using WallacVictor V 1420Multilabel HTS counter (PerkinElmer).All measurements all carry out three times.As shown in Figure 6, APX004 suppresses HUVEC propagation with dose-dependent manner.

Embodiment 5

The suppression of tumor growth in people lung cancer H460 model

APX004 is APX004 and mouse KDR cross reaction relative to an advantage of Ramucirumab.Therefore, APX004 directly can evaluate in the people's tumor xenograft models in mouse.In the body of APX004, anti-vasculogenesis and Anti-tumor effect show in many tumor xenograft models.

In order to evaluate effect in the body of APX004 in people H460 xenograft models, people NSCLC tumour H460 (KDR is negative) heterograft is by setting up people NSCLC clone H460 subcutaneous vaccination to the back side of female BAl BIc/c nude mice.When reaching average-volume 200mm 22 days tumor size ³time, carry the mouse of tumour random as represented by be divided into 3 treatment group (n=8-10).Then, random groups is to accept APX004 or Avastin that ip injects 5mg/kg/ dosage 3 times/week, 8 dosage altogether.Gross tumor volume and body weight is calculated: volume=(width) according to following equalities ²x length/2, every 2 to 3 days (Fig. 7 A).Carry out photo-optics (x400) to using the immunohistochemistry of anti-CD34mAb dyeing and send (brown) (Fig. 7 B) to describe microvasculature.

When stopping research (41 days), APX004 shows Anti-tumor activity (77% inhibiting rate) more more effective than Avastin (69% inhibiting rate).APX004 process causes tumor microvasculature to reduce (CD34 ⁺eC dyes).APX004 does not show remarkable toxicity (namely not changing relative to control group body weight).

Embodiment 6

Dose response in people lung cancer H460 model suppresses

In order to determine the effective dose of APX004 in people H460 xenograft models, people NSCLC tumour H460 heterograft is by setting up people NSCLC clone H460 subcutaneous vaccination to the back side of female BAl BIc/c nude mice.When tumour reaches average-volume 160mm ³time, carry the mouse of tumour random as represented by be divided into 3 treatment group (n=8-10).APX004 is used, altogether 8 dosage with 2.5mg/kg or 0.6mg/kg 3 times/all intraperitoneal.Every 2 to 3 days record tumor size and body weight.Gross tumor volume is calculated: volume=(width) according to following equalities ²x length/2.As shown in Figure 8, in H460 tumor model, when the dosage of 2.5mg/kg, APX004 confirms significance Anti-tumor activity (p<0.01).APX004 does not show significance toxicity (unchanged relative to control group body weight).

Embodiment 7

The suppression of tumor growth in human melanoma A375 model

In order to determine APX004 effect for tumor growth in people A375 xenograft models, human melanoma tumor xenogeneic graft is by setting up being inoculated into the back side of female BAl BIc/c nude mice under people A375 cell skin.When tumour reaches average-volume 100mm ³time, carry the mouse of tumour random as represented by be divided into 6 treatment group.Intraperitoneal uses the Avastin of APX004 or 3mg/kg of various dosage, twice weekly, 3 weeks, altogether 6 dosage.Every 3 days record tumor size and body weight.Vernier vernier is used to measure the vertical dimension of tumour.Gross tumor volume is calculated: volume=(width) according to following equalities ²x length/2.Symbol and rod value are mean value+standard deviations.As shown in Figure 9, A375 tumor growth is suppressed in 3mg/kg APX004 significance.At this dosage, the Anti-tumor that APX004 shows similar Avastin is active.Data show that A375 model is less and depend on VEGF-KDR approach.Therefore, the effect of obvious dose-dependency Anti-tumor is not observed.

Embodiment 8

The suppression of tumor growth in people rectum cancer HT29 model

In order to determine the dosage of APX004 and the relation of effect in human colorectal cancer HT29 xenograft models, human colorectal cancer heterograft is by setting up being inoculated into the back side of female BAl BIc/c nude mice under people HT29 cell skin.When tumour reaches average-volume 100mm ³time, carry the mouse of tumour random as represented by be divided into 4 treatment group.3 times/all intraperitoneal use the APX0043 week of various dosage.Every 3 days record tumor size.Vernier vernier is used to measure the vertical dimension of tumour.Gross tumor volume is calculated: volume=(width) according to following equalities ²x length/2.Symbol and rod value are mean value+standard deviations.As shown in Figure 10, HT29 tumor growth is suppressed in 5mg/kg and 10mg/kgAPX004 significance.The dose-dependency Anti-tumor observing APX004 in this tumor model is active.

In brief, as shown in embodiment above, APX004 is the humanization IgG1 antibody with high KDR binding affinity, and it is inhibiting angiogenesis effectively.In humanizing process, framework and CDR humanization are farthest to reduce potential immunogenicity.

APX004 is neutralizing antibodies, and it blocks the combination of KDR and its part and the KDR phosphorylation suppressing VEGF-to induce and endothelial cell proliferation.Because APX004 and mouse KDR cross reaction, therefore directly can evaluate APX004 in mouse model in the body of people's tumor xenogeneic graft, and without the need to producing alternative antibody.APX004 is formed and tumor growth with the microvasculature being similar to the Anti-tumor effect Tumor suppression-induction of Avastin in many people tumor xenogeneic graft.APX004 seems more more effective than the alternative DC-101 of ramucirumab in tumor model in vivo, wherein needs the DC-101 of more high dosage (>40mg) to carry out Tumor suppression growth.Contrary with TKI, APX004 is more specificity KDR target agent (not suppressing other vegf receptors).Like this, estimate that APX004 will have less side effect of missing the target.

Contrary with Avastin, Avastin is only in conjunction with the one (only VEGF-A) in VEGF family part, and APX004 blocks all known VEGF potentially in conjunction with KDR.Compared to only blocking VEGF-A, this can have for the more deep restraining effect of tumor-blood-vessel growth, and can contribute to reversing by the excessive Avastin resistance caused of part.Therefore, APX004 potential improve tumour patient treatment (it produces a large amount of VEGF families part in tumor environment) and the resistance of anti-VEGF therapy can be overcome.

And, tumor model (A375 and HT-27) the expressing K DR of use in above-mentioned research 2/3rds, and VEGF-KDR approach can be used to grow as autocrine loop.Therefore, antibody-mediated Anti-tumor effect as herein described can comprise at least 2 kinds of modes of action: (i) anti-vasculogenesis and (ii) directly Tumor suppression growth (46,47).

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Above-mentioned various embodiment can combine to provide further embodiment, and all United States Patent (USP)s that are that relate in this specification and/or that list in request for data page, U.S. Patent Application Publication, U.S. Patent application, foreign patent, foreign patent application and the non-patent document are all incorporated to herein all by way of reference.If needed, the aspect of embodiment can revise to utilize various patent, application and open source literature concept to provide other embodiments.

Consider above-mentioned explanation, can be carried out these and other to embodiment and change.Usually, in the dependent claims, the term used should not be construed and claim is restricted to disclosed particular in the specification and in the claims, but is construed as the four corner comprising all possible embodiment and the equivalents required by these claims.Therefore, claims can't help disclosure restriction.

Claims (38)

1. in conjunction with antibody or its antigen-binding fragment of the separation of KDR, comprise: (i) comprises the variable region of heavy chain in the VHCDR3 district that the VHCDR1 district of SEQ ID NO:3 or 11 expression, the VHCDR2 district of SEQ ID NO:4 or 12 expression and SEQ ID NO:5 represent; And the variable region of light chain in the VLCDR2 district that (ii) comprises VLCDR1 district that SEQ ID NO:6 represents, SEQ ID NO:7 represents and the VLCDR3 district that SEQ ID NO:8 represents;

Or the variant of described antibody or its antigen-binding fragment, comprise heavy chain and variable region of light chain, except replacing up to 8 seed amino acids in described CDR district, described heavy chain is identical with variable region of light chain with the described heavy chain of (ii) with (i) with variable region of light chain.

2. the antibody of separation according to claim 1 or its antigen-binding fragment, wherein said variable region of heavy chain comprises the aminoacid sequence that SEQID NO:1 represents.

3. the antibody of separation according to claim 1 or its antigen-binding fragment, wherein said variable region of light chain comprises the aminoacid sequence that SEQID NO:2 represents.

4., in conjunction with antibody or its antigen-binding fragment of the separation of KDR, comprise: (i) comprises the variable region of heavy chain of VHCDR1, VHCDR2 and VHCDR3 of antibody as shown in figure 11; And (ii) comprises the variable region of light chain of the correspondence of VLCDR1, VLCDR2 and VLCDR3 of antibody as shown in figure 11;

Or the variant of described antibody or its antigen-binding fragment, comprise heavy chain and variable region of light chain, except replacing up to 8 seed amino acids in described CDR district, described heavy chain is identical with variable region of light chain with the described heavy chain of (ii) with (i) with variable region of light chain.

5. the antibody of separation according to claim 4 or its antigen-binding fragment, wherein said variable region of heavy chain comprises any one of the aminoacid sequence that SEQID NO:17-42 represents.

6. the antibody of separation according to claim 4 or its antigen-binding fragment, wherein said variable region of light chain comprises any one of the aminoacid sequence that SEQID NO:43-68 represents.

7., in conjunction with antibody or its antigen-binding fragment of the separation of KDR, comprise variable region of heavy chain, described variable region of heavy chain comprises the aminoacid sequence that SEQ ID NO:1 represents.

8. the antibody of separation according to claim 7 or its antigen-binding fragment, comprise variable region of light chain, described variable region of light chain comprises the aminoacid sequence represented with SEQ ID NO:2 and has at least 90% conforming aminoacid sequence.

9. the antibody of separation according to claim 7 or its antigen-binding fragment, comprise variable region of light chain, described variable region of light chain comprises the aminoacid sequence that SEQ ID NO:2 represents.

10., in conjunction with antibody or its antigen-binding fragment of the separation of KDR, comprise variable region of light chain, described variable region of light chain comprises the aminoacid sequence that SEQ ID NO:2 represents.

11. the antibody of separation according to claim 10 or its Fab, comprise variable region of heavy chain, described variable region of heavy chain comprises the aminoacid sequence that the aminoacid sequence represented with SEQ ID NO:1 has at least 90% conforming aminoacid sequence.

The antibody of 12. separation according to claim 1, wherein said antibody is humanized.

The antibody of 13. separation according to claim 12, wherein VH district comprises the aminoacid sequence that SEQ ID NO:9 represents, and VL district comprises the aminoacid sequence that SEQ ID NO:10 represents.

The antibody of 14. separation according to claim 1, wherein said antibody is selected from single-chain antibody, ScFv, the univalent antibody of shortage hinge area and miniantibody.

The antibody of 15. separation according to claim 1, wherein said antibody is Fab or Fab ' fragment.

The antibody of 16. separation according to claim 1, wherein said antibody is F (ab ') 2 fragments.

The antibody of 17. separation according to claim 1, wherein said antibody is whole antibody.

The antibody of 18. separation according to claim 1, comprises human IgG constant domain.

The antibody of 19. separation according to claim 18, wherein said IgG constant domain comprises IgG1 CH1 structural domain.

The antibody of 20. separation according to claim 18, wherein said IgG constant domain comprises IgG1 Fc district.

21. the antibody be separated or its antigen-binding fragment, the combination of antibody competition and people KDR described in the antibody of described separation or its antigen-binding fragment and claim 1.

22. 1 kinds be separated antibody or its antigen-binding fragment, the antibody of described separation or its antigen-binding fragment with the KD of 5.3x10-11M or lower in conjunction with KDR.

23. 1 kinds of antibody or its antigen-binding fragment be separated, the antibody of wherein said separation or its antigen-binding fragment:

A. the combination of blocking VEGF and KDR;

B. KDR signal transmission is suppressed;

C. inhibition of endothelial cell proliferation;

D. Tumor suppression vasculogenesis;

E. inhibition tumor cell growth;

F.a.-e. any one of or multinomial combination.

24. the antibody of separation according to claim 23 or its antigen-binding fragment, the combination of the antibody of wherein said separation or its antigen-binding fragment blocking VEGF and KDR; Suppress KDR signal transmission; Inhibition of endothelial cell proliferation; Tumor suppression vasculogenesis; Grow with inhibition tumor cell.

25. 1 kinds of antibody or its antigen-binding fragment be separated, the antibody of described separation or the direct Tumor suppression growth of its antigen-binding fragment.

26. the antibody of claim 1,4 or 21, separation according to any one of 22-25, wherein said antibody and mouse KDR or non-human primates KDR cross reaction.

The polynucleotide of 27. 1 kinds of to encode according to claim 1 or claim 13 antibody of described separation or separation of its antigen-binding fragment.

28. 1 kinds of expression vectors, comprise the polynucleotide be separated described in claim 24.

29. 1 kinds of host cells be separated, comprise carrier according to claim 28.

30. a composition, comprise the antibody be separated according to any one of claim 1,13 or 21-26 or its antigen-binding fragment of physiologically acceptable vehicle and pharmaceutical effective amount.

Composition described in 31. claims 30 is in the purposes of the medicine of the cancer for the preparation for the treatment of patient.

Composition described in 32. claims 30 is in the purposes to the medicine of the cancer that VEGF or KDR of distortion expresses or activity is relevant that has for the preparation for the treatment of patient.

33. purposes according to claim 32, wherein said cancer is selected from: angiosarcoma, renal cell carcinoma, gastrointestinal cancer, transitivity stomach or gastroesophageal junction gland cancer, breast cancer, bladder cancer, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, nonsmall-cell lung cancer, neuroblastoma, ovarian cancer, melanoma, recurrent glioblastoma multiforme and leukemia.

Composition described in 34. claims 30 is in the purposes of the medicine of the inflammatory diseases for the preparation for the treatment of patient.

Composition described in 35. claims 30 is in the purposes of the medicine of the rheumatoid arthritis of preparation treatment patient.

Composition described in 36. claims 30 is in the purposes of the psoriatic medicine of preparation treatment patient.

The purposes of composition described in 37. claims 30 in the medicine of the disease of the vasculogenesis-mediation of preparation treatment patient.

38. purposes according to claim 37, wherein said patient suffer from age-relevant macular degeneration.